key: cord-0034153-fxtwkm9j
authors: nan
title: Scientific Abstracts
date: 2013-12-30
journal: Reprod Sci
DOI: 10.1177/1933719113482088
sha: 3c432fc46909d5cdeb51898f272b7de105527f0e
doc_id: 34153
cord_uid: fxtwkm9j

nan

Intergenerational Inheritance of Cardiovascular Disease Risk Induced by Chronic Fetal Hypoxia. Y Niu, BJ Allison, AD Kane, CM Lusby, EJ Camm, DA Giussani. Physiology, University of Cambridge, United Kingdom. The environment during early development can induce intergenerational non-genomically determined phentotypic changes in mammals. Studies have focussed on maternal behaviour, nutrition or endocrine manipulation, reporting, for instance, transgenerational effects of maternal low protein nutrition (Zambrano et al. J Phys 2005; 566:225; Torrens et al. BJN 2008; 100:760) or of pregnancies exposed to excess glucocorticoids (Drake et al. AJP 2005; 288: R34; Iqbal et al. Endo 2012; 153(7):3295; Long et al. AJOG 2012; 207(3) :203. e1-8) or of altered maternal attention to the neonate (Francis et al. Science1999; 286:1155) . The most common consequence of complicated pregnancy is chronic fetal hypoxia and we have reported that this environment can programme cardiovascular dysfunction (Giussani et al. PLoS One 2012; 7(2) :e31017). However, whether generational inheritance of disease risk can be induced by chronic fetal hypoxia is completely unknown. Methods: Pregnant Wistar rats (n=24, F0) underwent normoxic (N: 21% O2) or hypoxic (H: 14% O2) pregnancy from days 6-20 of gestation. This model does not affect maternal food intake. At 12 weeks, F1 males and F1 females were mated with partners from outside the colony to produce an F2 generation from both paternal and maternal lineages, which did not experience hypoxia. In both F1 and F2, cardiac (Langendorff) and femoral vascular (wire myography) function was assessed at 4 months of age in male offspring. Results: F1 offspring of hypoxic pregnancy showed enhanced left ventricular end diastolic pressure (LVEDP), impaired cardiac recovery from ischaemiareperfusion (I/R) and peripheral vascular endothelial dysfunction (Fig. 1) . The impaired cardiac recovery from I/R was transmitted to the F2 via the paternal line. The endothelial dysfunction was transmitted to the F2 via the maternal line ( Fig. 1) . Conclusions: We show intergenerational programming of cardiac and vascular dysfunction in rats following pregnancy complicated by chronic fetal hypoxia. The phenotypes in the F2 offspring from the paternal and maternal lineage suggest different mechanisms of generational transmission. Supported by the BHF.

Translating the Transcriptome To Develop Antenatal Treatments for Fetuses with Down Syndrome. Faycal Guedj, 1 Dustin Hines, 2 Jeanine C Foley, 2 Philip G Haydon, 2 Diana W Bianchi. 1 1 Mother Infant Research Institute, Tufts Medical Center, Boston, USA; 2 Neuroscience, Tufts University, Boston, USA. Background: Using a systems biology approach, we identified oxidative stress as the major functional abnormality in cell free RNA from amniotic fluid supernatant in fetuses with Down syndrome (DS) vs. euploid controls. (PNAS 2009; 106: 9425) . We hypothesized that apigenin, an FDA-approved antioxidant small molecule identified by the Connectivity Map database (www. broadinstitute.org/cmap), could potentially suppress oxidative stress in DS, thus enhancing neurogenesis. We tested our hypothesis in human cells and the Ts1Cje mouse model of DS. Methods: For in vitro studies, we evaluated the protective effect of apigenin on amniocytes from mid-trimester euploid (n=5) and karyotype confirmed DS (n=5) fetuses. We used the COMET assay to measure oxidative stress by calculating the average percentage of DNA in the comet tail (% DIT) in 300-500 cells. For in vivo studies, Ts1Cje mice and littermate controls were randomized to receive 200-250 mg/kg/day of apigenin in chow or only chow, starting with their mother's mating and continuing until 8-10 weeks of postnatal age. Exploratory behavior and locomotor activity were tested in an open-field (42 cm x 42 cm x 40 cm) for 60 min. The average speed and total distance traveled were recorded using video tracking.

Asn680Ser. Aimee Seungdamrong, Peter G McGovern, Andrea Wojtczuk, Laura T Goldsmith. Obstetrics, Gynecology, and Women's Health, New Jersey Medical School -UMDNJ, Newark, NJ, USA. Despite years of experience using FSH for ovarian stimulation, variability in ovarian response to FSH, even in young women with good prognosis for pregnancy success, remains unexplained. The FSH receptor (FSHR) single nucleotide polymorphism (SNP), which results in a change in amino acid 680 (intracellular domain) from asparagine to serine, affects ovarian response to FSH administered during controlled ovarian stimulation (COS) for in-vitro fertilization (IVF). (1, 2) We now examine live birth rates for IVF and present exciting differences in live birth rates associated with presence of FSHR SNP Asn680Ser. All IVF patients younger than 35 years old were screened for the study between 10/2009 and 12/2011. DNA was extracted from blood samples and genotyped with a Taqman SNP assay (C_2676874_10). Pregnant patients were interviewed by telephone at the time of expected delivery for birth information. Live birth rates were calculated as live births per IVF cycle and compared by Fisher's exact test. Seventy-nine of 194 patients agreed to participate. Four patients cancelled their IVF cycles for non-medical reasons. The genotype frequency was 0.32 (24/75) for the wild type (Asn/Asn), 0.48 (36/75) for the heterozygote (Asn/ Ser), and 0.20 (15/75) for the homozygote SNP (Ser/Ser). Median patient ages, 32.0 (Asn/Asn), 32.8 (Asn/Ser), and 31.6 (Ser/Ser) years old, were not different (p=0.26). We compared live birth rates between homozygous wild type patients, 21% (5/24) , and patients heterozygous and homozygous SNP, 49% (21/51). The live birth rates were significantly different, p-value = 0.0243. In women younger than 35 years old, presence of the FSH receptor SNP Asn680Ser, is associated with a significantly higher live birth rate than in women homozygous for the wild type FSHR. This FSHR SNP is a potentially clinically important mediator of variability in live birth rates in young, infertile patients. and a delay in parturition. Conversely RU486 treated animals demonstrate an upregulation of uterine BIP, a reciprocal downregulation of both CHOP and CASP3 and the onset of preterm birth. These data suggest that P4 regulates the timing of parturition by regulating the ERSR through PR action. In conclusion we speculate that activation of the uterine ERSR in the pregnant mouse uterus modulates CASP3 activation in a P4 and PR dependent manner thereby maintaining uterine quiescence. In addition we suggest that resolution of the ERSR via a withdrawal of PR action at term restores uterine contractility to allow for the onset of labor.

0mM Ca2+ and 2mM EGTA and pressurised in steps, and vessel dimensions recorded to construct stress-strain relationships to assess passive wall stiffness. Extracellular matrix (ECM) and adhesion molecule gene expression was determined in uterine arteries by QPCR and PCR Arrays. Fetal and placental weights were recorded on day 17.5 pregnancy. Results : Genotype had no effect on uterine artery compliance in 5 month mice at all time points measured (NP, day 12.5 and day 17.5 pregnancy). However, pregnant 8 month Rln-/-mice had significantly stiffer uterine arteries compared with Rln+/+ mice on day 17.5 pregnancy. Relaxin deficiency resulted in significantly stiffer mesenteric arteries at all time points measured in both 5 and 8 month mice. There was no effect of RLX deficiency on uterine artery Col1a1 and Col3a1 expression. We identified novel ECM and adhesion molecules that were significantly altered in uterine arteries of 8 month Rln-/-pregnant mice, including matrix metalloproteinases, intergrins and Eln. Fetal weight was significantly reduced by 18% in Rln-/-mice compared to Rln+/+ mice, whereas placental weight was unchanged. Summary: These data demonstrate that RLX deficiency compromises maternal arterial adaptation during pregnancy and impairs fetal growth.

Evidence In response to preovulatory LH surge, LH receptor (LHR) undergoes downregulation followed by a transient refractory period. This process can be mimicked by the administration of a pharmacological dose of LH/hCG. We have reported that downregulation is mediated by an RNA binding protein (LRBP). Purification and sequence analysis established LRBP as being mevalonate kinase (MVK) . Recently it was shown that miR122, a liver specific microRNA, is able to regulate MVK expression. The present study examined the potential role miR122 in LHR mRNA expression using a rodent model. Initial experiments focused on establishing the temporal relationship between miR122 expression and hCG-induced LHR mRNA downregulation. On day 5, PMSG-hCG-primed rats were treated with a single dose (50 IU) of hCG to induce LHR mRNA downregulation. miR122 expression was determined by real-time PCR at different time intervals after hCG injection. The expression of miR122 increased at 30 min (147 % vs. control, p< 0.05, n=7) and peaked around 1-2h (150-187 % vs. control, p< 0.05, n=7) following hCG treatment. An increase in LRBP levels was seen at 2h after hCG treatment and reached maximum level by 4h. The expected downregulation of LHR mRNA was seen after 4h and showed a time dependent decline reaching maximum downregulation by 12h.

These results clearly showed that downregulation of LHR mRNA expression was preceded by increases in miR122 followed by LRBP, consistent with a role for miR122 as an upstream initiator of LHR downregulation. The expression of miR122 and its upregulation following hCG treatment was then confirmed using fluorescent in situ hybridization. Furthermore, treatment with a LNA conjugated miR122-specific antagomir, prior to hCG injection, inhibited the hCG-induced increases in LRBP. Since SREBP-1a and SREBP-2 belong to a family of transcription factors known to regulate the expression of LRBP, their potential role as downstream activator/s of miR122 was examined. Results showed that the active form of SREBP-1a was increased by 1h following hCG treatment. The increase was time dependent showing maximum effect by 2-3h and declining by 4h. The same trend was observed for SREBP-2, where the activation was observed after 1h of hCG, and sustaining up to 4h. These results provide strong evidence to support the role of miR122 in mediating the downregulation of LHR expression in the ovary. We demonstrated that intraplacental gene therapy with adenoviral insulin growth factor (Ad-hIGF1) corrects impaired birth weight and glucose regulatory response in a mouse model of placental insufficiency. Fetal programming of liver insulin resistance has been proposed as a major mechanism. In addition, sex-dependent differences in adult body weight and the development of diabetes due to placental insufficiency are not clearly elucidated yet. We hypothesized that intra-placental Ad-hIGF1 reprograms the expression of genes involved in insulin signalling pathways such as PIK3R1, SLC2A2 (GLUT2), USP2, and AKT2 in female liver of a mouse model of placental insufficiency.

Laparotomy was performed on pregnant C57BL/6J mice at gestational day 18 and pups were divided into 3 groups. Control: Sham operated; IUGR: surgically induced by ligation of a branch of the uterine artery; IGF1Treated: intra-placental injection of Ad-hIGF1 after ligation. Pups were delivered on day 20, crossfostered to CD1 mice. At 32 weeks of age female offspring livers were dissected, weighed and RNA extracted for gene expression analysis by qPCR. Data were analysed using ANOVA.

Adult Female IUGR mice livers were significantly heavier (2.96 ±0.4 vs. 1.88± 0.31 vs. 2.1 ± 0.41, n= 5, p= 0.01) than SHAM and were restored to normal in the IGF1 treated group. AKT2 gene expression was significantly lower (0.63 ±0.09 vs. 1.2 ± 0.63 vs. 1.12 ± 0.6, n= 5, p= 0.05) compared to SHAM, but was restored to normal in the IGF1 treated group. No significant differences in liver gene expression of PIK3R1, USP2 and GLUT2 were seen between groups.

Intra-placental gene transfer of Ad-hIGF1 reprograms AKT2 gene expression in adult female IUGR liver and restores normal liver weight. These changes may represent a mechanism of in utero therapy reprogramming fetal liver to attenuate fetal predisposition to adult onset of diabetes.

Expression of Ovarian Cancer Antigens in Fetal and Adult Ovarian and Fallopian Tube Epithelium. Kara K Hoppe, Elizabeth M Swisher. Obstetrics & Gynecology, University of Washington, Seattle, WA, USA. Background: CA125 and HE4 are two antigens expressed in nearly all serous ovarian carcinomas; mesothelin and WT1 are expressed in a lower percent of cases. The variation of expression of these antigens in normal adult and fetal fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) is not known. We hypothesized that CA125 and HE4 are expressed in normal adult women at greater levels in FTE than OSE, and that tissue specific expression differences will be more pronounced in older women. We sought to determine the expression pattern of four different human ovarian cancer antigens (HE4, CA125, mesothelin, WT1) in normal adult and fetal FTE and OSE. We then compared their expression pattern in age matched FTE from subjects that were cancer free to those with serous ovarian or tubal carcinoma. Methods: Protein expression of HE4, CA125, mesothelin, and WT1 were assessed semi-quantitatively by immunohistochemistry on formalin fixed paraffin embedded sections from FTE and OSE of fetal and benign adult subjects. We evaluated fetal and adult cases stratifying by trimester and adult age. In addition, we performed the same analysis on age matched specimens with serous ovarian or tubal carcinoma. Protein expression was scored for intensity of staining and percent of positive epithelial cells. Results: HE4 was strongly expressed in all fetal FTE and OSE. However, in women without cancer, HE4 expression in OSE and FTE decreased with age starting in the perimenopausal category. However, in women with cancer, HE4 expression was retained in FTE regardless of age. In contrast, mesothelin was expressed in adult FTE and OSE but less in fetuses, and increased with gestational age. CA125 was expressed in FTE of all fetal and adult samples, but was not expressed in early fetal OSE. WT1 was strongly expressed in all fetal and adult FTE and OSE. Conclusions: HE4 and WT1 are fetal OSE antigens and CA125, HE4 and WT1 are fetal FTE antigens. HE4 expression decreases with increasing age in normal adult FTE in women without cancer, but is retained in the normal FTE of women with serous ovarian or tubal carcinoma. HE4 FTE expression might be an early biomarker to identify women at risk for serous carcinoma. A better understanding of normal expression of tumor antigens in at risk epithelium over the female lifespan may provide insight into why some women develop antibodies to these antigens and how that influences cancer development.

Role of Estrogen Receptor Alpha Enhancer RNAs in Estrogen-Regulated Transcriptional Responses in Breast Cancer Cells. Shino Murakami, 1 Nasun Hah, 2 Anusha Nagari, 1 W Lee Kraus. 1 Estrogen signaling controls a wide array of important physiological and pathological responses, and has been implicated in the onset and progression of breast cancers. Many of the effects of estrogens are mediated by the direct or indirect binding of estrogen receptors (ERs) to sites across the genome, which ultimately leads to regulated transcriptional responses at nearby or distallylocated target genes. Emerging evidence has indicated that the binding of transcription factors, such as ERs, to genomic DNA results in the bi-directional synthesis of short, non-coding RNAs in the vicinity of the binding sites. The function of these enhancer RNAs (eRNAs) is not well understood. We have used Global Run-On and sequencing (GRO-seq), a genomic method that maps the location and orientation of transcriptionally active RNA polymerases on the genome, in combination with a set of a novel bioinformatic approaches, to characterize the eRNAs arising from ER binding sites in ER-positive MCF-7 human breast cancer cells in response to estradiol. Using this approach, we identified more than 700 eRNAs originating at distal ER binding sites (i.e., >10 kb from gene promoters) in MCF-7 cells. We are now examining the mechanisms by which these transcripts are regulated, their effects on ER binding and chromatin-dependent gene loops, and the regulation of neighboring estrogen-regulated target genes. In addition, we are using these analyses to identify and map novel active enhancers in MCF-7 cells. These studies are revealing more complexity in the estrogen-regulated transcriptome than was previously known. Understanding the functions of eRNAs may aid in the discovery and development of better prognostic indicators and therapeutic targets in breast cancers, as well as a greater understanding of estrogen signaling in other estrogen target tissues. Proliferating cells utilize aerobic glycolysis to generate ATP and to allow the cell to form biomass and reducing equvilant. This metabolic phenotype is largely regulated by the expression and alternative splicing of pyruvate kinase M into PKM1 or PKM2 isoform. OBJECTIVES: Our objectives were to study the effects of 17β-estradiol ((E2) on PKM2 expression and its alternative splicing in primary human endometrial stromal cells and to investigate the potential biological functions of PKM2 in E2-induced cell proliferation. Design: We investigated the effects of E2 on PKM2 expression and posttranslational modification using immunoblotting, qRT-PCR, Co-IP and cell fractionation assays. The metabolic effects of PKM2 expression were evaluated by the assessment of glucose consumption lactate production rates, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).

Our results show that E2 up-regulates PKM2 with concomitant down regulation of PKM1 expression in endometrial cells through the c-Myc-hnRNPs axis. In addition, E2 enhances PKM2 phosphorylation, nuclear translocation and shifts its quaternary structure from tetrameric form to monomeric form. PKM2 expression directs glucose metabolism into pentose phosphate pathway. In addition, PKM2 physically interact with E2-liganded ER-α and act as ERα coactivator to promote the expression of cell cycle-regulating genes. Pretreatment of endometrial cells with small-molecule PKM2 activators abrogated the effects of E2 on PKM2 and ameliorated E2 mitogenic response in the endometrium. CONCLUSIONS: E2 up-regulates PKM2 expression and induces PKM2 phosphorylation and nuclear translocation in endometrial cells. These effects on PKM2 are critical for the E2 mitogenic response in endometrial cells. Conclusions HLA polymorphisms are associated with anti-tumor immunity and may influence clinical outcomes in EOC. The HLA-DRB1*13 and HLA-DRB4*01 alleles are significantly associated with the development of humoral immunity to ESO, but not necessarily with survival in EOC patients, most likely as a result of immunologic tolerance. Although, a relationship between the HLA-A*01 subtype and survival was seen, the underlying mechanism(s) will need to be determined. This study supports innovative vaccination strategies targeting HLA class I and II ESO epitopes, portending to a broad immunologic response. Background Epithelial progenitor cells have been identified in human endometrium and are thought to mediate endometrial regeneration. The canonical Wnt pathway is important in adult stem cell self-renewal, and has roles in estrogen-induced endometrial regeneration. A recent gene profiling study showed differential expression of 22 Wnt-associated genes between purified pre-and postmenopausal endometrial epithelial cells. 1 Further analysis identified an adhesion molecule (Adm2) as a candidate marker for human endometrial epithelial progenitor cells. The aim of this study was to examine the expression of 12 Wnt-associated genes in human endometrial Adm2+ and Adm2-epithelial cell subpopulations.

Endometrial epithelial cells were obtained from 11 hysterectomy samples from reproductive aged women not taking hormones. Tissue was dissociated to single cells and sorted into enriched and depleted fractions using Adm2 antibody coated magnetic beads. RNA was extracted and qPCR undertaken for the 12 Wnt-related genes using published primers. 1 Immunofluorescence for selected Wnt-related products was done on full thickness endometrium.

We found no differential expression of the 12 genes between proliferative (n=5) and secretory stage (n=6) samples. Neither was there any difference between enriched and depleted epithelial progenitor cell populations in the proliferative stage. Surprisingly, we found significantly higher expression (P<0.05) of WNT10A ligand, FZD9 receptor, GSK3B kinase, SOX9 transcription factor, and Wnt targets TLE1, SMO, MMP7 and VANGL2 in the Adm2-differentiated cells compared to the putative Adm2+ progenitor cells. Regulators of Wnt signalling, AXIN2 and β-catenin localised to both Adm2+ and Adm2-populations in pre and post-menopausal endometrium. Unexpectedly we found the potentially Wnt-regulated transcription factor SOX9 specifically localised to the cilia of Adm2-epithelial cells.

Our data suggest that Wnt-related genes are more highly expressed in differentiated endometrial epithelial cells rather than putative epithelial progenitor cells, indicating their possible role in mediating differentiation of epithelial cells into secretory glands. Past exposure to pelvic Chlamydia trachomatis (Ct) infection is a major risk factor for tubal ectopic pregnancy (EP), but the underlying mechanism of this association is not completely understood. In the human uterus, the putative 'window of receptivity' to the embryo in the mid-luteal phase of the menstrual cycle is accompanied by increased endometrial epithelial expression of the integrins alpha (1)beta (1), alpha(4)beta (1) and alpha(v)beta (3) . Unlike the uterus, all five integrin receptivity markers are constitutively transcribed and translated throughout the menstrual cycle in the epithelium of the FT. We hypothesized that past exposure to Ct predisposes to tubal implantation and EP by altering tubal integrin expression. We examined integrin expression in (a) FT collected from women with EP, and FT from non-pregnant women with and without serological evidence of past exposure to Ct (determined by serum levels of PgP3 protein); (b) human FT explants and immortalized epithelial cells (OE-E6/E7) exposed to short-term (8-24 hours) infection with Ct in-vitro; and (c) in oviduct isolated from a murine model mimicking past exposure of human FT to Ct infection. Expression of all 5 integrin subunits was higher (P<0.01) in FT from women with EP (n=18) compared to mid-luteal FT collected from non-pregnant women (n=19). Expression of integrins beta(1) and beta (3) was also higher (P<0.05) in FT from women with evidence of past exposure to Ct infection (n=18) compared to those without (n=19). Integrin expression was not significantly altered in FT explants (n=5) or in OE-E6/E7 cultures (n=6) after short-term infection with Ct in vitro. However, female C57BL/6 mice infected intra-vaginally with Ct (serovar E) and confirmed to have cleared the infection by day 30 post infection (n=6) showed increased (P<0.05) expression of oviductal integrin beta(1) compared to sham-infected controls (n=6) on day 63. We propose that past exposure to Ct infection increases tubal integrin expression thereby predisposing the microenvironment to ectopic implantation. Introduction miRNAs are short lenght non-coding RNAs expressed in a celular specific manner acting as negative regulators of gene expression for essential processes, such as celular differentiation & proliferation. Different laboratories have investigated the miRNA's signature of human endometrial receptivity with consensus about the up-regulation of hsa-miR-30d. This potential biomarker requires basic research to understand its role on endometrial receptivity.

The aim of this work is to analyze the transcriptomic effects of the overexpression of hsa-miR-30d in primary cultures of human endometrial epithelial cells (hEEC) .

Primary cultures of hEEC (n=4) obtained from biopsies at the day of LH peak were transiently transfected using HiPerfect with hsa-miR-30d Mimic or Scramble as a control. After 72 hours, RNA was extracted and quality assesed in the Agilent bioanalizer. Transfection efficiency determined by qPCR. Next, Agilent Human GE 4x44 v2 microarrays were performed, data normalized, log-transformed and analyzed using non-paired t-test for the differentially expressed genes (p-value<0.05 & Fold-change > |1.4|) between conditions using MeV software. Eight differentially expressed genes were confirmed by qPCR.

Pathways and Biological processes were assessed using DAVID & Cytoscape.

Hsa-miR-30d transfection induced ∼90-fold (89.95 ± 47.23) expression in transfected EEC vs control and produced a significant modification of the EEC transcriptome afecting the expression of 265 genes (94 over-expressed vs 171 under-expressed). Relevant up-regulated genes were interleukins (IL12A, IL23A and IL1RAP), adhesion molecules such as CADM2, CEACAM8, FZD9, PCDH8 and PTGER4. Relevant down-regulated molecules were FOS and TGFB3, secreted molecules such as PRL, MYBL2 a validated target for hsa-miR-30d, and LIF, a cytokine highly secreted by the glandular hEECs during the secretory phase.The most relevant pathway was the "cytokine-cytokine receptor interaction" and the GO term "Reproduction" highly represented.

In this work we have demonstrated that ectopic expression of hsa-miR-30d in hEEC induce transcriptomic modifications related to the immune response, cytokine production and proliferation that would potentially affect endometrial receptivity. *The first and second author have contributed equally.

In Vascular endothelial growth factor (VEGF) increases mitosis, angiogenesis, and permeability in human endometrium via a mitogen-activated protein kinase pathway initiated in adults primarily by activation of VEGF receptor 2 (VEGFR2). However, little is known about the regulation of VEGFR2 in the endometrium, and no studies have observed the effects of in vivo estradiol (E) on the expression of VEGFR2 in human endometrium. We evaluated the effect of E on VEGFR2 protein in human endometrial biopsies under controlled conditions of hormonal exposure. Methods: Endometrial biopsies were collected from participants during the proliferative phase of a spontaneous cycle on day 11 (CD11). Each participant then underwent ovarian suppression therapy with leuprolide depot (3.75mg IM) on CD25 for a minimum of 10 days. Following suppression, each participant was randomized to receive either 1)transcutaneous E replacement to mimic spontaneous cycle serum E, or 2)placebo (Plac). Successful E suppression and/or replacement was confirmed with serum E levels. Endometrial biopsies and serum E levels were again performed 2 and 11 days after randomization. Immunoblotting, following by densitometry, for VEGFR2 was performed on the endometrial biopsy lysates using GAPDH as a control. Student's t-test was used to determine significance. Data is expressed as mean±SE, p<.05 was accepted for significance. Results: 7 participants were studied, 4 randomized to E replacement and 3 to placebo. Women were aged 27±4 y, healthy and cycling regularly (26-35d), and of normal BMI (24±3kg/m2). The two groups did not differ in age or BMI. Eleven days of E replacement increased VEGFR2 signal intensity to 4-fold that of the Plac group (p=0.02) and levels of VEGFR2 were comparable between spontaneous CD 11 and E day 11 (p=.34). Within the E group, VEGFR2 signal was 4-fold higher on E day 11 vs E day 2 (p=.03). Serum E levels and VEGFR2 signal intensity were significantly correlated for all groups and cycle days (r=0.58, p=0.01). Conclusions: In this human in vivo trial we found a direct relationship between E and VEGFR2. Our study suggests that E is necessary for VEGFR2 expression, both physiologically and after ovarian suppression. Manipulation of the VEGFR2 signaling pathway through experimental changes in E exposure demonstrates a strong association between serum E levels and VEGFR2 protein in human endometrium.

The Impact of Estradiol on uNK-Mediated Endometrial Angiogenesis. Douglas A Gibson, Hilary OD Critchley, Philippa TK Saunders. MRC Centre for Reproductive Health, University of Edinburgh, Edinburgh, United Kingdom. Background: The human endometrium is a sex steroid target organ and during the secretory phase undergoes morphological transformation in preparation for implantation characterized by decidualization of endometrial stromal cells (ESC), vascular remodeling and an increase in number of resident immune cells. Uterine natural killer (uNK) cells accumulate in perivascular areas and are reported to secrete pro-angiogenic factors including vascular endothelial growth factor (VEGF) and placental growth factor (PLGF). Studies in mice have revealed that estrogen is a critical determinant of uterine receptivity during the window of implantation. We have recently obtained evidence that increased biosynthesis of estradiol (E2) parallels decidualization of ESC. In the present study we have used isolated uNK cells to investigate the impact of E2 on uNK-mediated endometrial angiogenesis in order to explore the role of this steroid in cell-cell interactions within the endometrial microenvironment. Methods: First trimester decidual samples (n=4) were obtained from women undergoing surgical termination of pregnancy (8-12 weeks gestation). uNK cells were isolated from decidua using the MACS system. uNK cells were treated with vehicle control and E2 with and without the anti-estrogen Fulvestrant (ICI 182, 780) . The impact of E2 on expression of angiogenic factors in uNK cells was investigated using human angiogenesis RT2 profiler PCR array (SABiosciences). The impact of E2-treated uNK-conditioned media (CM) on human endometrial endothelial cells (HEEC) angiogenesis was investigated using an endothelial network formation assay. Results: E2 treatment of uNK cells was associated with increased expression of mRNAs encoding CCL2, CXCL10 and interferon-γ. E2 treatment significantly decreased expression of transforming growth factor β1 (p=0.028), interleukin-1β (p=0.039) and the adhesion molecule thrombospondin 1 (p=0.02). Interestingly, expression of mRNAs encoding VEGFC and PLGF were not detected, while concentrations of VEGFA mRNA was not affected by E2 treatment. Incubation of HEEC with uNK-CM resulted in a significant increase in angiogenesis (network formation) using media from E2-treated uNK cells compared with those incubated in control media (P<0.05). Conclusion: E2 enhances the secretion of pro-angiogenic factors by uNK cells and therefore contributes to changes in the uterine microenvironment during the secretory phase in preparation for implantation. Chronic intrauterine hypoxia (HPX) reduces fetal growth and alters organ function by mechanisms associated with oxidative stress. Cell damage may be mediated by mechanisms that disrupt energy production by the mitochondria. We previously reported that prenatal HPX decreases mitochondrial enzyme activity and protein expression of cytochrome c oxidase (CCO), the terminal electron acceptor of complex IV, of fetal guinea pig (GP) heart ventricles. Since prenatal HPX has been shown to induce lasting changes in the cardiovascular system of the offspring, we propose that cardiac mitochondrial dysfunction of HPX fetuses is sustained in the offspring. To test this, we measured effects of chronic prenatal HPX on mitochondrial protein and enzyme activity levels of offspring heart ventricles. Methods. Pregnant GPs were exposed to either normoxia (room air, NMX, N=9) or HPX (10.5%O 2 , N=6) during the last 14d of pregnancy. Fetal GPs were allowed to deliver and both groups were housed in room air. At 90d of age, NMX and HPX offspring were anesthetized, and hearts excised for analysis. Left (LV) and right (RV) ventricles were dissected and frozen in liquid N 2 and stored at -80°C. Mitochondrial fractions of offspring heart ventricles were isolated and activity levels of CCO and MCAD (medium chain acyl dehydrogenase) assayed, protein expression of nuclear-and mitochondrial-encoded CCO subunits (COX4 and 1, respectively) quantified by Western, and mRNA expression of nuclear-encoded CCO subunits (4.1, 4.2 and 5b) , PGC1α (peroxisome proliferator-activated receptor coactivator) and NRF1 (nuclear respiratory factor) measured by real time RT-PCR. Results. Prenatal HPX decreased (P<0.05) CCO activity (CC/min/mg protein) in both LV (by 26.1%) and RV (by 16.3%) but had no effect on MCAD activity (reduced ferrocenium/min/mg protein) of offspring hearts. Consistent with these results, hypoxia decreased (P<0.05) LV COX4 (by 57%) and COX1 (by 51%) protein levels and reduced PGC1α (by 14%), COX5b (by 21%) and 4.1 (22%), but not 4.2 mRNA expression. Conclusion. Prenatal HPX downregulates cardiac mitochondrial function in GP offspring by sustained decreases in transcriptional regulation of mitochondrial protein subunits. This suggests that offspring exposed to prenatal HPX may be at risk of cardiac mitochondrial dysfunction, predisposing them to contractile dysfunction and heart failure.

from Hpx or Nmx dams at PN6, 9 and 14, plasma leptin was analyzed by ELISA. Data is presented as mean±SEM. . Actin has multiple crucial cellular functions and is a nitrative target; nitration of a single critical tyrosine residue can alter protein structure/function in a wide range of tissues/ organs (Amino Acids 42:65). One example is the transgenic mouse model of familial amyotropic lateral sclerosis where over-nitrated actin is found in presymptomatic spinal cord motor neurons (J Biol Chem 280:1304). We hypothesized that NT expression would be upregulated in the frontal cortex of IUGR baboon fetuses compared to controls. Methods: Pregnant baboons were fed as ad lib controls (CTR; n = 7) or 70% CTR global diet from 0.16 -0.9 gestation (IUGR n = 6) with fetuses recovered at c-section. NT peptide expression was determined by immunohistochemistry with image analysis for % area immunostained (Fraction) in neurons and astroglia. Statistical analysis was via Student's t-test with *p< 0.05 and data expressed as mean ± SEM. Results: At 0.9 gestation, fetal body wt was significantly (p< 0.05) decreased with IUGR (CTR -808.8 ± 40.5 vs. IUGR -668.5 ± 33.4g), while NT peptide expression was significantly (p< 0.05) increased with IUGR in both gray and white matter (Fig. 1 ). Conclusions: The observed increases in NT expression in astroglia and neurons with IUGR suggest that IUGR results in a pro-inflammatory state as opposed to post-natal calorie restriction which is known to reduce inflammation in animals including primates (Exp Gerontol 42:709). 

Antenatal Glucocorticoid Treatment Affects Long Term Hippocampal Development in Mice. CW Noorlander, 1 D Tijsseling, 2 PNE de Graan, 3 WB de Vries, 4 JB Derks, 2 GHA Visser. 2 1 Centre for Substances and Integrated Risk Assessment (SIR), National Institute for Public Health and the Environment (RIVM); 2 Obstetrics, University Medical Center Utrecht; 3 Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht; 4 Neonatology, University Medical Center Utrecht, Netherlands. Synthetic glucocorticoids (GCs) are administered to pregnant women at risk for preterm delivery, to enhance fetal lung maturation. The benefit of this treatment is well established, however caution is necessary because of possible unwanted side effects on development of different organ systems, including the brain. In the brain, the hippocampus is particularly sensitive to GCs, because of high expression of corticosteroid receptors. Therefore, we analyzed the effects of a single antenatal dexamethasone (dex) treatment on the development of the mouse hippocampus. Methods Pregnant mice were treated at embryonic day (E) 15.5 with a clinically relevant dose of dex (0.4 mg/kg) or an equal volume of saline. Pups were sacrificed and studied at seven different time points, n=8 per treatment group per time point. We investigated the effects of dex treatment on hippocampal volume, number of neurons in the CA and dentate gyrus, apoptosis and proliferation in the hippocampus at E16,E18, postnatal day 0 (P0), P5,P10,P20 and adult stage (6 months).

Results Dex treatment significantly reduced body weight, hippocampal volume and the number of neurons in the hippocampus transiently during development, these effects were no longer detected at adulthood. Dex treatment increased the number of apoptotic cells in the hippocampus until birth, postnatally no effects of dex treatment on apoptosis were found. Until birth, dex treatment decreased the number of proliferating cells in the subgranular zone of the dentate gyrus. The number of proliferative cells was increased at postnatal day 5 and 10, but decreased again at adulthood (Fig. 1) . Conclusion A clinically relevant dose of antenatal dex treatment transiently affects hippocampal volume, number of neurons and apoptosis in the hippocampus. However it also caused permanent deficits in proliferation in adulthood.

Transforming Growth Factor Beta1 Is a Potent Activator of Drug Transport in the Fetal Blood-Brain Barrier (BBB). Stephanie Baello, 1 Majid Iqbal, 1 Enrrico Bloise, 1 William Gibb, 2 Stephen Matthews. 1 1 Physiology, University of Toronto; 2 Obstetrics & Gynecology, University of Ottawa. The developing brain is protected from a range of xenobiotics and steroid hormones by multidrug resistance transporter, P-glycoprotein (P-gp). P-gp expression increases rapidly in the fetal brain BBB in late gestation. During this period, TGF-β1 is released by neurons and astrocytes in the developing brain. TGF-β1 has been shown to modulate P-gp activity in certain adult cell-types. However, little is known about how TGF-β1 affects P-gp in brain endothelial cell(BECs) in late gestation, when the brain is most vulnerable to teratogens. The objectives of this study were to determine the effect of TGF-β1 on P-gp expression and activity in the BBB at critical phases of brain development, and to determine the signaling pathways involved. We hypothesized that TGF-β1 will increase P-gp expression and activity but that the magnitude of effect will change with age. Background Significant data link intrauterine inflammation with perinatal brain injury, yet there is limited data regarding a) dose-dependent effect of lipopolysaccharide (LPS) and b) time course of neuronal injury. Our objective was to investigate time-and dose-dependent effects on fetal brain injury following maternal (Mat) inflammation. Methods Sprague-Dawley dams received saline (NS) or LPS (O55:B5) intraperitoneally (IP) at E20. Exposure time (2, 4 & 6 h) and dose (160, 320 & 640 µg/Kg) were tested (2 dams/arm). Core temperature, Mat plasma and pup plasma/brains were collected. Primary cortical cultures were established. IL-6 protein and RNA was assessed by ELISA and qPCR. Cell death was quantified by propidium iodide assay. Immunocytochemistry was performed (DIV) 3 for MAP-2. Means were compared with ANOVA and post hoc testing.

Results LPS did not induce delivery. Both 320 and 640 µg/Kg reduced dam temp within 2h by -1.1 and -1.6 ºC, (p<0.05 vs NS), persisting 4 h vs. 5.5h. Dam plasma IL-6 levels peaked 2h post LPS and response increased with dose (8758±2472 vs15861±5565 vs18712±8826 pg/mL, p=0.001) compared to NS (110±14 pg/mL). Fetal plasma IL-6 response mirrored maternal time course (2-4 h) but returned to NS levels by 6h. Baseline levels were similar but max fetal plasma IL-6 response (374±212 pg/mL) was significantly less than Mat. Fetal brain IL-6 RNA response was delayed with peak levels achieved 6h after dam IP injection. Max response was seen with 640 µg/Kg with minimal fetal brain response to 160 µg/Kg (Fig) . Pup neuronal cell death (Day 3 cortical cultures; NB/DMEM) was significant 4-6h post exposure to 640 µg/Kg LPS Conclusions Our model suggests that fetal inflammation lags 4-6 h after maternal stimulus. Models investigating multiple insults may be most effective when secondary insults (hyperthermia/hypoxia) are implemented no earlier than 6h after 640 µg/Kg O55:B5 IP LPS. Clinically, our results suggest a therapeutic window for intervention to block fetal response. Background POP is defined as the descent of one or more of the pelvic structures into the vagina and includes uterine, vaginal vault, anterior or posterior vaginal wall prolapses. The treatment of POP includes conservative surgical treatment and/ or implantation of synthetic/biological mesh. However, the long-term outcome of synthetic mesh surgery is unsatisfactory due to surgical failure and postsurgical complications. The aim of this study is to use a tissue engineering (TE) approach to improve the in vivo biocompatibility of a novel synthetic polyamide/gelatine composite mesh delivering a novel source of mesenchymal stem cells (MSC) in a rat model of wound repair.

Human endometrial MSC (eMSC) were isolated from hysterectomy tissue by W5C5-labelled magnetic beads (Masuda et al., Cell Transplantation, 2012) , culture expanded for 6 passages, labelled with a fluorescent dye (DIO), seeded onto scaffolds (PA/ 0.025% crosslinked gelatin, 25x10mm, 500.000 cells/mesh) and subcutaneously implanted dorsally in immunocompromised rats for 7, 30, 60 and 90 days (n=8/gp). Controls received mesh alone. Flow cytometry was used to detect eMSC after explantation. Immunohistochemical assessment of foreign body reaction and tissue integration was performed using collagen (I, III), CD31, CD45, CD68, and αSMA.

The implanted materials were well tolerated and there were no mesh erosions. eMSC could be detected on the mesh up to 14 days post-implantation. The eMSC meshes attracted significantly fewer leukocytes at 7 days (p< 0.05) and fewer macrophages at 30, 60 and 90 days. Meshes with eMSC promoted significantly more neovascularisation at 7 days (p< 0.05). New collagen production was observed at all timepoints in meshes with and without eMSC.

This TE approach significantly reduces the number of inflammatory cells around an implanted mesh and promotes neovascularisation suggesting that eMSC exert an anti-inflammatory effect and promote wound repair. eMSC delivered on PA composite mesh might be an alternative option for future treatment of POP.

Differential Expression of a Novel Protease and Protease Inhibitors in the Vagina: Role in Pelvic Organ Prolapse. Madhusudhan Budatha, 1 Teodoro I Montoya, 2 Ayako Suzuki, 3 Cecilia K Wieslander, 2 Masashi Yanagisawa, 3 Hiromi Yanagisawa, 1 Ruth A Word. 2 1 Molecular Biology, Univ TX Southwestern Medical Ctr, Dallas, TX, USA; Univ TX Southwestern Medical Ctr, Dallas, TX, USA; 3 Molecular Genetics, Univ TX Southwestern Medical Ctr, Dallas, TX, USA. Connective tissues of the pelvic floor undergo continuous remodeling, a process controlled by a delicate balance between synthesis and degradation of matrix components which is tightly regulated by proteases and their inhibitors. Mice deficient for the fibulin-5 gene (Fbln5 -/-) develop pelvic organ prolapse (POP) due to compromized elastic fibers and upregulation of MMP-9. Here, we tested the hypothesis that dysregulation of other proteases may contribute to POP in mice and humans. Expression of select protease inhibitors was analyzed in vaginal stroma and mucosa (epithelium) from the apex of the vagina of premenopausal controls (n = 17, epi = 3), premenopausal prolapse (n = 15, epi = 9), postmenopausal controls (n = 10), and postmenopausal (n = 46, epi = 11) prolapse. Results: Two caseinolytic proteases were upregulated in the vaginal wall of Fbln5 -/mice, V1 (25 kDa) and V2 (21 kDa). V1 activity was optimum at pH 8.0 and predominantly detected in estrogenized vaginal epithelium. Inhibitor profiling, affinity pull-down, and mass spectrometry data indicated that V1 was a serine protease with trypsin-like activity similar to PRSS3, a major extrapancreatic trypsinogen. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5 -/and WT, and (c) was able to cleave fibulin-5 in vitro. Serpina1a (a1-antitrypsin) and elafin were dysregulated in Fbln5 -/epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epi and not changed with prolapse. Interestingly, although Timp1 and Timp2 did not differ in stroma from women with prolapse, both were decreased significantly in epi from menopausal women regardless of prolapse status (P < 0.05). Elafin was expressed in epi predominantly, but decreased dramatically (P < 0.002) in both pre-and post-menopausal women with prolapse. The results suggest that loss of serine protease inhibitors such as elafin may result in increased secretion of bioactive serine proteases in the vaginal wall. These data collectively suggest that the balance between proteases and their inhibitors contributes to progression of POP and serves as a basis for protease-targeted therapy.

Chronic Vestibulitis Is Associated with Interleukin-4 Polymorphisms Linked to Interstitial Cystitis and Atopy. Dawn Pruett, Amy Schilling, Nicky Leeborg, Terry K Morgan. Pathology, Oregon Health & Science University. Background: Chronic vestibulitis, also known as provoked localized vulvodynia (PLV), occurs in the rim of mucosa between the labia minora and hymen. This common disease affects approximately 15% of women during their lifetime and is the leading cause of painful intercourse. The cause is unknown, but neurogenic inflammation appears to play a key role. Neurogenic inflammation is also a shared feature with interstitial cystitis, which is present in 20% of women with PLV. Many investigators suspect a genetic basis for interstitial cystitis. However, testing for a genetic predisposition for PLV is only in the early stages of discovery. We hypothesized that PLV would be associated with the IL-4 promoter (rs22432250) and intron 2 (rs2227284) variants commonly seen in patients with interstitial cystitis. Design: Retrospective analysis of 212 clinically confirmed cases of PLV diagnosed from 2002-2012 at Oregon Health & Science University. Subjects were classified into primary PLV (pain with first introital touch) and secondary PLV (de novo pain usually after childbirth or menopause). Clinical charts were also reviewed to screen for a history of interstitial cystitis and allergies.

Since patient race may significantly affect allele frequencies, only data from Caucasian women were included for genetic analysis (205/212). Subject DNA was available for 195 cases (91 primary and 104 secondary). IL-4 genotypes were determined using real-time PCR-based Taqman Allelic Discrimination (ABI). Data were analyzed by X 2 analysis. Results: Secondary PLV was associated with both IL-4 variants (p<0.01) known to increase IL-4 activity and lead to elevated serum IgE levels. The allele frequency of the T-allele at -589 (promoter) was 0.20 (Caucasian controls 0.14); the allele frequency of the T-allele at 3017 (intron 2) was 0.31 (controls 0.25). IL-4 allele frequencies in primary PLV were not significantly different than controls. The prevalence of interstitial cystitis was increased in both primary and secondary PLV (32%, 27%, respectively) compared with controls (6%). The odds ratio for atopy in secondary PLV was 2.34 [1.3-4.4 ] (p<0.01); it was not increased in primary PLV.

We have recently demonstrated that primary and secondary PLV may have different underlying causes. Our data now suggest IL-4 genetic variants associated with interstitial cystitis and atopy likely play a role in secondary PLV, but not primary disease.

Alison M Stuebe, 1 Alison Wise, 2 Thutrang Nguyen, 3 Samantha Meltzer-Brody, 4 Karen Grewen, 4 Anna-Maria Siega-Riz. 5 Objective: In animals, oxytocin infusion reduces diet-induced obesity, and oxytocin is implicated in appetite regulation. Recent studies have identified oxytocin receptor (OXTR) polymorphisms associated with differences in maternal behavior and circulating oxytocin. We sought to determine whether OXTR polymorphisms are associated with differences in gestational weight gain or postpartum weight retention. Methods: We genotyped 1283 women in the Pregnancy, Infection and Nutrition Study for three OXTR single-nucleotide polymorphisms (SNPs) that have been associated with differences in stress response or maternal behavior. We used linear regression to test whether OXTR genotype was associated with differences in gestational weight gain or with weight retention at 3 and 12 months postpartum. Self-identified Caucasion and African-American women were analyzed separately, with adjustment for probability of Yoruban ancestry to control for population stratification. Models were further adjusted for pregravid body mass index and breastfeeding duration.

Results: Gestational weight gain data were available for 1283 women, and postpartum data were available for 290 women. We found no association between OXTR risk allele carriage and gestational weight gain for either Caucasian or African-American women. Postpartum data were available for only 31 African American women, and we therefore limited our analysis to Caucasian women. At 3 months, women homozygous for the rs2254298 G risk allele retained 4.6 lbs (SE 1.5 lbs) more weight than A allele carriers (p <.01), adjusting for pregravid BMI and breastfeeding duration. At 12 months, rs2254298 G homozygotes retained 3.8 lbs (SE 1.8) more than than A allele carriers (p=.04). The rs2254298 GG has been associated with reduced parental sensitivity, insecure attachment, and lower circulating oxytocin levels. We found no association between rs53576 or rs1042778 and weight retention. Conclusion: Carriage of an oxytocin receptor polymorphism was associated with postpartum weight retention. Oxytocin signaling may play a role in postpartum weight regulation.

Small Artery Function in Women at Reproductive Age and with a History of Early-Onset Preeclampsia. Women with a history of preeclampsia (PE) have an increased risk of cardiovascular disease later in life. Our objective was to determine if the structure and function of peripheral resistance arteries are impaired in women without known vascular risk factors but with a history of severe early-onset PE. Such women (n=15) and controls that underwent a normal pregnancy (n=12) were studied ∼2 years postpartum. Pressure myography was used for a comparison of the structure and function of the isolated small subcutaneous resistance arteries and immunostaining for comparison of endothelial nitric oxide (NO) synthase and Lectin-like oxidized LDL-1 receptor (LOX-1) expressions. Women with a history of early-onset PE had higher blood pressure, insulin levels and HOMA index vs. controls. We found no evidence for early renal damage. The endothelium-dependent response to flow was significantly attenuated in the case group. The absence of NO contribution underlay an impaired response to flow in these women. Endothelium dependent dilatation to bradykinin and NO donor were however similar between the groups. The myogenic tone was enhanced in arteries from women with a history of PE and positively correlated with low-density lipoprotein to high-density lipoprotein (LDL/HDL) ratio. The basal tone in the case group was positively correlated with LDL/HDL ratio, triglycerides, glycated haemoglobin, high-sensitivity C-reactive protein and negatively correlated with HDL. Higher sensitivity to norepinephrine, angiotensin II and reduced distensibility were observed in the case group. Distensibility was negatively correlated with homocysteine levels in the case group only. The arteries from both groups had similar diameters, wall thickness, wall-lumen ratio and crossed sectional area. eNOS expression was similar between the groups, but LOX -1 expression was significantly enhanced in the case group. All observed vascular alterations might create prerequisites for the increased total peripheral resistance, which has important implications for the long-term cardiovascular health in young women with a history of earlyonset PE even if they do not have other concomitant risk factors.

The Intrauterine (i.u.) administration of LPS to elicit localised inflammation in the uterus is a well-established model for inducing preterm delivery in rodents. However, we have found that the control laparotomy with i.u. PBS also modifies labour, delaying it by up to 36 hours compared to non-treated pregnant controls. This effect may be related to inflammation of the peritoneum and/or uterus as a result of surgery, and hence is an important variable of the model to consider. To this end, we compared the local and systemic effects of the PBS-and LPS-laparotomy procedures, measuring inflammatory leukocyte (Ly-6C high monocytes and neutrophils) trafficking as an indicator of inflammation. Methods: On day 16 of gestation CD1 mice were anesthetized using isoflurane and a laparotomy was performed. The uterine horns were exteriorised and LPS Objective: Intrauterine infection confers an increased risk for adverse neurobehavioral disorders for exposed offspring. Using a mouse model, we have demonstrated that a low dose inflammatory challenge in the intrauterine cavity results in altered gene expression in the adult brains of exposed offspring. These studies sought to determine critical steps in the pathogenesis of postnatal brain injury, that occur during the fetal period, from exposure to intrauterine inflammation.

Methods: On E15 of gestation, CD-1 timed pregnant mice were randomized to intrauterine infusion of 50 ug of LPS or an equal volume of saline (N=6 per group). Maternal serum, amniotic fluid, fetal brains, uterine and placental tissues were harvested 48 hours after exposure to LPS or saline. Cytokine expression (IL1b, TNF, IL6) was assessed in the fetal brain, uterine and placental tissues by QPCR. Cytokine levels were assessed by ELISA in maternal serum and amniotic fluid. Fetal neuronal injury was assessed by counting dendritic processes in cortical cultures from fetal brains harvested at 48 hours. Gene expression profiling of neuronal-glial injury was also assessed in cortical cultures and whole fetal brains by QPCR.

Results: At 48 hours, 75% of dams remained pregnant with viable liters. Maternal IL6 levels were not significantly different between LPS and saline exposed dams. In the uterus, mRNA expression of IL1b and IL6 were mildly elevated in LPS exposed dams (P=0.03, P<0.01). In the placenta, mRNA expression of IL1b was increased 2-fold (P=0.03). In amniotic fluid, IL6 levels were increased 8-fold in LPS exposed dams. In whole fetal brains, genes involved in neuronal differentiation and synaptic plasticity were significantly altered in LPS-exposed. Likewise, the number of dendritic processes were significantly decreased in LPS-exposed cortical cultures compared to saline (4.02 +/-1.1 vs. 2.4 +/-0.87, P <0.001). In LPS-exposed cortical cultures, gene expression of neuronal differentitation and outgrowth were significantly altered compared to saline. Conclusions: The lack of a maternal immune response and/or preterm birth does not preclude the development of fetal brain injury from low level intrauterine infection. A mild inflammatory response appears sufficient to alter fetal neuronal development and may be causative in long term adverse neurobehavioral outcomes in neonates exposed to prenatal inflammation.

Model. Lisa F Stinson, Demelza J Ireland, Jeffrey A Keelan. School of Women's and Infants' Health, University of Western Australia, Perth, WA, Australia. Background and rationale: Intrauterine inflammation is a major cause of preterm labor and birth (PTB) prior to 32 weeks' gestation. Antibiotics plus cytokine-suppressive anti-inflammatory drugs (CSAIDs) may be useful in preventing PTB in some pregnancies. Intraamniotic delivery allows targeting of fetal membranes and tissues without risk of maternal immune modulation. The aim of this study was to evaluate a range of CSAIDs (N-acetyl cysteine [NAC, 5 mM] , SB239063 [20 µM], TPCA-1 [7 µM], NEMO binding domain inhibitor [NBD; 1 µM]) using an ex-vivo perfusion model to assess the ability of intraamniotic CSAID treatment to inhibit inflammatory activation in human fetal and maternal membranes. Methods: Fetal membranes with attached decidua were collected from healthy term placentas delivered by Caesarean section (n=5). Membranes were secured over Transwell inserts and placed in 6-well culture plates containing serumfree media plus azithromycin (5 µg/ml). To the inner (amniotic) compartment were added heat-killed E.coli (10 µg/ml), anti-inflammatory drugs (or DMSO vehicle; 1%) and fluorescent beads (40-60 nm) to assess membrane integrity and permeability. Accumulation of cytokines (IL-10, IL-6, TNF-α), MMP9 and PGE 2 was measured in conditioned media (fetal and maternal) after 3, 9 and 20 h incubation at 37 o C. Results: Membrane integrity was maintained in all Transwells. Cytokine accumulation in both compartments increased with time, whereas PGE 2 concentrations peaked at 9 h and declined by ∼40% at the 20 h time-point. Concentrations of PGE 2 and cytokines in the amniotic compartment were 40-90% of those in the decidual compartment. Of the four CSAIDs tested, TPCA-1 (an IKK2 inhibitor) and SB239063 (a p38MAPK inhibitor) were the most effective in inhibiting amniotic accumulation of cytokines and PGE 2 , with inhibitory effects of 85-90% seen at the 9 and 20 h time-points. NAC and NBD inhibited accumulation of PGE 2 , but not cytokines. SB239063 was also Our understanding of susceptibility factors that predispose certain women to infection-associated PTB remains unclear. Changes in (i) the genital tract mucosal immune environment or (ii) the population of pathogens that colonize the vagina are suggested to be primary triggers. HA is secreted by cervical fibroblasts, epithelial cells and immune cells. During cervical ripening, HA deposition is increased in the stromal matrix as well as in cervical mucus. Furthermore, cervical HA is increased after intrauterine injection of LPS in mice. Although it is believed that cervical HA plays an important role in disorganization of the extracellular matrix and increased tissue compliance, the physiological significance of increases in HA in the cervix are unknown.

To evaluate the specific functions of cervical HA in term and infection-induced PTB, mice that lack cervical HA synthases (Has) 1, 2 and 3 were generated by crossing 1 and 3 global KO mice with Has2 tissue-specific KO (Has2 flox/ flox ) under the control of progesterone receptor promoter (PR Cre/-). Cervical HA content was decreased >90% in HAS 1/2/3 KO mice. Immunohistochemical staining of HA revealed that cervical epithelia and mucus were devoid of HA with low HA in subepithelium and stroma. Surprisingly, biomechanical properties of cervical tissues were similar in WT and triple-KO mice on gestation d18.75 (0.37 ± 0.04 vs 0.44 ± 0.05 N, maximal load at failure; 6.7 ± 1.5 vs 8.4 ± 2.7 m 2 /N, distensibility; and 10.5 ± 1.4 vs 9.4 ± 1.1 mm, maximal distention). Further, reproductive function and timing of parturition in HAS 1/2/3 KO mice were indistinguishable from WT animals. Interestingly, however, vaginal application of 10 5 E.coli on d16.5 resulted in a 92% (10 of 11) PTB rate in HAS 1/2/3 KO compared with 21% in wild type mice (3 of 11, P<0.001) . Although epithelial thickness increased after E. coli administration, mucinar epithelia disappeared in cervices from KO, but not WT, animals. Electron microscopy, immunohistochemistry and gene expression analysis implicate epithelial dysfunction in the HA-depleted cervix. Collectively, these studies suggest that although HA is not obligatory for increased tissue compliance during cervical ripening and dilation, HA in cervical epithelia and mucus is crucial for limiting pathogen colonization in the lower reproductive tract thereby protecting against infection-mediated PTB.

Dietary Phytophenols as Potential Therapeutics in Preventing Infection/ Inflammation-Induced Preterm Birth. Ratana Lim, Courtney Wall, Gillian Barker, Martha Lappas. Obstetrics & Gynaecology, University of Melbourne. Background: The aetiology of a significant proportion of preterm births involves bacterial infection. Bacterial endotoxin induces pro-inflammatory cytokines, prostaglandins and extracellular matrix degrading enzymes (such as MMP-9), which play a key role in initiating uterine contractions and the rupture of fetal membranes. The pro-inflammatory transcription factors nuclear factor-κB (NF-κB) and activator protein (AP)-1 play a critical role in regulating these processes. In non-gestational tissues, natural dietary phytophenols exert anti-inflammatory properties via inhibition of NF-κB and AP-1. The aim of this study was to determine if curcumin (found in turmeric), naringenin (grapefruit), apigenin (parsley), luteolin (celery) and kaempferol (tea) regulate pro-inflammatory and pro-labour mediators in human gestational tissues. Methods: Human placenta and fetal membranes were treated with 60 µM curcumin, 400 µM naringenin, 20 µM apigenin, 20 µM luteolin and 100 µM kaempferol in the presence of 10 µg/ml lipopolysaccharide (LPS) (n=6 patients). Human pregnant primary myometrial cells and primary amnion cells were treated with 30 µM curcumin, 200 µM naringenin, 5 µM apigenin, 10 µM luteolin and 50 µM kaempferol in the presence of 1 ng/ml IL-1β (n=6 patients). Endpoint analysis was assessed by qRT-PCR, Western blot and ELISAs. Results: In placenta and combined fetal membranes, curcumin, naringenin, apigenin, luteolin and kaempferol significantly reduced LPS-stimulated release and gene expression of the pro-inflammatory cytokines IL-6 and IL-8; and cyclooxygenase (COX)-2 expression and subsequent prostaglandin release of PGE2 and PGF2α. In myometrial cells, all five treatments significantly attenuated IL-1β-induced COX-2 expression and resultant release of PGE2 and PGF2α. In primary amnion cells, all treatments significantly decreased MMP-9 mRNA expression and activity. Treatment with the five phytophenols decreased IL-1β-induced NF-κB p65 DNA binding activity and nuclear c-Jun expression. Conclusion: Our study demonstrates that the phytophenols curcumin, naringenin, apigenin, luteolin and kaempferol can reduce infection and inflammation-induced pro-labour mediators in human placenta, fetal membranes and myometrium. These effects appear to be mediated by both the NF-κB and AP-1 pathways. The efficacy of these dietary phytophenols in reducing infection-induced preterm birth in vivo is currently underway. Introduction Human beta defensins (HBD1, 2 and 3) are small cationic proteins with significant antimicrobial and immunomodulatory properties. HBDs are a component of the cervical mucus plug and may therefore contribute to local host defence. Treatment with vaginal progesterone can delay delivery in women with cervical shortening, but the potential mechanism(s) underlying this effect remain undetermined. A progesterone response element has been identified in the HBD gene cluster, and HBD expression in the endometrium has been shown to be cyclical. This study describes the expression of HBDs by endocervical and ectocervical epithelia in response to stimulation with classical infective and inflammatory agonists and progesterone.

The human endocervical cell-line End1/E6E7 and ectocervical cell-line Ect1/ E6E7 were stimulated with classical bacterial (Lipopolysaccharide, LPS; Peptidoglycan, PGN) and inflammatory (Interleukin 1 beta, IL-1β; Interferon gamma, IFNγ) agonists and progesterone for up to 24 hours. Post-stimulation, HBD1, 2 and 3 secretion was assessed by ELISA.

The expression of HBD1 by End1/E6E7 cells did not alter significantly in response to stimulation. In contrast, HBD1 secretion by Ect1/E6E7 cells more than doubled in response to IL-1β (p=0.0002) and IFNγ (p=0.02), and was slightly suppressed by progesterone (p=0.02). HBD2 release by End1/ E6E7 cells almost doubled in response to both LPS (p=0.02) and progesterone (p=0.01). HBD2 expression by Ect1/E6E7 cells doubled in response to LPS (p=0.005) and halved after stimulation with progesterone (p=0.006). Release of HBD3 by End1/E6E7 cells increased in response to LPS (p=0.0009), and almost doubled after stimulation with PGN (p=0.0003) and IFNγ (p=0.027). End1/E6E7 cells secreted almost three times as much HBD3 in response to incubation with progesterone (p=0.003). Conclusion Bacterial products LPS and PGN elicited a differential repertoire of antimicrobial peptides in the human cervical cell-lines End1/E6E7 and Ect1/ E6E7. Progesterone mediated a significant increase in HBD2 and HBD3 peptide secretion by endocervical cells but suppressed HBD1 and HBD2 secretion by ectocervical cells; this effect may augment cervical host defence, representing a potential novel mechanism by which progesterone may contribute to delaying the onset of preterm labour.

Mice through a Progesterone Independent Pathway. SW Yang, 1,2 W Li, 2 JRG Challis, 1 G Reid, 3 SO Kim, 3 AD Bocking. 1,2 1 Depts of Physiology and Obstetrics and Gynecology, University of Toronto; 2 Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto; 3 Lawson Health Research Institute, Dept of Microbiology and Immunology, Western University, London, Canada. Introduction: Intrauterine infection/inflammation is involved in 25-40% of preterm births (PTB). Probiotics have been proposed to protect against PTB, supported by our previous studies showing that Lactobacillus rhamnosus GR-1 supernatant (sGR-1) reduces lipopolysaccharide (LPS)-induced proinflammatory cytokine TNFα output by human placenta, decidua and amnion cells in vitro. We hypothesized that sGR-1 would prevent LPS-induced PTB in vivo. Methods: Pregnant CD-1 mice were given 2 doses of 0.2mL sGR-1 or saline intra-peritoneally on gestational day (gd) 14 and 15. Intra-uterine infusion of LPS (125ug or 65ug) or saline was given 1 hr after the last dose of sGR-1 on gd 15. Mice (n=10/ group) were monitored until term (gd 20) for PTB, defined as delivery of the first pup within 24 hours post LPS infusion. A second set of mice was sacrificed 8 hrs after LPS (125ug) infusion for measurement of cytokines and chemokines in the maternal plasma and amniotic fluid by Bioplex and progesterone in the maternal plasma by Enzyme-linked immunoassay (n=8/ group). Statistical significance (p<0.05) was assessed using One-Way ANOVA followed by Newman-Keuls test. Results: sGR-1 decreased LPS-induced PTB rate from 100% (LPS 125ug) to 58% (LPS 125ug + sGR-1) and from 80% (LPS 65ug) to 53% (LPS 65ug + sGR-1). Mice not delivering preterm delivered live pups at term. LPS (125ug) infusion significantly increased the concentrations of IL-6, TNFα, MIP-1α and MIP-1β in amniotic fluid compared to saline by 149-, 3-, 10-, and 39-fold respectively. sGR-1 pretreatment significantly decreased these LPS-induced elevations to 32-, 2-, 3-and 14-fold respectively, compared to saline. In the maternal plasma, sGR-1 pretreatment decreased significantly the LPS-induced increases in IL-6, TNFα and MIP-1β. Maternal plasma progesterone was significantly reduced following LPS infusion (42+/-7.4 ng/ml) compared to saline (68+/-4.6 ng/ml) and this was similar after pretreatment with sGR-1(38+/-4.5 ng/ml). Conclusion: We have shown for the first time in vivo that supernatant of probiotic Lactobacillus rhamnosus GR-1 attenuates LPS-induced PTB through a mechanism independent of the change in circulating progesterone.

Enhancing Vascular Contraction. Vijay Chinnathambi, Meena Balakrishnan, Chandra Yallampalli, Kunju Sathishkumar. Ob/Gyn, University of Texas Medical Branch, Galveston, TX, USA. Introduction: Among many factors, sex steroid hormones including estradiol and progesterone play an important role in vascular adaptations during pregnancy. However, little is known about the role of androgens. Plasma testosterone (T) levels are elevated in preeclampsia, PCOS mothers and pregnant African-American women, who often exhibit uteroplacental dysfunction and fetal growth restriction. Objectives: To test whether elevated T alters uterine vascular adaptations during pregnancy, and if these alterations depend upon endothelium-derived factors such as prostacyclin, EDHF and NO, or endothelium-independent mechanisms such as angiotensin II (Ang II). Methods: Pregnant SD rats were injected with vehicle or T propionate (0.5 mg/Kg/day from gestation day (GD) [15] [16] [17] [18] [19] to mimic gestational androgen exposure like in preeclampsia. On GD 20 rats, endothelium-dependent and -independent vascular reactivity with wire myograph were assessed in uterine arteries (UA). Placental expression of VEGF, HIF1α and hypoxia responsive genes, Egln1 and Ankrd37 were also examined. Results: Plasma T levels increased by 2-fold in T injected rats (2.1±0.17 vs 1.0±0.11 ng/ml in controls). Endothelium-dependent acetylcholine relaxation was lower in T-treated UA (E max = 52±4.9) compared to controls (E max =77±4.3). Further assessment of endothelial factors showed that T-treated UA exhibited blunted relaxation responses mediated by all 3 factors, NO, EDHF and prostacyclin. Relaxation to sodium nitroprusside was unaffected with T-treatment. In endothelium-denuded MA from T-treated rats, contractile responses to Ang II (logEC 50 =-9.1± 0.06 vs. -8.7±0.03 in controls) but not thromboxane and phenylephrine were significantly greater compared to controls. T treatment decreased placental weights (0.43±0.13 vs o.54±0.08 g in controls) and birth weight of pups (5.75±0.19 vs 6.30±0.19 g in controls) . Placental expression of HIF1α and hypoxia responsive genes, Egln1 and Ankrd37 were increased while VEGF expression was decreased in T placentas. Conclusion: These results suggest a novel role for testosterone as inducer of the increased uterine vascular resistance by blunting of endothelium-mediated vasodilation and exacerbating Ang II induced contractions, which may decrease blood flow to fetoplacental unit leading to placental ischemia and fetal growth restriction.

The Production of sFlt-1 Splice Variants Is Regulated by Jumonji Domain Containing Protein 6 (Jmjd6) in the Placenta. Kirsten R Palmer, Tu'uhevaha J Kaitu'u-Lino, Louie Ye, Laura Tuohey, Stephen Tong. Obstetrics & Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia. Background: Soluble fms-like tyrosine kinase 1 (sFlt-1) -a splice variant of the vascular endothelial growth factor receptor 1 (Flt-1) -is an anti-angiogenic factor with a key role in the pathogenesis of preeclampsia. Released at elevated levels from hypoxic preeclamptic placenta, it causes maternal endothelial dysfunction and end organ damage. Surprisingly, while there are numerous reports describing the association between sFlt-1 and preeclampsia, the molecular mechanism in placenta that results in the elevated production of sFlt-1 has not been discovered. Recently, jumonji domain containing protein 6 (Jmjd6) was shown to be an oxygen-dependent protein involved in angiogenic regulation (1) . In the setting of hypoxia, Jmjd6 is unable to interact and hydroxylate U2AF65, a spliceosome component that appears directly involved in Flt-1 splicing. This alters the splicing pattern to produce sFlt-1. Objective:

To determine whether Jmjd6 regulates sFlt-1 production in preeclamptic placenta. Methods: Placental Jmjd6-6 expression was determined by RT-PCR, western analysis and immunohistochemistry in severe preeclamptic (n=22) and gestationally matched preterm controls (n=8). Immunofluorescence was used to assess co-localisation of Jmjd-6 and U2AF65 within the placenta, with interaction between the splicing factor U2AF65 and sFlt-1 splice variant transcripts determined using RNA immunoprecipitation (RIP). The impact of hypoxia (1% oxygen) on Jmjd-6 and sFlt-1 i13 and e15a expression was determined in HUVEC, JEG-3 and syncytialised BeWo cells by RT-PCR. Results : Jmjd6 co-localises with the splicing factor U2AF65 to the syncytiotrophoblast layer. However, in pre-eclamptic placenta Jmjd6 expression is significantly reduced (p<0.0001). RIP confirmed that U2AF65 directly interacts with RNA transcripts for both sFlt-1 i13 and e15a splice variants in placenta. Hypoxia induced a significant decrease in Jmjd6 (p<0.0001) and an increase in both sFlt-1 variants (p<0.05) compared to normoxia (20% oxygen). Conclusion: Our data suggests that under hypoxic conditions, as seen in pre-eclamptic placenta, oxygen-dependent Jmjd6 is downregulated altering the splicing pattern of Flt-1 in favor of soluble transcripts. Therefore, Jmjd-6 may be the key molecular mechanism regulating sFlt-1 production in placenta. 

Placenta Expression of miR-517a and miR-517c Contributes to Trophoblast Dysfunction and Preeclampsia. Lauren Anton, 1 Anthony O Olarerin-George, 2 Amy Brown, 1 Sindhu Srinivas, 1 John B Hogenesch, 2 Michal A Elovitz. 1 1 Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA, USA; 2 Pharmacology and the Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA, USA. Introduction: MicroRNAs (miRNA) are small non-coding RNAs known to regulate gene transcription. miR-517a and miR-517c are highly expressed in the human placenta and are associated with the development of preeclampsia (PRE) . The objective of this study was to determine if miR-517a and miR-517c are altered in PRE placentas, if these miRNAs are regulated by hypoxia and to investigate their mechanistic role in early trophoblast dysfunction. Methods: Placental biopsies were collected from normal pregnant women delivering at term (n=14), with term PRE (≥37 weeks; n=18) or with preterm PRE (<37 weeks, n=13) and RNA was extracted. First trimester primary extravillous trophoblast (EVTs) cells (n=4) were treated with the hypoxia mimetic, CoCl 2 (0-400uM) for 6 hours. cDNA was generated for placentas and EVTs and miR-517a and miR-517c were measured by QPCR. EVTs were transfected with miR-517a, miR-517c and miR-negative control using Lipofectamine 1% RNAiMAX. Transfected EVTs were plated for matrigel invasion assays (n=3) or media was collected for sVEGFR1 measurement (n=4) by ELISA. Results: miR-517a was differentially expressed between PRE placentas and controls (p=0.0026). miR-517a was increased in preterm PRE vs control (p=0.0015) and in term PRE vs control (p=0.03). miR-517c was differentially expressed between PRE placentas and controls (p=0.023). miR-517c was increased only in preterm PRE vs controls (p=0.029). EVTs exposed to hypoxia (CoCl 2 , 100uM) had an increased expression of miR-517a (p<0.05) and miR-517c (p<0.001) vs vehicle. Transfection of EVTs with miR-517a (p<0.001) or miR-517c (p<0.001) resulted in a decrease in invasion compared to nontransfected cells. In EVTs, sVEGFR1 increased after miR-517a (p<0.001) and miR-517c (p<0.001) transfection. Conclusions: The results of this study demonstrate that miR-517a and miR-517c play a role in the development of PRE. These findings suggest that a prolonged hypoxic environment can increase miR-517a and miR-517c expression which lead to poor trophoblast invasion as a possible mechanism for the development Obesity is a significant and independent risk factor for preeclampsia. The underlying biological difference(s) between obese women who develop preeclampsia and obese women who do not develop preeclampsia is unclear. Retinol binding protein 4 (RBP4) is a novel cardiometabolic risk factor produced by hepatocytes and adipocytes. RBP4 induces insulin resistance, and RBP4 values are elevated in type 2 diabetes mellitus, obesity, and cardiovascular disease. We hypothesized RBP4 would be elevated in obese women who develop preeclampsia compared to similar obese women. Study Design: RBP4 was measured in maternal plasma samples from 15 lean pregnant women (BMI=22.9±2.3), 24 obese pregnant women without preeclampsia (BMI=38.2±6.9) and 20 obese pregnant women who later develop preeclampsia (BMI=37.5±7.2). Samples were collected at 10.3±3.1, 20.4±1.8 and 34.7±0.6 weeks gestation. RBP4 was quantified by ELISA, inter-assay variability <10%. Uric acid, insulin and glucose were also measured. Statistical analysis was by repeated measures ANOVA and Students t-test as appropriate. Correlation analysis was performed using Pearson product moment correlation coefficient. Statistical significance was accepted at p<0.05. Results: RBP4 is significantly elevated in the first trimester and across pregnancy in obese women who later develop preeclampsia compared to similarly obese women who do not develop preeclampsia (Table) . RBP4 was not associated with insulin sensitivity (as previously reported) or uric acid. Conclusion: RBP4 is a unique cardiometabolic risk factor that is significantly elevated across pregnancy in obese women who later develop preeclampsia. RBP4 may provide insight into the underlying differences between obese women who do and do not develop preeclampsia. RBP4 (µg/ml) First trimester Second trimester Third trimester Lean/ uncomplicated pregnancy 13. 0±3.7 [10.2, 14.7] 15.0±5. 5 [12.7, 16.2] 14.5±4. 2 [12.2, 16.1] Obese/ uncomplicated pregnancy 11. 1±3.7 [8.5, 14.5] 12. 6±4.4* [9.5, 14.1] 15.4±4. 4 [13.1, 18.0] Obese/ preeclampsia 14.0±3. 9# [10.5, 17.2] 16.1±4. 6# [12.8, 18.7] 18.5±5.9*# [12.8, 23 . Obstetrics & Gynecology, LSU Health Sciences Center, Shreveport, LA, USA. Objective: Emerging evidence has shown that vitamin D plays an important role in human pregnancy. Sufficient vitamin D intake during pregnancy reduces the risk of complications including gestational diabetes, preterm birth, and infection. On the other hand, vitamin D insufficiency/deficiency during pregnancy has been linked to several adverse pregnancy outcomes such as preeclampsia and low birth weight. Endothelial dysfunction is an underlying pathophysiological feature in preeclampsia. However, the impact of vitamin D on vascular endothelial cells (ECs) is largely unknown. This study was undertaken to determine effect of vitamin D on VEGF and superoxide dismutase (SOD) expressions in ECs. Methods: HUVECs were isolated from term placentas from normal pregnancies. Confluent ECs were treated with 1,25-dihydroxycholecalciferol (1.25(OH)2D3) at a concentration of 20nM for 6, 16, and 24hrs, or at different concentrations of 5, 20, and 100nM for 24hrs. Total cellular protein was collected at the end of the experiments. Protein expressions for vitamin D receptor (VDR), VEGF-A, and CuZn-SOD were determined by Western blot analysis. β-actin expression was determined as internal control for each sample. 1.25(OH)2D3 induced endothelial angiogenesis was also determined by wound healing assay. Results: We found that VDR was weakly expressed in untreated control ECs and endothelial VDR expression was dramatically up-regulated when cells were treated with 1.25(OH)2D3. The increased VDR expression in ECs induced by 1.25(OH)2D3 was in a dose-and time-dependent manner. Endothelial expressions of VEGF-A and VEGF receptor-2 (KDR) were increased in cells treated with 1.25(OH)2D3. 1.25(OH)2D3-induced endothelial angiogenesis (cell migration by wound healing assay) could be partially inhibited by VDR siRNA. Interestingly, CuZn-SOD expression was also up-regulated in ECs treated with 1.25(OH)2D3. Conclusions: Our results showed that 1.25(OH)2D3 up-regulates both VEGF and CuZn-SOD expressions in ECs. These observations suggest that promotion of endothelial angiogenesis and increased antioxidant activity could be the underlying beneficial effects of vitamin D on vascular endothelium.

The Role of Agonist-Induced Hyperpolarization in Enhanced Ca2+ Signaling in P-UAEC. Roxanne Alvarez, Fu-Xian Yi, Bikash Pattnaik, Ronald R Magness, Ian M Bird. Dept ObGyn and Pediatrics, Univ of Wisconsin, Madison, WI, USA. Uterine artery endothelial cells from pregnant ewes (P-UAEC) show periodic Ca2+ bursting in response to ATP via enhanced activation of TRPC channels. We have reported that ATP stimulates a change in membrane potential (V m ) in UAEC (SGI 2012) , and TRPC3 is known to be sensitive to V m . We now investigate if ATP-stimulated changes in V m play a role in regulating [Ca2+]i bursts. Objective: To determine if the dose dependent initial release of Ca2+ (from ER) or sustained Ca2+ bursting (TRPC Capacitative Ca2+ Entry) is related to changes in V m in P-UAEC. Methods: Ovine P-UAEC (passage 4) were grown in 35 mm glass bottom dishes (>90% density) and loaded with Fura-2 (Ca2+ dye) followed by DIBAC4 (V m dye). Simultaneous imaging of [Ca2+] i and V m was acquired for 5 min basal and 30 min ATP (0-100uM) stimulation. Area under the curve was evaluated for both the initial Ca2+ peak and the sustained Ca2+ phase. Changes in V m were reported as total change over 30 min. Dose response curves were constructed, and possible correlations between the initial Ca2+ peak, sustained phase and V m were identified. Results: The dose dependency for the initial peak and sustained phase Ca2+ were parallel, but not coincident. The EC 50 for the initial Ca2+ peak (18uM) was higher than the EC 50 for the sustained Ca2+ phase (3.8uM) . At 3uM ATP a submaximal sustained Ca2+ entry was observed, but there was barely an initial Ca2+ peak and no increase in V m was detected. At 10uM ATP the V m change sharply rose (EC 50 = 5.4uM) . From 10-100uM ATP the V m change slowly declined, the sustained Ca2+ phase plateaued, and the initial peak continued to increase. Conclusion: These results suggest submaximal Ca2+ bursting can occur without membrane potential change when there is minimal Ca2+ release from the ER, but a maximal Ca2+ CCE response coincides with both an initial peak and V m hyperpolarization ≥ 11mV. The possibility that changes in V m stimulate TRPC3 channel activation to enhance Ca2+ bursting may explain why the dose dependency for sustained phase Ca2+ entry is not always coincident with the initial Ca2+ release from the ER. Future research of the regulatory mechanism behind endothelial CCE can be achieved by targeting ion channels that regulate V m . In preeclampsia, and possibly other hypertensive disorders, future targeted therapies may be focused on manipulating endothelial V m to support Ca2+driven vasodilation. NIH HL079020, HL49210, HD38843.

TNF Alpha Induces Gap Junction Dysfunction in Sheep Uterine Artery Endothelial Cells; a Model for Preeclampsia. Amanda C Hankes, Fu X Yi, Mary A Grummer, Ronald R Magness, Ian M Bird. OBGyn, Univ Wisconsin, Madison, WI, USA. We have previously shown pregnancy adaptation of uterine artery endothelial function is due to an increase of capacitative Ca2+entry bursts in response to ATP, compared to non-pregnant derived cells. This pregnancy adaptation is dependent upon increased connexin 43 (Cx43) gap junction function. Preeclampsia (PE) is characterized by a loss of the normal vasodilation in pregnancy and is associated with increases in growth factors and cytokines such as VEGF and TNF, which are known to inhibit Cx43 function. While TNF is known to inhibit vasodilation in PE subjects via reactive oxygen species (ROS) production, in UAEC at 10ng/mL dose or below ROS production is not seen (Yi, SGI 2005) . VEGF is also known to phosphorylate Cx43 at s279/282, while TNF phosphorylates Cx43 via the Src sensitive site y265. Moreover, VEGF inhibits pregnancy adapted Ca2+ bursts in pregnant uterine artery endothelial cells (P-UAEC). It is unknown what the actions of TNF are on this response. Objective: To establish the inhibitory effects TNF may have on Ca2+ bursts and Cx43 phosphorylation in P-UAEC and if function can be rescued by the Src family inhibitor PP2. Methods: Ovine P-UAEC were grown in 35mm glass bottom dishes to 100% confluence. Cells were loaded with Fura-2 (Ca2+ dye). Cells were stimulated by ATP (100uM) and [Ca2+] i was monitored for 30 min. TNF was added (0.1, 1, or 10ng/mL) for 1h and re-stimulated with ATP. For rescue, 10uM PP2 was added 20 min prior to TNF addition. Cx43 phosphorylation was analyzed by Western Blot. Results: Cells pre-exposed to TNF showed a dose dependent inhibition of subsequent ATP stimulated Ca2+ bursts (to 63%, P<0.001 10ng/ml). This inhibition was more potent than VEGF (to 79%, 10ng/ml). With PP2, the cells recovered back to control levels of bursts. TNF also promoted inhibitory Cx43 phosphorylation at y265, which was fully reversed by PP2 (P<0.05). Conclusion: In UAEC TNF acts through Src to phosphorylate Cx43 at y265 and inhibit Cx43 function. While VEGF inhibits Cx43, TNF inhibition of bursting is greater and is rescued by PP2. This supports the hypothesis that inhibition of Cx43 in PE can be mediated via multiple factors and the TNF effect is predominantly mediated via the Src pathway. Thus, physiologic TNF inhibition occurs more through the Src pathway rather than ROS. Treatments aimed at Src may be more effective in PE subjects than antioxidants. Funded by NIH HL079020, HL49210, HD38843.

Mirn451 Deficiency Impairs Development of Endometriosis in an Experimental Mouse Model. Warren B Nothnick, 1 Amanda Graham, 1 Mitchell J Weiss. 2 1 Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA; 2 Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA. Endometriosis is a prevalent disease in women of reproductive age. Mirn451 is a putative regulator of cell proliferation whose expression is significantly reduced in endometriotic tissue from women with endometriosis and this reduction is mirrored in animal models of the disease. However, the functional significance of this reduction in endometriotic tissue is unknown. To determine if mirn451 expression levels influence the establishment and/or growth of endometriotic implant tissue, the following series of experiments was conducted. Experimental endometriosis was induced in reproductively intact, wild-type C57BL/6 mice by transplantation of endometrial fragments (N=8 fragments/mouse) from 22-24 day old PMSG-primed C57BL/6 donor mice which were either homozygous wild-type, heterozygous for mirn451 expression or deficient in mirn451 expression (mirn451 null). One month after induction, recipient mice were sacrificed and the number of established endometriotic implants was determined in each mouse. Recipients which received wild-type "implant" tissue contained significantly more endometriotic implants (3.2 +/-0.16 implants) compared to mice that received mirn451 heterozygous "implants" (2.4 +/-0.33 implants) or mirn451 deficient "implants" (0.9 +/-0.33 implants). To verify that the reduced number of endometriotic implants was due to an altered ability to establish ectopically, we again induced endometriosis as described above but with the exception that the wild-type uterine tissue from immature donors expressed green fluorescent protein (EGFP), while the donor tissue from mirn451 deficient mice did not. Six fragments of each genotype were transferred to the same wild-type recipient (N=5) and the number of implants assessed at 1 week post-induction. All recipients established endometriotic implants (range 2 -4 implants/mouse) and all implants expressed EGFP indicating that they were of wild-type origin. No (non-EGFP) mirn451 deficient implants had established in any of the mice (0/5). In summary, reduced mirn451 endometriotic implant levels are associated with implant growth. This reduction is not required for, but actually impairs, implant establishment. Future studies will examine the specific mechanisms by which mirn451 contributes to endometriotic implant growth and survival. Supported by NIH HD069043.

In Vivo Characterization of a Novel Disease-Protective Role of Transcription Factor KLF11 Arresting Progression of Endometriosis. Ye Zheng, 1 Zaid M Tabbaa, 1 John K Schoolmeester, 2 Gary Keeney, 2 Raul Urrutia, 3 Gaurang S Daftary. 1 1 Obstetrics and Gynecology, Mayo Clinic, Rochester, MN, USA; 2 Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA; 3 Epigenetics and Chromatin Dynamics Laboratory, Mayo Clinic, Rochester, MN, USA. Objective: The Sp/KLF transcription factor family regulates most expressed genes. KLF9 has been extensively characterized in the endometrium as a progesterone receptor cofactor and is necessary for implantation. KLF11 is associated with several human diseases as diabetes, cancer and is known to arrest leiomyoma growth. Here we investigate in vivo using knockout animals, a specific role for Klf11 in contrast to Klf9, in the pathogenesis of endometriosis. Methods: A Tissue microarray containing human eutopic and ectopic endometrium (N=40) was plated. Endometriotic lesions (0.5 cm) were surgically induced in Klf9-/-and Klf11-/-animals using established protocols (N=10/genotype). Immunohistochemistry was used to evaluate KLF11 expression in human and Ki67 and Collagen1 expression in mouse tissues respectively. In vitro characterization of regulation of an array of fibrogenic markers by KLF11 in human endometrial stromal cells was assessed by qPCR, ChIP, EMSA and Luciferase Assay. Results: KLF11 expression was diminished in human endometriosis lesions compared to eutopic endometrium (p<0.05) from the same subject. In contrast to wildtype animals, Klf11 -/-mice there was proliferation of endometriotic cells (0.68 vs. 0.44cm; p<0.05), increased Ki-67 as well as a robust fibrotic response reminiscent of advanced human disease (adhesion score 2.71 vs. 0.43; p<0.05). Concomitantly, Lesional Collagen1 mRNA was only increased in Klf11-/-mice. In contrast to Klf11-/-animals, endometriotic lesions regressed with minimal fibrosis in Klf9-/-mice, similar to those in wildtype (p>0.05). Conclusion: KLF11 expression is diminished in human endometriosis. Using an animal model we show for the first time that loss of Klf11 resulted in lesion growth and fibrosis as seen in advanced human disease. KLF11 repressed several pro-fibrogenic target genes. Aberrant regulation of these targets likely results in the observed phenotype. Interestingly, KLF11 recruits several epigenetic co-factors to repress its targets. This robust mouse model uncovers another disease-specific, biomedically relevant role of Klf11. Furthermore, it offers the potential to test novel epigenetic modulators in the treatment of endometriosis.

Vitamin D3 Reduces Tumor-Promoting mTOR Signaling Targets by Inducing DDIT4 in Human Uterine Fibroid Cells. Sunil K Halder, 1 Archana Laknaur, 1 Kevin G Osteen, 2 Ayman Al-Hendy. 1 1 Department of Obstetrics and Gynecology, Meharry Medical College, Nashville, TN, USA; 2 Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, TN, USA. Background: Uterine fibroids (leiomyoma) are benign tumors that cause major health issues in reproductive age women. These tumors are three to four times more prevalent in Black women who have also ten times more hypovitaminosis D than white women. Recently, we have presented that Med12 gene exon 2 somatic mutations, that can activate Wnt/β-Catenin signaling, were detected in more than 70% of fibroid lesions in our tissue collection from southern USA. However, vitamin D3 regulation of Wnt/βcatenin and downstream growth-promoting mammalian target of rapamycin (mTOR) signaling in uterine fibroids is yet to be studied. Objective: To study the effect of bioactive 1,25-dihydroxyvitamin D3 on Wnt and downstream growth-promoting mammalian target of rapamycin (mTOR) signaling targets in human uterine fibroid cells. Design: Immortalized human uterine fibroid cells (HuLM) and primary human fibroid cells were used to analyze the expression and localization of Wnt4, Wisp1, DDIT4 (DNA-damage induced transcription 4), and mTOR signaling targets after treatment with different concentration of 1,25-dihydroxyvitamin D3. Effects are analyzed by western blot and immunofluorescence staining. Results: 1,25-dihydroxyvitamin D3 decreased Wnt4, Wisp1, and mTOR protein expression in a concentrationdependent manner in HuLM cells. On the other hand, 1,25-dihydroxyvitamin D3 induced DDIT4 in a concentration-dependent manner in HuLM cells. Moreover, it reduced phosphorylation of p70 S6 Kinase while the level of total p70 S6 kinase was unaltered. Immunofluorescence analyses using HuLM and primary human fibroid cells showed that Wnt4, DDIT4, and Wisp 1 proteins were mainly localized in the nucleus. 1,25-dihydroxyvitamin D3 decreased nuclear Wnt4 and Wisp1 and induced nuclear DDIT4 in above cells. The level of nuclear Med12 protein was not notably affected by 1,25-dihydroxyvitamin D3. Our results suggest that 1,25-dihydroxyvitamin D3 suppressed Wnt4 and downstream mTOR signaling by inducing DDIT4 and by reducing p70 S6 Kinase activity, and that ultimately reduced fibroid tumorigenicity. Conclusions: Vitamin D3 and/or analogues could be potential therapeutic options for safe non-surgical treatment of uterine fibroids. Support: RCMI grant 2G12RR003032-26 to S.K.H.

Targeting of the Cannabinoid Signalling Pathways Affect Endometrial Cell Survival, Proliferation and Inflammation in a Mouse Model of Endometriosis. AM Sanchez, 1 E Somigliana, 2 A Mugione, 1 G Velasco, 1 E Papaleo, 1 M Candiani, 1 P Vigano, 1 P Panina. 1 1 Reproductive Sciences, San Raffaele Scientific Institute, Milan, Italy; 2 Ob/Gyn Dept, Fondazione Policlinico Cà Granda, Milan, Italy. INTRODUCTION . Cannabinoids have been shown to affect inflammatory responses, cell proliferation and survival that are critical in endometriosis development. The components of the endocannabinoid system are differentially regulated throughout the menstrual cycle in normal endometrium and are expressed in deep endometriotic nodules and in sensory and sympathetic neurons innervating the lesions. The regulation and function of the endocannabinoid signaling in endometriosis is, however, largely unknown. In this study we have analyzed the effect of a known modulator of the endocannabinoid system, methanadamide (MET), and one the CB1 receptor, in the development of endometriosis in a validated mouse model of the disease. METHODS. MET (5mg/kg), was injected i.p. at the time of endometriosis induction in Balb/c female mice, and treatment was repeated 5 d/wk for 2 wks. Endometriosis was also induced in C57Bl/6 CB1 -/mice and in their wild-type (WT) littermates. On day 15 lesion number and size were evaluated. Gene expression analysis of inflammatory cytokines, survival factors and adhesion molecules were evaluated in eutopic and ectopic endometrium by qRT-PCR. RESULTS. Treatment with MET was able to significantly increase the total volume of endometriotic lesions (0.042±0.009 cm 3 ) compared to that of vehicle-treated mice (0.020±0.003 cm 3 ; p=0.04). Expression of transcripts for survivin, N-cadherin and b1-integrin in ectopic endometrium from MET-treated mice were increased compared with vehicle treated mice (p=0.045, p=0.029, p=0.040 respectively), consistently with a pattern of active proliferation of ectopic endometrial cells. No difference in eutopic endometrium transcript expression was found between treated and untreated mice. Expression of IL-6 in ectopic endometrium was up-regulated in MET-treated mice compared with vehicle-treated mice (p=0.03). When endometriosis was induced in CB1 -/mice, lesion size was reduced (0.019±0.0025 cm 3 ) compared to their WT littermates (0.029±0.002 cm 3 ) (p=0.027). Survivin and N-Cadherin were down-regulated in ectopic endometrium of CB1 -/mice. CONCLUSIONS. These results suggest an involvement of the endocannabinoid signalling pathways in the development of endometriosis leading to the regulation of survival, adhesion and inflammatory factors.

Maternal Age: A Modifiable Risk Factor for Congenital Heart Disease. Claire E Schulkey, Suk D Regmi, Patrick Y Jay. Pediatrics, Washington University School of Medicine, Saint Louis, MO, USA. Introduction: Epidemiologic studies consistently associate maternal age with the risk of congenital heart disease (CHD) even after excluding cases of chromosomal abnormality. We previously reported a similar maternal age effect in a mouse model that is haploinsufficient for Nkx2-5, a cardiac transcription factor. Human mutations cause non-syndromic CHD. Hypothesis: A modifiable factor in the mother is the basis of the maternal age effect. Methods: To localize the basis of the maternal age effect to either the mother or oocyte, we transplanted ovaries between old and young mothers. The offspring were genotyped and phenotyped for heart defects for this and subsequent experiments by PCR and histological sectioning. Insulin resistance and obesity increase as mice age. To assess its role in the maternal age effect, females were placed on a life-long, high-fat diet at age three weeks. Mating commenced at seven weeks. Glucose tolerance was assessed at twelve weeks in non-gravid mothers.

To reduce the age-related increase in insulin resistance and obesity, running wheels were put in cages. Mothers could exercise voluntarily, which led to a significant improvement in weight and insulin sensitivity. Statistical analysis: The incidences of heart defects in each experimental group were compared by a chi-square test. Results: Ovarian transfer experiments reveal that the incidence of defects is related to the age of the mother and not the oocyte (chi-square test, p < 0.01). An age-related maternal factor must interact with embryonic cardiac development. Impaired glucose tolerance in young mothers has no effect, so the maternal age effect is not due to insulin resistance alone. Of note, exercise by old mothers reduces the incidence of CHD to that of sedentary, young mothers. The incidence of membranous VSD in old, sedentary mothers, ∼16%, falls to ∼6% with exercise (chi-square test, p<0.01). Conclusions: Contrary to conventional wisdom, the effect of maternal aging on the risk of CHD is related to a modifiable factor in the mother. Thus, even though the embryo carries the deleterious mutation, one may target a maternal pathway to prevent disease. The findings may have broad implications for embryonic development and the fetal origins of adult disease.

Offspring. Guadalupe L Rodriguez-Gonzalez, 1 Claudia Vega, 1 Luis Reyes, 1 Lourdes Boeck, 1 Carlos Ibanez, 1 Peter W Nathanielsz, 2 Fernando Larrea, 1 Elena Zambrano. 1 1 Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición SZ, Mexico, Mexico City, Mexico; 2 Center for Pregnancy and Newborn Research, University of Texas Health Sciences Center, San Antonio, TX, USA. Objective. We have shown that MPR in pregnancy impairs male reproductive capacity 1 . It is suggested that early life nutritional adversity is associated with increased ROS 2 . ROS is a major cause of male infertility, damaging spermatogenesis and sperm function 3 . We hypothesized that MPR in rat pregnancy increases offspring ROS production in sperm and adversely impacts fertility. Methods. We studied male offspring of mothers fed either control (C) (20% casein) or restricted (R) (10% casein) isocaloric diet in pregnancy. After birth rats ate C diet and euthanized at postnatal day (PND) 110 and sperm was collected from the tail of epididymis and vas deferens to measure: 1) ROS (by fluorescence), malonaldialdehyde (MDA -by spectrophotometry) and superoxide dismutase (SOD -by xanthinoxidase assay) and glutathione peroxidase (GPx -chemical colorimetry) activity; 2) sperm quality (concentration, viability and motility). At 110 and 450 males offspring were mated with no related female young rats and fertily rate (FR) was evaluated. Data M ± SEM; analysis by t-test; n=6, p<0.05. Results. At PND 110, sperm obtained from R offspring had higher ROS and MDA and decreased SOD activity and sperm quality vs C and no changes in GPx activity and motility. FR was lower at PND 450 in R vs C (fig 1) . glucocorticoid (GR) receptor to affect metabolism. We therefore hypothesized that CRTC2 might influence the transcriptional activity of PR and GR, mediating nutritional signals to the action of these receptors. Methods: Transient transfection-based reporter assays tested the effect of CRTC2 over-expression on the transcriptional activity of PR and GR. HeLa and T47D breast cancer cells were treated with progesterone-and glucocorticoidresponsive mouse mammary tumor virus luciferase gene, CRTC2-expressing plasmids, and progesterone or dexamethasone. Knockdown experiments using CRTC2 siRNA in T47D examined mRNA expression of the endogenous progesterone-and glucocorticoid-responsive genes with rtPCR. To elucidate the pathways activating CRTC2, cells were treated with a constitutively active mutant of AMPK, cAMP analogue forskolin, or transient transfection of activating mutant CRTC2 plasmids with alanine substitutions at serine sites 171, 275, and 307. Results: CRTC2 overexpression enhanced the transcriptional activity of GR and decreased the PR activity. CRCT2 overexpression shifted dexamethasone and progesterone titration reporter assay curve activity up-and down-ward, respectively. CRTC2 knockdown with its siRNA decreased GR responsive glucocorticoid-induced leucine zipper mRNA expression but increased PR responsive Kreuppel-like factor 5 mRNA expression. Overexpression of AMPK decreased PR activity whether CRTC2 was overexpressed or knocked down. CRTC2 overexpression of activating mutations at phosphorylation sites 275 and 307 increased GR activity as compared to wild type CRTC2. Conclusions: Our results suggest that CRTC2 acts as a coregulator for some steroid hormone receptors, possibly mediating nutritional signals to these receptors. CRTC2 might play a role in progesterone resistance observed in some reproductive disease. This research was supported, in part, by Intramural research program of the Program in Reproductive and Adult Endocrinology, NICHD, NIH.

Peptidoglycan-Induced Trophoblast Apoptosis. Melissa J Mulla, 1 Kledia Myrtolli, 1 Serkalem Tadesse, 1,2 Seth Guller, 1 Errol R Norwitz, 2 Vikki M Abrahams. 1 Objective: There is a strong correlation between bacterial infection and pregnancy complications, such as preterm labor. While inflammation is a common mechanism, pathogens also represent a potential trigger for placental apoptosis, and certain Toll-like receptors (TLR) can mediate this response. We previously demonstrated that TLR2 activation by gram-positive bacterial peptidoglycan (PDG) triggers first trimester trophoblast apoptosis and decreased constitutive IL-6 secretion. This is dependent upon the presence of TLR1 and the absence TLR6, both TLR2 co-receptors. Since TLR10 has also been identified as a co-receptor for TLR2, the objective of this study was to determine its expression and function in the trophoblast. Methods: First and third trimester human placental tissue and isolated trophoblast were evaluated for TLR10 by immunohistochemistry (IHC) and Western blot. A first trimester human trophoblast cell line (3A) expressing TLR1, TLR2 and TLR10, but lacking TLR6 was used. This cell line was transfected with either a TLR10 dominant negative (TLR10-DN) or a vector control. Cells were treated with or without PDG (80µg/ml) for 48hrs. Cell lysates were analyzed for apoptosis by measuring caspase-3 activity. Supernatants were analyzed for IL-6 by ELISA. Results: IHC showed that first and third trimester placenta expressed TLR10 which was localized to the syncytiotrophoblast, cytotrophoblast and extravillous trophoblast. Western blot analysis of isolated first and third trimester trophoblast, and the first trimester trophoblast cell line confirmed TLR10 protein expression (94kDa). Treatment of the vector control trophoblast cells with PDG induced a significant increase in caspase-3 activity (p<0.05; n=3) and this was significantly inhibited by the presence of the TLR10-DN (p<0.05). In contrast treatment of both the vector control and TLR10-DN cells with PDG similarly reduced IL-6 secretion (p<0.05; n=3). Conclusion: This study demonstrates that TLR10 is expressed by the trophoblast across gestation and that in early pregnancy TLR10 plays a role in promoting PDG-induced apoptosis, but does not regulate PDG modulation of trophoblast IL-6. Together these findings suggest that TLR10 may regulate trophoblast TLR2-mediated apoptosis, and thus, the balance between trophoblast survival and cell death.

Role of Arcuate KNDy Neurons in Estrogenic Feedback on GnRH Neurons. Courtney A Marsh, 1 Megan Greenwald-Yarnell, 2 Martin G Myers, Jr.. 3 1 Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA; 2 Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA; 3 Internal Medicine, University of Michigan, Ann Arbor, MI, USA. Since GnRH neurons do not express estrogen receptor-alpha (ERa), feedback is thought to be indirect, via ERa-containing neurons that synapse on GnRH neurons. While two distinct kisspeptin (Kp) populations may stimulate GnRH, estrogen is postulated to promote positive feedback on Kp neurons in the hypothalamic preoptic region, while arcuate nucleus (ARC) Kp neurons mediate estrogenic negative feedback. ARC Kp neurons also contain neurokinin B (NKB; aka Tac2) and dynorphin (Dyn) (hence, KNDy neurons), while preoptic Kp neurons do not contain Tac2 or Dyn. We crossed Kpcre and Tac2cre mice onto the ERaflox background (ERaKpKO and ERaTac2KO mice) to permit the genetic ablation of ERa expression from all Kp neurons and ARC KNDy neurons only, respectively. We studied the phenotypes of these mouse lines to test the hypothesis that ARC KNDy neurons mediate estrogenic negative feedback on GnRH. We confirmed that Tac2cre-expression did not overlap with preoptic Kp neurons, and did not substantially delete from ERa-expressing neurons other than KNDy neurons. Both ERaKpKO and ERaTac2KO mice displayed early pubarche, with persistent vaginal cornification apparent from the time of vaginal opening. Both ERaKpKO and ERaTac2KO mice displayed evidence of gonadal hyperstimulation: uteri were large and fluid-filled; ovaries exhibited early folliculogenesis and had no corpora lutea. Most measured parameters were similar between both genotypes, but serum LH concentrations were higher in ERaTac2KO females than in control or ERaKpKO mice. These data suggest that estrogen action via ERa in Kp neurons (including ARC KNDy neurons) promotes negative feedback on the hypothalamic reproductive system. Furthermore, KNDy neurons may mediate effects distinct from those of preoptic Kp neurons, since LH was elevated in the ERaTac2KO compared to ERaKpKO females.

Low Serum Adiponectin at Mid Pregnancy Is Associated with the Development of Gestational Diabetes in Obese Hispanic Women. Theresa L Powell, Evelyn Miller, Christiane Meireles, Vanessa I Ramirez. Obstetrics and Gynecology, University of Texas Health Science Center San Antonio, San Antonio, TX, USA. Background Gestational diabetes (GDM) is more prevalent in pregnancies complicated by maternal obesity and both conditions have elevated risk for fetal overgrowth and long-term health consequences for the mother and her child. Obese Hispanic mothers have high rates of GDM (13%) and previous studies have implicated adiponectin in the development of GDM in normal and overweight women. We characterized the mid-gestation metabolic profile of obese Hispanic women in order to identify factors of importance for the development of GDM. Specifically, we hypothesized that low adiponectin is associated with the development of GDM. Methods Seventy-two obese, predominantly Hispanic (92%), women were recruited at 24-28 weeks gestation. Fasting serum samples were collected and we measured glucose, insulin, lipids, and metabolic factors associated with obesity and insulin resistance. Thirty women had been recently diagnosed with GDM. At the time of sampling six were on glyburide, one on insulin, 23 were diet-treated. Obese (n=42) and Obese + GDM (n=30) groups were compared statistically using Student's T-test.

Results Body mass index, tricep skinfold and mid-arm circumference did not differ in the two groups. Obese women with GDM had significantly higher fasting glucose (89.9 ± 16.1 vs 79.3 ± 5.8 mg/dl, p<0.001), triglycerides (230.4 ± 67.1 vs 182. 6 ± 48.4 mg/dl, p<0 .001), VLDL (46.1 ± 13.5 vs 36.6 ± 9.7 mg/dl, p<0.001) and lower HDL (58.1 ± 14.2 vs 67. 5 ± 15.4 mg/dl, p<0 .02) and adiponectin (4.4 ± 1.5 vs 7.6 ± 3.8 mg/ml, p<0.001). Fasting insulin was elevated in obese GDM mothers (16.4 ± 10.0 vs 13.0 ± 6.1 mIU/ml) but the difference was not statistically significant. The two groups had similar HbA1c, suggesting gestational diabetes rather than T2DM. Furthermore, serum leptin was not different in the two groups, reflecting equivalent degrees of adiposity. Maternal TNF-a and IL-6 were similar in the two groups.

Conclusions At 24-28 weeks gestation, obese Hispanic pregnant women with recently diagnosed GDM had a exacerbated metabolic profile compared to obesity alone. Adiponectin is known to increase insulin sensitivity, therefore the markedly lower adiponectin in obese women with GDM compared to obese women without GDM is consistent with the possibility that low circulating adiponectin is mechanistically linked to the development of GDM.

Previous Cesarean Delivery and the Risk of Unexplained Stillbirth: Analysis of 128,680 Second Pregnancies in Scotland, 1999-2008. Gordon C Smith. Obstetrics & Gynaecology, Cambridge University, United Kingdom. Background An analysis of Scottish data from 1992-1998 demonstrated that women with a previous cesarean delivery were at increased risk of unexplained stillbirth in their 2nd pregnancy (Lancet 2003; 362:1779-84) . Some studies from other parts of the world reported similar associations, whereas others did not. Given the inconsistency, we analysed the next 10 years of Scottish data.

We applied the same inclusion/exclusion criteria and analytic approach as the Lancet study and identified records for 128,680 eligible singleton 2nd births between 1999 and 2008. There were 89 stillbirths among 23,708 women with a previous cesarean delivery (2.36 per 10,000 women per week) and 289 stillbirths in 104,972 women previously delivered vaginally (1.68 per 10,000 women per week, P=0.002). When analyzed by cause, women with a previous cesarean had an increased risk (hazard ratio [95% CI] P) of unexplained stillbirth (1.48 [1.12-1.95 ] P=0.005) and the excess risk was apparent from 34 weeks onwards (1.70 [1.21-2.39 ] P=0.002). When the analysis was confined to 101,929 women where the records from the 1st and 2nd birth could be linked (allowing direct confirmation of the previous mode of delivery) the association was stronger (2.39 [1.66-3.44 ] P<0.001). Adjustment for maternal characteristics, previous birth weight percentile, previous perinatal death and previous preterm birth was without material effect (2.29 [1.57-3.36 ] P<0.001).

The association was similar if the analysis was confined to women whose first birth was at term (2.45 [1.65-3 .63] P<0.001). The association was also similar whether the previous cesarean was performed prior to labor (2.11 [1.21-3.70] P=0.009), was performed after <9 hours of labour (2.10 [1.01-4.37 ] P=0.047), or was performed after ≥10 hours of labor (3.18 [1.85-5.47 ] P<0.001). There was no association between previous operative vaginal delivery and the risk of stillbirth (0.74 [0.44-1.25] P=0.3). The overall pattern of results was very similar to the previous report.

Previous cesarean delivery is confirmed as a risk factor for unexplained stillbirth in Scotland. The association is independent of maternal characteristics, obstetric outcome or the indication for the cesarean delivery. Negative studies in other parts of the world may represent true biological variation, but some may reflect combinations of poor data quality and inappropriate analytic approach and interpretation of results.

Decrease of Uterine Blood Flow (UBF). SJ Bischoff, 2 R Schiffner, 1 F Rakers, 1 S Rupprecht, 1 H Schubert, 2 PW Nathanielsz, 3 M Schwab. 1 Acute stress in pregnant sheep leads to a catecholamine mediated decrease in UBF and induces a prolonged fetal lactate increase and O2 sat decrease. As a result, chronic exposure to maternal stress is considered to induce fetal growth retardation in sheep and humans. Objective: We hypothesized, chronic maternal stress results in adaptation to stress and attenuates the UBF decrease and fetal lactacidosis to acute episodes of stress. Methods: Ten pregnant sheep underwent repeated isolation stress between 0.2 and 0.66 gestation (G 30 and 100d gestational age, dGA, term 150 dGA) resulting in a reproducible cortisol increases with only slight habituation. Ten pregnant ewes functioned as controls. Animals were instrumented with maternal and fetal carotid artery and jugular vein catheters and a uterine ultrasound flow probe five d before acute isolation stress at 0.70 or 0.87 G (105 or 130 dGA).

Results: Acute maternal stress at 0.70 and 0.87 gestation transiently increased maternal blood pressure (MBP) and maternal heart rate (MHR) and decreased UBF (p<0.05). UBF decrease was more prolonged at 0.87 than at 0.70 G (p<0.05). At both gestational ages, the UBF decrease was more accentuated and prolonged in chronically stressed ewes than in controls (p<0.05, Fig. 1 ). All fetuses responded with a lactate increase (p<0.05). Fetal pH and O2 sat decreased in controls at 0.87 but not at 0.70 G. Preceding stress prevented the drop in pH (p<0.05) probably because of hyperventilation-mediated maternal hypocapnia. Conclusions: Maternal stress induces a UBF decrease during the third trimester that is more prolonged with advancing gestational age. The UBF decrease is prolonged by preceding chronic stress. The prolonged UBF decrease is still detectable 4 weeks after discontinuation of chronic stress. Background: Infant birth weight increased through the 1990s but may now be decreasing. Rates of obesity have increased and smoking has decreased, both associated with higher birth weight and excess macrosomia. Obesity is also associated with excess hypertension and concomitant risk of growth restriction. We hypothesized that increasing maternal adiposity and lower rates of smoking would be associated with excess fetal under and overgrowth. Methods: We utilized a registry of all singleton term births at Magee-Womens Hospital (Pittsburgh PA) delivered 1997-2010 at >= 37 weeks (n=68,164) to model changes in birth weight. Models were adjusted for week of delivery, race, age, smoking, gestational weight gain, pre-pregnancy BMI, parity, hypertension (chronic, preeclampsia, gestational) , and gestational diabetes. Small (SGA, <10 th %) and large (LGA, >90 th %) for gestational age births were evaluated using estimated fetal weight standards. Results were stratified by each maternal characteristic to examine trends in subgroups. Results: Since 1997 rates of maternal smoking decreased and births to African American or Hispanic women increased. Rates of overweight, obesity, primiparity, chronic hypertension, preeclampsia, gestational hypertension, and gestational diabetes increased. SGA increased (9.5% to 10.2%, p=<.01), and

LGA decreased (8.4% to 7.5%, p=.01). Mean birth weight at term decreased, on average, each year by 4.91 g ([0.44], p<0.0001). Adjustment for gestational week attenuated this to 3.51 g ([0.42], p<0.0001), and adjustment for all maternal characteristics attenuated this to 2.52 g per year ([0.96], p=.009). Quantile regression indicated that birth weight decreased consistently across the entire birth weight distribution. Birth weight decreased yearly among most subgroups: smokers and nonsmokers, normal and overweight, White and African American, every category of gestational weight gain, and those delivering ≥38 weeks. The only two groups with increasing birth weight were preeclamptics (14g [5.7] ) and those delivered at 37 weeks (9 g [3.5] Conclusions: CD at EP compared to term CD is associated with an increased risk for uterine rupture in a subsequent pregnancy, even after adjustment for incision type. This may reflect that a poorly developed lower uterine segment at EP may prevent access to truly non-contractile myometrium. These data inform counseling after EP CD and support judicious use of CD at such gestational ages.

The The same results remained significant when analyzed separately for women who smoked <5, 5-10 and >10 cigarettes per day. Using multiple logistic regression models while controlling for confounders, smoking remained a significant independent predictor of preterm delivery (OR 1.79, 95%CI 1.56-2.06), NICU admission (OR 1.35, 95%CI 1.13-1.61) and intrauterine fetal demise (OR 1.96, .

Smoking does not only reduce birthweight but also appears to harm the fetus in multiple other dangerous ways. As pregnancy may be a 'window of opportunity' for behavioural changes, efforts to promote smoking cessation should be firmly encouraged. 

Mortality from Common Malignancies Following Hysterectomy with Ovarian Conservation: A Cohort Study from the Rochester Epidemiology Project (REP). Zaraq Khan, 1 Shannon K Laughlin-Tommaso, 1 Amy L Weaver, 2 Cathy D Schleck, 2 Adil E Bharucha, 4 Elizabeth A Stewart, 1 Walter A Rocca. 3 1 Obstetrics & Gynecology, Mayo Clinic, Rochester, MN, USA; 2 Biomedical Statistics, Mayo Clinic, Rochester, MN, USA; 3 Epidemiology, Mayo Clinic, Rochester, MN, USA; 4 Gastroenterology, Mayo Clinic, Rochester, MN, USA. Objective: Although short-term outcomes after hysterectomy with ovarian conservation (HS-OC) are favorable, there is limited data on long-term outcomes. We studied mortality from common malignancies in women after HS-OC and in age-matched referent women (REFW). Methods:We included all women who underwent HS-OC for benign indications from 1 /1/1965-12/31/2002 in Olmsted County MN. Each woman was agematched (±1 year) to a referent woman who had not undergone hysterectomy as of the index year. Data were abstracted from the Rochester Epidemiology Project medical records linkage system. We estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazard models. Subjects were censored if they were lost to follow-up or were alive at the end of the study (3/18/2012) . Results:A total of 3,816 women had HS-OC during the study period. After a median follow-up of 21.4 years (IQR: 10.9-31.3 years), women with HS-OC had lower (i) mortality from all cancers and (ii) mortality from lung cancer when compared to REFW. The mortality from breast and other gynecologic cancers was reduced but not significantly. Mortality from ovarian cancer was not reduced. However, statistical power was limited for rare cancers. Conclusion:Our findings suggest that women undergoing HS-OC have lower mortality from cancer compared to REFW. Mortality was significantly reduced for lung cancer. This association deserves further investigation.

Interpregnancy Weight Change and Risk for Adverse Perinatal Outcomes: An Epidemiological Study. Annick FL Bogaerts, 1,2 Lieveke Ameye, 3 Bea RH Van den Bergh, 4 Ingrid Witters, 5 Evelyne Martens, 6 Roland Devlieger. 7 1 Healthcare, KHLim, Limburg Catholic University College; 2 Healthcare Research, PHL, University College; 3 Development and Regeneration, KU Leuven; 4 Psychology, Tilburg University; 5 Center of Human Genetics, KU Leuven; 6 Perinatal Epidemiology, Flemish Government Brussel; 7 Obstetrics and Gynaecology, KU Leuven. Aim:We examined interaction between pre-pregnancy body mass index(BMI) from the first to the second pregnancy and the risk for adverse perinatal outcomes during the second pregnancy.Method:A Belgian cohort of women who had their first two consecutive singleton births between 2009 and 2011 was used(N=7468).Interpregnancy weight change was operationalized as difference between pre-pregnancy BMI of the first and second pregnancy. Multivariate logistic regression models, adjusting for confounding variables were performed.Results:Mean pre-pregnancy BMI increased between two consecutive pregnancies from 23.3kg/m²(SD4.0) to 23.9kg/m²(SD4.4) (p0.0001).Weight gain between pregnancies decreased with maternal age and height, was lower with longer interpregnancy time interval; and higher in case of adverse outcomes during the first pregnancy including pregnancy induced hypertension(PIH), induction of labour(IOL), caesarean delivery(CS) and macrosomia.The risk for perinatal complications during second pregnancy, including PIH(AOR1.10,95%CI1.02-1.19), GDM(AOR1.11,95%CI1.03-1.20), CS(AOR1.07,95%CI1.03-1.12), IOL(AOR 1.04,95%CI1.01-1.07) and ma crosomia(AOR1.05,95%CI1.00-1.08) increased with an increase in BMI between two consecutive pregnancies. Furthermore, the risk of PIH decreased with gestational weight gain(GWG) below recommendation(AOR0.51,95 %CI0.30-0.87) and longer duration(AOR= 0.71,95%CI0.61-0.84) of first pregnancy. Risk for IOL(AOR0.84,95%CI0.73-0.95) and macrosomia(AO R0.61,95%CI0.50-0.74) decreased with low GWG during that pregnancy. In addition to the influence of interpregnancy weight gain, the adjusted odds for PIH(AOR14.48, , GDM(AOR39.77, , CS(AOR48.94, and IOL(AOR3.15, were influenced by the occurence of this complication during their first pregnancy. Conclusion:This study shows that weight retention between the first and second pregnancy is associated with more perinatal complications. Besides the prevention of maternal obesity before pregnancy, stabilizing interpregnancy maternal weight appears an important contributor to perinatal outcomes.

Deprived Neighbourhoods and Adverse Pregnancy Outcomes: A Systematic Review and Meta-Analysis of Observational Studies. Amber A Vos, Anke G Posthumus, Gouke J Bonsel, Eric AP Steegers, Semiha Denktas. Obstetrics and Gynecology, Erasmus Medical Center, Rotterdam, Netherlands. Objective To summarize evidence on the association between neighbourhood deprivation and the risks for preterm birth, fetal growth restriction and perinatal mortality. Design Systematic review and meta-analysis of observational studies. Data sources Searches of Medline, Embase and Web of Science for articles published up to April 2012, reference list screening, and email contact with authors. Study selection Observational studies that directly compared the risk of living in the lowest deprived neighbourhood quintile with the highest deprived neighbouhood quintile regarding the outcomes preterm birth (<37 weeks of gestation), small-for-gestational age (< p10), and perinatal mortality were included. Data synthesis Data was extracted by two independent investigators. Random effects model was used to calculate the odds ratios for preterm birth, small for gestational age and perinatal mortality. A meta-analysis assessed the risk of adverse birth outcomes by comparing highest deprived neighbourhood quintiles with lowest deprived quintiles. Results Perinatal outcomes from seven cohort studies, including 2,579,032 pregnancies, were evaluated. Compared to the lowest deprived neighbourhoods quintile, odds ratios for adverse pregnancy outcomes in the highest deprived neighbourhood quintile were significantly increased for preterm birth (n = 7 studies, odds ratio = 1.23, 95% confidence interval 1.18 to 1.28; I 2 77%), small for gestational age (n = 3 studies, odds ratio = 1.31, 95% confidence interval 1.28 to 1.34; I 2 99%), and perinatal mortality (n = 3 studies, odds ratio = 1.33, 95% confidence interval 1.21 to 1.45; I 2 0%). For the 5 studies using neighbourhood income as measure for deprivation, a positive association was also found between the highest and lowest deprived neighbourhood quintile for preterm birth (OR 1.21, 95% confidence interval 1.15 to 1.27), I 2 78%).

Conclusion For pregnant women, living in deprived neighbourhoods is associated with significantly higher risks for preterm birth, SGA and perinatal mortality.

Preparing for In-Vitro Fertilization Cycles. Tia Jackson-Bey, 1 Alan M Martinez, 1 Julie M Sroga, 1 Krystene B Dipaola, 1 Michael A Thomas, 1 Steven R Lindheim. 2 1 Obstetrics & Gyncecology, University of Cincinnati, Cincinnati, OH, USA; 2 Obstetrics & Gynecology, Arizona Reproductive Institute, Tucson, AZ, USA. Objective: Genetic considerations are an important component of preconception planning. Currently the American Congress of Obstetricians & Gynecologists recommends genetic screening for all couples considering family planning, including screening for cystic fibrosis (CF) . Despite this recommendation, many patients choose to forgo genetic screening. The aim of this study was to examine factors that influence the decision to pursue CF screening when preparing for IVF treatment. Design: Retrospective chart review in an academic center. Methods: From January 2011 through June 2012, a total of 223 IVF charts were reviewed. As per protocol, patients were given written and verbal preconception genetic information at both their initial consultation and subsequent IVF education session. All patients were given the option to screen for individual common genetic disorders, including CF, Tay-Sachs, and sickle cell trait/ thalassemia. Primary outcome included number of patients who completed CF carrier screening. Logistic regression was used to compare this outcome with multiple parameters, including age, race, infertility diagnosis, family history of inherited genetic disease, insurance coverage of fertility treatment, and insurance coverage of CF screening. Chi Square was used for categorical comparisons. Results: A total of 49 patients (21.9%) completed CF carrier screening, of which 36 are Caucasian, 6 African American, 4 Asian, 2 Hispanic, and 1 other. Regression analysis revealed that preconception CF carrier screening significantly correlated with insurance coverage of CF screening (OR=1.369, 95%CI 1.369 to 5.011, p<0.002) and inversely correlated with family history of inheritable genetic disease (OR=0.387, 95%CI 0.177 to 0.848, p<0.018). Chi-square for the model was 14.925 (P<.001) . No other factors correlated with the decision to undergo CF screening. Conclusions: Preconception genetic screening is routinely offered to all patients undergoing infertility treatment. CF insurance coverage appears to be an incentive for testing, whereas a family history of inheritable genetic disease is not an important incentive in the decision to complete CF screening. These results highlight the need for insurance coverage to increase participation in routine CF screening.

Growth Restricted but Enhanced Adipocyte Proliferation: Mechanism of Programmed Adipogenesis. Mina Desai, 1 Juanita Jellyman, 1 Guang Han, 1 Marie H Beall, 2 Michael G Ross. 1 1 Ob/Gyn, Ctr.; 2 OB, LA Perinatal Assoc. OBJECTIVE: Enhanced adipocyte proliferation is one of key features of programmed obesity. We have established a rat model of maternal undernutrition that results in low birth weight (LBW) newborns which paradoxically develop adult obesity. LBW newborns have an upregulated adipogenesis signaling pathway with an increase in mature adipocytes, though it is unclear as to whether this occurs simply as a result of increased fat storage or is secondary to an enhanced, programmed proliferation. We hypothesized that increased preadipocyte proliferation results in an increased population of mature adipocytes. We determined in vivo preadipocyte proliferation and expression of the adipocyte progenitor marker preadipocyte factor-1 (Pref1), in LWB and Control male newborns at 1 day of age. STUDY DESIGN: From pregnancy day 10 to term, Control dams received ad libitum food, whereas study dams were 50% undernourished to produce LBW newborns. To assess in vivo preadipocyte cell proliferation, pregnant dams were injected with bromodeoxyuridine (BrdU, 50mg/kg/day, i.p.) from e17-e19. At 1 day of age birth, subcutaneous adipose tissue was collected from newborn males to determine protein expression of Pref1 (Western analysis) and tissue was immunostained for cell markers of preadipocytes (Pref1) and proliferation (BrdU). The total number of Pref1/BrdU positive cells per field was counted using Image-J software and values given as means±SE. RESULTS: At 1 day of age, LBW were significant smaller than controls (6.8±0.2 vs. 7.5±0.1). Nonetheless, LBW showed significantly increased expression of subcutaneous adipose Pref1 (2-fold) as compared to Control males. Furthermore, LBW newborns adipose tissue exhibited significantly increased BrdU staining in Pref1 cells (250±15 vs. 175±10 AU) as compared to Controls. CONCLUSION: Increased Pref1 protein expression in LBW adipose tissue indicates increased number of adipocyte progenitor cells. Despite being growth restricted at birth, the 40% increase in BrdU staining in LBW adipose tissue is consistent with an intrinsic, enhanced adipocyte proliferation on day 1 of life. These finding suggest that programmed adipose cell proliferation contributes to increased adipocyte differentiation and adult obesity in LBW offspring.

was lower in T offspring of both sexes compared to respective controls. Further assessment of endothelial factors showed that EDHF-mediated relaxation was selectively blunted in T males while only NO-mediated relaxations was impaired in T females. Assessment of EDHF/NO components showed that expression of endothelial SK3 channels and connexin43 were lower in T males while eNOS expression was lower in T females compared to controls. In endothelium-denuded MA, contraction to PKC activator, PDBu was greater in T offspring of both sexes but with a greater magnitude in males than females. Assessment of vascular PKC isoenzymes showed unique and differential expression profile, with upregulated PKCα, β and ζ in males but only upregulated PKCδ in T females. Conclusion: These results suggest a novel role for T as an intrauterine programming agent of sex-specific dysfunction of vascular endothelium (EDHF in males and NO in females) and smooth muscle (PKCα, β and ζ in males and PKCδ in females) contributing for differing onset and severity of hypertension in male and female offspring.

The Intrauterine Environment: Not a Trivial Risk Factor for Cardiovascular Disease. JA Thompson, 1 R Gros, 2 O Sarr, 2 TRH Regnault. 2 1 Physiology, GHSU, Augusta, GA, USA; 2 Physiology, UWO, London, ON, Canada. Intrauterine growth restriction (IUGR) is a known risk factor for cardiovascular disease (CVD) but is not yet fully integrated into clinical risk profiling. We've demonstrated aberrant aortic remodelling in IUGR fetuses and consequent vascular dysfunction in adulthood. The models used recapitulate placental insufficiency (PI), a major cause of IUGR in developed countries. In such societies, the nutrient deprived IUGR fetus is often confronted with a postnatal environment of nutrient overabundance. Thus, this study examined the independent and interactive effects of IUGR and postnatal diet on vascular function. Uterine artery ablation in pregnant guinea pigs was used to induce PI and IUGR. After weaning, normal birth weight (NBW) and low birth weight (LBW) offspring were pair-fed either a control diet (CD, 18% fat) or a western diet (WD, 46% fat, high fructose). Animals were sacrificed in young adulthood. Dose-response curves for phenylephrine (Phe), methalcholine (MCh) and sodium nitropusside (SNP) were generated in aortic and carotid rings mounted on a wire myograph. Three additional aortic rings were used for length-tension curves. mRNA levels of superoxide dismutase (SOD) were measured using real-time PCR. Curves were compared by 2-way ANOVA and mRNA levels compared using a t-test. Results are expressed as mean ±SEM. Birth weight of NBW and LBW pups was 105±1.6g and 77.8±1.5g, respectively (p<.0001). Abdominal circumference (AC) was lower (p<.05) and crown-rump length-to-AC was higher (p=0.07) in LBW relative to NBW pups. In male carotids, the EC 50 from the MCh [ ] response curve was higher in LBW/WD relative to NBW/ CD (p<.01). The aorta of both LBW/CD and LBW/WD had blunted responses to MCh. Responses to Phe and SNP were not different. The length-tension curve (male and female) was shifted to the left in NBW/WD (n=13), LBW/CD (n=5) and LBW/WD (n=8), compared to NBW/CD (n=15) (all p<.001). The slope of LBW/CD was markedly greater than NBW/WD (p<.001), while the LBW/ WD curve was further shifted to the left. LBW/WD had reduced mRNA levels of Mn SOD (p<.05) and Cu/Zn SOD (=0.06), relative to NBW/CD (p<.05). Therefore, in young adulthood, vascular function is influenced more by the intrauterine environment than by poor postnatal diet. WD exacerbates vascular dysfunction in IUGR offspring, possibly through oxidative stress. Thus, prenatal factors are powerful players in CVD.

Single Course Antenatal Glucocorticoid Has Multigenerational Effects on Growth, Endocrine Function and Behavior. Vasilis G Moisiadis, 1 Paul Blakeley, 1 Alisa Kostaki, 1 Stephen G Matthews. 1,2 1 Physiology, University of Toronto, Toronto, ON, Canada; 2 Ob-Gyn and Medicine, University of Toronto, Toronto, ON, Canada. OBJECTIVE: Antenatal synthetic glucocorticoid (sGC) exposure can permanently 'program' metabolic, cardiovascular and neurologic function in offspring. Further, we have recently shown that multiple course antenatal sGC treatment leads to profound effects on hypothalamic-pituitary-adrenal (HPA) function and behavior in second generation (F 2 ) offspring. While clinical use of multiple course therapy remains controversial in the management of preterm labour, single course treatment represents the 'gold-standard' of care. In the present study, we hypothesized that single course sGC therapy would significantly affect growth, HPA function and behavior in juvenile F 2 offspring. METHODS: Pregnant guinea pigs were subcutaneously treated with betamethasone (Beta; 1mg/kg; n=12) or saline (C; n=11) at 75% of gestation (term ∼69 days). Adult F 1 females (Beta & C) were mated with control males to produce F 2 offspring; there was no manipulation during pregnancy. Anthropometric measures were taken at birth and on postnatal day (PND) 20 (weaning). F 2 offspring were tested in an open-field (30 min) to assess locomotor activity and anxiety on PND19 and 24. Attention was assessed by prepulse inhibition (PPI) testing on PND23. HPA function was assessed under activated (PND19 & 24) and basal conditions (PND26). RESULTS: Single course sGC exposure significantly reduced birth weight (P<0.01) and abdominal circumference (P<0.05) in F 2 males, with no effect in females. At PND19 and 24, Beta female offspring showed habituation to the open-field (P<0.05); Beta males did not. Beta female and male offspring mounted more rapid HPA responses to stress (P<0.05). At PND26, only Beta males demonstrated a diurnal change in basal cortisol through the subjective day (P<0.05). There was no effect of sGC on attention in either sex. CONCLUSIONS: Prenatal treatment with single course sGC leads to multigenerational effects on growth, HPA function and behaviors. Interestingly, males appear to be more susceptible to these effects than females. We are currently investigating the molecular mechanisms that underlie multigenerational programming by sGC. Given that approximately 10% of all children in the developed world have been prenatally exposed to sGC, it is critical that we understand the long-term implications of such therapy. Funded By: Canadian Institutes for Health Research. Introduction Poor maternal nutrition during fetal life leads to increased risk of metabolic diseases in the offspring. Here we determined the effects of maternal diet on ageing of the offspring reproductive system and explored DNA damage as an underlying mechanism.

We utilised an established rat model where dams are fed an 8 % protein diet during pregnancy and offspring suckled by control-fed mothers (recuperated). Offspring were studied at 3 and 6 months of age. Telomere length was measured using Southern blotting and mitochondrial DNA (MtDNA) copy number by q-RTPCR. Oxidative stress was assessed through measurement of 3-NT and 4-HNE.

Recuperated females had a lower weight at birth than controls (P<0.01) but caught up during lactation. At 3 and 6 months of age, ovaries and oviducts of recuperated offspring had increased (P<0.01) mtDNA copy number (P<0.01). Germ-line cells showed no difference in mtDNA copy number. Ovarian telomere length tended (P=0.08) to be shorter in recuperated animals at 3 months but by 6 months telomeres were significantly (P<0.01) shorter in the recuperated group. In the oviducts, increased telomere shortening was detected at both 3 (P<0.05) and 6 months (P<0.01). Oxidative stress markers were increased in recuperated animals in ovaries at 6 months.

Suboptimal early nutrition induces accelerated ageing of ovaries and oviducts, via oxidative-stress mediated DNA damage. Both the mitochondrial and nuclear genomes are affected, although cells from the germ-line appear to be preferentially protected. This has important implications for fertility and the health of further generations.

Lipoxin A4 (LXA4) Synthesis Pathway Is Cycle Regulated in Human Endometrium and Dysregulated in Women with Endometriosis. Tolga B Mesen, 1 Bruce A Lessey, 2 Geraldine O Canny, 3 Lingwen Yuan, 1 Doug D Taylor, 4 Steven L Young. 1 1 Ob/Gyn, U. of North Carolina, Chapel Hill, NC, USA; 2 Ob/Gyn, Greenville Hospital System, Greenville, SC, USA; 3 Ob/Gyn, Centre Hospitalier Universitaire Vaudois; 4 Ob/Gyn, U. Louisville, Louisville, KY, USA. Background: LXA4, an eicosanoid produced by sequential actions of ALOX15B and ALOX5, acts as an anti-inflammatory factor and a selective estrogen receptor modulator. These functions suggest a role for LXA4 in endometrial physiology and endometriosis pathophysiology. The regulation of these enzymes in endometrium has not been previously studied. Objective: To determine cycle regulation of ALOX15B and ALOX5 in the endometrium of normal subjects and those with endometriosis. Materials and Methods: Normal subjects (n=16) and those with endometriosis (n=17) were randomized to endometrial sampling during the proliferative or urine LH-timed midsecretory phase. Additional normal subjects (n=20) underwent a modeled cycle with experimentally determined serum progesterone (P) concentrations. Relative ALOX15B and ALOX5 mRNA expression was determined by real-time RT-PCR and protein was localized using immunohistochemistry (IHC). Effects of estrogen (E) and the progestin, medroxyprogesterone acetate (MPA), were studied in vitro using Ishikawa cells. Differences between groups were assessed using Kruskal-Wallis and Mann-Whitney tests. Results: In normal endometrium, ALOX15B and ALOX5 expression are increased 11-and 3-fold in the mid-secretory phase (p=0.008, p=0.038). Expression of both was also induced by P in vivo (p<0.001 and p=0.04). ALOX-15B (p=0.01 and p=0.039) was increased in both cycles phases in subjects with endometriosis, while ALOX5 was only increased in the proliferative phase (p=0.002) (Figure) . In vitro treatment with MPA, but not E also stimulated ALOX15B expression. IHC localized ALOX15B protein to endometrial epithelial cells.

Conclusion: Key components for LXA4 synthesis are present and P regulated in human endometrium. In subjects with endometriosis, endometrial ALOX-15B and ALOX5 expression is significantly increased, suggesting possible utility as a diagnostic marker and a role in endometriosis pathophysiology. and Gynecology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. We conducted a meta-analysis of genome-wide association (GWA) studies of 4,604 endometriosis cases and 9,393 controls from Japanese 1 and European 2 ancestry. Our results show that rs12700667 on chromosome 7p15.2 previously found in Europeans replicates in Japanese (P = 3.6 × 10 -3 ) and we confirmed association of rs7521902 on 1p36.12 near WNT4. We also demonstrated association of rs13394619 in GREB1 on 2p25.1, and identified a novel locus on 12q22 near VEZT (rs10859871). Excluding endometriosis cases with minimal (rAFS stage I-II) or unknown severity, we identified additional novel loci on 2p14 (rs4141819), 6p22.3 (rs7739264) and 9p21. 3 (rs1537377) . All seven SNP effects were replicated in an independent cohort and produced P < 5 × 10 -8 in a combined analysis. We have identified novel variants associated with risk for endometriosis and demonstrated strong overlap in association signals for significant SNPs in both Japanese and European populations. In future, risk prediction and targeted disease therapy may be transferred across these populations. 

Wnt/β-Catenin: Paracrine Signaling between Stem Cells and Main Population in Uterine Fibroid Cell Growth. Masanori Ono, 1 Ping Yin, 1 Antonia Navarro, 1 John S Coon, 1 David C Brooks, 1 Saurabh Malpani, 1 Jiajia Ma, 1 Navdha Mittal, 1 Wenan Qiang, 1 Vanida A Serna, 1 Stacy A Druschitz, 1 Takeshi Kurita, 1 Cara J Gottardi, 2 Serdar E Bulun. 1 1 Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA; 2 Medicine, Northwestern University, Chicago, IL, USA. Each uterine leiomyoma is thought to be a benign monoclonal tumor arising from a single transformed myometrial smooth muscle cell, however, it is not known what leiomyoma cell type is responsible for tumor growth. Recently, we have reported that leiomyoma side population cells (LMSP), which have tumor initiating cell characteristics, are necessary for in vivo growth of leiomyoma xenograft tumors. Lower estrogen (E) and progesterone (P) receptor (R) levels in LMSP suggest an indirect paracrine effect of steroid hormones on stem cells via the mature neighboring cells. ERα and PR levels were strikingly lower (p<0.05) in LMSP compared with main population cells of leiomyoma tissues. Xenografts under the kidney capsule of immunodeficient mice made of freshly-isolated leiomyoma side population and myometrial smooth muscle cells grew to relatively large tumors (1.5 mm 3 ), whereas leiomyoma main population/myometrial smooth muscle cell xenografts produced smaller tumors (0.3 mm 3 , p<0.05, n=8), in the presence of E+P. LMSP by itself did not grow on cell culture plates. Intriguingly, E+P induced minimal growth of leiomyoma side population when co-cultured with myometrial cells maintained in a separate insert. However, LMSP showed a robust growth when in direct contact with myometrial cells in mixed cultures; E+P further stimulated this growth. Co-culture experiments in the presence of E+P further revealed that myometrial cell-derived paracrine factors stimulate the canonical Wnt pathway in LMSP leading to transcriptional activation of Wnt target genes. Interestingly, it has been reported that dysregulated Wnt signaling in the uteri causes mesenchymal tumorigenesis. Activation of β-catenin via E+P promoted LMSP proliferation. Separately, a Wnt ligand array was performed on cultured myometrial cells treated with or without E+P. Validation of select genes from the PCR profiling data demonstrated significantly enhanced expression of Wnt11 and Wnt16 (p<0.05) in myometrial cells treated with E+P. Our findings are suggestive of a paracrine role of Wnt ligand that arises in response to E+P from myometrial cells and activates Wnt signaling in leiomyoma tumor initiating cells, eventually leading to tumor growth.

Epigenome and Risk of Uterine Fibroids (UF): Altered Phosphorylation of Histone Methyltransferase EZH2 in Stem Cells from UF & Adjacent Myometrium. Sangeeta Nair, Chakradhari Sharan, Waseem Khoder, Sarbani Maitra, Ayman Al-Hendy. CWHR, Obstetrics and Gynecology, Meharry Medical College, Nashville, TN, USA. Background: Uterine fibroids are common benign tumors of premenopausal women with higher incidence in African-Americans. They originate from the myometrium and are estrogen and progesterone dependent. Previous reports by Walker C et al (2010 Walker C et al ( , 2012 have shown that in Eker fibroid rat model, exposure to exogenous estrogens brings about developmental reprogramming via histone modification of myometrium and enhanced estrogen-responsive gene expression with consequent fibroid formation. Objective: To test if histone modification is involved in human fibroid tumorigenesis. Methods: Stem cells were isolated from fibroids (F) and adjacent myometrium tissue (MyoF) from women undergoing hysterectomy at Nashville General Hospital approved by local IRB. Cells were selected from collagenase treated tissue, using CD44 & STRO1 antibody-coated biotinylated Dynabeads (Invitrogen).The stemness phenotype of the cells was confirmed via the expression of a panel of specific markers/genes as tested by flow cytometry and PCR. Cell lysates were prepared for western blot analysis. Similarly tissue lysates were prepared from archived fibroid and matched myometrium tissue. Western blot was used to determine expression of phosphorylated EZH2, (Enhancer of Zeste Homologue 2, histone methyltransferase) in the MyoF and F stem cells and corresponding tissues. Statistical analysis was done using student's t test. Results: Expression of phosphorylated histone methyltransferase EZH2 was 2 fold higher in F stem cells compared to MyoF stem cells (P<0.05). Similar significant differences were seen in phosphorylated EZH2 expression in F versus MyoF tissue. No difference was observed in total EZH2 protein levels among all tested samples. The results suggest an increased phosphorylation of histone methyltransferase EZH2 in fibroids compared to adjacent myometrium that may be originating from the corresponding stem cell compartment. Conclusions: Phosphorylation of EZH2 brings about chromatin remodeling via reduction of H3K27Me3 levels and consequent up-regulation of estrogen regulated gene expression which is conducive of uterine fibroid formation. Further research is needed to determine the pathways through which epigenetic changes in histone modification can explain the role of epigenetics in higher incidence of UF in African-Americans. BACKGROUND: Uterine fibroids(UFs) affect 77% of women by menopause and account for $9.4 billion in healthcare costs each year. Although UFs are highly heritable, genetic risk is poorly understood. The first UF genomewide association study(GWAS) was recently performed in a Japanese population(1,607 cases and 1,428 controls), with reported genome-wide level significance for gene variants across 10q24.33, 11p15.5, and 22q13.1. We tested for association of these variants and UF risk in U.S. populations. METHODS: Women were enrolled in the Right from the Start cohort (RFTS, 2001 (RFTS, -2012 and the BioVU DNA repository (2007-present) . We examined 65 single nucleotide polymorphisms(SNPs) tagging these three regions for association with UFs. UF status was determined by pelvic imaging. Logistic regression was used to test for SNP association with UF presence. We also combined association results from both cohorts using meta-analysis. RESULTS: 1,111 UF cases and 1,617 controls were used in analyses. We observed statistically significant associations among European Americans(EAs) across the three regions, but only two prior associated SNPs replicated(blocked 1.35, Q=0.24, I=28.4, p=8.7x10-3) . We did not find evidence for association within African Americans, however, when we metaanalyzed across races we observed associations at a SNP in BET1L(rs939917) and TNRC6B(rs4821942). Meta-analyses of RFTS, BioVU, and the prior GWAS showed little heterogeneity in effect sizes across studies with meta p values ranging between 1.32x10-8 to 3.13x10-9, which were much stronger than the prior GWAS, and supported the associations observed for all three previously identified loci. CONCLUSIONS: We independently replicated GWAS associations previously observed at BET1L and TNRC6B in a U.S. population. These data suggest that common variants may increase risk for UF in both EA and Japanese populations. However, further research is needed to assess the role of these and other genes across other racial groups. Background. Activation of blood pressure control mechanisms such as the renin angiotensin system (RAS) and Endothelin-1 (ET-1) have been shown to support proliferation of uterine leiomyoma (fibroid) cells. Activation of angiotensin type 1 receptor (AT1R) upregulate endothelial cell ET-1 secretion, however, the effects of activation of the AT1R on ET-1 in the uterine lieomyoma is unknown. Objective. This study was designed to test the hypothesis that AT1R blockade in fibroid smooth muscle cell (fSMC) inhibits cell proliferation and decreases fibroid ET-1. Methods. Immediately following hysterectomy, uterine fibroids and myometrium are excised, washed in PBS and collagenase digested, plated in petri dishes and incubated until confluency was achieved. Confluent cells were passed into 6 well plates seeded at 5x10 5 /well and incubated DMEM containing 10% fetal bovine serum, 5% penicillin/streptomyocin with or without 0.1µM of Losartan (an AT1R antagonist) and cultured for 72hrs. Fibroid and myometrium tissues (0.5mg/well) were cultured in duplicate with or without 0.1µM of Losartan for 72hrs. Secreted ET-1 was measured from cell culture media via ELISA, and normalized to total cell protein or tissue weight. Individual cell counts were used determine effects losartan to decrease cell growth. Results. Tissues were collected from 11 patients, ages 31-49yrs with an average blood pressure of 134/84mmHg. Losartan significantly decreased fSMC proliferation by approximately 50% compared to untreated fSMC cultures (0.547x10 6 vs 1.129x10 6 ; p=0.012; n=11). Importantly, administration of losartan had no effect on myometrium smooth muscle cell proliferation (p=0.299). Furthermore, losartan decreased fSMC ET-1 secretion compared to the untreated cells (2.15 vs 2.74 pg/mg) as well as fibroid ET-1tissue secretion compared to untreated fibroid tissue (4.18 vs 5.14 pg/mg). Conclusions. AT1 R blockade significantly decreased fSMC proliferation and fibroid secretion of ET-1, thereby supporting our hypothesis that activation of vasoactive mechanisms, RAS and ET-1, are important pathways in the development of uterine lieomyoma in prehypertensive premenopausal patients. These data further support a possible link between development of hypertension and potential cardiovascular disease with uterine fibroids in premenopausal women.

Differential Interaction between Progesterone Receptors and AP-1 Members -A Potential Regulatory Switch between Uterine Quiescence and Activation. Lubna Nadeem, 1 Oksana Synlova, 1 Dong Xuesen, 3 Stephen Lye. 1, 2 1 SLRI, Mount Sinai Hospital, Toronto, ON, Canada; 2 Physiology, Ob/ Gyn, U of Toronto, Canada; 3 Dept of Urologic Sciences, Vancouver Prostate Center, BC, Canada. BACKGROUND: Increased expression of the gap junction protein, connexin43 (Cx43), is essential for timely labour, however, the mechanisms by which Cx43 gene transcription is regulated remains unclear. We previously showed that AP-1 dimers differentially activate Cx43 transcription: Jun/Fos heterodimers providing strong and Jun/Jun homodimers weak transactivation of Cx43. We have shown that the ligand-bound progesterone receptor recruits p54nrb/ mSin3A co-suppressor and suppress Cx43 transcription through interactions with c-Jun at AP-1 sites. Since the PRA and PRB isoforms have different transcriptional activities, we hypothesized that these isoforms differentially interact with AP-1 dimers to modulate Cx43 transcription. METHODS: Immunoblotting was used to determine expression levels of AP-1 proteins in human myometrial cell line (hTERT-HM), stably expressing PRA or PRB and stimulated with progesterone (MPA, 100nM). Interaction between PR isoforms and AP-1 was examined in-situ by Proximity Ligation Assay (PLA) and further confirmed by co-immunoprecipitation. We used an AP-1 containing Cx43 promoter-luciferase reporter together with the expression of different Jun and Fos members, in SHM cells, to determine the transactivating potential of PRA and PRB. RESULTS: Stable expression of PRA significantly increased nuclear JunD, Fra-1 and Fra-2 levels, while PRB increased nuclear c-Jun and JunB levels. MPA enhanced PRA interaction with all Jun and Fos family members except FosB, while PRB interacted with all Jun members, but only cFos and Fra-2. Co-IP analysis of nuclear extracts confirmed those interactions. In preliminary studies, cells transfected with PRA and PRB alone showed similar induction of Cx43 transcription, but in the presence of cJun, JunD or Fra-2, PRA increased Cx43 transcription. Importantly, PR co-suppressors P54nrb and mSin3A interacted exclusively with PRB and not PRA, suggesting that PRA might participate in the induction of Cx43 gene transcription. CONCLUSIONS: Suppression of Cx43 during pregnancy may result from an interaction of PRB with Jun/Jun homodimers. Upregulation of PRA at term increases JunD, Fra-1 and Fra-2 expression, with higher probability of PRA-Jun/Fos heterodimer complex formation and increased Cx43 transcription. OBJECTIVE: We used semi-quantitative proteomics analyses to identify serum biomarkers of early spontaneous preterm delivery (sPTD) in a nested casecontrol study within a prospective longitudinal cohort of multiparous women with previous preterm deliveries (n=500) enrolled at three sites in the US (Genomic and Proteomic Network for Preterm Birth Research, U01-HD05088). METHODS: Maternal blood was collected at 19-24 and 28-32 weeks gestation, and serum aliquots were analyzed by LC-MRM-MS (two peptides/protein, three MRM/peptide). Targeted proteomics (stable isotope-labeled proteins secreted by trophoblast, endometrial, endocervical, and vaginal epithelial cell lines) and shotgun proteomics were employed to identify a panel of 32 candidate proteins that were differentially expressed in pooled serum samples from five women with sPTD at <34 weeks and five controls who delivered at 39-41 weeks. The expression of these 32 candidate proteins in individual serum samples was then compared between 35 cases and 35 controls matched by age, race, and clinical site. RESULTS: Only serpinB7 had serum concentrations that were different in women with and without early sPTD. The mean concentration of serpinB7 at 28-32 weeks was 15-fold higher (P=0.021) in women with subsequent sPTD compared to controls; there was no difference at the earlier time point (19-24 weeks) . Higher levels of serpin B7 at 19-24 weeks and at 28-32 weeks were associated with a shorter interval to delivery (P=0.019 and 0.001, respectively), and higher levels of serpin B7 in samples from 28-32 weeks were associated with a lower gestational age at delivery (P=0.004). Changes in serpinB7 protein levels in the same individual between 19-24 weeks and 28-32 weeks were not associated with sPTD. CONCLUSIONS: Targeted and shotgun proteomics analyses using maternal serum samples identified one protein, serpin B7, associated with early sPTD. Our results require validation in other cohorts and analysis of the possible mechanistic role of serpin B7 in parturition. Meanwhile, a similar approach can be utilized with other maternal specimens (e.g., urine, cervico-vaginal fluid, saliva) to identify biomarkers associated with sPTD. Obstetrics & Gynecology, University of Alberta. Background Numerous studies, including our own, have demonstrated that maternal stress -chronic or prenatal -can increase the risk of preterm birth. Genetic predisposition plays a major role in the etiology of both preterm birth and common mental disorders such as major depressive disorder. Objective To investigate whether single nucleotide polymorphisms (SNPs) in genes involved in the stress response are associated with spontaneous preterm birth using a candidate gene approach. Methods Maternal DNA from saliva of 190 cases (singleton spontaneous preterm birth at <37 weeks) and 369 controls (singleton term birth at 38-42 weeks without a history of preterm birth) were compared. Sociodemographic and medical data were collected. Sixteen SNPs -either tag SNPs located in key genes involved in the stress response identified in the Preterm Birth Genome Project database or SNPs found to be associated with adverse mental health outcomes in the published literature -were selected for genotyping and sequencing. SNPs were genotyped using Taqman® SNP genotyping assays. Univariate and multivariate logistic regression was performed. Results Four tag SNPs, all located in the mineralocorticoid receptor (MR) gene were associated with spontaneous preterm birth: rs17484063, rs2883929, rs4835136, and rs7680420 (p<0.05) Univariate analysis showed that maternal age, smoking, alcohol use, educational status, and a history of spontaneous miscarriage were associated with preterm birth (p<0.05) and were included as covariates in the multivariate model. After adjustment, two SNPs remained significantly associated with spontaneous preterm birth: rs17484063 (OR 0.50, p=0.038) and rs2883929 (OR 0.49, p=0.017). For each additional minor allele, the risk of preterm birth was halved. Conclusions Using a candidate gene approach, we discovered two novel polymorphisms, rs17484063 and rs2883929, both located in the MR gene that associate with spontaneous preterm birth. This is the first report to our knowledge demonstrating an association between these polymorphisms and preterm birth supporting a potential role for genetics in the association between chronic maternal stress and preterm birth.

Effects of Leptin on Human Myometrial Cell Proliferation. M Barrichon, 1 T Hadi, 1 M Wendremaire, 1 F Goirand, 1 M Bardou, 1,2 P Mourtialon, 3 P Sagot, 3 F Lirussi. 1, 2 1 U866, INSERM; INSERM; 3 Obstetrics and Gynecology, CHU, Dijon. BACKGROUND: Maternal obesity is associated with higher rates of post-dates pregnancies. Physiology of labor is poorly understood but it has been demonstrated that at the end of pregnancy, myometrial cells undergo phenotypic changes, from a proliferative phase to a contractile state in order to initiate labor. It has been shown that leptin, an adipokine increased in obese women inhibits, in vitro, myometrial contractility. Moreover it has been shown that leptin induces proliferation in many cell lines by activating ERK1/2 signaling pathway. This study was aimed to investigate the ability of leptin to induce human myometrial cell proliferation. METHODS: Primary cell lines were established from myometrial biopsies obtained from women with uncomplicated pregnancies, at term, before the onset of labor. After 24h adhesion and 72h starvation, cells were stimulated by leptin (6.25, 12.5, 25, 50, 100ng/ml) . Proliferation was assessed by cell counting using flow cytometry and by studying the cell cycle. Percentage of cell in S-phase was investigated by flow cytometry and cyclin D1 and E expression by western blotting. ERK1/2 activation and its nuclear translocation were assessed by western blotting and immunofluorescence respectively. Specificity of the effect was assessed using a specific leptin receptor antagonist. RESULTS: We showed that leptin increased both total cell number (increase vs. control, +49.8% and +50.1% for 6.25 and 50ng/ml respectively) and percentage of cells in S-phase (+73.8% vs. Ctrl, for leptin 50ng/ml). Leptin also up-regulated cyclin E (G1-S transition) expression (expressed in arbitrary density units, ADU: 1.00 (ref), 1.74±0.28, 1.34±0.08 for control, for 8h and 24h stimulations at 25ng/ml respectively). Finally, we suggest that leptin-induced myometrial cell proliferation is mediated through ERK1/2 signaling pathway, as an increase in ERK1/2 phosphorylation associated with its nuclear translocation was observed after 40min of leptin treatment at a concentration of 25ng/ml (ADU: 1.00 (ref), 2.33±0.43, for control and leptin respectively). CONCLUSION: Our preliminary results suggest that leptin induces myometrial cell proliferation by activating the cell cycle in an ERK1/2 dependant manner. These data suggest a contribution of leptin, by maintaining uterine quiescence, in the development of parturition-related disorders observed in obese women. Background: Functional progesterone (P4) withdrawal leading to labor is achieved in part by regulation of the ratio of nuclear progesterone receptor (nPR) A to B. The role of membrane PR (mPR) in labor is not known. Methods: To describe the expression of transcripts for mPRα and P4-targeted contraction-associated proteins during inflammation-induced preterm labor, uteri and placentas were harvested 8hrs after intrauterine injection of either PBS, heat killed E. coli or peptidoglycan (PGN)+polyinosinic:cytidylic acid (PIC) on day 15 of gestation. To study the interaction of mPRs and toll-like receptors (TLRs), a murine macrophage cell line (RAW264.7) was treated with lipopolysaccharide (LPS, a TLR4 ligand, 10ng/mL), P4 modified to be active only extracellularly by conjugation to BSA (P4-BSA, 100nM), both or neither for up to 4hrs. Gene expression was measured by RT-PCR normalized to GAPDH or by western blotting. Results: Intrauterine administration of either E. coli or PGN+PIC to pregnant mice significantly decreased expression of mPRα in uterus but not in placenta, and increased expression of COX2 in both uterus and placenta. No changes were seen for oxytocin receptor (OXTR) and connexin-43. In macrophages cultured in vitro, both LPS and P4-BSA caused significant down-regulation of mPRα, steroid receptor coactivator 2 (SRC2), and OXTR, with no interaction between LPS and P4-BSA. Both P4-BSA and LPS induced the expression of COX2, IL-1β, and cAMP-response elementbinding protein 3 (CREB3). Both P4-BSA and LPS induced phosphorylation of p38 MAPK in a time-dependent manner, with maximal effects at 30 mins of stimulation. Protein levels of IκBα (an indicator of NF-κB activation) and phosphorylation of ERK were unaltered by LPS and/or P4-BSA with up to 60 mins of treatment. Conclusion: Inflammation leads to down-regulation of mPRα both in pregnant mouse uterus and in a macrophage cell line. Activation of mPR signaling in macrophages in vitro induces a pro-inflammatory profile resembling that of LPS, a highly potent inflammatory ligand. These data suggest that binding of membrane progesterone receptors by progesterone induces inflammatory responses. Down-regulation of mPRα following exposure to pro-inflammatory danger signals may be a mechanism by which progesteronedependent inflammation can be minimized.

Sterile Intraamniotic Injection of IL1 Alters the Microbiome in a Primate Model of Chronic Inflammatory Preterm Birth. Kjersti Aagaard, 1 Jun Ma, 1 Lisa Miller, 2 Alan Jobe, 3 Suhas Kallapur, 3 Claire Chougnet. Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, USA; 2 California National Primate Research Center, UC Davis, Davis, CA, USA; 3 Pediatrics, Cincinnati Children's Hospital Research Foundation, Cincinatti, OH, USA. Objective: Chorioamnionitis is frequently associated with infectious related preterm birth, despite the fact that pathogenic causative microbes are infrequently cultured. We recently observed that sterile intraamniotic rhIL1β in Rhesus macaques resulted in Grade I/II chorioamnionits (Fig. A, B) , and sought to now exhaustively reconstruct shifts in the microbiome gene catalogue of the chorioamnion and placenta. Study design: Aided by our recent advances with the Human Microbiome Project (Nature doi10.1038), we undertook whole genome shotgun (WGS) metagenomic approaches to robustly characterize the amnion and placental microbiome in primates. Briefly, at prescribed preterm intervals (126-132 days) Rhesus macaques were sterile injected with saline (n=3) or 10µg rhIL1β (Peprotech; n=9). Dams underwent cesarean either 24 or 72 hours later, and chorioamnion and placental samples were collected by sterile dissection. DNA was extracted for WGS (Illumina HiSeq) and binned microbial seuqnecs were annotated and exhaustively analyzed on HUMAnN and MGRAST for gene cataloguing. Results: Sterile injection of intramniotic rhIL1β significantly increases IL6 in the placenta, plasma, and developing fetal lung. In association with these markers of chorioamnionitis, we observed structuring of the microbiome (comprised of 342 species representing 150 genus). Using computational metabolic reconstruction modeling, from >300 million reads of generated WGS data we constructed a microbial catalogue comprised of 6973 genes differentially associated with histologic chorioamnionitis. Conclusion: Histologic chorioamnionits induced by sterile injection of rhILβ structures the primate amniotic and placental microbiome community. We speculate that these findings may inform clinical care in the future, and potentially diminish the administration of broad spectrum antibiotics for presumptive chorioamnionitis.

Metabolic Adaptation of Syncytiotrophoblast Mitochondria with Maternal Obesity. J Mele, A Maloyan, L Myatt. Center for Pregnancy and Newborn Research, UTHSCSA, San Antonio, TX, USA. Background: Obesity is associated with mitochondrial dysfunction resulting from chronic inflammation and oxidative stress. We have previously shown placental mitochondrial dysfunction with increasing maternal adiposity. The placenta can rapidly switch between oxidative phosphorylation and glycolysis. The ability to control metabolic reliance in this manner may be a protective mechanism against the pathological consequences of disease states. It is not known how chronic inflammation and the oxidative stress of obesity affect the ability of the highly metabolically active syncytiotrophoblast (ST) to adapt its metabolic capacity. We hypothesized that metabolic flexibility of syncytiotrophoblast is diminished in placentas from women of increased adiposity. Methods: Cytotrophoblasts were isolated from placentae collected by c-section at term (no labor) from lean (LN; BMI range 18-23), overweight (OW; 24-26) and obese (OB; 34-44) women (n=4 each group) and syncytialized over 72hrs. For the final 24hrs, media contained either galactose (10mM) or glucose (17mM). Mitochondrial function was then assessed using a XF24 analyzer (Seahorse Bioscience). In galactose, ST must rely exclusively on mitochondrial respiration for energy. Basal oxygen consumption rate, ATP coupled respiration, maximum respiration, spare capacity, proton leak, nonmitochondrial respiration and coupling efficiency were calculated using ETC inhibitors oligomycin (0.5µM), FCCP (0.75µM) and rotenone (3µM)/antimycin A (1.5µM). Results: Maximal respiration and spare capacity of ST decreased significantly (p<0.05, ANOVA) with increasing adiposity whether cultured in media containing glucose or galactose. When challenged with galactose, ST from LN women were able to significantly increase (p<0.01) basal respiration from 15±2.3 (glucose) to 34±4.5pmoles (galactose) O 2 /min/µg protein without significantly affecting other mitochondrial parameters. No effect of galactose challenge on respiratory parameters was seen in ST of OW or OB women. Conclusions: Mitochondrial dysfunction in ST from placentas with increasing maternal adiposity is evident by reduction in maximal respiration and spare capacity. ST from lean women are metabolically flexible as they can respond to metabolic challenge by increasing oxidative phosphorylation. This response is lost in OW or OB women. In vivo this may result in placental and fetal compromise in the obese mother when faced with further stressors. Glucocorticoids play a vital role in fetal organ growth. However, over-exposure of the fetus to glucocorticoids leads to fetal growth retardation, and programs the development of adult diseases. Low glucocorticoid levels in the fetal body are ensured by the abundant expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in the placenta, which inactivates maternal cortisol to inactive cortisone. During cytotrophoblast syncytialization, 11β-HSD2 expression is markedly increased. However, the underlying mechanisms remain largely unknown. We have demonstrated that the transcription factor Sp1 plays a significant role in 11β-HSD2 expression during syncytialization. P300, a histone acetyltransferase, is a common transcriptional cofactor for Sp1. Whether p300 could interact with Sp1 to induce 11β-HSD2 expression during syncytialization is not known. Objectives: To examine p300 and its interaction with Sp1 in the induction of 11β-HSD2 expression during syncytialization. Design: The distribution of 11β-HSD2, p300 and Sp1 in human placenta at term was examined with immunohistochemistry. Placental cytotrophoblasts were prepared from term placentae in the absence of labor for the study of 11β-HSD2, p300 and Sp1 expression during syncytialization in vitro with real time PCR and Western blotting. P300 in 11β-HSD2 expression was studied with siRNA-mediated knock-down and over-expression of p300 as well as p300 inhibitor C646. Chromatin immunoprecipitation (ChIP) was conducted to study the acetylation of histone 3 lysine 9 and 27 on 11β-HSD2 promoter. The interaction of Sp1 and p300 were examined with co-immunoprecipitation (co-IP) and ChIP. Results: Stronger stainings of 11β-HSD2, p300 and Sp1 were found in syncytiotrophoblasts than in cytotrophoblasts. The expression of 11β-HSD2, p300 and Sp1 were increased along with enhanced acetylation of H3K9 and H3K27 on 11β-HSD2 promoter during syncytialization. Over-expression of p300 increased 11β-HSD2 expression, while p300 knock-down or C646 attenuated acetylation of H3K9 and H3K27 and 11β-HSD2 expression. ChIP revealed the binding of both p300 and Sp1 to 11β-HSD2 promoter. Co-IP showed Sp1 and p300 in the same protein complex in the syncytiotrophoblasts. Conclusions: Recruitment of p300 by Sp1 enhances the acetylation of H3K9 and H3K27 on 11β-HSD2 promoter, which plays a crucial role in the upregulation of 11β-HSD2 during syncytialization. Background Placental amino acid transport is increased in obese women giving birth to large babies. Obese pregnant women have elevated circulating markers of inflammation and pro-inflammatory cytokines raise placental amino acid transport in vitro. The omega 3 fatty acid docosahexaenoic acid (DHA) has anti-inflammatory properties, partly via its receptor GPR120. We hypothesized that DHA supplementation in obese pregnancies decreases placental amino acid transport activity. Methods Obese women (n=35) were enrolled at 26 wks gestation and assigned to placebo (corn/soy oil) or DHA (800 mg/day). Blood samples were collected at 26 and 36 wks gestation and placentas at term. Maternal red blood cell (RBC) and placental membrane DHA levels were measured by GC/MS. Placental microvillous (MVM) and basal plasma (BM) membranes were isolated (n=31). Expression of amino acid, fatty acid and glucose transporters was determined by Western blot. MVM amino acid uptake was assessed by isotope labeled tracers. Effect of DHA on amino acid uptake in cultured primary trophoblast cells was investigated. Results Maternal RBC DHA levels did not differ at baseline (5.7% vs. 5.9%) and increased at 36 wks in the DHA supplemented group (6.2% vs. 10.0%; p<0.01). Placental DHA levels (r=0.372) and MVM GPR120 expression (r=0.357) correlated with maternal RBC DHA (p<0.05). Expression of BM glucose transporter 1 (r=0.433) and MVM fatty acid transporter 4 (r=0.494) correlated with placental DHA levels (p<0.05). In contrast, MVM System L transporter LAT1 (r=-0.640) expression and amino acid uptakes (System A, r=-0.413; System L, r=-0.580) correlated inversely with placental DHA levels (p<0.05). Birth weights were not affected. In trophoblast cells, DHA exposure (25 or 50µM for 24h) reduced System A activity (-36% and -61%; p<0.05, n=7).

Conclusion DHA supplementation reduces placental amino acid transporter activity in obese women and we confirmed this in cultured trophoblast cells. We speculate that DHA supplementation in obese pregnancies may be beneficial by attenuating increased amino acid transport, which otherwise could contribute to fetal overgrowth. In contrast, increased placental DHA was associated with up-regulated fatty acid and glucose transporter expressions. Thus, DHA supplementation appears to differentially modulate nutrient delivery to the fetus.

Mechanism of Uptake of Cationic Nanoparticles by Human Placental Syncytiotrophoblast Cells. Gautam Kaul, 1 Tristan D Clemons, 2 Killigudi S Iyer, 2 Kamali Pugazhenthi, 1 Jeffrey A Keelan. 1 1 School of Women's and Infants' Health, University of Western Australia, Perth, WA, Australia; 2 Centre for Startegic Nano-fabrication, University of Western Australia, Perth, WA, Australia. Background and rationale: Nanoparticle-mediated drug delivery has enormous potential for the treatment of placental disorders in pregnancy. The placental syncytiotrophoblast layer is bathed in maternal blood and accessible to circulating nanomaterials. Studies suggest that some nanomaterials are taken up by the syncytium and cross the placenta, while others are not. The aim of the present study was to explore, using functionalised, fluorescent nanoparticles, the effect of surface composition on placental nanoparticle uptake and the mechanism of cellular uptake. Methods: Nanoparticles (100-150 nm) doped with rhodamine B and magnetite (iron oxide) for visualisation purposes were prepared from polyglycidylmethacrylate (PGMA) polymer, with/without surface modification via polyethyleneimide (PEI) conjugation. Trophoblasts were extracted from normal term placentas (n=5) and allowed to syncytialise in culture for 3-5 days, then exposed to nanoparticles for 0.5 -6 h at concentrations of 2-200 mg/mL in the presence/absence of the following pinocytosis inhibitors: filipin III (7.6 uM), cytochalasin B (4 uM), ameloride (50 uM), nystatin (50 uM), ikarugamycin (5 uM) , chlorpromazine (28 uM) and dynasore (80 uM). Uptake was assessed using fluorescent microscopy and quantitative fluorometry Results: Syncytial uptake of nanoparticles was entirely dependent on the PEI (cationic) surface modification; anionic uncoated particles were excluded. Uptake of PEI-coated nanoparticles was time-and concentration-dependent, with levels of uptake increasing across the entire experimental time period. Of the 7 inhibitors tested, dynasore (an inhibitor of dynamin-mediated endocytosis) was the most potent uptake inhibitor, blocking nanoparticle uptake at 6 h by ∼89% vs. vehicle control (P<0.01); ikarugamycin and chlorpromazine were also effective, albeit to a lesser extent (∼50% inhibition; P<0.01). Conclusions: Cationic surface modification with PEI greatly enhances the ability of polymeric nanoparticles to be taken up by the human placenta. Uptake of PEI-PGMA nanoparticles into placental syncytiotrophoblasts appears to be via a dynamin-mediated, clathrin-dependent endocytotic process, similar to that used for the uptake of transferrin. Background and Objective: ZNF403 is an evolutionarily conserved member of the zinc finger protein family containing a C2H2 zinc finger motif at the N-terminus and an LxxLL nuclear receptor binding domain at the C-terminus. It was originally identified as a dioxin-induced factor in mouse embryonic stem cells but its function is not yet clear. This study was to investigate the role of ZNF403 in mammalian physiology. Methods and Results: A Znf403 mutant mouse line in the C57BL/6 genetic background was generated using gene-trap technology. The Znf403 null mutant embryos die in utero during E13.5 to E15.5. Gross anatomical examination of Znf403 null embryos showed neither deformity of appearance nor malformation of the hearts and brains with the exception of dysmorphic placentae, characterized by proliferative nests of trophoblastic tissue. Immunohistochemistry, morphometric analyses and rhodamine dye perfusion tests revealed that lethality of Znf403 null embryos was likely caused by a defect in placental perfusion due to remarkable decreases in both fetal and maternal blood vessels in the labyrinth. Significant elevation of Stat3 phosphorylation and a dramatic increase in the number of trophoblast stem cells (TSCs) were detected in the E15.5 of Znf403 null placentae. BrdU labeling indicated a marked increase in placental trophoblast proliferation, while trophoblast apoptosis was unaffected. In vitro studies using murine TSCs revealed that deficiency of Znf403 promoted TSC proliferation while their differentiation was inhibited. ZNF403 was localized in both the cytoplasm and nuclei of trophoblasts in mice and human placentae. Western blot analysis of normal human placentae demonstrated decreasing expression of ZNF403 with progression of gestation. Conclusions and Implications: Data indicate for the first time that ZNF403 is an essential factor for pregnancy success through its role in maintenance of a balance of TSC proliferation and differentiation during placental development. Conditions related to the over-and under-development of trophoblast may be relevant targets to further study the role of ZNF403 in humans, including hypertensive disorders of pregnancy, intrauterine growth restriction, invasive placentation, and late trimester demise. Objective: Fatty acids, particularly essential fatty acids, are critical for proper fetal growth and development. As a part of our ongoing studies to define pathways underlying placental fat uptake and trafficking, we sought to examine trophoblastic neutral lipid accumulation and the function of the lipid droplet associated PLIN family of proteins. We tested the hypothesis that PLIN2 plays a key role in fat accumulation in human trophoblasts. Methods: Primary human trophoblasts (PHT cells) isolated from term human placentas and BeWo cells were cultured under standard (O2=20%) or hypoxic conditions (O2<1%), or with or without fatty acids (linoleic acid: oleic acid=2:1) for 24 h. BODIPY493/503 was used to visualize intracellular lipid droplets. PLIN family gene expression was determined by RT-qPCR and confirmed by western immunoblotting. PLIN2 and/or PLIN3 were knocked down by lentiviral-based RNA interference. Results: Among PLIN family members, PLIN2 and PLIN3 were expressed in PHT cells. PLIN4 mRNA, in addition to PLIN2 and PLIN3, was detected in BeWo and JEG-3 cells. PLIN1 and PLIN5 mRNA expression was below detectable levels. PLIN2 was markedly upregulated during lipid droplet formation that was induced by hypoxia or fatty acids in trophoblasts, and associated with increased concentration of lipid droplets. PLIN3 expression was unchanged. Intriguingly, knock down of PLIN2 had no effect on lipid droplet morphology in BeWo or PHT cells, but was associated with a reciprocal increase in PLIN3 expression along a range of PLIN2 expression levels (y=-7.8709x + 4.1605, R²=0.79437, p<0.0001, N=12) . In contrast, knock down of PLIN3 had no affect on PLIN2 expression or cell phenotype (N=8). Importantly, PLIN2/ PLIN3 double knock down induced cell death the most of all; cell death was greater in PLIN2 knock down than PLIN3 knock down in PHT cells. PLIN2/ PLIN3 double knock down in BeWo cells led to peripheral sequestration of lipid droplets. Conclusions: PLIN2 and PLIN3 play a key role in lipid droplet formation and mobilization in human trophoblasts. Reduced expression of trophoblastic PLIN2 is compensated by reciprocal increase in PLIN3 expression, likely bolstering cell protection against lipotoxicity. However, PLIN 3 is not likely to able to fully compensate PLIN2 function alone. Supported by a scholarship from Kobe University, Japan (MM), and NIH R01ES11597 and P01HD069316 (YS).

Monocytes of Women with Pre-Eclampsia Alter the Inflammatory Response of Human Vascular Endothelial Cells in Co-Culture. Ebtisam Al-ofi, 1 Seth Coffelt, 2 Dilly Anumba. 1 1 Academic Unit of Reproductive and Developmental Medicine, University of Sheffield Medical School, United Kingdom; 2 Division of Immunology, Netherlands Cancer Institute, Netherlands. Introduction: Preeclampsia (PE) is characterized by an exaggerated systemic inflammatory response and generalized endothelial dysfunction. We recently demonstrated that monocyte subpopulations from women with PE are abnormally skewed and exhibit exaggerated responses to toll-like receptor (TLR-2 and -4) ligands. Objective: We sought to determine the role of activated monocytes in the generalized vascular endothelial dysfunction in PE by carrying out co-culture experiments of primary human monocytes with vascular endothelial cells. We investigated the inflammatory responses of our co-culture model following stimulation with TLR-2 and -4 ligands. Methods: Human vascular endothelial cells (HUVECs) were isolated from umbilical cords, cultured and passaged. HUVECs were seeded onto gelatincoated 12 well tissue culture plates until they reached 70-90% of confluence. Then monocytes, isolated from 9 PE (GA=33.18 ± 5.8 wks) and 9 normal pregnant (GA= 33.15 ± 4.0) women, were plated on top of HUVECs at the fractional rate of 1 monocyte to 5 HUVECs. Mono-and co-cultures were stimulated with lipopolysaccharides (TLR4 ligand), peptidoglycan (TLR2 ligand) and fibrinogen (an endogenous TLR4 ligand) for 24 hours. Flow cytometry was used to confirm monocyte and endothelial cell populations in the model. Cytokines and chemokines were measured in supernatant medium by cytometric array. Results: In comparison with NP, co-cultures of PE monocytes with endothelial cells demonstrated profound secretion of inflammatory cytokines (IL-6 and IL-1β) and chemokines (IL-8 and MCP-1) (P<0.05), but reduced production of the anti-inflammatory cytokine (IL-10, P<0.01). Compared to NP, stimulation with TLR-2 and -4 ligands increased the expression of IL-6 and IL-1β from PE co-cultures (P<0.05), reduced expression levels of IL-8 and MCP1 (P<0.05), whilst IL-10 was unchanged. Conclusion: Our findings suggest that PE monocytes play a key role in the modulation of endothelial dysfunction in pre-eclampsia and the systemic inflammatory response in this condition, observations which may be mediated by TLR -2 and -4.

Fibrinogen Attenuates sFlt-Induced Dys-Angiogenesis and Increases Vascular Adhesion Molecule Expression in Pre-Eclampsia. Ebtisam Alofi, 1 Seth Coffelt, 2 Dilly Anumba. 1 1 Reproductive & Developmental Medicine, University of Sheffield; 2 Division of Immunology, Netherlands Cancer Institute. Introduction: Vascular endothelial dysfunction characterizes pre-eclampsia (PE). The imbalance between the angiogenic factors vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF), and the anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1), may play an important role in mediating endothelial cell dysfunction. Objective: Using an experimental co-culture model of primary monocytes and vascular endothelial cells we sought to determine the effects of TLR ligands on the expression levels of VEGF, sFlt-1 and cell adhesion molecule (sVCAM-1) in PE vs normotensive pregnant women (NP). Methods: Human vascular endothelial cells (HUVECs) were isolated from umbilical cords, cultured and passaged, and then seeded onto gelatin-coated 12 well tissue culture plates until they reached 70-90% of confluence. Then monocytes, isolated from 9 PE (GA=33.18 ± 5.8 wks) and 9 NP (GA= 33.15 ± 4.0) women, were plated on top of HUVECs at the fractional rate. Cultures were stimulated with lipopolysaccharides (TLR4 ligand), peptidoglycan (TLR2 ligand) and fibrinogen (an endogenous TLR4 ligand) for 24 h. sVCAM-1 and VEGF were measured in supernatant by cytometric array, and s Flt-1 by ELISA. Results: VEGF production by monocyte-HUVEC co cultures was lower in PE compared to NP (P<0.05). Expression levels of sFlt-1 from HUVECs were downregulated by co-culture with monocytes from both PE and NP (P<0.001); downregulation of sFlt-1 by NP monocytes was significantly more than by PE monocytes (P<0.05). Exposure of PE monocytes in co-culture with HUVEC to fibrinogen resulted in downregulation of sFlt-1 production whilst this was unchanged for NP (P<0.05). However, stimulation of co-cultures by bacterial ligands induced sFlt-1 production in NP and PE (P<0.05). Compared to NP, monocytes-HUVEC co-culture in PE demonstrated higher basal expression levels of sVCAM-1 (P<0.05), and their exposure to TLR ligands was also associated with an exaggerated sVCAM-1 response (P<0.05).

Fibrinogen is implicated in TLR4-mediated angiogenesis, coagulation and immunomodulation, but its vascular role in PE is ill understood. Our observations suggest that fibrinogen may contribute to endothelial repair in PE by enhancing angiogenesis through mechanisms that may include suppression of sFlt-1 production, in addition to its well established role in vascular haemostasis.

Choline Modulates Soluble-Endoglin in Cultured Human Trophoblasts. Benjamin Y Andrew, Xinyin Jiang, Sara Jones, Marie A Caudill, Patsy M Brannon. Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA. Anti-angiogenic factors, soluble fms-related tyrosine kinase-1 (sFLT1) and soluble endoglin (sEng), contribute to the pathogenesis of preeclampsia (PE) through their proposed sequestration and inhibition of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-ß), respectively. This reduction of available pro-angiogenic factors leads to vascular dysfunction and PE. Circulating sEng results from the cleavage of the transmembrane endoglin (Eng) by matrix metalloproteinase-14 . Recently, we showed that supplemental choline reduced placental sFLT1 mRNA and maternal circulating sFLT1 in pregnant women and in cultured placental trophoblasts (HTR-8/SVneo). To determine if choline also plays a role in the regulation of sEng production independently or interactively with hypoxia (Hx), HTR-8/SVneo cells were cultured in a 2x4 factorial design in either normoxia (20% O 2 , Nx) or Hx (1% O 2 ) and one of four choline concentrations [13 µM, 18 µM, 28 µM (control), and 48 µM] for 96 hours. Data were analyzed by two-way ANOVA and appropriate post-hoc comparisons (SigmaPlot). Levels of mRNA (determined by qRT-PCR) were reduced under Hx for MMP-14 (39.63%, p<0.001), and Eng (21.21%, p<0.001). Low choline (5 µM) under Nx, but not Hx, increased mRNA levels of MMP-14 (19.7%, p=0.016) compared to control. Choline did not affect Eng mRNA under Nx or Hx. Low choline increased media sEng (determined by ELISA) under Nx (14%, p=0.036) compared to control and Hx increased media sEng (9.25%, p<0.001) across all choline levels. Thus, low choline modulates, under Nx but not Hx, the anti-angiogenic sEng similarly to its reported effects on the other anti-angiogenic factor, sFLT1. Low choline under Nx does not affect Eng mRNA (the precursor to sEng), but increases MMP-14 mRNA, suggesting that the increase in sEng may be partially due to enhanced MMP-14 cleavage. Conversely, Hx decreases MMP-14 and Eng mRNA levels but paradoxically increases media sEng, irrespective of choline level. Thus, pathways other than MMP-14 cleavage and independent of choline status may mediate the modulation of sEng release under Hx. Together these results suggest the potential for low choline status as an initiator of angiogenic imbalance and subsequent placental Hx, which may continue to promote an anti-angiogenic state, eventually leading to PE.

The Prostacyclin Analog, Beraprost, Can Paradoxically Increase Vascular Tone. Yurij P Vedernikov, Ancizar Betancourt, Rina Ow, Daren T Tanchico, Hossein Golabbakhsh, Michael A Belfort. Obstetrics & Gynecology, Baylor College of Medicine, Houston, TX, USA. OBJECTIVE: The Vascular endothelium can produce both vasodilating and vasoconstrictor factors in response to mechanical and chemical stimuli. This study was designed to evaluate the concentration response relationship (CRR) of human omental arteries (OA) to beraprost (BER), an analog of prostacyclin (PGI 2 ) and a PGI 2 receptor antagonist CAY 10441 (CAY) STUDY DESIGN: Omentum samples were obtained during gynecological surgical procedures from 15 patients. OA were isolated and 2 mm rings were mounted onto a wire myograph 620 M (DMT) between two 25-µ nitinol wires in PSS, 37 o C. The mounted rings were equilibrated and normalized, vascular reactivity was tested with KCL. The integrity of the endothelium function was checked, and after washing and resting, the rings were exposed to 10 -7 M of a thromboxane analog (U 46619). CRR to BER was obtained in the absence and presence of CAY at 10 -8 , 10 -7 and 10 -6 M. All experiments were run concurrently. Paired t-test was used for statistical analysis. Asterisks show at what concentration the difference with control is significant. RESULTS: 1) The OA from 7 women responded to BER with concentrationdependent relaxation (CDR), (IC 50 =-8.29±0.14) followed by an increase in tension at higher BER concentrations (Group I: left graph in circles); 2) The OA from 8 patients reacted to BER with CDR only (IC 50 =-7.83±0.17, no significant difference), (Group II: left graph in squares); 3) In Group I, CAY decreased BER-induced relaxation and depressed BER-induced increase in tension at 10 -7 and 10 -6 M (middle graph); In Group II, CAY decreased BER-induced relaxation at 10 -7 and 10 -6 M (right graph). CONCLUSION: 1). Beraprost, a PGI 2 agonist, paradoxically increased the vascular tension at higher concentration in about half of the study samples; 2) both vascular relaxation and increase in tension induced by BER can be significantly reduced by a PGI 2 receptor antagonist. These observations may provide important insight to our understanding of dissimilar patient responses to vasoactive agents.

Variation in Endoglin Pathway Genes Is Associated with Preeclampsia. Mandy J Bell, 1,3 James M Roberts, 2,3 Sandra A Founds, 1,3 Arun Jeyabalan, 2, 3 Carl A Hubel, 2,3 Lauren Terhorst, 1 Yvette P Conley. 1 1 School of Nursing, Univ. Pittsburgh; 2 Obstetrics, Gynecology & Reprod. Sciences, Univ. Pittsburgh; 3 Magee-Womens Research Institute, Univ. Pittsburgh, Pittsburgh, PA, USA. Background: The pathophysiology of preeclampsia (PE) is unclear. Endoglin (ENG) is a co-receptor of the transforming growth factor beta family (TGFβ). Elevations in circulating soluble ENG protein and placental/blood ENG mRNA expression precede the clinical presentation of PE, suggesting a role for this anti-angiogenic factor in PE. Methods: This case-control study investigated ENG's potential role in PE by examining the association between ENG pathway genetic variation and PE in 355 white (181 cases/174 controls) and 60 black (30 cases/30 controls) women matched on ancestry, age, and parity. Tagging single nucleotide polymorphisms (tSNPs) and potentially functional SNPs of the TGFβ family (ENG, TGFβ1, TGFβR1, ALK1, and TGFβR2) were evaluated with iPLEX® and TaqMan® technologies. Allele/genotype/haplotype tests were conducted separately in white/black subgroups with χ2 or Fisher's exact tests. Odds ratios were computed with binary logistic regression for tSNPs with significant genotype tests. Results: Of the 49 SNPs evaluated, variation in two ENG tSNPs (rs11792480, rs10121110) and one TGFβR2 tSNP (rs6550005) was associated with PE in white women (P <0.05, each). In black women, variation in two TGFβ1 tSNPs (rs4803455, rs4803457), one TGFβR1 tSNP (rs10739778), and three TGFβR2 tSNPs (rs6550005, rs1346907, rs877572) was associated with PE (P <0.05, each). Notably, white women that inherited the AA genotype for ENG tSNP rs10121110 were 2.29 times more likely to develop PE compared to those that inherited the GG genotype (P=0.008, [99% CI: 1.02 to 5.13]). Moreover, ENG haplotype TACGA, which contains the risk alleles for rs11792480 and rs10121110, was over-represented in white women with preeclampsia (P=0.02). In black women that inherited the CT genotype for TGFβ1 tSNP rs4803457, they were 7.44 times more likely to develop PE compared to those that inherited the CC genotype (P=0.005, [99% CI: 1.19 to 46.41]). Conclusions: These results suggest that ENG pathway genetic variation is associated with PE and that the pathway's involvement in PE may differ in whites and blacks. Funding:T32NR009759, F31NR011379, P01HD30367, Magee CRC grant #5M01RR00056, Eta Chapter of STTI Honor Society of Nursing, ISONG

Plasma Levels of S100B in Women with Preeclampsia. Lina Bergman, Anna-Karin Wikstrom, Tansim Akhter, Tord Naessen, Helena Akerud. Institute for Women's and Children's Health, Uppsala University, Uppsala, Sweden. Objective: S100B is suggested to be a peripheral biomarker of central nervous system injury with increased blood-brain barrier permeability. The aim of this study was to investigate if serum-S100B is elevated among women with preeclampsia compared to women with normal pregnancies. We wanted furthermore to evaluate if levels of S100B correlate to symptoms of preeclampsia and severtity of the disease. Study design: A case-control study with a cross-sectional design was performed. Women with early onset-and late onset preeclampsia were recruited at time of diagnosis and individually matched to controls in aspects of gestational age. S100B was analysed with an ELISA assay and laboratory parameters and premonitory symptoms were collected from the medical records. Results: Levels of S100B were significantly higher among women with preeclampsia compared to controls. There was no difference between women with early onset preeclampsia compared to those with late onset preeclampsia.

Women with facial oedema and women with proteinuria >5g/24 hours had significantly higher levels of S100B compared to the other women in the case group.

Conclusion: S100B levels are higher among women with preeclampsia compared to controls which might indicate a defect in the cerebrovascular endothelium in women with preeclampsia. There seems furthermore to be a correlation between levels of S100B and premonitory symptoms as well as laboratory parameters, which might be of relevance indicating that severity of the disease and levels of S100B associates.

Growth Factor-Induced Endothelial Cell Functional Disruption as a Model of Preeclampsia: Translation from Sheep to Human. Jennifer L Krupp, 1, 2 Derek S Boeldt, 1 Fu-Xian Yi, 1 Dinesh M Shah, 1 Ian M Bird. 1 1 Ob/ Gyn, Perinatal Research Labs, Univ Wisconsin Madison, WI, USA; 2 Ob/Gyn, Univ Iowa, Iowa City, IA, USA. Introduction: In pregnancy, enhanced vasodilation occurs in response to Ca2+ stimulating hormones, and in preeclampsia a corresponding loss of adapted function is associated with elevated circulating VEGF. Prior studies in pregnant sheep uterine artery endothelial cells (UAEC) showed this sustained vasodilator production response to ATP is dependent on sustained Ca2+ responses in the form of transient bursts, which are in turn dependent on enhanced cell-cell communication via connexin 43 gap junctions. VEGF-165 pretreatment of UAEC can inhibit gap junctions by phosphorylation and so reverse adaptation. In human umbilical vein endothelial cells (HUVEC), sustained burst responses are also observed and closely resemble those of UAEC. We hypothesize that HUVEC also depend on gap junction communication for sustained Ca2+ responses and are similarly inhibited by VEGF-165. Objective: As previously studied in UAEC, we use a 30-minute pretreatment of primary HUVEC from normal pregnancies with VEGF-165 to induce a preeclampsia-like phenotype of disrupted capacity to produce vasodilators, and compare to the effect of gap junction inhibitors. In so doing we validate primary HUVEC as a translational model of endothelial dysfunction in pregnancy. Methods: Like UAEC, HUVEC were grown to 95% on 35mm glass bottom dishes. Cells were then loaded with Fura-2 and stimulated with 100uM ATP for 30 minutes (control). After washing, cells were pretreated as below, followed by a second ATP stimulation. Results: VEGF-165 pretreatment inhibits ATP-stimulated Ca2+ bursts in HUVEC by 40% (p<0.001) (vs 21% for UAEC (p<0.001)). Pretreatment with the connexin 43-specific inhibitory peptide, Gap27, inhibits ATP-stimulated Ca2+ bursts in HUVEC by 30% (p<0.001) (vs 54% for UAEC (p<0.0001)). Conclusion: As in UAEC, HUVEC depend on connexin 43 gap junction function for sustained Ca2+ burst responses, and are thus also subject to negative regulation by VEGF-165 pretreatment. It is therefore highly likely that both cell types undergo similar negative regulation of Ca2+ mobilization and so vasodilator production by VEGF-165. As such, HUVEC can potentially serve as a translational model to aid in the discovery of novel endothelial-targeted therapies to treat diseases of endothelial disruption, such as preeclampsia. Introduction: Preeclampsia is a common disorder of human pregnancy diagnosed by the onset of hypertension and proteinuria after the 20th week of gestation and associated with endothelial dysfunction. Importantly, women with preeclampsia are at a greater risk for cardiovascular disease later in life. We have previously shown that lectin-like oxidized low density lipoprotein (oxLDL) and its receptor (LOX-1), are increased in the vasculature of women with preeclampsia. LOX-1 contributes to oxidative stress, which may result in reduced bioavailability of nitric oxide (NO) and increased endothelin (ET-1)-mediated vasoconstriction. Furthermore, ET-1 induces LOX-1 expression, encouraging the maintenance of oxidative stress. We hypothesize that changes in vascular function persist postpartum and contribute to increased cardiovascular risk following a preeclamptic pregnancy. Methods: The postpartum vascular effects of preeclamptic-like symptoms were assessed in a rat model of reduced utero-placental perfusion pressure (RUPP). Pregnant Sprague Dawley rats underwent surgery on day 14 of gestation, during which restrictive clips were placed around the abdominal aorta and ovarian arteries, reducing utero-placental blood flow. Sham-operated rats were used as controls. At 1 month postpartum, vascular function was analyzed using wire myography. Phenylephrine (PE)-induced vasoconstriction and methylcholine (MCh)-induced vasodilation were assessed in thoracic aorta and mesenteric arteries. In addition, responses to big ET-1 were assessed in mesenteric arteries and the inhibitory action of oxLDL on vasodilation was assessed in thoracic aorta, a vessel prone to increased LOX-1 expression. Results: Preliminary analyses of RUPP (n=3) and Sham (n=4) animals at 1 month postpartum show no difference in relaxation or constriction in the thoracic aorta. Constriction with PE or big ET-1 in mesenteric arteries was also unchanged. At low doses of MCh (10 -8 M), reduced relaxation was observed in mesenteric arteries from RUPP compared to Sham animals (31.7 ± 5.6% vs 63.7 ± 6.7%, p<0.05). Discussion: Our data suggest that vascular function largely recovers in our RUPP model. Deficits in relaxation observed in resistance arteries may be an indicator of an underlying phenotype which further manifests under the stress of ageing. Future studies will focus on evaluating vascular function beyond 1 month postpartum.

AT1-AA Enhances ANGII Induced Hypertension and Renal Vascular Sensitivity. Justin M Brewer, 1 Kedra Wallace, 1 Florian Herse, 2 Janae N Moseley, 1 Ralf Dechend, 2 Gerd Wallukat, 2 James N Martin, Jr., 1 Babbette B LaMarca. 1 1 Department of OBGYN, University of Mississippi Medical Center, Jackson, MS, USA; 2 Experimental and Clinical Research Center, Charite, Berlin, Germany. Objective: Recent work has shown that autoantibodies to the angiotensin II (ANG II) type 1 receptor (AT1-AA) play an important role in the hypertension and increased vascular sensitivity seen in preeclampsia.We have shown that the AT1-AA appears to act synergistically with ANG II to lead to increased mean arterial pressure (MAP) and increased renal vasoconstriction in pregnant animals and drastically activate the endothelin-1 (ET-1) system in endothelial cells.This leads us to hypothesize that the blood pressure effect observed with AT1-AA and ANGII during pregnancy occurs via activation of ET-1 pathways. The purpose of this study was to determine if blockade of ET-1 using an endothelin type A receptor antagonist (ETA) could blunt these changes seen in pregnant rats chronically treated with AT1-AA and ANGII. Study Design: Three groups of pregnant rats were examined: normal pregnant (NP)rats (n=18); AT1-AA+ANGII (n=11) treated with AT1-AA (1:50) and ANG II (50 ng/kg/min) infused via mini-osmotic pumps from day 12 to 19 of gestation; and AT1-AA+ANGII+ ETA blockade administered via drinking water (ETA 5 mg/day, n=21) beginning on day 12.Renal vascular sensitivity was assessed on day 18 using a Vevo 770 unit with a 30 Hz transducer and insonating angle <30°.On day 18 power Doppler velocimetry was used to determine the resistive index (RI) of both renal arteries, and carotid catheters were placed in all animals and MAP determined on day 19. Results: MAP was significantly elevated in the AT1-AA+ANGII group (118 +/-4.5mmHg) compared to NP animals (96+/-7.3 mmHg) (p<0.001).This response was significantly blunted in the ETA treated group (105 +/-8.7mmHg) compared to the control AT1-AA+ANGII group (p<0.01).The renal RI was 0.671 in NP animals, 0.74 with chronic AT1-AA+ANGII but was unchanged in ETA treated dams (0.753) (p=0.44). Conclusions: These results show that the addition of ETA drastically blunts the hypertension that is seen in pregnant animals exposed to AT1-AA + ANG II. However, ETA blockade did not decrease the renal vascular sensitivity seen with AT1-AA + ANGII.Although ETA blockade blunted the blood pressure in response to AT1-AA+ANGII, surprisingly, these changes occurred independently of renal artery resistance. Background: Pre-eclampsia (PE) is associated with endothelial dysfunction initiated by excessive generation of reactive oxygen species. Oxidative stress converts circulating low density lipoprotein (LDL) to oxidised LDL (oxLDL). Lectin-like oxidised low density lipoprotein receptor 1 (LOX-1) and its soluble equivalent, sLOX-1, are scavenger receptors for oxLDL and a host of other molecules including activated platelets, leukocytes and other unidentified molecules. We hypothesised that upregulated LOX-1 ligands in PE plasma alter LOX-1 activity in normal pregnant (NP) human vessels incubated in PE plasma. Methods: Control matched PE plasma was obtained from a multicentre pregnancy biobank (SCOPE Study) (n=6). Plasma oxLDL and sLOX-1 concentrations were determined by ELISA. Human omental arteries were incubated in 3% NP or PE plasma. LOX-1 expression was determined by immunohistochemistry. Vascular function was assessed using wire myography with vessels were exposed to oxLDL (50µg/ml) and LOX-1 inhibitor (TS20) (10µg/ml) and suitable controls. Results: No significant difference in oxLDL concentration was found in PE plasma when compared to NP plasma (3.35 ± 0.78 vs. 3.5 ± 0.70µg/ml; P>0.05; NS; n=6). Plasma sLOX-1 concentration and omental vessel LOX-1 expression were not significantly different in any group. Incubation of normal pregnant vessels in PE plasma impaired relaxation to bradykinin (BK) when compared with vessels incubated in NP plasma (Rmax 50 ± 3% vs. 96 ± 1%; P<0.001; LogEC50 -6.83 ± 0.18 vs. -7.19 ± 0.15mol/L; P<0.001; n=6). oxLDL exposure further impaired relaxation to BK in vessels incubated with PE plasma (Rmax 30 ± 4% vs. 50 ± 3%; P<0.001; LogEC50 -7.15 ± 0.24 vs. -6.83 ± 0.18mol/L; P<0.001 n=6). LOX-1 inhibition protected against PE plasma induced impaired relaxation (Rmax 94 ± 2% vs. 30 ± 4%; P<0.001; LogEC50 -7.25 ± 0.10 vs. -7.16 ± 0.24mol/L; P<0.001; n=6). Conclusion: Deleterious LOX-1 activity is mediated through a heterogeneous pathway as inhibition of the receptor prevents endothelial dysfunction in the absence of oxLDL upregulation in this in-vitro model of pre-eclampsia.

Preeclampsia Is Associated with Significant Differences in Omental Venous and Arterial Contractility. Ancizar Betancourt, Yurij P Vedernikov, Jimmy Espinoza, Daren T Tanchico, Hossein Golabbakhsh, Michael A Belfort.

Obstetrics & Gynecology, Baylor College of Medicine, Houston, TX, USA. OBJECTIVE: Preeclampsia is associated with increased arterial vascular response to vasoactive agents. However, there is limited evidence of changes in venous contractility during pregnancy and preeclampsia. This study compares the vascular contractility of omental vein (OV) and arteries (OA) from non-pregnant (NONPR), normal pregnant (NORPR), and preeclamptic (PRE) women. STUDY DESIGN: Omentum samples were obtained during surgical procedures from 15 NONPR, 14 patients with NORPR and 14 women with PRE. Omental vessels were isolated and 2 mm rings were mounted onto wire myographs. The concentration-contraction relationships (CCR) were compared between the omental artery and vein among study groups when the vascular rings were exposed to stable thromboxane A 2 analog U46619 (U46), serotonin (5-HT), norepinephrine (NE), and prostaglandin F2α (PGF2α). Paired t-test was used for statistical analysis. A p<0.05 was considered significant. RESULTS: 1) There were no significant differences in the IC 50 or maximal vascular responses between the omental artery and vein of NONPR to any vasoactive agent; 2) In contrast, in the NORPR group contractility and maximum response of OV to U46619 was significantly lower compared to OA when the vascular rings were exposed to U46, but not the other agents (first graph); 3) OA and OV rings from preeclamptic women also showed significant differences in the IC 50 when exposed to U46 (second graph); 4)Furthermore, OV rings from preeclamptic women showed less response to 5-HT and PGF2α than OA rings (Third and fourth graphs). CONCLUSION: 1) Pregnancy is associated with significant differences in vascular reactivity between omental arteries and veins compared to nonpregnant women; 2) These differences are more pronounced in omental vessels from preeclamptic women. It is possible that abnormal venous and arterial response to vasoactive agents may participate in the pathogenesis of preeclampsia. OBJECTIVE: Normal pregnancy is associated with changes in vascular reactivity. This study was designed to compare the effect of a prostacyclin (PGI 2 ) analog Beraprost (BER) and a PGI 2 receptor antagonist (CAY 10441) on the vascular reactivity of isolated omental arteries (OA) from normal pregnant (NORPR), and preeclamptic (PRE) women compared to those from non-pregnant (NONPR) women STUDY DESIGN: Omentum samples were obtained during surgical procedures from 15 NONPR, 14 NORPR and 14 PRE women. OA were isolated and 2 mm rings were mounted onto wire myographs. OA were exposed to a thromboxane analog (U 46619, 10 -7 M) and concentration-response relationships to BER (10 -10 to 10 -5 M) were compared in the presence or absence of CAY 10441 at 10 -8 , 10 -7 , and 10 -6 M. A comparison was also performed among the subset of vascular rings responding with an increase in tension after initial relaxation by BER (7 from NONPR, 8 from NORPR and 8 from PRE). Paired t-test was used for analysis. A p<0.05 was considered significant (denoted by the asterisks in the figure). RESULTS: 1) There were no significant differences in the IC 50 ' s for BERinduced OA relaxation among groups (see table) ; 2) In the NONPR group, CAY inhibited BER-induced vascular relaxation and inhibited the increase in vascular tension at 10 -7 and 10 -6 M (figure on the left); 3) In the NORPR group, CAY exerted this inhibitory effect only at the highest concentration tested (Figure in the center); 4) Of note, in the vascular rings from preeclamptic women no inhibitory effects of CAY on BER-induced relaxation were observed. However, CAY inhibited the increase in vascular tension at 10 -8 M in the subset of rings with increased tension following BER-induced relaxation. CONCLUSIONS: Normal pregnancy is associated with a decreased response to prostacyclin antagonism, which is more prominent in preeclampsia. Future studies will determine if these changes in vascular reactivity predate the clinical presentation of preeclampsia. Little is known about the role of membrane-bound VEGF receptors in the pathogenesis of hypertension in pregnancy. VEGF receptor type 2 (FLK) has been linked to abnormal persistence of the syncytiotrophoblast and to increased vascular resistance of the placenta. We explored the difference in expression of membrane-bound VEGF receptors among placentas from patients with PE, chronic hypertension (HTN), and normal controls. Materials and methods: We analyzed 28 third-trimester placentas (32-41 weeks) from patients with PE (10), HTN (8), and controls (10). A full thickness biopsy taken at the time of delivery was analyzed by light microscopy, western blot and immunohistochemistry. Results: FLT was upregulated in placentas of PE patients, compared to those with HTN and controls. This excess protein was concentrated in the extracellular matrix around the spiral arterioles. Placental FLK was upregulated in HTN, compared to PE and controls. The signal was located in the extravillous trophoblast and related to the spiral arterioles, including their endothelial cells.Western blot analysis demonstrated a specific increase in VEGF receptor expression consistent with the immunohistochemistry findings.

Placental VEGF receptors are upregulated in hypertensive disorders of pregnancy. There was enhanced expression of FLT in PE, and of FLK in HTN. Each of these membrane-bound receptors was upregulated in different morphologic areas of the placenta. Our findings are consistent with the hypothesis that PE and chronic hypertension, although they may be difficult to distinguish clinically, develop through different molecular pathways.

Influence of Prepregnancy Physiology on Cardiovascular Adaptation during Pregnancy. Sarah A Hale, Carole McBride, Ira M Bernstein. Ob/Gyn and Reproductive Sci, University of Vermont, Burlington, VT, USA. Introduction: Cardiovascular adaptation is critical for successful pregnancy outcome and includes decreased vascular resistance and increased vascular compliance. Our laboratory evaluates the relationship between prepregnancy physiology and pregnancy adaptations. Previous studies from our lab suggest increased pulse wave velocity (PWV), a measure of arterial stiffness, prior to pregnancy in women destined to develop preeclampsia. While cardiac output (CO) increases and arterial stiffness decreases during pregnancy, the relationship between changes in these physiologic variables and prepregnancy status is less defined. Here, we evaluated the relationship between CO and PWV, prior to pregnancy and changes in these parameters that attend pregnancy. Methods: 23 healthy women of normal BMI (23.74 ± 0.79 kg/m2), aged 29.6 ± 0.7 yrs, were enrolled in the study and evaluated after an overnight fast and an hour of supine rest. All women conceived singleton gestations. Prepregnancy evaluations were performed during the follicular phase (Prepreg) and late pregnant (LP) evaluations performed at 31.8 ± 0.3 wks. CO was determined using Doppler echocardiography. PWV was measured using simultaneous EKG tracings and ultrasound determined arterial flow waveforms. Data are reported as mean±SE. P<0.05 was considered significant. Results: All pregnancies were normotensive and women delivered at term (39.7 ± 0.3 wks). Birth weight was 3500 ± 104 g. PWV tended to decrease from Prepreg to LP (Prepreg: 2.63 ± 0.04, LP: 2.57 ± 0.05 m/s, p = 0.06). As expected, CO increased from Prepreg to LP (Prepreg: 4.7 ± 0.2, LP: 5.9 ± 0.2 L/min, p<.001). Increased Prepreg CO was associated with a larger decrease in PWV from Prepreg to LP (r = -0.45, p=0.03). Higher Prepreg PWV was associated with a greater reduction in PWV during pregnancy (Prepreg to LP, r = -0.635, p = 0.001). However, there was no association of Prepreg PWV or CO with change in CO during pregnancy.

Conclusions: In a group of women with normal first pregnancy outcomes, higher Prepreg CO and arterial stiffness are associated with a larger decrease in arterial stiffness during pregnancy that may suggest a normalizing effect of pregnancy in women who otherwise might be at risk for hypertensive disease during pregnancy. This cardiovascular normalization likely facilitates increased vascular compliance required for normal pregnancy outcome.

Direct Activation of sGC Improves Some but Not All Deleterious Effects of NOS Inhibition during Pregnancy. Nicole Maille, Richard Adams, Renju Raj, George Osol. Ob/Gyn, University of Vermont College of Medicine, Burlington, VT, USA. Nitric oxide (NO) signaling is essential for normal uterine arterial remodeling during pregnancy, as NOS inhibition (chemical or genetic) significantly attenuates this process. Although NO is most often associated with activation of soluble guanylate cyclase (sGC) and cGMP-PKG signaling, several other targets for this molecule have been recently identified. The goal of this study was to see whether co-treatment of NOS-inhibited animals with a direct activator of sGC (BAY: BAY 41-2272) would ameliorate some of the cardiovascular effects of NOS inhibition, e.g. hypertension and remodeling. BAY (1 mg/kg/ day) was administered via a subcutaneous osmotic pump while L-NAME (0.5 g/L) was added to drinking water during the second half of pregnancy. Measurements of mean arterial pressure (MAP) were made on day 14/22 of pregnancy using tail cuff oscillometry, and main uterine artery (MUA) diameters and pup and placental weights were measured on day 20/22 of pregnancy in Control (C: n=5), L-NAME (L: n=10), and L-NAME+BAY (L+B: n=6) treated Sprague-Dawley rats. Co-treatment with BAY prevented the development of hypertension (C: 93 ± 5.6 mmHg; L: 128 ± 3.4 mmHg; L+B: 98 ± 8.7 mmHg; p<0.05) and fully restored MUA remodeling (C: 214 ± 12 µm; L: 185 ± 16 µm, L+B: 217 ± 13 µm). Spiral artery remodeling (depth of trophoblast invasion, evaluated with image-measuring software) also improved with BAY co-treatment. Despite the beneficial effects on MAP and small and large artery remodeling, BAY did not significantly improve the reduction in pup weights associated with NOS inhibition(C: 2.44 ± 0.07 g, L: 2.21 ± 0.05 g, L+B: 2.15 ± 0.07 g). There were no between-group differences in placental weights. In summary, activation of sGC in pregnant NOS-inhibited rats appears to have complex effects. Our results highlight a novel a role for BAY as potential treatment for disorders associated with hypertension and/or impaired vascular remodeling, but not as treatment for fetal growth restriction.

Plasma Volume Expansion and Uterine Blood Flow as Predictors of Birth Weight. Carole A McBride, 1 Gary J Badger, 2 Sarah A Hale, 1 Ira M Bernstein. 1 1 Ob/Gyn and Reproductive Sci, UVM, Burlington, VT, USA; 2 Medical Biostatistics, UVM, Burlington, VT, USA. Objective: There is growing evidence that women's prepregnancy physiology contributes significantly to pregnancy outcome. These prepregnancy indicators contribute to the body's adaptations to fluid changes over pregnancy, and effect overall pregnancy outcomes. We hypothesize that vascular accommodation of increased plasma volume expansion and uterine blood flow (UBF) would contribute to newborn gestational age (GA) and birth weight (BW), at delivery. Methods: 36 healthy, normotensive women, were enrolled in a longitudinal study prior to pregnancy. Body mass index (BMI), plasma volume corrected for prepregnant BMI (PV/BMI), and cardiac output were evaluated prior to pregnancy during the follicular phase and during pregnancy at 12-14 and 30-32 weeks. Volumetric uterine blood flow was measured employing transvaginal Doppler ultrasound and estimated prior to pregnancy and at 12-14 weeks. Plasma volume was evaluated using the Evan's blue dye dilution method. Univariate and stepwise multivariate regressions were used to determine association between prepregnancy BMI, infant sex, PV/BMI, UBF, cardiac output and infant BW corrected for gestational age. Data are expressed as mean± standard deviation, with significance at p<.05. Results: The mean age of participants was 30.4±4.1 years, with mean BMI of 24.1±5.1 kg/m2. Mean GA at delivery was 277±11 days, with a mean BW of 3409±611 grams. Significant independent contributors to birth weight corrected for gestational age included fetal sex with male infants larger than female (males: mean 3604±492; females: 3235±665 grams; p=.003), maternal volume expansion during pregnancy (range 18-106, mean 57±21 mL/BMI, p=.01) and maternal prepregnancy uterine blood flow (range 8-136, mean 32±9 mL/min, p<.03). The overall regression model accounted for 76% of variation in birth weight. We identified no independent contribution to either prepregnancy BMI or cardiac output or the change in cardiac output through the 3rd trimester. Conclusion: Maternal volume expansion during pregnancy and prepregnancy uterine blood flow contribute significantly to birth weight corrected for gestational age and newborn sex. The specific association of prepregnancy uterine blood flow to birth weight supports the broad hypothesis that maternal prepregnancy physiology plays a significant role contributing to birth outcome. Introduction: Preeclampsia is a common disorder of pregnancy, resulting in increased maternal morbidity/mortality and induced preterm delivery, for which there is no current therapy beyond delivery of the fetus. Tanshinone IIA (TS) is a lipid-soluble component of Danshen widely used in traditional Chinese medicine known to cause vasodilation, inhibition of inflammatory mediators and scavenging of peroxyl radicals. We discovered that both oxidized LDL and its receptor (LOX-1) are increased in the systemic vasculature of women with preeclampsia. LOX-1 is known to be up-regulated by TNF and peroxynitrite and its activation could contribute to vascular dysfunction via reduced bioavailability of nitric oxide (NO). We hypothesized that TS would prevent up-regulation of the oxLDL/LOX-1 receptor pathway in a rat model of reduced utero-placental perfusion pressure (RUPP). Methods: The RUPP model was used to investigate involvement of the LOX-1 pathway and treatment with TS in the pathophysiology of preeclampsia. Uteroplacental blood flow was reduced via the surgical insertion of restrictive clips on the abdominal aorta and both uterine arteries on d14 of pregnancy. Sham surgeries were performed as controls. Both groups were treated with TS in drinking water (approx. 27mg/kg/day) from the day of surgery onwards. On d20 (term 21d), animals were euthanized and ex vivo vascular experiments were performed using agonists and inhibitors of the oxLDL, LOX-1 and platelet activating factor receptor (PAFR) pathways (an alternate receptor for oxLDL). Results: TS treatment significantly increased (p<0.001) vasodilator responses of the thoracic aorta to methylcholine (MCh) in both the Sham (10%) and RUPP (15%) groups. No differences were found between the groups in regards to the involvement of the oxLDL/LOX-1 receptor pathway. Our preliminary data suggests, however, that contrary to our original hypothesis, oxLDL enhanced relaxation in the RUPP group via activation of the PAFR pathway. Discussion: Our preliminary data show that TS is effective in improving vasodilation. Interestingly, oxLDL may reduce vasodilation via the LOX-1 receptor or alternatively increase vasodilation via the PAF receptor. The mechanism by which this improvement is mediated in vascular complications of preeclampsia, however, requires more thorough investigation. Background: Abnormal spiral artery remodeling has been implicated as a root cause of preeclampsia and intrauterine growth restriction (IUGR). AGT genetic variants, causing slightly elevated angiotensin II, are associated with preeclampsia, IUGR, and abnormal spiral artery remodeling. To isolate the effects of a 20% increase in AGT expression, we study a murine AGT gene titration (3-copy) model (TG). This mouse develops a preeclampsia-like syndrome with IUGR. Using microbubble-enhanced ultrasound imaging we have determined that uteroplacental blood flow in TG dams has a higher flow velocity and reduced spiral artery transit time than controls (WT). Our objective now was to test if there are differences in spiral artery remodeling between TG and WT dams. Methods: Uteroplacental vasculature from 3 WT and 2 TG dams was perfused with x-ray contrast at embryonic day (E)17 and 3D microcomputed tomography images were obtained (n=7 placentas/group). Arterial vessel numbers, diameters, and the degree of spiral artery coiling were measured using Amira 3D visualization software. Results: WT dams exhibited a complex network of spiral arteries (4 to 6 per tree) that merged into large canals feeding the intervillous space. In contrast, the number of spiral arteries in TG dams was reduced by 38% (4.6 +/-0.4 (SEM) vs. 2.8 +/-0.3, p=0.003), coiling was reduced by 22% (p=0.01), and 3D surface renderings revealed further reductions in length and branching ( Figure 1 , scale=1 mm), consistent with microbubble echo studies. Spiral artery diameters were 40% wider in TG dams compared with controls (p<0.001).

Conclusions: Imaging data suggest rapid uteroplacental blood flow in our mouse model of preeclampsia/IUGR is related to reduced spiral artery numbers, length, and coiling. Distal spiral artery dilation in these TG mice may be a response to shear stress related to higher flow velocities. We suspect this may also damage the placental labyrinth. During pregnancy, uterine angiogenesis is partly regulated by estrogens and estrogen metabolites. 4-Metoxyestradiol (4-ME 2 ), the direct metabolite of 4-hydoxyestradiol (4-OHE 2 ) synthesized by catechol-O-methyltransferase (COMT), induces proliferation in pregnant uterine artery endothelial cells (P-UAECs). We reported (Jobe et al, 2011) that 4-OHE 2 induces P-UAEC proliferation via β-adrenergic receptors (β-AR) and independent of estrogen receptors (ERs).We thus asked if 4-ME 2 will follow its two immediate precursors by stimulating angiogenic activity via ERs or ARs. Methods: P-UAECs from late pregnant ewes were treated with or without blockers (10µmol/L) against α-AR (phentolamine), β-ARs (propranolol) or ERs (ICI 182, 780) . To determine signaling pathways for 4-ME 2 actions, we evaluated activation of mitogen-activated protein kinases (MAPK) or PI3K signaling cascades. Functionality of MAPK signaling pathways was also determined by treatment of P-UAECs with protein kinase blockers (2.5/5 µmol/L) against p42/44 (PD98059), P38 (SB203580), JNK (SP600125) and the PI3K (LY294002) pathway followed by 4-ME 2 (0.1 µmol/L). Cell proliferation was evaluated via 5-Bromodeoxyuridine assays. Phosphorylated and total p42/44, p38, JNK, MAPKs and PI3K were evaluated using western blots. Results: Blocking α-AR, β-AR and ERs with phentolamine, propranolol or ICI 182,780 did not affect proliferation responses to 4-ME 2 . Pretreatment with PD98059, SB203580 and SP600125 all abrogated (P<0.05) 4-ME 2 -induced P-UAEC proliferation. However, LY294002 pretreatment (P>0.05) did not alter 4-ME 2stimulated proliferation. Western blots revealed proliferation-specific and time-dependent activation of phosphorylated p42/44, p38 and JNK MAPKs, but not PI3K. Total p42/44, p38, JNK and PI3K were unchanged by 4-ME 2 . Conclusions: Thus 4-ME 2 stimulates P-UAEC proliferation independent of either ERs or ARs. The 4-ME 2 mechanism for proliferation converges at the level of p42/44, p38 and JNK MAPKs and is independent of PI3K signaling. These findings provide insights into complexities of estrogen metabolite signaling during pregnancy via 4-ME 2 , whose activity is independent of classic ERs or ARs, but still activates distinct MAPK signaling cascades indicating that estrogen metabolites have the capacity to bypass these common receptors and yet still induce angiogenesis. HL49210, HD38843, HL87144, R25GM083252.

Cells. Yang Gu, Qin Dong, Lynn J Groome, Yuping Wang. Obstetrics & Gynecology, LSU Health Sciences Center, Shreveport, LA, USA. Objective: MicroRNAs (miRNAs) have emerged as important posttranscriptional regulators of gene expression and play critical roles in cancer biology and cardiovascular diseases. miR-203 is an inflammatory miRNA. Suppressor of cytokine-singlaing-3 (SOCS-3) is an important cellular antiinflammatory regulator and is a target of miR-203. This study was undertaken to determine if miR-203 mediates SOCS-3 expression and increases inflammatory response in endothelial cells (ECs).

Methods: SOCS-3 anti-inflammatory activity was determined by measuring endothelial adhesion molecule ICAM expression and neutrophil-endothelial adhesion in ECs transfected with SOCS-3. Increased miR-203 production was induced by transfection of miR-203 precursor into ECs and expressions of miR-203, SOCS-3, and ICAM were then determined. miR-203 expression was determined by real-time PCR and SOCS-3 expression by Western blot. ICAM expression was determined by cell-based immunoassay. ICAM production was measured by ELISA. miR-203 induced increased inflammatory response was also determined by neutrophil-endothelial adhesion measured by MPO assay. Data are expressed as mean ± SE and analyzed by t-test or ANOVA. A p <0.05 was set as statistically significant. Results: ICAM expression and neutrophil-endothelial adhesion were significantly reduced in ECs transfected with SOCS-3, p<0.01. In contrast, ECs transfected with miR-203 precursor showed increased miR-203 production, p<0.01, and increased ICAM expression, p<0.01. Higher MPO levels were detected in neutrophil-endothelial co-cultures in which ECs were transfected with miR-203 precursor, p<0.01. Conclusions: We demonstrated that miR-203 inhibits anti-inflammatory mediator SOCS-3 expression and increases endothelial inflammatory response. Since down-regulation of SOCS-3 expression is associated with increased endothelial inflammatory response in preeclampsia, aberrant miR-203 expression may contribute to increased inflammatory responses in vascular endothelium in preeclampsia and other inflammatory related cardiovascular diseases as well.

Preeclampsia-Upregulated Angiogenesis-Associated microRNA-17 Family miRNAs (17, 20a and 20b) Inhibit Cytotrophoblast Endovascular Transformation. Wen Wang, Dong-bao Chen. Ob/Gyn, University of California Irvine, Irvine, CA, USA. Introduction: The pathogenesis of preeclampsia is associated with inadequate spiral artery invasion of cytotrophoblast that undergoes an epithelial-endothelial transformation (i.e., endovascular transformation) during human placentation. MicroRNAs (miRNAs) are a class of noncoding 21-25 nucleotide RNAs that negatively regulate gene expression post-transcriptionally. We have shown that angiogenesis-associated miRNA-17 family miRNAs (miR-17, 20a, and 20b ) are upregulated in preeclamptic placentas compared to controls. Hypothesis: MiRNA-17 family miRNAs inhibit cytotrophoblast endovascular transformation in association with downregulation of target genes important for angiogenesis and spiral arteriole remodeling during early human placentation. Methods: Each miRNA precursor of miR-17, 20a and 20b was amplified and cloned into the lentiviral pGIPZ vector. The miR lentiviral vectors were packaged and tittered in 293T cells and then used for overexpressing miR-17, 20a and 20b in human extravillous trophoblast derived HTR-8/SVneo cells. Stable HTR-8/SVneo cell lines overexpressing miRNAs were selected by puromycin. Total RNAs were extracted from the transduced cells and real-time qPCR was used for determining the expression of miR-17 family and their downstream target genes including HIF1A, VEGFA, ephrin-B2, EPHB4 and MMP2. Endovascular transformation of HTR-8/SVneo cells was assessed by expression of markers of epithelial and endothelial cells as well as the ability of the cells to form tube-like structure on growth factor-reduced matrigel. Results: Overexpression of miRNA-20b significantly decreased the levels of HIF1A, VEGFA, MMP2, and EPHB4 mRNAs in HTR-8/SVneo cells. When cultured on matrigel-coated plates, HTR-8/SVneo cells forms tube-like structures spontaneously; overexpression of miR-17, 20a and 20b significantly inhibited the ability of HTR-8/SVneo cells to form tube-like structures in association with a shift of epithelial and endothelial marker gene expression. Conclusion: Preeclampsia-upregulated angiogenesis-associated miRNA-17 family miRNAs (miR-17, 20a and 20b) inhibit cytotrophoblast endovascular transformation that involves down-regulation of target genes important for angiogenesis (supported by RO1 HL70562 & R21HL98746). and angiogenesis. Nevertheless, it is unclear if IL6 and IL8 regulate expression and activation of fetoplacental endothelial NO synthase (eNOS), which is a major NOS isoform responsive for NO production in placenta. Hypothesis: IL6 and IL8 play important roles in regulating cell viability and migration as well as eNOS expression and phosphorylation in fetoplacental endothelial cells during pregnancy. Methods: Viability and death of human umbilical cord vein endothelial (HUVE) cells after serum starvation were determined using positive staining of acetomethoxy derivate of calcein (calcein AM) and ethidium homodimer-1 (EthD-1) as indexes, respectively. HUVE cell migration was evaluated using the BD FluoroBlok Trans-wells system. Expression and phosphorylation of eNOS were evaluated using Western blotting. Results: IL6 and IL8 did not affect HUVE cell viability and death. As compared to the control, IL6 increased HUVE cell migration by ∼ 30%, while IL-8 slightly decreased it by ∼ 10%. We also observed that while IL6 had no effect on the phospho-eNOS (Thr495) (indicative of eNOS inactivation) and phospho-eNOS (Ser1177) (indicative activation), IL-8 time-dependently increased the levels of phospho-eNOS (Thr495) (∼2.2 folds), but not phospho-eNOS (Ser1177 Background: Immunoglobulin G (IgG) is the major serum immunoglobulin, accounting for roughly 75% of all immunoglobulins. IgG is the only class of immunoglobulin that crosses the placenta and it serves as the main immunologic barrier between the fetus and external environments. There has not been a clear consensus on what the normal values of IgG are throughout pregnancy. The aim of this study was to measure serum immunoglobulin G in each trimester of the pregnant female to determine a normal IgG profile throughout all trimesters in normal pregnancy. Methods: De-identified maternal blood samples were obtained from our Maternal Fetal Tissue Bank (MFTB). The maternal plasma samples were obtained from 61 subjects within the MFTB that had a sample in each trimester throughout their pregnancy. The determination of IgG in the maternal plasma samples was determined by Enzyme-Linked Immunosorbent Assay (ELISA). Absorbance was measured at 405λ (BioRad xMark plate reader). Data was analyzed by Analysis of Variance (ANOVA) for repeated measures and by regression analysis. The statistical analysis was performed with SigmaStat 3.0 software (Systat Software, Inc, California). Results: The mean values of IgG concentration in maternal plasma showed a statistically significant drop throughout pregnancy. The mean IgG concentration in the first trimester was 18.5 mg/ml ± 0.88 and gradually declined to the second trimester mean concentration of 17.4 mg/ml ± 0.86. The mean IgG concentration at term was 16.5 mg/ml ± 0.82. It was determined that the term IgG concentration (>37 weeks) is 90% of the first trimester. Maternal age, body mass index, infant birth weight, and infant Apgars were not related to the extent of decrease of immunoglobulin G during pregnancy. Conclusion: While previous studies have revealed contradictory findings regarding the concentration of IgG in pregnancy and its trend we have found a significant drop in IgG throughout pregnancy with the lowest concentration occurring at term. This drop may be due to the transfer of IgG to the fetus. Our study determined that normal values of IgG in pregnancy ranged from a mean in the first trimester of 18.5 mg/ml to 16.5 mg/ml at term. Conclusion: TEG and PFA are practical, point-of-care assays that evaluate whole blood haemostatic properties. Clinically, they are already used in the obstetric setting to establish candidates for neuraxial analgesia and to assess coagulation status in postpartum hemorrhage. However, reference ranges relevant to the pregnant population have not yet been established. This study demonstrates that some TEG and PFA parameters are significantly different among pregnant subjects compared to non-pregnant controls, and the findings are consistent with a hypercoagulable state.

Non-Esterified Fatty Acids and Spontaneous Preterm Birth: Factor Analysis To Identify Possible Patterns of Risk. Janet M Catov, 1 Marnie Bertolet, 2 Yi-Fan Chen, 2 Rhobert Evans. 2 1 OBGYN, U of Pittsburgh, Pittsburgh, PA, USA; 2 Epidemiology, U of Pittsburgh, Pittsburgh, PA, USA. Background: Triglycerides increase dramatically as part of normal gestation, but excess concentrations have been associated with preterm birth (PTB). Triglycerides promote the release of inflammatory non-esterified free fatty acids (NEFA) which are markers of tissue damage and directly perturb membranes. Thus, the accumulation of NEFAs could provide a mechanistic link between elevated lipids and spontaneous PTB. Methods: In a nested case control study NEFA were assayed in serum collected at mean gestational week 10.2 (SD 3.9) using standard enzymatic procedures in 111 women with spontaneous PTB (<37 weeks after preterm labor or premature membrane rupture) and 212 women with term births (>=37 weeks). C-reactive protein (CRP), total and HDL cholesterol, triglycerides, and uric acid were also measured, and LDL-cholesterol was estimated. Polytomous logistic regression models evaluated the association between 1 st trimester tertiles of NEFA and PTB <34 weeks and 34-36 weeks. We also used factor analysis to characterize different patterns of biomarkers and maternal characteristics that may be associated with PTB. Results: Women with NEFA in the highest tertile (>= 0.33 mmol/L) were 2.0 (95% CI 1.1, 3.6) times more likely to deliver preterm compared to those with NEFA in the lowest tertile (<0.19 mmol/L) adjusted for age, race, BMI, family history of preeclampsia, periconceptional vitamin use and smoking. Risk of PTB <34 weeks was particularly high among women with high NEFA (OR 3.7 [1.3, 10.4] ); no excess risk was detected for PTB 34-36 weeks (OR 1.6 [0.8. 3.0] ). Factor analysis identified three clusters of biomarkers and risk factors that were associated with sPTB: smoking and education, lipids (NEFA and HDL-cholesterol) , and family history of preeclampsia. Each SD increase in the factor dominated by NEFA and HDL was associated with a 1.5-fold increase in PTB 34-36 weeks and a 1.9-fold increase in PTB <34 weeks. Conclusions: Non-esterified fatty acids in the 1 st trimester are independently associated with spontaneous PTB, particularly sPTB <34 weeks. Factor analysis detected patterns of risk factors and biomarkers that are correlated, and appropriately accounting for this correlation structure suggests that NEFA together with HDL-cholesterol may contribute to PTB.

Lipoprotein Heterogeneity in Black and White Women during Early Pregnancy and Preterm Birth. Janet M Catov, 1 Rachel H Mackey, 2 Christina M Scifres, 1 Hyagriv N Simhan. 1 1 OBGYN, U of Pittsburgh, Pittsburgh, PA, USA; 2 Epidemiology, U of Pittsburgh, Pittsburgh, PA, USA. Background Differences in the size and quantity of lipoprotein particles are associated with inflammation and disease outside of pregnancy, particularly when discordant from traditional cholesterol contents. Discordance is common with high triglycerides, making pregnancy particularly susceptible. We considered that lipoprotein particle size and number would be related to preterm birth (PTB), and that associations may vary by maternal race. Methods: Serum and plasma were collected at mean gestational age of 9.1 weeks (range 5-18) from 22 women with PTB (<37 wks) and 42 women who delivered at term (>=37 wks). Cholesterol content was assayed in serum using standard enzymatic techniques, and HDL, LDL, and VLDL particles (HDL-P, LDL-P, and VLDL-P), subclasses and particle sizes were quantified in plasma using NMR spectroscopy. Results were evaluated according to PTB, and stratified by maternal race given the evidence of race differences in lipids and PTB. Results: Overall, black v. white women had fewer total, large and medium VLDL-particles (all p<0.05), fewer LDL particles (897 v. 1076 nmol/L, p=0.05), more .7 mg/dl, P<0.01) and larger HDL particles (9.6 v. 9.3 nm, p=0.02). LDL cholesterol was unrelated to gestational age at sampling in the 1 st trimester (r=0.03, p=0.88) regardless of maternal race. Among black women, the concentration of total LDL particles and small LDL particles increased as gestational age at sampling increased (for small LDL-P, r=0.52, p<0.01). This was not detected in White women (r=0.10, p=0.66). Black women with PTB had excess large HDL particles compared to Blacks with term births (9.4 v. 6.7, p=0.02), independent of pre-pregnancy BMI, age, and smoking. Each additional large HDL particle was associated with 1.62-fold increased risk of PTB (95% CI 1.04, 2.54). White women with PTB tended to have fewer large VLDL particles compared to whites delivered at term. Conclusions: Black-white differences in the lipoprotein profile in the 1 st trimester (lower VLDL-P, LDL-P and higher large HDL-P) are similar to findings in non-pregnant adults. However, these lipoprotein concentrations had race-specific associations with gestational age and PTB. Outside of pregnancy this lipoprotein profile is associated with less atherosclerosis, but during pregnancy this may signal an impaired lipoprotein response that is required for healthy gestation.

Inhibition of IL-6 by Curcumin in Uterine Decidual Cells. Y Sangeeta Devi, Susan Ferguson, Asgerally Fazleabas, Justin DeKuiper. OB/ GYN & Reproductive Biol, Michigan State University, Grand Rapids, MI, USA. IL-6 is a multifunctional pro-inflammatory cytokine and has been implicated in many gestational disorders such as unexplained infertility, recurrent miscarriage, preeclampsia, fetal brain injury and preterm birth. Expression of IL-6 is very low or undetectable in mid-gestation during the normal pregnancy, but induced in the uterine decidual cells upon infection or IL-1b treatment leading to aberrant expression and pregnancy complications. However, there are no appropriate therapeutic interventions available to circumvent inflammatory mediated gestational disorders. Therefore, the goal of this study is to identify a safe and effective pharmacological compound to counterbalance inflammatory responses in the uterus. Curcumin, a naturally occuring polyphenolic compound, has been widely used in ayurvedic medicine to treat inflammatory and infectious diseases. However, to the best of our knowledge, the antiinflammatory effect of curcumin has never been studied in uterine decidual cells. Therefore, we examined the effect of curcumin on IL-6 expression using two types of uterine decidual cells 1) HuF cells, primary human fibroblast cells obtained from decidua parietalis; 2) UIII cells, a rodent non-transformed decidual cell line. Curcumin treatment dramatically inhibited the expression of endogenous as well as IL-1b induced IL-6 in these cells. Curcumin also sharply inhibited expression of gp130, a critical molecule in IL-6 signaling, whereas expression of IL-6 receptor was not affected. To examine whether this inhibition involves transcriptional regulation, we transfected an IL-6 promoter-luciferase construct spanning -276 to +20 bp region which contains a putative NFkB site. IL-1b treatment markedly stimulated the promoter activity; however curcumin treatment completely abrogated this stimulation. To further understand whether NFkB is involved in this inhibition, we examined the effect of curcumin on the expression of p50 and p65 RelA subunits of NFkB in HuF and UIII cells. P50 NFkB expression is dramatically repressed by curcumin treatment while p65 RelA show moderate downregulation suggesting that the inhibition of IL-6 by curcumin is most likely mediated through NFkB. Taken together these results suggest that curcumin is a potent inhibitor of IL-6 in uterine decidual cells and this compound may have therapeutic potential for the prevention of inflammatory mediated gestational disorders.

The Ability of Progesterone To Regulate the Inflammatory Monocyte Subset during Pregnancy. Lydia F Edey, Kieran P O'Dea, Bronwen R Herbert, Masao Takata, Mark R Johnson. Surgery and Cancer, Imperial College, London, United Kingdom. Introduction: During pregnancy it has been suggested that immune responses are biased towards an anti-inflammatory state. Progesterone (P4) is a key regulator of the immune response during pregnancy having both suppressive and activating roles. Murine monocytes exist as at least two phenotypically and functionally distinct subsets, distinguished by levels of the surface marker, Ly-6C. The Ly-6C high subset has been shown in various infection and inflammation models to be preferentially recruited to inflamed sites. In mice, this 'inflammatory subset' has recently been shown to be recruited to the uterus from the blood in the early stages of gestation. We have identified an Ly-6C high monocyte-like population in the myometrium of pregnant CD1 mice in the later stages of gestation. Here we investigated how P4 influences the trafficking of the local and systemic Ly-6C high monocyte populations and other leukocytes during pregnancy and labour. Methods: CD1 pregnant mice were treated with P4 or a vehicle from E14. Myometrium, placenta, lungs, liver and blood were collected at E16, E17, E18 and labour in vehicle-treated and E16-E20 in P4-treated mice. The P4 and glucocorticoid receptor antagonist, RU486, was administered on E16 and tissues were collected at 4.5h, 9h and 13.5h after administration and at labour. Tissue and blood samples were analysed using flow cytometry (n=6 for each condition). Results: In the myometrium, clear effects with P4 treatment were observed. Continuous P4 administration prevented the increase in Ly-6C high cell densities that took place in vehicle-treated mice up to E18 (1.03±0.69 ×10 5 ; 2.85 ± 1.01 x10 5 ; 3.54± 1.51 x10 5 ; vs. 1.14± 0.26 x10 5 ; 1.94 ± 0.65 x10 5 ; 1.55 ± 0.46 x10 5 cells/g myometrium at E16, E17 and E18 in control and P4 treated animals, respectively; mean ± SD). However, administration of P4 up to E20 produced a marked increase in Ly-6C high cells at E19 and E20 (38% and 521% on E19 and E20, respectively compared to E18 control; p= 0.0029). On E16 there was a 343% increase in the Ly-6C high cell population in the myometrium 13.5h after RU486 compared to E16 controls. Conclusions: These results indicate that continued exposure to P4 is able to substantially modify infiltration or in situ proliferation of Ly-6C high monocytes in the myometrium around term. Blocking P4 activity at E16 with RU486 resulted in greater numbers of neutrophils, Ly6C high cells, and macrophages in the myometrium.

Haemodynamic Profiling throughout Pregnancy in Normal and Lipopolysaccharide-Treated Mice. Laura Howe, 1 Zhen Wang, 2 Lydia F Edey, 1 Leiper James, 2 Mark R Johnson. 1 1 Surgery and Cancer, Imperial College London, London, United Kingdom; 2 NOS Signalling Group, Medical Research Council Clinical Science Centre, London, United Kingdom. Introduction:Infection and inflammation during pregnancy have been associated with preterm birth in over 40% of cases. Immunomodulatory agents have shown promise in the prevention of preterm labour (PTL) and improvement of neonatal outcome in animal models; however the potential of these agents to adversely affect maternal susceptibility to infection has not so far been addressed. We have been able to establish a mouse model in which telemetric measurements can be taken throughout the course of gestation allowing us to monitor haemodynamic changes in response to endotoxaemia. Methods:Female CD1 mice were implanted with a PA-C10 radiotelemetry probe (Data Science International) into the left common carotid artery. Nonpregnant baseline recordings were taken for 48h, after 7-10 days post-surgery. Mice were then timed-mated and recordings were resumed upon detection of a copulatory plug until 3 days post-partum. Further cohorts of mice were used to identify the dose and serotype of LPS needed to induce pre-term birth with no maternal mortality, and to harvest maternal tissues at several time points for analysis of cytokines and inflammatory markers. Results:In female CD1 mice it was seen that mean arterial pressure fell from 109.4 ± 1.86 mmHg (non-pregnant baseline) to post-implantation nadir at E8 (100.8 ± 1.3 mmHg, P<0.01, repeated measure ANOVA), which then gradually increased until just before labour. Similar trends were also observed in systolic and diastolic arterial pressures. During gestation, heart rate and activity levels were maintained, with activity only significantly decreasing at labour and post-partum. Intraperitoneal injection of 10µg of 0111:B4 LPS on E16 was found to induce preterm labour 21.43 ± 1.5 hours (n=5) post-injection (PBS controls laboured 51.95 ± 3.5 hours post-injection, P<0.05), causing 98 ± 1.8% pup death but no maternal mortality. Conclusions:These data will enable us to draw comparisons between mice injected with LPS at E16 and non-pregnant controls, and therefore to ascertain whether the haemodynamic response to endotoxaemia is altered during gestation, and the bearing this may have on maternal susceptibility to infection. Objective: Triglyceride transport proteins have remarkably stable expression patterns in the human plasma proteome. In this study, we developed a targeted proteomics approach to measure the rate of change in a panel of 14 unique plasma apolipoprotein subtypes from early to late gestation, and we used this panel to interrogate differential expression patterns in 3 distinct pregnancy complications. Methods: Plasma was prospectively collected from 311 nulligravids at 5 gestational ages. The cohort included 6 cases each of severe preeclampisa (sPE), gestational diabetes (GDM), and preterm premature rupture of the membranes (PPROM), with 11 age-matched uncomplicated controls. Two complementary mass spectrometry assays measured relative concentrations of apolipoprotein subtypes. ELISA and enzymatic detection assays quantified plasma apolipoproteins and cholesterols. Kruskal Wallis ANOVA with post hoc Dunn's test assessed statistical significance among cohorts (p<0.05). Results: Of the 14 apolipoprotein subtypes investigated, 3 were differentially expressed among the 3 cohorts. The ratios of sialylated variants of ApoCIII to mature, truncated ApoCII were significantly elevated in sPE, with truncated ApoCII significantly lower in this cohort relative to controls (p<0.05). The rate of change of of ApoAII cysteinylated dimer was significantly elevated in PPROM patients (156±6.8% of control), whereas this protein was lower in GDM patients (71±11% of control). LDL/VLDL cholesterols were differentially expressed in the GDM and PPROM cohorts. Wherease these cholesterols increased by about 34±17% in controls, GDM cases showed essentially no change over gestational time (11±12% increase from trimester 1 to 3). PPROM VLDL/LDL levels were approximately 40% lower than controls in both first and second trimester. Discussion: The rate of change of plasma cholesterols and apolipoproteins was positively correlated with gestational time in normal individuals, but cysteinylated ApoAII dimer, truncated ApoCII, sialylated ApoCIII, and the VLDL/HDL cholesterols varied differentially among individuals with PPROM, sPE, and GDM. These results suggest improved sensitivity of the mass spectrometry method over biochemical techniques in detecting early, differentiable indicators of complicated pregnancies.

Activation of NF-κB in Human Amnion Is Mediated through G αi and Is Mimicked by the Oxytocin Receptor Antagonist -Atosiban. SH Kim, 1 A Blanks, 2 S Thornton, 3 M Johnson, 1 PR Bennett, 1 V Terzidou. 1 1 Surgery & Cancer, Imperial College London, United Kingdom; 2 Clinical Sciences Research, Warwick Medical School, United Kingdom; 3 Medicine & Dentistry, University of Exeter Medical School, United Kingdom. Inflammation is recognised as one of the key characteristics of both preterm and term labour. There is accumulating evidence suggesting that NF-κB, a transcription factor associated with inflammation, plays a significant role in the physiology and pathophysiology of human labour. In human amnion cells, NF-κB has been shown to increase in association with labour at term. We have previously reported that oxytocin (OT) activates NF-κB, resulting in increased expression of COX-2 and prostaglandin synthesis in human amnion cells. Here we investigate the relevant G-protein coupling of OTR, the downstream effect upon cytokine synthesis and the effect of the oxytocin antagonist -atosiban. In term pre-labour amnion epithelial cells, OT treatment increases degradation of IκBα, phosporylation and nuclear translocation of p65. There is also increased phosporylation of ERK and p38 but not JNK MAPkinases (p<0.05). Pretreatment with pertussis toxin (PTX) reduced the effect of OT upon NF-κB, ERK and p38 indicating that the effect is mediated through G αi . RT-PCR studies showed that human amnion expresses the G αi2 and G αi3 but not G αi1 isoforms. siRNA knockdown of G αi2 reduced the effect of OT upon NF-κB, ERK and p38 and inhibited the increased expression of COX-2. siRNA knockdown of G αi3 reduced p38 and ERK but not NF-κB and inhibited COX-2 upregulation. Neither pretreatment with any of three downstream inhibitors of G αq (GFX, Go6893, U73122) nor siRNA knockdown of G αq had any effect upon OT mediated NF-κB activation. OT stimulation caused a significant increase in mRNA expression of the NF-κB-regulated genes IL-8, IL-6, CCL2, CCL5, SOD2 and COX-2. Pretreating amnion cells with atosiban prior to OT stimulation did not suppress OT-mediated activation of NF-κB or the upregulation of COX-2. Atosiban alone resulted in activation of NF-κB, ERK and p38 to the same extent as OT (p<0.05). We conclude that OT couples with G αi2 and G αi3 but not G αq to induce activation of NF-κB and increase expression of downstream genes. COX-2 expression requires both NF-κB and MAPkinase activation. Counterintuitively atosiban does not inhibit, but stimulates NF-κB in amnion. This could have implications for the role of atosiban as an acute tocolytic drug in the management of preterm labour. Thomas' Hospital -King's College, London, United Kingdom. Objective: to determine whether a policy of serial measurement of cervical length during pregnancy to assess the need for cervical cerclage, followed by targeted cerclage in those found to be at risk, will reduce the risk of preterm labour (prior to 37 weeks) in comparison to routine antenatal care in women who have had a prior cervical cone for cervical precancer.

Material & Methods: Design: Retrospective cohort Setting: Imperial College -Kings College Period: 1996 -2010 Population: Pregnant women who had excisional treatment prior to their first pregnancy Interventions: serial measurement of cervical length during pregnancy to assess the need for cervical cercalge, followed by targeted cerclage in those found at risk versus routine antenatal care. Outcomes: Primary: preterm labour (<37 weeks) -Secondary: severe (<34w), extreme (<28) prematurity, low-birth weight (<2500g, <2000g, <1500g, <1000g) and correlation with the depth of excision. Results: A total of 495 women were included, 315 in the intervention and 180 in the routine care group. Cervical length indicated cerclage significantly reduced the risk of preterm birth (<37 weeks) (8.2% vs 16.3%, RR: 2, p=0.04) and lowbirth weight (<2500g) (5.4% vs 13%, RR: 2.4, p=0.004) . The risk of preterm birth at <28, <32, <34 weeks of gestation and birth weight <2000g, <1500g, <1000g was not significantly different amongst the compared populations (p<0.05). The analysis of the correlation between cone depth and outcomes will be presented. Conclusions: The policy of cervical length surveillance in specialist prematurity clinics with targeted cervical cerclage reduces the risk of preterm birth in women after CIN treatmenr. Further well-designed randomized controlled trials are urgently required to assess the value of these interventions. Preterm birth (PTB) remains one of the most important issues facing perinatal medicine today. Approximately one-third of all cases of spontaneous PTBs are due to the untimely rupture of the fetal membranes, with chronic inflammation and/or infection being the biggest aetiological factor. The nucleotide oligomerization domain (NOD) intracellular molecules recognise a wide range of microbial products as well as other intracellular danger signals, thereby initiating inflammation through the activation of nuclear factor κB (NF-κB). NF-κB play a central role in the terminal processes of human labour and delivery including regulating mediators of fetal membrane rupture, including cytokines, and the extracellular matrix degrading enzyme MMP-9. The aims of this study were to determine the effect of (1) human term and preterm labour, pro-inflammatory cytokines and bacterial endotoxin LPS on NOD1 and NOD2 gene and protein expression; (2) NOD1 and NOD2 activation on the expression of pro-labour mediators in human fetal membranes. We found that NOD1 and NOD2 expression was significantly higher in fetal membranes after spontaneous labour when compared to non-labouring tissues. Bacterial endotoxin LPS and the pro-inflammatory cytokines TNF-α and IL-1β significantly increased NOD1 and NOD2 expression. Furthermore, LPSinduced NOD1 and NOD2 expression was decreased by the NF-κB inhibitor BAY 11-7082. The NOD1 agonist C12-iE-DAP and the NOD2 agonist L18-MDP significantly increased the expression and secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) and the expression and activity of MMP-9. The effects of NOD1 and NOD2 were mediated via NF-κB as (i) C12-iE-DAP and L18-MDP increased NF-κB activation, and (ii) the NF-κB inhibitor BAY 11-7082 significantly attenuated C12-iE-DAP and L18-MDP induced expression and secretion of pro-inflammatory cytokines and MMP-9.

In conclusion, our data show that NOD1 and NOD2 are increased in fetal membranes after labour and bacterial infection. Agonist activation of NOD1 and NOD2 leads to NF-κB activation and transcription of NF-κB induced genes, including TNF-α and IL-1β which can then also induce NOD1 and NOD2, thus forming an amplifying feed-forward loop. We suggest that NOD1 and NOD2 may be therapeutic targets for preterm premature rupture of the membranes (PPROM), a major antecedent of PTB.

Changes in Progesterone Receptor Membrane Component 1 (PGRMC1) Expression in Fetal Membrane Cells with Progesterone, TNF-α and Oxidative Stress Stimulus. Yan Meng, David Schomberg, Amy Murtha, Liping Feng. OB-GYN, Duke University, Durham, NC, USA. Objective: Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in fetal membrane cells and is involved in fetal membrane cell survival. Our previous work demonstrated that PGRMC1 expression appears to be differentially expressed in fetal membrane layers in pregnant subjects with a significant reduction in expression in the fetal membranes of PPROM subjects. Altered expression of PGRMC1 may contribute to fetal membrane cell death and ultimately membrane rupture. The present study was designed to elucidate the factors that could influence PGRMC1 expression in fetal membrane cells. Methods: Fetal membranes were collected from women with uncomplicated term pregnancies undergoing elective cesarean delivery (n=3). Primary amnion, chorion and decidua cells are isolated and cultured as described previously. Cells were exposed to ethanol (vehicle), progesterone (P4), 17 α-hydroxyprogesterone hexanoate (17P), medroxyprogesterone 17 acetate (MPA) and promegestone (R5020) at 10 -7 in DMEM/F-12 media without serum for 48 and 72 h after the cells reach 90% confluence. Cells were also treated with tumor necrosis factor (TNF)-α at 1ng, 10ng and 50ng and the reactive oxygen species H 2 O 2 at 1 µM, 10 µM and 100 µM under the same conditions. PGRMC1 protein level was quantified using Western blotting normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PGRMC1 expression in all progestin treated groups was compared to ethanol (vehicle) control. Results: PGRMC1 protein expression is up-regulated by P4 and 17P treatment in fetally derived amnion and chorion cells (P=.03) but not decidua cells. Treatment with MPA or R5020 did not change PGRMC1 expression in any of the three cell types. TNF-α and H 2 O 2 treatment also did not influence PGRMC1 protein expression in primary cultured fetal membrane cells.

These results indicate that PGRMC1 expression is induced by P4 and 17P rather than MPA and R5020 in fetal membrane cells, but this induction is specific to amnion and chorion cells. PGRMC1 expression in fetal membranes may not be regulated by TNF-α and reactive oxygen species H 2 O 2 (oxidative stress) although additional work is required to confirm our findings. Objective: Spontaneous preterm birth (PTB) is a major public health concern due its complex etiology and pathophysiology. Children born preterm have a higher risk of morbidity and mortality during early as well as later on in their life. Recent studies suggest that DNA methylation of several genes vary in neonates across a range of gestational ages, and fetal DNA methylation changes may impact PTB. The objective of this study is to evaluate whether methylation of CpG sites differ in fetal DNA from early PTB (<34 weeks). Methods: DNA methylation of umbilical cord blood from African Americans with early PTB (24-34 weeks; N=22) or term birth (39-41 weeks; N=28) was assessed using the HumanMethylation450k BeadChip. The associations between proportion of DNA methylated and PTB were evaluated by fitting a separate linear model for each CpG site, adjusting for the appropriate covariates (sex and batch effects). Results: We found 6498 CpG sites associated with PTB (FDR<.05; 1.05x10 -11 <p<6.71x10 -3 ). Of the PTB-associated CpG sites, 61% (3959 CpG sites) increased in methylation and 39% decreased (2539). PTB-associated CpG sites were depleted in the promoter and first exon and enriched in the 5'UTR, gene body and intragenic regions (p<2.2x10 -16 ). As expected, associated CpG sites were also depleted in CpG islands (16.3% vs. 31.1%; p<2.2x10 -16 ) and enriched in CpG shores (32.7% vs. 22.9%; p<2.2x10 -16 ). Many of the top PTBassociated CpG sites were in genes involved in parturition (CALCA and MYLK), the immune system (IL10, HLA-A and HLA-B), cell proliferation and migration (BCL9 and TGFBR3), and fetal development (ESR1). We also identified CpG sites that were previously associated with gestational age (PIK3CD). Conclusions: Dramatic differences in fetal DNA methylation across the genome associate with PTB, and multiple CpG sites reside in genes associated with PTB pathophysiology. Further validation of these data and functional studies may provide insight into pathways related to the etiology or consequences of PTB. Introduction Spontaneous preterm birth (sPTB) remains a challenging problem in obstetrics, and is associated with significant neonatal morbidity and mortality. Fetal Fibronectin (fFN) has become a useful test as a strong negative predictor for spontaneous PTB (Berghella et al. 2008) . Studies have emerged suggesting that concentrations of fFN within cervicovaginal secretions correlate with actual risk of spontaneous PTB (Goepfert et al. 2000; Kurtzman et al. 2009; Lu et al. 2001) . Therefore quantitative measurements of fFN may provide additional discriminatory information and be more clinically beneficial in predicting risk and outcome. The aim of this study was to determine if risk of sPTB in asymptomatic women correlated with absolute concentration of fFN. Study Design: A prospective blinded study of CVF fFN concentration (ng/ mL) in asymptomatic women considered at high risk of spontaneous preterm labor (n=744; 22-27 +6 weeks') using a 10Q analyser (Hologic). Clinicians were blinded to the quantitative result until post-delivery but the qualitative TLI IQ (Hologic) fFN result was made available. Results: The rate of sPTB (<34 weeks') was lowest (2%) for women with concentrations 0-9 ng/ml, and highest for those with concentrations ≥200 ng/ mL (31%). Compared to <10 ng/mL fFN, the relative risk of delivery was: (10-49 ng/ml) 4.3 (95% CI 0.03 to 0.13), (50-199 ng/mL) 4.3 (95% CI 0.005 to 0.15), (≥200 ng/mL) 13 (95% CI 0.16 to 0.42). The positive predictive value for sPTB (<34 weeks') increased from 15, 19, 31% with increasing thresholds (10, 50, 200 ng/mL respectively), yet NPV remained >95%. Conclusions: Risk of sPTB is increased for concentrations above 10 ng/mL. Quantitative fFN provides additional thresholds (10 and 200 ng/mL) over the qualitative method (50 ng/mL) to discriminate risk of sPTB in high-risk asymptomatic women. Objective: Preterm premature rupture of membranes (PPROM) is a common complication in patients with a cervical cerclage, and the approach of this scenario is controversial The potential benefit of a retained cerclage to prolong latency and decrease complications related to prematurity needs to be balanced with the risk of adverse neonatal outcomes related to infection. Inflammation markers in the amniotic fluid (AF) such as interleukin 6 (IL-6), glucose and white blood cells (WBC) are indicators of infection. Methods: A retrospective cohort study included pregnancies presenting with PPROM after cerclage placement and diagnostic amniocentesis. The unexposed group consisted of cases with immediate cerclage removal after PPROM, and the exposed group had cerclage retention > 12 hours after PPROM. Adverse outcomes were evaluated including the interval from PPROM to delivery, perinatal mortality, neonatal cumulative morbidity, and histological chorioamnionitis. Analyses were performed using AF IL-6, glucose and WBC levels to measure the relationship with outcomes. Results: The patient population included 40 cases; unexposed group (n=22) and espoxed group (n=18). Interval from PPROM to delivery was decreased significantly in the group with immediate removal of cerclage (p<0.005). Latency >48 hours and >7 days were associated with cerclage retention, (p<0.001) and (p<0.01) respectively. Histological chorioamnionitis was associated with retained cerclage (p<0.05). Neonatal outcomes were not significantly different between the study and control groups. Although, elevated IL-6 was associated with neonatal cumulative morbidity (p<0.05). Low AF IL-6 and low AF WBC were associated with latency >7 days (p<0.001) and (p<0.01) respectively. Conclusions: Cerclage retention for more than 12 hours after PPROM was found to prolong latency, but neonatal outcomes did not differ significantly between immediate removal of cerclage versus retention after PPROM. However, AF IL-6 and WBC levels may be of clinical value to individualize the management of patients. Introduction: Prolonged second stage of labor has been associated with a higher risk of several maternal complications, especially postpartum hemorrhage related to uterine atony. Delayed recognition of postpartum hemorrhage is a major cause of maternal morbidity and mortality. The present study was undertaken to better define the association of second stage labor duration and blood loss. Methods: A cross-sectional descriptive study was performed. The patient population included term pregnancies with vaginal delivery. Duration of second stage of labor (minutes) and estimated blood lost (milliliters) were recorded. Pearson correlation test with 2-tailed was performed. Results: The population studied included 7,486 cases. No statistical significant correlation was found between the duration of the second stage of labor and the quantification of the estimated blood loss (p=0.725). Conclusions: We did not find a correlation between the duration of the second stage of labor and the estimated blood loss after a vaginal delivery. Figure 1 . Correlation between duration of second stage of labor in minutes and estimated blood loss in milliliters.

Intrauterine Contraction Patterns at Baseline and during the Pre-Epidural Intravascular Fluid Bolus in Term Labor. Rebecca Benfield, 1 Dawn Daniels, 2 Sarah Buck, 2 Joy Shepard, 1 Jan Salstrom, 3 Denise Brigham, 4 Melydia Edge, 1 Melvin Swanson, 1 Edward Newton. 3 1 Graduate Nursing Science, East Carolina University, Greenville, NC, USA; 2 Women's Health Services, Vidant Medical Center, Greenville, NC, USA; 3 Obstetrics and Gynecology, East Carolina University, Greenville, NC, USA; 4 Medicine Hematology Oncology, East Carolina University, Greenville, NC, USA. Background: Immersion to the chest (hydrotherapy) in term labor results in decreased contraction frequency and the duration is positively correlated with percent plasma volume shift. Significant decreases in plasma vasopressin and oxytocin also occur (Benfield, et al, 2010) . Immersion to the neck produces central volume expansion similar to that induced with 1.5 L of saline infused intravenously within 21 min (Johansen, 2000) . Might the intravascular preepidural fluid bolus used to offset maternal hypotension during epidural analgesia affect uterine contractility similarly to immersion? Objective: We compared uterine contraction parameters including Montevideo Units (MVUs), frequency, duration, peak amplitude and resting tone at baseline (BL) and during the intravascular pre-epidural fluid bolus (bolus) in laboring women at term with intrauterine pressure catheter (IUPC) monitoring. Design and Methods: Retrospective review of 2065 charts, identified 16 healthy women (10 nullipara, 6 multipara) with IUPC monitoring prior to and during epidural analgesia initiation who met study criteria (exclusion of magnesium sulfate therapy or chorioamnionitis). Thirteen women received Pitocin during both epochs and three did not. The BL epoch began up to 60 min prior to bolus initiation: the bolus epoch occurred during the 1 L Lactated Ringers infusion. Epoch length varied (all had at least 4 contractions). Mitutoy Absolute Digimatic Calipers were used to measure duration. Amplitude and resting tone (mmHg) was extracted from the monitor strip grid. All measurements were double checked and randomly cross-checked by an independent observer. The paired t-test, significant at p < .05*, compared BL to bolus epochs. Results: Contraction frequency (M=.38, p=.03*); resting tone (M=2.9, p=.02*); MVUs (M=11. 4, p=.19) ; and duration (M=1.5, p=.50) increased while amplitude declined slightly (M=.10, p=.96). Conclusion: Unlike hydrotherapy, contraction frequency and resting tone were significantly increased with the bolus while amplitude decreased slightly. Future research will focus on the discrepancy.

A Silk-Based Biomaterial for Cervical Injection: A Pilot Study of Biocompatibility in a Pregnant Rat Model. Agatha S Critchfield, 1 Jeannie C Kelley, 1 Reid McCabe, 2 Lauren Richey, 3 Simona Socrate, 4 Errol R Norwitz, 5 David L Kaplan, 2 Michael House. 5 1 Division of Maternal Fetal Medicine, Tufts Medical Center, Boston, MA, USA; 2 Department of Biomedical Engineering, Tufts University, Medford, MA, USA; 3 Division of Laboratory Animal Medicine, Tufts University, Boston, MA, USA; 4 Health Sciences Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; 5 Mother Infant Research Institute, Tufts Medical Center, Boston, MA, USA. Objective: To develop an injectable, silk-based biomaterial as an alternative to cervical cerclage. Here, we performed a pilot study of biocompatibility of the biomaterial in a pregnant rat model. Methods: A purified silk solution (6% w/w) was prepared and sterilized as previously reported. Aliquots (2 mL in 3 mL syringes) were sonicated for 10-20s to accelerate gelation (Branson 450 Sonifier, 1/8" diameter-tapered microtip, 15% amplitude setting) and stored on ice until injection 4h later. Timed-pregnant Sprague-Dawley rats (n=4) at gestational day 15 underwent laparotomy and 300µL was injected into the cervix with a 27g needle under direct visualization. A saline control (n=1) was injected by the same procedure. Unused silk solutions were incubated at 37 o C to assess time to gelation in vitro. Sacrifice occurred on gestational day 19. Histological evaluation was performed on the harvested cervices. Results: The animals tolerated the procedure well and none experienced preterm delivery. On histological evaluation, the silk-based biomaterial was identified in the cervical stroma in three of four study animals. A mild to moderate granulomatous to eosinophilic inflammatory response was seen surrounding the silk material, consistent with a mild foreign body response. This response was not seen in the saline control. No neutrophil response was seen. Gelation of silk solutions occurred by 12-24h in vitro. Conclusions: A novel rat model for studying cervical injections is presented. The injectable, silk-based biomaterial provoked a mild foreign body response. Future studies will compare the biocompatibility of the silk-based biomaterial to other biomaterials used for cervical surgery and investigate the ability of this intervention to delay spontaneous delivery. Objective: To determine if epidural anesthesia and histologic chorioamnionitis are independent predictors of intrapartum fever. Study Design: Secondary analysis of a retrospective cohort study designed to determine the accuracy of clinical signs in prediction of histologic chorioamnionitis. Subjects were term parturients with placental examination in 2005. Chorioamnionitis was classified according to the Amniotic Fluid Infection Syndrome Nosology Guidelines. Cohorts with and without epidural anesthesia and with and without intrapartum fever (≥ 38°C) were compared with regard to maternal, intrapartum and newborn data. A forward stepwise multiple logistic regression model was constructed with fever as the dependent variable and epidural anesthesia and histologic chorioamnionitis included among the independent variables. Significance was set at p ≤ 0.05. Results: There were 641 term parturients who had the placenta examined out of a total of 2124. Epidural anesthesia was used in 69% of all term parturients and 76% of the study subjects. Intrapartum fever occurred in 37% with epidural versus 7% without (OR 7.62; ). Histologic chorioamnionitis occurred in 62% with epidural anesthesia and 42% without (OR 2.24; . Histologic chorioamnionitis was more likely to occur in the presence of intrapartum fever (OR 4.63,95% CI: 3.04-7.08). Forward stepwise multiple logistic regression was performed with fever as the dependent variable and 12 independent variables. Independent predictors of intrapartum fever are given in the Essential study data included details of cerclage placement and outcome data for the pregnancy. Results: There were 24 patients with a multiple pregnancy and 212 with a singleton pregnancy. Of these 212 patients: 110 (51.9%) patients underwent prophylactic cerclage, 61 (28.8%) indicated cerclage placement for a short cervix and 41 (19.3%) rescue cerclage placement for an open cervix. Prophylactic cerclages were placed between 10 and 22 weeks and were removed between 15 and 39 weeks. Deliveries occurred at less than 24 weeks in 8 (7.3%), less than 28 weeks in 15 (13.6%), less than 32 weeks in 23 (20.9%) and less than 37 weeks in 48 (43.6%). 8 fetuses (7.3%) were stillborn or died shortly after birth. Indicated cerclages were placed between 11 and 24 weeks and were removed between 20 and 40 weeks. Deliveries occurred at less than 24 weeks in 3 (4.9%), less than 28 weeks in 7 (11.5%), less than 32 weeks in 11 (18.0%) and less than 37 weeks in 28 (45.9%). 4 fetuses (6.6%) were stillborn or died shortly after birth. Rescue cerclages were placed between 13 and 23 weeks. They were removed between 16 and 38 weeks. Deliveries occurred at less than 24 weeks in 11 (26.8%), less than 28 weeks in 15 (36.6%), less than 32 weeks in 22 (53.7%) and less than 37 weeks in 27 (65.9%). 11 fetuses (26.8%) were stillborn or died shortly after birth. Conclusion: Though outcomes are favorable in most cases of 'prophylactic' and 'indicated' cerclage, there is an association with preterm birth. The majority of patients undergoing a 'rescue' cerclage will deliver prematurely, with one quarter delivering before viability and one third before 28 weeks. Methods: This was a prospective randomized trial of patients presenting for induction of labor. Randomization was performed by the University of South Carolina Department of Epidemiology and Biostatistics and transferred to the password-locked Pyxis system in the nursing station. Allocation was concealed from the PI and recruitment staff until after patient enrollment when the next sequentially-numbered opaque envelope was removed from the Pyxis. This pilot study had planned enrollment of 90 patients. 30 patients were randomized each to Foley alone, Prepidil alone or Foley catheter with Prepidil injected through the catheter into the lower uterine segment.

Success of cervical ripening was assessed by change in Bishop score. The combined arm had superior cervical ripening. The average change in Bishop score was 1.41 (range 0-4) for Prepidil alone, 2.6 (range 0-6) for Foley alone and 3.76 (range 1-9) for combined Foley Catheter and Prepidil. When assessing change in Bishop score of at least three points, statistical significance was reached comparing the combined method to Prepidil alone 72% vs 21% (p 0.00007 CI 2.75 -35.61) but not when comparing the combined method to Foley catheter alone 72% vs 56% (p 0.1196 CI 0.61 -6.81). Time to vaginal delivery was longest in the Prepidil alone arm, average 1403 minutes (range 613-2943). The Foley alone and combined arms had similar times to vaginal delivery at 1058 minutes (range 340-1723) and 1075 minutes (range 371-2094), respectively. Statistical significance was not reached when assessing for delivery within 24 hours between groups. Conclusion: This study confirms the efficacy of combining Foley catheter and Prepidil gel for cervical ripening. Change in Bishop score was improved when combining these 2 methods, as compared to using either method separately. Time to delivery was improved when using the combined method as opposed to Prepidil alone.

Are Clean Gloves on Labor and Delivery Truly Clean? Laura Houston, 1 Scott Sullivan, 1 David Soper, 1 Laura Goetzl, 1 Lisa Steed. 2 1 Obstetrics and Gynecology, Medical University of South Carolina, Charleston, SC, USA; 2 Pathology & Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA. Background: There is no consensus regarding the use of sterile and clean gloves for intrapartum cervical exams. A recent retrospective study on our labor and delivery unit suggested that clean glove use may be a safe, cost-effective alternative to routine sterile gloves. Objective: To compare culture results between clean and sterile gloves on labor and delivery. Study design: An observational prospective study was performed. 20 sterile and 20 clean gloves from separate boxes were randomly cultured on blood and anaerobic agar culture media by pressing one surface of each glove against the agar for 20 seconds.

The plates were incubated under aerobic and anaerobic conditions for 48-72 hours. Following incubation, the total number of colonies was determined, and the microorganisms were identified to the Genus level.

No growth was noted from all 20 sterile gloves or all 5 gloves from newly opened clean boxes. 3/5 clean gloves from the first third of their boxes showed no growth, while coagulase negative Staphylococcus and Bacillus sp. grew from 2/5 gloves. From clean gloves obtained at the middle third of used boxes, 4/5 had no growth, with Bacillus sp. growth from one glove. 3/5 boxed gloves from the last third showed no growth, while coagulase negative Staphylococcus and Bacillus sp. grew from 2 gloves. Conclusion: Most gloves cultured did not exhibit bacterial growth. Environmental and skin contaminants (coagulase negative Staphylococcus and various Bacillus species) were noted on several boxed gloves at subsequent stages of use. Based on our microbiological evidence, clean gloves appear to pose no significant pathogenic infection risk to patients having vaginal examinations on labor and delivery. Objective: The aim of this study was to evaluate the effect of inflatable obstetric belts on uterine fundal pressure in the management of the second stage of labor. Methods: One hundred eighty-eight nulliparous with a singleton pregnancy at term were enrolled. Standard care was performed in the control group, and uterine fundal pressure by the Labor Assister™ (Baidy M-420/Curexo, Inc., Seoul, Korea) was applied during the second stage of labor in addition to standard care in the active group. The Labor Assister™ is an inflatable obstetric belt that synchronized to apply constant fundal pressure during a uterine contraction. Participants received patient-controlled epidural analgesia. Results: The ninety-seven women in the active group spent less time in the second stage of labor when compared to the ninety-one women in the control group (46.51±28.01 min vs. 76.26±36.87 min, p<0.01). There was no significant difference in perinatal morbidities, perineal laceration, and analyses of cord blood between the two groups. Conclusion: The inflatable obstetric belt on uterine fundal pressure reduces the duration of the second stage of labor without complications in women who receive patient-controlled epidural analgesia. Objective: Induction of labor (IOL) increases the risk of cesarean delivery (CD), specifically in nulliparas. This risk is believed to be attenuated in multiparas, however, there is a paucity of data evaluating this. Our objective is to evaluate the risk of CD among multiparas undergoing IOL compared to those presenting in spontaneous labor (SpL). Methods: We performed a large retrospective cohort study of women with 2 consecutive deliveries from 2005-2010. This secondary analysis evaluated the first of these 2 pregnancies and was restricted to term women (≥37 wks) that had an IOL or presented in SpL. Prior CD were excluded. Maternal data were obtained through chart abstraction. Multiparity was defined as a prior delivery ≥20 weeks. SpL was defined by presenting at 5cm dilation or documented cervical change to 4cm. Associations between IOL and CD rate were determined using logistic regression. Results: 867 women were analyzed (609 IOL, 258 SpL). Overall CD rate was 18%. CD rate was higher for IOL vs. SpL (23 vs. 7%, ], p<0.001). Of the 867 women, 310 were multiparas (33% had IOL); 557 were nulliparas (67% had IOL). There was an increased risk of CD in multiparas undergoing IOL vs. SpL (15 vs. 5%; OR 4.0 [1.3-11 .8], p=0.01) as well as in nulliparas (27 vs. 11%; ], p<0.001). The increased risk of CD remains after controlling for race, age, and BMI (multiparas: aOR 3.6 [1.2-10.8 ], p=0.02; nulliparas: aOR 3.0 [1.7-5.5] , p<0.001). Among multiparas, risk of CD is higher with starting cervix ≤2cm for IOL ], p=0.01) compared to SpL and there is a trend towards increased risk of CD with cervix >2cm ], p=0.07). Among IOL multiparas, there was no difference in CD risk when comparing IOL for maternal/fetal indications vs. PROM/postterm (p=0.8). Discussion: IOL increases risk of CD among nulliparas. Novel to this study, IOL also increases risk of CD among term multiparas, regardless of indication for IOL. This increased risk of CD is important to acknowledge when considering IOL in all patients, regardless of parity. Objective: The rate of women undergoing an induction of labor (IOL) is increasing. Simultaneously, the preterm birth (PTB) rate is largely unchanged. The impact of IOL on spontaneous PTB (sPTB) rates in a subsequent pregnancy is unknown. It is plausible that IOL may affect cervical integrity, altering it for future pregnancies. Our objective was to determine if IOL in one pregnancy is associated with sPTB in the subsequent pregnancy. Methods: A retrospective cohort study of women with 2 consecutive deliveries from 2005-2010 was performed. Maternal data were obtained through chart abstraction. Women were selected based on term IOL or term spontaneous labor (SpL) in the index pregnancy. Prior history of sPTB was excluded. SpL was defined by 5cm dilation or cervical change to 4cm. Primary outcome was sPTB (<37wks) in subsequent pregnancy. Categorical variables were compared with χ2 analyses and logistic regression was used to calculate odds. Results: 887 women were included (622 IOL, 265 SpL). Overall PTB rate was 11.4%; sPTB rate was 7.2%. Of the IOLs, 57% were for maternal/fetal indications (M/F); 32% PROM/postterm (P/Pt); 11% elective. Women with term IOL were less likely to have a subsequent sPTB when compared to term SpL (6 vs. 11% sPTB; .81], p=0.005). The reduced sPTB rate remained when restricted to M/F IOL (6.4%, ], p=0.05) and P/Pt IOL ], p=0.005), each compared to SpL. A decreased sPTB rate remained after adjusting for race, drug use, PNC, BMI, and mode of delivery in index pregnancy ], p=0.04). Subsequent sPTB risk was modified by the starting IOL cervical exam. Compared to SpL, when the cervix at IOL was ≤2cm, there was a more pronounced reduced risk of subsequent sPTB ], p=0.001). This was not significant for a cervix >2cm ], p=0.7). Discussion: Term IOL in an index pregnancy, specifically among women with an unfavorable cervix, decreases the risk of sPTB in a subsequent pregnancy when compared to term SpL. These findings suggest that failure to go into SpL may reflect cervical integrity and be associated with a reduced risk of sPTB. Understanding this decreased risk could have large implications in understanding cervical biology and aid in preventing sPTB. Objective: Induction of labor (IOL) increases the risk of cesarean delivery (CD). With initiatives to decrease the primary CD rate, there needs to be a focus on understanding length of labor, particularly latent labor (LL), in IOL patients. The definition of "failed IOL" remains controversial although 12-18hrs of LL has been suggested to define it. The objective of this study is to evaluate the duration of LL and mode of delivery among multiparas and nulliparas undergoing term IOL. Methods: We performed a retrospective cohort study of women with 2 consecutive deliveries from 2005-2010. This analysis evaluated the first of these pregnancies and was restricted to term (≥37 weeks) IOL with no prior CD. Maternal data were obtained through chart abstraction. IOL was defined by (1) use of ripening agent, (2) rupture of membranes/pitocin use at ≤4cm. Multiparity was defined as prior delivery ≥20 wks. Associations between IOL and mode of delivery were evaluated using χ2 statistics. Results: 610 women were included (194 multiparas, 416 nulliparas). Overall CD rate was 23%. Among multiparas, most had LL <12hrs and the majority had LL <24hrs. The majority had a vaginal delivery (VD) [table 1]. Of multiparas that had a CD with LL <24hrs, 45% were for non-reassuring heart tones (NRHT); 48% were for arrest disorders. Among nulliparas, approximately half had LL <12hrs but >90% had LL <24hrs. Of nulliparas that had a CD with LL <24hrs, 49% were for NRHT; 48% were for arrest disorders. Total number of women delivered within 24hrs was higher for multiparas than for nulliparas (95% vs. 85%, p<0.001). Discussion: Regardless of parity, the majority of women have latent labor and total delivery time <24hrs. However, there is an incremental gain in the number of VDs after this time point making it difficult to establish a strict cut off for failed IOL. Duration of latent labor should be individualized and risks of CD must be weighed against risks of remaining undelivered. Background: Inflammation is believed to be a major cause of early preterm birth but the origins and mechanisms by which this occurs remain uncertain. We sought to determine the relationships between lifestyle features in pregnancy and the level of inflammatory cytokines in the amniotic fluid early in the second trimester. Methods: Amniotic fluid was collected with consent from 398 women with singleton pregnancies undergoing genetic amniocentesis between 15 and 20 weeks gestation. The cytokines IL-6, IL-10, TNF-a and MCP-1 were determined by multiplex assay using the Luminex MAGPIX system. Confidential lifestyle data were collected by questionnaire in a private setting. Levels between groups were compared using Mann-Whitney and Kruskal-Wallis tests. Results: The median maternal age was 35 years (range 19-48). Fifty one women (14.8%) reported smoking in the current pregnancy. Sexual activity during pregnancy was reported as: none (n=61, 15.3%), <once a week (n=149, 37.4%), once a week (n=98, 24.6%), 2 to 3 times per week (n=69, 17.3%) and daily (n=15, 3.8%). IL-6 and IL-10 levels were significantly elevated in women who smoked (P=0.001, P=0.035).

There were no significant associations between amniotic fluid cytokine levels and sexual activity in pregnancy, or history of threatened abortion (n=9, 2.3%), urinary tract infection (n=22, 5.5%) or antibiotic usage (n=48, 12.1%). Conclusions: Smoking is associated with increased levels of some inflammatory cytokines in the amniotic fluid. Similar effects of smoking have been observed in other organ systems. These cytokine responses may play a central role in the complications of pregnancy known to be associated with smoking. In contrast, sexual activity in mid-pregnancy is not associated with an inflammatory response in the amniotic fluid. Any role for sexual activity in predisposing women to early preterm birth may be more likely to result from risk of infection than from a direct effect on inflammatory cytokines.

Manual Objective: In active labor, women with persistent occiput posterior or transverse (OP/OT) positions have an increased rate of cesarean delivery compared with those in occiput anterior position. The purpose of this study is to determine the cost-effectiveness of manual rotation in reducing cesarean delivery in persistent fetal OP/OT positions. Study Design: A decision analytic model was designed to compare manual rotation and expectant management of fetuses in the OP/OT position. Baseline estimates for outcomes and costs associated with malposition and manual rotation were derived from the literature. Costs were adjusted to 2012 dollars using the medical component of the CPI and a discount rate of 3% was applied to all future outcomes and costs. A cost-effectiveness threshold of less than $100,000 per quality-adjusted life year (QALY) was considered cost effective. Results: Manual rotation was cost effective at reducing cesarean delivery in persistent OP/OT position. In a theoretical population of 100,000 women with fetal OP/OT position, 8.2% of women after successful manual rotation versus 34.3% of women after no attempted manual rotation will proceed to cesarean delivery. The cost difference between manual rotation ($7,155) versus expectant management ($7,812) was $657 per parturient. In our sensitivity analysis, manual rotation remained cost saving down to a success rate of 2.6% and up to a cost of $657.

It was cost effective up to a cost of $7,080. Conclusion: We found that manual rotation was a dominant strategy (less costly and better outcomes) when compared to expectant management. It remained cost effective even at a success rate far below 50%. Because this simple procedure appears to reduce costs, incentives to providers to learn and utilize manual rotation should be considered.

The . Transvaginal ultrasonographic assessment of cervical length (CL) provides information regarding SPTB risk in women without cerclage. The utility of CL measurements and serial CL assessments following cerclage is uncertain. STUDY DESIGN: Retrospective cohort of women with singleton pregnancies from 2 tertiary centers 2009-2012. Women undergoing cervical cerclage placement for any indication <24 wks gestation were included. Medical records were abstracted, and stored CL images reviewed with the investigator blinded to outcomes. Women with ≥2 CL measurements after cerclage placement and <28 wks gestation were compared to those with 0-1 CL. The primary outcome was composite neonatal morbidity (neonatal death, severe IVH, NEC requiring surgery, or BPD). Data were analyzed using Chi-square and Student's t-test. RESULTS: 87 women were included; 55 (63%) had ≥2 CL. The majority (91%) of women received all or some prenatal care with a Perinatologist. Demographics, including the number of prior SPTB and gestational age of the earliest prior SPTB were similar between groups. CL parameters did not vary between groups, but women with ≥2 CL delivered later and had lower rates of neonatal morbidity and mortality.

There were non-significant trends towards increased utilization of antenatal steroids, tocolysis, and magnesium for neuroprotection among women with ≥2 CL. CONCLUSIONS: Women with cervical cerclage have a high risk of SPTB. Serial CL monitoring early in pregnancy (prior to 28 wks) after cerclage placement may provide additional information regarding PTB risk, and may be used to guide clinical decisions including need for inpatient admission, tocolysis, and antenatal corticosteroids. Methods: An extensive search for all human proteomic studies on biomarkers for PTB was conducted in 4 electronic databases The data extraction identified a) general characteristics of the study b) participant characteristics, c) type of proteomic approach.

Results: A total of 108 references were identified, 43 were selected for full text reading and based on our stringent selection criteria, data were extracted from 9 studies. In summary; 1) A total of 82 dysregulated proteins associated with PTB were reported in maternal serum (n=3), amniotic fluid (n=2), cervico-vaginal fluid (n=2), placental tissue (n=1), and in chorio-decidual tissue (n=1).

2) The depletion of the most abundant proteins prior to proteomic analyses were performed in two studies.

3) The most frequently employed proteomic approach was two-dimensional polyacrylamide gel electrophoresis. 4) The results were confirmed with complementary proteomic or antibody techniques in only two studies. 5) Potential biomarkers, identified in proteomic studies, represent a broad spectrum of intracellular and matrix proteins that play an important role in the inflammatory process, coagulation cascade, and proteolytic process. 6) No studies have replicated data in an independent cohort. Conclusion: Our search of proteomics literature did not yield biomarkers that predict PTB. This is partially due to heterogeneity in the existing studies. Proteomics offer an opportunity to identify novel biomarkers and pathways for PTB. Designing of future studies can be improved based on the issues identified in this review. 

Multiple studies now suggest that PTL is more likely to be caused by placental insufficiency than intra-amniotic infection. This working hypothesis is supported by our observation that the same AGT genetic variant more prevalent in women with preeclampsia and IUGR is also associated with PTL. We suspect the underlying cause may be related to differences in spiral artery remodeling. Preterm labor may be the least "severe" pleiotropic manifestation of this disease process, which may explain why demands on placental efficiency in male babies (larger than females) strengthens this genetic association (second hit). Objective To assess fetal cerebral myelination after exposure to chronic maternal psychological stress. Method Pregnant ewes were isolated twice a week for 3 h between 30 and 100 days of gestation (dG, term 150 dG). Fetal brains (stress n = 12, controls n = 11) harvested at 130 dG, were fixed with 4 % paraformaldehyde. Paraffin slices were cut at the level of the primary motor cortex. Immunostaining against myelin basic protein (MBP) was performed in order to evaluate myelin formation. Golgi staining was added on adjacent sections to relate possible changes of myelination to process formation. Sections were then examined using light microscopy and image analysis.

Results MBP immunoreactivity was reduced in the cortical grey matter (cortical laminae LII-VI), in the descending superficial white matter (sfwm), and in the subcortical white matter (swm) by 33.0 %, 41.7 % and 19.0 %, respectively (p < 0.01, Figure 1 ). Axonal formation was not affected by maternal stress (Figure 1 ). Conclusions Chronic maternal psychological stress impairs cerebral development in fetal sheep. In particular, stress affected neuronal myelination but not axonal formation. Reduced myelination of primary motor cortex fibers may contribute to impaired motor development described in infants of mothers who had experienced stress during pregnancy.

Effect of chronic maternal stress on myelination of fetal sheep brain. mean ± SEM, ***p < 0.001, **p < 0.01 vs. control fetuses (two-tailed Mann-Whitney U test). Supported by the European Community FP7 HEALTH, Project 279281 (BRAINAGE).

Measuring p-value 1 7.1 (5.7-10.2) 6.5 (5.3-8.0) 6.3 (5.3-8.6) 6.3 (4.6-9.7) 6.4 (4.9-8.2) <0.001 2 8.9 (5.3-11.9) 8.7 (7.4-11.0) 8.6 (5.7-10.7) ** 7.8 (4.9-9.9) <0.001 3 9.0 (6.7-11.3) 11.3 (9.8-13.0) 9.2 (7.8-12.6) 8.9 (6.4-11.3) 10.2 (8.9-11.3) <0.001 In four of five patients, L90 produced the greatest mean CO and maximal range in CO. In one patient, R90 produced maximal CO output. When CO and contractions are matched, discreet increases in CO occur 10-30 seconds after contraction when patients are placed in or near their optimal position.

Conclusions: Aortocaval compression can be assessed in labor by measuring CO non-invasively using electric cardiometry. Minimizing aortocaval compression may improve labor outcomes by reducing uterine venous congestion, improving cardiac venous return and maximizing cardiac output. OBJECTIVE: Maternal stress has previously been implicated in preterm labor among women carrying both singleton and multiple gestations, yet little is known about the correlation between pregnancy-specific anxiety with other markers of stress. The objective of this study was to examine the association between pregnancy-specific anxiety and other markers of stress and social support specifically among pregnant women carrying multiple gestations. Moreover, we sought to assess the role that pregnancy-specific anxiety and social support play in preterm birth and other adverse pregnancy outcomes in such pregnancies. METHODS: A prospective cohort study of women with multiple gestations was conducted. Comprehensive stress, anxiety, depression, and social support surveys were administered during the first and third trimesters. Results from these surveys and adverse pregnancy outcome data were analyzed and compared. RESULTS: 20 patients were enrolled in the study. One patient underwent fetal reduction at 20 weeks due to fetal anomaly. Successful completion of both first and third trimester surveys as well as pregnancy outcome data were available for the remaining 19. In this cohort, pregnancy-specific anxiety was strongly correlated with the other forms of distress -specifically depressive symptoms (r=0.58, p<0.01), PTSD symptoms (r=0.68, p<0.01), and perceived stress (r=0.60, p<0.01), but not with satisfaction with overall social support (r=-0.18) or received support from spouse (r=0.03). When comparing pregnancyspecific anxiety with adverse pregnancy outcomes including preterm labor, pre-eclampsia and PPROM no correlation was found. However, gestational age at delivery was strongly and positively correlated with perceived support from the spouse (r=0.54, p<0.02). DISCUSSION: Women with pregnancy-specific anxiety should be screened for other forms of distress. Those with poor spousal support may be at greater risk for adverse pregnancy outcomes in multiple gestations. To compare the perinatal outcome of early (7-8 weeks of gestation) transvaginal multifetal pregnancy reduction (MPR) from triplets and higher order multiples to twins with nonreduced twin gestations.

A prospective cohort study of 61 (44 triplets and 17 higher order) multiple pregnancies who underwent early first trimester transvaginal reduction were matched with 90 bichorionic-biamniotic nonreduced twins. The rates of early miscarriage, pregnancy loss before 24 weeks, preterm delivery prior to 32 weeks and 37 weeks of gestation as well as pregnancy complications such as, gestational diabetes, hypertension and IUGR were compared between the two groups.

In general, the outcome of triplets and greater multiple pregnancies reduced to twins was favorable and comparable to the outcome of non reduced twins. 3 (5%) pregnancies in the study group and 1 (1%) in the control group were lost prior to 24 weeks (p=NS). The rates of early spontaneous miscarriage of one of the fetuses (8%vs7%), preterm delivery prior to 32 weeks (10% vs 4% p=NS) and 37 weeks of gestation (54% vs. 53%) were similar among both groups. Similarly, there was no significant difference in the incidence of gestational diabetes (13% vs. 14%) and hypertension (13% vs 8%) between the 2 groups. However, the incidence of IUGR was significantly higher among the non-reduced twins (0% vs 8%, P=0.02). When comparing triplets reduced to twins (N=44) with high order multiple pregnancies reduced to twins (N=17) there was no significant difference in the incidence of early miscarriage and pregnancy loss before 24 weeks, but a significant higher rate of preterm delivery prior to 32 weeks was demonstrated for the latter group (2% vs 31% pv=0.005).

The outcome of twin pregnancies following early transvaginal multiple pregnancy reduction seems to be comparable to the outcome of nonreduced twins. However, when the starting number of fetuses is 4 or more the perinatal outcome is not as favorable. Fig  1A) and end of lactation ( Fig 1B) . Ex decreased both C and MO M cort but did not normalize values. MO increased cort in pooled male and female blood of 2 d neonates ( Fig 1C) , Ex partially reduced cort in neonates. 

Our data address the controversy whether glucocorticoids play a significant and even possibly common role across species in programming responses to different challenges e.g. maternal stress, under and over nutrition. We have shown in other studies that maternal Ex improves but does not completely prevent adverse OFF outcomes. Our data presented show for the first time, increased cort in mothers and OFF in MO and potential prevention by MEx. Cushing's Disease in pregnancy is associated with increased maternal and fetal morbidities, however the mechanisms of the detrimental cortisol effects are poorly understood. To determine effects of chronically increased maternal cortisol in late gestation, pregnant ewes (115d gestation) either continuously infused with cortisol (1 mg/kg/d; CORT1), infused for 12h overnight (0.5 mg/ kg/d; CORT0.5), or were not treated (control). Samples for plasma cortisol, insulin and glucose were collected, and uterine blood flow (UBF) measured on days 120, 125, 130, 135 and 140. A glucose tolerance test was also performed. Cortisol infusion increased maternal cortisol to levels similar to mild stress. Of the first 6 ewes studied in the CORT1 group, 4 delivered stillborn lambs at 140-146 days gestation; one lamb was born live at 143 days. Of the first 5 control ewes studied, all delivered live lambs. In the CORT0.5 ewes, 4 of 6 ewes delivered live lambs. In subsequent CORT1 and control ewes, the study was terminated at 142-143d before or at the start of labor. Of 8 control ewes, one delivered a live lamb at 142d and one a dead lamb after prolonged labor at 143d, and 6 fetuses were alive at the time of sacrifice. Of 10 CORT1 ewes, 5 were live at 142d, and 5 had died or were stillborn. Subsequent post hoc analysis of UBF, plasma cortisol, glucose and insulin and responses to the glucose tolerance test revealed differences among the groups. UBF was increased in the CORT1 ewes who had live fetuses (652±55) as compared to the control (457±43) and those with stillborn lambs (390±55); flow in CORT0.5 ewes was similar to control (432±62 ml/min). Basal glucose concentrations in the CORT1 ewes, both those with live and stillborn lambs (66.0±2.6; 69.3±2.1 mg% were increased as compared to control ewes (58.6±1.7). CORT0.5 glucose concentrations (64.7±3.0) were not different than control. In contrast, plasma insulin was higher in CORT1 ewes with stillborn lambs (2.25±0.26 ng/ml) as compared to those with live lambs (1.36±0.30) or control ewes (1.23±0.21). There were also differential effects on the response to glucose: the insulin response was reduced compared to control ewes in the CORT1 with live fetuses, but was increased in CORT1 ewes which later gave birth to stillborn lambs. These results suggest in the presence of increased maternal cortisol in late gestation, insulin resistance may predict fetal demise. Objective: Given the most recent changes in obstetric demographics (increasing maternal age and increasing prevalence of medical comorbidities) we expected to find an increased number of CVE in this population. The goals of our study are to characterize and compare the outcomes for pregnant and postpartum women with cerebrovascular events requiring ICU admission. Study Design: This is a single institution historical cohort study of pregnant and postpartum patients admitted to an ICU at our institution for a cerebrovascular event from 2007-2012. Women were identified from a database search of all ICU admissions. The postpartum period was considered up to 42 days. Data collected included demographic information, details of cerebrovascular event history and diagnosis, maternal outcomes. Comparisons of outcomes include postpartum versus antepartum, ischemic versus hemorrhagic events, timing of delivery. Results: During the study period there were 174 women admitted to the ICU in our institution: 23 (13.2%) were due to CVE. The table below describes the women based on type of CVE and timing of admission as it related to delivery. Only one-third of these women had an underlying medical comorbidity with the most common being hypertension (4/23). There were no reported maternal deaths in our cohort, but 22% (5/23) required prolonged rehabilitation or skilled nursing care. CVE subtype was not predictive of the morbidity outcome. Conclusion: In our population, CVE was a common indication for admission to the ICU. There appears to be an increasing trend in these admissions and we expect this may be attributed to advancing maternal age and medical comorbidities in our obstetric populations. MRI was cost effective compared to serial ultrasounds with a savings of $167.88 and was associated with an overall increase in 0.01 QALYs. MRI would remain cost effective if the probability of a CNS/NTD anomaly was as low as 1 in 10,000 and if the MRI cost up to $1,800. Conclusion: When considering the increased risk of CNS anomalies in obese women, the use of MRI in those who have inadequate visualization on screening ultrasound appears to be a cost-effective strategy for prenatal diagnosis. Objective: To ascertain the rate of utilization of amniocentesis (AC) in preterm labor (PTL) in the US and the association with pediatric outcomes including mortality rates. Study Design: Data from the National Center for Health Statistics at the Centers for Disease Control and Prevention were analyzed from the years 2004 to 2006. Nineteen states, representing 49% of the population, reported data. There were a total of 8,405,654 births. Analysis was limited to singleton cases between 24-29 weeks with documented preterm labor. Maternal age ≥ 35 years and congenital anomaly cases were excluded to minimize the chances that AC was performed for genetic reasons. Only cases reporting whether or not AC was performed were included. The frequency of AC overall and in different GA groups was determined, as was the correlation between AC and different maternal and newborn outcomes. Odds Ratios (OR) were calculated and then adjusted for GA at delivery, maternal race and age, diabetes and hypertensive disorders; the non-AC served as the reference group. Results: There were 19,909 eligible PTL cases of which 273 (1.4%) underwent AC. There was no difference in mean birth weight (p=0.20), mean maternal age (p=0.06), or race (0.83) in cases with and without AC. The rate of AC utilization did not vary with GA. No differences were found in early onset neonatal morbidities such as hyaline membrane disease, ventilation, anemia, fever. AC was associated with a higher adjusted OR for death at less than 7 days of life (2.16, 95% CI 1.60, 2.90), neonatal death (2.07, 95% CI 1.54, 2.77), and infant death (2.24, 95% CI 1.71, 2.94) . Conclusions: These data suggest low rates of AC utilization in potentially eligible patients with PTL. There were no significant differences in maternal age or race for rates of AC utilization or in neonatal morbidities. However, death at less than 7 days of life, neonatal death, and infant death were positively associated with AC, possibly suggesting increased utilization of AC in pregnancies at the highest risk.

A Scoring System for Severity of Placenta Accreta. Sam Nasseri, 1 Adam Bogomol, 2 Manuel Alvarez, 2 Themba Nyirenda, 2 Stacy Zamudio, 2 Abdulla Al-Khan. 2 1 School of Medicine, St. Georges Univ., Grenada; 2 Radiology, HUMC; 3 Ob/Gyn, HUMC; 4 Research, HUMC; 5 Ob/Gyn, HUMC; 6 Obstetrics and Gynecology/MFM Division, Hackensack University Medical Center (HUMC), NJ, USA. Introduction: Placenta accreta is a significant cause of morbidity and mortality in pregnancy. Ultrasound (US) and Magnetic Resonance Imaging (MRI) have improved antenatal detection of placental pathologies. Our goal is to develop an accreta severity scoring system using past obstetrical history, US and MRI to predict the degree of placental invasion (accreta vs. increta, percreta). Methods: We retrospectively analyzed 40 cases of suspected accreta. All had US and MRI assessments. A Maternal-Fetal Medicine specialist and a Radiologist, blinded to histopathologic diagnosis, evaluated the presence/absence of literature-derived indicators of accreta. We weighted prior C-sections as 1 (for a single C-section) or 2 (for >2 C-sections). Four US and four MRI indicators were weighted equally for a possible high score of 10. Results: There were 5 accretas, 8 incretas,10 percretas and 17 normal cases. Logistic regression indicated that MRI findings were of greater significance than US findings (OR range 18.7-144.5 vs. 7.3-9.9 for US). Univariate analyses confirmed this: MRI findings of bladder wall irregularity (87% of all accreta-related pathologies, 0% of normal), myometrial irregularity (91% accretas, 24% normal) and uterine wall irregularity (87% accretas, 18% normal) performed better than any of the ultrasound measures (e.g. placenta previa and increased vascularity at the uterine-bladder interface was present in 91% of accretas and 59% of normal patients). Median score and interquartile range in normal cases was 3 (2, 4) , in accreta 7 (4, 8, p=NS vs normal) , in increta 9 (7, 9, p<0.001) and in percreta 9 (9, 9, p <0.001). For a score ≥7, sensitivity and specificity was 86.9% and 88.2% for any type of accreta. Conclusions: A scoring system for accreta severity has been developed, but requires refinement via differential weighting of individual risk factors and highly correlated MRI/US findings. Our future work is directed at weighting the factors using multivariate modeling and prospectively testing the utility of the scoring system.

Neonatal Antioxidant Treatment Prevents Impaired Cardiovascular Function at Adulthood Following Neonatal Glucocorticoid Therapy. Y Niu, 1 EA Herrera, 1 RD Evans, 2 DA Giussani. 1 (5): e1282, 2012). Therefore, there is accumulating interest in modifying NICU dexamethasone therapy to maintain benefits, but prevent adverse effects. The mechanisms underlying the adverse effects of dexamethasone are not understood but oxidative stress may play a role. We investigated whether neonatal antioxidant therapy ameliorates the cardiovascular dysfunction at adulthood induced by neonatal dexamethasone treatment in NICU-relevant doses. Methods: Rat pups received daily i.p. injections of a tapering dose of dexamethasone (D; n=8; 0.5, 0.3, 0.1 µg/g) or D with vitamins C and E (DCE; n=8; 200 and 100 mg/kg, respectively) on postnatal days 1-3 (P1-3); vitamins were continued from P4-6. Controls received equal volumes of saline from P1-6 (C; n=8). A fourth group received vitamins alone (CCE; n=8). At P100, hearts were assessed under both working and Langendorff preparations. Peripheral vascular reactivity was determined in femoral arteries via myography. Results: Dexamethasone pups at adulthhod had an increased left ventricular (LV) mass (70±2 vs. 63±1 %), enhanced LV end diastolic pressure (14±2 vs. 8±1 mmHg) and these hearts failed to adapt cardiac output with increased preload or afterload (Fig 1.A,B) . Neonatal dexamethasone markedly impaired NOdependent femoral relaxation at adulthood (Fig 1.C 

Sciences Center, San Antonio, TX, USA. Objective:Adverse uterine environments during fetal development predispose offspring to metabolic disease but few studies address behavioral outcomes. We showed effects of MO on offspring anxiety behavior (1) and that outcomes may be related to increased maternal reactive oxygen species (ROS). We hypothesized that maternal treatment with the anti-oxidant Rvt before and during pregnancy would improve OFF anxiety behavior. Methods: From weaning through pregnancy and lactation We female Wistar rats ate chow (C) or high energy obesogenic diet (MO). Half of C and MO mothers given 20mg/kg Rvt orally for 30 d before and during pregnancy (C+Rvt and MO+Rvt). All rats were bred at PND 120 and continued on the same diet. Litters were adjusted to 10 pups. All OFF ate chow from weaning until the end of the study. At 90d old, one male and female from each litter was tested in elevated plus maze (EPM) with activity video analysis. Data M±SEM. Statistical analysis by ANOVA; p set <0.05. Results: Both males and femaleMO OFF had fewer entries and spent less time in the open arms vs. C. In female OFF, number of entries and time spent in the open arms increased in MO+Rvt (Fig 1) .

Elevated plus maze (EPM) end points. Open arm entries A) male, B) female and time spent (sec) C) male and D)) female.M ± SEM. n=6 from different litters. * p<0.05 vs C. Data not sharing a letter are different in the same maternal diet group. Conclusion:MO increased male and female OFF anxiety behavior, suggestive of lower impulsiveness to explore unknown spaces. Maternal Rvt administration recovers the innate tendency to explore novel environments in females but not males, suggesting differences in ROS activation or Rvt sensitivity in response to MO in male and male fetuses. Objective: There is a weak association between abnormal serum analytes and adverse obstetrical outcomes such as preeclampsia (PET) and small for gestational age (SGA). Our objective was to determine if uterine artery (UtA) Doppler studies would be clinically useful for risk stratification in a cohort of women with abnormal serum analytes. Study Design: We retrospectively reviewed outcomes of patients referred to our placenta function clinic with at least one abnormal analyte on prenatal genetic screening (PAPP-A<0.3, hCG>3.0, AFP>2.5, inhibin>2.0, or unconjugated estriol<0.3 MoM). Uterine artery pulsatility index (PI) and notching were assessed by Doppler velocimetry at approximately 24 weeks. Medical records were abstracted for outcomes including any PET, preterm PET (pPET, < 37 weeks), SGA (BW < 10%) and IUGR (BW < 3%). Results: 69 patients were identified with ≥ one abnormal analyte, UtA Doppler screening, and complete delivery outcomes. The percentage of patients with 1, 2, or 3 abnormal analytes was 75%, 23%, and 2% respectively. 8(11%) had PET and 13(19%) delivered a SGA neonate. Sixteen patients (23%) had abnormal mean UtA PI (PI>1.6) and/or notching. The odds ratios for adverse outcomes predicted by UtA Doppler are listed below in table 1. Abnormal UtA Doppler studies increased the likelihood of a composite outcome of PET or SGA from 12% to 55%; a negative UtA Doppler study reduced the likelihood from 10% to 4%. UtA Doppler studies increased or decreased the likelihood of a more clinically severe composite outcome of pPET and IUGR from 22% to 64% and 4% respectively. Conclusion: There is a significant association between UtA Dopplers and the subsequent development of PET or SGA in patients with abnormal analytes. UtA Doppler screening improves the prediction of these adverse outcomes by 4-8 fold and can be incorporated into an abbreviated surveillance regimen for these high risk patients. Providers can be reassured that a normal study decreases the risk of a severe early adverse outcome to lower than the baseline rate. (Rudich et al. 1997 ; Am J Physiol 272:E935), the hypothesis that combined dexamethasone (DEX) and antioxidant therapy will prevent the development of insulin resistance was also investigated. Methods: One male Wistar rat pup per litter (32 litters) received i.p. either saline or DEX in tapering doses (0.5, 0.3 and 0.1 µg/g) on postnatal days (P) 1-3 in addition to either i.p. saline or vitamins C and E (200mg/kg and 100mg/ kg respectively) during P1-6. At adulthood (P100), hepatic insulin signalling proteins were assessed by Western blotting.

The expression of molecules involved in insulin signalling (IRS-1, AKT1, PKCξ) was significantly decreased in the livers of rats treated with DEX ( Fig.1 ). Co-administration of DEX with antioxidants in some cases reversed the changes (AKT1), and in other cases did not (IRS-1, PKCξ). Treatment with antioxidants alone induced a decrease in IRS-1 and PKCξ. Conclusion: Postnatal glucocorticoid treatment in clinically relevant doses decreases the expression of key proteins involved in insulin signalling in the liver, suggestive of insulin resistance. Oxidative stress does not appear to be the only mechanism underlying this effect. Figure 1 : Changes in expression of insulin receptor substrate-1 (IRS-1), the protein kinases AKT1 and AKT2 and protein kinase C ξ (PKCξ) from the livers of P100 rats. Different letters indicate a significant difference between groups (ANOVA and Student Newman Keuls post hoc test). C=control, D=DEX,CE=vitamins C and E; n=8 per group.

Investigating the Role of Tbx4 in the Germline. Nataki C Douglas, 1 Ripla Arora, 2 Mark V Sauer, 1 Virginia E Papaioannou. 2 To investigate the requirement of Tbx4 for development and differentiation of the internal reproductive system, we analyzed germ cell development in the absence of Tbx4. METHODS: Whole mount immunofluorescence with an antibody against SSEA-1 was used to assess primordial germ cell (PGC) development in Tbx4 +/and Tbx4 -/embryos at embryonic day (E)10.0. As Tbx4 -/embryos die by E10.5, a tamoxifen inducible estrogen receptor Cre was used to delete conditional Tbx4 alleles (Tbx4 fl ) in vitro to analyze gonad differentiation between E11.5 and E13.5. GDF9Cre and ZP3Cre, two oocyte-specific Cre recombinases, were used to assess postnatal ovarian follicle formation and female fertility in the absence of Tbx4. RESULTS: The mean number of PGCs is reduced in Tbx4 -/embryos compared to Tbx4 +/littermates at E10.0 (P=0.03). In vitro differentiation of the gonad into morphologically distinct testes and ovaries occurs normally when Tbx4 is deleted starting at E11.5. In GDF9Cre;Tbx4 fl/and ZP3Cre;Tbx4 fl/mice, Tbx4 is deleted from oocytes postnatally. Nonetheless, in GDF9Cre;Tbx4 fl/and ZP3Cre;Tbx4 fl/adult females, primordial, primary, secondary, and antral follicles form, ovulation occurs, corpus luteum formation is normal and the mice are fertile. CONCLUSIONS: During embryonic development, loss of Tbx4 results in fewer PGCs. However, in the absence of Tbx4, embryonic male germ cells in the urogenital ridge can organize into testis cords and ovarian follicle formation and fertility are not obviously impaired in adult female mice. However, it is possible that this reduced PGC number could affect long term fertility in adults. Thus, our data set the stage for performing TBX4 mutation analyses in women with premature ovarian insufficiency.

The Objective: Consumption of sugar-sweetened beverages is steadily increasing coincident with an increase in adult obesity and diabetes. Exposure to high glucose before birth increases the risk of childhood obesity and diabetes, however little is known on the long-term effects of sucrose (cane /table sugar) and high fructose corn syrup (HFCS). This study will test the impact of maternal sucrose and HFCS consumption during pregnancy on the development of the rat offspring. Methods: Albino Wistar rats were randomized to either the control (C, n=12 dams), sucrose (S; n=7 dams) or high fructose corn syrup (HFCS; n=9 dams) groups. Dams were given ad libitum access to a standard rat chow and either a 10% w/v sucrose (S group) or HFCS (HFCS group) drink, or plain water for 4 weeks prior to mating and throughout pregnancy. Within 24h of birth, all pups were weighed, and a post mortem was performed on pups within a subset of litter (C, n=4; S, n=3; HFCS, n=5 litters).

Results: Preliminary analysis suggests that maternal S or HFCS consumption during pregnancy has no effect on litter size (C, 13.6±0.5; S, 11.9±0.9; HFCS, 12.7±1.1 pups per litter), or male:female gender ratio per litter (C, 1:1.3; S, 1:1; HFCS, 1:1.3). There was no effect of treatment on birth weight (P=0.51), however there was a significant effect of sex (P<0.001) and an interaction between the effects of treatment and sex on birth weight (C male, 6.13±0.07; C female, 5.94±0.06; S male, 6.37±0.09; S female, 5.82±0.08; HFCS male,6.21±0.10; HFCS female 5.77±0.06 g; P=0.05). Interestingly we report a significant effect of treatment on relative heart weight at birth (C, 0.00457 ±0.00008; S, 0.00476±0.00014; HFCS 0.00509±0.00017 g; P<0.05). There was no effect of treatment or sex on relative liver, kidney, adrenal or brain weights. Conclusions: These data suggest that maternal sucrose and HFCS consumption during pregnancy have a sex specific effect on pup growth during pregnancy. Furthermore, HFCS has a tissue specific effect on fetal growth such that relative heart weight was higher in the HFCS group, however this was not seen in other tissues examined. Further studies are underway to establish whether these early life changes persist throughout the post-weaning and adolescent period. Such effects could have significant outcomes on the postnatal metabolic heath of the offspring. Objective H19 is a highly conserved, paternally imprinted, maternally expressed gene encoding a long noncoding RNA. H19 regulates mammalian development; it is highly expressed during fetal life and downregulated after birth, except in a few adult tissues including skeletal muscle, heart, and the ovary, where it varies with the menstrual cycle. H19 acts as a molecular "sponge" for the let-7 family of miRNAs, which are known to play critical roles in development, cancer, and metabolism. We aim to characterize the role of H19 in the female reproductive tract, particularly in steroid hormone production and fertility.

To evaluate H19 expression in adult ovary, human cumulus cells (CCs) were obtained after controlled ovarian hyperstimulation, followed by RNA extraction and RT-qPCR. To determine whether H19 affects expression of steroidogenic enzymes whose mRNAs are predicted to contain binding sites for let-7, CCs were transfected with H19-specific siRNAs or non-targeting control siRNAs and expression of key steroidogenic pathway enzymes, including STAR and cyp450scc, were compared by RT-qPCR. Reciprocal studies were performed by ectopically expressing H19 in human granulosa cell (GC) line KGN cells (which do not express endogenous H19) by transfection of an H19-expressing vector or empty negative control vector. Expression levels of STAR and cyp450scc were measured by RT-qPCR and Western blot. Cell growth in response to ectopic H19 expression was assessed using cell viability assays.

Bioinformatic analysis predicted let-7 binding sites at the 3'-UTRs of STAR and cyp450scc mRNAs. H19 expression is high in gonadotropin-stimulated CCs. In addition, H19 knockdown in cumulus cells resulted in decreased expression of STAR and cyp450scc. Ectopic expression of H19 in KGN cells led to significant upregulation of STAR and cyp450scc, recapitulating H19-let-7-mediated regulation of steroidogenic gene expression in CCs. Conclusion H19 is abundantly expressed in stimulated cumulus cells. siRNA-mediated downregulation of H19 in CCs leads to reduced expression of STAR and cyp450scc, whereas ectopic expression of H19 in KGN cells results in increased expression of both enzymes and promotes KGN cell growth. These results not only suggest a regulatory role for H19 in the steroid pathway and GC growth, but also identify KGN cells as an ideal model system for future studies of H19-mediated regulation of gene expression in GCs. OBJECTIVE: Placental CRH is involved in the timing of labor. In human placenta, CRH expression is upregulated at both a transcriptional and translational level by glucocorticoids, and the mechanism involves the activation and nuclear translocation of RelB and NF-κB2 (p52) which then bind to an NF-κB enhancer in the CRH gene promoter (Wang, Mol Endocrinol 2012; 26:1356) . Whether this is true of all primate species is unknown. This study examines the expression of CRH and NF-κB signaling molecules in the placentas of non-human primates. STUDY DESIGN: Placentas were obtained from several non-human primate species delivered at term: (i) marmoset, (ii) rhesus monkey, and (iii) baboon.

Tissues were sampled and paraffin embedded. Immunohistochemistry was used to localize expression of CRH and subunits of NF-κB signaling molecules (RelB, NF-kB2) in each placenta (minimum n=3 for each) using speciesspecific antibodies. Expression was examined using a Zeiss light microscope. Appropriate positive controls were included. RESULTS: All data refer to expression in villous cytotrophoblast cells. In rhesus monkey and baboon placentas, robust expression of CRH (cytoplasm) as well as RelB (cytoplasm + nuclear) and NF-κB2 (cytoplasm + nuclear) was seen. This is similar to data in humans. In marmoset placentas, CRH staining was weakly positive in cytoplasm. RelB had strong cytoplasm staining and weak nuclear staining with only 2-7% of villous trophoblasts containing positive for nuclear RelB. In contrast, marmoset placentas did not express NF-kB2. CONCLUSIONS: While CRH and molecules regulating its expression are present in all primate placentas, species-specific differences exist. Given the critical role of placental CRH in the timing of labor, further studies are needed to better understand the significance of these species-specific differences. The absence of NF-kB2 in marmoset placentas suggests that rhesus monkeys or baboons may serve as more appropriate animal models for studying the role of the non-canonical NF-KB pathway in the timing of parturition in humans. The proteomic analysis from human IVF culture media has so far provided limited clinical information due to the small volume and the confounding influence of serum proteins used in culture supplements. Here we used the murine model with the aim to identify and compare the secretomes from murine embryos grown in vitro in protein-free and protein-supplemented media. Methods: A total of 432 2-cell embryos were randomly allocated to culture up to the blastocyst stage in protein-free and in 3% BSA-supplemented media. Four conditions were tested: (a) conditioned media of embryos grown in Krebs plus BSA; (b) conditioned media of embryos grown in Krebs without BSA; (c) Krebs medium plus BSA alone, as control; and (d) Krebs medium alone, as control. Proteins were separated by SDS-PAGE; bands were visualized by coomassie staining, followed by in-gel trypsin digestion and liquid chromatography-tandem MS. The processed MS/MS data were searched with MASCOT Daemon server combined with SwissProt protein database for the presence of membrane-associated and secreted proteins. RT-PCR was used to detect gene expression from mouse blastocysts. String analysis was applied to demonstrate functional relationships among the identified genes.

Results: Blastocyst formation rates were >95% in both groups. A total of 92 proteins were identified, from which, 25 proteins were common to both culture conditions; 35 and 32 proteins were only found under BSA-free and BSA-grown conditions, respectively. The BSA-free group had 7 unique signals corresponding to membrane-associated/secreted mouse proteins. Gene expression was confirmed from the blastocysts cultured with and without BSA for 2 of these proteins, prolow-density lipoprotein receptor-related protein 1 and integrin alpha-6. Indirect biological network connections among the 7 identified genes were established, revealing robust functional associations.

The secretome fingerprinting of mouse embryos during the preimplantation stage was characterized, speculating that without the confounding influence of serum proteins, enhanced protein identification could be achieved. Because of homology between murine and human, these studies could provide information to be translated to the clinical setting. Semin Reprod Med. 2011; 29:197; Camm et al. FASEB J. 2011; 25(1) :420-7), but the mechanism remains uncertain. We showed that programming of cardiovascular dysfunction in hypoxic pregnancy is prevented by maternal treatment with antioxidants (Giussani et al. PLoS One 2012;7(2):e31017), however the source of oxidative stress in hypoxic pregnancy remains unknown.

Here, we investigated the role of xanthine oxidase in programming cardiac and endothelial dysfunction in hypoxic pregnancy in both young adult and elderly rats. Methods: Female Wistar rats (n=48) were randomly divided into normoxic (N: 21% O2) or hypoxic (H: 14% O2) pregnancy, with or without maternal treatment with allopurinol (30 mg/Kg in jelly) from days 6-20 of gestation. This experimental model of hypoxia does not affect maternal food intake. At 4 and 15 months, 1 male per litter was culled for assessment of cardiac (Langendorff preparation) and endothelial (in vitro wire myography) function.

Aging impaired basal peripheral vascular endothelial and cardiac diastolic function (Fig 1 A&B) . Offspring of hypoxic pregnancies showed persistent endothelial and cardiac diastolic dysfunction at 4 and 15 months, and enhanced myocardial contractility at 4 but not 15 months (Fig 1 C) . Treatment of hypoxic pregnancies with allopurinol prevented cardiac dysfunction at 4 and 15 months, and the endothelial dysfunction at 15 months.

The data suggest that aging impairs basal cardiovascular function and developmental hypoxia amplifies this process, in part, via enhanced activity of the xanthine oxidase pathway. Med. 2011; 29:197; Giussani et al. PLoS One 2012: 7(2) :e31017). Here, we investigated whether altered cardiac mitochondrial function is a mechanistic component of this programming. Methods: Female Wistar rats (n=17) were mated in normoxia. From day 6 to day 20 of pregnancy, rats were exposed to normoxia (n=8) or hypoxia (13% O2; n=9), and allowed to deliver. Litters were reduced to 8 pups. Male offspring were weaned and housed in normoxia throughout their lives. At 4 months of age, permeabilised cardiac muscle fibre bundles were prepared from 1 rat per litter for mitochondrial respiratory measurements. Results: Prenatal hypoxia programmed electron transport chain (ETC) dysfunction, alterations in fatty acid oxidation and in the respiratory control ratio in hearts of adult offspring (Fig.1) . State III respiration rates were 53% lower (p<0.001) with complex I substrates (glutamate/malate) and 54% lower (p<0.01) with complex II substrates (succinate/rotenone). Prenatal hypoxia did not significantly alter mitochondrial respiration rates in the presence of pyruvate as substrate, but state III respiration rates in the presence of palmitoyl-carnitine were 40% lower (p<0.01) than in controls. Respiratory control ratios (RCRs) were lower with glutamate/malate (p<0.05), pyruvate (p<0.05) and palmitoylcarnitine (p<0.001) as substrates. Conclusion and implication: Prenatal hypoxia can programme cardiac mitochondrial dysfunction in the adult rat. This mechanism may underlie the increased incidence of metabolic disease in adults born following complications of pregnancy. [Ortiz, Kidney Int 2001] . The effects on cerebrovascular system are not known. This is important because stroke is the third most common cause for disability and invalidism. Objective: To examine the effects of prenatal DEX exposure and early postnatal stress on stroke outcome in later life.

Methods: Mice were treated with saline or 2x170µg/kg body weight DEX 24h apart corresponding to 2x12mg DEX administered to a 70kg pregnant woman to enhance fetal lung maturation at either E16/17 (one course) or E14-17 (two courses, term E19). Litter size was reduced to six pups. One group of pups of untreated mothers underwent maternal separation at P3-13 for 3h daily. Estrus was synchronized in females. At six months of age, mice underwent transient middle cerebral artery occlusion (MCAO) modeling media infarction as the most common form of embolic stroke. Infarct volume and the injury-related gliotic zone were estimated after 7 days using immunostaining of microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Results: Infarct size following MCAO was similar in males and females ( Fig.  1 ). One course of prenatal DEX treatment (E16/17) resulted in larger infarctions and gliotic zones in female and male mice (p>0.05, Fig. 1 ). Two courses of prenatal DEX (E14-17) did not further increase infarct size and gliotic zone ( Fig. 1 ). Postnatal stress did not affect infarct size and astrogliosis. Conclusions: Prenatal DEX exposure but not postnatal stress in mice worsens stroke outcome in later life. Two and four days of DEX treatment have the same effects. The DEX effects might be due to programming of cerebrovascular reactivity resulting in a higher cerebrovascular tone and the inability of the cerebral vasculature to dilate maximally as we have shown previously in rats using myography [Schwab, Reprod Sci 2010; 292A] . The fetal and neonatal pancreas show developmental plasticity to their metabolic environment. We have previously demonstrated significant developmental differences in the endocrine pancreas in fetal and neonatal baboons (PMID:22723715); we hypothesized that perinatal environmental exposures will lead to morphological changes on α-β-δ pancreatic cells in non-human primate fetuses. Objective: To examine histological differences in the endocrine pancreas of fetuses exposed to antenatal corticosteroids (ANS), maternal obesity (MO) and maternal nutrient restriction (MNR). Methods: Fetal baboons were delivered via c-section at 0.9 gestation to healthy (CTR), MO, MNR or ANS exposed mothers (n=4 group). Consecutive sections from pancreatic tissue were immunostained for insulin, glucagon, and somatostatin. Relative volumes and absolute mass of the microscopic structures were calculated utilizing the Computer Assisted Sterology Toolbox (CAST) 2.0 system. Plasma insulin and glucagon were measured by ELISA. Statistical calculations were performed utilizing SPSS version 17.0. Results: Birth weight was significantly lower in MNR and MO fetuses compared to CTR and ANS fetuses (p<0.01); however, the total pancreas weight as a percent of body weight was similar between all groups. Fetal α-β-δ cell percent area comprised 10.3±1.6, 11.7±1.3 and 13±2.5 (mean, ±S.E.) of endocrine pancreas in CTR animals respectively. Alpha cell percent area was significantly increased in fetuses exposed to ANS (1.7 fold) and MNR (1.8 fold) when compared to CTR fetuses (p=0.01). Beta cell area was increased by 1.5 fold and 1.7 fold in MNR and MO exposed fetuses but did not reach statistical significance (p=0.1, p=0.09 respectively). Delta cell percent area was not altered. No differences in baseline fetal plasma insulin and glucagon were found between groups so far. Conclusions: Fetal pancreatic endocrine cell development is specifically altered depending on the type of environmental exposure in non-human primates; these alterations may have long lasting consequences depending on the cell line affected. Whether these changes persist and correlate with metabolic derangements remain to be determined.

Maternal Controlled Exercise Improves Glucose Disposal in Mouse Offspring. Lindsay G Carter, Brice Kinney, Kevin J Pearson. Graduate Center for Nutritional Science, University of Kentucky, Lexington, KY, USA. Subtle changes in the intrauterine environment can influence long-term health outcomes in offspring. Previous studies from our laboratory have shown that voluntary exercise prior to and during pregnancy and nursing can improve adult offspring glucose tolerance and insulin sensitivity. With the voluntary exercise intervention, animals ran an average of 7 hours/day at a speed of 10 meters/minute; we therefore set out to determine if controlled maternal exercise (shorter duration and slower running speed) could also enhance offspring glucose tolerance and insulin sensitivity. Female ICR mice were separated into sedentary or exercise cohorts. Exercise mice were placed in running wheels (8 meters/minute) on a rotating platform for 1 hour daily for 4 weeks prior to, and during pregnancy and nursing. Body composition was measured in the female mice prior to mating while body weight was monitored over the entire study. Pregnancy rates, litter size and survival, and offspring body weights were also measured. After weaning, a glucose tolerance test was performed in 3 month old male and female offspring. Controlled exercise resulted in significantly decreased fat mass and body weight in the female mice prior to mating (P < 0.05 for both). Body weight differences were maintained during pregnancy but were diminished during nursing. Exercise did not significantly affect pregnancy rates, litter size or survival, or offspring body weight. At 3 months of age, male offspring from exercised dams had significantly improved glucose disposal (P < 0.01) following an oral glucose challenge compared to offspring from sedentary dams. There were no differences in female offspring glucose disposal at this age.

The current findings suggest that maternal exercise can improve offspring glucose regulation. Glucose and insulin tolerance testing will be performed as the mice age in order to assess the life-long effects of controlled maternal exercise on offspring glucose metabolism. This will be especially important in the female offspring because the effects of controlled maternal exercise may only occur at later ages, similar to results seen in our voluntary exercise model. Exercise during pregnancy could be a short-term intervention that positively impacts offspring metabolic health and decreases susceptibility of future generations to several diseases. The 'Developmental Origin of Health and Disease (DOHaD)' paradigm proposes that environmental factors in pregnancy act in utero to program the risk for adverse health outcomes. Despite its short duration, gestational diabetes mellitus (GDM) has long term consequences for the offspring and confers an increased risk for endothelial dysfunction. As a result of maternal GDM the feto-placental compartment is affected by the diabetic environment. The placental endothelium is continuous with the fetal endothelium and represents a model to study fetal endothelial function. We hypothesized that primary human arterial endothelial cells isolated from term placentas after healthy pregnancies (normal AEC) and pregnancies complicated with GDM (diabetic AEC) would differ in their intrinsic biological program. We focused on two key endothelial processes i.e., proliferation and angiogenesis.

Normal and diabetic AEC were cultured at 24, 48 and 96h. Viable and dead cells were counted to determine proliferation. In vitro angiogenesis (2-D network formation) was studied in media containing 2% normal or diabetic cord blood serum (CBS). In addition, the global DNA methylation profile was determined by 450k methylation arrays. Diabetic AEC had reduced proliferation (ANOVA p<0.003) with 36±10% fewer viable and 33±8% fewer dead cells after 96h culture as compared to normal AEC. In the presence of normal CBS total tube length was increased by 45±10%, the number of branching points by 311±28% and the number of meshes by 163±50% in diabetic vs. normal AEC (for all parameters ANOVA p<0.001). Diabetic CBS did not influence network formation potential neither in normal nor in diabetic AEC. Thus the difference in proliferation and tube formation is the result of an intrinsic program of the cells. In fact principal component analysis revealed differences in the global methylation pattern between normal and diabetic AEC. The altered proliferation and network formation potential of diabetic placental AEC even when cultured under identical conditions as normal AEC argues for changes in the cells, which are independent of the acute environmental conditions. These changes appear to reflect an intrinsic program to which epigenetic modifications contribute. Thus GDM-associated programming of endothelial cells of the offspring may begin in utero. To test our hypothesis, pregnant mice were randomly placed in one of three treatment groups: ad libitum feed + saline injection (control), 50% food restriction + saline (restricted), or 50% food restriction + 1 mg/kg•d leptin injection (restricted + leptin). Mice were treated from d1.5 to d11.5 after conception then returned to normal diet sans injections. Previously, we found that female offspring of restricted + leptin mothers were more obese following a high fat diet than offspring from other treatment groups. Male offspring from restricted mothers had the same weight, but less body fat than those of control or restricted + leptin mothers. Analysis of offspring liver gene expression revealed alterations in IGF family members and prompted investigation of fetal livers to determine how early these alterations are apparent. For the current study, mothers from the three treatment groups (n=5) were sacrificed at pregnancy d18.5. In male fetuses, Hsd11β1 was down regulated in livers from restricted + leptin mothers (p= 0.0398). Ghr was up regulated in livers from restricted compared to restricted + leptin mothers (p= 0.0417). In females, fatty acid transporter CD36 was up regulated in fetal livers from restricted (p= 0.0259) and restricted + leptin mothers (p= 0.0099), perhaps consistent with increased obesity in adult restricted + leptin offspring. However, there were no differences among treatment groups in glucose metabolism or fatty acid synthesis genes investigated. These changes in gene expression provide insight into mechanisms by which maternal diet influences offspring health in a sexually dimorphic manner when mothers are under-or over-nourished during pregnancy. The infantile rapid growth after undernourishment in utero was reported to be a risk factor of adult obesity. Recently, it was highlighted that the chronic inflammation-associated adipose tissue remodeling, with a relative increase of small size adipocytes among enlarged adipocytes, in the white adipose tissues (WAT) plays as a "metabolically morbidly obese". In this study, we hypothesized that rapid infantile growth after undernourishment in utero accelerates adipose tissue remodeling and constitutes a risk of metabolic syndrome. To prove the hypothesis, we developed a mouse animal model of undernourishment in utero and compared between the growth at weaning and parameters of adipose tissue remodeling at 17wks. Methods; Maternal caloric restriction (30% reduction) was apply to C57/BL6 pregnant mice. At 3 wks, body weight distribution of the offspring was assessed by Z-score [(body weight) -(mean body weight)] / standard deviation (SD) of body weight ] at weaning. After a HFD (60% fat) from 9 to 17 wks, the body weight and weight of subcutaneous WAT, serum levels of glucose and total cholesterol, rate of small adipocyte (less than 30 um in diameter), immunohistochemical detection rate of macrophage specific F4/80 in WAT were assessed. The gene expression of inflammatory M1 macrophage-specific CD11c and anti-inflammatory M2 macrophage-specific CD163 in WAT were measured by quantitative RT-PCR. Result; At 17 wks, all of the body weight (r=0.85), weight of subcutaneous WAT (r=0.07), serum glucose levels (r=0.67), total cholesterol levels (r=0.46), the rate of small adipocyte (r=0.49), immunohistochemical detection rate of macrophage specific F4/80 (r=0.54), the M1/M2 macrophage ratio of CD11c/ CD163 gene expression were positively correlated with Z-score at weaning (3 wks) in undernourished offspring (all P<0.001), but not in normally nourished offspring. Conclusion; Undernourishment in utero and rapid infantile growth may play a role of "Two Hits Theory" in the development of metabolic syndrome by transforming a "metabolically healthy obese" into a "metabolically morbidly obese" with adipose tissue remodeling. The intrauterine environment of the obese woman programs her offspring for cardiovascular and metabolic disease in later life. Animal models have shown that maternal obesity promotes cardiovascular abnormalities in offspring including left ventricular hypertrophy and cardiac fibrosis. Consistently, we found a significant 11-fold accumulation (p=0.003) of interstitial and perivascular fibrosis in the fetal hearts collected from baboon model of maternal obesity developed in our Center. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through translational repression and mRNA degradation. We have previously performed miRNA microarray analysis using fetal hearts from the offspring of baboons fed a HFD or regular diet (RD) in pregnancy. Among the miRNAs identified, we found significant changes in miR-18a, which targets the extracellular matrix (ECM) genes, and whose downregulation has been previously linked to cardiac fibrosis. We found 1.5-fold reduction (p=0.001) in miR-18a expression in the hearts of baboon fetuses born to HFD fed mothers compared to those fed a RD. As inflammation, oxidative stress and high levels of free fatty acids (FFA) are the hallmarks of the obese intrauterine environment, we hypothesized that downregulation of miR-18a in the fetal hearts is a direct effect of these factors.

To address this hypothesis we studied the effect of exposure of human primary embryonic cardiomyocyte cultures to inflammatory (TNFα, 2-20 nmol/l) and oxidative (H2O2, 10-50 µM) stresses and to high levels of FFA (palmitate, 0.1-0.4 µM) for 24 hours on expression of miR-18a. While inflammatory and oxidative stresses had no significant effect, exposure to 0.1µM and higher concentrations of palmitate was sufficient to cause a 1.5-fold reduction in the expression of miR-18a (p<0.05). We also studied the effect of a HFD for 8 weeks in adult C57/B6 mice (45% of calories from fat) vs. RD (13% calories from fat). This resulted in a 5-fold downregulation (n=6 each, p=0.02) in cardiac miR-18a further suggesting a potential link between dietary fat and cardiac miR-18a expression. In summary, using three approaches we show that the high levels of FFA such as exist in the intrauterine environment of the obese woman may cause a reduction in fetal cardiac miR-18a expression potentially promoting cardiovascular abnormalities in the offspring. Introduction: Intrauterine growth restriction, resulting in low birth weight (LBW) or pathological cardiac hypertrophy can induce a fetal phenotype of energy metabolism in the newborn, which is characterised by increased reliance on glucose rather than fatty acids, for energy production. We have investigated the effect of LBW on the development of left ventricular hypertrophy in lambs at 21 d after birth. Methods: Carunclectomy was performed 10 weeks prior to mating to induce LBW. Hearts were collected from average birth weight (ABW) and LBW lambs at 21d of age. We used qRT-PCR to quantify cardiac mRNA expression of molecules involved in glucose and fatty acid metabolism normalised to the geometric mean of 3 stable housekeeper genes. We used Western blotting to quantify cardiac protein expression of molecules involved in glucose and fatty acid metabolism. A section of the left ventricle was fixed for Periodic Acid Schiff staining, to determine tissue glycogen content. Results: There was increased abundance of insulin signalling pathway proteins (phospho-insulin receptor, insulin receptor and phospho-Akt) and the glucose transporter (GLUT)-1, but no change in GLUT-4 or glycogen content in the heart of LBW compared to ABW lambs. There was, however, increased abundance of cardiac pyruvate dehydrogenase kinase 4 (PDK-4) in LBW compared to ABW lambs. There were no significant changes in the mRNA expression of components of the peroxisome proliferator activated receptor regulatory complex or proteins involved in fatty acid metabolism. Conclusion: We concluded that LBW induced left ventricular hypertrophy was associated with increased GLUT-1 and PDK-4, suggesting increased glucose uptake, but decreased efficacy for the conversion of glucose to ATP. A reduced capacity for energy conversion could have significant implications for vulnerability to cardiovascular disease in adults who are born LBW. Previous studies in sheep have demonstrated that in vitro embryo culture and transfer result in an increased relative heart weight in singletons, but not in twins in late gestation. This increase in heart weight may be a result of cardiac hypertrophy or an increase in afterload. This study aims to determine (a) if the increase in relative heart weight in the offspring of in vitro embryo culture and transfer is due to an increase in blood pressure in late gestation and (b) if the increase in relative heart weight persists into postnatal life and whether it is due to an increase in blood pressure. Embryos were collected 24h after artificial insemination of superovulated donor ewes, and either transferred to an intermediate ewe (ET) or cultured in vitro in the absence (IVC) or the presence of human serum (IVCHS) for 6 days before transfer to recipient ewes. Naturally mated (NM) ewes formed a control group. An additional cohort was lambed normally. Baseline recordings were carried out at 120-135 day gestation in fetal cohort, and 19-20 weeks after birth in postnatal cohort. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were calculated as maximum and minimum pressure respectively. Heart Rate (HR) was derived from the blood pressure signal. Mean arterial pressure (MAP) and rate pressure product (RPP) were calculated using the formulae: DBP+ (0.4*(SBP-DBP)) and SBP*HR respectively. There was no significant change in SBP, DBP, MAP, HR and RPP in ET, IVC and IVCHS groups when compared to NM in fetal life. There was an increase in heart weight in the IVC group only in males (P< 0.05) in postnatal life but no change in relative heart weight. There was no significant difference in SBP, DBP, MAP, HR and RPP in ET, IVC and IVCHS when compared to NM in postnatal life. This study demonstrates that the increase in relative heart weight in embryo culture and transfer groups is not caused by an increase in blood pressure in fetal life. The findings also highlight the fact that in vitro embryo culture and transfer can potentially have long term effects on cardiac development that is not associated with hypertension. Maternal obesity and high fat diet consumption during pregnancy influence a number of disease states in the next generation. In rodent models, offspring born to high fat diet fed dams are at heightened risk for obesity and impaired glucose tolerance. Experimental high fat diets are typically either lard or butter based and contain 30 to 60% fat. Therefore the purpose of this study was to determine which experimental maternal diet composition would induce the most severe obesity and glucose intolerance outcomes in offspring. Primiparous ICR mice were fed either lard (10, 45, 60% fat calories) or butter (10, 32, 60% fat calories) based purified diets for 4 weeks prior to mating, throughout pregnancy, and for the first 14 days of lactation. Control diets were also purified rather than chow based. Prior to breeding, body weight, body composition, and glucose tolerance were measured in the female mice after 3 weeks on the respective diets. Female body weight was also monitored throughout mating, pregnancy, and nursing. Offspring body weight was measured regularly prior to and after weaning. Body composition and glucose tolerance were measured in 12 week old offspring.

Prior to breeding, both groups of mice fed the 60% fat diets gained significantly more weight than the controls, while all of the high fat diet fed mice had significantly increased fat mass. Further, the 60% lard mice had elevated fasting glucose levels but normal glucose tolerance while the 60% butter based mice had normal fasting glucose levels but impaired glucose tolerance. Male and female offspring body weight, body composition, and glucose tolerance were not significantly affected by any of the maternal high fat diets at 12 weeks of age. However, other studies exploring maternal effects on offspring metabolic outcomes have not revealed differences until later in life so we will continue to monitor the offspring as they age.

In conclusion, experienced female ICR mice fed high fat diets exhibit altered body composition, but offspring have normal body weight, body composition and glucose tolerance at 12 weeks of age. Future studies will compare primiparous and nulliparous female mice fed control chow and purified diets to determine whether these parameters could diminish the observed effects of maternal high fat diet consumption. Introduction: Maternal obesity in humans is associated with fetal overgrowth, however the underlying mechanisms are not well established. The purpose of this study was to develop a mouse model of diet-induced maternal obesity with metabolic and fetal growth characteristics resembling the clinical condition. We hypothesized that maternal obesity is associated with activation of placental insulin/IGF-I/mTOR and leptin signaling pathways, which are positive regulators of amino acid transporters. Methods: C57BL/6J female mice were fed a control (C, n=29) or a high fat/ high sugar (HF/HS, n=30) pelleted diet supplemented by ad libitum access to sucrose (20%) solution. When body weight had increased 25% in the HF/ HS group, body composition (n=5 per group) was determined using DEXA and animals were mated. At E18.5, a glucose tolerance test (GTT, 2g/kg) was performed in a subgroup of animals (n=4-5 per group). In another subgroup, maternal serum, fetuses and placentas were collected (n=20 per group).

The activity of placental insulin, mTOR, leptin, and inflammatory signaling pathways was assessed by determining the phosphorylation of key signaling molecules using Western blot. Results: Maternal fat mass was increased by 126% (p=0.0001) in HF/HS fed animals as compared to controls, with no change in lean body mass. Daily caloric intake during pregnancy was 54% higher (p=0.0001) in the HF/HS group. Obese dams were glucose intolerant as determined by an increase in the glucose AUC (p=0.0007) following GTT. Maternal circulating leptin, cholesterol, and non-esterified fatty acids were increased (p<0.05) in the HF/HS group, whereas adiponectin was decreased (p=0.054) as compared to the control group. Maternal obesity increased fetal weight (+18%, p=0.0005) without altering placental weight or litter size. Phosphorylation of placental ERK1/2 (T-202/Y-204), S6 ribosomal protein (S-235/236), 4E-BP1 (T-37/46) and STAT-3 (Y-705) was increased in obese dams. In contrast, expression of placental caspase-1, IκBα, IL-1β and phosphorylated-p46 SAPK/JNK was unaltered. Conclusion: This novel mouse model of maternal obesity shows clear similarities to the human condition with respect to maternal metabolic changes and fetal growth. Activation of placental insulin/IGF-I/mTOR and leptin signaling pathways in obese mice may stimulate placental amino acid transport and contribute to increased fetal growth. Results: Systolic BP was increased by 22±8mmHg (p≤0.02) and diastolic BP by 19±7mmHg (p≤0.02) in offspring of DM treated animals. Renal nonreceptor mediated vasoconstriction (v/c) to KCl and receptor-mediated v/c to endothelin-1 was increased in DM exposed offspring ( Fig.1, p≤0 .05). Renal receptor mediated v/c to norepinephrine, endothelial (ACh) and non-endothelial (PGE 2 , NO) vasodilation did not differ in DM and control offspring. Conclusions: One-third the clinical DM dose programs arterial hypertension in adult female sheep offspring. Arterial hypertension is probably mediated by enhanced renal vascular tone which may affect renal cortical blood flow and activity of the renin-angiotensin system. Objective: Betamethasone is administered to accelerate lung development and improve survival of premature infants but may be associated with hypertension later in life. Prenatal exposure to clinically relevant doses of Beta reduces nephron number in animals. Several mechanisms have been proposed to explain the development of hypertension including changing in the handling of Na+ by the kidney. In proximal tubules, the Na/H exchanger (NHE3) and the Na/K-ATPase pump regulate sodium reabsorption. The objective of this study was to examine the consequences of antenatal Beta exposure on postnatal expression the Na/H exchanger (NHE3) and the Na/K-ATPase mRNA in kidney. We also wished to determine if antenatal glucocorticoid exposure combined with a second insult (unilateral nephrectomy) would alter NHE3 and the Na/K-ATPase genes expression in adult sheep kidney. Materials and Methods: Male and female sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1years) and underwent unilateral nephrectomy followed by removal of the remaining kidney 3 weeks later at necropsy. Renal cortex samples were collected for analysis of NHE3) and the Na/K-ATPase mRNA expression by quantitative real-time RT-PCR. Results: Prenatal steroid exposure increased NHE3 mRNA expression in male sheep compared with vehicle treatment and following nephrectomy (F=7.32; P<0.01). There was no significant difference in NHE3 mRNA expression in female sheep exposed to Beta. There was no difference in Na/K-ATPase mRNA expression in Beta exposed male sheep; however, following nephrectomy, Na/K-ATPase mRNA expression was increased in male sheep(F=7.26, P<0.01); Incontrast, both Beta (F=9.83,P<0.006) and unilateral nephrectomy (F=18.98,P<0.005) increased Na K ATPase mRNA expression in female sheep. Conclusions: The data suggest that hypertension induced by antenatal Beta may be linked to increased NHE3 expression in male but not in female sheep. However the expression of Na/K-ATPase appears more responsive to effect of prenatal Beta in females. Thus, there are gender related differences in the effects of antenatal Beta exposure on genes related to Na transport in the kidney. Supported by NIH grants 17644 and 47584. the first, directly exposed generation but can have transgenerational effects. We determined whether cardio-renal and metabolic deficits and dysfunction are transmitted to the next generation from mothers born small, and the role of the maternal environment vs embryo-specific effects in mediating this risk. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) in WKY rats. Restricted and Control female offspring (F1) were mated with normal males. Embryo transfer was performed at embryonic day (E) 1 with F2 embryo gestated in either a Control or Restricted mother. Non-transfer offspring were also generated. Physiological measures were performed at 6mo (blood pressure, intraperitoneal glucose tolerance test, insulin challenge). Nephron number was determined at E20 and postnatal day (PN) 35. Pancreatic β-cell mass was determined at E20 and 6mo. Body weights were not different at E20 and birth (non-transfer groups). Growth rate was slowed at PN14-2mo and accelerated at 2-3mo in non-transfer F2 Restricted offspring compared with Controls (p<0.05). Nephron number was reduced (-15-22 %; p<0.05) in non-transfer F2 Restricted offspring at E20 but was restored by PN35. At 6mo, non-transfer F2 Restricted males, but not females, had increased blood pressure compared with F2 Controls (+18 mmHg; p<0.05). No fetal pancreatic β-cell deficits were present. Non-transfer F2 Restricted male and females had reduced first phase insulin secretion but normal glucose tolerance associated with reduced β-cell mass in males but increased β-cell mass in females (p<0.05). F2 offspring from growth restricted mothers were born of normal weights but experienced slowed, followed by accelerated, growth. Metabolic and cardio-renal deficits and dysfunction were transmitted to the next generation via the maternal line. The complexities of embryo-transfer procedures resulted in a lack of ability to delineate a clear pathway (maternal vs embryo-specific) for transgenerational effects.

Intrauterine Background: Maternal consumption of a high fat, high cholesterol diet (mHFCD) during pregnancy predisposes the newborn towards non-alcoholic fatty liver disease (NAFLD) at birth. Intrauterine growth restriction (IUGR) similarly predisposes the infant towards NAFLD in adolescence. Little is known about how mHFCD and IUGR combined affect NAFLD. High hepatic cholesterol characterizes NAFLD. Hepatic cholesterol depends on the expression of sterol-responsive element binding protein 2 (SREBP2) and its target HMGCoA reductase (HMGCR). We subsequently hypothesized that IUGR and mHFCD would each increase hepatic cholesterol and decrease SREBP2 and HMGCR mRNA in rat pups at birth. Further, we hypothesize that the combination of IUGR and mHFCD would further increase hepatic cholesterol and decrease SREBP2 and HMGCR mRNA compared to either IUGR or mHFCD alone. Methods: Virgin female rats were fed either a regular diet (RD) or a HFCD for 5 weeks prior to mating. IUGR was induced by bilateral uterine artery ligation at day 19 of a 21 day gestation. Pup liver lipids and mRNA were isolated at birth. Hepatic cholesterol was quantified with a colorimetric kit. Real-time RT PCR was used to determine mRNA levels of SREBP2 and HMGCR. Results: Compared to control offspring of RD fed dams (Con-RD), control offspring of maternal HFCD fed dams (Con-mHFCD) and IUGR offspring of RD fed dams (IUGR-RD) did not have increased liver cholesterol levels or decreased SREBP2 or HMGCR mRNA levels. Compared to Con-mHFCD, female IUGR pups from mHFCD dams (IUGR-mHFCD) increased hepatic cholesterol at birth (131+/-16%, p<0.05). Compared to Con-mHFCD offspring, IUGR-mHFCD decreased SREBP2 mRNA (77 +/-8%, p<0.05 in female, and 67 +/-7%, p<0.01 in male) and HMGCR mRNA (66 +/-7%, p<0.05 in female). Conclusion/Speculation: We conclude that IUGR-mHFCD increased hepatic cholesterol and decreased SREBP2 and HMGCR mRNA levels more than IUGR or mHFCD consumption alone. Maternal HFCD and IUGR disrupt the nutritional environment of the developing fetal pup. Combined in utero insults of mHFCD and IUGR may alter SREBP2 and HMGCR expression thereby predisposing to increased hepatic cholesterol levels in IUGR offspring. . Sham rats had sutures only. Some ENDO rats were also exposed to either an uncontrollable or controllable (platform) swim stress protocol for 10 days. Blood samples were collected on days 1, 5, and 10 during stress for corticosterone levels, and fecal pellet numbers (FPC) were counted as an indirect measure of anxiety. All rat weights were recorded from surgery to sacrifice. At sacrifice (day 60), all rats were examined for presence of endometriotic cysts, and colon and uterine tissue were analyzed for macroscopic, microscopic and myeloperoxidase (MPO) levels.

Only ENDO rats developed cysts, which were increased in number and size by stress. Stressed animals gained less weight than those without stress (p<0.05). Rats receiving uncontrollable stress had higher anxiety (FPC and thigmotaxis) than those exposed to controllable stress or no stress (p<0.05), and higher corticosterone levels. Uncontrollable stress rats had higher colonic damage (p<0.05) and uterine MPO compared to no stress, while controllable stress showed no difference. CONCLUSION: The level of stress controllability appears to modulate behavior and the pathophysiology of endometriosis, and may offer possibilities for therapeutic interventions. GRANT SUPPORT: 1R15AT006373 (CBA); GM082406 (SH). Objectives: Endometriosis is in part, regulated by progesterone (P4) via its interaction with the principal P4 receptor (PR) isoforms (PR-A and PR-B) in ectopic endometrial cells. Effects of P4 are determined by the PR-A:PR-B ratio such that a PR-A-dominant state mediated pro-inflammatory actions whereas a PR-B-dominant state mediated ani-inflammatory actions. The objective of this study was to measure PR-A and PR-B levels and their cellular localization in eutopic endometrium from women with and without endometriosis, peritoneal endometriotic lesions and endometriomas. Methods: Tissue was collected from women (18-45y) undergoing laparoscopy for endometriosis (n=18) associated with pelvic pain, dysmenorrhea and infertility and controls (n=14) undergoing laparoscopic tubal ligation. No patients received hormonal treatment for at least 3 months before the surgery and all women had regular menstrual cycles. At laparoscopy samples of eutopic endometrium, peritoneal endometriosis, disease-free peritoneum and endometrioma cyst wall were obtained. Fixed and frozen tissues were subjected, respectively, to immunohistochemical and immunoblot analyses for PR-A and PR-B. Results: PR-A and PR-B were detected exclusively in the glandular epithelial cell in eutopic endometrium and in peritoneal endometriosis and endometrioma. In peritoneal endometriosis PR-A was the predominant isoform detected whereas both receptors were detected in eutopic endometrium and endometrioma. Immunoblot analyses showed that levels of PR-A and PR-B were markedly (∼10-fold) elevated and in a PR-A-dominant state in eutopic endometrium from women with endometriosis compared with women without disease regardless of menstrual phase. Similarly a PR-A-dominant state was detected peritoneal endometriosis and endometrioma. Conclusion: The PR-A dominant status of eutopic endometrium in women with endometriosis could in part explain the initiation of the disease. We propose that efflux of a PR-A-dominant endometrial tissue to the peritoneal cavity may be responsible for the initiation of the inflammatory cascade and progression of the disease. Our data suggest that susceptibility to endometriosis may be intrinsic to the eutopic endometrium based on the PR-A:PR-B status.

B Cell Lymphoma Protein 6 (BCL-6), Suppressor of Cytokine Signaling 2 (SOCS2) and Their Potential Role in Endometriosis. Emily Evans-Hoeker, 1 Lingwen Yuan, 1 Bruce A Lessey, 2 Steven L Young. 1 1 Obestetrics and Gynecology, University of North Carolina, Chapel Hill, NC, USA; 2 Obstetrics and Gynecology, Greenville Hospital, Greenville, SC, USA. BACKGROUND: BCL-6 is a transcription factor involved in immunomodulation, cell cycle control and apoptosis inhibition. Knockout mice exhibit increased inflammation and cell cycle activity, both thought to be key in endometriosis (osis). We have previously shown that BCL-6 is increased in human endometrium at the time of implantation and decreased in osis. SOCS2, an immunomodulator, inhibits the JAK/STAT pathway but its role in the endometrium and relationship to BCL-6 has not previously been explored. PURPOSE: Investigate expression patterns and regulation of SOCS2 by BCL-6 in normal human endometrium and osis. MATERIALS AND METHODS: Endometrial biopsies were obtained from normal (NL, n=20) and infertile subjects with osis (OS, n=21) randomized to proliferative (PRO) or midsecretory (MS) phase, by urine LH timing. Normal subjects (n=10) were sampled on the 10th day of exposure to experimentally-defined levels of progesterone (P) during a modeled cycle. In vitro regulation was studied by treatment of Ishikawa cells with estradiol plus medroxyprogesterone acetate (EP). Relative mRNA expression was assessed with real-time RT-PCR and compared using Kruskal-Wallis and Wilcoxon Rank Sum tests. Proteins were localized by immunohistochemistry (IHC).

In vivo, NL BCL-6 expression was increased in MS vs. PRO and by P dose (p<0.001). IHC revealed decreased BCL-6 and increased SOCS2 in OS compared to NL. In vitro, EP treatment resulted in increased BCL-6 and decreased SOCS2 (Figure) . BCL-6 knockdown resulted in increased SOCS2, while over expression resulted in SOCS2 suppression ( Figure) .

Transcription factor BCL-6 is progesterone and cycle regulated in human endometrial cells in vivo and in vitro. BCL-6 can suppress SOCS2, providing a mechanism for the observed inverse expression pattern seen in vivo and in vitro. These findings suggest a role for altered BCL-6 expression in the endometrial dysfunction seen in endometriosis. Supported by NIH/NICHD R01 HD067721 Purpose: Epithelial-mesenchymal transition (EMT) is a process that is important in both normal organ development and cancer progression. EMT is characterized by the loss of E-cadherin and the loss of E-cadherin correlates closely with poor prognosis in malignancy. As seen in the progression of neoplastic lesions, implanted endometrial cells possess a new capacity for migration that is characteristic of mesenchymal cells. We hypothesized that an imbalance of epithelial and mesenchymal characteristics may induce the pathogenesis of endometriosis. The aim of this study is to investigate the epithelial and mesenchymal characteristics of human endometriotic lesion. Materials-Methods: We analyzed the expression of several epithelial and mesenchymal markers, including ZEB1, vimentin, Snail, and E-cadherin by immunohistochemistry. Fifteen patients with endometriosis and 9 patients without endometriosis undergoing surgery for benign indications were included in this study. The expression of each marker was evaluated the intensity and extent of staining especially in the glandular epithelial cells of both endometriotic lesions and normal endometrium. Results and Conclusions: Eutopic endometrium exhibited mesenchymal markers on proliferative phase and epithelial expression pattern on secretory phase. The expression levels of E-cadherin were lower in several endometriotic lesions than in eutopic endometrium. This study identified ZEB1 expression in the glandular epithelial cells of endometriotic lesions. ZEB1 expression was more frequently seen in invasive endometriosis, occurring concurrently with high rASRM scores or serum CA125 levels. In conclusion, glandular epithelial cells within endometriotic lesions have increased mesenchymal features over that seen in eutopic endometrium. We demonstrate for the first time that ZEB1 was expressed by epithelial cells of benign tissue. These results suggest that development of enhanced mesenchymal characteristics of endometriotic epithelial cells may play an important role in the pathogenesis of endometriosis. Endometriosis, the presence of endometrial tissue outside the uterine cavity, is a common benign gynaecological condition with unknown pathogenesis. Dislocation of the menstrual endometrium to the pelvic cavity via a trans-tubal route, retrograde menstruation, may play a role in its pathogenesis. In vivo studies have shown more frequent contractions in women with endometriosis. This increased myometrial contractility may contribute to the retrograde menstruation. Intracellular calcium entry via L-type Calcium Channels (LTCCs) is crucial to the excitation-contraction coupling process which ultimately leads to contraction. We therefore investigated in vitro myometrial contractility with respect to the influence of ovarian hormone and the presence of endometriosis and examined the role of LTCC to determine if differences in contractility were inherent and explain differences in contractility. Methods Spontaneous contraction of myometrial strips from 28 non-pregnant women undergoing hysterectomy were studied and the contractility patterns compared in women with (n=7) /without (n=16) endometriosis and in 5 post menopausal (PM) women. Biopsies from the same 3 groups (n=15 per group) were studied for the myometrial LTCC by immunohistochemistry. Results The amplitude of myometrial contractions from PM women was significantly decreased (10±3%, p=0.02) compared to healthy pre-menopausal women (100%). The frequency of contractions in women with endometriosis was significantly faster (3-times greater, p=0.04) compared to pre-menopausal women and their amplitude reduced (62±18%, p=0.20) Immunostaining intensity scores for myometrial LTCC was lowest at the mid-secretory phase of the cycle in pre-menopausal women (p=0.04) and the highest scores were seen in the pre-menopausal, menstrual and PM myometrium (p=0.01) with no significant difference observed between the myometria of women with and without endometriosis. Conclusions This data demonstrates (i) diminished amplitude of contractions in PM uterus, (ii) endometriosis myometrium has an increased frequency of contractions compared to matched controls, consistent with in vivo data, (iii) Significantly low LTCC scores in the mid-secretory phase, suggesting a progestogenic effect, (iv) Changes in contraction amplitude cannot be explained by a parallel reduction in Ca entry via LTCC.

Stress Enhances Uterine and Colonic Contractility in Endometriosis. Siomara Hernandez, Myrella L Cruz, Antonio E Ramirez, Anixa Hernandez, Caroline B Appleyard. Physiology and Pharmacology, Ponce School of Medicine and Health Science, Ponce, Puerto Rico. PURPOSE: Endometriosis is defined as the presence of endometrium-like tissue outside the uterus, including the gastrointestinal (GI) tract and is characterized by peritoneal inflammation, adhesions, infertility, chronic pelvic pain and GI symptoms. We have previously found increased uterine and colonic contractility in an animal model of endometriosis, however the impact of stress is unclear. DESIGN METHODS: Endometriosis was induced in female Sprague-Dawley rats by suturing uterine horn tissue next to the intestinal mesentery (endo-no stress). One group of rats was also exposed to an uncontrollable stress protocol for 10 days (endo-stress). Fecal Pellet Counts (FPC) and corticosterone levels were measured. All animals were sacrificed after 60 days and examined for cysts, and colonic damage. Uterine and colonic strips were mounted in organ baths to study in vitro isometric contractile responses of longitudinal and circular smooth muscle to carbachol (10-8-10-3M). The tissue was weighed and desiccated, calculating tissue cross-sectional area and force exerted (mN/ cm2). RESULTS: All rats developed cysts, which were more numerous and of greater size in endo-stress. These animals had greater FPC (p<0.01) and corticosterone levels (p<0.05) than endo-no stress indicating increased anxiety. Endo-stress animals also gained less weight (p<0.01), and had higher colonic damage (p<0.05) and uterine myeloperoxidase activity. Endo-stress had increased contractility in longitudinal and circular muscle of both the colon (p<0.05) and the uterus (p<0.05) compared to endo-no stress. CONCLUSION: Our data suggest that stress can influence the colonic and uterine contractility in an animal model of endometriosis, perhaps contributing to pelvic pain and GI effects. GRANT SUPPORT: GM082406 (SH); 1R15AT006373 (CBA). Introduction Endometriosis is a common condition in which the immune response is dysregulated within the eutopic endometrium and at ectopic sites [1] . The baboon (Papio anubis) is generally considered the best model of endometriosis pathogenesis, however, little is known regarding the immune response in this model [2] . Objective To characterise immune cell populations in eutopic endometrium and peritoneal endometriotic lesions from a baboon model of induced-endometriosis. Materials and methods Uterine and peritoneal samples were obtained from normally cycling female baboons with (n=5) and without (n=5) induced endometriosis, on days 9-12 post ovulation. Immunohistochemical staining was performed with antibodies for all T cells (CD3), effector T cells (CD4), cytotoxic T cells (CD8), B cells (CD20) and natural killer cells (NK; CD56). Results CD3+ and CD20+ cell aggregates were present in the functional layer of the endometrium in some specimens, while in the basal layer, these cells often surrounded large vessels. NK numbers in the uterus varied greatly between specimens but were most numerous in the functional layer of the endometrium. Effector T cells and cytotoxic T cells were rare in the endometrium. CD3+ T cells were abundant and effector T cells present in endometriotic stroma of most lesions, with some infiltrating the glandular epithelium. Rare cytotoxic T, B and NK cells were observed in and around some lesions. Conclusions In the baboon model of induced-endometriosis, a range of immune cells are present in varying densities in the endometrium and myometrium, and in peritoneal endometriotic lesions. In women, the failure of the immune system to clear shed endometrial tissue and to protect the peritoneum from its adhesion and invasion is hypothesised to be involved in endometriosis establishment [1] . Detailed studies of the early immune response to inoculated menstrual tissue in the baboon endometriosis model will be useful to determine the role of the immune system in endometriosis pathogenesis. 

The Relationship between Serum Eicosapentaenoic Acid Levels Objective: Previous work shows omega-3 fatty acids may be involved in the pathophysiology of endometriosis; however, an association between physiological omega-3 fatty acid levels and endometriosis in women has not been established. Our objective was to determine if omega-3 fatty acid levels are associated with endometriosis. Methods: We designed a cross-sectional study of two hundred and seven women undergoing in vitro fertilization (IVF) at one institution whose serum specimens and clinical data were available from an ongoing prospective tissue bank. Fasting serum free fatty acid (FFA) levels were measured with mass spectrometry. FFAs measured were palmitic acid, linoleic acid, oleic acid, stearic acid, alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), arachidonic acid (AA), and docosahexaenoic acid (DHA). We investigated the relationship between serum FFA levels and the diagnosis of endometriosis and the chance of pregnancy in women with endometriosis. Data were analyzed using student's t-test and Fisher's exact test. Age was adjusted for using logistic regression. Results: Women with the lowest quartile of serum EPA levels were 80% more likely to have a diagnosis of endometriosis compared to women with the highest serum EPA levels (95% CI 0.038-0.91). This relationship persisted after adjusting for age (OR=0.4, 95% CI 0.04-0.93). Interestingly, among the twenty-three women in the study with endometriosis, EPA was negatively associated with the chance of pregnancy after IVF (RR 0.3, 95% CI 0.11-0.79). Conclusions: EPA is an essential omega-3 FFA obtained through the diet and it is negatively associated with the diagnosis of endometriosis. It is worth exploring if increased omega-3 fatty acid intake is beneficial in women with symptomatic endometriosis. For subfertile women with endometriosis further work is needed to determine if omega-3 fatty acid intake affects pregnancy.

Human Endometrium with Deficiencies in Endometriosis. Sahar Houshdaran, Zara Zelenko, Juan C Irwin, Linda C Giudice. Obstetrics, Gynecology & Reproductive Science, UCSF, San Francisco, CA, USA. Objective: Human endometrium undergoes major transcriptomic changes in response to cyclic changes in estradiol (E2) and progesterone (P4), resulting in proliferation, angiogenesis, differentiation, and apoptosis. Endometriosis, characterized by endometrial tissue outside the uterus, is an E2-dependent, P4-resistant disorder. We recently found that DNA methylation profiles of human eutopic endometrium change across the cycle with abnormalities in women with endometriosis. Herein, we investigated the relationship between DNA methylation and gene expression to elucidate epigenetic regulation of transcription in cycling endometrium normally and in disease. Methods: Eutopic endometrial tissues included 6 samples each in the proliferative, early secretory, and mid-secretory phases from women with severe endometriosis and without disease (total 36). DNA methylation and gene expression were assessed on the same samples by the 27K Infinium platform and U133 Affymetrix microarrays, respectively. Corresponding genes on both platforms were selected and their relationship investigated in the proliferative-to-secretory transition where highest transcriptome changes occur, using Spearman correlation using R. Pathway analysis was done using DAVID database. Results: In control samples, genes with differential methylation between the phases, showed a negative correlation between methylation and gene expression levels, suggesting DNA methylation is involved in their transcriptional regulation, and pathways included regulation of apoptosis, blood vessel development, surface receptor signal transduction, and regulation of proliferation. Neither the same genes nor the negative correlation was observed in disease and different pathways were found including cell adhesion, extracellular matrix interaction, and regulation of neurogenesis. Conclusion: Our data provide further evidence that epigenetic mechanisms are involved in gene expression regulation of the endometrial response to E2 and P4 and that epigenetic abnormalities may play a crucial role in eutopic endometrium and P4-resistance in endometriosis. Support: NIH SCCPIR U54HD 055764-06 (LCG)

Expression of FKBP52 in Human Adenomyosis. Chih-Feng Yen, 1, 2 Frederick Schatz, 3 Umit Kayisli, 3 Chyi-Long Lee, 1 Charles J Lockwood, 3 S Joseph Huang. 3 Adenomyosis, identified by abnormal growth of endometrium in the myometrium, is found to have reduced progesterone responsiveness. The FK506-binding protein, FKBP52, acts as a co-chaperone with heat shock protein 90 to interact with the ligand-binding domain of the PR and enhances progestin-initiated transcription. Objective: Determine the relative abundance of FKBP52 in eutopic (EUT) and ectopic (ECT) endometrial from patients with adenomyosis compared to control endometrium (CON) from patients with myoma obtained across the menstrual cycle. Methods: Biopsies of eutopic endometria and adenomyosis as well as CON across menstrual cycle were obtained. RT-PCR was performed using specific primer sets for FKBP52 and GAPDH. Two-micrometer formalin-fixed paraffinembedded sections were immunostained using primary monoclonal mouse anti-human antibody against FKBP52 followed by IgG rodamine-conjugated anti-mouse secondary antibody. Relative abundance of FKBP52 was determined by evaluating individual fluorescence intensity. Results: Expression of FKBP52 in secretory phase is consistently higher than that in proliferative phase in both CON and EUT. FKBP52 is mainly expressed in cytoplasm of glandular epithelial cells (GEC) and stromal cells (SC) across the menstrual cycle. In secretory phase, FKBP52 expression in EUT and ECT is consistently higher in GEC than that in SC. Its expression in EUT and ECT is greater than that in CON irrespective cell types. However, FKBP52 expression is higher in EUT compared to ECT, specifically in SC. Conclusions: This study reveals a differentially complicated expression pattern of FKBP52 in endometrium from patients with adenomyosis. Endometriotic lesion development in a mouse model of endometriosis appears to be linked with the activation status of resident macrophages (Bacci et al. 2009 ). Classically activated (inflammatory) macrophages suppressed, whereas alternatively activated (tissue-remodelling) macrophages promoted lesion development. Lesions harvested from the mouse model of endometriosis initially showed inflammatory activity but this was followed several days later by wound healing and tissue remodelling. We hypothesise that the activation status of macrophages is dynamically regulated during different phases of endometriotic lesion development. Methods: To address this hypothesis mice expressing GFP on the macrophage surface (MacGreen mice) were bred onto a SCID background to construct a MacGreen mouse model of endometriosis. Human eutopic endometrial tissue was subcutaneously injected into transgenic mice (n=44 in 5 experiments). The lesions were harvested 4 (n=10), 7 (n=12), 10 (n=10) and 14 (n=12) days after implantation. Immunohistochemical markers of macrophage activation including MHC Class II (a marker of classical activation), Arginase1 (Arg1) and scavenger receptor (CD204) (markers of alternative activation) were used to identify the macrophage phenotypes in xenografts. A morphometric analysis of 10 lesions harvested at day 10 was performed to assess glandular volume fraction and macrophage phenotypes. Results: The median weight and size of the resulting lesions did not change across the 4 time points although an increased glandular component was noted over time. At day 10, 35.7% of GFP-positive macrophages expressed MHC Class II antigens, 40.7% expressed Arg1 and 50.8% expressed CD204 in day 10 lesions. A similar analysis is ongoing in lesions from other time points. Heterogeneous expression of different macrophage markers within the GFPpositive macrophage population suggests that macrophages have a variety of biological functions in endometriosis-like lesions. Macrophages in lesions developed over 10 days showed a predominance of the alternatively activated markers (Arg1 & CD204) although some still retained MHC Class II marker consistent with classical activation. Our study will continue to explore macrophage phenotypes before and after this time point. Manipulation of macrophage phenotype in endometriotic lesions could alter tissue growth and survival and might be a useful tool to modulate endometriotic disease in women. Main Outcome Measure(s): DNMT1, DNMT3A, and DNMT3B expression in E-IUM and E-OSIS were assessed by RT-PCR and immunoblot analysis. DNMT3B recruitment to the SF-1 gene, essential for estrogen biosynthesis, and ESR1, which encodes estrogen receptor-α, was examined by chromatin immunoprecipitation (ChIP). Results: IVD treatment reduced DNMT3B mRNA (74%) and protein levels (81%) only in E-IUM. DNMT1 and DNMT3A were unchanged in both cell types. Significantly more DNMT3B bound to the SF-1 promoter in E-IUM compared with E-OSIS, and IVD treatment reduced binding in E-IUM to levels similar to those in E-OSIS. DNMT3B enrichment across three ESR1 promoters was reduced in E-IUM after IVD, although the more distal promoter showed increased DNMT3B enrichment in E-OSIS after IVD. Conclusions: The inability to downregulate DNMT3B expression in E-OSIS may contribute to an altered epigenetic fingerprint that misdirects gene expression in endometriosis and contributes to its estrogen-dependent phenotype.

Endometrial Stromal Stem Cells from Women with Endometriosis Abnormally Express COX-2, StAR, CYP19A1 and Apoptotic Markers. Graciela Krikun, 1 Balamuthu Kadalmani, 2 Karthikeyan Palanivel, 2 Hugh S Taylor. 1 1 Ob/Gyn & Rep. Sci., Yale University, SOM, New Haven, CT, USA; 2 Department of Animal Science, Bharathidasan University, Tamilnadu, Tiruchirappalli -620 024, India. Introduction: Endometriosis is an invasive gynecological disease characterized by diminished apoptosis, sustained ectopic survival of dysfunctional endometrial cells and implantation of endometriotic lesions outside of the uterus. The present study determined the expression pattern of apoptotic and steroidogenic genes in the progenitor stem cell population of endometrium. The expression of these genes was determined in a subpopulation of endometrial cells that displayed CD90 positivity (CD +ve 90) and colony forming capacity. Methods: We obtained endometrial samples from 12 women +/-endometriosis. Endometrial stromal cells (ESCs) were isolated and cultured in DMEM for 15 days. Purified ESCs were sorted by CD90 positivity using multi-color flow cytometry. Subsequently, single cell cloning was carried out by serial dilution in 96-micro well plates. Fifteen days later, cell colonies were identified (CFUs) and cultured in complete medium. mRNA expression of apoptotic genes, mitogen activated kinase 14 (MAPK14), nuclear factor kappa B (NFkB), steroidogenic acute regulatory protein (StAR), aromatase (CYP19A1) and cycloxygenease-2 (COX-2) were determined by quantitative RT-PCR. The protein levels of StAR, CYP19A1 and COX-2 were determined by western blotting. Results: A subset of stromal cells derived from women with endometriosis were isolated and identified as progenitor stem cells based on CD90 positivity and colony forming ability. The cells displayed increased levels of MAPK14, NFkB, COX-2, StAR and CYP19A1 both at the mRNA and protein level. Conclusions: CD90 +ve endometrial progenitor stem cells from women with endometriosis displayed aberrant expression of apoptotic regulators and steroidogenic enzymes. These aberrations likely contribute to sustained survival of stem cells in women with endometriosis. Endometriosis is a disease characterized by altered stem cells. Introduction. In endometriosis (E),regurgitating endometrial cells implant outside the uterus causing changes leading to abnormal recruitment of immune cells.The released cytokines drive the differentiation programs of CD4+T cells toward Th1,Th2,Th17 and Tregs.Th1 are characterized by T-bet and STAT1 and 6,and the production of IL-2,IFN-γ,and TNF-α.Th2 are characterized by GATA-3 and STAT 5 and 6,and the production of IL-4,IL-5,IL-13.Th17 are characterized by RORC and produce IL-17A,IL-17F and IL-22.Tregs are characterized by FoxP3,and produce IL-10 and TGF-β.In E,endometriotic cells provokes changes in the microenvironment leading to abnormal recruitment of immune cells.Aim of this work is to verify the presence and levels of T-bet+,GATA-3+,RORC+ or FoxP3+ cells and IFN-γ,IL-4,IL-17A or IL-10 producing cells by evaluating gene expression at mRNA production in ovarian endometrioma samples (OEs) from women with E and ovarian functional cysts (OFC) from women without E as controls (C). Materials and Methods. We collected OEs from 60 women with E at severe stages and 10 OFC as C.Total RNA was isolated from tissues for RT-PCR analysis of all markers and β-actin as endogenous control. Results. Our results showed that T-bet mRNA was present in 92% of OEs and slightly higher than C.60% of OE was IFN-γ mRNA+ and upregulated.All the OEs were GATA-3 mRNA+ and slightly higher than C. 81% of OEs was IL-4 mRNA+ and upregulated.RORC and IL-17A mRNA were present and slightly upregulated respect to C in 61% and 68% of OEs,respectively.Foxp3 mRNA was present in 52% of OEs with levels almost overlapping to C.Indeed, IL-10 mRNA was present and strongly overexpressed in 100% of OEs. Conclusions. We hypothesize that,in the positive OEs,T-bet,RORC, FN-γ,IL-17A mRNA presence may represent a hallmark of inflammation.Since only half of OEs comprised FoxP3+ cells,the upregulated presence of GATA-3 mRNA may account for IL-10 mRNA overexpression.This together with IL-4 mRNA overexpression could represent a sign of Th2 polarization.These apparently contradictory results could be due to coexistence of inflammatory and reparative phenomena in E,responsible of the disturbance of the immunological equilibrium in ectopic endometrium.

Suppressor of Cytokine Signaling (SOCS) Expression in Normal and Abnormal Endometrium. Bruce A Lessey, 1 Jung-Yoon Yoo, 2 Jae-Wook Jeong, 2 Sarah Gill, 1 Lingwen Yuan, 3 Steven L Young, 1 Asgerally T Fazleabas. 2 1 Obstetrics and Gynecology, University of South Carolina Greenville, Greenvile, SC, USA; 2 Obstetrics and Gynecology and Reprod Biology, Michigan State University, College of Human Medicine, Grand Rapids, MI, USA; 3 Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Introduction: Dysregulation of endometrial cytokine expression contributes to the pathogenesis and pathophysiology of endometriosis, an inflammatory disease affecting > 175 million women worldwide and may underlie endometriosis-associated infertility. Suppressor of cytokine signaling (SOCS) members, are a family of SH2-domain containing proteins that modulate inflammation by inhibiting cytokine-induced JAK-STAT signaling. This study is the first evaluation of SOCS expression in the endometrium of healthy women and those with endometriosis. Methods: Using IRB approved protocols, samples of normal and endometriotic endometrial tissues (n =40) from women were examined throughout the menstrual cycle by western blot (WB) and qRT-PCR for all 8 SOCS proteins (CISH, SOCS 1-7). Additional samples of endometrium from the baboon endometriosis model were studied over the time course of the disease (1,3,6,9 & 15 months) . Expression pattern and location for selected SOCS proteins was confirmed by immunohistochemistry (IHC). Results: SOCS2, SOC3 & SOCS6 are regulated during the menstrual cycle in both human and baboon endometrium. Higher expression of CISH, SOCS1, SOCS2, & SOCS3 was observed in women with endometriosis using IHC, WB, and qRT-PCR, with more expression seen in more advanced disease.

In the baboon model, endometrial SOCS1, SOCS2, SOCS3, SOCS4 and SOCS6 expression increased over time after endometriosis induction. Conclusions: SOCS family members are key modulators of inflammation and several are regulated during the menstrual cycle. Over-expression of CISH, SOCS2 and SOCS3 could inhibit STAT3 and STAT5, used in leukemia inhibitory factor (LIF) and prolactin signaling, accounting for the decidualization defects associated with endometriosis and implantation failure. NIH R01 HD067721 & U54 HD40093 Microarray studies have revealed a suite of microRNAs that are differentially expressed in endometriosis. However, before functional cell culture studies can be performed, it is imperative to localise these miRNAs to the cell type of origin. We hypothesised that members of the miR-99 and 125 families would localise to immune cells in the endometrial stroma, because these miRNAs are known to promote cell survival and suppress differentiation in the haematopoietic system. Locked Nucleic Acid -In Situ Hybridisation (LNA-ISH) was performed for miR-99a, miR-100, and miR-125b in 8 paired eutopic and ectopic tissues from women with endometriosis; 4 in the proliferative and 4 in the secretory phase. Using serial sections, immunohistochemistry was performed for markers of macrophages (CD68), endometrial stromal cells (CD10) and myofibroblasts (alpha smooth muscle actin). Additionally, LNA-ISH was used to identify miR-99a, -100, and -125b positive cells in human xenografts from the severe combined immunodeficient mouse model of endometriosis, harvested 4, 7, 10 and 14 days after tissue injection. In eutopic endometrium, miR-99a, miR-100 & miR-125b were localised to stromal cells & absent from epithelial cells, with no obvious cycle phase differences. In ectopic lesions, these miRNAs were predominantly confined to sub-epithelial stromal cells. A similar distribution was observed for all three miRNAs, but miR-125b was more abundant than miR-99a and miR-100. Immunohistochemical analysis revealed that miR-99a, miR-100, & miR-125b localisation was distinct from regions of alpha smooth muscle actin staining, but coincided with areas of CD10 and CD68 immunoreactivity. Divergence in the localisation and abundance of miR-125b was observed when day 4, 7, 10 and 14 ectopic endometrial xenografts were compared. These findings indicate that miR-99a, -100 and -125b are consistently present in the stroma of human eutopic and ectopic tissues in a cycle-independent manner. These miRNAs appear to co-localise with endometrial stromal and/ or immune cells, and may promote their survival and suppress differentiation. The activity of these microRNAs may change between different phases of lesion development. Modifying miRNA function could provide a therapeutic avenue to inhibit endometriotic lesion development.

Sustained Improvement in Endometriosis-Related Pelvic Pain with Leuprolide or Norethindrone Treatment. Ozgul Muneyyirci-Delale, 1,2 Cassandra Charles, 1 Ninet Sinaii, 3 Jenny Anopa, 1 Mudar Dalloul, 1 Pamela Stratton. 4 1 Obstetrics and Gynecology, SUNY Downstate Medical Center, Brooklyn, NY, USA; 2 Obstetrics and Gynecology, Kings County Hospital Center, Brooklyn, NY, USA; 3 Biostatistics & Clinical Epidemiology Service, NIH Clinical Center, Bethesda, MD, USA; 4 Program in Reproductive and Adult Endocrinology, NICHD/NIH, Bethesda, MD, USA. Objective: To compare the effectiveness of 24 weeks Leuprolide Depot 11.25mg (LD) vs Norethindrone Acetate 5mg (NA) followed by NA alone for 28 weeks in relieving pain from endometriosis. Design: Prospective double-masked randomized clinical trial. Materials and Methods: 62 women with symptomatic endometriosis were randomized to LD (N=31) or NA (N=31) for 24 weeks in phase I; then all were given NA for 28 weeks in phase II (total 1 year of treatment). For this ongoing study, subjects were defined as Group A or Group B. Visual analog scale (VAS) was used to assess menstrual (MP), coital (CP) and non-menstrual pelvic pain (NMP). VAS were evaluated at baseline and compared to Week 12, 24 and 52 to assess improvement. Data were analyzed as intent to treat using paired t-test, Fisher's exact and Wilcoxon two-sample tests. Results: Women were predominantly Black (81%) and single (66%), had a mean age of 34.1 ±6.7 yr and reported pelvic pain for 14.3 ±8.0 yr. Group A and B did not differ in these characteristics, stage of endometriosis, gravidity, or BMI. More women in Group A than B continued the study at week 52 (A:27 vs B:24, p=0.834). Group A and B did not differ in baseline pain scores. In phase I, MP subsided in both groups; only those in A reported menses and pain (N= 6, p=0.027). VAS scores significantly decreased for MP and NMP by Week 12 (A: p<0.0001 and p=0.008, respectively; B: p<0.0001 for both) through Week 24 (A: p<0.0001 and p=0.015, respectively; B: p<0.0001 for both). Group B reported greater reductions in MP and NMP (p=0.020 and p=0.038, respectively) in Phase I although fewer Group B women completed 24 weeks of treatment (A:29 vs B:24, p=0.625).

VAS for MP and NMP did not change from Phase I to Phase II. There was no change in CP with treatment in the few women that were sexually active. Conclusions: Menstrual and non-menstrual pelvic pain significantly decreased with both norethindrone acetate and leuprolide. Regardless of initial treatment, pain relief continued when transitioned to only norethindrone acetate. There was no significant difference in lesion size between BZA & BZA/CE treatment groups or between different doses of either treatment. Ovarian cyst formation was not observed. BZA treatment also led to lower estrogen receptor alpha(Esr1) expression compared to the control group. BZA dosages of 2, 3 or 5mg/kg reduced Esr1 expression by 1.60, 1.97 & 2.04-fold, respectively (p<0.05). Treatment with the TSEC containing higher BZA dosages(3mg/ kg & 5mg/kg) led to significantly lower levels of Esr1 expression compared to controls; BZA(3mg/kg)/CE treatment resulted in a 1.94-fold decrease in Esr1 expression compared to BZA(1mg/Kg)/CE or to control (both p<0.05). BZA(5mg/kg)/CE resulted in 2.36-fold decrease in Esr1 expression compared to low dose treatment (p<0.05). No differences were observed in expression of progesterone receptor (Pgr). IHC analysis demonstrated Esr1 & Pgr protein levels to be consistent with the changes seen in RNA levels. CONCLUSION: BZA/CE treatment led to decreased endometriosis lesion size. The mechanism may involve a decreased Esr1 expression. The combination of CE & BZA may prove to be a novel treatment option for endometriosis.

How Sensory Innervation Impacts the Intensity of Endometriosis-Associated Pain. Kathleen M Peters, 1 Paul Wrigley, 2 Ian S Fraser. 1 1 ObGyn, University of Sydney, Sydney, Australia; 2 Pain Research Institute, University of Sydney, Sydney, Australia. Background Pelvic pain is the most common presenting symptom of endometriosis. General pain thresholds appear to be lowered in women suffering painful endometriosis. Nerve conduction studies to elucidate contributors to this phenomenon have often investigated large, myelinated peripheral nerves and a loss of function. Recently, increased sensory innervation has been observed in peritoneal lesions, myometrium and endometrium of women with endometriosis [1] . Therefore, exploring how various sensory inputs and a gain in sensory function contribute to endometriosis-associated pain may highlight previously unknown aspects of disease progression. Aim This study aimed to examine pelvic pain intensity and alterations in sensory input in the pelvis of women with and without endometriosis. Methods We utilised Quantitative Sensory Testing (QST): the application of a standardised battery of potentially noxious stimuli to endometriosis subjects, with considerable pain, and to pain-free, aged-matched controls, as outlined by the German Research Network on Neuropathic Pain [2] . QST assesses the different types of sensory nerves (afferents) through self-reported, detectionand pain-thresholds of applied stimuli. Stimuli are grouped as thermal, mechanical, vibratory and pressure. Sites included the (control) dorsal hand and (test) lower, midline abdominal (T11 dermatome) and lumbosacral spine (L4-S1). Subjects rated the intensity of their pelvic pain on the day of QST using an 11-point numerical rating scale (NRS) [3] . Results Currently, 4 endometriosis subjects have been tested. Higher pain intensity scores (>4) appear to align with lowered pain thresholds to a number of the applied QST stimuli. The study and analysis are continuing. Discussion Contributions to neuronal excitability occur through the different sub-types of sensory fibres. Identifying which sensory fibres are hyper-sensitive in endometriosis will help build a somatosensory profile of the afferents potentially involved in peripheral and central sensitization; both thought to impact the pain associated with endometriosis. 

Evaluating the Psychological Contributors to the Amplification of Endometriosis-Associated Pain. Kathleen M Peters, 1 Michael K Nicholas, 2 Ian S Fraser, 1 Paul Wrigley. 2 1 ObGyn, University of Sydney, Sydney, NSW, Australia; 2 Pain Research Institute, University of Sydney, Sydney, NSW, Australia. Background Pelvic pain, the most common symptom of endometriosis, is notoriously variable in presentation. Variability may be due, not only to disparate indications, but also the diverse psychological aspects each woman brings. The subjective nature of pain with its psychological amplification makes valuable assessments difficult. Recently, semi-quantitative questionnaires have been refined to provide a more objective evaluation of pain. Consensus has been achieved on the essential outcome measurements for the assessment of pain. How best to apply these to endometriosis-associated pain remain to be determined. Aim To identify a core set of validated questionnaires suitable for use in research and clinical practice that assess the physical, psychological and social impact of endometriosis-associated pain. Methods A qualitative appraisal was undertaken of questionnaires assessing general health, physical and emotional functioning. Endometriosis-specific and pain-specific questionnaires were also examined. Questionnaires were found via literature search and through consultation with pain content experts. Selection criteria focused on questionnaires with published normative data to aid interpretation of outcomes measurements. Questionnaire burden was considered. Results Suitable questionnaires were identified: including general Quality of Life, Activity Interference and Mood Disturbance estimations [1] [2] [3] . To capture the relationship between intensity of pain, decreased physical and emotional functionality and increased psychological contributors, a Pain Intensity Scale and Pain Coping and Pain Catastrophizing Questionnaires were incorporated [4] [5] [6] . Conclusions Accumulating evidence suggests that, in a chronic pain state, the perception of pain influences response to treatment. Endometriosis may be thought of as a chronic pain condition. Quantifying the contributors to the amplification of pain may expand available treatment options to reduce painrelated distress and disability in women with endometriosis. 1. Roland, M.et al, Spine, 1983 Background: Women suffering from chronic pelvic pain and endometriosis have high rates of sensitization (83% vs 15%) and myofascial dysfunction (94% vs 15%) compared to healthy volunteers. Objective: To evaluate the interrelationships among levator spasm, sensitization, myofascial dysfunction (MyoDys), pain severity, anxiety and depression in women with chronic pelvic pain and endometriosis (CPP+E), chronic pelvic pain only (CPP), and healthy volunteers (HV).

Design: Prospective cohort study Materials and Methods: Allodynia and hyperalgesia were assessed bilaterally from T9 to S2. Sensitization was defined as >6 abnormal segments/side. Myofascial trigger points (TrP) were examined in 7 muscles/side. MyoDys was defined as having TrP in >4 muscles/side. Pelvic floor muscle spasm, termed levator spasm, was assessed on pelvic exam. Headaches, body pain, pelvic pain, anxiety and depression were assessed by questionnaire. All CPP had symptoms suggesting endometriosis and underwent surgery for histologic diagnosis and treatment of any endometriosis lesions. Data were analyzed using Kruskal-Wallis test for trend and logistic regression modeling. Results: Subjects included CPP+E (n=18), CPP (n=11) and HV (n=20). 61% of women with CPP+E had levator spasm compared to 36% CPP vs 0% HV (p<0.0001). 87% with levator spasm had MyoDys whereas 45% with MyoDys had levator spasm. After adjusting for group, those with MyoDys were more likely to be sensitized (OR=6.81,95%CI:1.04-44.36;p=0.045). In considering any history of endometriosis instead of group, those with MyoDys were more likely to be sensitized (OR=9.41,95% CI:1.77-50.08;p=0.009). After adjusting for group, those with more severe headaches, body pain, and pelvic pain (OR=2.29, 95% CI:1.14-4.59;p=0.02), anxiety (OR=1.05, 95% CI: 1.004-1.099;p=0.03) or depression (OR=1.06,95%CI: 1.005-1.113; p=0.03) were more likely to be sensitized. Conclusions: Levator spasm, myofascial dysfunction, sensitization, overall pain severity, anxiety and depression appear to be interrelated in women with chronic pelvic pain associated with endometriosis. Assessment of these factors may be important in the treatment of women with endometriosis and chronic pelvic pain. Support: Intramural Program, PRAE/NICHD, BCES/CC, and RM/CC, NIH; NCT00073801

Estradiol and Inflammatory Cytokines Induce Neuroangiogenic Responses in Endometriosis Stromal Cells. Antonio MC Francisco, 1 Jie Yu, 1 Tamer Yalcinkaya, 1 Erika B Johnston-MacAnanny, 1 Neil Sidell, 2 Sarah L Berga, 1 Robert N Taylor. 1 1 Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, NC, USA; 2 Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, GA, USA. Endometriosis lesions contain endometrial glands and stroma, along with nerves, capillaries and leukocytes. We are studying the recruitment of tissue macrophages into endometriosis implants, and the influences of macrophagederived cytokines on epithelial, stromal, neural and endothelial cell growth. Their precise interactions are not well understood, but elucidation of the complex paracrine cross-talk may inform new therapies for the dysmenorrhea, dyspareunia, chronic pelvic pain or infertility that characterize the syndrome. Innervation and capillary infiltration of endometriosis implants are well documented and we have proposed that these processes are molecularly coordinated ("neuroangiogenesis" [Asante & Taylor, 2010] ). Using proteomics methods to identify endometrial neurotrophins (NTs), we discovered that brainderived neurotrophic factor (BDNF) and NT4/5 were differentially expressed in eutopic endometrium from subjects with endometriosis compared to normal controls (Browne et al., 2012) . The objective of this study was to evaluate the effects of estradiol (E2) and two macrophage-derived cytokines, TNFα and IL1β, on BDNF, NT4/5, VEGF and IL6 expression in stromal cells isolated from eutopic endometrium of subjects with endometriosis. Eutopic endometrial stromal cells (ESC) were isolated from women with endometriosis (n=3) using established methods and cultured for 48 h in the absence or presence of E2, TNFα or IL1β in concentrations ranging from 1-100 nM. Cell supernatants and lysates were prepared for ELISA and Western blots, respectively. E2, TNFα and IL1β each increased BDNF and NT4/5 production (≥3-fold, P<0.05). Of the angiogenic factors, VEGF secretion increased 2-fold in response to E2, but not to cytokines, and IL6 increased ≥8-fold in response to TNFα and IL1β, but not to E2 (P<0.02). Regulation at the mRNA level is under investigation currently. The findings support the hypothesis that E2, possibly generated via local aromatase activation, and macrophage-derived cytokines contribute to neuroangiogenesis in endometriosis and represent potential therapeutic targets. Supported by the Eunice Kennedy Shriver NICHD through U01 HD66439 and a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil.

GWAS Identify Novel Loci Associated with Endometriosis. Hans Albertsen, Rakesh Chettier, Pamela Farrington, Kenneth Ward. Research, Juneau Biosciences, LLC, Salt Lake City, UT, USA. BACKGROUND: On average, diagnosis and treatment of endometriosis is delayed by 7-10 years from the onset of symptoms. Absence of a timely and non-invasive diagnostic tool is presently the greatest barrier to the identification and treatment of endometriosis. OBJECTIVE: Although endometriosis is a heterogeneous disease with complex etiology, the high heritability of the condition suggests that genetic markers will be discovered. Using a genome-wide association study (GWAS) we set out to identify predictive markers of endometriosis. METHODS: A two-stage GWAS (discovery and replication) was performed. Patients with surgically confirmed endometriosis cases (n=2,019) were compared to population controls (n=14,471). All subjects were of European ancestry (>95%). Illumina OmniExpress was used for genotyping. PLINK (ver 1.07) was used for genetic analysis. ADMIXTURE (ver. 1.22) was used to determine each subject's ancestry. RESULTS: We identify four novel endometriosis loci including LINC00339-WNT4 on 1p36.12 (rs2235529; P=3.05×10-9, OR=1.30), RND3-RBM43 on 2q23.3 (rs6757804; P=6.45×10-8, OR=1.20), RNF144B-ID4 on 6p22. 3 (rs6907340; OR=1.20) , and HNRNPA3P1-LOC100130539 on 10q11.21 (rs10508881; P=4.08×10-7, OR=1.19) that reach genome-wide significance. Further evidence suggests the involvement of nine additional genomic regions. Our data confirm the previously reported association of endometriosis to the WNT4-region. CONCLUSION: We have identified four novel loci strongly associated with endometriosis and confirmed the involvement of a region around WNT4, which previously has been suggested as being associated to endometriosis. Nine other regions hold promise as candidate loci for endometriosis. Utmost care was taken in the clinical classification of patients and only surgically confirmed patients with > 95% European ancestry were considered in this large GWAS of endometriosis. The study is well powered (>90%) to identify markers with at least a 10% minor allele frequency and an odds-ratio >1.20. Our data suggest the top 5 loci explain only a small portion of the heritability of endometriosis. We conclude that endometriosis loci that contribute to the "missing heritability" must involve less common alleles or more recent mutations than the ancestral loci marked by the single nucleotide polymorphism assayed in this GWAS.

Exploration of Urinary Biomarkers as a Possible Screening Tool in Endometriosis. Fred TK Wong, 1 Robert Markham, 1 Ben Crossett, 2 Ian Fraser, 1 Cecilia Ng. 1 1 ObGyn, University of Sydney, Sydney, NSW, Australia; 2 Molecular Bioscience, University of Sydney, Sydney, NSW, Australia. Background: Endometriosis, a benign gynaecological disease affects 10-15 % of women of reproductive age. The "gold standard' for diagnosis, still remains laparoscopy or laparotomy coupled with histological confirmation. Despite numerous research efforts, pathogenesis and a simple and definitive screening tool for endometriosis have remained elusive. Aims & Objectives: Tokushige et al. (2011) described the finding of a novel urinary protein biomarker, cytokeratin 19, to be higher in abundance in women with endometriosis. The successful identification of additional urinary biomarkers may help to elucidate key molecular networks responsible for the pathogenic development of endometriosis. We wished to establish whether a difference exists in the urinary proteome from first pass morning samples versus urine collected during surgery. Overall, our objective was to identify specific and sensitive urinary biomarkers capable of screening for different stages of endometriosis. Materials &Methods: Protein extraction and concentration (10kDa molecular weight cut-off filters, Amicon Ultra 15) were performed (n = 3 patients).Liquid off-gel fractionation was utilised to obtain three separate pH fractions (pH4. 6-5.4, 5.4-6.2 and 6.2-7 .0), followed by 2D gel electrophoresis (2DE). Following image analysis (Progenesis SameSpot, Nonlinear Dynamics), proteins of interest were subjected to trypsin digestion and identification by MALDI-TOF mass spectrometry (MS; Voyager, AB Sciex, Australia) and MASCOT (MatrixScience,UK).

Preliminary gel image analysis has identified an average of (17) protein spots across the three fractions. Proteins of interest (7 out of 17) are currently undergoing MS identification. It is our hypothesis that further analyses of results will lead us to assess and nominate biomarkers, which may be sufficiently specific and sensitive for the diagnosis and staging of endometriosis.

Liquid-off gel fraction, reducing proteome complexity, coupled with 2DE, offered clear protein spot separation from patients with and without endometriosis. It is hoped that successful identification should allow development of a non-invasive screening tool for endometriosis. REFERENCES 1.Tokushige, N., et al. Fertil Steril. 2011. 95 (1) Objective: The cost effectiveness of abdominal (AM) or laparoscopic myomectomy (LM) prior to ART has not been reported. IM fibroids can impair fertility, and myomectomy is often performed prior to administering assisted reproductive technologies (ART). We sought to determine whether removal of IM fibroids prior to ART lowers cost/ongoing pregnancy. Design: Decision tree mathematical model with sensitivity analysis utilizing published data. Methods: A PubMed search determined: (1) the likelihood of ART ongoing pregnancy (OPR) in patients with IM fibroids in situ vs. post-AM and post-LM; and (2) mean perioperative costs (surgery and hospital stay) of AM and LM. ART charges were obtained from individual clinic websites. Adjusted to 2012 dollars, mean ART costs were estimated at $15,223, and mean AM+ART and LM+ART costs at $23,381 and $25,676, respectively. These acted as surrogates for clinical costs. A decision tree compared cost/ongoing pregnancy via ART in the setting of IM myoma(s) in situ vs. post-myomectomy. Sensitivity analyses were performed over the range of OPRs. Results: Likelihood of ART ongoing pregnancy in patients with IM fibroids in situ vs. post-myomectomy from four published studies were 18.5%-35.0% (in situ) vs. 33.9%-52% (AM) and 25%-86% (LM). Sensitivity analyses determined that pre-ART AM was cost effective when cost/ongoing pregnancy was >$68,971 (i.e. when OPR among women with in situ myomas was </=22.0%). If the OPR among women with in situ myomas was >/=33.9% (cost/OP</=$44,963), AM was not cost effective. In the intermediate range of OPRs with in situ myoma(s), AM was only cost effective if it increased OPR by >/= 11.9%. LM was only cost effective when it increased OPR to >59.0%, which though reported in the literature, is significantly above the national mean, even among good prognosis patients. Conclusions: AM was cost effective only when fibroid(s) greatly impaired OPR (OPR≤22%) or when OPR was between 22 and 33.9% and surgery improved OPR by 11.9% or more. A laparoscopic approach was even less cost effective than an abdominal one. From a cost perspective, surgery for IM fibroids prior to ART should be undertaken judiciously, i.e. only when perceived detriment is great and expected postoperative outcome is excellent. Support: Intramural Research Program and the Program in Reproductive and Adult Endocrinology, NICHD, NIH.

Prolactin as a Biomarker for Tumor Burden and Response to UPA Therapy in African American Women with Symptomatic Leiomyoma. Karenne N Fru, 1 Gary Levy, 1 Robert Wesley, 2 Lynnette Neiman, 1 Aradhana Venkatesan, 3 Alicia Armstrong. 1 Objective: There are considerable ethnic differences in the molecular characteristics, prevalence, and symptomatology of uterine fibroids. Studies have demonstrated that the selective progesterone receptor modulator, ulipristal acetate (UPA), effectively controls bleeding and reduces fibroid volume in women with uterine leiomyomas. Studies of pharmacologic therapies for uterine leiomyomas are limited by small numbers of African American (AA) patients and medical therapies have been shown to have different efficacy in AA women limiting the applicability to AA patients. This study aims to determine if prolactin is a biomarker that can be applied to AA women with uterine fibroids treated with UPA. Materials and Methods: This study is a subgroup analysis of a previously published placebo-controlled, double blind study evaluating the efficacy of UPA for treatment of uterine fibroids. African American patients receiving UPA or placebo were identified and compared to non-AA patients. The primary outcome was the change in serum prolactin from before and after treatment with 3 months of UPA therapy. Results: Thirty-eight premenopausal women (32 AA and 6 non AA) underwent treatment with UPA at 10 mg, 20 mg or placebo. AA patients receiving UPA demonstrated a reduction in fibroid volume (-23±19.1) compared to AA women receiving placebo (+8.2% ± 24.8) (P<0.05). The mean change in PRL before and after treatment in AA and non AA patients was (-10.36±3.55) and (-0.116±11.8) (p=0.0073). All patients receiving UPA were likely to have a decrease in prolactin compared to placebo (p=0.043). However when AA patients receiving UPA were compared to non-AA patients undergoing treatment, the change in prolactin prior to and after treatment was not significant (p=0.27). Fibroid volumes did not correlate with serum prolactin (r=0.14, p=0.40). Conclusions: PRL is a potential biomarker for the therapeutic response of leiomyomas to pharmacologic therapy. However, this study did not demonstrate a difference in the change in PRL between AA and non-AA women.

Vitamin D3 Reverse Developmental Reprogramming by Modulating EZH2 Histone Methyltransferase Activities in Human Fibroid Stem Cells. Sunil K Halder, Sangeeta Nair, Chakradhari Sharan, Ayman Al-Hendy. Obstetrics and Gynecology, Meharry Medical College, Nashville, TN, USA. Background: Hypovitaminosis D may contribute to the epigenetic changes in the developing uteri during fetal and neonatal periods leading to E2 hyperresponsiveness and increased risk of uterine fibroids in adulthood. Epigenetic changes such as histone methylation can be inherited and can cause repression or induction of gene expression. Recently, Dr. C. Walker and co-workers (Mol Cancer Res; 2012:546-557) showed that neonatal exposure to certain environmental estronens can stimulate AKT signaling which phosphorylate the enhancer of zeste homolog 2 (EZH2), and as a result decreased EZH2 activity and levels of trimethylated lysine 27 on histone 3 (H3K27me3). Reduction of H3K27me3 results in increased expression of estrogen responsive-genes to enhance responsiveness to steroid hormone and increase fibroid incidence. Objective: To evaluate whether the biologically active 1,25-dihydroxyvitamin D3 can reverse developmental reprogramming that occurs in human uterine fibroids stem cells. Design: We have isolated stem cell populations from normal myometrium from a non-fibroid uteri (myoN), myometrium adjacent to fibroid lesions (myoF) and fibroid tissues (F). Tissue samples were chopped into small pieces, digested with collagenase and then filtered through 100µm pore size sterile filters. These populations of cells were passed through columns containing magnetic beds coated with stem cell specific antibodies. Captured cells were eluted from beds, cultured in coated dishes and then treated with 1,25-dihydroxyvitamin D3 (10nM, 100nM and 1000nM) for 48 hrs. Protein extract were analyzed by western blots and compared for the expression of EZH2 (phospho versus unphospho) between vitamin D3treated versus untreated stem cells. Results: 1,25-dihydroxyvitamin D3 decreased p-EZH2 in myometrium (myoN and myoF) and fibroid stem cells in a dose-dependent fashion, while the total EZH2 levels were not changed. These cells also expressed VDR (vitamin D receptor) which was induced by 1,25-dihydroxyvitamin D3. Conclusions: 1,25-dihydroxyvitamin D3 can possibly reverse the developmental reprogramming by modulating EZH2 activity that in turn leads to reduction of estrogen-responsive gene expression in human uterine fibroids. Vitamin D and analogues can possibly be novel agents for prevention of uterine fibroids in high risk groups. Support: 5R01HD046228 to AHA; RCMI grant 2G12RR003032-26 to SKH.

The Effect of Menses on the Assessment of Sexual Function. Shannon K Laughlin-Tommaso, 1 Bijan J Borah, 2 Elizabeth A Stewart. 1 1 Obstetrics and Gynecology, Mayo Clinic, Rochester, MN, USA; 2 Health Sciences Research, Mayo Clinic, Rochester, MN, USA. Background: Sexual functioning perception has been related to health condition and may be influenced by timing of the menstrual cycle. Our objective was to determine if reporting of sexual dysfunction is related to timing of the menstrual cycle.

Methods: As part of the FIRSTT trial (NCT00995878), women complete the Female Sexual Functioning Index (FSFI) survey twice prior to treatment of fibroids: once during menses and once when not menstruating. Questions ask about sexual functioning over the past 4 weeks. Scoring on the FSFI combines questions into 6 sexual function domains (see Table) each in a Likert scale of 0 (or 1) to 5. The scores for each domain during menses and non-menses periods were calculated and compared using a paired t-test. A discordance score for each woman was calculated as the difference between their two scores for each domain. The discordance score was then categorized into no change, improvement or worsening relative to the score reported at the time of non-menses. Results: Twenty-five women completed the FSFI twice. The mean FSFI scores for each of the 6 domains did not differ between menses and non-menses reporting. Discordance between the two responses was highest for arousal domain (68%) and lowest for pain domain (42%). Higher percentage of women reported improvement in pain and desire from non-menses to menses responses, the opposite was the case for arousal, lubrication, and satisfaction domains. Similar numbers of women reported no sexual activity at menses and non-menses report. (58) 7 (29) 3 (13) Conclusion: Although the mean values of the domain scores were not different between the two responses, women did report sexual functioning differently between menses and non-menses reporting. Timing of the questionnaire should be considered in studies. Objective: Uterine fibroids are the most prevalent tumors in reproductive aged women and produce excessive and abnormal extracellular matrix (ECM) induced by TGFβ3. Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM) that has been demonstrated to clinically decrease fibroid size and is currently approved in Europe as a pre-operative adjunctive medical therapy. TGFβ3 acts through Smads to stimulate ECM expression including MMPs, collagen 1A1, versican and fibronectin. These ECM components are ubiquitously over-expressed in leiomyoma cells compared to myometrium. The purpose of this study was to evaluate the effect of UPA therapy on leiomyoma TGFβ3 expression, signaling and resultant ECM expression. Methods: Laboratory study with immortalized human leiomyoma and patientmatched myometrial cells. After obtaining IRB approval, immortalized human leiomyoma and myometrial cells in culture were exposed to UPA at concentrations of 10-8-10-6 molar for 72 hours in cell culture followed by RNA and protein isolation and real time RT-PCR and western blot analysis. Results Objective: Transforming growth factor β (TGF-β3) controls the cellular proliferation in numerous cell types, including leiomyomas. Studies have shown that TGF-β3 is threefold to fivefold overexpressed in leiomyoma cells when compared to normal myometrial cells. We investigate the effects of peroxisome proliferators-activated receptor (PPAR) agonists on TGF-β3 expression. We examine the effects of PPAR agonists on leiomyoma cell growth. Methods: Human leiomyoma cells were isolated from surgical specimens. Women taking any hormonally active medications, such as GnRH agonists and oral contraceptive pills were excluded. The cultured cells were treated with serum free, phenol red-free media prior to treatment with increasing concentrations of PPAR agonists, rosiglitazone and troglitazone. After 24 hours of incubation, qRT PCR was utilized to determine TGF-β3 mRNA expression in treated cells compared to untreated cells. Data was analyzed using student's t-test. Results: TGF-β3 expression was significantly downregulated by treatment of leiomyoma cells with rosiglitazone. Cultured cells exposed to rosiglitazone had a 6-fold decrease in TGF-β3 expression (p < 0.05) compared to untreated cells. Utilizing concentrations of 1 and 10 uM rosiglitazone, no significant dose responsive decrease in TGF-β3 was noted with both concentrations demonstrating a similar decrease in TGF-β3 expression. After treatment with 10uM of troglitazone, there was no significant change in TGF-β3 expression (0.79, p>0.05) compared to untreated cells. PPAR expression increased (2.63 fold, p< 0.05). Conclusion: Our findings suggest that non-hormonal interventions aimed at downregulation of TGF-β3 offer a novel treatment option for leiomyomas. PPAR agonists, typically utilized to treat diabetes mellitus, offer the dual advantage of potentially inhibiting leiomyoma growth. With the appropriate patient selection and further studies, these findings offer potential new strategies for myoma treatment.

Fibroids; MyoF as a Pre-Tumor State. Sangeeta Nair, Chakradhari Sharan, Jyotsna Thota, Ahsen Chaudhury, Sarbani Maitra, Ayman Al-Hendy. CWHR, Ob Gyn, Meharry Medical College, Nashville, TN, USA. Background: Uterine fibroids are benign, hormone dependent, monoclonal tumors originating from the myometrium in premenopausal women.We have recently shown that low vitamin D levels are associated with increased risk of uterine fibroids. Lately, new theories have evolved based on the isolation of a side population of somatic stem cells in fibroids and adjacent myometrium which may help in understanding the pathophysiology of uterine fibroids. Objective: To investigate the expression of estrogen (ER), progesterone (PR) and vitamin D receptors (VDR) in human stem cells isolated from uterine myometrium without any microscopically-detected fibroids (MyoN) versus uteri with fibroids (MyoF) and from fibroid lesions (F). Methods: Stem cells from F, MyoF and MyoN were isolated from women undergoing hysterectomy at Nashville General Hospital approved by local IRB. Cells were selected from collagenase treated tissue, using CD44 & STRO1 antibody-coated biotinylated Dynabeads (Invitrogen) and cultured in suitable media. The stemness phenotype of the cells was confirmed via the expression of a panel of specific markers/genes tested by flow cytometry and PCR. Total RNA and cDNA were prepared using RNeasy kit (Qiagen There is growing evidence that 25-OH Vitamin D plays a critical role in human physiology and may be involved in several disorders. In recent years, experimental evidence consistently supported a role of this element also in the pathogenesis of uterine fibroids. However, to our knowledge, there is no study comparing serum levels of 25-OH Vitamin D in affected women and unaffected controls. Experimental difficulties may explain the lack of this evidence. Of relevance, age and parity markedly affect both the development of fibroids and sun exposure habits. Design Cross sectional observational analysis of women scheduled for assisted reproductive technologies (ART).

Between January and June 2012, infertile women scheduled for ART were invited to participate. Subjects agreeing to enter the study, underwent a transvaginal ultrasound for the identification of uterine fibroids. Two controls were matched to each case and corresponded to the two following infertile women of similar age whose uterus resulted unremarkable at ultrasound. Selected women provided a serum sample that was tested for 25-OH Vitamin D. The quantitative detection of 25-OH Vitamin D was performed using a commercially available kit based on a chemiluminescence technology.

Seventy-six patients with uterine fibroids were selected; they were matched to 152 controls. The mean level of serum 25-OH Vitamin D in affected women was significantly lower than in controls (16.8 ± 7.9 and 19.6 ± 8.1 ng/ml, respectively, p=0.014). A condition of severe 25-OH Vitamin D deficiency (< 10 ng/ml) was observed in 22% (17/76) of women with fibroids and in 10% (15/152) of controls (p=0.015). The Odds Ratio for carrying fibroids in women with severe deficiency was 2.63 (95% Confidence Interval: 1.15-6.05). Moreover, a significant inverse correlation was observed between the number of uterine fibroids and the serum value of 25-OH Vitamin D (Spearman's Rho correlation=-0.15, p=0.025).

Our data provides evidence that a significant correlation exists between serum 25-OH Vitamin D levels and the presence of uterine fibroids. The decision to select cases and controls from a population of infertile women and to match them by age should have protected our results from the confounding effects of age and parity. Further studies are however needed to definitely support a causal relationship.

Health Background: Fibroids are the most common tumor in reproductive aged females with higher prevalence and surgical burden in African-American women (AAW). Fibroids remain the leading cause of hysterectomy in this country, and are responsible for $34.4 billion in health costs annually. Health literacy and knowledge of a given disease have been shown to be correlated with positive health behaviors. To our knowledge, no studies have been done assessing women's knowledge of fibroids and their sequelae.

Objective: The goal of this pilot study is to explore knowledge of fibroids and their clinical manifestations amongst AAW and to assess need for larger studies. Methods: English-speaking women between the ages of 18-70 attending a community fair in Chicago were recruited. Demographic information was obtained and subjects completed a questionnaire assessing health literacy, general reproductive health knowledge, and knowledge of fibroid prevalence, diagnosis and clinical manifestations.

Results: 210 women with a mean age of 48 yrs participated. 95% self-identified as black/African-American, 32% had at least a college degree, 65% had a household income <$50,000 and 21% had no health insurance. The prevalence of inadequate health literacy was 13%. The majority of subjects knew that fibroids are more common in AAW (75%), can cause menorrhagia (85%) and can increase the risk of miscarriage (77%). However, a significant number thought that fibroids are cancerous (41%) or increase the risk of heart disease (21%). 38% believed that fibroids can be diagnosed with a blood test. Internet usage, partner status and education level had the highest correlation with knowledge of fibroids and their systemic sequelae. Of note, health insurance status and health literacy status showed no significant correlation with fibroid knowledge.

Conclusions: The majority of women at an urban community fair were aware of the increased prevalence of fibroids in AAW and the associated clinical symptoms. However, there was some degree of misconception of the systemic sequelae of uterine fibroids. There is a need to raise fibroid awareness and correct any misconceptions of their systemic manifestations or diagnosis. "Ever having used the internet" and knowledge of uterine fibroids suggests that technological interventions may improve health knowledge and health behaviors in this population. Objective: To develop a FACS-based protocol for isolation of fibroblast and smooth muscle cell populations from human myometrium and fibroid tissues, and to use x-chromosome inactivation to determine the clonality of these different cell populations. Hypothesis: One or more fibroblast sub-populations isolated from human fibroid tissues will not be clonally derived. Methods: Human myometrial and fibroid tissues from hysterectomy (n=57, mean age 47) were collected following informed consent. Isolated cells were prepared for FACS and sorted into 4 subpopulations based on CD90 and ALDH expression. Genomic DNA and cDNA from whole tissues and sorted cells were used to determine clonality based on x-inactivation using the HUMARA assay.

Results: Four different cell populations were consistently identified in fibroid and myometrium by FACS, with an additional ALDH 'bright' population in many of the fibroids. Conventional HUMARA using genomic DNA gave confusing although repeatable results on clonality. By contrast, HUMARA performed on cDNA from the same samples clearly demonstrated that in the majority of cases fibroids, and all their constituent sub-populations of cells were clonally derived, while myometrium was not.

We have developed a protocol for separating different cell phenotypes from fibroid and myometrium, based on CD90 and ALDH expression. Despite cells with quite different phenotypes appearing in similar proportions in both myometrium and fibroid, we have shown that the fibroid cells are all clonally derived. This result demonstrates that following initiation of a fibroid from a single cell, there appears to be a relatively normal differentiation of daughter cells into the cellular phenotypes found in myometrium. These findings have implications for our understanding of how fibroids grow and develop.

Paricalcitol, None-Hypercalcemic Vitamin D Receptor Activator, Inhibits Proliferation of Human Uterine Leiomyoma Cells. Chakradhari Sharan, Sunil K Halder, Sangeeta Nair, Ayman Al-Hendy. Obstetrics and Gynecology, Meharry Medical College, Nashville, TN, USA. Introduction: Uterine leiomyomas (ULMs) are the most common (77%) benign tumor among women and are associated with many health complications. ULMs are 3-4 times more common in black women than in white women, and as such constitute a major health disparity challenge. Blacks also suffer from vitamin D deficiency about 10 times (about 45%) more than whites (4%). A major factor in this phenomenon is that dark skin pigmentation decreases UV absorption and thus limits sun-induced vitamin D synthesis in blacks. This observation along with decreased milk consumption due to lactose intolerance reduces the levels of Vitamin D in black Americans. Additionally we have recently reported that Vitamin D3 decrease expression of COMT enzyme (F&S, 2011), TGF-Binduced fibrosis (JCEM, 2011) and shrinks uterine fibroids in Eker Rat model (BOR, 2012) . Objective: In this study, we wanted to evaluate the effects of Paricalcitol, a potent none-hypercalcemic VDR (vitamin D receptor) activator, on human leiomyoma cells. Methods: Human immortalized leiomyoma cells (HuLM) were cultured in SmBM medium supplemented with 5% FBS, 0.1% insulin, 0.2%hFGF-B, (Lonza). The monolayer cultures at approximately 70% confluence were treated with various concentrations (1nM -1µM) of Paricalcitol (Zemplar, Abbott, Deerfield, IL) for 7 days. Medium were changed every other day. Cell proliferation was analyzed by CyQuant cell proliferation assay kit (Life Technology). Results: Paricalcitol exhibited marked antiproliferative effect on the growth of HuLM cells. Compared with untreated control, the inhibitory effect of Paricalcitol on HULM was observed at 1 nM as early as 24 hours (10%) and peaked at 1 µM concentration at 168 hours (63%). The difference was statistically significant (p=0.005). Evaluation of the in vivo utility of Paricalcitol in various fibroid animal models is currently under investigation in our laboratory. Conclusions: Paricalcitol (Zemplar), potent none-hypercalcemic VDR activator, demonstrates effective dosedependent anti-proliferative utility against human uterine leiomyoma cells. Vitamin D 3 and its analogues might be a new family of compounds to provide alternative safe simple and effective oral non-surgical treatment option for management of uterine fibroids, which would have a major positive effect on women health worldwide. Grant support: NIH grant 5RO1-HD46228 and RCMI grant 2G12RR003032.

Weakening. Corinne Belville, 1,2 Gael Clairefond, 2 Sabine Chauveau, 2 Denis Gallot, 2 Vincent Sapin, 2 Loic Blanchon. 2 1 GReD, Auvergne University, Clermont-fd, France; 2 R2D2-EA7281, Auvergne University, Clermont-fd, France. Introduction: Epigenetic modifications influence gene expression by acting at different levels including DNA methylation. The fetal membranes enclose the developing embryo and a precise control of gene regulations is essential for the homeostasis of this transitory but indispensable fetal annex. Today, the epigenetic components of pathological pregnancies have been linked to the placenta (preeclampsia, IUGR) but only recently and poorly to the fetal membranes (chorioamnionitis, preterm premature rupture of membranes: PPROM). The aim of our work was to precisely detail the expression levels of DNA methyltransferases (DNMTs) and DNA demethylases (GADD45/TET) in amnion and chorion from intact-ZIM to altered-ZAM. Methods: Human fetal membranes were obtained from 9 different women with healthy pregnancy (39,10 ± 0,075 weeks of gestation) undergoing planned caesarean section after informed consent in accordance with the Declaration of Helsinki. mRNA extractions were done with RNeasy lipid tissue kit from amnion, chorion or total membrane. mRNA quality was checked on Bioanalyser. mRNA quantification of DNMT, GADD45 and TET family members was done by qPCR (Roche). Housekeeping genes (36B4 and RPS17) quantification was also performed. All repeated experiments (n=6) were done according to the MIQE requirements. Relative expression was expressed as a ratio between gene interest mean and the geometric mean of both housekeeping genes. For the protein level, immunohistochemistry were done for DNMT/GADD45 and TET family members demonstrated as differentially express by qPCR. Results: A differential ZIM/ZAM expression (Wilcoxon pair-test) was observed for Dnmt3B in amnion and DNMT2 and 3A in chorion. Furthermore, a difference is also noted for the GADD45 family member (except alpha) in both amnion and chorion; whereas no difference could be underlined for TET protein. Beside this, a difference on the expression levels was also established between amnion and chorion for TET1 and 2, GADD45β and γ.

Conclusion: The expression of the DNMT, GADD45 and TET family members is tissue specific (amnion and chorion) and could be correlated to methylation levels on genes, as we will study by a global methylation study. This step is essential to understand in case of PPROM the transcriptional mechanism leading to a fetal membrane zone weakening. Introduction: Lin28 nuclear protein and let-7 miRNAs participate in signaling pathways involved in cell developmental and differentiation. Lin28 expression is higher in the placenta than any other organ, however, gestational tissuespecific expression and the effects of gestational age have yet to be established. Let-7 miRNAs are differentially expressed in early development and their expression peaks during the transition from oocyte to the 2-cell stage in the embryo.

Objective: The aim of this study was to establish the tissue-specific expression of Lin28 and Let-7 miRNAs in term human gestational tissues. Methods: Term placenta, choriodecidua, and amnion (n=18) were obtained from women at term, before the onset of labour (Caesarean section, n=6), during active labor (Caesarean section, n=6) and after labor and delivery (n=6). Total tissue protein and miRNA were extracted. Lin28 expression was assessed by Western blot analysis. The expression of Let-7 miRNAs (let-7a, b, c, d, e, and f) was determined using real-time PCR. Data were expressed as mean ± SEM. The tissue-specific and labor-associated changes in analyte expression were assessed by ANOVA. A p-value <0.05 was considered statistically significant. Results: No statistically significant effects of labour status on either Lin 28 or let-7 miRNA expression were identified, therefore, data were combined to assess tissue specific expression. The expression of Lin28 in term gestational tissues displayed tissue specific expression (p < 0.05) with placenta = choriodecidua > amnion (placenta 14.33±5.826 fold change over amnion, choriodecidua 13.15±2.619, amnion 1.00±0.09). With the exception of let-7e (choriodecidua > placenta=amnion, p <0.05), let-7 miRNAs were expressed similarly in term gestational tissues. Discussion: Although no differences in their expression was found between these delivery conditions, tissue-specific expression of Lin28 was observed. Of the isoforms, let-7e miRNA was found to be differentially expressed between gestational tissue types. Let-7e is implicated in cell growth pathways. Future studies will studies to determine whether or not both Lin28 and let-7 undergo placental expression changes throughout gestation, and investigate possible roles in placental and fetal growth. Introduction: Conditions that cause any alteration in normal embryonic gene expression patterns may have harmful effects on the embryo. DNA methylation patterns are a major determinant of gene transcription. However, the DNA methylation pattern and its maintenance by DNA methyltransferase in in vivo grown hamster preimplantation embryos have not been investigated.

Hamsters are a good model because estrogen, required for blastocyst formation, is provided by the preimplantation embryo similar to primates and possibly humans.

Objective: To examine global DNA methylation and DNA methyltransferases expression patterns in various stages of in vivo developed hamster preimplantation embryos. Results: We observed clear DNA methylation in both pronuclei at the zygotic and the 2-cell embryo stage. However, immunofluorescence signals were reduced in 4-cell and 8-cell stage embryos. Morula stage embryos showed strong DNA methylation in outside cells compared with the inside cells. In the blastocyst stage, stronger methylation was observed in outer trophoblast cells compared to inner cell mass cells. Dnmt1 mRNA was present in all stages of preimplantation embryos by RT-PCR. Dnmt1 protein expression was observed in both the nucleus and cytoplasm of in vivo grown zygotes to the blastocyst stage. However, at the blastocyst stage, its signal became weaker in the inner cell mass cells compared to trophoblast cells. Expression of Dnmt3a mRNA was found in the 1-cell and 2-cell stage embryos but not in the 4-cell stage. However, it reappeared again in the 8-cell and blastocyst stages. Conclusions: We have shown for the first time reprogramming of DNA methylation in hamster preimplantation embryos. We were able to detect noticeable change in only DNA methylation pattern from the two-cell stage to the blastocyst stage. The observed change in DNA methylation/Dnmt1 patterns at the blastocyst stage may be associated with blastomere lineage specification to inner cell mass and trophoblast cells. The absence of Dnmt3a mRNA at the 4-cell stage suggests lack of de novo DNA methylation at this embryonic stage. Overall, these findings in hamster preimplantation embryos provide a temporal pattern of DNA methylation that is required to regulate stage-specific gene expression and cell lineage specification. (Supported by NIH grants: RO1HD044741 to BCP and T32 to Judy Aschner)

Studies. Amy L Creekmore, Kerry L Sanders, Men-Jean Lee, Jill L Reiter. Dept of OB/GYN, Indiana Univ School of Medicine, Indianapolis, IN, USA. Objective: Stable reference genes are essential for appropriate normalization and accurate measurement of relative gene expression studies. Reference genes commonly used in human placental gene expression studies include ACTB, GAPDH, TBP, YWHAZ, and 18S rRNA, despite evidence for considerable variation in their expression amongst different samples. Our aim was to determine whether a survey of several placental gene expression microarray data sets would identify novel reference genes with greater stability than commonly used genes. Study Design: Genevestigator, a web-based software tool, was used to screen a set of six microarray data sets including samples from normal 1st, 2nd, and 3rd trimester placentas, as well as placentas from pregnant mothers who smoked or suffered from preeclampsia or malaria. We identified 10 putative reference genes (DNAJC8, FKBP15, HMGCL, PEX16, RAB11B, RING1, TBC1D13, TRADD, USP4, and VPS18), with the lowest standard deviation (SD) of signal intensities across all samples (N=54). The stability of these putative and common reference genes was compared using RT-qPCR with high integrity RNA (RQI mean±SD: 8.7±0.48; N=24) isolated from four quadrants of six normal term placentas. Each RNA sample was analyzed in duplicate RT reactions primed with either oligo-dT (dT) or a mixture of dT with random hexamers (dT+RH). The stability of Cq values was compared with four software programs, BestKeeper, Normfinder, geNorm, and RefFinder. Results: USP4, RING1, PEX16, HMGCL, and TBP were consistently found to be the most stable reference genes using each software program. NormFinder stability values for these genes were 0.004-0.006 (lower values indicate more stability), whereas commonly used reference genes had stability values of 0.012-0.031. When dT alone was used as the RT priming strategy, gene-specific SD of Cq values increased 104-309%, which altered the order of the highest ranked reference genes: DNAJC8, VPS18, TBC1D13, USP4, and TBP. This set of reference genes had stability values between 0.08-0.011, indicating a twofold decrease in stability from the top reference genes in the dT+RH RT priming condition.

We have identified a new set of genes with greater than twofold apparent stability in expression compared to common reference genes. In addition, RT priming conditions influence reference gene stability; thus, appropriate reference gene validation is warranted when RT-qPCR assay conditions are altered. Objective: Endometriosis affects 10% of reproductive age women. Although, therapeutically altering sex-steroid levels is the mainstay of treatment, it can be unfocused and unsatisfactory. Disease-heterogeneity may contribute to clinical diversity, necessitating development of novel, individualized, targeted treatments. KLF11, a Sp/KLF transcription-factor is highly expressed in urogenital organs, is mutated in diabetes and is anti-proliferative in fibroids. Induced endometriotic lesions in Klf11-/-mice develop scarring resembling advanced human disease. Here, we characterize in vitro and in vivo, underlying dysregulated molecular pathways with potential translational implications. Methods: Lesions were induced in Klf11-/-or wildtype(wt) mice by implanting autologous uterine segments to the peritoneum using published protocols (N=10/group). In crossover experiments Klf11-/-uterine implants were transplanted in wt animals and vice versa. qPCR and Immunohistochemistry were used to determine lesional Collagen1 (COL1) expression. ChIP, EMSA, mutagenesis and Luciferase-assays were used to characterize gene regulation in human endometrial stromal cells (HESC). Results: Lesion growth and prolific scarring were found in Klf11-/-animals.

In contrast, wildtype(wt) animals had minimal adhesions and lesion-regression (p<0.05). When Klf11-/-uterine segments were transplanted in wt animals, extensive fibrosis developed. In contrast, wt implants in Klf11-/-animals had minimal scarring, suggesting that aberrant regulation of scar tissue-specific COL1 by Klf11 in endometriotic stromal cells caused fibrosis. In stromal cells, KLF11 bound the COL1 promoter, repressed luciferase and mRNA levels. When KLF11 was mutated and unable to bind Sin3/HDAC, COL1 repression was reversed, likely from non-deacetylation of COL1 promoter histones.

In this study we demonstrate regulation of Collagen1 by the human disease-related gene KLF11. We demonstrate in vitro that KLF11 regulates COL1 by recruiting the Sin3/HDAC cofactor. We then show in vivo the phenotypic consequences resulting from aberration of this regulatory pathway. Unlike pharmacologically-resistant inherited gene mutations, novel epigenetic-modulators selectively target histone modifications and thereby can reverse disease progression.

Evolutionary Genetics of Dizygotic Twinning and Small Body Size in Callitrichine Primates. R Alan Harris, 1,2 Suzette D Tardif, 3 Tomas Vinar, 4 Derek E Wildman, 5, 6 Julienne Rutherford, 7 Jeffrey Rogers, 1, 8 New World monkeys (NWM) are characterized by an extensive size range, and callitrichines such as marmosets and tamarins manifest diminutive size and unique reproductive adaptations. While other primates are capable of multiple gestations, callitrichines are unique in both the common occurrence of twins and higher order multiple gestations as well as adaptations to reduce complications of twinning observed in humans, such as preterm birth, growth discordance, and twin-twin transfusion. Evolutionary explanations for twinning in callitrichines have been put forth that are related to their diminutive size, and with the availability of the common marmoset (Callithrix jacchus) genome assembly the genetic underpinnings of these traits can now be explored. Likelihood ratio tests identified five positively selected genes (GHSR, IGF2, IGF1R, and IGFBP2/7) coding proteins in the growth hormone/insulin-like growth factor (GH-IGF) axis. Numerous GHSR and IGF1R mutations cause growth reduction in humans. Sequencing of these genes in other callitrichines and NWM families revealed a large number of nonsynonymous substitutions (NS) specific to NWM compared to other primates. Callitrichine specific NS were identified in GDF9, BMP15, BMP4 and WFIKKN1. Polymorphisms in GDF9 occur among human cohorts with a propensity for dizygotic twins, and polymorphisms in GDF9 and BMP15 are associated with twinning in sheep. Immunohistochemistry was performed to detect these proteins in bilateral ovaries from marmosets and non-callitrichine NWM. We postulate that positive selection affected NWM growth patterns, with callitrichine miniaturization coevolving with a series of reproductive adaptations.

The Mullerian aplasia, also known as Mayer-Rokitansky-Kuster-Hauser syndrome, consists of the congenital absence of the uterus and upper vagina in 46,XX females. This disorder affects approximately 1 in 4500 female live births and 10% of women with primary amenorrhea. In addition, unilateral renal agenesis and skeletal abnormalities commonly occur, but deafness and cardiac defects may be present in some patients. The molecular basis for mullerian aplasia is largely unknown as WNT4 mutations only comprise a small percentage of affected patients. More recently, copy number variants (CNVs) have been described in mullerian aplasia patients, and some of these have been identified repetitively. We hypothesized that mullerian aplasia patients will have heterozygous, de novo mutations that will not be found in their parents. The purpose of the present study was to test mullerian aplasia patients for mutations in candidate genes identified by several different complementary methods employed by our laboratory. In addition to known gene WNT4, five genes near derivative chromosome breakpoints in a patient with a balanced translocation (CNOT10, TRIM71, ZNF213, OR1F1, and ZNF200) and five genes within or near CNVs detected by array comparative genomic hybridization (CTNNA3, LRRTM3, FAM190A, HNF1B, and CHSY3) were studied for mutations. DNAs extracted from 37 mullerian aplasia patients were subjected to PCR-based DNA sequencing of the protein coding exons and splice junctions from each of these 11 genes. Interestingly, no WNT4 mutations were identified in any mullerian aplasia patients. However, rare heterozygous sequence variants were identified in CTNNA3 and FAM190A that were not present in the SNP database and 1,000 genomes database. These are currently being confirmed and tested in available family members to determine their segregation with the phenotype.

Our preliminary findings suggest that the use of chromosomal translocations and CNV analysis provide reasonable candidate genes for testing in patients with mullerian aplasia.

Temporal Objective: Heterochromatin protein 1 (HP1) proteins are "gatekeepers" of epigenetic signals through methyl-K9-histone H3 silencing. HP1γ plays a critical role in germ cell proliferation and meiotic spermatogenesis, and mouse knockouts show both sexes to be infertile with very few progeny. We previously reported the significant role of Serine 83 HP1γ phosphorylation (P-Ser83-HP1γ) in somatic mitosis. However, these signals have yet to be described in oocytes and early embryonic mitotic divisions. Our objective was to define these epigenetic signals in oocytes and the initial stages of embryo development. Methods: P-Ser83-HP1γ and pan HP1γ antibodies were used in immunofluorescence (IF), immunohistochemistry (IHC) and immunogoldbased electron microscopy (EM). Human ovary sections were used for IHC. Protein localization was observed in human and mouse oocytes by IF and EM. IF was performed on mouse embryos every 24 hours from pronuclear to the hatching blastocyst stage. Wild type HP1γ, S83A and S83D HP1γ mutantcarrying adenoviruses were used to infect cells for Affymetrix whole genome microarray analysis to identify genes significantly regulated by phosphorylation of this protein. S83A abolishes Aurora A-mediated HP1γ phosphorylation, while S83D mimics constitutive phosphorylation. Results: Meiotic centrosomes of M2 oocytes did not exhibit high levels of HP1γ nor its phosphorylated form. IHC on human ovary sections demonstrated that P-Ser83-HP1γ is more prominent in highly proliferative cells, concentrating in granulosa cells of antral follicles. Mouse embryos from pronuclear to blastocyst stages demonstrated P-Ser83-HP1γ localization to centrosomes during early embryonic cleavage divisions that coincide with embryonic genome activation. Concordantly, expression profiling implicated phosphorylation of HP1γ in the regulation of follicle development, oogenesis and pre-implantation embryonic development. Conclusion: Our data provide evidence that P-Ser-83-HP1γ is not present in the meiotic plate of the M2 oocyte but is activated in early embryonic cell divisions. Disruption of this pathway may also play a role in embryonic cleavage abnormalities resulting in infertility or early pregnancy loss. Regulation of pertinent genetic networks confirms the importance in early embryonic development. Leptin is an adipokine involved in food intake regulation and its level correlates with adipose tissue storage. Recently, this hormone has been recognized as a novel endocrine player in developmental biology. During pregnancy, leptin is produced by maternal, fetal and placental tissues emphasizing its importance during this sensitive period. Somatic epigenetic modifications such as DNA methylation are established during this period and can serve as molecular markers of intrauterine environment disruption. Hence, investigating leptin epigenetic effects on pregnancy outcomes like birthweight is relevant to increase our understanding of maternal-infant morbidity and lifetime disease risk. Using the resources of the Rhode Island Child Health Study (RICHS), a birth cohort centered in Providence, RI, we sought to investigate the extent of leptin ( LEP) promoter DNA methylation in 60 human cord blood samples and paired placental tissue in relation with maternal and infant characteristics. We measured DNA methylation using the gold-standard bisulfite pyrosequencing method. In preliminary analysis, we found that cord blood LEP methylation inversely correlates with infant birthweigth, length and head circumference in a sex dependent manner; stronger effects were observed in female infants. Moreover, LEP methylation is significantly greater in infants considered small for gestational age (SGA) compared to those considered adequate for gestational age (AGA). Additionally, cord blood LEP methylation directly correlates with maternal weight gain during pregnancy whereas in paired placental tissue we observed and inverse association. Identifying epigenetic effects of maternal and fetal factors of hormones such as leptin during pregnancy is important to provide insights into molecular mechanisms that can influence development and potentially infant lifelong health outcomes. (EP) is the end result of abnormal selection of an implantation site. It is associated with risk factors including pelvic inflammation, smoking and in vitro fertilization. Epigenetic changes can result from any of these risk factors and therefore be present in the maternal tissue that hosts an EP. Objective: To investigate epigenetic aspects of implantation in EP by comparing DNA methylation patterns between fallopian tube tissue from patients with EP and fallopian tube tissue from patients with a normally implanted pregnancy. Design: Prospective observational study. Materials and Methods: Fallopian tube tissue was prospectively collected from 15 cases of EP in which a salpingectomy was performed and from tubal ligation procedures during cesarean section cases. Biopsies of the fallopian tube tissue adjacent to the implantation site of the EP and of the ampullar portion of the fallopian tubes from tubal ligation cases were collected intraoperatively and fresh frozen. Whole genome DNA methylation profiling was performed on the tubal biopsies using the Illumina Infinium Human Methylation450 BeadChip kit and statistically analyzed. Results: Significant differences in DNA methylation patterns were found in the comparison between the fallopian tube from EP cases and patients with and intrauterine pregnancy. Immune response related genes as well as tissue identity related genes were differentially methylated in the EP fallopian tubes when compared with tissue from patients with a normally implanted pregnancy.

The fallopian tubes from EP cases showed significantly different epigenetic marks compared to fallopian tubes from normal pregnancy cases.

The differently methylated genes represent inflammatory pathways and genes responsible for maternal tissue identity. These changes may be responsible for conflicting molecular signaling between maternal tissues and the embryo during early implantation.

Research, Dept of Ob/Gyn, University of Texas Health Science Center at San Antonio, TX, USA. Introduction: Over 60% of women of child bearing age in the US are overweight or obese. Maternal obesity has both immediate effects on pregnancy outcome but also programs the offspring for obesity, metabolic syndrome and cardiovascular disease in adult life. The programming effect of the adverse intrauterine environment is transduced through the placenta. We have provided evidence of altered placental function with increasing maternal adiposity. The interaction of environmental factors such as maternal diet with the genome may cause epigenetic changes that alter placental function. Objective: We hypothesized that increased maternal adiposity would affect the placental epigenome at the level of DNA methylation. Methods: Five random samples of villous tissue were collected from each placenta and flash frozen following cesarean section at term (39.31±0.72, mean±SD) prior to labor from normal weight (pre-pregnancy or 1st trimester BMI=23.4±2.3) and obese women (BMI=34.0±2.9) (n=10 each group). Random tissue samples were combined and genomic DNA was isolated from each of the 10 placentas per group, combined into two pools (normal and obese) and then subjected to the MeDIP-chip analysis using NimbleGen 2.1M human DNA methylation arrays. Results: MeDIP-chip analysis identified 23,082 and 24,399 methylated regions in normal and obese placentas, respectively. DNA methylation was detected at the testis-specific positive control and imprinted genes but not at the negative control genes. DNA methylation at the DSCR4 gene, predominantly expressed and unmethylated in the placenta was not seen, indicating no detectable contamination with maternal blood or tissue. We identified a total of 21 genes in the placenta where DNA methylation was significantly altered (18 increased and 3 decreased) in response to maternal adiposity in the region from -1 to +1kb of transcription start sites. The differentially methylated genes identified include those involved in placental development (6 genes), cellular invasive

properties (2), cancer metastasis (2), vascular remodeling (4), increasing BMI (1), lipid transport (2), energy metabolism (1), immune and/or inflammatory responses (7). Conclusion: We find increased overall methylation in placentas of obese women and direct evidence that the maternal obesogenic environment may specifically alter the placental epigenome, potentially changing placental function.

Genome-Wide Methylation Profile Identifies Genes Involved in the Pathogenesis of Endometriosis. Hanyia Naqvi, Hugh S Taylor. Reproductive Sciences, Yale University, New Haven, CT, USA.

Objective: Endometriosis has been associated with aberrant methylation in the eutopic endometrium. Using a genome wide methylation screen, we identified methylated genes from endometrium obtained from controls or women with endometriosis. Aberrant epigenetic modification may lead to alteration in the expression of genes contributing to the pathology of endometriosis. Methods: Endometrial biopsies were obtained from subjects undergoing surgical treatment of endometriosis (n=7) or endometriosis-free controls (n=6). DNA was extracted from the eutopic endometrial tissue. 500ng of the genomic DNA were bisulphate converted using the Zymo Bisulphate conversion kit. Illumina Infinium HumanMethylation27, RevB Beadchip was used to survey the genome-wide methylation profile. 200ng of the converted DNA were amplified using the Illumina Infinium protocol and hybridized to the microarray for 16 hours. Hybridization was followed by a single base extension and fluorescent amplification using Tecan Freedom Evo. Chips were dried, scanned (Illumina iScan System) and analyzed. GenomeStudio was used for bioinformatics and statistical analysis. Results: Of the 27,578 genes on the methylation array, 120 genes were significantly altered by > 1.5 fold. 59 genes were significantly hypermethylated and 61 genes were significantly hypomethylated (p <0.05). Examples of hypermethylated genes include Cell division cycle associated 2 (CDCA2) and Inhibitor of DNA binding 2 (ID2). Genes with decreased methylation include Zinc finger proteins protein receptor (ZNF681) and Insulin Receptor Substrate 2 (IRS2). Gene expression associated with altered methylation status were validated using quantitative real time PCR (qPCR). Conclusion: Genomic methylation has been recognized to play a role in the epigenetic regulation of gene expression and development of endometrial lesions. Global methylation profiling reveals a limited number of candidate genes in the endometrium of women with endometriosis. CDCA2 (regulates chromosome structure), ID2 (negative cell differentiation regulator), ZNF681 (of the Kruppel-like zinc finger protein family) and IRS2 are all aberrantly methylated in endometriosis; aberrant DNA methylation and gene expression of these genes may contribute to abnormal regulation of the endometrial cell proliferation and function in women. Serotonin is synthesized within the placenta during crucial developmental periods and plays an important role in the development of the HPA axis by guiding the formation of fetal brain circuits. The genes involved in this pathway are targets of epigenetic control, particularly DNA methylation, and maternal factors, including depressed mood, have been linked to methylation. This project sought to analyze placental promoter methylation of genes involved in the serotonin response pathway and to correlate methylation, gene expression and neurodevelopmental outcomes in a human population. Participants were infants enrolled in the Rhode Island Child Health Study, an ongoing, population-based birth cohort in Providence, Rhode Island. Placental tissue, extensive maternal and infant clinical characteristics and exposure histories are collected, and infants are assessed for neurodevelopment using the NICU Network Neurobehavioral Scales (NNNS). Promoter CpG methylation from placental DNA was analyzed through quantitative bisulfite pyrosequencing of the serotonin transporter, SLC6A4 (N=60), and the serotonin receptor, HTR2A (N=221). The SLC6A4 promoter was relatively unmethylated and there was little variability between patient samples, although moderate correlation between expression of this gene and methylation was observed.

There was greater variability in methylation within the HTR2A promoter.

HTR2A methylation was generally lower in female infants, higher in infants whose mothers reported depression or anxiety/OCD during pregnancy, and was positively correlated with the NNNS attention score. These results suggest that methylation of the HTR2A gene can be environmentally modulated and play an important role in neurodevelopment, specifically infant attention, which may be indicative of later behavioral outcomes. Ongoing work includes expanding the sample size of our examinations, and demonstrating the relationship between gene expression and methylation of HTR2A in the placenta. We also are examining the potential modification of the relationship between methylation and neurodevelopmental outcomes by genetic variation a SNP which lies within the promoter region of HTR2A. BACKGROUND: Mice deficient in the peptidyl-prolyl isomerase Pin1 exhibit profound fertility defects and a reduced number of oocytes, owing to Pin1's role in the regulation of primordial germ cell cycle progression during embryonic development. Furthermore, expression of Pin1 mRNA in the follicles of bovine ovaries appears to be responsive to FSH and may help regulate granulosa cell proliferation during follicular development. Given these findings, we hypothesized that mutations in the human PIN1 gene might play a role in premature ovarian failure (POF) by decreasing cell divisions that give rise to the primordial germ cell pool and by disrupting follicular development.

Genomic DNA was collected from predominantly North American Caucasian women with POF, with POF defined as amenorrhea prior to age 40 with elevated FSH >20 IU/L. Primer sets specific for all 4 exons of PIN1 were designed, DNA from each sample was amplified by polymerase chain reaction (PCR), and PCR products underwent Sanger sequencing on a commercial platform. Sequencing results were systematically compared to each other and to a reference human genome using Sequencher 5.0 (Gene Codes Corporation, Ann Arbor, MI). Chromatograms were also manually examined to identify sequence variants.

Our results show no variation in our patients, with only two synonymous mutations across all samples. CONCLUSION: Our pilot study, which to the best of our knowledge is the first to study PIN1 in the context of POF, suggests that mutation of this gene is not a common cause of POF in our study population. Objective: Risk for adverse neonatal outcome increases with declining gestational age (GA) though the mechanisms responsible for this remain unknown. An emerging body of evidence supports the role of epigenetic alterations, such as changes in DNA methylation, as molecular mediators of adverse postnatal phenotypes. A recent study reports an association between CpG sites in 25 genes and gestational age in primarily term deliveries. This study will replicate and extend these findings to a PTB cohort. Methods: GA was determined by obstetrician report and maternal last menstrual period. DNA methylation of umbilical cord blood from African Americans with early PTB (24-34 weeks; N=22) or term birth (39-41weeks; N=28) was assessed using the HumanMethylation450k BeadChip. CpG sites in GA-associated genes were extracted from this data. Each of these CpG sites was examined for association with GA by fitting a separate linear model for each CpG site, adjusting for the appropriate covariates (sex and batch effects). Results: For all 25 genes, the CpG sites associated with GA in term deliveries also associated with GA in this cohort (.0052<p<7.5x10-11). Fine mapping of an additional 569 CpG sites in the GA-associated genes revealed that almost 25% also associated with GA (.05<p<7.5x10-11) independent of location in the gene. The most prominent of these differentially methylated CpG sites were in genes already implicated in the pathophysiology of PTB including AVP, ESR1 and OXT.

Conclusions: The results of this study provide support for these genes in human development and also provide insight into the molecular mechanisms underlying PTB.

Yuping Zhou, 1 Geraldine Brichant, 2 Huan Yang, 3 Sirisha Gudavalli, 4 Hugh S Taylor. 5 In another abstract, we present our most recent genome wide association study (GWAS) that identified several loci strongly associated with endometriosis. GWAS typically use a very conservative correction for multiple hypothesis testing, and some have suggested that important disease markers can also be found in the markers trending toward significance. This investigation tests additive model using a large number of the most highly associated polymorphisms (as proposed by Stahl et al. Nat Genet 2012) METHODS: Illumina OmniExpress was used for genotyping. PLINK (ver 1.07) was used for genetic analysis. ADMIXTURE (ver. 1.22) was used to confirm that each subject has >95% European white ancestry. Single nucleotide polymorphism (SNP) genotypes were filtered for quality and logistic regression (LR) was performed whereby the risk score, serving as a proxy for the risk of the subject developing endometriosis, was treated as a single predictor for training and validation. From the discovery GWAS (1,514 surgically confirmed endometriosis cases and 12,660 population controls), we selected sets of markers (100; 500; 1,000; 10,000 and 20,000) and trained the LR algorithm. We then tested the performed each model on the validation set of 505 cases and 1811 controls.

The performance of each model in the independent validation cohort resulted in a poor prediction as shown by area under the curve (AUC). 

We conclude that a simple polygenic risk model using the most associated common single nucleotide polymorphisms in this GWAS does not allow prediction of endometriosis. Too many of the "trending toward association" markers represent false-positive noise rather than truly associated disease markers. We are currently investigating whether inclusion of copy number and rare variants improve the predictive performance. was characterized in DNA from maternal blood longitudinally in 14 women at <16 gestational weeks, at delivery and postpartum; in 14 nulligravid controls; and in 14 preeclamptics at delivery. All women were non-smokers and the three groups were matched for age and BMI. Genomic DNA was derived from buffy coat, purified, and bisulfite modified then run on the Illumina platform. Mean methylation levels at each CpG site were compared using a t-test. RESULTS: Methylation patterns in all inhibin/activin genes did not differ significantly between the non-pregnant groups -postpartum vs nulliparous. In contrast, the two CpG sites in the INHBE gene, but none of the other genes, were significantly hypo-methylated during early pregnancy as compared with the non-pregnant state (p< 10 -3 ; FDR < 0.10). Methylation patterns were not significantly different in any of the inhibin/activin genes in preeclamptic women at delivery compared with normotensive women. CONCLUSION: Our results demonstrate that early pregnancy is associated with decreased methylation at two CpG sites in INHBE, but preeclampsia at delivery does not appear to have a significant association with differential methylation in any of the inhibin/activin genes. Although not as well-studied as inhibin-A and B, the inhibin-BE subunit has been demonstrated to be expressed in liver, endometrium, ovary, cervix, placenta and leukocytes, with putative functions including tissue remodeling and angiogenesis. Altered methylation may be an epigenetic mechanism in normal early pregnancy to control inhibin/activinE production -a potential novel chemokine deserving further study.

Detecting Our proofof-concept experiment suggests comparisons between reference epigenomes for leukocyte subpopulations and experimental differential methylation data may be an effective methodology for detecting confounding due to cellular heterogeneity.

Study Design: We extracted iron use data from total of 30,406, term (26,902) and preterm births (3, 504) in a retrospective study, from 2007 to 2009 in five university hospitals of CEE. Results: The data was analyzed using SAS to investigate iron use frequencies for each core. The rates of iron use and rates of anemia vary from 2.2% (Ukraine) to 39% (Slovakia). There was no relationship in iron use and diabetes except for Hungary and Slovakia where iron use was higher in subjects who developed diabetes. Logistic models indicate that anemia is a very strong predictor in all the participating CEE countries except Ukraine and smoking is an additional predictor in all these countries except Hungary. In Slovakia, current (pregnancy induced or exsisting) diabetes and BMI are also significant predictors of Ironuse. Conclusion: Biomarkers index could be more sensitive to changes in maternal iron status rather than the overall outcome measures. The results of the Iron use in the retrospective study are crucial to make adjustments in the ongoing prospective study and able to design iron supplementation schemes for the safe prevention and correction of iron deficiency or excess. . Ten women (67%) were smokers, 12 (80%) were using combined anti-retroviral therapy, 6 (40%) had a CD4 cell count ≤200 cells/µl, and 12 (80%) had a detectable viral load at the time of diagnosis. Eleven women (73%) diagnosed as stage I, 1 (7%) stage II, 2 (13%) stage III, and 1 (7%) stage IV. Twelve patients (80%) had keratinizing squamos cell carcinoma (SCC), 2 (13%) with basaloid SCC, and 1 (7%) had verrucous SCC. Thirteen (87%) women received surgery. Median tumor diameter was 2 cm (0.1-8) and median depth of invasion was 2 mm (0.5-44). Surgical margins were negative in 5(33%), positive for carcinoma in-situ in 2 (13%), and in 5 patients (33%) there was invasive disease within 8 mm of the surgical margin. Two (13%) women were treated with radiation. Three (20%) women were recommended to receive chemotherapy, but none were medically eligible to initiate treatment. Four deaths occurred, 3 (20%) died of HIV related causes and 1 (7%) died of progression. The median surveillance of our cohort was 37 months . The five-year progression free survival (PFS) rate was 65% and five-year overall survival (OS) rate was 54%. Five-year OS rate was significantly better in women with CD4 cell count >200 compared to CD4 ≤200 (75% vs. 27%, p=0.01). Conclusions: Among women with vulvar cancer and HIV, more deaths were attributed to HIV related causes than from vulvar cancer. A CD4 cell count >200 was associated with a significantly improved overall survival. 

We retrospectively reviewed charts of all patients with VIN III between January 2000 and July 2012 to identify those who were HIV+. All women received a wide local excision at the time of diagnosis. They were then followed every six months with repeat examination and biopsies as indicated. Comprehensive exams included cervical, vaginal, vulvar, and anal examinations. Chi-square and Fisher's exact tests were used as appropriate.

Results: Thirty four HIV+ women were identified with VIN III and followed for a median of 68.5 months (5-119). Median age was 41 years (24-61) and median duration of HIV infection was 15.5 years (8-21). Eighteen (53%) patients were smokers, 31 (92%) were using combined anti-retroviral therapy (cART), 18 (53%) had a CD4 cell count ≤200 cells/µl (CD4 ≤200), and 19 (56%) had a detectable viral load (VL) at the time of diagnosis. Seventeen patients (50%) had cervical intraepithelial neoplasia II or greater. Twenty (59%) patients had dysplasia of the cervix or anus requiring excision. Six (18%) patients progressed from VIN III to invasive disease with a median time to progression of 30.5 months (11-97). In patients with invasion, 1 patient was stage IA, 3 were stage IB, and 2 patients were diagnosed with stage III. Of note, both patients with advanced disease were non-compliant with cART and regular surveillance. Neither a detectable VL (p=0.69), nor CD4 ≤200 (p=0.39) was significantly associated with increased risk of progression to cancer. CD4 ≤200 was associated with concomitant cervical and/or anal dysplasia requiring excision (p=0.05), while detectable VL was not (p=0.15). Conclusions: Over 80% of HIV positive women with VIN III did not progress to cancer. Nearly 60% of patients had synchronous cervical or anal dysplasia requiring excision. A CD4 ≤200 was not associated with increased risk of progression to cancer, but was associated with multi-focal disease. Among compliant patients, conservative management of VIN III in HIV positive women is feasible with routine and comprehensive surveillance.

Feasibility recommends peritoneal washings to be performed at the beginning of surgical procedure for cancer staging. Laparoscopic and robotic techniques, have raised concern of the increased risk of positive cytology compared to the classical open surgery. This has been attributed to the use of intrauterine manipulator. Methods: A retrospective chart analysis was carried out to evaluate the difference in peritoneal washing cytology obtained pre-operatively and postoperative for gynecologic malignancies. Pelvic washing was collected at the time of abdominal entry and at the end of the procedure. All cytology results were correlated with the histology results of cell blocks. Results: Of the 54 patients who had hysterectomy with malignant conditions of the endometrium, ovary, and cervix, 15 (28%) patients has a positive washing. Six were positive for pre-operative and 5 post-operatively. 4 patients had positive, or atypical cells both pre and post-operative samples. 9 (60%) were endometrial, 5 (33%) were ovarian and 1 (6%) was of cervical origin. Robotic procedures were performed on 5 patients and the remaining 10 had open procedures. All pathology specimens were reported as satisfactory for evaluation. Conclusion: Intraoperative pre and post surgical washing cytology can be evaluated adequately regardless of presence of a higher number of mesothelial, inflammatory and blood cells. Surgical procedures for women with gynecologic cancer can expose exfoliation of the tumoral cells intra-peritoneally at the conclusion of the procedure, regardless of the surgical technique. Combined pre-and post-operative washing may be the best practice to evaluate peritoneal cytology Objective: Reproductive health is a significant concern for female cancer survivors. While cancer therapies are known to impact ovarian function, limited data exist regarding long term impact on androgen production. The objective of this study is to assess androgen levels in female cancer survivors and to determine if androgen levels influence sexual function in this population. Design: Cross-sectional analysis of data from a prospective longitudinal cohort study Methods: Young women with a history of cancer were compared to healthy, reproductive age controls. Total testosterone and DHEAS were measured in the early follicular phase. Sexual function was assessed using the sexual function subscore from the Quality of Life in Adult Cancer Survivors (QLACS) scale.

Libido, vaginal dryness and frequency of sexual intercourse were also assessed. Results: Twenty nine cancer survivors were compared to 42 healthy controls. Survivors were significantly younger (mean age 25.7 vs. 29.5 years, p=0.01), but there was no difference in race, BMI, marital status, parity, or use of hormonal contraception. Adjusted for age, DHEAS levels were significantly lower in female cancer survivors (102.1 vs. 158.6 mcg/dL, p= 0.001.) This association remained significant after controlling for hormone use. There was no difference in age-adjusted testosterone levels between the groups (9.31 vs. 11.29 ng/dL, p=0.25.) Cancer survivors were more likely to report vaginal dryness (28% vs. 7%, p=0.02), but there was no difference in frequency of intercourse or in the QLACS sexual function score. Nineteen percent of cancer survivors endorsed low libido compared to 7.5% of controls, but this difference did not reach statistical significance (p=0.16). There was a significant negative association between DHEAS and vaginal dryness (Spearman r= -0.25, p=0.038), but no correlation between DHEAS and libido or DHEAS and sexual function score. Conclusion: We demonstrated that female cancer survivors have lower DHEAS levels, higher rates of vaginal dryness and a trend toward lower libido. There was no correlation between androgen levels and sexual function. Further studies are needed to examine the impact of cancer therapy on androgen production and female sexual function. Background: Cancer incidence and Mortality is higher in males than females, suggesting that some gender-related factors are behind such a difference. However, this phenomenon is poorly investigated and the realted cause are unknown. Methods: The last available Surveillance, Epidemiology and End Results (SEER) database served to access survival data of the US population. Around 1.2 millions of cases were included in the analysis. Cox and Kaplan-Meier method tested the impact of gender on servival across age and calculated the by gender hazard ratio of dying from cancer 5 years following diagnosis. The distribution of hazard ratio across age was then compared with the sistribution of 130 variables assessed in another large US populatio study (NHANES III). Kolmogornov-Smirvon test assessed the homology. Finding: Cancer survival was lower in males than in females in the age range 17 to 61. The risk of death from cancer in males was about 30% higher than that of females at the same age. The effect was more evident in African-Americans, in solid tumors and in patients with metastatic disease. Intrestingly, when compared to the 130 variables assessed in the NHANES III study, hazard ratio exactly matched the distribution of free testosterone, whereas none of the other 129 parameters investigated exhibited a similar homology. Interpretation: Our findings suggest that male sex hormones are responsible for agressive cancer in patients younger than 61 years. Therefore, anti-anderogen therapies may be tested to reduce cancer mortality in male patients developing cancer under 61 years of age. Bevacizumab. Both studies were followed by lymphodepletion using high-dose Cyclophosphamide and Fludarabine and transfer of 5-30 x10e9 autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood T cells, in combination with antiangiogenesis therapy and vaccination. Feasibility, safety, and biological and clinical efficacy were evaluated. Results Eight subjects have completed vaccination and T cell transfer to date, while 13 other subjects completed vaccination only. Vaccination was well tolerated caused only grade 1 toxicities. It elicited tumor-specific T cell responses against various ovarian tumor antigens and clinical responses correlated with the immune response with 66% of patients achieving clinical benefit (prolonged PFS or remission inversion). After lymphodepletion, adoptive transfer of autologous T cells produced durable reduction of CD4 + FoxP3 + T regulatory cells, increased total lymphocyte counts and restored vaccine-induced antitumor immunity in patients who experienced clinical benefit. One patient exhibited no evidence of disease at end of study. Stable disease was observed in 3 subjects to date. In one subject where adoptive T cell transfer was not followed by restoration of vaccine-induced antitumor immunity disease progression was observed.

Conclusions The use of combinatorial immunotherapy comprising DC vaccination with whole tumor antigen and adoptive lymphocyte transfer using tumor antigen-specific T cells for the treatment of recurrent ovarian cancer is promising but warrants further investigation. Our second case is a 50yo G3P2012 who presents with a history of right flank pain for 3 days. Pelvic exam revealed tender right adnexa with transvaginal ultrasound findings significant for a complex right adnexal cyst with solid intra-cystic echogenic nodules. Final pathology revealed serous borderline tumor of a right paratubal cyst. Later surgery for this patient also included similar comprehensive surgical staging. All subsequent final pathology was revealed to be negative for residual malignancy in both cases described. Discussion: Serous borderline tumors are tumors of low malignant potential defined by atypical epithelial proliferation of serous type cells in greater number than surrounding benign cells and without stromal invasion. Clinical features of these tumors may present with abdominal pain due to a ruptured cyst or torsion. Conclusion: Paratubal serous borderline tumors are exceedingly rare with these cases being the 5th and 6th reported cases in the literature. The prognosis of paratubal serous borderline tumors is difficult to estimate secondary to the extreme rarity of this condition and unknown etiology. Management is generally extrapolated from data surrounding the treatment of ovarian borderline serous carcinoma. Continued reporting of these rare cases is paramount to our future understanding of these tumors. Objective: Aggressive angiomyxoma is a rare soft-tissue neoplasm of the female pelvis and perineum with a high local recurrence rate. These tumors displace rather than invade adjacent viscera, and therefore, have rare metastatic potential. A series of patients with aggressive angiomyxomas of the vagina and /or pelvis were identified in order to review the clinical presentation, pathology and managment. Study Design: Patients with a diagnosis of aggressive angiomyxoma of the vagina and/or pelvis, inclusive of Danbury and Yale-New Haven Hospitals, between 1985 and 2011 were identified.

Results: A total of 8 patients with histologically confirmed aggressive angiomyxoma of the vagina and/or pelvis were detected. The mean age was 50.6 years old. All of the patients were multiparous; three of the patients had body mass indexes (BMI) >37. Three patients were post menopausal. Five patients presented with symptoms of the tumor including vaginal swelling, vaginal prolapse, abdominal pain. All patients underwent surgical removal of the tumors. The size of the tumors ranged from 3cm to 15cm in size. Conclusion: Aggressive angiomyxoma should be considered in the differntial diagnosis of any female with a perineal, vaginal, and pelvic mass. MRI is the best imagining modality to evaluate aggressive angimyxomas, Surgical resection with clear margins remains the treatment of choice. The utility of homonal therapy in the treatment of aggressive angimyxomas based on ER and PR status is unclear; however, various hormonal treatments have been used to decrease tumor recurrence.

Enigmatic Role of Interleukin (IL)-1β in the Timing of Delivery in the Long-Evans Rat. Background: Prior to both normal and preterm birth, IL-1β levels increase in gestational tissues and invading leukocytes; it is considered to be an essential event of the birth cascade. However, administration of IL-1β induces preterm delivery in mice and rabbits, but not in Sprague-Dawley rats bringing into question its role in parturition in this species. Recently a new, highly specific hepta-peptide allosteric antagonist to the IL-1 receptor (1) was developed, named 101.10. We reported earlier that it delayed IL-1β-induced preterm delivery in mice (Reprod Sci 16 (3): 111A, Abst. 148). Since the Long-Evans rat is nearly 50% larger than white rats and is increasingly being used for parturition studies, we explored the role of IL-1β in its parturition process. Hypothesis: IL-1β is involved in parturition in the Long-Evans rat. Methods: We injected increasing doses of IL-1β (5-20 µg/dam, i.p., gestational day (GD 17), or 101.10 (1.5mg/kg/d x 3d, s.c., GD 18-20), or saline as control to pregnant Long-Evans dams and observed their gestational length. Uterine tissues and peripheral blood leukocytes were isolated from rats on GD 17, 20 and 22. Regression analysis or Student's t-test was used to evaluate results. Results: Gestational length did not change due to IL-1β administration (n=4 for each dose, y=0.138x + 535.6, p=0.888). In contrast, 101.10 significantly delayed delivery (saline vs. 101.10; 533.9 h ±1.19 (n=5) vs. 551.0 h ± 6.54 (n=4), p=0.023). Conclusion: These data suggest that IL-1β is a critical element of the birth cascade in pregnant Long-Evans dams, although on its own it cannot induce labour and delivery. Further information regarding the IL-1β role in parturition will be revealed when tissue and leukocyte analysis is completed. Funding: Supported by Juntendo Medical University and AIHS ITG PreHOT.

Adipokinins Objective: To evaluate adipokinin levels in women at risk for preterm delivery (PTD) Methods: Serum levels of leptin, adiponectin, and resistin were prospectively studied at 3 times (23-34weeks, 35-36weeks, at delivery) in pregnant women <35weeks who had preterm labor (PTL) in the current pregnancy (n=47) or a prior PTD (n=45). The levels were compared across time and between PTL and prior PTD groups and stratified by body mass index (BMI, kg/m 2 ). Covariates included parity, progesterone use, and corticosteroids for fetal lung maturity. Adipokinin levels were compared between pregnancies that resulted in PTD (n=32) and term delivery (TD, n=46) in each of the 3 times.

Results: The PTL group was younger with more nulliparas and corticosteroid use, but had less progesterone use; p<0.006 for comparisons to the prior PTD group. There were differences in leptin (p=0.002), but not in adiponectin or resistin levels over the 3 times between the PTL and PTD groups, after controlling for covariates.

When stratified by BMI, there were differences in leptin (p<0.001 for BMI<30; p=0.04 for BMI≥30) and adiponectin (p=0.02 for BMI<30; p=0.16 for BMI≥30), but not in resitin between the PTL and prior PTD groups. There were no differences in the adipokinin levels for each of the times, p>0.05 for all comparisons between PTD and TD. Conclusion: Prior studies suggest that adipokinins such as leptin inhibit uterine contractions. Higher mid-trimester leptin levels in PTL compared to prior PTD may represent a compensatory response to PTL and requires further evaluation in the study of PTL treatment. Introduction: Human parturition resembles an inflammatory reaction, where progesterone (P4) has been suggested to suppress pro-inflammatory genes expression to prolong pregnancy. Previously, we have confirmed the antiinflammatory effect of P4 on human myometrial cells and shown that this is regulated by both progesterone receptor (PR) and glucocorticoid receptor (GR). We revealed a novel P4 action in our microarray studies that some of the IL-1β-associated genes were enhanced by progesterone. Although it is still unclear which nuclear receptors are mediating this inflammatory effect of P4, it may provide a broader view of P4 action and help us understand more about the role of P4 and its relevant receptors in human parturition. Methods: Primary cultures of human myometrial cells were grown from myometrial biopsies obtained at the time of elective LSCS. Cells were exposed to different stimuli, IL-1β (5ng/mL), MPA (1 uM) and P4 (10 uM), either alone or in combination for 6 h, and then total RNA and protein were extracted from each culture. The concentration and purity of RNA were determined by value of OD260:280. Three samples were chosen as representatives, which had various endogenous PR level, but similar GR levels, for Affymetrix Human Genome U133 plus 2.0 Array. To confirm the results from this cDNA microarray, independent studies by qPCR was performed on the candidate genes. Results: Three gene lists were generated by combining the top 50 up-regulated and the top 50 down-regulated genes from the comparison of each stimulus to the vehicle control. In each list, genes were further filtered and only those with p value <0.05 and fold change >1.5 were analysed. In the list of IL-1βregulated genes, 36 genes were up-regulated and 7 genes were down-regulated. P4 reversed the effect of IL-1β on these genes expression in most of the cases. However, P4 showed enhancement of 6 IL-1β-induced genes (IRAK3, CXCL10, CCL8, IL7R, CXCL11 and HSD11β1) and 2 IL-1β-repressed genes (MMP16 and EGR1), whereas MPA failed to show the same effects on these genes except on IRAK3 and HSD11β1.

This study has shown that P4 not only plays anti-inflammatory roles in human myometrial cells but that it also enhances the effect of IL-1β on certain genes. These data also suggest that MPA cannot be used as an alternative to P4 in in vitro studies. Further studies will be carried out to investigate which receptors are involved in this enhancement. Background: Vascular Endothelial Growth Factor (VEGF) is a secreted polypeptide growth factor and a multifunctional cytokine that interacts with tyrosine kinase receptors VEGFR1 (FLT) and VEGFR2 (KDR). VEGFR1 enhances monocyte migration and tissue factor production, and can negatively regulate VEGFR2, which is involved in vascular proliferation and angiogenesis. PGF2α is a contractile agonist that also has multiple essential signaling roles in parturition including a significant role in the amplification of the inflammatory response, making a potential relationship between PGF2α and VEGF interesting. Hypothesis: PGF2α induces VEGF expression and modulates expression of its receptors in HMSMC. Methods: Cultured HMSMC from the upper (US) and lower (LS) uterine segments were treated with PGF2α (0.01 to 10 µM) for 24 h, and mRNA expression was quantified using Real-Time PCR. Results: VEGF148 did not respond significantly to PGF2α treatment. PGF2α at 10µM stimulated VEGF121 and VEGF165 expression (P<0.001). VEGF145 expression gradually increased with ascending concentrations of PGF2α, with significant (P<0.001) 2.9 and 3.6-fold increases in expression at 10µM in the LS and US, respectively. Increasing PGF2α induced an increase in FLT (VEGFR1) expression (LS P<0.001), but conversely decreased KDR (VEGFR2) (LS P<0.05), increasing the R1/R2 ratio. Conclusion and Significance: These data show that PGF2α increases the expression of three VEGF isoforms in HMSMC, and increases the R1 to R2 ratio. R1 contributes to chemotaxis by increasing monocyte migration and the release of tissue factors. Raised levels of pro-inflammatory cytokines and chemokines are associated with both preterm and term labor, with or without the presence of infection. The potential ability of VEGF to increase leukocyte activation and therefore cytokine release in HMSMC suggests VEGF could be an important participant in parturition, but needs further study. PGF2α may contribute to leukocyte activation and migration via VEGF, amplifying the inflammatory response for parturition. Objective: Omentin is a novel adipokine which can modulate an antiinflammatory pathway and is strongly expressed by the placenta. The function of omentin during pregnancy is unknown. The purpose of this study was to define the natural history of omentin longitudinally in human pregnancy and correlate observation with inflammatory cytokines. Study Design: This is a secondary analysis from a prospective multicenter study of culturally and ethnically diverse pregnant women with repeated measures design. Pregnant women greater than 16 years of age with a singleton gestation were recruited in the first trimester for specimen collection in three periods: 1) 5-13 weeks; 2) 14-26 weeks; and 3) 27-36 weeks gestation. At each collection point, serum concentrations of omentin-1 and markers of inflammation were determined by ELISA or Luminex. Statistical analysis was performed by paired t-test and regression analysis.

Results: Eighty eight women provided samples in the first trimester compared to 67 in the first and second trimesters and 61 in all three trimesters. Omentin averaged 16.3 ± 11 ng/ml in the first trimester, 12.7 ± 8 ng/ml in the second trimester, and 13.9 ± 9 ng/ml in the third trimester. In paired specimen comparison, the omentin concentration was significantly higher in the first trimester than in the second (p=0.0001) or third (p=0.0005) trimesters. Omentin was negatively correlated with BMI in the first trimester (p=0.017) and third trimester (p=0.0035), and negatively correlated with CRP in the second trimester (p=0.0369). Omentin concentrations were not correlated with CRP, TNF alpha, IL-6, IL-8, or IL-10 at any other time point. Conclusion: Omentin is significantly higher in the first trimester than in the remainder of pregnancy. Further study is needed to elucidate the significance of this finding and its potential correlation to the inflammatory state in pregnancy.

The Correlation of Serum Leptin and Recurrent Pregnancy Loss. Stephanie T Romero, Margarita Sharshiner, David W Branch, Robert M Silver. Obstetrics and Gynecology, University of Utah, Salt Lake City, UT, USA.

Objective: Obesity is associated with an elevated risk of pregnancy loss, and with elevated levels of the hormone leptin. However, it is unclear whether increases in leptin are associated with pregnancy loss, especially recurrent pregnancy loss (RPL). Thus, our objective was to compare maternal serum leptin in patients with and without RPL. Study design: Case-control study design with 134 women with unexplained RPL, defined as two or more pregnancy losses with no more than one live birth and 134 age-matched controls with at least one full term uncomplicated pregnancy and no more than one pregnancy loss. Maternal serum was analyzed for leptin using quantitative spectrophotometry. Results Conclusion: Leptin levels in women with RPL are lower than controls, even when controlling for BMI. The association between early pregnancy loss and obesity is unlikely due to increased leptin levels. The significance of relatively lower leptin levels in RPL patients warrants further investigation.

Interpregnancy Interval and Anti-Inflammatory Cervical Cytokine Milieu among Women with Prior Preterm Birth. Raj Shree, Hyagriv Simhan. Obstetrics, Gynecology, and Women's Health, Magee-Womens Hospital of the University of Pittsburgh, Pittsburgh, PA, USA. Objective: A history of prior preterm birth (PTB) is an established risk factor for PTB, and the risk of recurrence is increased with shorter interpregnancy interval (IPI). The mechanisms underlying the association between short IPI and recurrent PTB are unknown. We have previously demonstrated that higher concentrations of cervical anti-inflammatory cytokines in early pregnancy is a risk factor for subsequent early spontaneous PTB and upper genital tract inflammation. We sought to examine the association between IPI and concentrations of cervical anti-inflammatory cytokines in early pregnancy among women with previous spontaneous PTB. Study Design: Prospective cohort of 76 women with previous spontaneous PTB who had cervical fluid interleukin (IL)-4, IL-10, and IL-13 measured at less than 13 weeks were included in this analysis. Using published principal factor analysis methods (Paediatr Perinat Epidemiol. 2011 May;25 (3):277-82.), the cervical anti-inflammatory score (ANTI) was calculated. From our previous work, the higher the ANTI score, the higher the subsequent risk of preterm birth. IPI was calculated as the length in time from a previous spontaneous preterm birth and the conception date of the current pregnancy. Confounders included, level of education, marital status, gonorrhea, chlamydia, BMI, race, and cigarette smoking. We analyzed the association between IPI and ANTI using univariable and multivariable methods Results: There was a significant negative linear relation between IPI and ANTI (beta = -0.075, p=0.017, see Figure) . This association persisted after adjustment for confounders (p=0.02).

Among women with prior spontaneous preterm birth, there was a significant negative linear relation between IPI and ANTI, an established risk factor for preterm birth. Based on our previous work and the current finding, as IPI decreases by 1 month, the ANTI-associated risk of PTB is estimated to increase by 4%. Further investigation is necessary to determine whether this association can be utilized to develop recurrence prevention strategies. In western countries heart disease is one of the leading causes of death. The use of guinea pigs as an animal model to study heart disease is well established. To assess cardiac hypertrophy further a model of cardiac pressure overload induced by transverse aortic constriction (TAC) was developed in guinea pigs (GP). We aimed to study myocardial expression level of hypertrophic marker genes involved in cardiac hypertrophy in male and female GP and compared these to a sham operated control group. Methods: 4 groups of GP were used: GP subjected to sham surgery, male (n=8) and female (n=4) to act as controls; GP subjected to TAC, male (n=13) and female (n=8) to act as a pathological model of hypertrophy. Left ventricles were excised, placed in RNAlater, equilibriated overnight at 4 0 C and stored at -70 0 C. Total RNA was extracted from these tissues using the RNeasy kit from Qiagen and the mRNA converted to cDNA. Copy numbers of atrial nutriuretic peptide (ANP), brain nutriuretic factor (BNF), beta-myosin heavy chain (β-MHC), sarco endoplasmic reticulum calcium atpase (SERCA) and endothelial nitric oxide synthase (eNOS) were measured by qPCR using a Rotor-Gene TM (Corbett Research, Australia). The results are presented as mean ± SEM and as a ratio of GAPDH. Results: Aortic constriction increased ANP and BNF, in particular in the female group. ANP was raised in male from 16.30 ± 5.94 to 101.19 ± 132.74 and in females from 0.95 ± 0.22 to 1037.52 ± 682.97. BNF was raised in male from 14.64 ± 5.01 to 183.80 ± 93.48 and in females from 3.44 ± 0.65 to 1981.32 ± 1150.63 (p<0.05 in both cases). β-MHC, SERCA and eNOS were not affected by aortic constriction in male animals but the expression of all these genes were increased by TAC in the female group. Conclusions: Aortic constriction in the female group demonstrates an increased response in the up-regulation of traditional markers associated with cardiac hypertrophy in comparison to males. There is evidence to suggest that this response is further exaggerated in pregnancy as cardiac output increases and the heart compensates by developing a reversible physiological hypertrophic growth. We propose to use this animal model to study pregnant and nonpregnant GP to investigate the influence of pregnancy on maternal cardiac function and fetal outcome further.

Is Placental Drug Transport Compromised Following Acute and Chronic Prenatal Endotoxemia? Enrrico Bloise, 1 Melaine C Audette, 1 Sophie Petropoulos, 1 Mohsen Javam, 1 Manzerul Bhuiyan, 1 William Gibb, 3 Stephen G Matthews. Acute exposure to very high doses of lipopolysaccharide (LPS), inhibits placental multidrug resistance P-glycoprotein (P-gp; encoded by Abcb1a/b) and breast cancer resistance protein (BCRP; encoded by Abcg2) in the rat. This would impair fetal protection against harmful factors in the maternal circulation. However, it is unknown whether acute or chronic LPS exposure, in doses that mimic sub-lethal clinical infection, alters placental multidrug resistance. We hypothesized that acute or chronic sub-lethal LPS exposure decreases placental P-gp and BCRP expression resulting in reduced fetal protection to potential teratogens. METHODS: Sub-lethal acute and chronic LPS doses were titrated in C57/B6 mice by quantifying the ratio of dead/reabsorbed fetuses in a litter. Acute LPS (150ug/kg; ip) or vehicle (n=6/gp) was given at E15.5 and E17.5 and placentas and fetal units collected after 4 & 24h. Chronic LPS (5ug/ kg/day) or vehicle (n=6/gp) was administered from E11.5-15.5 and tissues collected 4h after last treatment. P-gp activity was assessed by measuring [ (3) H]digoxin accumulation (a P-gp substrate). Placental Abcb1a, Abcb1b and Abcg2 and interleukin-6 (IL-6) mRNA were measured by RT-PCR. P-gp and BCRP were assessed by Westerns. Maternal plasma IL-6 was also determined. RESULTS: At E15.5, maternal IL-6 was significantly elevated 4h after single (p<0.01) and chronic (p<0.05) LPS exposure, but levels had returned to baseline by 24h. Placental IL-6 mRNA levels were also significantly increased after acute and chronic LPS treatments (p<0.05). Conversely, Abcb1a, Abcb1b and Abcg2 mRNA and P-gp and BCRP protein expression were unaffected. Fetal digoxin accumulation was not different between groups. Of note, [ (3)H]digoxin accumulation was increased (p<0.05) in maternal hearts of mice exposed to chronic LPS treatments. CONCLUSIONS: Acute and chronic sub-lethal LPS exposure, resulted in robust inflammatory responses in the systemic circulation and in the placenta. However, in this model of infection there was no effect on the expression of placental P-gp and BCRP expression or P-gp activity.

Collectively, we demonstrate that sub-lethal LPS exposure during pregnancy does not impair fetal protection against potentially harmful xenobiotics in the maternal circulation. Introduction: Maternal elevated TSH concentration has been associated with adverse pregnancy outcomes. We hypothesise that TSH may have a direct impact on uteroplacental development. We sought to describe the ontogeny of the TSH receptor (TSHR) in human placenta and decidua, and elucidate a functional role for TSH in uteroplacental development. Methods: TSHR expression in human placenta (n=90) and decidua (n=51) (7-42 weeks gestation) was assessed by TaqMan RT-PCR, Western immunoblotting and immunohistochemistry. Trophoblast proliferation (MTT assay) and invasion through Matrigel® in response to TSH (0-100U/L) were assessed in vitro using the cell lines JEG3 (cytotrophoblast-like) and SGHPL-4 (extravillous trophoblast-like).

Results: TSHR mRNA was detectable in 66% of human placental samples (53% in first trimester, 83% at term) and 45% of decidual samples. Compared with first and second trimester placentae, term samples expressed 14 and 21-fold more TSHR mRNA respectively (p<0.05). There was no significant change in decidual TSHR mRNA expression across gestation. TSHR protein was present in placenta and decidua from the first trimester until term. Immunohistochemistry localised TSHR to syncytiotrophoblast, cytotrophoblasts, extravillous trophoblasts, fetal capillaries and decidual stroma. JEG3 cells demonstrated an inverted bell-shaped dose response with a trough in proliferation with 0.3U/L TSH compared with 0U/L (15%, p<0.001) after 24h. Proliferation remained reduced by 25% (p<0.001) and 17% (p<0.05) with 0.1 and 0.3U/L TSH respectively compared with 100U/L after 48h. In contrast, SGHPL-4 cells, showed a bell-shaped dose response in proliferation with a peak increase of 27% and 35% with 0.3U/L TSH compared with 0U/L after 24h and 48h respectively (p<0.001 for both). SGHPL-4 invasion increased 3-fold with 50U/L TSH compared with 0U/L (p<0.001) after 48h. Conclusion: TSH may have a direct physiological role in human trophoblast proliferation and invasion and could have an effect independently of thyroid hormone. Methods: Three choriocarcinoma cell lines (BEWO, JAR, and JEG-3), a first trimester placental cell line (HTR-8/SVneo), and a positive control (SCC-MT1) were used to induce MDSC from healthy donor PBMC using in vitro co-culture methods. A suppression assay was then performed and flow cytometry was used to measure their ability to inhibit T cell proliferation. All 5 cell lines were tested for both CD33+ and CD11b+ MDSC subsets and repeated with PBMCs from two different donors. The mean and SEM of each sample was analyzed by ANOVA followed by Dunnett's post-test for comparison to T cells only.

Results: All cell lines generated the CD11b+ MDSC subset with a significant frequency of induction of suppression. No induction of suppression was observed with the CD33+ subpopulation.

Conclusion: This study demonstrates the ability to induce suppressive CD11b+ MDSCs with human placental cell lines suggesting a possible novel mechanism for fetomaternal immunologic tolerance and a possible target for future studies of common placentally-related obstetrical complications. Inflammation during pregnancy has been associated with fetal growth restriction (FGR), stillbirth and later onset diseases in surviving newborns. Those pathologies are prominent in women experiencing reduced fetal movements (RFM) and our recent study has found evidence of placental dysfunction in cases of RFM (Warrander et al., 2012) . We hypothesised that inflammation occurs in pregnancies with RFM and contributes to placental dysfunction. Our aim was to determine the inflammatory profile in placentas from pregnancies with RFM compared to other pathologies of pregnancy. Methods: Placentas were collected from women experiencing RFM, FGR, or preeclampsia (PE) in the third trimester and compared with term uncomplicated pregnancies (n=9-12/condition). First trimester samples were also included to identify changes with gestation. ELISA was used to determine cytokine levels in placental lysate. Immunohistochemistry was used to assess cytokines localization and cellular origin. Results: Placental levels of interleukin (IL)-1β and receptor antagonist (IL-1Ra) were decreased at term compared to first trimester tissue (p<0.001). Conversely, IL-10, IL-6 and TNF-α levels were unchanged. Placental concentrations of both IL-1 agonists (α and β) as well as IL-1Ra were increased in RFM pregnancies as compared to normal (α: p<0.01, β and Ra: p<0.001). This induction of the IL-1 system was specific to RFM placentas since levels in placentas from both FGR and PE pregnancies were the same as in normal term placentas. Furthermore, IL-10 was decreased in RFM (p<0.001), but not in FGR or PE samples. IL-1Ra was localised mainly to the maternal facing syncytiotrophoblast throughout the villous tree.

Conclusion: This study reveals a unique cytokine profile in placentas from RFM pregnancies compared to other pregnancy pathologies, with a predominant increased in the IL-1 system and a concomitant decreased in anti-inflammatory IL-10. Elevated IL-1Ra is indicative of prior inflammatory stimulus and together with reduced levels of IL-10 suggests a bias towards an inflammatory phenotype in RFM pregnancies. Our data are consistent with our hypothesis that there is a contribution of inflammatory mediators to placental dysfunction in RFM pregnancies, leading to perinatal mortality and morbidity

Role of Inflammatory Chemokines in Gestational Diabetes Pathogenesis. D Giuffrida, 1 A Rolfo, 1 AM Nuzzo, 1 E Piccoli, 1 S Cardaropoli, 1 A Piazzese, 1 N Di Simone, 2 T Todros. 1 1 Obstet. and Gynaecol, University of Turin, Turin, Italy; 2 Obstet. and Gynaecol, Catholic University, Rome, Italy. Objectives: Gestational diabetes mellitus (GDM), defined as glucose intolerance of variable severity with onset or first recognition in pregnancy, is associated with increased risk of maternal and neonatal morbidity. It has been suggested that inflammatory chemokines might be implicated in the pathophysiology of GDM. Our hypothesis is that aberrations of expression and/or functionality of D6 and DUFFY decoy receptors, deputed to degradation of chemokines, could cause or contribute to chemokines accumulation typical of diabetes. The aims of the present study are to evaluate maternal serum levels of D6/DUFFYrelated chemokines and to investigate D6 and DUFFY spatial localization and expression in GDM placentae, in order to assess the contribution of these receptors to GDM pathogenesis. Methods: Placental, umbilical cord and placental membranes biopsies were collected from GDM (n=10) and control (CTRL, n=10) pregnancies. Maternal serum samples were obtained from the same study population. D6 and DUFFY mRNA levels was assessed by Real Time PCR while their spatial localization and expression were investigated by immunohistochemistry (IHC . Paraffin-embedded sections of processed placental biopsies were incubated for antigen retrieval. Immunohistochemistry was performed with primary rabbit polyclonal antibody to leptin and leptin receptor (LR) and for negative control normal rabbit immunoglobulin was used. Slides were digitized and morphimetric analysis was performed with Definiens' Tissue Studio to determine percentage of leptin and LR staining. Statistical analysis was conducted using the Student's t-test.

We found a 2-fold increase in placental LR expression in pregnancies affected by idiopathic macrosomia (p=.002). LR staining was positively correlated with fetal birth weight (r=.723). Leptin hormone staining was not significantly different between macrosomic infants and controls. LR was localized in cytotrophoblasts with intense staining found in extravillous trophoblasts when compared to CK-7, a known marker for cytotrophoblasts.

Idiopathic macrosomia is associated with an increase of placental leptin receptor (LR) expression as compared to normal pregnancy. Presumably, increased LR may lead to increased materno-fetal transport of nutrients, supporting excess fetal growth. Further studies are planned to elucidate the role of placental leptin signaling since idiopathic macrosomia may arise secondary to maternal and/or fetal signaling.

Effects of Combination Antiretroviral Therapy on Progesterone Levels and Birth Outcome in a Mouse Model. Eszter Papp, Lena Serghides. Centre for Global Health, University Health Network, Toronto, ON, Canada. Background: HIV-positive pregnant women are exposed to combination antiretroviral therapy (cART) to prevent mother-to-child transmission of the virus. cART typically consists of nucleoside analogues and protease inhibitors. This regimen has been associated with small birth weight and increased rate of preterm delivery. A widely used combination in pregnancy is zidovudine (AZT), lamivudine (3TC), with ritonavir-boosted lopinavir (LPV/r). Protease inhibitors can inhibit enzymes of progesterone synthesis. Decreased progesterone levels during pregnancy have been linked to small birth weight and preterm delivery. Objective: to investigate the effects of exposure to LPV/r-containing cART in pregnancy on birth outcomes and progesterone levels in mice. Hypothesis: exposure to LPV/r -containing cART will result in lower progesterone levels and lower fetal weight in pregnant mice. Methods: Pregnant C57BL/6 mice were exposed to human-equivalent doses of AZT+3TC+LPV/r during pregnancy. Group 1 was exposed from gestational day 1 (G1) till term (G18). Group 2 was exposed from G1 till G6 (pre-implantation). Group 3 was exposed post-implantation (G6 -G13). Maternal plasma was collected, number of implantations, fetal and placental weight, fetal viability and number of resorptions were recorded. Progesterone levels were quantified by EIA. Data were analyzed with Student's t-test and Pearson correlation. Results: in Group 1, cART exposure resulted in increased rate of pregnancy loss (38% loss in exposed vs. 0% in control). At G18, significantly more fetal demise (15.2% vs. 3.8 %) and decreased progesterone levels (578±28 vs. 619±18 ng/mL) were present in the exposed group compared to control. Mean placental and fetal weight was also significantly decreased in the exposed group (0.76 vs. 0.81 g and 0.085 vs 0.101 g, respectively). Progesterone levels correlated with fetal weight (R=0.677). In Group 2, number of implantations was unchanged, but progesterone levels were significantly lower in the exposed group (964±212 vs. 1119±94 ng/mL). In Group 3, drug exposure was associated with a significant increase in fetal demise (28% vs. 2.6%) without affecting fetal, placental weights or progesterone levels.

Conclusions: Delayed cART exposure shows benefit in maintaining normal fetal and placental weights and optimal progesterone levels in mice. Further studies aim to clarify the possible benefit of progesterone-supplementation to antagonize the adverse effects of cART. Background: Placental myostatin production is negatively correlated with gestational age and has been implicated in the control of glucose uptake. The roles of myostatin in placental development and functions are yet to be established. The localization of myostatin in the human placenta is unknown.

Objective: To determine the localisation of myostatin in first and third trimester placenta. Methods: Tissue selection: First and third trimester placental tissue collection was approved by the Human Research Ethics Committees of the Royal Brisbane and Women's Hospital, and the University of Queensland. Women gave written informed consent for the use of placental tissue for research purposes. First trimester (10 week) placentae were collected after clinically-indicated termination of pregnancy (n=3) and third trimester placentae (n=3) from elective caesarean section. Immunohistochemistry: Villous tissue from first and third trimester placentae were selected and processed by methods established and in routine use in our laboratory. Results: Localisation of cytokeratin 7, vimentin, and myostatin as well as isotype controls for antibodies used and secondary only control were conducted on serial sections. Isotype control and secondary only control were negative for staining on both first and third trimester placental sections. Trophoblast and cells of mesenchymal origin were identified by cytokeratin 7 and vimentin staining respectively. Myostatin in first trimester placenta was localised to cytotrophoblast, endothelial and stromal cells. Myostatin in third trimester placentae was localised to cytotrophoblast, intermediate trophoblast, endothelial and stromal cells.

Conclusions: Myostatin is localised in cells essential for the development and function of the human placenta. We postulate myostatin to be a key regulator of placental formation, development and function. Further research on the regulation and functions of placental myostatin could not only advance our understanding of placental development but also create new avenues for therapeutic intervention and diagnostic tools for complications of pregnancy.

Ovarian Preeclampsia (PE), a pregnancy-specific disorder characterized by late gestational hypertension and proteinuria, is one of the leading causes of perinatal and maternal morbidity and mortality. The inbred mouse strain BPH/5 spontaneously develops these cardinal signs of PE during pregnancy, along with early placental defects, decreased litter size, and low-birth-weight pups. Abnormal placentation in early gestation is thought to be responsible for the cascade of events leading to the clinical syndrome of PE, although the precise mechanisms are still unclear. Since proper placental development begins with synchronous implantation of an activated blastocyst into a hormonally-primed, receptive uterus, we hypothesized that defects in the implantation process would characterize the BPH/5 model of PE. BPH/5 mice exhibit a delay in timing of implantation marked by a complete absence of implantation sites at 3.5pm (n=6) and significantly fewer implantation sites still by 4.5am (6.5±1.5, n=6 vs C57 8.75±0.96, n=4; p<0.05). BPH/5 also show embryo clustering at e5.5 (43±8% clustered, n=5 vs. 4±4% for C57, n=6; p<0.05), a sign of dysregulated implantation in mice. This is associated with an aberrant preimplantation estrogen profile characterized by a premature estrogen surge at 2.5am (13.6±1.6pg/mL, n=6) that then drops significantly at the time C57 surge at 2.5pm (5.85±1.1, n=9 vs 16.97±2pg/mL for C57, n=7, p<0.05). Expression of the estrogen-sensitive implantation signaling molecules, Lif and Cox2, are delayed in BPH/5 implantation sites as measured by qRT-PCR. Significant Lif expression is not detected in BPH/5 until 4.5am (n=8) as compared to C57 12 hrs earlier at 3.5pm (n=8). Cox2 expression is also delayed by 12 hrs in BPH/5 as compared to C57, as is positive-staining for the decidual cell marker, alkaline phosphatase. Together, this points towards a delay in uterine receptivity and decidualization in BPH/5. Interestingly, BPH/5 females have irregular estrous cycles and deficient circulating estrogen levels prior to pregnancy (2.4±0.5pg/ mL, n=5 vs C57 7.5±0.9pg/mL, n=3; p<0.05). Our data suggest that BPH/5 mice have asynchronous and dysregulated implantation events, which are associated with inadequate circulating estrogen before and during early pregnancy.

Differential VEGF mRNA Splicing in Preeclampsia and Normotensive Human Placentas. (121, 145, 165, 189 and 346) , alternative splicing at a distal site in the terminal exon 8 of VEGF gene produce novel VEGF isoforms (VEGF xxx b) that translate proteins of the same length as the normal VEGF isoforms, but with different sequence and anti-angiogenic properties. However, whether these alternative VEGF splice variants are differentially expressed in preeclampsia is not fully understood. Objectives: To evaluate the differential expression of VEGF mRNA splicing variants in human placentas of various phenotypes of preeclampsia in comparison to normotensive controls. Methods: Placentas from normotensive deliveries (n=10/ group), severe preeclampsia (n=5-10/group) of early-onset, preeclampsia with chronic hypertension, IUGR or preterm birth, and paired controls were obtained via caesarean section. The placental samples were snapfrozen and total RNA was extracted using Trizol reagent. RNA (2 ug/sample) was reversed transcribed for PCR amplification of normal VEGF variants with a forward primer (exon 4, 5'-GAGATGAGCTTCCTACAGCAC-3') and a reverse primer (exon 8a, 5'-CTCACCGCCTCGGCTTGTCAC-3). VEGF xxx b variants were amplified with the same forward primer and a reverse primer (8b, 5'-TCAGTCTTTCCTGGTGAGAGATCTGCA-3). The amplicons were analyzed by agarose gel electrophoresis and sequencing. Placental protein was extracted for analyzing VEGF165 and VEGF165b by immunoblotting with specific antibodies. Results: Five VEGF amplicons (346 bp, 295 bp, 188 bp, and 91 bp) amplified with the exon 8a primer were detected in both normal and preeclamptic placentas, and the levels of VEGF165 mRNA and protein were found to be increased in preeclamptic placentas. The VEGF 165 b mRNAs was detected in both normal and preeclamptic placentas whereas VEGF 121 b was only detected in preeclamptic samples. Moreover, the ratio of placental VEGF/VEGFb was altered by preeclampsia. Detailed analysis of VEGF mRNA splicing with various phenotypes of preeclampsia is underway. Conclusion: Although both VEGF and VEGFb expressions are altered in human placentas by preeclampsia via mRNA splicing, changes of VEGF/VEGFb ratio suggest that alternative VEGF splicing is a crucial mechanism for placental angiogenesis that is deranged in preeclampsia (Supported by HL70562 & HL98746) . Objective: Recently, we have been developing and optimizing an imaging system based on a matrix -assisted laser desorption ionization (MALDI)based mass spectrometer, which provides clear two-dimensional molecular identification with highly sensitive mass spectrometry from mixtures of ions generated on tissue surface. In the present study, we applied this technology to investigate the difference of phospholipids molecular species of human placenta between preeclampsia and normal cases. Method: Placenta tissue blocks were obtained from Japanese pregnant women at Hamamatsu Medical University Hospital. Immediately after delivery, the blocks were frozen in liquid nitrogen. The tissues section were sliced into 8µm, mounted on to indium tin -oxide(ITO) glass slides, and sprayed matrix. By using MALDI-TOF/YOF type mass spectrometer (ultraflexII), we selected laser the tissue section, and got two-dimensional ion images. Two-dimensional ion images were compared to the hematoxylin and eosin (HE) (90%) were different from those induced by acute exposure to hypoxia. Ingenuity Pathway Analysis surprisingly revealed that only 5 of these differentially expressed genes were directly regulated by HIF1A and HIF2A. Rather, gene ontology analysis indicated that most of PCN-regulated genes were related to cell cycle and cellular movement, which were in agreement with bench-top functional bioassays that PCN significantly enhanced FGF2-and VEGFA-stimulated cell proliferation and migration. Interestingly, pre-exposing the PCN cells to 21% O2 up to 5 days did not completely diminished cell proliferation and migration. These PCN-enhanced cell proliferation and migration responses to FGF2 and VEGFA were mediated via augmented activation of the MEK/ERK1/2 and/or PI3K/AKT1 pathways. Importantly, these PCN-enhanced cellular responses were associated with an increase in the activation of VEGFR2, but not FGFR1 without altering their expression. Conclusion: PCN programs endothelial cells to undergo dramatic changes in transcriptomes and cellular proliferative and migrative responses to FGF2 and VEGFA. These PCN endothelial cells likely offer a unique endothelial model, more closely mimicking the in utero state.

(NIH P01-HD38843). Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis.

Resistance of Syndecan-1-deficient mice to experimentally induced tumorigenesis has been linked to an alteration in Wnt-responsive precursor cell pools suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. In this study, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human MDA-MB-231 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown substantially reduces putative cancer stem cell pools compared to controls. Moreover, the CD44(+)CD24(-/low) phenotype decreased upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated in Syndecan-1-silenced cells. Furthermore, activation of STAT-3 and NFkB transcription factors and expression of a coreceptor for Wnt signaling, LRP-6, were reduced in Syndecan-1-depleted cells compared to controls. Our study demonstrates the viability of flow cytometric approaches in analyzing cancer stem cell function. As Syndecan-1 modulates the breast cancer stem cell phenotype via regulation of the IL-6/STAT3 and Wnt signaling pathways, it emerges as a promising novel target for therapeutic approaches. Objective. LC-PUFAs are synthesized in the lactating MG. Docosahexaenoic (DHA) and Arachidonic (AA) acids are important in neonatal brain development. We evaluated effects of low maternal protein intake on MG development at different states of lactation and analyzed the milk fatty acid composition.

Methods. Pregnant rats received control (C -20% casein) or a restricted (R -10% casein) isocaloric diet during pregnancy and lactation. At 13 and 20 days of lactation (dL) pups from one set of mothers were removed from their mothers for 4h after which the mothers received 0.8U oxytocin (ip) and milk expressed 15 min later. Milk samples were assayed by Folch procedure and DHA and AA were determined by gas chromatography. Pups from a second set of mothers were removed from their mothers for 4h and were weighed individually immediately before they were returned to the mothers for nursing and again 1h later to quantify relative milk, fat, AA and DHA intake. At 14 and 21 dL, mothers were weighed and their MG removed for histological analysis and lipid extraction. Data are Mean ± SEM and analysis was by Student's T-test. Significance set at p< 0.05. Results. Total fat, DHA and AA milk offspring intake were lower in R group at 20 dL; at 13 dL DHA was lower in R (Table 1) . Maternal body weight decreased at 14dL and 21dL in R group. Total MG weight and fat content were lower in R group in comparison with C at the 2 different ages, lovulo-alveolar development were lower in R group. (n=3), UV-light exposed (n=3), obese (n=4), and non-obese (n=4). Additionally, four groups of non-pregnant primates were evaluated: Obese (n=6), Obese after 15% weight reduction (n=6), non-Obese (n=4), and UV-light exposed (n=6). Daily intake of Vitamin D was calculated based on the content in the total daily food consumption. The Vitamin D levels were was evaluated in the serum and plasma as described in previous studies. Bonferonni and Student t-test were applied for statistical analyses. P value < 0.05 was considered significant.

Results: The daily Vitamin D consumption for non-pregnant and pregnant primates was 1634.57 ±164 IU/day and 2406 ± 273 IU/day respectively. Vitamin D concentrations were not significantly decreased in non-pregnant obese primates compared to non-obese. However, in the pregnant primates, this decrease was significant. The concentrations of Vitamin D were not different in UV-light deprived and UV-light exposed pregnant primates. Conclusion: There was an almost four-fold increase in the daily intake of Vitamin D compared to the daily-recommended dosage for humans (4000IU/ day). Furthermore, the serum Vitamin D levels were two-fold higher in comparison to humans. The significantly lower Vitamin D concentration in obese primates, even in the presence of UV-light exposure, is likely due to abnormal Vitamin D metabolism. This is supported also by the fact that weight reduction alone did not increase Vitamin D concentrations in the obese.

The Objectives: Both osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) play an important role in the regulation of bone turnover, but the contribution of these two cytokines to the pathogenesis of osteoporosis remains controversial. The present study assess the relationship between the plasma levels of OPG, RANKL, bone turnover markers (BTMs) and bone mineral density (BMD) among postmenopausal women with and without osteoporosis. Methods: A total of 340 postmenopausal women diagnosed with osteoporosis and compared with age-matched healthy controls (n=340) with normal bone mineral density (BMD). Each women completed a questionnaire and provided fasting blood and second-void urine samples. Plasma OPG and RANKL were measured by using two-site sandwich ELISA -(Biomedical Gruppe Kits, Austria). Other analytes including PTH, FSH, LH, E 2 , 25(OH)D, bone turnover markers [serum osteocalcin (s-OC), serum procollagen type 1 N-terminal propeptide (s-PINP), urinary N-terminal cross-linked telopeptide of type 1 collagen (u-NTX) and cross-linked C-terminal telopeptide of type-1 collagen (s-CTX)] and interleukin-6 (IL-6) were measured by commercially available kits and reagents on autoanalyzers. Results: Plasma OPG was significantly higher in women with osteoporosis (19.26 ± 8.44 pmol/L) than in age-matched controls (11.15 ± 4.66 pmol/L) (P<0.0001). Similarly, plasma RANKL levels were significantly higher in women with osteoporosis (0.71 ± 0.48 pmol/L) than in age-matched controls (0.34 ± 0.29 pmol/L) (P<0.0001), respectively. The OPG/RANKL ratio was higher in women with osteoporosis vs age-matched controls (P<0.001). Women with osteoporosis showed higher levels of BTMs, IL-6 and PTH but lower serum 25(OH)D and E 2 than corresponding controls. Multiple regression analysis showed that BMD values for lumbar spine (L 1 -L 4 ) and neck femur were predicted by OPG (by 17.6%) and RANKL (by 12.3%), respectively. Conclusions: Both circulating OPG and RANKL levels inversely related to BMD among postmenopausal women through increased bone resorption.

Bringing Background: G protein-coupled receptor 30 (GPR30) has been proposed to mediate acute estrogenic responses on the cardiovascular system. Studies in animals suggest its involvement in the regulation of vascular tone via nitric oxide (NO) signaling, however little is known about its function in human arteries.

Objective: We hypothesized that GPR30 receptor expression and function differs in isolated small arteries from women and men. We aimed to study acute vasodilatory effects of GPR30 agonist G-1 and to assess GPR30 expression in isolated small arteries from pregnant and non-pregnant women, as well as from age-matched men. Methods: Human subcutaneous arteries dissected from three groups were mounted on a wire myography system. After pre-constriction with NE(3µM), concentration-response curves to incremental concentrations of GPR30 agonist (G1) (10 -8 -3´10 -6 M) were obtained before and after NO synthase (NOS) inhibition. Immunohistochemistry was performed on frozen sections to detect the GPR30 expression in the artery wall from all three groups. Immuno stains were then scored blindly using an arbitrary scale 0-3 by three investigators.

The highest relaxation to G1 was observed in arteries from postmenopausal women (% max relaxation from initial preconstriction (40±5, n=12), which differed significantly from that in arteries from men (20±5%, n=5) and pregnant women (20±5%, n=11). NOS inhibition reduced relaxation to G1 in arteries from postmenopausal women (48±5% versus 27±5%, n=10 p< 0.05), totally abolished dilatation in arteries from pregnant women (n=5) and had no effect in arteries from men (n=4). The highest staining intensity was observed in arteries from pregnant women (2.55±0.4, n=6) , while reduced staining was observed in arteries from postmenopausal women (1.93±0.4, n=6) and men (1.16±0.3, n=6, p< 0.05). Conclusions: G-1 acutely dilates human peripheral resistance arteries with its greatest effect in postmenopausal women. NO significantly contributes to G1 induced dilatation in arteries from post-menopausal and pregnant women only; however, other mechanisms confer G1 induced relaxation in male arteries. GPR30 protein is present in arteries from women and men. Acute activation of this receptor may emerge as a candidate therapeutic target for cardiovascular disease.

Bazedoxifene Conclusion: Bazeodoxifene treatment led to significantly decreased expression of BRCA2 in mouse mammary tissue. BZA is unique among currently available SERMs and TSECs in its ability to suppress BRCA2. BRCA2 has a well known function in DNA repair and cell cycle control. Low BRCA2 expression is seen in quiescent cells and here may indicate beneficial effect of BZA on the mammary gland.

Tapering and Low Dose Maintenance GnRH Agonist Therapy for Symptomatic Uterine Leiomyoma in Perimenopausal Women: "Draw-Back Therapy." Toshihiro Yoshimura. OBGYN, Kumamoto Shinto General Hospital, Kumamoto, Japan. Introduction: The efficacy of long-term, gradually decreasing gonadotropin releasing hormone agonist (GnRHa) therapy, so-called draw-back therapy, for the treatment of uterine leiomyoma was investigated. Menopause is delayed in patients with leiomyoma, but GnRHa accelerates bone loss. Its use for longterm treatment of leiomyoma may thus be indicated only in perimenopausal patients with an absolute contraindication to surgery. Patients and Protocols: This retrospective study covers the period from June 2008 to August 2012. The subjects were 21 women with uterine leiomyoma who underwent drawback therapy. All were between 48 and 55 years old, weighed 43 to 72 kg, and had medical or surgical complications. Menstrual cycles were regular, but all showed profuse uterine bleeding. Hysterectomy was recommended, but longterm GnRHa therapy was thought justified. In 13 patients, leuprolide acetate depot injections, 1.88 mg once monthly, were given 2-3 times until uterine bleeding stopped. The patients then received nafarelin, 400 µg daily. In 8 patients, nafarelin 400 µg/day was given from the beginning. Uterine bleeding stopped in all cases after 3 months of treatment. Standard high-dose GnRHa treatment was followed by lower doses. A daily dose of 200 µg (a single nasal spray) was used for 2-3 months, followed by 200 µg every other day for 2-3 months, and then 200 µg every 3 days was continued. Serum E2 and FSH were monitored. After 2 years, treatment was discontinued, and the patients were observed to determine whether menopause occurred. Bone mineral density was within the normal range. Results: In most patients, uterine bleeding stopped with a very low maintenance dose of GnRHa, and E2 (<10 pg/ml) and FSH (<10 miu/ml) levels were suppressed. Four patients reached menopause by the end of treatment, and 7 soon after treatment. Four patents are still under treatment, and 5 exhibited menorrhagia, so nafarelin was restarted. In one patient, massive uterine bleeding occurred at the lowest dose. Serum E2 was 120 pg/ml, and hysterectomy was performed. A few patients complained of mild hot flushes. Conclusion: This study shows that GnRHa draw-back therapy suppresses the pituitary ovarian axis, and that uterine bleeding is controlled at a very low dose. It was thus an effective and economical therapy that led to natural menopause in selected cases. However, the potential for bone loss and prophylactic administration of bisphosphonates needs further study.

Myometrial Artery Calcifications Increase with Aging. Sarah C Hessler, Gerson Weiss, Debra S Heller, Peter G McGovern, Sara S Morelli, Laura T Goldsmith. Obstetrics, Gynecology and Women's Health, New Jersey Medical School, Newark, NJ, USA.

In women, risks for developing coronary heart disease increase with age.

Identifying markers and models for study of vascular aging is required to distinguish the roles of age from menopausal status on cardiovascular disease risk. The presence of vascular calcifications seen in imaging studies, including mammography, x-ray, CT, or ultrasound, has been correlated with increased risk of cardiovascular disease. Vascular calcifications of peripheral vessels, including breast arterial calcifications on mammography, correlate with increased prevalence of coronary heart disease. However, no prior studies have examined myometrial artery calcifications and whether the prevalence is related to aging. Since greater than 500,000 hysterectomies are performed annually in the US, myometrial arteries may be a convenient source of material for assessment of vascular changes. We hypothesized that the prevalence of myometrial artery calcifications increases with age and if so hysterectomy samples can serve as a useful model for vascular aging studies. Medical records of all women older than 45 years who had a hysterectomy for a benign indication at University Hospital in Newark, NJ, between July 1, 2009 and June 1, 2012 were reviewed (n=172). Hematoxylin and eosin stained slides of the myometrium prepared at the time of hysterectomy were evaluated by a single pathologist blinded to the patients' characteristics. Myometrial artery calcifications, defined as the presence of acellular densely basophilic material within the media of the vessels, were designated as present or absent. Data were analyzed using multiple logistic regression.

The prevalence of myometrial artery calcifications increased with advancing chronologic age (p=0.022). 2 of 51 women age 50-59 (age 56 and 58, 3.9%), 10 of 27 women age 60-69 (37%), and 5 of 10 women age 70 or greater (50%) had myometrial artery calcifications. Myometrial artery calcifications did not correlate with number of years since menopause. These data demonstrate that myometrial artery calcifications are strongly correlated with advancing age. This is the first study which shows that histologic sections from hysterectomy specimens are a useful model for the evaluation of vascular aging. The presence of myometrial artery calcifications may present a unique opportunity for women undergoing hysterectomy to be counseled regarding the potentially increased risk of developing cardiovascular disease. Objective: The H1FOO gene encodes the member O protein of the histone H1 family. Its expression is restricted to the growing/maturing oocyte and to the zygote in mice. The upregulation of the oocyte specific H1FOO transcript and protein was shown to be linked to the recruitment of primordial follicles, therefore, to the onset of oocyte development. The human H1FOO gene is located on chromosome 6 and the corresponding transcript gives rise to two distinct, alternatively spliced mRNA species (H1foo α and H1foo β ). We investigated whether nucleotide variants were present in the H1FOO gene in women with premature ovarian failure (POF) as compared to control women.

We collected genomic DNA from predominantly North American Caucasian women with POF defined as amenorrhea prior to age 40 with elevated FSH >20 IU/L. Primer sets specific for all 5 exons of transcript one and 2 exons of transcript two of H1FOO were designed for polymerase chain reaction (PCR). The PCR products were then sequenced using Sanger sequencing method and systematically compared to the human genome database sequence (ENSG00000178804) using Sequencher v5.0 (Gene Codes Corporation, Ann Arbor, MI). Chromatograms were also manually examined to identify sequence variants.

In 60 women with POF, we detected two novel sequence variants in 3' untranslated region (UTR) and one novel variant (c.712G>C, p.Lys192Asn) in exon 4 of transcript two of the H1FOO gene. These variants were not found in controls (N=65). The effect of non-synonymous coding region variant c.712G>C (p.Lys192Asn) on protein function was predicted to be tolerated by using the SIFT (Sorting Intolerant from Tolerant) algorithm. The significance of the novel variants in the 3' UTR region is unclear. Discussion: We found three novel variants in the H1FOO gene in women with POF. However, the frequency of these variants appears to be low in our study population. Moreover, the functional impact of these variants remains unclear.

Further studies are necessary to confirm these findings and to determine their impact on the gene's function.

Kisspeptin: Switching Gears from the Brain to the Ovary. Background: The G-protein coupled receptor (KISS-1R), whose ligand is the excitatory neuropeptide kisspeptin (KISS-1), is essential for the regulation of GnRH neurons and puberty. Recent studies have demonstrated that the gonadotropin hypersecretion in postmenopausal women is secondary to an increase in KISS-1 mRNA in the hypothalamus neurons, which encodes kisspeptin peptides. Interestingly, the kisspeptinergic system is also found in the ovary where it plays a role in ovulation and oocyte maturation. However, the role of human granulosa cell (GC) kisspeptin signaling in reproductive aging is still unclear. The objective of this study was to test the hypothesis that reproductive aging alters GC kisspeptin signaling by altering KISS-1 and/or KISS-1R mRNA expression in human GCs. Materials and Methods: 12 women (mean age=34.6 ± 1.5 years, mean BMI=28.5 ± 1.9 kg/m 2 ) who underwent controlled ovarian hyperstimulation followed by oocyte retrieval were enrolled. Cumulus GCs were collected. The RNA was isolated, reverse transcribed, then KISS-1 and KISS-1R mRNA expression was quantified using RT-PCR. Relative gene expression was calculated using the 2 -∆∆CT method with GAPDH as reference gene. Kruskall Wallis and Spearman correlation were used as appropriate.

Results: There was no amplification of the KISS-1 mRNA in our samples. Among all participants, KISS-1R mRNA levels were positively correlated with age (r= 0.71, p=0.009). When subdivided into 3 age groups, KISS-1R mRNA levels were significantly increased with age.

KISS-1R mRNA among three age groups Age (years) <35 (n=4) 35-39 (n=6) >40 (n=2) P value KISS-1R mRNA 4.3 (3.8-4.8) 18 (21-127) 196 (379-562) 0.004 Data are expressed as median (25th-75th percentile) Conclusion: These preliminary data strongly suggest that in an infertile population, age-related upregulation in KISS-1R expression may alter kisspeptin sensitivity in human GCs indicating a possible age-related physiologic role for the kisspeptin system in human follicular dynamics. Understanding the role of kisspeptin in the ovary may offer opportunities for innovative therapeutic options for the treatment of ovarian aging. The strength of uterine contractions during labor is sufficient to occlude uterine blood vessels, leading to transient hypoxic episodes. Despite this, uterine contractions are maintained, their strength increases and labor progresses. Previously, we showed that repeated 5 minute episodes of hypoxia significantly increase the force of contraction in pregnant rat uterus, suggesting that 'hypoxic preconditioning' may be occurring. We have now investigated if this phenomenon occurs in human myometrium, and at different stages of gestation and what the underlying mechanism may be. Methods: Uterine strips (2mm×10mm) from rats (non-pregnant, day18, and term-pregnant (n=8-11) and women undergoing term elective (n=7) and in-labor cesarean section (n=4) were dissected and mounted in organ baths, bubbled with HEPES-buffered, oxygenated physiological saline solution, in the presence of oxytocin for isometric force recording. The effect of repeated 2, 5 or 10min hypoxia was studied by replacing the O 2 for N 2 . The effect of varying O 2 concentration (2-20%) was also studied. Paired control experiments were bubbled with O 2 throughout. Contractions were analyzed during and after each hypoxic episode.

In some experiments strips were loaded with Indo-1 AM for simultaneous measurement of force and intracellular Ca (n=5). Results: Hypoxia abolished or significantly reduced contraction force in all tissues. In non-pregnant and day18 pregnant rats there was no increase in contractility with repeated hypoxic episodes. Term pregnant rats showed an increasing contractile strength over 5 hypoxic episodes. The effect was largest with the briefer hypoxic episodes and required the O 2 to be below 10% in the tissue bath. Preliminary data with Indo-1 suggest that hypoxia significantly increases basal Ca but the increased contractility after hypoxic episodes is not due to increased Ca signalling. Similar increases in contractility after hypoxic episodes were found in laboring human myometrium (after 1 episode of hypoxia, p<0.05), but not non-laboring tissues. Conclusions: There is a gestationally dependent mechanism in rat and human uterus that leads to increases in contraction amplitude triggered by brief episodes of hypoxia. These changes appear not to be due to increased Ca transients and suggest increased sensitivity of the myofilaments. These changes will help maintain myometrial contractility in the face of adverse metabolic changes during labor.

Hormonal Regulation of Hydrogen Sulfide-Producing Enzymes in the Mouse Uterus. Kelsey E Breen, Robert Valdez, Brian Wakefield, K Joseph Hurt. Obstetrics & Gynecology, University of Colorado, Aurora, CO, USA. Background: The small molecule gaseous messengers, nitric oxide (NO) and carbon monoxide (CO), are capable of eliciting uterine relaxation. Hydrogen sulfide (H 2 S) is the newest gasotransmitter and also relaxes smooth muscle; however, its role in uterine tocolysis is not well understood. NO production in the reproductive tract is modulated by estrogen and progesterone, but hormonal regulation of H 2 S synthesis is not described. We investigated the expression of H 2 S-producing enzymes, cystathionine β-synthase (CBS) and cystathione γ-lyase (CSE), in the mouse reproductive tract after exogenous hormone treatments to determine the possible role of endogenous H 2 S in parturition and in the menstrual cycle. Methods: Immunohistochemical and immunofluorescent staining were used to localize CBS and CSE expression in mouse uterine tissue. C57/Bl6 virgin female mice were ovariectomized and injected two weeks later with sesame oil (vehicle), estradiol (.5 mg/kg), progesterone (.5 mg/kg), or estradiol plus progesterone (.5 mg/kg each) for two days. The female reproductive tract was harvested on the third day. Hormonal effects were determined by vaginal swab cytology and uterine morphology. CBS and CSE expression were evaluated by qPCR normalized to the housekeeping gene RPL13A and western blot normalized to βactin. Results: Immunolocalization demonstrated CBS and CSE expression in the myometrium. Estrogen significantly upregulated CBS mRNA compared to vehicle-(p<0.05) or to progesterone-(p<0.05) treated tissues (N=8-10) as assessed by qPCR. There were no changes in CSE mRNA levels across treatment groups (p=0.67; N= 7-10). CBS protein expression was not significantly different across treatment groups, but progesterone dramatically upregulated CSE protein expression in the upper uterus as compared to sesame oil (p<0.05), estradiol-(p<0.01), or estradiol plus progesterone-(p<0.01) treated tissues (N= 5). Conclusions: H 2 S-producing enzymes are regulated by estrogen and progesterone in mouse uterus. Sex hormones may inversely regulate CBS and CSE expression via transcription and post-translational mechanisms, respectively. Taken together, these results suggest that the hormonal changes throughout pregnancy or the menstrual cycle may directly influence H 2 S production.

Relaxant Effect of Selective Estrogen Receptor Modulators (SERMs) on Human Pregnant Myometrial Contraction. Noppamart Whangteeranon, 1 Apilada Ojaroen, 1 Areepan Sophonsritsuk, 1 Patama Promsonthi, 2 Boonsri Chanrachakul. 2 1 Obstetrics and Gynecology, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 2 Obstetrics and Gynecology, Bumrungrad International Hospital, Bangkok, Thailand. Objective: To investigate the relaxant effect of selective estrogen receptor modulators (SERMs), tamoxifen, raloxifene, and fulvestrant, on oxytocininduced human pregnant myometrial contraction. Methods: This study was approved by the Ethical Committee and writen informed consent was obtained from each participant. Myometrial biopsies were obtained from 18 low-risk term pregnant women, undergoing elective cesarean section. Tissue stripes were placed in the organ chambers under physiologic condition for isometric tension recordings. Myometrial contractions were induced by oxytocin (10 -9 mol/L). The cumulative effect of SERMs, tamoxifen, raloxifene, and fulvestrant, (from 10 -9 to 10 -4 mol/L) were recorded and evaluated. Results: Tamoxifen, raloxifene, and fulvestrant have significant relaxant effect on oxytocin-induced contraction of human pregnant myometrium (P< 0.05). The values of log-transformed half maximal inhibitory concentration (log IC 50) for tamoxifen, raloxifene and fulvestrant were -6.0 ± 0.85, -5.4 ± 0.79, -5.9 ±1.67 mol/L, respectively. There was no significant difference in the relaxant effect among these drugs. Conclusion: Tamoxifen, raloxifene, and fulvestrant exert significant concentration-dependent relaxant effect on human term pregnant myometrial contraction. These results demonstrate the selective effect of SERMs on pregnant human myometrium which may raise the possibility of using SERMs as the tocolytic agents.

Myometrium. Jeanette Chin, 1 Erin Zinkhan, 2 Jennifer Zalla, 2 Ben Numpang, 2 Robert Lane, 2 Lisa Joss-Moore. 2 1 Obstetrics and Gynecology, Maternal Fetal Medicine, University of Utah, Salt Lake City, UT, USA; 2 Pediatrics, Neonatology, University of Utah, Salt Lake City, UT, USA. Objective: Maternal obesity increases the risk for dysfunctional labor, failure-to-progress in labor, and postpartum hemorrhage. Such labor abnormalities are indicative of impaired myometrial contractility. Ex-vivo , myometrial contractility is impaired by lipid accumulation. A key contributor to lipid accumulation, particularly in the context of a high-fat, high-cholesterol diet (HFCD), is the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). This lipid accumulation is mediated in part by PPARγ target genes, including the muscle-specific fatty-acid binding protein-3 (FABP-3). However, it is unknown whether a maternal HFCD affects mRNA levels of PPARγ and FABP-3 in myometrium. We hypothesized that a maternal HFCD would increase myometrial PPARγ and FABP-3 mRNA transcript levels. Methods: Female Sprague-Dawley rats were fed either a HFCD (n=6) or regular diet (n=5) for 5-8 weeks and then mated. Their respective diets were maintained throughout gestation. The dams were sacrificed at term (embryonic day 21), but prior to the onset of labor. Maternal serum and myometrial tissue were harvested. Quantification of serum lipid levels was performed at ARUP Laboratories. Levels of myometrial PPARγ-1, PPARγ-2 and FABP-3 transcripts were determined with real-time RT-PCR.

Results are reported as HFCD as % of regular diet control. The HFCD increased maternal serum total cholesterol (855%, p<0.01). LDL was only detectable in serum from the rats fed the HFCD (562.6 mg/dL). The HFCD increased mRNA levels of the PPARγ-1 transcript (294%, p=0.05). PPARγ-2 mRNA was undetectable in myometrium from either group. The HFCD increased mRNA FABP-3 levels (392%, p=0.06).

A maternal HFCD increases myometrial mRNA levels of PPARγ-1 and its target gene FABP-3 in the rat. We speculate that protein levels will reflect these observed changes in mRNA levels. Furthermore, in the setting of a HFCD, PPARγ-1 signaling may contribute to myometrial lipid accumulation with resultant labor abnormalities.

Contractile Mechanisms in the Cervix and Uterus. Emma S Darios, Stephanie W Watts. Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA. Both the uterus and cervix express smooth muscle that is pharmacologically sensitive to contractile agonists. Due to the difference in function of these two tissues, we hypothesized that their basic contractile mechanisms would be different. Strips and rings of virgin Sprague Dawley uterus and cervix tissues, respectively, were mounted in isolated tissue baths for measurement of isometric contraction. We studied two agonists, which activate heptahelical G protein linked receptors, oxytocin (OT) and the muscarinic cholinergic agonist carbamylcholine (carbachol). The contractile mechanisms investigated have been validated in vascular smooth muscle. The Erk MAPK pathway was inhibited by PD098059 (1 mM), but did not modify contraction (potency or efficacy) in either tissue to either agonist (table) . Similarly, the phosphoinositide-3-kinase inhibitor LY294002 (10 µM) and reactive oxygen species inhibitor 4-hydroxy Tempo (1 mM) were without effect on agonistinduced contraction in the uterus. 4-hydroxytempo reduced OT-induced contraction 59% (vs. vehicle max) in the cervix. By contrast, the L-type voltage dependent calcium channel antagonist Nifedipine (1 µM) inhibited maximal contraction to OT in the uterus 32% and cervix 20%. A similar inhibition was observed with carbachol as the agonist in the uterus (26%) and cervix (33%). Notably, the Rho Kinase inhibitor Y-27632 (10 µM) was ineffective in reducing carbachol-induced contraction in the uterus and cervix, but significantly reduced contraction to OT in the uterus (39%) and cervix (47%). These findings illustrate that reproductive smooth muscle mechanisms are not identical to those in vascular muscle and contractile agonists do not activate the same pathways. There are subtle differences in mechanisms activated within the uterus and cervix. Understanding these mechanisms is an important step to understanding how cervix and uterus function might be independently influenced. The RNA binding protein p54nrb exerts multiple functions and participates in a variety of nuclear processes, including transcription initiation, RNA processing, and DNA repair. p54 binds steroid receptors and further recruits the Sin3A/HDAC transcription repressor complex to inhibit downstream gene transcription initiation. p54 also forms a protein complex with the spliceosome to play essential roles in pre-mRNA splicing. These multiple functions of p54 could regulate gene transcription both positively and negatively, but the molecular mechanism is unclear.

Our previous work demonstrates that protein phosphatase-1 dephosphorylates p54 at serine and threonine residues, alters p54 protein associations and therefore modulates p54 transcription corepression and RNA splicing activities.

In this report, we further demonstrate that the tyrosine residue 267 is also important for the many functions of p54. Using luciferase reporter assays, we

show that p54wt induces 50% decreases in both the androgen receptor (AR) and the progesterone receptor (PR) transactivation. Mutation of tyrosine 267 into phenylalanine, p54(Y267F), results in 2 and 6 fold increases in PR and AR transactivation, respectively. In our RNA splicing assays, we show that p54wt induces 30-40% increases of both ARwt and ARv7 (an AR splice variant) transcripts, while p54(Y267F) results in a 70% decrease of ARv7 transcripts. In addition, we show that p54(Y267F) decreases protein interactions with the HDAC1 corepressor, and U1A and U2AF RNA splicing factors when compared to p54wt. We conclude from these data that tyrosine residue 267 plays important roles in regulating the multiple functions of p54. (Figure) .

We surveyed the pregnant mouse and human uterus for splice factors that may be utilized for alternate ERα splicing and regulated by P4 action. Western analysis demonstrated the presence of SF2/ASF, hnRPA1 and BARX2 that allow alternative pre-mRNA splicing. SF2/ASF has been shown to be essential for differential isoform expression and functions by preventing exon skipping in pre-mRNA processing. We show a proportional increase of SF2/ASF ( Figure) associated with the increased ERα66 and decreased ERα46 isoform in the term mouse uterus, indicating a potential regulatory role of SF2/ASF in ERα isoform production.

In conclusion we speculate that understanding the regulatory mechanisms involved in alternate splice variant generation with respect to uterine ERα isoforms and P4 action will provide a clear understanding of how E2 actions during pregnancy are regulated in a tissue specific manner. Dystocia accounts for ∼20% of all caesarean sections (CS) in the western world. The only treatment available is oxytocin (OT), which only works in 50% of cases. Lactate is significantly increased in myometrial capillary blood during dystocia suggesting it may be impairing force production, but there are no functional data on the effect of lactate on the myometrium. We have therefore investigated its effect on contractility. Methods Myometrial strips were taken from non-pregnant or late pregnant rats and biopsies obtained with consent from women undergoing CS at term or an hysterectomy. The effects on spontaneous contractility in response to sodium lactate and another weak acid, propionate (1-20mM) were investigated. OT-stimulated contractions (0.1nM rat, 0.5nM human) were also investigated. In some experiments simultaneous force and intracellular Ca signalling or intracellular pH (pHi) were measured. Statistical differences were tested using non-parametric tests and significance taken as P<0.05. Results Lactate dose dependently, significantly decreased spontaneous contractility; e.g. at 5mM the integral of force was 32%±12% and 28%±13% relative to control (100%), in rat (n=6) and human (n=5) respectively. Propionate also significantly reduced contractions. The effect on pregnant myometrium was greater than in non-pregnant (n=5 rat, n=4 human). The effects of lactate were significantly reduced in the presence of OT; 5mM lactate reduced force to 68%±17% in rats (n=6) and in human to 64±15% (n=5). Significant reductions in force in labouring myometrium were seen and in the presence of OT, were also found (amplitude 81%±17%, n=6). Lactate decreased pHi (n=5) and also inhibited Ca transients (n=3) and its effects mirrored those of force.

Conclusions Lactate in the physiological range potently decreases spontaneous contractility in both rat and human myometrium. The effects of lactate were reduced in the presence of OT or labour but still produced significant decreases.

Other weak acids produce similar effects to lactate suggesting its mechanism of action is not via lactate's role in metabolism. Lactate inhibited Ca transients, which could be due to a fall of pHi. We suggest that differences in myometrial lactate production/efflux in women can lead to accumulation of extracellular lactate, which as we have shown, will reduce myometrial contractions and could therefore contribute to labour dystocia. We recently reported that myometrial distension in nonpregnant rats stimulates growth of the uterine vasculature, which may be one mechanism that underlies the expansive vascular remodeling that occurs during pregnancy. Following abdominal incision in adult Sprague-Dawley rats (n= 16), medical grade silicone (∼0.2 ml) was infused into one randomly selected uterine horn, and the uterine corpus was ligated near the cervix and below the oviduct to prevent leakage. While the initial distension is modest, uterine stretch and/or silicone stimulates the secretion of an exudate into the uterine lumen causing further distension and a progressive increase in uterine volume from 2.3 to 12.7 ml in four weeks. The aim of the current study was to evaluate the composition of the exudate for proangiogenic factors, inflammatory cells, and cytokines. After sacrificing the animals, the uterine fluid was collected by syringe, centrifuged to separate out the silicone, and then analyzed for its molecular (ELISA; multiplex assay) and cellular (automated hematology system; cytospin) components. A myometrial segment and a serum sample were also collected for analysis. There were no detectable elevations of growth factor or cytokine content within the myometrium or in the serum. In the exudate, VEGF and PDGF concentrations were increased 23-fold and 15-fold above circulating levels. There were also modest increases in neutrophils, activated and inactivated macrophages, and in three cytokines (RANTES, CCL20 and GRO-KC). Conversely, PlGF, TGFβ, IL-2, IL-6, and IL-17 were not detectable. In summary, myometrial stretch triggered by silicone injection leads to the secretion of a lumenal exudate, presumably from the uterine epithelium, which is highly enriched in several proangiogenic growth factors relative to the blood. The clear, straw-colored fluid also contains modest elevations of monocytes and cytokines indicative of a mild inflammatory response. We speculate that reabsorption of angiogenic growth factors into the venous circulation may stimulate venous growth directly, and facilitate arterial growth indirectly via venoarterial exchange, in view of the close apposition of arteries and veins within the mesometrium. This concept is supported by previous studies showing that the uterine venous wall is permeable to large (70 kDa) tracers, and that solute permeability is significantly augmented during pregnancy. The maintenance of uterine quiescence throughout gestation is vital for a successful pregnancy. We previously demonstrated a tocolytic role for uterine caspase 3 (CASP3) during pregnancy and this current study defines the endoplasmic reticulum stress response (ERSR) as a mediator of uterine CASP3 activity in vivo. We have identified that the myometrium utilizes the ERSR to monitor, tolerate and adapt to intrinsic and extrinsic contractile stimuli throughout pregnancy via the activation of tocolytic CASP3. Furthermore, elevated levels of myometrial CASP3 activity targets the gap junction protein Connexin 43 (Cx43) for cleavage and sequestration from the myometrial cell membrane thus reducing the myocyte contractile potential. In vitro analysis +/-CASP3 inhibitors confirmed Cx43 is a direct target for uterine CASP3 action.

Additionally, this study reveals that a failure to elicit an appropriate ERSR results in a premature ablation of CASP3 which leads to a precocious increase in Cx43, enhanced myometrial contractile potential and the onset of pre-term birth.

Utilizing the pregnant mouse we manipulated the ERSR utilizing Tunicamycin (TM), an inducer of the ERSR and 4-Phenylbutyrate (PBA) a chemical chaperone which attenuates the ERSR. Mice were administered a single intraperitoneal injection of TM (0 -1.0 mg/kg b.w) on E15. Administration of TM (> 0.2 mg/kg b.w) resulted in the onset of preterm labor at E17 (independent of a decline in P4 levels). Western blot analysis revealed a massive induction of the uterine ERSR within 8hr of TM exposure, which was unexpectedly ablated by 24hrs. Decreased uterine ERSR resulted in reduced CASP3 activation, a reciprocal recovery of Cx43 levels and increased uterine contractility as confirmed by force transduction analysis. In contrast, attenuation of ERSR relaxed the uterine myocyte. These data confirms the ability of pregnant uterus to utilize the ERSR as a mechanism for determining myometrial contractility and consequently gestational length via CASP3 mediated cleavage and sequestration of Cx43.

With the growing recognition of an association between ERSR and human disease novel targets and new strategies for therapeutic intervention are beginning to emerge. These agents may ultimately be utilized to reveal indicators for and the resolution of pre-term birth. Abnormal uterine contractility presenting as inadequate contraction remains understudied. We previously found cfP RNA that alter myometrial contractility. Thus, we hypothesize that term nulliparas who with an active phase labor arrest have an altered plasma transcriptome. We seek to compare the plasma transcriptome of nullipara with labor dystocia leading to cesarean delivery to other clinically relevant groups using genomic transcriptome microarrays. A prospective cohort was designed using a case-control study. Six patients (3 cases, 3 controls) were recruited and 2ml plasma was obtained either at recognition of complete cervical dilation or when the diagnosis of protracted or arrested labor was made. CFP RNA was isolated using a proprietary method developed in our laboratory. RNA yield, purity and integrity were determined by nanospectrometer and Agilent Bioanalyzer. Affymetrix genome transcript microarrays were analyzed by MetaCore. Gene validation and QC evaluation were performed. Of the 27,116 genes sought, 332 genes were altered in association with dystocia-67 down regulated and 265 up regulated. These genes were sorted into the top 10 diseases consisting of Hypertension, Neurologic Manifestations, Neurobehavioral Manifestations, Intellectual Disability, Obesity, Chorioamnionitis, Craniomandibular Disorders, Temproomandibular Joint Disorders, Ovarian Diseases, and Acromegaly. The up regulated genes involved G-protein coupling cGMP nucleotide second messenger, positive regulation of amine transport, negative regulation of systemic arterial blood pressure, regulation of urine volume, regulation of neurotransmitter transport, positive regulation of the force of heart contraction, vasodilation, multicellular organismal process, etc. Of note, we found for the first time that SUMO is significantly down regulated in dystocia; SUMO contributes to DNA damage mediated by p53/NF-κB activation. Our study reveals the SUMO-pathway may play an important role in dystocia.

Optimal Genomic DNA was isolated from each myometrial tissue sample and then samples in each group were combined and subjected to MeDIP-chip analysis using NimbleGen 2.1M human DNA methylation array.

We found no differences in methylation in the promoter regions of the contraction associated genes CX43, OXTR, PTGFR, PTGES2, PTGES3, PTGIS, PTGER2, PTGER3, and PTGER4. However the 5'-region of the PTGES gene was unmethylated in all groups of tissues from patients with established labor but specifically methylated in the two groups without labor. The PTGS2/COX2 gene is heavily methylated at the 5'-region upstream of the CpG island in the three groups with labor but with less or no methylation in the groups without labor. Conclusion: This study provides preliminary evidence for differences in the myometrial epigenome at the level of DNA methylation that may regulate differences in gene expression associated with the length of gestation and the onset of labor. ER∆7 has been shown to act in a dominant negative fashion repressing full length ERα66 action. In our study we have observed a decrease in ER∆7 as term approaches and speculate that ER∆7 may suppress the uterotonic action of uterine ERα66 across gestation. Both human and mouse myometrial tissue exhibit increased levels of ERα46 across gestation but at term ERα66 becomes the dominant nuclear isoform regulating increased CAP expression.

In order to define the molecular origins of these alternate transcripts we surveyed all ERα transcripts across gestation in the mouse and human uterus. We observed five ERα 5' untranslated regions associated with unique promoter regions. RT-PCR confirmed alternative transcript usage across gestation in the mouse revealing the presence of transcripts that produce a C-terminally truncated protein (C-TERP) that parallels human ER∆7 function. We have also determined that the uterine ERα46 transcript produced by skipping exon 1 is always associated with the F promoter. Utilizing q-PCR and RPAs, we have quantified the alternative ERα promoter usage and uterine transcript expression across gestation. We propose that uterine ERα action is gestationally regulated by alternate uterine ERα isoforms, which are expressed through differential promoters. These events allow for tissue specific regulation of ERα action during pregnancy. Uterine smooth muscle exhibits rhythmic contractions in the absence of hormonal or nervous stimuli. The frequency of these contractions is regulated by pacemaker potentials that all myometrial cells intrinsically produce. These potentials are hypothesized to be initiated by a depolarizing leak current in the myometrium, but the cationic leak channel responsible for this current remains to be elucidated. Because many leak channels are inhibited by gadolinium (Gd 3+ ), we sought evidence for existence of a leak current in myometrial cells by performing whole-cell patch clamp analysis on human myometrial cell lines in the absence or presence of Gd 3+ . We found a Gd 3+ -sensitive leak current in 60% of cells. By replacing Na + in the extracellular bath solution with a nonpermissive cation, NMDG, we demonstrated that the leak current was Na +dependent. In support of the hypothesis that activation of this channel mediates increases in contraction at the onset of labor, patch clamp analysis indicated that the myometrial leak current was enhanced by oxytocin and that this effect may be pregnancy-dependent. Furthermore, Gd 3+ was able to significantly reduce the frequency of contraction in mouse and human uterine strips. In an effort to identify the channel responsible for this current, we used qPCR and Western blot analysis to look for expression of a candidate Na + leak channel, NALCN. We found that NALCN is expressed in the myometrium and that its expression changes during pregnancy. Together, our results indicate that NALCN may be responsible for a Na + -dependent, Gd 3+ -sensitive leak current in the uterus. This current is enhanced by oxytocin and contributes to the frequency of contractions in the uterus. This work has important implications for uterine dysfunctions such as preterm labor and dystocia.

Effects mouse M1 and M2 protein expression was examined by immunoblotting. M2 immunolocalization was examined by immunohistochemistry in formalinfixed mouse uterus. Mouse and human skeletal muscle samples were used as a positive control. Results: MYOM2 mRNA was detected in pregnant human myometrium. Mouse myom2 gene was high in pregnant myometrium and decreased during term and preterm labour. Immunoblot revealed a strong expression of M1 and M2 proteins in mouse heart and skeletal muscle tissues at the predicted size of 185kD and 165kD. Distinct protein expression of M1 and M2 proteins in the mouse myometrium revealed two weak bands at 185kD and 165kD and a strong new protein band at approximately 125kD. Conclusion: These data 1) suggest the existence of a myometrial-specific form of myomesin proteins and indicate that 2) reductions in M2 expression is associated with labour, and that 3) M2 expression is regulated positively by progesterone. We propose that myomesins are a new class of smooth muscle proteins that contribute to the timing of labour. Funding: CIHR (MOP-37775) Myometrial tissue samples were collected from consented patients undergoing hysterectomy (non-pregnant; NP), elective caesarean section at term before the onset of labour (term not in labour; NIL), or emergency caesarean section after the onset of labour (term in labour; TIL). For term samples (NIL and TIL) tissue was collected from upper and lower myometrial segments (U and L). Total RNA was extracted from myometrial tissue and gene expression/AS analysed by RT-PCR and immunoblotting (n=10 for each group). A high throughput PCR based system, the Layered and Integrated system for Splicing Annotation (LISA), was used to assess global patterns of AS in myometrial tissue. Results: RT-PCR and immunoblotting analyses demonstrate a spatiotemporal variation in expression of genes encoding the splicing regulatory RNA binding proteins SRSF1-6 inclusive and RBFOX2 in NIL and TIL tissues. Furthermore, a distinct spatial variation in the pattern of AS of genes regulated by RBFOX2 (SYNE2, NFYA and TAK1) is also observed in these tissues. The pattern of AS affecting MYL6, encoding smooth muscle myosin light chain (MLC)17, shows a distinct temporal change from NP to NIL/TL tissues. Conclusions: AS regulates the expression of key smooth muscle and contractility associated genes in the myometrium and may contribute to the temporal and spatial changes in myometrial function observed during pregnancy. Transglutaminases (TG) are a family of enzymes best known for protein crosslinking. Through an acyl-transfer reaction between glutamine and lysine, TG creates a covalent isopeptide bond and thus remodels protein. Tissue remodeling takes place in pregnancy and the risk of preterm labor is significantly higher in women with celiac disease who are also seropositive for TG2 IgA antibodies. We hypothesized that TG would be present and active in the non-pregnant and pregnant rat uterus and cervix. Real-time RT-PCR suggested that TGs 1 through 4 would be present in uterine and cervical tissues. Immunohistochemical techniques were used in tissue from female Sprague-Dawley rats. TGs 1 through 4 were present in the pregnant and non-pregnant rat uterus and cervix. In these same tissues, active TG1 and TG2 were localized in situ using FITC-labeled peptides, which interact with specific TG isoforms (K5 for TG1, and T26 for TG2). QN mutant peptides acted as respective controls, and showed no signal.

In the non-pregnant rat cervix, TG1 activity was prominent in the epithelium while TG2 activity was prominent throughout the body. In the pregnant rat cervix, TG1 activity was reduced as compared to the non-pregnant cervix (16.8% signal density (px/cm), p < 0.05, two-way ANOVA) in the epithelium, but TG2 activity was not statistically reduced (18.2% (px/cm), p > 0.05). In the non-pregnant rat uterus, TG1 activity was observed throughout the perimetrium, while TG2 activity was noted in the myometrium and endometrium. In the pregnant rat uterus, TG1 activity was decreased in the perimetrium when compared to the non-pregnant uterus (1.9% (px/cm), p < 0.05), while TG2 activity was maintained throughout the endometrium and myometrium (74% (px/cm), p > 0.05). TGs participate in the contraction of the cervix and uterus, supported by findings in isometric experiments. Cystamine (a non-specific TG inhibitor) concentration-dependently inhibited the maximal uterine contraction to oxytocin (1 mM = 64.15% ± 5.81% vehicle contraction; 5 mM = 17.43% ± 1.20% vehicle contraction). Between pregnant and non-pregnant tissues, uterine maximum contraction to oxytocin was similarly reduced by cystamine (1 mM, p > 0.05, two-way ANOVA). These findings highlight functions of TGs never before considered in reproductive tissue, namely remodeling of the cervix and contraction of the uterus.

A New Mechanism for Cooperative Effects in Myometrium; Oscillations of Interstitial Potassium. Roger C Young, Gabriela Goloman. Obstetrics, Gynecology and Reproductive Sciences, University of Vermont, Burlington, VT, USA. Background: Individual myometrial myocytes contract in concert with near neighbor myocytes. The primary mechanism of this cellular cooperativity is the electrical syncytium. To raise intracellular Ca, enough Ca needs to come into the cells from the interstitial space to overcome intracellular Ca buffering. These inward currents may be large, and if so, would require charge balancing outward currents. If outward K currents participate, then K concentrations may rise in the narrow, restricted, interstitial space. Here, we tested this hypothesis by measuring K on the outside of the cells (Ko) with K-sensitive microelectrodes. Methods: K-sensitive electrodes were constructed from patch clamp-type micropipettes. Tips were treated with silane, and then filled with K ionophore solution, Cocktail A (Sigma-Aldrich). Late pregnant myometrial strips from rat and human were placed in a muscle bath, and then impaled with a K-sensitive electrode. Contractile activity was monitored with an FT-03 force transducer.

In KCl solutions the voltage sensitivity of the K-sensitive electrodes was +49 mV/decade, and was linear from 2.5 to 40 mM. Non-K-sensitive electrodes were used to test for motion artifacts. Using K-sensitive electrodes, phasic rises of Ko were observed in human and rat myometrium that began briskly near the onset of the contraction. Elevations of Ko persisted into the period between contractions. In 9 experiments in the rat, 68 contractions were analyzed. Peak rises of Ko averaged 19.0 ± 9.9 mM.

Conclusion: K-sensitive electrodes were used to measure interstitial Ko in pregnant human and rat myometrium. We observed Ko concentrations high enough to modulate the electrical activities of the cells that share the same interstitial space. Local Ko elevations likely create a cooperative effect among the cells, suggesting metabolism-based syncystial effects. Long-lasting elevations of Ko may participate in setting the period between contractions. These studies define a new mechanism for the contribution of outward K currents to myometrial contractility.

Outward K Currents Are Expressed throughout the Entire Action Potential in Pregnant Rat Myometrium. Roger C Young, Gabriela Goloman. Obstetrics and Gynecology, Univ. of Vermont, Burlington, VT, USA. Background: In an accompanying abstract, we find interstitial K (Ko) rises during myometrial contractions. Rises of Ko result from cellular outward K currents in association with electrical activity. Previously, there has been no direct method to experimentally determine in tissue where K currents are expressed in relation to the action potential. Methods: Double-barreled microelectrodes were made with tip openings of 1-2 microns, separated by 15 microns. The back-fill end of one capillary tube was shortened. Suction and pressure was applied to only the long capillary tube. The tip of this electrode was silanized, filled with K ionophore solution Cocktail A (Sigma-Aldrich), then back-filled with .2 M KCl. The short electrode was filled with bath solution. Pregnant rat myometrium was placed in a muscle bath and impaled.

The long electrodes responded to changes of KCl (49 mV/decade). We observed repetitive bioelectrical bursts from the short electrode, indicating local action potential activity. In 6 experiments (23 contractions), the bioelectrical activity always started before the onset of the voltage rises of the K-sensitive electrode. The time delay was 0.32 ± 0.09 seconds. Conclusion: Because electrical activity started before Ko began to rise, we conclude that depolarization of the tissue-level action potential, or events downstream from the action potential, cause outward K currents. The very short time delay indicates that K currents are active for nearly the entire duration of the action potential. These data offer direct evidence that outward K currents are not solely expressed during repolarization, but likely serve as counter currents for inward Ca currents.

Testing ) while ATR and PRA had no effect. Androsterone was decreased by SIM and LOV, respectively, by 52 and 40% (P<0.05) while ATR and PRA had no effect. Progesterone concentrations were not affected by any of the statins. CYP17 mRNA was reduced by SIM and LOV, respectively, by 78% and 49% (P<0.01) whereas ATR and PRA had no effect. Conclusions: SIM, the most lipophilic of the statins had the greatest effect on reduction of growth and inhibition of androgen production while PRA, the most hydrophilic statin, had little or no effect. Direct ovarian effects of statins are likely related to their ability to access tissues in the absence of specific transporter proteins.

Metformin OBJECTIVE -To determine if treatment with metformin, an insulin sensitizing agent, reverses the endothelial dysfunction observed in the mesenteric resistance vessels of rats with dihydrotestosterone (DHT)-induced PCOS. MATERIALS/METHODS -Female rats were randomized at 4 weeks to implantation of a 7.5 mg, 90-day DHT pellet or no pellet, utilizing a novel rat model exhibiting the metabolic and ovarian characteristics of PCOS. At 12 weeks, DHT treated animals were randomized to DHT (DHT) or DHT and metformin (DHT-MET). DHT-MET animals received 300 mg/kg/day of metformin in drinking water. Animal weight, feed and water consumption were monitored throughout the treatment period.

At 16 weeks experiments were performed on isolated mesenteric resistance arteries using a pressurized arteriograph. Endothelial function was assessed by the vasodilatory response (efficacy) to acetylcholine (ACh). Serum steroid concentrations and insulin were analyzed by sensitive RIA. Fasting blood glucose was obtained. Differences between groups were determined by ANOVA. Data is expressed as mean ± SEM. Treatment with metformin normalized blood pressure; however, had no effect on weight. DHT animals demonstrated reduced efficacy to Ach when compared to control and DHT-MET animals. Treatment of adult DHT rats with metformin restored normal vasodilatory efficacy. CONCLUSIONS -This study expands on our previous work and shows that treatment with metformin reverses endothelial dysfunction and normalizes blood pressure in adult PCOS rats after the onset of clinically evident disease. These data offer insight allowing for the development of more targeted research focusing on the relationship between IR and the development of altered vascular reactivity in PCOS. , in addition to HgbA1c and OGTT with fasting and 2 hr glucose and insulin levels. Metformin was discontinued for 2-4 weeks prior to CGM in women with diagnosed glucose intolerance. Patients with type 1 or 2 diabetes were excluded. Percent of time of glucose above 120 and 140 mg/dL, highest and average glucose levels during CGM were compared to BMI, HgbA1c, estimated average glucose and OGTT results using Spearman correlation and linear regression analyses. Results: The percent of time of glucose values above 120 mg/dL (15.1 +/-19.9 mg/dL) and 140 mg/dL (7.4 +/-10.6 mg/dL) measured by the iPro compared significantly to FPG (90.7 +/-9.1 mg/dL), but not to BMI, HgbA1c, estimated glucose, fasting and 2 hr insulin and 2 hr glucose levels (p<0.05). The iPro high (155.4 +/-43.3 mg/dL) and average glucose (102.2 +/-12.0 mg/dL) values correlated significantly to FPG but not to BMI, HgbA1c, estimated glucose, fasting and 2 hr insulin, and 2 hr glucose levels (p<0.05). Conclusions: The iPro CGM correlated with FPG but not the 2 hr glucose following glucose loading. The overall cost of CGM may be greater than the cost of an OGTT, with more inconvenience to patients. This study consists of a relatively small sample size and is confounded by inherent patient variation in daily food intake habits, but may provide extensive information regarding differences in glycemic control in women with PCOS. Studies comparing CGM before and during Metformin treatment are in progress. Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder encountered in the reproductive age female population, affecting approximately 5-10% of these women. Hyperinsulinemia and/or insulin-resistance (IR) have a high prevalence in women with PCOS and when associated with obesity are recognized as part of a syndrome associated with several cardiovascular risk factors, such as dyslipidemia, hypertension, dysfibrinolysis and glucose intolerance.

The gold standard methods currently used to assess insulin sensitivity are expensive, time-consuming and difficult to apply in large scale clinical studies, therefore the objective of the study was to develop and validate a simple measure of IR using OGTT values in nonobese (non-ob) PCOS women, the HOMA-M 120 (G 120 (mg/dL) x I 120 (µIU/mL) / 405), (Phase 1). Our secondary objective was to verify the validity of different IR indexes in overweight-obese (ovob) PCOS women (Phase 2) by comparing them with data obtained from euglycemic hyperinsulinemic clamp. 201 lean and 198 overweight obese non-diabetic PCOS women, aged 18-35 years, were selected from our database among patients who attended the Gynaecological Outpatient Clinic of our Hospital between January 2010 and April 2012, in a retrospective observational 2-Phases study. All patients underwent OGTT, euglycemic hyperinsulinemic clamp and androgenic and biochemical assays. The predictive performance of each IR index was analyzed by receiver operating characteristic (ROC) curves.

The best correlation coefficients with clamp studies were obtained with the Belfiore Area (R(s) = 0.579) and the HOMA-M120 (R(s) = -0.576) in non-ob PCOS patients and the Sib (R(s) = 0.697) in ov-ob PCOS patients. The best predictive index of IR in non-ob PCOS was a HOMA-M 120 value of 12.8 or more (AUC ROC = 92.4%). In the ov-ob PCOS population, the best predictive performance was obtained by a Sib of 10.2 or less (AUC ROC = 85.7%).

The simple evaluation of the HOMA-M 120 and the Sib in non-ob and ovob PCOS patients, respectively, seems to effectively replace the euglycemic hyperinsulinemic clamp in primary prevention practice.

Low Serum adiponectin and plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels were measured by enzyme immunoassays in both groups. In addition, we calculated the homeostasis model assessment insulin resistance index (HOMA-IR) and the Lipid Accumulation Product (LAP) index and tested the linear correlation between these metabolic indexes and the plasma natriuretic peptide concentrations.

Results: The levels of ANP and adiponectin were reduced in the PCOS group compared to the control group (p <0.0001 and p = 0.0143, respectively). BNP concentrations did not differ between the two groups (p = 0.979). ANP levels correlated with those of adiponectin and BNP in the overall analysis (r = 0.35 and p = 0.006, r = 0.26 and p = 0.040, respectively) but not within each group (PCOS or control) when analyzed separately. There was no correlation between ANP and metabolic parameters (blood glucose, insulin, or lipids), HOMA-IR or LAP index. Conclusion: Women with PCOS have lower ANP and adiponectin levels compared to controls matched for age and BMI. Thus, the mechanisms that affect ANP and adiponectin production and/or clearance may be altered in PCOS regardless of adiposity. These hormones may be involved in the metabolic features of PCOS.

Metabolic Polycystic Ovarian Syndrome (PCOS) represents the most common endocrine disorder in reproductive age women, is the most common cause of female infertility, and these patients have miscarriage rates similar to other subfertile populations. Prior data shows that markers of uterine receptivity are decreased in PCOS patients. Recent work from our laboratory suggests that elevated DHEA levels in vitro inhibit a key pathway in glucose metabolism resulting in the inhibition of endometrial decidualization. As DHEA levels are elevated in 50% of PCOS patients this suggests that the endometrial metabolic milieu in PCOS patients may be abnormal, contribute to impaired endometrial function, and thus subfertility. Two groups of women were recruited to participate. Controls were normoovulatory women with proven fertility. Cases consisted of patients with primary infertility and PCOS with exclusion criteria of known endometriosis or other endocrine disorders. Both groups underwent endometrial biopsy, received a blood draw for DHEA and Testosterone and completed a survey. To complete the study, 20 patients will be enrolled per group based on P value (lt) 0.05 and power of 80%. To induce decidualization, primary endometrial stromal cells (ESCs) isolated from the biopsies were exposed to 1 mM medroxyprogesterone acetate and 0.5 mM cAMP for nine days. The mRNA expression levels of known markers of decidualization; prolactin, IGFBP1, and interleukin-15, were measured by quantitative RT-PCR. The fold change in gene expression between non-decidualized and decidualized samples was calculated using the comparative cycle threshold method with a housekeeping gene. To date, samples from 8 PCOS patients and 14 controls have been analyzed. The fold change in gene expression of decidual markers exhibit a decrease in PCOS patients as compared to controls. By controlling the in vitro hormonal environment and measuring proven markers of endometrial decidualization, we found that women with PCOS have ESCs that demonstrate reduced ability to undergo normal decidualization compared to non-PCOS women. With further elucidation of the multiple processes contributing to poor decidualization, targeted therapies can be designed to improve fertility outcomes in patients with this common endocrinopathy. To compare insulin-resistance and pancreatic β-cell function of women with polycystic ovary syndrome (PCOS) based on their Body Mass Index (BMI) and ethnicity. Materials and Methods: In a retrospective study conducted on 51 women with PCOS of reproductive age, demographic and laboratory data were abstracted from our computerized database. BMI was stratified as: healthy weight (BMI 18.5 -24.9 kg/m²), overweight (BMI 25 -29.9 kg/m²) and obese (BMI ≥30 kg/m²) groups. PCOS was diagnosed by the Rotterdam criteria 2003 consensus workshop. Insulin resistance and β-cell function were measured using the Homeostatic Model Assessment Insulin Resistance (HOMA-IR) and β-cell function (HOMA-β%). HOMA-IR and HOMA-β% were compared among the 3 BMI categories. Statistical analyses were performed using the SPSS 15.0. Quantitative variables were compared by one-way ANOVA and Post Hoc tests using the Tukey method. The level of significance was set at 5% in all analyses. Results: Fifty one patients participated in the study: 57% (29/51) healthy weight, 21.5% (11/51) overweight, 21.5% (11/51) obese. The age distribution of the three BMI groups was (mean ± S.D.): 34.95±5.32, 35.44±3.26, 34.87±5.97, respectively. Within the study population, 100% of the Asian patients had a normal BMI, and of the Caucasian women 62.8% were normal weight. The distribution of the African Americans was equal between the three BMI groups and 62.5% of the Hispanic patients were obese. HOMA-IR was significantly higher in obese women compared to overweight and healthy weight patients, (mean ±S.D. : 2.88±2.09, 1.13±0.73, 0.84±0.49, respectively, p<0.0001). Moreover, HOMA-β% was significantly increased in obese women in comparison to the other two groups, (mean ±S.D. : 186.89±131.62, 106.83±46.77, 86.60±40.91, respectively, p<0.0001) . In contrast, no difference was found between healthy weight and overweight subjects. Conclusions: Our data suggest higher insulin resistance andβ-cell function in obese PCOS patients as compared to non-obese PCOS patients using the HOMA-IR and HOMA-β% calculations. In contrast, no differences in these parameters were observed between healthy weight and overweight PCOS patients. Based on our data, Asian ethnicity is associated to a lesser degree with increased insulin resistance and β-cell function in PCOS patients than Caucasian, African American and Hispanic ethnicities. This Retrospective case-control study was conducted on fifty-one women with PCOS of reproductive age compared to their BMI and age matched non-PCOS control group. Demographic and laboratory data were abstracted from our computerized database. BMI was stratified as: healthy weight (BMI 18.5 -24.9 kg/m²), overweight (BMI 25 -29.9 kg/m²) and obese (BMI ≥30 kg/m²) categories. PCOS was diagnosed by the Rotterdam criteria 2003 consensus workshop. Insulin resistance and ß-cell function were measured using the Homeostatic Model Assessment Insulin Resistance (HOMA-IR) and β-cell function (HOMA-β%). HOMA-IR and HOMA-β% were compared among the three BMI categories. Medians and interquartile ranges are presented for continuous distributions. The Kruskal-Wallis one-way analysis of variance was used to compare medians among three BMI groups. A two sample Chi-Square test was used to compare BMI distributions between PCOS cases and controls. Logistic regression was used to estimate odds ratios of PCOS per unit increase in HOMA-IR and HOMA-β%. All statistical analyses were done using SAS Version 9.2 (SAS Institute, Cary, NC). All hypothesis testing was done at the 5% level of significance. Results: Fifty one patients participated in each group of the study: 57% (29/51) healthy weight, 21.5% (11/51) overweight, 21.5% (11/51) obese. The age distribution of the 3 BMI groups was (mean±S.D.): 34.95±5.32, 35.44±3.26, 34.87±5.97, respectively. There was an overall difference in median HOMA-IR values among the three BMI categories in the PCOS and control groups (p=0.0008, p<0.0001, respectively). While there was an overall significant difference in median HOMA-β% values among the three BMI categories in the PCOS group, no such difference was found in the control group (p=0.0092, p=0.0971, respectively). The Odds Ratio (OR) for HOMA-IR and HOMA-β% were 1.18 (p=0.339) and 1.04 (p=0.821) respectively. Conclusions: Our data suggest that there is no significant difference between PCOS patients and controls with respect to median HOMA-IR or HOMA-β% based on their BMI. Median HOMA-IR and HOMA-β% values are higher among PCOS patients, but not significantly higher. Background: Placental growth factor (PLGF) is an angiogenic factor which belongs to the vascular endothelial growth factor (VEGF) subfamily. VEGF is an important mediator of ovarian hyperstimulation syndrome (OHSS) and has been shown to be increased in polycystic ovarian syndrome (PCOS) women, suggesting angiogenic imbalance in this disorder. Soluble fms-like tyrosine kinase-1 (sFLT-1) is the circulating receptor for PLGF and VEGF, decreasing their bioavailability. PLGF expression has not been previously characterized in PCOS, neither has its relationship to sFLT-1. Objective: We aimed to examine serum and follicular fluid levels of PLGF and its inhibitor sFLT-1 in PCOS compared to control patients during controlled ovarian stimulation. Design: Prospective case-control study. Materials and Methods: Serum was collected from PCOS (n=14) (Rotterdam criteria) or matched control (n=15) patients undergoing controlled ovarian stimulation at baseline (day 3), day of HCG administration, and day of oocyte retrieval. Follicular fluid was collected on day of oocyte retrieval. PLGF and sFLT-1 protein levels were measured by ELISA. Statistical analysis was performed using student's t-test or Mann-Whitney test. Results: PLGF was increased in follicular fluid of PCOS compared with control women (58.7 vs. 42.5 pg/ml, p<0.01). PCOS women were found to have lower follicular fluid levels of the circulating receptor sFLT-1 compared with control patients (2.7 vs. 3.6 ng/ml, p<0.03). The bioavailability of PLGF (PLGF/sFLT-1 ratio) was markedly increased in follicular fluid of PCOS vs. control women (1.8-fold, p<0.01) . No differences in either factor were found in serum at baseline, HCG day and oocyte retrieval day between PCOS and control women. Conclusions: This is the first study examining PLGF expression in PCOS. The increased follicular fluid PLGF/sFLT-1 ratio results in increased PLGF bioavailability in PCOS. This proangiogenic state may contribute to the pathogenesis of PCOS and ovarian hyperstimulation syndrome, which most often occurs in patients with PCOS. Background: While the first-line medical treatment for anovulation-related infertility in polycystic ovary syndrome (PCOS) women is clomiphene citrate (CC), 25% of patients are CC resistant and fail to form a dominant follicle. Although not currently a diagnostic criteria for PCOS, an elevated ratio of luteinizing hormone (LH) to follicle stimulating hormone (FSH) has long been recognized as a key feature in the local androgen excess of PCOS. We hypothesize that PCOS women with an elevated LH/FSH ratio have an increased rate of CC resistance and may be more responsive to letrozole. Objective: To assess the value of an elevated LH/FSH ratio in predicting the formation of a dominant follicle when ovulation induction is implemented with CC or letrozole in PCOS women. Design: Retrospective case series. Materials and Methods: A chart review identified 312 monitored cycles in 104 women between 2007-2012. All patients met the diagnostic criteria set by the 2006 Androgen Excess-PCOS Society and had a cycle day 3 LH and FSH levels drawn. Only ovulation induction with CC or letrozole was included. Primary outcome was formation of a dominant follicle of at least 18mm in diameter prior to triggering ovulation. Clinical pregnancy was defined as positive fetal heart motion on ultrasound.

A total of 236 cycles (76%) achieved a dominant follicle. The formation of a dominant follicle was higher in the letrozole group compared to CC(n=154 vs 82, P <0.001, with an adjusted OR of 5.55, CI 3.0-10.25). Thirty-three percent of cycles with letrozole developed two or more dominant follicles compared to 19% in the CC group (P=0.006). The clinical pregnancy rate was not significantly different between letrozole or CC (13% vs 9%, P=0.26). LH/FSH ratio <1 or ≥1 was not associated with formation of a dominant follicle (n=63 vs 173, P=0.77). However, upon treatment with letrozole, the odds of forming a dominant follicle was greater than with CC (OR: 6.57, CI: 3.2-13.49) when LH/FSH ≥ 1. When LH/FSH <1, letrozole had no significant effect on dominant follicle formation (OR: 3.55, CI: 0.96-13.08). Conclusion: LH/FSH ratio ≥1 is a good predictor for which PCOS patient will be successful in forming a dominant follicle using letrozole as compared to clomiphene citrate. Objective: Pertussis infections have risen exponentially in the United States with the highest incidence of morbidities and mortalities in infants less than one year of age. Current recommendations are to give Tdap to all gravidas after 20 weeks gestation or immediately postpartum. Vaccine "cocooning" of close contacts is also advocated to reduce transmission to newborns. This study was undertaken to determine Michigan birthing hospitals' Tdap vaccination policies and practices. Methods: A telephone survey of Michigan birthing hospital administrators was conducted May-June of 2012 using an established questionnaire. Statistical analysis was by Fisher exact test for categorical variables. Results: Response rate was 83% of Michigan's 84 birthing hospitals. Fifty-one (73%) reported a process in place to assess gravida Tdap vaccine status. Only 14 (27%) had a written policy. Vaccine cost was most often (20%) cited as a barrier to policy implementation. The majority (91%) of hospitals surveyed indicated that they offered Tdap. Physician/patient initiation of the vaccine process was required in 27%; nurse-physician contact 25%; standing orders 16%. The minority (11%) evaluate antepartum-admitted gravidas. Only 4% reported vaccinating household contacts. All hospitals documented vaccine administration in hospital records, but only 53% entered this data into the Michigan Care Improvement Registry (MCIR). Most (77%) documented patient vaccination refusal; few (6%) record this in MCIR. No significant difference (p > 0.05) was seen for urban settings, affiliated hospitals or institutions associated with an Ob/Gyn residency teaching program. Conclusions: Based on recall data, many Michigan birthing hospitals have not adequately taken measures to address pertussis vaccine surveillance in an obstetrical patient population. A majority of hospitals reported assessing postpartum patients for vaccine status; however, few had a written policy. Few inquire about Tdap status during the antepartum period. Likewise, vaccine opportunities are being missed for other close contacts. Additionally, despite having a robust statewide vaccine registry, suboptimal utilization of this system compromises the long term care of these patients.

Organizational Applicability of a Shared Care Approach in the Obstetric Collaborations in the Netherlands. Anke G Posthumus, 1 Vera LN Scholmerich, 1,2 Adja JM Waelput, 1 Amber A Vos, 1 Lieke C De Jong-Potjer, 1 Rachel Bakker, 1 Gouke J Bonsel, 1, 3, 4 Peter Groenewegen, 2 Eric AP Steegers, 1 Semiha Denktas. 1 1 Obstetrics and Gynaecology, Erasmus Medical Center, Rotterdam, Netherlands; 2 Organization Science, Faculty of Social Sciences, VU University, Amsterdam, Netherlands; 3 The School of Midwifery, University of Applied Sciences, Rotterdam, Netherlands; 4 Public Health, Erasmus Medical Center, Rotterdam, Netherlands. Relatively high perinatal mortality rates in the Netherlands have required a critical assessment of the current national obstetric system. In the levelled care system that is currently in place, professionals in midwifery and obstetrics are (mainly) working autonomously. In the evaluation of this system, the need for organizational improvement -in particular the closer collaboration between midwives and obstetricians-was emphasized. The focus in these changes is on the Obstetric Collaborations (OC's, consisting of obstetricians in a single hospital and all surrounding community midwives). This closer collaboration necessitates a new form of care, Shared Care. Aims of this project are to establish a model for collaboration based on Shared Care for the OC's and to monitor the implementation of this model, primarily focussing on the care providers.

The project will take place in five OC's. The intervention will consist of a shared risk selection instrument (the Rotterdam Reproductive Risk Reduction Checklist (R4U), focusing on medical and non-medical risks), corresponding care pathways for each risk and OC-meetings to further discuss women with a high score on the R4U checklist. Evaluation of interdisciplinary communication and referral, care provider opinion and care provider compliance to the intervention will take place using medical record investigation, questionnaires and diaries.

Outcome measures are changes in communication frequency and referrals, barriers and incentives for participation and care provider compliance. Insights to the study methodology will be presented.

Based on these outcomes from practice the Shared care model will be further developed to suite the need of the medical practice and may also provide useful knowledge for the national project focussing on patient outcomes.

An Evidence Based Infrastructure with Methods and Tools To Implement Preconception Care. Sevilay Temel, 1 Toon AJJ Voorham, 2 Eric AP Steegers, 1 Semiha Denktas. 1 1 Obstetrics and Gynaecology, ErasmusMedical Centre, Rotterdam, Netherlands; 2 'Rochussenstraat Building', Midwifery Academy, Rotterdam, Netherlands. Background Studies on the effectiveness of preconception care (PC) programs indicate 'incomplete outreach' as a major barrier. An outreaching PC was developed with three approach pillars: 1. collective PC awareness through public campaigns, 2. tailored target group-specific PC peer education, and 3. individual PC by health care providers for candidate parents using standardized and evidencebased instruments.

The three pillars were evaluated by using: 1. a cross sectional survey in the general population with measurements between 2007-2011, 2. questionnaires filled out by participants of the target group-specific education sessions, and 3. in-person interviews with participating midwives and GPs. Results 1. In total 5543 residents filled out the survey. Correct knowledge of preconceptional FA supplementation was increased from 30% in 2007 to 36% in 2009 (p=0.001), with no further increase in 2010 (36%). Regarding to whom a woman should consult when she thinks she is pregnant, in 2011 20% answered to consult a midwife directly and 66% to consult a GP for a referral to the midwife. 2. 86 (of 250) men and 219 women attending a target group-specific education session filled out a questionnaire. The majority was middle aged and lived in deprived neighbourhoods. Smoking ranged from 34% in Turkish, 38% in Dutch to 43% in the Caribbean group in contrast to 1% in the Moroccan group. 36% of Turkish, 50% of Moroccan and 50% of Caribbean women were over weighted (25-30 kg/m2). Reasons to participate were to receive information about how to become healthy pregnant, an existing child wish, and information about miscarriages in the past. 3. 43 individual PC consultations took place between July 2009 and March 2012. The health professionals mentioned the following main reasons for the low number of consultations: 1. few women actively seek for PC consultation, 2. a low (GP) or no (midwife) fee for PC consultations, c. extra administration required, d. collaboration between midwives and GPs as a point of attention.

The public campaign enhanced increase in PC knowledge. The target groupspecific education sessions were well attended by men and women; risk factors were prevalent. PC is not actively sought by women and practical difficulties in the implementation of PC consultation still needs to be solved. Obesity is associated with adverse cardiovascular outcomes later in life. We have previously shown that a normal pregnancy is protective against hypertension later in life in both obese and non-obese animals. The objective of this study was to investigate if alterations in the renin-angiotensin system (RAS) could explain the protective effect of pregnancy. Methods: Virgin CD-1 female mice were placed on standard (SF) or high fat (HF) diet. After 3 months, mice were randomly allocated to a breeding versus non breeding group resulting in 4 groups: primigravid on SF (SF-PG) or HF (HF-PG) and nulligravid on SF (SF-NG) or HF (HF-NG). The primigravid group proceeded through a normal pregnancy and delivery. After weaning of the primigravid group, all animals were contemporaneously placed on SF diet. Visceral adipose tissue (VAT) and kidney were collected from PG at 6 months post partum and from age-matched NG mice. Protein expressions of angiotensin (ANG) and its receptors, angiotensin receptor 1 and 2 (AT1 and AT2, respectively) were determined using Western blot analysis. One-way ANOVA and Kruskall Wallis test with appropriate posthoc tests were used for statistical analysis (significance P<0.05). INTRODUCTION In this study we compared biochemical cardiovascular risk factors and 30-year cardiovascular disease (CVD) risks between women with previous term hypertensive pregnancy disorders (HTP cohort), women with previous severe very early onset preeclampsia (SPP cohort) and women with previous uncomplicated pregnancies (NTP cohort). METHODS Women with previous term PE or PIH defined according to the ISSHP were included in the HTP cohort. Women with previous severe early PE with an onset before 24 weeks' gestation were included in the SPP cohort. Controls were friends of HTP women who had an uncomplicated pregnancy. The Framingham risk score was used for individual 30-year CVD risk estimation. Potential differences between groups were compared using One-way ANOVA (Bonferroni correction) and the Kruskal-Wallis test. P values <.05 were considered to indicate significant difference.

The median (range) follow-up period was 29.2 (16-52) months for HTP's, 66.0 (49-120) months for SSP's and 29.3 (11-86) months for NTP's. 

Both HTP and SPP women have an increased risk for cardiovascular disease within 30 years compared with NTP women. Women with term hypertensive disorders have similar age-adjusted risk profiles as women with severe very early onset preeclampsia.

Adherence to Post Partum Diabetes Screening in a Regional High Risk Perinatal Center. Faro Revital, Schwebel Marlene, Distefano Valeria. Obstetrics, Gynecology, and Reproductive Sciences, UMDNJ-RWJMS, New Brunswick, NJ, USA. OBJECTIVE: Gestational diabetes is defined as impaired glucose tolerance that is first identified during pregnancy. Up to 5% of pregnancies in the US are affected by gestational diabetes and up to 70% of these women will develop overt diabetes mellitus over their lifetime. Early detection of overt diabetes mellitus immediately following the post partum period can improve lifelong outcomes in women. We sought to determine the rate of adherence to postpartum glycemic testing in women with gestational diabetes, and the factors associated with nonadherence, in a multidisciplinary high risk perinatal center. STUDY DESIGN: The study was a retrospective review 180 women with diabetes in pregnancy presenting for care between 2006 and 2011. During the study period 122 gestational diabetics were cared for in our center. We reviewed patient adherence to post-partum diabetes screening and used logistic regression analysis to determine if different characteristics influenced adherence to testing over others. RESULTS: Of 122 women with gestational diabetes 14% were diet controlled, and 86% required medication to control their gestational diabetes. Only 38 patients completed post partum diabetes testing, which comprised 24% of patients. Maternal characteristics are outlined in [ Table 1 ]. Similar adherence rates were found among women in all groups. (1 per 134). All were done at the time of cesarean section. The indications for sutures were placenta previa in 18 (44%), uterine atony in 15 (37%), placental abruption in 5 (12%), partial placenta accreta in 2 (5%), low lying placenta in 2 (5%) and uterine torsion in 1 (2%). Hysterectomy was avoided in 38 of 41 women (92.7%). The average total hemorrhage was 2534g (710-8840g) and blood transfusion was required in 20 cases (48.8%). In 2 cases, both involving partial placenta accreta by histological examination, hysterectomy was unavoidable. There were no adverse events due to B-lynch sutures; there were neither complaints of menstrual irregularities nor severe dysmenorrhea. 4 women out of 38 (10.5%) had subsequent uncomplicated term pregnancies, all delivered by elective cesarean section. Conclusion B-Lynch compression suture for postpartum hemorrhage is an effective conservative surgical method to avoid hysterectomy. Currently, there are no complications due to B-Lynch compression suture, including obvious secondary infertility.

Extraperitoneal Cesarean Section: Introduction of a "New" Surgical Technique. Surgical technique: Preoperatively a catheter is placed and blocked to better visualize and to ease preparation of the bladder. The bladder is exposed by Pfannenstiel incision and a blunt separation of the rectus muscles in the midline. The superior ramus of the left pubis is palpated digitally to indicate the direction of further preparation towards the left paravesical space until the left lateral umbilical ligament (obliterated umbilical artery) is identified. The bladder is separated from the lower uterine segment with blunt dissection. At this point the peritoneal vesicouterine fold becomes visible. The bladder can be further pushed to the right and inferiorly. The exposed lower uterine segment must now be incised below the peritoneal vesicouterine fold. The incision is dilated digitally towards both sides. The baby is delivered and the placenta is removed as it is done in TCS. The uterine incision is closed with a continuous suture, the abdominal wall is closed in the usual manner. Discussion: ECS is technically feasible and could be developed as a safe and potentially beneficial anternative to the usual transperitoneal section. Objective: We aim to model invasive extravillous trophoblast cells (EVT) using human embryonic stem cells (hESC). EVT have the unique ability to invade endometrium and modify spiral arteries to ensure adequate blood supply to the placenta. Poorly functioning EVT can lead to shallow placentation and to spontaneous pregnancy loss, preeclampsia or intrauterine growth restriction. An appropriate in vitro model might allow testing of potential therapies for these disorders. Methods: hESC were transferred to matrigel invasion chambers with 8 µm pores and cultured in medium "conditioned" with mouse embryonic fibroblasts and containing three distinct differentiation additives: 1) 4ng/ml FGF2, 2) 10 ng/ml BMP4 or 3) 10 ng/ml BMP4 plus 1µM of an activin A signaling inhibitor (A83-1) and 0.1µM of an FGF receptor inhibitor (PD173074) (BAP). After 5 days, invasive cells on the basilar membrane were fixed and stained. Nine microscopic fields from each membrane were photographed and the invasive cells counted. In parallel experiments, invasive cells were immunostained for the trophoblast marker, KRT7, and the EVT marker, HLAG. Progression of differentiation was followed at days 2, 4, 6 and 8. Results: hESC maintained in the presence of FGF2 invaded matrigel minimally while those treated with BMP4 and with BAP (P<0.05) displayed robust invasive capacities. Although no differences were observed between the raw invasion data obtained from cells treated with BMP4 and BAP, these treatments induced a 64-fold and 159-fold increase in invasiveness, respectively, when compared to controls with FGF2 alone if the data were normalized for cell growth rates. The majority of invasive cells were positive for HLAG and KRT7, suggesting that differentiation was occurring along the EVT pathway. Cells grown in BAP lost stem cell-specific POU5F1 staining within 48h, those grown under BMP4 alone retained POU5F1 positivity for as long as 4-5 days, depending on colony size. HLAG positive cells also appeared more quickly under BAP conditions, when compared to BMP4 exposure alone. Conclusion: Human ESC differentiate into trophoblast cells in response to BMP4 alone but do so more rapidly when exposed to BAP. A subpopulation of these cells is invasive and displays an EVT phenotype. Supported by NIH Grant NIH: 1R01HD067759 to RMR. Recently we have shown that surface glycolipid SSEA-1, is a marker of human endometrial basal glandular epithelial cells (hEBGECs) that harbours endometrial stem/progenitor cells (SPCs). Endometrial SPCs are linked to the pathogenesis of endometriosis and SSEA-1 + cells are present in ectopic endometriotic lesions. We aimed to further characterise the SSEA-1 + hEBGEC sub-population, assessing their gene expression profile for common markers of stemness, and endometrial cell differentiation. Methods: Single-cell suspensions of endometrial epithelial cells were obtained from 20 women not on hormonal therapy, with regular menstrual cycles undergoing hysterectomy in women with (n=10) and without endometriosis (n=10) for benign conditions. Epithelial cells were sorted with FACS or MACS into SSEA-1 enriched and SSEA-1 depleted fractions; cultured in serum-free 2D (n=7/group) or 3D matrix (n=3/group); and qPCR was performed comparing gene expression between the two populations. Markers of stemness included OCT4, NANOG, SOX2, CD133 and PODXL. Sex steroid receptors (PR and ERα), were also investigated as markers of endometrial differentiation. Data was statistically analysed using the Wilcoxen signed-rank test. Results: SSEA-1 + cells from all women showed significant down-regulation of PR (p=0.016) and ERα (p=0.016) in 2D culture when compared with SSEA-1epithelial cells. SSEA-1 + cells from women with endometriosis displayed significant up-regulation of OCT4 (p=0.047), NANOG (p=0.031), and FUT4 (p=0.047) compared to women without endometriosis. SSEA-1 + cells grown in 3D culture showed distinct up-regulation of all markers of stemness compared to the SSEA-1populations taken from healthy fertile women. This up-regulation was amplified in the same cell populations originating from women with endometriosis. Conclusion: This study showed: 1) The gene expression profile of SSEA-1 + hEBGECs is consistent with a more primitive cell type to SSEA-1 -.

2) SSEA-1 + cells exhibit SPC-like qualities when cultured in 3D culture, an environment which mimics the in-vivo niche and preserves stemness.

3) SSEA-1 + hEBGECs from women with endometriosis show differential gene expression profile to women without endometriosis. BACKGROUND: Endometrium is highly dynamic with an extraordinary capacity for self-renewal. Recently, our laboratory reported on generating megakaryocytes (MKs) from differentiating human endometrial stromal stem cells (hESSCs). MKs released platelets functionally and morphologically identical to control platelets. We sought to characterize ultrastructural features of generated platelets. METHODS: Isolated hESSCs were cultured; purity confirmed by flow cytometry and immunocytochemistry. Flow: hESSCs showed strong, positive expression of stromal cell specific markers CD90, CD29 & negative expression of hematopoietic lineage markers CD45, CD34. Immunocytochemistry: hESSCs were positive for CD90 & negative for epithelial specific marker cytokeratin, indicating passage 4, 6 hESSCs were an homogenous cell population devoid of hematopoietic and epithelial cells. To induce hESSC differentiation into MKs, hESSCs were cultured in serum-free medium with thrombopoietin x 18d. MK differentiation was analyzed by flow cytometry & confocal microscopy. Platelets were collected from MK differentiation medium supernatant (d.10-18) and were analyzed by transmission electron microscopy (TEM). RESULTS: MKs were successfully generated & expressed characteristic phenotypic markers CD41a, CD42b. Platelets expressed strong positive CD41a immunoreactivity. Platelet TEM identified alpha granules, dense tubular systems, mitochondria, ribosomes, large vacuoles and glycogen deposits intracellularly with outer cytoplasmic membrane showing irregular blebbing, all identical to human control platelets. Dense granules were not identified as this required a separate preparation. CONCLUSIONS:Released platelets share identical ultrastructural features with normal human platelets adding additional support that it is feasible to generate platelets that are not only functionally similar, but also structurally identical to normal human platelets. These results also suggest that hESSCs could be a potential source for cell-based therapy in regenerative medicine. Introduction: Poor placentation developed in early pregnancy leads to placental diseases such as preclampsia. Understanding the pathogenesis of the diseases has been hampered by inaccessibility to the early stages of human placental tissue. Human embryonic stem cells (hESC) treated with BMP4, are possible in vitro models of early placental development. Maintaining pluripotency of hESC is dependent on FGF and TGFB/activin signaling. Perturbing this cell signaling network leads to exit from the pluripotent state. Here we have improved the differentiation procedures to generate trophoblast (TB) cells by including inhibitors of activin A signaling (A83-01) and a FGF receptor inhibitor (PD173074) to the BMP4 system (termed BAP). Method: We performed the following experiments using hESC. 1) Monitoring cellular morphological changes in cultured hESC in the presence of BMP4 or BAP. 2) Immunoassay for syncytiotrophoblast (STB) markers, hCG, P4, PGF in medium after culturing hESC with BMP4, BAP, or inhibitors alone (AP). 3) Immunohistochemistry by using anti-KRT7 (a general marker of TB) and anti-HLA-G (extravillous trophoblast (EVTB) marker) antibodies for differentiated hESC. 4) FACS analysis to measure KRT7 positive cells in the cells differentiated with BMP4 or BAP. 5) Western blotting analysis and PCR analysis to detect proteins or gene expressions related to TB, STB and EVTB cells. Result: A morphological switch to an epithelial phenotype started by day 2, and the process occurred more rapidly under BAP than with BMP4. STB markers hCG, P4 and PGF, were significantly (P<0.05) higher in BAP than BMP4. BMP4 treatment progressively led to the appearance of KRT7 positive cells by day 4 when about 95 % of the cells had converted. In the case of BAP, almost all cells were KRT7 positive by 48 h. While BMP4 treatment led to expression of HLA-G, brachyury (T), a mesoderm marker, was not detectable in the cells treated with BAP. Conclusion: Blocking the signaling systems essential for maintaining pluripotency improves the differentiation of hESC to trophoblast derived by BMP4. Our longer term goal is to generate iPSC from umbilical cords of new-born babies of preeclampsia patients to generate STB and EVTB as abnormal models to uncover the mechanisms of disease. Background:Adult human endometrium undergoes cyclic shedding and subsequent regeneration that likely involves stem/progenitor cells. Perivascular cells co-expressing CD146 and PDGFRB surface markers within the human endometrium are clonogenic multipotent endometrial mesenchymal stem cells (eMSC). eMSC can be induced to differentiate into endometrial stromal fibroblast (eSF)-like cells which respond to progesterone (P4) with IGFBP1 secretion, suggesting eSF lineage differentiation. We investigated whether CD146+/PDGFRB+ eMSC differentiate to CD146-/PDGFRB+ eSF lineage cells with a broadly similar P4-responsive secretion profile, supporting a tissue progenitor physiologic role for eMSC. Methods:Endometrial tissues procured through the NIH UCSF Human Endometrial Tissue Bank. Endometrial cells were enzymatically dissociated, then CD146+/PDGFRB+ (eMSC) and CD146-/PDGFRB+ (eSF) populations isolated by fluorescence activated cell sorting (FACS), and expanded in primary clonal culture in eSF medium (DMEM/MCDB-105+10% FBS and insulin). eMSC cultures were phenotyped by FACS, and tested for P4-responsiveness with IGFBP1 (ELISA) and cytokine (IL1α, 4, 6 

Is Endometriosis an Inevitable Consequence of the Unique Differentiation Potential of SSEA-1 Expressing Human Endometrial Epithelial Cell Subpopulation? Louise da Silva, 1,2 Anthony Valentijn, 1 Jo Drury, 1 Patricia Murray, 2 Dharani Hapangama. 1 1 Women and Children's Health, Liverpool University, United Kingdom; 2 Cellular and Molecular Biology, Liverpool University, United Kingdom. Introduction: During a woman's reproductive years, human endometrium undergoes a monthly cycle of menstruation, re-growth and differentiation. Such plasticity suggests the presence of endometrial stem cells. Recently, we proposed stage specific embryonic antigen-1 (SSEA-1) as a candidate marker for human endometrial epithelial adult stem/progenitor cells (hEEp-ASPCs). This study aimed to investigate the key stem/progenitor cell feature of differentiation of the SSEA-1 expressing hEEp-ASPCs. Methods: Single-cell suspensions of endometrial epithelial cells were obtained from 19 women not on hormonal therapy, with regular menstrual cycles, undergoing hysterectomy for benign conditions. Epithelial cells were sorted via MACS into SSEA-1 enriched and SSEA-1 depleted fractions, and cultured in serum-free 3D matrix (n=8) or in differentiation-induction media (n=11). hMSCs were employed as the control for adipogenic and osteogenic differentiation. Differentiation was assessed by qPCR for changes in expression of adipocyte markers (PPARγ2 and LPL) and osteocyte markers (ALP and OSX), and confirmed by Oil Red O or Alkaline Phosphatase staining respectively. Results: SSEA-1+ cells had a significantly greater ability to produce polarized, single lumen gland-like structures when compared to SSEA-1-fractions after 10-14 days (p<0.0001). These gland-like structures consisted of cells expressing Cytokeratin, β-catenin, and steroid receptors ERα, ERβ, and PR, resembling the endometrial glandular architecture. As expected, hMSCs showed adipogenic and osteogenic differentiation confirmed by qPCR and Oil Red O or Alkaline Phosphatase staining. However, neither SSEA-1+ nor SSEA-1-cells were able to differentiate into adipocytes or osteocytes. Conclusions: Our data suggest SSEA-1+ hEEp-ASPCs are able to produce mature endometrial gland-like structures and possibly are unipotent only. This is likely to prevent the development of maternal microchimerism into fetal tissue, averting potential damage to progeny. Furthermore, we propose that endometriosis may be an inevitable consequence of the existence of a unipotent hEEp-ASPC, and is integral to the theory of retrograde menstruation. Objective: Circulating CD34+CD133+ hematopoietic proangiogenic progenitor cells are derived from the bone marrow. Previous studies have suggested that estradiol levels mediate circulating CD34+CD133+ cell numbers corresponding to phases of the menstrual cycle. Our objective was to determine levels of CD34+CD133+ cells in women undergoing in vitro fertilization (IVF), as this represents a unique opportunity to correlate angiogenic stem cell levels with folliculogenesis and conception. Design: Prospective cohort Materials and Methods: This pilot analysis included 14 patients undergoing fresh cycle IVF between January and September 2012. Peripheral blood samples were acquired at IVF baseline, maximum stimulation, and pregnancy testing. Mononuclear cells were isolated and labeled with anti-human CD34-FITC and CD133-PE monoclonal antibodies. Flow cytometry was performed to quantify the CD34+CD133+ subset. Progenitor cell levels were correlated with IVF outcomes using Spearman and Pearson correlations. A linear mixed effects model was used to test the association between stem cell levels and estradiol levels. Results: Progenitor cell levels did not appear to significantly differ between IVF baseline and maximal stimulation (mean change: .01, T=0.43) and estradiol levels did not influence mobilization of CD34+CD133+ cells (multivariable model T-value, T=-.10; significance by absolute T>= 2.0). Absolute numbers of CD34+CD133+ cells did not significantly correlate with age, anti-mullerian hormone level, length of stimulation, follicle counts, endometrial thickness, oocytes retrieved, fertilization rates, or clinical pregnancy. Greater changes in CD34+CD133+ progenitor cell number between baseline and maximal stimulation showed a trend towards fewer embryos transferred (-0.67, CI: -1.28, -0.07, p=0.048) and more day 5 embryo transfers (0.64, CI: 0.01, 1.27, p=0.064). Conclusions: Absolute circulating proangiogenic progenitor cell numbers did not strongly correlate with estradiol levels or IVF characteristics. The associations between change in CD34+CD133+ progenitor cell number and embryo number and transfer day may be suggestive of better embryo quality. Data collection is ongoing to clarify the role of these angiogenic stem cells in female reproduction.

The For this study, 10-day (short-term cultured) and 30-day old (long-term cultured) EBs were subjected to estradiol (E2), estriol (E3), selective estrogen receptor modulator (raloxifene, RLX), bisphenol A (BPA) and 1,3,5-tris(4hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) for 7 days. To confirm the effects of estrogen treatment, ICI 182780 was added to the respective EBs for additional 7 days following estrogen treatment. The expression of 7 marker genes, which included α-fetoprotein (AFP), hepatocyte nuclear factor (HNF)-3β, HNF-4α, brachyury, cardiac actin (cACT), nestin, and Oct-4, was measured by quantitative reverse transcription PCR.

Significantly lower expression of HNF-4α in both short-term and long-term cultured EBs was observed after treatment of estrogen compounds compared to control. The expression of HNF-3β in short-term cultured EBs has been positively affected by E2, E3 and RLX. The lower expressions of HNF-4α by E2 and RLX were negated by ICI 182780 treatment, although these findings were not statistically significant in E3-treated group. These findings suggest that estrogen compounds have effects on endodermal and mesodermal differentiation of human EBs.

("The final, definitive version of this paper will be published in Reproductive Sciences by SAGE Publications Ltd, All rights reserved.)

Alterations Cardiac features of differentiated cells were confirmed by expression of specific markers at each stage of differentiation. Middle-and late-stage hPSC-derived CMs showed an increase of pigmented cells and demonstrated slower beating rates along the in vitro cultivation period. SA-β-gal staining showed that day 18 CMs showed a larger stain-positive population than at day 12, this trend was more prominent at day 24. A decrease of functionality, i.e. mitochondrial membrane potential, was observed after JC-1 staining. Slowing and disappearance of beating coincided with these changes.

In this study, we demonstrated aging phenomena in hPSC-derived CMs using cellular senescence pigment staining and mitochondrial potential assessment.

Our findings may provide a basis to develop tools to screen out senescent cells to be clinically applied (A111539).

Effects [3] [4] [5] and late-stage (day 11-13) cardiac progenitors were treated with 10 µM of Ginsenosides, Rb1 and Re, for 24 and 48 hrs. Expression of cardiac-specific genes and proportion of differentiated cell population were evaluated. Also, the effects on functionality of hESC-derived CMs were analyzed using mitochondrial potential measurement. The expression of mesodermal marker Brachyury and cardiac-specific genes Nkx2.5, Tbx5 were significantly up-regulated when ginsenoside Re was treated at early stage. Beating lasted for a longer period when treated both Rb1 and Re at late stage of differentiation. These results indicated that Ginsenoside can be used to facilitate differentiation and to support for the functional longevity of hESC-derived CMs (A111539). The cellular source(s) needed for cyclic regeneration of the endometrium remains unknown. Limited data suggest the bone marrow (BM) is a source of endometrial cells. The objective of the current studies was to determine whether the BM is a source of multiple endometrial parenchymal cell types using a murine BM transplant model. BM was harvested from transgenic mice which express Green Fluorescent Protein (GFP). Recipient syngeneic female C57BL/6 mice, 4-6 weeks of age, were lethally irradiated, followed by tail vein injection of whole BM cells. Flow cytometry was performed on peripheral blood to assess BM reconstitution with GFP+ cells. Mice with successful BM reconstitution (>90% chimerism) were sacrificed at 3, 5, 9 and 12 months post transplant and hysterectomy was performed. Numbers of GFP+ cells in the endometrial stromal and epithelial compartments were determined using confocal laser microscopy. To distinguish BM-derived parenchymal cells from leukocytes, immunohistochemistry using anti-CD45 (pan leukocyte marker) was also performed on uterine sections from mice sacrificed at 5 and 12 months. BM-derived parenchymal cells were identified as GFP+/CD45-, and leukocytes were identified as GFP+/CD45+. In the stroma, BM-derived cells were detectable at the first time point examined and increased with time ( Figure 1 ). In contrast, BM-derived epithelial cells were not detected until 12 months post transplant (range 0-27 GFP+ epithelial cells detected per 15 high power fields assessed per animal). Of BM-derived cells detected in the stroma, 94-97% (5 months) and 48-76% (12 months) were nonhematopoietic parenchymal cells. Of BM-derived cells detected in the epithelium, 25-70% (12 months) were nonhematopoietic parenchymal cells.

These data demonstrate that the BM is a significant source of nonhematopoietic endometrial parenchymal stromal cells, and contributes to a much smaller extent to nonhematopoietic parenchymal epithelial cells. That BM cells give rise to critical structural components of the endometrium implicates the use of BM-derived cells in treatment of disorders of endometrial function.

The were studied: (i) Lambs were Ventilated (n=7) for 2 hours prior to tissue collection: for the first 15 minutes a high tidal volume and no positive end-expiratory pressure were used to cause VILI. Three ml of PBS was instilled into the endotracheal tube immediately prior to ventilation and 3 ml PBS was administered via an umbilical vein catheter within 5 min of commencing ventilation, and serial physiological measurements were made; (ii) Ventilation+hAECs lambs (n=8) were treated identically to Ventilated lambs but received intratracheal and intravenous administration of 90 million hAECs each, suspended in 3 ml PBS; (iii) Tissues were collected immediately after delivery from unventilated Control lambs (n=8). Samples of lung tissue were frozen for subsequent measurement of pro-inflammatory cytokine mRNA expression using RT-PCR. Repeated measures ANOVA was used to analyze serial physiological data; mRNA levels were compared by Kruskal-Wallis ANOVA and Dunn's multiple comparison test. Results: Airway resistance was higher in Ventilated+hAECs lambs than in the Ventilated group (p=0.046); other indices of respiratory function and physiological variables were not different between groups. IL-6 and IL-8 mRNA levels were higher in Ventilated lambs than Control (p<0.05); levels in Ventilation+hAECs lambs were not significantly different from Control or Ventilated+hAECs groups. Conclusion: Prophylactic hAEC administration did not compromise wellbeing but did not attenuate the initial lung inflammation induced by VILI. Background: Human amnion epithelial cells (hAECs) are immunomodulatory and anti-inflammatory in models of lung injury. Inadvertent lung injury of preterm neonates during the immediate neonatal period can cause brain damage that likely contributes to adverse neurological outcomes. In preterm lambs aggressive ventilation causes brain inflammation and injury. We hypothesised hAECs would reduce brain inflammation caused by aggressive ventilation. Methods: Three groups of lambs (126 days of gestation: term is 147 days) were studied: (i) Ventilated lambs (n=5) were mechanically ventilated for 2 hours prior to tissue collection: for the first 15 minutes aggressive ventilation with a high tidal volume and no positive end-expiratory pressure was used. Three ml of PBS was instilled into the endotracheal tube immediately prior to ventilation and 3 ml PBS was administered via an umbilical vein catheter within 5 min of commencing ventilation; (ii) Ventilation+hAECs lambs (n=5) were treated identically to Ventilated lambs but received intratracheal and intravenous administration of 90 million hAECs each, suspended in 3 ml PBS; (iii) Tissues were collected immediately after delivery from unventilated Control lambs (n=5). Brains were dissected and samples of subcortical and periventricular white matter from the frontal and parietal lobes were processed for immunohistochemistry using an antibody to ionized calcium binding adaptor molecule 1 (Iba1) to quantify microglia/macrophage density. Groups were compared by ANOVA, multiple comparisons were made with Fisher's LSD test. Results: White matter Iba1-positive cell density was higher in Ventilated lambs than Control in specific brain regions (Figure) . In these regions Iba1-positive cell density in the Ventilation+hAECs group was lower than in the Ventilation group and was not different from Control. Conclusion: Brain inflammation resulting from aggressive ventilation of preterm newborn lambs is prevented by hAECs. Methods: Ten DsRed(+) mice were used as BM donors. 30 recipient animals were irradiated to induce complete bone marrow ablation, and were transplanted with BM from DsRed(+) mice. All recipient animals were given physiologic levels of estrogen replacement. The recipient mice were randomly divided into 3 groups, and uteri were harvested at 2, 6, and 16 weeks. Uteri were digested into a suspension, and sorted by FACS. Conclusions: BM derived cells contribute to uterine epithelium. There is a progressive conversion to BM derived stem cells to endometrial cells that is completed within 16 weeks. BM derived stem cells produce progeny that acquire a distribution of epithelial and stromal cells similar to that seen in endogenous endometrium. BM cell fate in the uterus is carefully regulated to recapitulate normal cell allocation. Results: NβC and SOX9 expression was largely confined to the basal epithelial cells throughout the cycle. Cytoplasmic NAP1L1 was seen in the functional and basal glands. 3.1% of basal and 0.8% of functional epithelial cells stained positively for NβC and these cells also expressed SSEA-1 (p=0.002). Cytoplasmic NAP1L1 immunostaining was significantly stronger in the basalis glands (p=0.01). SOX9 modified quickscores were significantly increased in the basalis over the functionalis (6.5 vs 2.5, p<0.0001). IHC findings were confirmed by WB.

No NβC expression was seen in the GnRH or the functionalis of CP groups but was present in 1.5% of thin basalis-like endometrium in the Mirena group. High modified quickscores for SOX9 were observed in GnRH and Mirena groups (8.3, 8.7) but were absent from the functionalis of CP group. NAP1L1 staining was unchanged in the GnRH or Mirena groups but appeared to be increased in the functional glands of CP groups. Conclusion: Wnt/β-catenin signalling appears to be differentially regulated in the epithelial cells of basalis, and functionalis and may be altered by hormonal therapy. The rare NβC expressing endometrial epithelial cells that were located in the SSEA-1 expressing endometrial basal glands may represent human endometrial epithelial SPC population.

Oogonial Since the 1950s, the obstetrical dilemma hypothesis (OD) has dominated evolutionary reconstructions of human reproductive biology and behavior. The OD holds that antagonistic selection for a large neonatal brain and a narrow, bipedal-adapted birth canal poses a problem for childbirth; the solution during human evolutionary history was to truncate gestation, resulting in the birth of an underdeveloped, relatively altricial neonate for a primate.

However, the OD does not hold up to current evidence. First, there is no support for the assumption that a wider human pelvis is detrimental to bipedalism. In human walkers and runners, effective mechanical advantage is not predicted by biacetabular breadth (evidence comes from tests of kinematics, kinetics and oxygen consumption during walking and running as well as lower body MRI to determine how pelvic anatomy influences locomotor biomechanics). Second, human gestation is not truncated; it is longer than expected when controlling for maternal body mass (z-score=0.6 calculated from log/log trend line for 21 primate species, r2=0.56). And, third, pregnant humans invest more, not less than expected (relative neonatal body mass: r2=0.84, z-score=1.29; relative neonatal brain mass: r2=0.87, z-score=1.01). According to our model of fetal, neonatal, and maternal energetics, built with published measures of energy expenditure, a longer human gestation is simply not feasible. Thus, we offer an alternative hypothesis to the pelvic-based OD, called the EGG (energetics, gestation and growth) hypothesis, to explain the evolution of human gestation length. This human model is hypothesized to apply to other primates and eutherian mammals as well.

In conclusion, although pelvic remodeling during hominin evolution may have contributed to the present parturitional difficulty, this has not altered the timing of birth and is not the cause of the seemingly underdeveloped state of neonates.

Human altriciality reflects just how much more encephalization remains to be accomplished after parturition compared to other primates. This new energetic perspective (EGG) on the evolution of human gestation has important implications for understanding gestational variation within our species. This abstract was adopted from one that was presented at the annual meeting of the Society of Vertebrate Paleontology, on November 4, 2011, held in Las Vegas, NV. Correlations between pain and EMG activity were evaluated with crosscorrelograms. Movement of abdominal muscles and pelvic organs were evaluated by optic flow analysis of ultrasound video recordings.

Results: Abdominal muscle activity consistently (97%) preceded elevations in pain in 4 out of 9 subjects, implying that the mechanisms underlying abdominal muscle activity are responsible for menstrual pain perception in some subjects. Cross-correlograms confirmed that abdominal muscle activity predicted pain magnitude (p<0.05). After partial alleviation of pain in these subjects with naproxen (28%, p<0.05), SMG activity time-locked to cramp onset was also significantly reduced (p<0.05). In 2 subjects abdominal muscle activity occurred only after pain onset; in 3 subjects abdominal muscle activity did not occur before or after pain onset. To reassess these observations using an alternative data capture method, we performed an optic flow analysis on the same ultrasound videos used to capture the SMG. In a subject with abdominal muscle activity preceding spontaneous menstrual pain, we detected pixel motion primarily in the abdominal surface muscles using optic flow analysis. Other phenotypes were either associated with bladder, bowel or uterine movements. Discussion: These findings demonstrate the feasibility of SMG to differentiate menstrual pain phenotypes. Some forms of menstrual pain appear to be mediated by abdominal muscle activity implying that abdominal somatic afferents/ efferents may be a critical target for reducing menstrual pain. Additional data from this ongoing study will facilitate development of SMG for validated research and clinical tools. . Surprisingly, billed costs were only 11% higher in abdominal than laparoscopic surgeries, due to a near 4-fold greater cost in surgical supplies that offset overnight hospital stays. Moreover insurance reimbursement for abdominal vs. laparoscopic surgery was greater (23 ± 9% vs. 17 ± 9%, mean ± SD, p <0.0001). The incidence of complications requiring additional medical intervention did not differ. Conclusions: From the viewpoint of institutional reimbursement, laparoscopic hysterectomy results in lower reimbursement, both of outpatient surgery and disposable materials used during surgery. This study was not powered, nor is the medical record adequate to determine why abdominal rather than laparoscopic surgery was chosen on an individual doctor/patient basis. However it is disturbing that more patients undergoing abdominal hysterectomy had private rather than public insurance, and that insurance reimbursement is higher for an inpatient than outpatient procedure despite obvious benefits to the latter.

Optimizing the Modified Laparoscopic Vecchietti Procedure. BACKGROUND: Ectopic pregnancy accounts for 1-2% of pregnancies, however, only 25-30% are eligible for medical treatment, largely due to the limited efficacy of methotrexate. Gefitinib inhibits the epidermal growth factor receptor (EGFR), which is highly expressed in placenta. Our preclinical work shows that combination gefitinib and methotrexate supra-additively regresses placental tissues. We have translated this combination treatment approach to a phase II human clinical study. OBJECTIVE: To assess the safety and efficacy of combination gefitinib and methotrexate in a larger cohort of women with ectopic pregnancy. METHODS: We are recruiting 40 women with hemodynamically stable ectopic pregnancies and treating them with 50mg/m2 IM methotrexate and 7 days of oral 250mg gefitinib. Safety is assessed through regular clinical reviews and blood tests. Efficacy will be measured as rate of serum hCG decline and time taken to achieve cure compared to a contemporaneous cohort of women with ectopic pregnancies treated at our institutions with methotrexate only. RESULTS: We have recruited 20 participants and 17 have completed treatment.

The combination is well tolerated with no serious adverse events reported. Common side effects are rash in 15/17 (88%) and diarrhoea in 9/17 (53%) participants. An interim data analysis of participants with pre-treatment hCGs of between 1000-3000IU/L (n=13) shows that the relative change in participants' hCG levels between days 1 and 4 is 0.77 (95% CI 0.53-1.01) compared to controls 1.07 (95% CI 0.97-1.16) (p=<0.02) who were treated with methotrexate only (n=85). There is a trend for mean hCG levels at days 4 and 7 to be lower in participants compared to controls. Mean GA at emergency cerclage was 20.9 ± 2.7 weeks. In the cerclage group there was a significantly higher rate of multiple pathogens at cervicovaginal swabs at admission and/or before delivery (p =0.001). Among short and long term infant outcomes only neonatal sepsis was more frequent in the cerclage group (41% vs 14%, p=0.03). Conclusion: Neonatal sepsis is significantly higher in cases of emergency cerclage that deliver before 34 weeks without difference in long term infant outcome. Changes in the vaginal microbiome may result in complications of pregnancy such as preterm delivery, the single most significant factor contributing to infant morbidity and mortality. To begin exploring the role of the vaginal microbiome in this and possibly other complications of pregnancy, we conducted a longitudinal assessment of the vaginal microbiome during pregnancy. Here we report the results of twelve subjects who had a vaginal swab of the posterior fornix and cervix at 8-12, 17-21, 27-31, and 36-38 weeks gestation. Asymptomatic women with uncomplicated pregnancies were enrolled in the study following an IRB approved protocol. One of the exclusion criteria was any antibiotic treatment during the preceding 8 weeks. Total DNA from the samples was extracted and 16S rDNA amplified (V3-V5 region) and sequenced using high-throughput next generation sequencing. In addition to the microbiome swab, subjects completed a sexual history questionnaire and microscopy was performed to assess bacterial vaginosis ( helveticus. This persisted in the remaining time points in all but one subject who had a mix of L. helveticus (58%) and L. iners (31%) at 36 weeks gestation. We have found that the vaginal microbiome remained stable throughout pregnancy and even tended toward a monoculture. This is in contrast to what has been seen in women during the menstrual cycle and may reflect the more stable hormonal milieu during pregnancy. There were no complications of pregnancy in the women enrolled for this analysis, but the stability of the microbiome and the tendency toward a single dominant species may facilitate the identification of changes that could herald complications of pregnancy. Objective: There has been a steady increase in the primary cesarean rate (CS). A first CS sets the path for successive CS's and the associated costs and complications. One of the main indications for a primary CS is "failure to progress" or "cephalo-pelvic-disproportion", and the diagnosis are made after oxytocin use for augmentation of labor. Thus, we decided to examine our use of oxytocin during labor. Methods: We reviewed 434 consecutive charts of patients admitted between May-July 2012 to the labor and delivery at ARMC, the county hospital at San Bernardino, CA. Inclusion criteria were singleton live pregnancies at term and vertex presentation. Exclusion criteria were patients with previous CS, multiple pregnancies, abnormal placentation or presentation, and elective primary CS for maternal or fetal indications. Chart were reviewed and the following data were obtained: age, parity, BMI(weight/ height 2) at time of delivery, method Friday of delivery, use of oxytocin (augmentation versus induction), cervical exam at time of admission, Apgar scores, and birth weight. Spontaneous labor was defined as patient having regular uterine contractions and a cervix = > 3 cm of dilatation, regardless of effacement. The maternity unit is covered 24/7 by in-house attending, OB residents, and physician assistants. Attending decided if to use oxytocin and the rate of infusion. Mean and SD were obtained, and compared by paired and unpaired t-test.

Results: 63% of patients were admitted in spontaneous labor, of which 37% received augmentation. A 6% primary CS was observed in this group. In the induction group (n=160), all received oxytocin. The primary CS rate was a 13%, a significant difference from the spontaneous labor (p <0.01). There was no difference on the groups BMI, except in the primary CS in the induction group (BMI=37.7 ± 9.7 n=21) versus the spontaneous labor group (BMI = 32 + /-5.6, n=6). We did not observe a difference in birthweight, Apgar score, or umbilical artery pH among those delivering vaginal versus CS. Conclusion: Our hospital has a relatively low primary cesarean section rate. We did not find an increase failure to progress rate in patients with higher BMI. Furthermore, we were unable to confirm a higher birthweight in patients undergoing CS; however, the maternal BMI in those patients undergoing CS in the induction was significantly higher (p< 0.01). Threatened preterm labour (TPTL) accounts for about 30% of pregnancy-related hospital admissions. Only 5% of these symptomatic women will deliver a premature baby (<37 weeks gestation) within 2-10 days. Increasing evidence demonstrates that peripheral leukocytes can be used to monitor a variety of biochemical and physiological processes occurring in the body. Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) 8-plex coupled to LC-MS/MS was employed to quantify and investigate differentially expressed leukocyte lysate proteins obtained from peripheral blood in women with TPTL.

Blood was collected at point of hospital admission from 16 women who had preterm birth within 48 hr and 24 women who did not deliver within 48 hr. Women were matched for maternal and gestational age at presentation, gravidity and parity. Leukocyte lysates were digested, reduced and cysteine-blocked. An aliquot from each sample was combined to make a superpool as the reference control. For each 8-plex, 7 individual samples randomly selected from each group of women were labelled (25ug) using channels 113 to 119. The reference superpool (25 ug) was labelled with channel 121. After cation exchange and zip-tip clean up, six 8-plex runs were analysed using the TripleTOF 5600. Data was processed using ProteinPilot and supplementary software. A total of 763 proteins were identified using UniProt/Swiss Prot database (Global FDR 1%); 354 proteins were quantified; and 14 proteins (HNRPD, ALBU, CAN1, RAP1B, HXK3, RS15A, COX5A, ADDA, MIF, IQGA2, ST1A1, ST1A2, UBIQ and UBQL1) were differentially expressed between the two groups (p<0.05, Mann-Whitney U test). These leukocyte proteins are involved in various cellular metabolic processes, particularly in mRNA translation. These data provide further insights into the pathophysiology of TPTL. High levels of stress in pregnancy increase the risk of preterm birth (PTB). This may be due to altered hypothalamic-pituitary-adrenocortical axis (HPA axis) activation but the biologic mechanism remains poorly understood. Extraction of cortisol from human hair may be used as a marker of chronic HPA axis function. Ultrasound measurement of the fetal adrenal gland is another potential non-invasive marker of stress. Premature enlargement of the fetal adrenal gland has been strongly correlated with PTB. It is unknown whether fetal adrenal gland measurements correlate with fetal cortisol exposure or whether early enlargement of this gland may be predictive of PTB. These differences persisted even after adjusting for antenatal steroid use.

Conclusion: Meta-analysis data indicate no relationship between latency period and adverse outcomes in PPROM. Our findings linking low GA at delivery to increased mortality suggests that the practice of elective delivery in stable PPROM cases should now be reassessed. Conclusions: The results suggest that women with total S25OHD concentrations below 30nmol/L had increased risk of SPB, which was more strongly evident in women with S25OHD3 below 30nmol/L, whose risk of SPB was increased more than 2-fold.

Analysis of Fecal Microflora Composition in Preterm Infants Using 16S rRNA. Masako Kitsunezaki, 1 Manabu Kemmochi, 2 Mari Ooka, 2 Takashi Ito, 2 Masahiko Nowatari, 2 Yuki Bando, 2 Masahiro Ishii. 2 1 Pediatrics, Sagamihara Kyodo Hospital, Sagamihara, Kanagawa, Japan; 2 Pediatrics, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan. Background:Commensal bacterial colonization affect the maturation of mucosal immunity, but study on the development pattern of the intestinal microflora in early life is limited. The composition of microflora may be influenced by various factors, such as nutrition, delivery method and antibiotics treatment, etc. The aim of this study is to understand the sequential change of microbial flora in the feces from preterm baby through 16S rRNA PCR assay. Material and Method:The fecal samples were obtained from 33 infants born at our hospital, between June-August 2011. Preterm babies (31.6±0.5weeks gestation, 1398±83g birth body weight) were cared for the NICU for the period of sample collection. Fresh fecal samples were collected four times during the age of 1-30days (day1.5±0.2, day7.1±0.2, day14.2±0.3, day30.3±0.8). The samples were refrigerated under anaerobic conditions. After bacterial DNA was extracted using a QIamp DNA stool Mini Kit, subjected to PCR amplification using the species-specific primers (Bifidobacterium, Enterobacter, Enterococci and S. aureus). The effect of gestational length, feeding materials, supplement of antibiotics was compared to the flora composition. Results:In the group of probiotics treatment and the group of breast milk, the colony of Bifidobacterium was found at very early postnatal day. However, the total titers Bifidobacterium on the late postnatal day (day 30) samples were not different between the group. S.Aureus was not colonized until the first week of life in the group of breast milk. Enterobacter, Enterococci were predominant in the feces from day 30 infant of the breast milk group. Conclusion:Mucosal immune function in preterm infant is under discussion. Although probiotics and breast milk may work to compose the balanced microflora for infants in NICU, long term observation is required to determine if this contribute to adverse outcome in future.

Outcomes Aim of the study was to assess maternal and fetal outcomes and independent predictors of caesarean delivery(CD)in an obstetric cohort of post-term pregnancies beyond 41 gestational wks managed expectantly until 41.3wks. Retrospective study of all singleton pregnancies undelivered at 41 wks and managed expectantly according to a standardized protocol, that delivered in our Dep in the period between June 2011 and May 2012. Antenatal fetal testing with biophysical profile was performed at 41, 41.3, 41.5 wks. Stripping of membranes was performed at 41.3wks and induction at 41.5. Asphyxia was defined as a 5 minutes Apgar score<7 and/or umbilical artery pH≤7 and/or BE≥-16 and/or need for intubation and/or resuscitation. We compared deliveries between 41 and 41.2 wks and beyond 41.2 wks (41. and evaluated independent predictors of need for CD. We included 508 pregnancies:n=272(53%) between 41 and 41.2 wks and n=236 (47%)at41.3-42. The overall rate of spontaneous labor was significantly higher between 41 and 41.2 wks vs 41.3-42 wks (p<0.01;66%vs46%). The rate of CD (p=0.14;13%vs18%), of operative vaginal deliveries (p=0.51;7%vs8%), and of spontaneous delivery (p=0.09;79%vs74%) were not significantly different in the two groups. No differences in birth weight, umbilical artery pH (p=0.19, 7.3±0.1 vs 7.2±0.07) and asphyxia (p=0.49, 0.3%vs0.4%) were found in the two groups. One stillbirth occurred at 41.4 weeks. At univariate analysis parity (p<0.001) and maternal height (p=0.002) were associated with CD, whereas maternal BMI (p=0.16), gestational age at birth (p=0.08), induction (16% vs 10%,p= 0.04) and use of prostaglandin (18%vs 15%,p=0.41) were not. At multivariate analysis only parity (OR 0.17, 95%CI0.1-0.4) and maternal height (OR0.92,95%CI0.87-0.96) were independently associated with lower risk of CS whereas induction of labor and advancing gestational age were not.

Expectant management between 41 and 42 wks enables a great number of patients to achieve spontaneous onset of labor without increasing the need of CS and neonatal morbidity. The only independent predictors of CS are nulliparity and maternal height. After adjusting for confounders, anesthesia was found to be associated with decreased odds of severe perineal laceration (aOR 0.61 [0.41-0.9]) while Asian race was found to be associated with increased odds of severe perineal laceration. Oxytocin and advanced maternal age did not appear to affect the odds of perineal laceration.

The now widespread use of epidural anesthesia and oxytocin induction or augmentation of labor does not appear to increase the odds of severe perineal lacerations. 

From 2010-2012 we enrolled pregnant women with influenza-like illness and laboratory-confirmed influenza prior to treatment. A comparison group of women without influenza-illness matched 1:1 for maternal age (within 5 years), ethnicity, and gestational age (within 2 weeks) to women with influenza was also enrolled. Maternal blood was collected at enrollment. A commercially available 12-plex panel was used to quantify 12 pro-or anti-inflammatory cytokines applying microsphere-based Luminex technology on sera. For viral load, frozen maternal whole blood specimens were thawed and RNA was extracted on the Qiagen EZ one. Purified influenza virus RNA was quantified by RealTime PCR using separate primers and probes specific for Influenza A and B strains and synthetic RNA dilutions to produce standard curves for each assay. The Wilcoxon signed-rank test was used to compare groups. RESULTS: 11 pregnant women with confirmed influenza (type A=5 and type B=6) and 11 matched women without influenza are analyzed. Mean gestational age at enrollment was 16.4±7.6 vs.16.7±7.0 wks respectively. Of the 12 cytokines, 2 pro-inflammatory markers, Interleukin (IL) 1-beta and macrophage inflammatory protein 1-alpha, were undetectable; concentrations of the other 10 cytokines are compared (Table 1) . Several pro-inflammatory cytokines including TNF-alpha and IL 1-alpha were increased; anti-inflammatory IL-10 was also increased. There was no detectable viremia (influenza RNA) in women with or without influenza (as a control, a negative blood sample was spiked with influenza virus and it was readily detected). CONCLUSION: During pregnancy, influenza infection is not associated with a viremia but it induces both a pro-and anti-inflammatory systemic response involving selected cytokines. Variations in the inflammatory changes in relation to type of influenza, clinical severity, gestational age, and fetal outcomes warrant further study. Intrapartum changes in maternal temperature are known. The relation of epidural analgesia (EA) on these temperature changes, as well as the mechanisms leading to these changes, remain unclear. We performed a prospective cohort study to investigate the effect of EA on intrapartum temperature changes.

We included 43 healthy, term, parous women in spontaneous labor with singleton pregnancies ≥37 weeks delivering at our hospital in a 2 months period. Women with documented prelabor rupture of membranes (ROM) > 18 hours and fever at admission were excluded. Maternal tympanic temperature was measured 2-hourly during labor. Fever was defined as a temperature of ≥38°C.

Maternal and neonatal outcomes were compared between women receiving EA (n = 30) and not receiving EA (n = 13). Differences between groups were analysed with T-test and ANOVA. A p value < 0.05 was considered significant.

No differences in maternal temperature at admission and intrapartum duration of ROM were noted between both groups. Intrapartum fever occured in 40% of women with EA as compared to women without EA, p<0.006. Women with EA had a higher temperature during the second stage of labor as compared to women without EA, p<0,003. Subgroup analysis of women with EA who developed intrapartum fever had a higher temperature at the onset of labor as compared to women with EA without fever, resp. 37,3°C vs. 36,8°C, p<0,006. Instrumental delivery occured in 30% of women in the EA group while none were performed in women without EA, p<0,03. The overal length of labor was greater in women with EA as compared to women without EA, resp. 9,4 hrs vs. 5,1 hrs, p<0,005. Neonatal outcome at birth did not differ between both groups. CONCLUSIONS: EA is associated with intrapartum fever without affecting neonatal outcome.

The higher temperature at the onset of labor seen in women with EA seems to rule out infection as a cause of EA-associated fever. Whether the latter is triggered by maternal infection or pharmacological properties of EA remains to be elucidated.

Neonatal Outcomes after Fetal Acidemia. Emilie L Vander Haar, Jaclyn M Coletta, Mary E D'Alton, Cynthia Gyamfi-Bannerman. Obstetrics and Gynecology, Columbia University Medical Center. Objective: Little is known regarding outcomes of infants after peripartum fetal acidemia, defined as pH <7.0, however, acidemic fetuses are at risk for neonatal encephalopathy (NE), a heterogeneous syndrome characterized by symptoms of central nervous system dysfunction after late preterm or term birth. We sought to determine short-term neonatal outcomes including the incidence of NE in fetuses born with a pH less than 7.0. Methods: We conducted a retrospective cohort study of all deliveries between January 2004 to December 2011 with a fetal cord blood umbilical artery pH of less than 7.0. Routine delivery cord blood is collected at our institution. Data were abstracted from medical records to determine neonatal outcomes including birthweight, Apgar score, admission to the neonatal intensive care unit (NICU), length of stay, head imaging and outcome at discharge. In addition, baseline and subsequent physical exam documents were reviewed to determine whether the neonate fulfilled the clinical definition of NE. Babies with NE were then compared to babies without NE. Results: Of 33,295 deliveries in our study period, 55 neonates had a cord blood pH <7.0 (0.17%). Of these, 33 (60%) were admitted to the NICU and 9 (16.4%) were diagnosed with NE. Outcomes by diagnosis are noted in the table.

Outcomes in Neonates with and without NE Diagnosis NE Non-NE Mean NICU length of stay (days) 13 (4-48) 2.7 (1-15) Median NICU length of stay (days) 11 4 (1-15) Median 5 min Apgar score 4 (2-9) 8 (5-9) Mean pH 6.83 (6.67-6.99) 6.95 (6.78-6.99) Mean base excess 18 (11-26) 11.5 (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) Within the NE cases, 4/9 (44%) had seizures, 3/9 (33%) had abnormal MRI findings and 3/9 (33%) underwent head cooling. When NE was not diagnosed, none of those outcomes occurred. The mean pH for neonates who developed seizures was 6.74 (range 6.67-6.81), with a mean base excess of 20.8 (range 15-26). There was one neonatal death after withdrawal of care in the group with NE. None of the neonates were transferred to a long-term rehabilitation facility, and importantly, of the neonates without NE, none of these morbidities were noted. Conclusion: Only a small proportion of neonates with fetal acidemia went on to develop neonatal encephalopathy. Adverse outcomes were more common in the group meeting criteria for NE. Longterm outcomes of these neonates are necessary to determine the consequences of perinatal acidemia.

Outcomes Introduction: Alterations in the human vaginal microbiota and the absence of lactobacilli are characteristics of bacterial vaginosis (BV), which is a known risk factor for preterm birth (PTB). Lactobacilli are a common commensal of both the human and mouse vagina. Oral supplementation with probiotic L. rhamnosus GR-1 (GR-1) and L. reuteri RC-14 restores lactobacilli in the human vagina; reduces BV recurrence and confers benefits to urinary and reproductive tract health. To date, the mouse vaginal microbiome has been characterized only by culture-based methods. We hypothesize that oral administration of GR-1 will colonize and alter the bacterial composition of the mouse vaginal environment. Methods: A daily dose of saline or GR-1 (10 9 cfu) was given orally for 3, 5 and 7 days until gestational day 15. Bacterial DNA was extracted from vaginal tissues (MoBio DNA) on day 16. We monitored the mouse vaginal microbiota using next-generation Ion-Torrent sequencing by sequencing the V6 region of the 16S rRNA gene. Statistical analyses were performed using Krustal-Wallis followed by Dunn's test (significance was defined as p < 0.05). Results: Ion torrent sequencing revealed a reduction in Gammaproteobacteria in the saline control mice from 66% Gammaproteobacteria to 10.5%, 10.4% and 23.4% in mice oral gavaged with GR-1 for 3, 5 and 7 days respectively. Conclusions: Despite advances in obstetric care including BMZ, aggressive use of antibiotics and close fetal monitoring, the majority of parturients diagnosed with PPROM will deliver within one week. There may be room for repeat course of BMZ in the population of parturients with a latency period greater than 14 days to the reduce risk of neonatal morbidity. In utero exposure to intrauterine inflammation is a major cause of adverse neurological outcomes. Utilizing a mouse model of intrauterine inflammation, we demonstrated fetal and neonatal brain injury. The objectives of this study were to elucidate long-term neurologic sequelae in the offspring: 1)behavioral; 2)MRI changes and 3)immunohistochemistry of brain tissue.

Methods: A mouse model of intrauterine inflammation was utilized. CD-1 mice were randomized to intrauterine infusion of lipopolysaccharide (LPS) or saline (NS) at E17. At postnatal days (PND) 5, 9 and 13, offspring were tested by the following developmental behavioral tests: geotaxis, surface righting, cliff aversion and open field (n=11 LPS and n=4 NS litters). Open field was repeated at PND 60 (n=8 LPS and n=4 NS litters). MRI was performed using Brucker 11.7 T horizontal bore scanner under general anesthesia at PND14. T2 weighted images were acquired for evaluation of: whole brain, neocortex and ventricles. At PND 14 and 60, mice were sacrificed for immunohistochemistry of periventricular white matter and neocortex. The following staining was performed for confocal microscopy evaluation: Iba1(microglia), GFAP (astrocytes), MBP (oligodendrocytes) and NeuN (neurons).

In utero exposure to LPS resulted in decreased motor activity and increased time to perform developmental tests (geotaxis, cliff aversion, open field and surface righting (p<0.05 for all)). Furthermore, LPS-exposed adult mice exhibited hyperactivity on open field test at PND 60 (p<0.05). These findings were associated with generalized brain edema on MRI with increased volumes of whole brain and neocortex (p<0.05 for both) on PND14. On immunohistochemistry, LPS-exposed mice, demonstrated persistent microglial activation in periventricular white matter and neuronal loss in neocortex at PND 14 and 60. Conclusion: Exposure to intrauterine inflammation in utero appears to lead to persistent microglial activation, neuronal loss in adult brain and is associated with neurologic sequelae. Future fetal and neonatal therapeutic interventions may be tested in the mouse model by attempting to decrease the long-term neurological outcomes. to mimic gestational androgen exposure as in preeclampsia. Six month old female offspring were examined for plasma T levels and ovarian steroidogenic enzymes expression. Some rats were treated with flutamide @ 15 mg/kg/sc, BID for 10 days and radio telemetric blood pressures (BP) were recorded. Endothelium-dependent and -independent vascular reactivity with wire myograph were assessed in mesenteric arteries (MA). Results: Plasma T levels were higher in TP offspring (0.84±0.03 vs 0.45±0.09 ng/ml in controls). Ovariectomy of TP rats prevented T increase. StAR and cyp11A1 were upregulated while aromatase was downregulated in TP ovaries. Baseline BP was higher in the TP females. Flutamide reduced BP in TP offspring but not in controls. Cessation of flutamide reversed BP to pretreatment levels.

Endothelium-dependent acetylcholine relaxation were lower in MA of TP offspring (E max =68±7.9 vs 88±3.0% in controls). Further assessment of endothelial factors showed that only NO-mediated relaxations were blunted in TP MA but prostacyclin-and EDHF-mediated relaxations were unaffected. Flutamide reversed the reduced NO-mediated relaxation responses. In endothelium-denuded MA from TP offspring, PKC-mediated contractions were greater and these enhanced responses in the TP offspring were reversed by flutamide.

This study shows that prenatal T excess acts as an endocrine disruptor to cause defective gonadal steroidogenesis, generating increases in plasma T levels in adult females, which causes defective vascular function hypertension. 

Preadipocytes showed dose-dependent increased proliferation in response to BPA (Fig A) . Despite no change in the medium lipid content, BPA-exposed adipocytes exhibited dramatic increase in lipid staining (Fig B) .

CONCLUSION: Adipocyte progenitor cell exposure to BPA causes increased proliferation and a marked increase in intracellular lipid. Mechanisms for enhanced lipid storage may include upregulation of lipogenic enzymes or BPA-mediated differentiation to terminal adipocytes. These results suggest that early developmental exposure to BPA may contribute to programmed adipogenesis and obesity. Maternal obesity is related to insulin resistance (IR) in the offspring. Multiple signalling branches of endoplasmic reticulum (ER) stress pathway have been implicated on IR development in different models of obesity. Thus, we evaluated cellular insulin response and activation of ER stress markers in human umbilical vein endothelial cells (HUVEC) from pregnancies with maternal obesity. Ethics Committee approval and informed consent were obtained. Primary cultured cells from pregnancies with maternal obesity (HUVEC-OB) and normal nutritional status (HUVEC-N) were exposed (0-60 min) to physiological levels (1 nM) of insulin. Total and phosphorylated Akt (P-Akt), p44/42 mapk (P-p44/42 mapk ) were assayed by western blot. Inhibitory phosphorylation (serine 307) of IRS-1 (P-ser307-IRS-1) was also measured. ER stress activation was evaluated by phosphorylation of PERK (P-PERK) and ATF-6 (western blot), and XBP-1 splicing (non-quantitative RT-PCR). HUVEC-N exposed to insulin showed an early increase of P-Akt and P-p44/42 mapk (∼12 and ∼20-fold in relation to basal level, respectively), with a maximal response at 1 minute. HUVEC-OB exhibit retarded and reduced P-Akt and P-p44/42 mapk (∼5 and ∼8-fold, respectively) in response to insulin, with a maximal increase at 15 minutes. Increased levels of P-ser307-IRS-1/total IRS-1 ratio (2.6 ± 0.25 vs 1.0 ± 0.32, p<0.05) and P-PERK/total PERK ratio (3.2 ± 0.44 vs 1.0 ± 0.11, (31) in the years 1993-2011. Exclusion criteria were: recurrent pregnancies or family members with NTDs/OFC, maternal insulin-dependent diabetes mellitus, maternal intake of clomiphene or anticonvulsants. We used as control group a sample of 56000 pregnancies managed at our hospital in the same period.

According to the month of conception, both case and control groups were divided into 4 subgroups, corresponding to the four seasons. The conception dates were determined by estimation based on the reported maternal last menstrual period. We then compared for every season the rates of conception between each defect group and control group using the Fisher test, with p < 0,05 considered significant. RESULTS: Of the 106 women, 6 met the exclusion criteria and were not included in the analysis and no woman took the preconceptional supplement of folic acid. Fisher Test analysis showed a significantly seasonality only in SB group with an higher prevalence of conceptions in fall than in control group (19/41, 46,3%, p = 0,003) and a lower prevalence in summer (4/41, 9.7%, p=0,019). [ Table 1 ]. CONCLUSION: The seasonality in the prevalence of isolated SB suggests that environmental factors play a greater role in the development of malformation compared to the other two fetal anomalies. Moreover, all three defects are folic acid-preventable but isolated SB appears more correlated with folate status, which may vary with season, because of changes in diet and for heat (cooking methods, climatic condition and maternal fever). greater increase in visceral adipose tissue AGT expression (9-fold). In both post-weaning diets, subcutaneous adipose tissue AGT was similar in OB and Control males.

The adult male offspring of obese pregnancies have notably increased adiposity and upregulated visceral adipose AGT. As expected, postweaning high fat diet increased adiposity in both groups, but exaggerated the increased adipose tissue AGT expression in OB offspring. These findings suggest that programmed adiposity and depot specific enhanced AGT may contribute to hypertension in offspring of obese dams.

In Utero Magnesium (Mg) Deficiency Programs Brain Development, Growth, Immune Function, and Behavior in the Offspring. OBJECTIVE: To use a genome-wide cytosine methylation assay as a discovery platform to identify loci of interest (LOI) linking adverse intrauterine environment (exposure to maternal western diet and calorie restriction) with an aging phenotype in female offspring. STUDY DESIGN: SD dams were fed 1 of 3 diets: standard chow (Con), calorie restricted (pair-fed 60% kcal/d of Con) from gestational day 11 through lactation (CR) or western diet from 3 wks through gestation/lactation (WD). Litters culled (8/lit) and fed standard chow post weaning. Massively parallel sequencing-based HELP assay used to examine cytosine methylation levels at >1.65 million loci in the liver of the three female offspring groups at 9 wks (6/gp) and compared to aged females (Old, n=4) at 19-20 months. LOI were determined using two-sided t-test for locus-specific difference in average methylation between groups and control (p<0.05) and magnitude of methylation difference between group means (angle >25 or <-25). Identified LOI in promoter regions uploaded to Ingenuity Pathway Analysis (Redwood City, CA) to determine ontology and protein interactions. 13 LOI relevant to metabolism and aging were selected for validation with quantitative pcr. RESULTS: While Old demonstrated global hypomethylation compared to Con, no global methylation differences were seen between WD, CR, and Con. Self sorting Heatmap demonstrate significant regional differences between CR and WD compared to Con with corresponding loci from Old ( Figure 1 ). Statistically significant changes in gene expression between study groups compared to Con were noted in 7/13 LOI relevant to insulin resistance and cardiovascular disease.

CONCLUSION: This study demonstrates that a set of functionally significant loci predictive of an aging phenotype may be identified utilizing a genome wide epigenomic assay as a discovery platform. Further validation of other LOI may provide insight into the mechanisms responsible for developmental programming. Objective: To characterize the renal phenotype and genome-wide DNA methylation dysregulation in male offspring exposed to maternal calorie restriction. Study Design: SD dams were fed standard chow (Con) or calorie restricted diet (pair-fed 60% kcal/d of Con) from gestational day 11 through lactation (CR). Litters culled (8/lit) and fed standard chow post weaning. Non-invasive tail-cuff blood pressure (bp) and urine albumin levels (via ELISA) were obtained from 2 and 6 mo old male pups (8/gp). Kidneys were harvested at 2 mo for histology, glomeruli counts and DNA extraction. Massively parallel sequencing-based HELP assay was used as a discovery platform to examine cytosine methylation levels at >1.65 million loci (4/gp). Results: At 2 months, CR had similar mean bp (145±3/94±3 vs 149±2/97±2 mmHg, p=NS) and albumin/ creatinine ratios (0.37±0.07 v 0.34±0.09 mg/mg, p=NS) compared to Con. While no significant differences in proportion of cells were noted on hystology, CR demonstrated decreased glomerular number (20,177±933 vs 31,680±1126 glomeruli/kidney, p<0.01) compared to Con. By 6 months CR developed systolic and diastolic hypertension (161±5/109±4 v 140±5/94±5 mmHg, p<0.05) and increased albumin/creatinine ratios (0.69±0.06 v 0.42±0.13 mg/mg, p=0.06) compared to Con. While no global methylation differences were seen among groups, a heat map of the top 500 differentially methylated loci in the 2 month kidney demonstrate distinct global regional differences ( Figure 1 ).

In utero exposure to maternal CR lead to phenotypic and epigenetic renal dysregulation in young male rats and suggests that the underlying pathophysiology for development of chronic kidney disease and hypertension later in life may originate from developmental programming. Assessment of genome-wide epigenetic alterations in the kidney may provide insight into the mechanisms responsible for developmental programming. We have shown that antenatal exposure to glucocorticoids (GC) induces hypertension in adult life and it is associated with an increase responsiveness to endothelin (ET) in vitro. The aim of this study was to investigate the mechanisms responsible for the increase response to ET-1 in isolated resistance arteries. METHODS: Pregnant sheep were treated with two IM doses of betamethasone (BM, 0.17 mg/kg) or vehicle (V) 24 h apart at 80 days of gestational age and allowed to deliver at term. Sheep were euthanized at 1.5 yr of age. Arteries (300 µm) obtained from the brachial vascular bed in V (n=8) and BM animals (n=10) were mounted on a wire myograph for measuring isometric force. Responses to eight increasing concentrations of Endothelin-1 (ET-1; 10 -9 -10 -7 ) and KCl were assessed in the absence and in presence of inhibitors of cADPR synthesis, i.e. niacinamide (NIA; 10 mM) and 2,2'-Dihydroxyazobenzene (DAB; 60µM). NIA and sodium nitroprusside (SNP; 10 -8.5 -10 -4 M) were used to study the relaxing capacity of cADPR inhibition on KCl preconstricted arteries. Data are expressed as Mean±SEM and were analyzed by ANOVA. RESULTS: ET-1 significantly increased wall tension in both V (Panel A) and BM (Panel B). Arteries from BM animals displayed a higher sensitivity to ET-1 compared to V treated sheep (B). Pretreatment with NIA or DAB resulted in an significant decrease of the BM effect on the ET-1 response (B). NIAC was more effective in reducing KCl induced tension in V compared to BM. In contrast, SNP was equaly potent in both groups.

CONCLUSION: We have shown that antenatal GC treatment significantly increases blood pressure in sheep and also increases vascular reactivity. Here we show that the enhanced vascular response to ET-1 is mediated by the cADPR/RyR pathway. Inhibition of the cADPR pathway not only affects the response to agonist, but it also affects the relaxation responses. We propose that enhanced vascular reactivity contributes to the increased blood pressure observed. HL 68728

Effects Background: Early life lead exposure is common in the US with over 500,000 children aged 1-5 having blood lead levels exceeding 5µg/dL, the level recommended for intervention by the CDC. The underlying mechanism(s) that link lead exposure to adverse health outcomes are unclear. Epigenetic deregulation may explain these effects, and imprinted genes may be particularly vulnerable. Imprinted insulin-like growth factor 2 (IGF2) encodes a mitogenic factor critical to normal growth and neural function and is overexpressed in many cancers. IGF2 transcription is controlled in part by a differentially methylated region (DMR) whose methylation state is vulnerable to some early life exposures, but vulnerability to lead exposure is currently unexplored. Objective: To determine if physiologically relevant exposure to lead acetate in vitro alters DNA methylation at the IGF2 DMR. Methods: Immortalized normal human embryonic kidney cells (HEK293) were exposed to 0-25 µg/dL lead acetate in triplicate for 72 hours. The experiment was performed in quadruplicate. DNAs were bisulfite modified and analyzed by bisulfite pyrosequencing. One-way analysis of variance was used to compare mean methylation values across exposure groups. Results: IGF2 DMR methylation significantly differed across lead exposure groups (p=0.005) with a maximal decrease of 3.2% methylation at the 25 µg/ dL dose relative to the mock treated control cells.

Lead exposure in vitro is associated with decreased methylation at the IGF2 DMR at lead levels that are physiologically relevant to human exposure. These results support that lead-induced shifts in DNA methylation occur in human cells and furthermore suggest that IGF2, a gene highly relevant to growth, neural function and cancer, is vulnerable to these effects. Further work is required to confirm these findings in vivo. OBJECTIVE: Increases in a diversity of childhood and adult diseases (eg, autism, behavioral/learning abnormalities, obesity) have been attributed, in part, to programming effects resulting from developmental exposures. Bisphenol A (BPA) is a ubiquitous chemical widely used in plastics (eg, water bottles, food can liners) and paper industries. BPA is an endocrine disruptor (EDC) chemical which has estrogen receptor effects, and significant levels are consistently observed in pregnant women and fetal plasma and amniotic fluid. We sought to determine if fetal/newborn BPA exposure modifies neurogenesis, potentially resulting in altered cerebral structure or function. STUDY DESIGN: Newborn rats born from normal dams were sacrificed and brains dissected. Hypothalamic neuroprogenitor cells (NPCs) from control newborns were cultured in both complete and differentiation medium and treated with BPA (1, 10, 20 µM) or DMSO (control) for 5 days. NPC proliferation (MTT assay) was assessed, and protein expression (Western Blot) of NPC markers (Nestin), NPC proliferative and neurogenic factors (Hes1, Mash1), and markers neuronal and astrocyte cell types (Tuj1, GFAP, respectively) were analyzed. *P<0.05 vs. untreated cells. RESULTS: In response to BPA exposure, NPC proliferation markedly increased, as measured by MTT assay as well as Nestin and Hes1. In differentiation media, BPA (10 µM) increased NPC expression of Mash1, Tuj1 and GFAP (Fig A, B) , with a greater increase in Tuj1 as compared to GFAP.

CONCLUSION: Exposure to BPA causes increased NPC proliferation, in part via upregulated Hes1. Increased NPC differentiation to neurons as compared to astrocytes may alter cerebral structure and/or function and thus contribute to neurobehavioral abnormalities. Non alcoholic fatty liver (NAFLD) and obesity have reached epidemic proportions worldwide and an adverse in utero environment leading especially to small for gestational age (SGA) as well as high fat/high sugar foods may represent important risk factors for these adult diseases. Our primary focus was to investigate the effects of an adverse in utero (placental insufficiency) in combination with an adverse postnatal (a high-fat/high-fructose diet, HFHFr) environment upon adipose tissue and liver in young adult guinea pig offspring. Male newborns with a normal birth weight (NBW) and SGA pups (obtained by uterine artery ablation) were nursed by their dams who continued to receive a standard diet during lactation period. At postnatal day 15, pups were weaned and fed either the control diet (CONT, 18.3% fat, 8% fructose) or the HFHFr (45% fat, 16% fructose) until 140 days (young adulthood). Offspring were sacrificed at 140 days and epididymal adipose tissue (EPI) and liver phenotypes and functions were investigated. On the CONT diet, SGA offspring exhibited increased relative weight of EPI (P < 0.05). Interestingly, on the HFHFr, relative weight of EPI was similar in both SGA and NBW offsprings. In addition, on the CONT diet, the SGA displayed hypertrophied epididymal adipocytes compared to NBW offspring (P < 0.05) whereas HFHFr had no effect on adipocyte diameter in SGA and NBW offsprings. On the CONT diet, lipid content in EPI was also higher in SGA than in NBW (P < 0.05). Moreover, increased mRNA levels of transcriptional regulators of lipogenesis i.e. SREBP-1c and PPARγ and of lipogenic ACC gene were observed in the EPI from SGA fed on the CONT diet. SGA offspring fed the CONT diet, showed signs of an initiation of a fatty liver (increased hepatic cholesterol, lower gene expression of microsomal triglyceride transfer protein, MTTP). However, they didn't display any changes in hepatic triglyceride content. On the HFHFr, both NBW and SGA exhibited a fatty liver (increased hepatic triglycerides + cholesterol). Taken together, the in utero environment appears to play a major role in the setting of visceral obesity in young adulthood while initiating the development of a possible later fatty liver. HFHFr had no impact upon adipose development at this age, but promotes similar markers of NAFLD in NBW offspring as SGA alone.

Increased Introduction: Incidence of preterm birth (PTB) is significantly higher in blacks, who also have higher lifelong exposure to environmental toxins. In our earlier studies we reported glandular and stromal hyperplasia in rats exposed in utero to Benzo(a)Pyrene (B[a]P), a ubiquitous polycyclic aromatic hydrocarbon. We have previously reported that rats exposed to B(a)P exhibit PTB. Objective: To assess the changes in expression of genes encoding the enzymes involved in DNA methylation and histones modifications, in uterine myometrium of adult rats exposed in utero to B(a)P versus unexposed healthy controls. Methods: Female Long Evan rats exposed in utero to B(a) P were grown to adulthood (60 days) and euthanized. Uterus was dissected, endometrium removed and cDNA prepared from myometrium was subjected to epigenetic chromatin modification enzymes RT² Profiler PCR array (Qiagen) for enzymes known or predicted to modify genomic DNA and histones which in turn regulate chromatin accessibility and therefore gene expression. To understand the correlation between epigenetic modifications and cytokine and inflammatory genes expression, we performed Real time (RT)-PCR analysis for rat IL-1 β, IL-6, TNF α and iNOS genes using corresponding primers. Results: We observed more than 2 fold down regulation in SET domain genes Setd5, Setd6; Histone acetyltransferase genes Brca2, Edf1, Hat1, MII1, Med24, and Myst1; DNA methyltransferase gene Dnmt1; Histone Deacetylase genes Hdac1, Hdac3, Hdac5, Hdac6, Hdac10 and Mta2; Histone phosphorylation gene Aurka and DNA/Histone demethylase genes Jmjd6, Mbd2 and up regulation in Histone Methyltransferase gene Prdm2 in myometrium of rats exposed in utero to B(a)P vs controls. RT-PCR analysis showed a significant increase in mRNA expression of IL-1 β and TNF α and decrease in iNOS and IL-6 (p<.05) in myometrium of rats exposed in utero to B(a)P vs control.

Conclusions: This data suggests that in utero exposure of rats to B(a)P leads to permanent developmental reprogramming of PTB-relevant genes such as pro-inflammatory cytokines, likely via chromatin modification. Further research is needed to understand the contribution of epigenetics to the health disparity in PTB. Funding: RCMI 5G12 RR003032-27. Our approach is to automatically extract and refresh clinical data from the hospital EMR systems (EPIC and GE-IDX) into our research database to make the process more efficient, reduce errors, and protect patient privacy.

Our biobank team collaborated with the ICTS data team to develop our Biobank data registry. This registry dynamically extracts and links the EMR data with the specimen data, de-identifies it, provides a web portal for access, and automates the data refresh from the EMR. We will maintain our study patient list in EPIC, which is referred by Extraction, Transformation and Load programs daily to refresh our study registry. The purpose is two-fold: to help us avoid the manual phenotypic data entry, and to load the EMR data directly into a database located in a secured environment. A web portal built on top of the secured database provides an interactive interface to view the list of all the patients enrolled in four biorepositories, their episodic data, samples associated with each patient, clinical outcomes, and some basic graphs to evaluate the biobank in terms of samples and subjects. The software allows for de-identified information to be shared with other researchers using a web based secure application. Biorepositories are critical to the successful conduct of translational research because they provide more focused and appropriate samples. However, it is necessary to develop dynamic tools to link specimen information with EMR data. Critical to biobank solutions are security, ability to query data, and accurate clinical and specimen data. Many EMRs have limited ability to interface with biospecimen management software and struggle with attaching phenotypic information, however, our software will provide detailed EMR data integrated with specimen data through automated integration of our biobank with one of the most used EMRs and enhance the functionality of our Women's Health Tissue Repository.

Maternal Vitamin D deficiency is associated with poor muscle performance, but evidence relating maternal vitamin D status to cesarean delivery is mixed and difficult to interpret in modern cohorts where thresholds to intervene in labor are low. Our objective was to examine the association between maternal 25-hydroxyvitamin D (25(OH)D) status at ≤26 week gestation and primary cesarean section, indicated instrumental delivery, and duration of labor in a random subset of women with singleton pregnancies in the Collaborative Perinatal Project (1959-65) who labored (n=2773). Maternal serum 25(OH)D was measured by liquid chromatography mass spectrometry. We adjusted for maternal race/ ethnicity, season of blood draw, gestational age at blood draw, prepregnancy BMI, height, smoking status, socioeconomic status, infant sex, and study site in linear and logistic regression models. There were 52 (1.9%) primary cesarean deliveries and 192 (7.3%) indicated instrumental deliveries among women who labored. There was no difference in the incidence of primary cesarean among those with 25(OH)D ≥50 vs. <50 nmol/L (2.1% vs. 1.7%, p=0.5) either before or after adjustment for confounders (adjusted OR: 0.7, 95% CI: 0.4, 1.4). Further, instrumental delivery and length of labor for stages 1 and 2 were not associated with maternal 25(OH)D in confounder adjusted models. In an era when cesarean rates were low and instrumental delivery was high, we observed that maternal vitamin D status in early pregnancy was not associated with primary cesarean, instrumental delivery, or length of labor. Future studies should examine maternal vitamin D status at multiple points in gestation with these labor and delivery outcomes. Conclusion: The prevalence of maternal obesity increased during the three years of registration and more than 50% of overweight and obese women continued to gain weight above recommendations. Perinatal complications increased by pre-pregnancy BMI and prevalence of GDM in obese women even increased during the three years of registration.

Results: The overall preterm birth rate increased from 9.1% in 1999- We developed and implemented recruitment methods including both direct outreach efforts such as Google and Facebook advertising and partnering with SB Associations to promote the research on their websites and to their members. Both outreach approaches directed interested participants to an enrollment website. Following completion of an on-line survey, selfadministered saliva collection kits were mailed to eligible mothers and their children. To support return of the biospecimens, the participants were reminded via automated emails and through Spina Bifida Association outreach activities. Results: Recruitment resulted in a total of 1,881 mothers expressing interest in the study by beginning the online survey; 1,415 (75.2%) registered, completed the survey, and provided contact information for saliva kit distribution. Of these eligible mothers, 1,091 (77.1%) returned the kits, along with saliva from 999 of their SB-affected children (2,090 samples total, 98% with sufficient DNA for analysis). This entire process took less than a year. Enrollment via SB associations accounted for 76% of samples collected vs. only 24% of samples through other methods. In addition, emails and follow-up telephone calls contributed signficantly to rapid return. To validate this approach, we completed recruitment of a second, independent population in October 2012 for replication of our genetic results. Within 10 months, we have successfully recruited an additional 1,240 mothers and 1,100 of their SB-affected children (70% recruitment rate among eligible mothers). In addition, we also recruited 3,900 ″controls″ mothers within a 6 month period leveraging an internet-based approach.

Discussion: This novel approach combining partnering, outreach, the internet, and self-collection of saliva allows for quickly recruiting large numbers of subjects for genetic epidemiologic research projects. 

Alcohol consumption in early pregnancy is a major public health issue. The data suggested that alcohol consumption during pregnancy, in particular binge drinking, was associated with an approximately 20% increased risk of SGA and reduced BW. There was no significant association between alcohol consumption and SpPTB but alcohol use was associated with a mild reduction in PE.

Center, Odessa, TX, USA. Women will frequently present late for their first prenatal visit (FPV) and this is commonly seen in our predominately Mexican-American Clinic. This is a chart review to determine when pregnant women present for their FPV. We have also recorded clinical data that may represent barriers to this first visit which includes (1) adiposity (BMI); (2) ethnicity; (3) socioeconomic status; (4) gravidity; and (5) maternal age. There are 397 subjects in this study. When our entire study population is examined for their FPV, 38 % were in the 1 st Trimester; 46 % in 2 nd trimester and 16 % in the 3 rd trimester. When subjects were examined for adiposity (BMI) a similar pattern is seen for lean, overweight and obese subjects, with most subjects seen in the 2 nd trimester for all BMI groups. When ethnicity was examined, in the 1 st trimester, Caucasians (35%) and Mexican-Americans (39%) had similar numbers; both groups were slightly higher in the 2 nd trimester. By the 3 rd trimester, numbers were similar to the total group. Numbers for other ethnic groups were too small to be reliable. Maternal age was divided into 5 year windows between 15-19 years to 36-39 years. Similar distributions (32-43%) are seen for all ages in the first trimester, except for the oldest group where only 26% showed up in the first trimester. Similar distributions are seen in the second trimester, although %s are generally higher for each group. In the third trimester most ages present between 123-15%, except for the oldest group, where 30% are seen for their FPV. When categorized by gravida (1, 2, 3-5, and >6) , visits in the first trimester were generally higher (31-45%) and for 2 pregnancies, were higher in the 1 st trimester than in the 2 nd trimester. Gravidas 1, 2, and 3-6 have 13-22% for FPV in the third trimester. Socioeconomic status was determined by paysource, with Insurance as the best; Medicaid medium; and Self-pay and Chips as the lowest form. Distribution based on paysource for FPV are remarkably similar to the total picture. The proposed barriers to the FPV did not enlighten our understanding for this late obstetrical visit. Conclusion:Prior Cesarean delivery,particularly elective & maternally requested Cesareans were associated with an increased time to next birth.An increased hazard ratio of stillbirth in subsequent deliveries but not ectopic pregnancy or miscarriage among women with an index Cesarean was also found. (1945 ( -March 2012 ,using a detailed search-strategy and crosschecking of references.Cohort,case-control and cross-sectional studies were eligible.Two assessors individually reviewed titles,abstracts and full articles to identify eligible studies,using a standardized data-abstraction form and assessed study quality.A meta-analysis was not suitable due to between-study heterogeneity. Data synthesis:Over 9,184 titles were screened,with 11 articles included.

Main findings of the studies which reported effect estimates are summarized. Six studies reported a longer time to next pregnancy and a lower subsequent pregnancy rate following Cesarean section.Five studies reported no difference in time to next birth or subsequent pregnancy rates in the Cesarean group compared to the vaginal delivery group.Lack of adjustment for confounders and indication for Cesarean was common reducing the study quality. Conclusion:Evidence on the relationship between Cesarean delivery and pregnancy interval is conflicting.Residual confounding is possible and further research of better methodological quality is required to assess whether any delay in pregnancy interval is causal or as a result of parental choice to delay childbirth. Background:In the United States, United Kingdom, Australia and Ireland between 30% and 81% of unborn babies are exposed to alcohol in utero. However, evidence on the consequences of prenatal alcohol consumption at low or moderate levels is conflicting and international guidelines have not reached consensus on alcohol recommendations for pregnant women.The aim of this review was to determine the relationship between low to moderate levels of prenatal alcohol exposure on speech and language development in infants. Search Strategy:Using medical subject headings, PubMed, Web of knowledge, Scopus, Embase, Cinahl, and the Cochrane Library were searched up to February 29th 2012. Case control and cohort studies were included if they compared low or moderate drinking in pregnancy (< 70 grams of alcohol/5 standard drinks per week) to no drinking and reported speech and language outcomes in infants. Two assessors independently reviewed titles, abstracts and fulltext articles, extracted data and assessed quality. (2) and Australia (1)] totaling 10,642 women between 1990 and 2009 met the inclusion criteria. Due to methodological heterogeneity between studies, results could not be combined using meta-analysis. Greene et al. 1990 cohort study included 618 disadvantaged women in Cleveland, USA and reported no association between low alcohol consumption and language outcomes in infants at 1, 2 or 3 years. Faden et al. 2000 cohort study of 8,885 American women reported no association between decreased language achievement at age 3 and mean number of drinks per day in pregnancy. O Leary et al. 2009 cohort study of 1,759 non-indigenous Australian women reported no increased odds of language delay for light drinking in pregnancy. However, studies were judged to be of low quality based on a 6 item bias classification tool. Conclusion:Studies included in this review do not provide sufficient robust evidence to confirm or refute an association between low or moderate alcohol use during pregnancy and speech and language development in infants. There is a need for adequately powered, high quality, population based epidemiological studies. Until such evidence becomes available it remains prudent to advice women to abstain from alcohol consumption during pregnancy. Background:In the United States, United Kingdom, Australia and Ireland between 30% and 81% of unborn babies are exposed to alcohol in utero. However, evidence on the consequences of prenatal alcohol consumption at low or moderate levels is conflicting and international guidelines have not reached consensus on alcohol recommendations for pregnant women. Methods:Data was collected on 10,953 infant-mother pairs in the Growing up in Ireland Infant Cohort Study through face to face interviews between 9 months and 1 year after birth. We conducted a retrospective cohort study to examine the association between moderate alcohol consumption (consuming 3 standard drinks or less per week) in each trimester of pregnancy and preterm birth (birth occurring before 37 weeks gestation), birth weight in grams and small for gestational age (defined as birth weight below the 10 th customised centile). Logistic and linear regression adjusting for age, ethnicity, parity, smoking status, body mass index, education and gestational age were used to generate crude and adjusted odds ratios and mean birth weight differences. Preliminary results show that women in urban areas are older (31 vs 30 years), more often of non-Western ethnicity (35 vs 2%) and of low socioeconomical status (38 vs 21%; all p<0.001). Urban woman had lower gestational age during screening (10 vs 28 weeks) and were more often nulliparous (49 vs 41%; p<0.05). Psychotropic medication use was equal in both groups (4%). Among urban women 18% had EDS scores of 12 or more compared to 8% among the rural women (p<0.001). Prevalence of psychosocial problems did not significantly differ for insufficient support, relational problems and physical/sexual abuse, but did differ for single status (4 vs 1% p<0.05) and financial problems (24 vs 15% p<0.001). Substance use during pregnancy was comparable regarding smoking and alcohol consumption (10% until pregnancy was known for both substances in both groups). Illicit drug use was more prevalent among urban women (1 vs 0% p<0.05). Full predictive analysis will be completed within the next 2 months. Conclusion Pregnant women from urban and rural areas show substantial differences in background risks and the prevalence of PPS. Urban pregnant women show higher prevalence of depression, psychosocial problems and substance use during pregnancy. We hypothesized smoking would be associated with increased BV prevalence in FF or Ff, but not ff, genotypes of the Fok1SNP.

Methods: BV-IDEAS is a cross-sectional study of BV in a ethnically diverse non-pregnant urban clinic population. Women completed surveys on demographics and sexual/medical history and gave vaginal swab and lavage specimens during routine GYN visits. DNA was extracted from lavage samples and analyzed by PCR for the Fok1SNP. Nugent score 7-10 indicated BV. Race and current smoking were self-reported. Only women self-identified as white, black, or Hispanic were included in this analysis. Prevalence ratios (PR) and differences (PD) and 95% confidence intervals (95%CI) were calculated, adjusted for race/ethnicity. Results: 400 women had complete information and identified as white, black, or Hispanic. 50% had BV, 17% were white, 34% black, 49% Hispanic, 17% smoked, and 12% had ff genotype. ff was most frequent in whites (19%) and least frequent in blacks (4%). Compared with women who did not smoke and had FF or Ff genotype, BV prevalence was not significantly increased in women who smoked and had ff genotype Background: An inverse association between birth weight and the risk of developing attention problems in childhood has been reported repeatedly. However, this association could be subject to confounding. Mendelian randomization, in which genetic variants are used as a surrogate for measuring the exposure, is an approach which may help better understand this association. Aim: To assess the causal nature of the observed association between reduced birth weight and risk of attention problems in childhood. Methods: In a population-based birth cohort, birth weight was assessed in 2,141 children of north-European descent. Child attention problems at age 6 were assessed by parental report with the Child Behavioural Checklist. From previous meta-analyses of genome wide analysis, we identified 15 single nucleotide polymorphisms (SNPs) that predict birth weight. First, we examined the association between birth weight and attention problems and corrected for potential confounders. Second, we used regression models to examine the associations between child birth weght and behavioural problems. Next, joint significance of the selected SNPs on birth weight and childhood attention problems were analysed using F-statistics. Results: Low birth weight corrected for gestational age was not associated with attention problems. We observed a curvilinear association between birth weight and attention problems that remained after adjustment (β 0.02 95%CI 0.00; 0.05, P=0.04). The negative association between birth weight below the turning point was reduced after correction for numerous confounders. Selected SNPs associated with birth weight (F: 3.098, P<0.001) significantly predicted problems of attention (F: 1.743, P=0.04). However, the directions of the effects were not consistent and this association was therefore not explained by birth weight. Conclusion: The association between birth weight and attention problems is subject to confounding. Genetic variants associated with birth weight were significantly associated with attention problems but our observations suggest a potential pleiotrophic effect of the selected SNPs. Further studies are needed to explore the mechanisms underlying this association. Objective: The aims of our study are 1) characterize the indication for antepartum admission to the ICU and 2) report the maternal outcomes of those requiring ICU admission. Study Design: This is a single center retrospective review of all obstetric patients admitted to an ICU, prior to delivery, over a 5-year period. Subjects were identified from a database of ICU admissions during the study period. Inclusion criteria were antepartum admission to one of four intensive care units at our institution. Data collected included demographic information, indication for ICU admission, medical comorbidities, interventions required and maternal outcomes. Results: We studied 88 women (age 27 +/-6.5 yrs, GA at admission 25 +/-8.2 wks, length of ICU stay 6.7 +/-8 days). 50% of subjects were transferred from outside institutions. Indications for admission were cardiac failure/arrhythmia (18%), respiratory failure (16%), trauma/burns (13.6%), and cerebrovascular event (8%). Additional less common indications are listed in [ Table 1 ]. Conclusion: In our study the primary indications for antepartum ICU admission differed from those reported in other studies. These findings may be partially attributed to our status as a neurosurgical and trauma center, but should also emphasize the diverse medical complications in contemporary obstetric populations. Conclusion: Increased postnatal growth rates in Ad.VEGF-treated lambs most likely reflect their relative size advantage at birth rather than altered epigenetic status of key somatotropic genes. We previously determined fetal PAI-1 is inversely related to venous cord blood oxygen in pregnancy. We hypothesized that changing PAI-1 levels in hypoxic pregnancies with placental insufficiency may be contributing to the pathological vascular changes of the placenta. We sought to determine if (i) PAI-1 is increasing or decreasing in the plasma, (ii) its relationship to oxygen, VEGF and FGF-2 levels, and (iii) determine the effect of its changing levels on angiogenic regulation. Methods: Using the blood immediately effluent from the placenta collected from the venous umbilical cord following delivery, we measured PAI-1 from controls (n=12) and in severe cases of placental insufficiency (n=18). We tested PAI-1's relevance to angiogenic regulation. Results: PAI-1 expression in effluent placental blood plasma was dependent on VEGF and FGF-2 regulation in controls (r=0.78, p<0.01 and r=0.89, p<0.001, respectively) but not in FGR. Instead PAI-1 was found to be highly elevated in effluent placental plasma of newborns with severe fetal growth restriction (FGR) caused by placental insufficiency. PAI-1 was increased in a reverse correlation to blood oxygen level (-0.68, p<0.0001). Effluent placental plasma from FGR induced ∼2 fold greater in vitro angiogenesis (p<0.001), which was fully attenuated by PAI-1 inhibition. PAI-1 was highly correlative to angiogenesis overall (0.75, p<0.001). VEGF and FGF-2 regulated angiogenesis in controls (r=0.78, p<0.01 and r=0.89, p<0.001, respectively) but did not account for increased angiogenesis in FGR.

The results identifying circulating PAI-1 as a pathologically upregulated protein by hypoxia, eclipsing normal VEGF/FGF-2 mediated angiogenic regulation are noteworthy. We find PAI-1 is pro angiogenic in nature and may be contributing to the vascular pathology of the placental chorionic villi vasculature in FGR by accelerating non-branching angiogenesis and thereby promoting vascular resistance.

Sarah A Hopkins, 1 James C Baldi, 2 Wayne S Cutfield, 3 Sex-specific alterations in fetal growth have been characterized in response to adverse changes in the intrauterine environment, but have not been described in non-pathological conditions. We have reported an association between regular aerobic exercise in pregnancy and reductions in birth weight and cord growth peptides, suggesting adaptations in nutrient partitioning to the fetus leading to decreased stimulation of fetal growth. The female fetus is thought to respond to an adverse environment by reducing growth. In contrast, pregnancies with a male fetus adapt to continue normal growth, but are at increased risk of growth restriction if conditions deteriorate. Objective: Our aim was to test the hypothesis that the reduction in birth size and cord IGF-I in exercise offspring was due to greater growth reduction in female offspring than in males. Methods: Offspring size was assessed in a cohort of nulliparous women (30±4 yrs, BMI 25.5±4 kg/m 2 ) randomized to exercise training (n=47) or control (n=37) from 20 weeks gestation to term. Neonatal auxology was assessed within 48 hours of birth. Cord growth peptides were measured by ELISA. Results: Male exercise offspring had reduced BMI SDS compared to control males (p=0.02). The reduction in cord insulin-like growth factor(IGF)-I was four-fold greater in males than females (-39% vs. -9%). Cord leptin decreased in male, but increased in female, exercise offspring (NS). Conclusions: In contrast to our hypothesis, aerobic exercise during pregnancy had subtle effects on fetal growth that were more pronounced in male offspring. These findings warrant investigation in a larger study to assess sex-specific responses to maternal exercise. Nuclei of mononucleated fetal cardiac myocytes can divide and progress into two new mononucleated or one binucleated cell; binucleated cells only rarely divide. Since cardiac myocytes are almost all mononucleated at 115d gestational age (GA; term 147d), but most are binucleated at 14d postnatally (PN), we tested the hypothesis that rapid postnatal ventricular growth is almost entirely due to an increase in cell volume and is not due to a further increase in the number of cells. METHODS: We studied 27 neonatal sheep between birth and 59d PN. Hearts were dissected into components (n=11), or enzymatically dissociated (n=16). Myocyte sizes were measured from photomicrographs. Analysis includes previously published data from 91 fetuses (dissected n=28, dissociated n=63) aged 92-145d GA. Segmental linear regression was performed by least mean squares method, with an unconstrained breakpoint (intercept1+slope1*d, intercept2+slope2*d, breakpoint where d is days post-conception). RESULTS: The cell volume per nucleus -which rose slowly prenatally -started to rise rapidly just before birth, even faster for the left ventricle (LV) than for the right (RV; figure) . Consequently, myocyte volumes per nucleus that were much larger in the RV than in the LV before birth became smaller at about 10d PN. In contrast, the numbers of nuclei increased rapidly before birth, but after birth rose very much more slowly in the RV and not at all in the LV (in 10 9 nuclei per freewall, LV:-3.029+0.03753d, 1.9325-0.002746(d-132.2), 132.2d, r 2 =0.6129; RV:-1.305+0.01724d, 1.0396+0.002007(d-136), 136d, r 2 =0.5971). The number of nuclei in the LV freewall was at all times almost two times larger than in the RV.

CONCLUSION: Around the time of birth there is a sudden change in the cellular dynamics of cardiac growth. Although the cellular developments of the LV and RV are somewhat different, the ventricles respond similarly to the challenge of birth. In large mammals, the numbers of myocyte nuclei and the number of myocytes increase rapidly before birth but appear to be mostly fixed once born. Objectives: The purpose of our study was to investigate the effects of the mode of delivery on the oxidant and antioxidant system via umbilical cord blood. Methods: We examined gas analysis of umbilical venous blood and umbilical arterial blood immediately after delivery. 18 pregnant women had vaginal delivery and 20 pregnant women had cesarean section after gestational age 37 weeks. We checked lipid peroxide concentration by thiobarbituric acid reaction, protein carbonyl content by 2,4-dinitrophenylhydrazine(DNPH) reaction, and total antioxidative ability by oxygen radical absorbance capacity(ORAC) assay. Results: Lipid peroxidation levels in umbilical venous blood were significantly higher in patients delivering with planned cesarean section (1.81±0.05 nmol/ mg protein) than those with vaginal delivery (1.04 ±0.07 nmol/mg protein) (p<0.05). Antioxidative ability in umbilical venous blood were significantly higher in patients delivering with planned cesarean section (119.70±0.27 uM/ uL) than those with vaginal delivery (118.29±0.57 uM/uL) (p<0.05). There was no significant difference in carbonyl content of umbilical venous blood and in lipid peroxide, carbonyl content, and total antioxidative of umbilical arterial blood. Conclusion: Lipid peroxidation levels and antioxidative ability in umbilical venous blood were higher in patients delivering with planned cesarean section than those with vaginal delivery. It can be proposed that both the mother and neonate are exposed to higher oxidative stress during cesarean section. Objective: To examine risks of intrauterine growth restriction (IUGR) and poor perinatal outcomes in patients with estimated fetal weights (EFW) between 10-20% percentile. Study Design: Retrospective, cohort study evaluating pregnancies with an ultrasound EFW between the 10th-20th percentile between January 2002-January 2012. Exclusion criteria were delivery at outside institution, gastroschisis, higher order multiple gestations and lethal fetal anomalies. Cases were identified with EFW percentile between 10 and 20. A control with estimated weight greater than 20th percentile was obtained for each case, matched by gestational age and date of ultrasound. Ultrasound reports were reviewed and the estimated fetal weight and abdominal circumference recorded, as were intrapartum events and neonatal outcomes ascertained from the medical record. Chi-square was used for categorical variables and logistic regression for continuous. Multiple logistic regression controlled for maternal age, gestational age, birth weight and mode of delivery. Results: 1093 cases were identified, with 178 excluded delivering elsewhere, 16 for gastroschisis, 9 for fetal demise, 11 for multiple anomalies,1 triplet gestation and 1 conjoined twins. Matched controls were obtained for the remaining 877 cases. Of the infants with estimated fetal weights between 10-20 percentile, there was a statistically significant incidence of intrauterine growth restriction, neonatal intensive care unit (NICU) admissions and composite perinatal morbidity defined as cord pH <7.0, 5-minute Apgar <7, NICU admission, intubation and IUGR. 

Certain fetuses with growth delay may be unidentified by defining intrauterine growth restriction as estimated fetal weight below the 10th percentile. We have shown those with estimated fetal weight between the 10th and 20th percentile are at significantly increased risk of IUGR, NICU admissions and composite perinatal morbidity. Cortisol's effects are due to both circulating and local cortisol generation and metabolism by 11βHSD-1 and 2. Tissue cortisol is kept low by conversion to cortisone by 11β HSD-2. Cortisol increases 11βHSD-1, We hypothesized 1) 11β HSD1 and 2 would be present in the fetal baboon pancreas at 0.9G and 2) the 11βHSD 1:2 ratio would increase in IUGR. METHODS: Pregnant baboons were fed ad lib (CTR n=7) or 70% CTR global diet (IUGR n=6,) from 0.16 -0.9 G with IUGR fetuses obtained at CS. Pancreatic islet 11 β-HSD1/2 protein was quantified by immunohistochemistry for fraction immunostained. Analysis was by Student's t-test. RESULTS: Fig 1 11βHSD1 (A -CTR n=7, B -IUGR n=6, C) increases and 11 βHSD2 (D -CTR n=7, E -IUGR n=6, F) decreases. Distribution of 11βHSD1/2: Triple staining for insulin, glucagon and either 11βSD1 or 2 (not shown) indicated that the enzymes were not present in alpha or beta islet cells.IUGR increased islet 11βHSD-1and decreased 11 βHSD2.in the IUGR fetal pancreas. Mean ± SEM, *p< 0.05. CONCLUSIONS: 11β HSD1 is increased in IUGR baboon fetal peri-renal adipose tissue (In Press in Diabetes). Adipose tissue cells are present in the islet and we propose that it is adipose cells that contain the 11βHSD. Combined with higher circulating fetal cortisol, increased local cortisol production would expose the fetal pancreatic islets to higher levels of cortisol than normal for the current stage of maturation. Cortisol stilulates 11BHSD1 and thus the stage would be set for potentially triggering a cycle of feed-forward induction of 11β -HSD1 with increased risk of later life failure of β-cell function as has been shown in the setting of IUGR. (HD 21350).

Insufficiency. Antoni R Macko, Xiaochuan Chen, Miranda J Anderson, Amy C Kelly, Sean W Limesand. Animal Sciences, The University of Arizona, Tucson, AZ, USA. Placental insufficiency and intrauterine growth restriction (PI-IUGR) complicates pregnancy and increases incidence of postnatal metabolic diseases. Chronic fetal hypoglycemia and hypoxemia stimulate secretion of norepinephrine (NE) into circulation from the fetal adrenal medulla to suppress insulin secretion. Previous studies In an ovine model of PI-IUGR at 0.9 gestation reported chronic and severe hypoxemia and hypoglycemia in 50% growth restricted fetuses associated with four-fold elevated plasma NE concentrations and reduced basal and glucose-stimulated plasma insulin concentrations. A pharmacological adrenergic blockade augmented insulin secretion responsiveness in PI-IUGR but not control fetuses, indicating the presence of a compensatory ß-cell stimulus-secretion adaptation apparently mediated by chronically elevated NE. The objective of the current study was to determine the etiology of NE dysregulation of fetal ß-cell function.

In the current study, basal, glucose-, and arginine-stimulated fetal plasma insulin concentrations were measured in the absence and presence of a pharmacological adrenergic blockade in control (n=8) and PI-IUGR (n=10) sheep fetuses at 0.7 gestation, prior to measurable anatomical fetal growth restriction. Placental weights were 34% lower (P<0.05) in PI-IUGR animals (233.0+20.9 vs. 345.3+17.9 g, P<0.05), but fetal weights were not different (1.2 0.1 vs. 1.2+0.1 kg, P<0.05) and no asymmetry of fetal growth was detected. Fetal plasma glucose concentrations were also not different between groups, however, in PI-IUGR fetuses compared to controls, blood oxygen content was lower ( Objective: Folic acid supplementation is an effective prophylactic agent against congenital anomalies, yet little is known regarding fetal utilization of folate. Such information may optimize current repletion strategies. This study aims to characterize the concentrations of folate species in umbilical cord serum relative to maternal supplementation and race.

Methods: This is a single-center, prospective cohort of cord blood samples obtained from 40 uncomplicated term deliveries at Saint Luke's Hospital in Kansas City. HPLC mass spectrometry quantitated the following concentrations in extracted serum samples: total folate, 5 methyltetrahydrofolate (5MTHF), 5,10-methenyl-tetrahydrofolate (5,10-MeTHF), tetrahydrofolate (THF), and unmetabolized folic acid. Results: Folate concentrations in the umbilical cord serum are presented in Figure 1 . Prenatal supplementation increased the concentrations of total folate (p<0.0133). However, African American cord serum demonstrated a lower total folate concentration, in comparison to Latina (p<0.0016) and Caucasian (p< 0.0008) samples. When quantitating specific folate species, ethnicity and supplementation affected the concentration of only 5MTHF, while all other metabolites remained unchanged. Furthermore, supplementation increased the total folate concentrations of Latina and Caucasian cord serums combined (61 vs. 82.3 nmol/L, p<0.03), while African American cord serum levels changed less (44.6 vs. 53.9 nmol/L, p<0.33).

Conclusions: This study implicates the importance of 5MTHF in affecting variation in fetal folate metabolism. Furthermore, racial differences exist not only in total folate concentrations but also in response to supplementation, suggesting a possible genetic etiology. This finding interestingly corroborates with our previous studies performed on maternal blood. Eventually, this study emphasizes the need for further investigation regarding maternal-fetal correlations in elucidating fetal folate utilization. For meta-analysis of longitudinal growth trajectories, cross-sectional data was extracted at the 2 time points (20 & 30 wks) where there was the greatest overlap in number of ultrasound scans between participating cohorts. There were no common SNPs or overlapping regions between the 3 biometric measures nor with recently published common genetic variants associated with childhood obesity and HC.

These data suggest that genetic influences on fetal growth may be different for each biometric measure. A discovery meta-analysis between RAINE and Generation R is in progress and replication will be completed in the INMA birth cohort and Southampton Women's Study. Validated signals will be further explored in maternal DNA. Friday analyzed for cytokines (IL-6, IL-1β, CXCL1, CCL2 and TNFα; sensitivity <3pg/ml) using multiplex. Data was analyzed using ANOVA and t-test.

The average fetal and placental weights were significantly lower in the BL and MBL dams compared to SH and MSH dams, respectively (P<0.001) ( Pregnancy is associated with maternal immunologic changes. Mean maternal B cell count is stable or increased in pregnancy. The fetal immune system begins to develop in the first trimester and matures throughout gestation to prepare for extra-uterine life. BLyS (also known as BAFF) is a potent B cell survival factor that inhibits B cell apoptosis and has, to our knowledge, never been studied in pregnancy. Objective: To evaluate BLyS levels in maternal and cord blood samples at delivery and postpartum. Methods: Women receiving prenatal care at Los Angeles County + University of Southern Ca from October 2011-January 2012 were eligible to participate in this study. After obtaining consent, patients were stratified by presence or absence of a medical condition into "healthy" and "disease" groups. Maternal samples were collected prior to delivery and postpartum. Cord blood samples were collected at delivery. Serum was isolated, and BLyS levels were determined by ELISA (Human Genome Sciences, Rockville, MD). Data were analyzed with SigmaPlot, using paired t-test and Spearman Correlation as appropriate. P-value<0.05 was considered significant. Results: Forty-one cord blood samples were included. In 29 patients, matched maternal antepartum and postpartum and cord blood samples were available. Median maternal age (25% and 75% IQ range) was 28 (24.7, 37.0); median gravidity was 3.0 (2.0, 3.0). Median gestational age at delivery was 38.9 weeks (36. 6, 39.2) . Median maternal antepartum and postpartum BLyS levels (ng/mL) were 0.611 (0.450, 0.726) and 0.760 (0.546, 0.968) respectively, and median cord blood levels were 1.637 (1.199, 2. 317) (p< 0.001). Cord blood levels were significantly higher in the "healthy" group compared to the "disease" group (p=0.027) and were significantly associated in both cohorts collectively (n=41) with gestational age at delivery (R = 0.593, p< 0.0001). (Figure 1 )

Maternal and cord blood BLyS levels are significantly different. Cord blood levels are directly correlated with gestational age. BLyS levels may be correlated with maturation of the fetal immune system. Epidemiological studies indicate that MNR in pregnant women predisposes offspring to obesity and central fat distribution phenotype. METHODS:We examined in vitro adipocyte differentiation in omental, subcutaneous abdominal and femoral adipose tissue depots in control normally grown fetuses (CTR) and MNR fetuses of baboon mothers fed 30% global diet of CTR from 30 d pregnancy to term -5 males and 5 females in each group. Adipocyte differentiation was assessed by gene expression (qRT-PCR) of peroxisome proliferator-activated receptor gamma (PPARγ) and its target genes fatty acid binding protein 4 (FABP4, a.k.a. aP2) and adiponectin in differentiated primary adipose-derived stromal/stem cell cultures. As fetal adipose tissue exhibits brown adipose tissue features, we also measured uncoupling protein 1 (UCP1) mRNA. RESULTS: MNR male fetuses were IUGR -MNR 762 ± 24g vs. 884 ± 49 g CTR. There was no significant difference in the adipogenic transcription factors among depots. There was a proportional increase in white and brown-specific adipogenic factors in male IUGR but not female fetuses (Fig 1) .

Sex-dependent expression of white (PPARγ, adiponectin, FABP4) and brown (UCP1) adipogenic genes in differentiated cultures from omental, subcutaneous abdominal and femoral adipose tissue depots combined in control and MNR baboon fetuses CONCLUSIONS: Male IUGR appear adapted to meet the extra-uterine cold challenge by increasing increased thermogenic activity. Whether IUGR fetuses are predisposed to later enhanced adiposity will be dependent on rate of UCP1postnatal loss. HD 060158 and HD 21350 Suboptimal intrauterine nutrition predisposes to central obesity and metabolic syndrome in adulthood suggesting interplay between maternal nutrition and regional fetal fat tissue development. METHODS: To elucidate the underlying mechanisms, we compared the genomic expression profiles (Illumina® microarrays) of preadipocytes from omental (OM), subcutaneous abdominal and femoral (scA, scF) fat depots in female fetuses from mothers fed control or 30% nutrient-restricted diets (CTR vs. MNR; n=3/group). RESULTS:Both groups had comparable body weight. We found 100 differentially expressed genes and validated (RT-PCR) nine genes that varied between the diets, depots and/or diet × depot interactions (Fig.1A-C) .

Horizontal bars marks depots with significantly different gene expression, p <0.05), and diet × depot (C; Horizontal bars denotes significant differences between the depots within a treatment group, p <0.05; *marks the specific depots with a significant alteration in the respective gene expression by the maternal diet, p <0.05) CONCLUSIONS: Functional considerations of the encoded proteins implies that MNR reprograms preadipocytes to increased adipogenesis (ETV4, ITGA8, LSP1, and TP53BP2) with increased brown fat features in sc depots (ITGA and RASL11A) and increased stemness in OM adipocyte precursor cells that may contribute to adult abdominal obesity. 

Research, University of Texas Health Science Center, San Antonio, TX, USA. Introduction: Human epidemiology studies of the Dutch Hunger Winter and Chinese Famine indicate an increased risk of schizophrenia and mood disorders in intrauterine growth restricted (IUGR) offspring whose mothers experienced maternal under nutrition (MNR) during pregnancy. As altered serotonergic signaling has been implicated in the etiology of involved in numerous neuropsychiatric and developmental disorders, including major depression, anxiety and autism, it is possible that disrupted development of the serotonergic nervous system underlies the increased risk of mood disorders observed in humans exposed to MNR/IUGR. We hypothesize that MNR/ IUGR results in disrupted development of the serotonergic nervous system, which contributes to the risk of mood disorders in human offspring subjected to adverse intrauterine conditions. Methods: Fetal baboons received a 30% reduction in maternal nutrition beginning at day 30 of gestation and continuing until C-section. This model has been demonstrated to result in offspring with IUGR. mRNA expression was assessed utilizing quantitative Real-Time PCR (qRT-PCR). Results: Previously we described decreased immunoreactivity of tryptophan hydroxylase (TPH) in 165dg MNR fetuses. Utilizing qRT-PCR we now demonstrate decreased mRNA expression of the 5-HT1a receptors (HTR1a) and TPH at both 120dg and 165dg. Conclusion: MNR results in observable decreases in multiple proteins involved in serotonergic neurotransmission. These changes are measurable by 120d gestation and persist to birth. Further study is needed to determine if these changes persist into postnatal life. The PORTO Trial is a prospective study conducted at the seven largest obstetric centers in Ireland. The objective of this analysis is to characterise growth velocity in IUGR (defined as EFW<10th centile) and to correlate this with perinatal outcomes to allow for better distinction between physiological and pathological IUGR. Study Design Over 1,000 consecutive ultrasound-dated singleton pregnancies with EFW<10th centile were recruited between 24 0/7 and 36 6/7 weeks gestation (2010-2012). Pregnancies were followed with serial sonographic assessment of fetal biometry and multivessel Doppler until birth. Fetal growth velocity patterns and associated Doppler changes were analysed based on adverse and normal perinatal outcomes. Adverse perinatal outcome was defined as composite morbidity outcome of IVH, PVL, HIE, NEC, BPD, sepsis and perinatal mortality.

Of the 1,036 pregnancies recruited, 980 (95%) fetuses with normal perinatal outcomes showed good interval growth and followed their centiles until term (mean GA at birth: 38 weeks, mean birthweight: 2553g). 56 (5%) pregnancies resulted in adverse perinatal outcome (mean GA at birth: 32 weeks, mean birthweight:1321g); an EFW below the 10 th centile in addition to an increased umbilical artery PI> 95 th was associated with an increased risk of perinatal morbidity (p=0.0006). Those with adverse perinatal outcomes were found to have an elevated UA PI > 95 th centile as early as 24 weeks gestation. In addition, fetuses with adverse outcomes had a negative growth velocity as shown in the figure below.

Our data demonstrate that sonographic evaluation of EFW, umbilical and middle cerebral artery Doppler at 24 weeks' gestation can be used to stratify IUGR pregnancies to increased surveillance or routine care. This may impact on appropriate resource allocation, reassure those with normal pregnancies and select fetuses at risk of adverse outcome. Overweight adolescents entering pregnancy are at increased risk of developing obesity; however, the effects of maternal overweight or excessive gestational weight gain (GWG) on the fetus are less understood in this population. Specifically, the effects of pre-pregnancy body mass index (ppBMI), and GWG on fetal fat accretion are unknown among this age group and these effects may differ from those observed among adults. The objective of this analysis was to identify dietary determinants of fetal abdominal fat thickness during the third trimester of pregnancy in a racially diverse group of pregnant adolescents. A total of 171 pregnant teens (ages 13-19y) entering prenatal care at 12-30 weeks of gestation were enrolled in a study designed to investigate relationships between vitamin D status and fetal bone growth. Of the teens studied, 66.7% were African American, 33.3% were Caucasian, and 23.6% were Hispanic.

Teens had a mean of 11.3 ± 3.1 prenatal care visits across pregnancy which included attendance at up to three study visits, completed during early, mid-, and late gestation. At each visit, anthropometrics were measured, a 24-h dietary recall was administered and measures of fetal biometry were obtained. Fetal abdominal wall thickness (Fetal AbFat), a measure of fetal subcutaneous fat, was calculated from sonogram data during each study visit. Simple and stepwise multiple linear regression were used to assess statistical determinants of fetal fat accretion during late pregnancy. At the time of the third study visit (34.7 ± 2.7 wks; range 26.9 -41.0 wks) mean fetal abdominal fat and GWG were 0.47 ± 0.14 cm and 14.3 ± 6.9 kg, respectively, and mean birth weights were 3285.6 ± 472.6 g and 3373.6 ± 478.6 g for African American and Caucasian teens, respectively. No differences in fetal fat or GWG were observed as a function of race or ethnicity. Stepwise regression was used to identify a model that included determinants of Fetal AbFat. Significant determinants included birth weight, gestational age, maternal race, and total dietary sugar intake. The positive association between total sugar intake and fetal abdominal fat suggests that high sugar intakes among pregnant adolescents may contribute to the accumulation of excess abdominal fat in utero, of which the health implications in later life are unknown. Background: Customization of fetal growth assessment with epidemiologic (EPI) data has been suggested to improve identification of intrauterine growth restricted (IUGR) fetuses at risk of adverse perinatal outcomes. To date no one has evaluated customized growth charts to predict adult consequences of IUGR. Aims: To compare 3 methods of customizing fetal growth assessment to predict metabolic dysfunction in young adults: 1) Gardosi population EPI adjusted birth weight; 2) study specific EPI adjusted birth weight and prenatal biometry (head, abdomen and femur); and 3) study specific genetic and EPI adjusted birth weight and prenatal biometry. Methods: Mother-child pairs (n=1377) from the Western Australian Raine Study underwent serial ultrasound during pregnancy, birth anthropometry and physical/biochemical assessment (lipids, glucose and insulin) at age 17 and 20. The proportion of actual to predicted birth weight was calculated for each individual using the three methods. Maternal and fetal genetic variants (n=26) previously associated with fetal growth were used for model 3. Individualized prenatal biometric curves were developed (models 2 and 3). Multivariate linear regression was used to assess associations between fetal growth and adult outcomes. Results: Customized fetal growth assessment was better than non-customized birth weight to identify associations with total cholesterol (p=0.001), LDLcholesterol (p<0.001) and triglyceride (p<0.001), with trends to associations with HDL-cholesterol and body mass index. Adding genetic variants did not improve the identification of associations with metabolic outcomes but altered prenatal curves substantially for some individuals. Conclusion: Epidemiological customization of fetal growth assessment improves identification of individuals at risk of adult metabolic abnormalities compared to non-customized birth weight. The customized prenatal biometric curves developed may improve the ability to identify IUGR fetuses prenatally in clinical settings. Further evaluation in larger cohorts is required, in particular to assess the prediction of rare adverse perinatal outcomes and to assess any additional benefit of genetic customization. Objective: Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. Fetal growth is regulated by the insulin-like growth factor (IGF) system, and therefore dysregulation of this system may have profound effects on fetal and placental growth. mRNA of placental origin is detectable in the maternal blood and may provide a non-invasive insight into placental function. Therefore we sought to examine whether mRNA transcripts in maternal blood coding genes regulating fetal growth are differentially expressed in two independent cohorts of samples collected prospectively: 1) severe preterm FGR, and 2) at 28 weeks gestation in pregnancies destined to develop FGR at term. Methods: Real time PCR analysis was performed on maternal blood samples and placenta from women with severe preterm FGR and term FGR. IGF RNA transcripts were first analyzed in women with diagnosed severe preterm FGR (n=20) compared to gestation matched healthy controls (n=15). IGF RNA transcripts were then analyzed at 28 weeks and 36 weeks in a prospectively collected longitudinal study of low risk women (n=52) undergoing serial ultrasound assessment of fetal growth. Circulating IGF RNA were compared in pregnancies with term FGR and well-grown controls.

Results: In women with severe preterm FGR there was increased expression of placental growth hormone (6.3 fold), IGFs (IGF1 3.4 fold, IGF2 5.0 fold), IGF receptors (2.1 fold) and IGF binding proteins (3.0 fold), and reduced expression of ADAM12 (0.5 fold) in both placenta and maternal blood when compared to controls (p<0.05). Furthermore, IGF transcript expression correlated with severity of FGR, as evidenced by worsening umbilical artery Dopplers. Notably, at 28 weeks gestation there was increased IGF2 (3.9 fold), PGH (3.2 fold) and IGFBP2 (3.0 fold) expression in maternal blood in women destined to develop FGR at term (p<0.05). Conclusion: IGF transcripts in the maternal blood are dysregulated at 28 weeks gestation in pregnancies affected by preterm FGR and those destined to develop FGR. Therefore a non-invasive test measuring differential expression of IGF transcripts in maternal blood may 1) detect unsuspected severe preterm FGR present in utero and 2) be performed at 28 weeks to predict term FGR. Such tests could be used to identify unsuspected FGR and allow for timely delivery, preventing cases of stillbirth.

Ketamine Decreases Apoptotic and Inflammatory Gene Expression in Fetal Hippocampus Exposed to Global Acute Hypoxic Hypoxia. Eileen I Chang, Charles E Wood. Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL, USA. Introduction: Acute hypoxic hypoxia (HH) is a mild form of fetal stress where the maternal and fetal partial pressure of oxygen is decreased, resulting in activation of fetal neuroendocrine stress responses. We have hypothesized that hypoxia causes inflammation and apoptosis in the fetal brain, caused by increased glutamatergic signaling. Previously, we have shown that HH increased both fetal plasma ACTH and cortisol, and that pretreatment of the fetus with ketamine, a noncompetitive N-Methyl-D-aspartate receptor antagonist, partially inhibited the ACTH response. Using an ovine gene array, we have also previously shown that HH stimulates a genomic response that is consistent with inflammation and apoptosis. We propose that treatment with ketamine during HH will interrupt inflammatory and apoptotic signaling pathways in the fetal hippocampus. Methods: Fetal sheep were chronically catheterized at gestational day 125 (n=3-5/group), and tracheostomy was performed on the ewe. Ketamine (3 mg/ kg) was administered intravenously to the fetus 10 min prior to the induction of hypoxia; fetal HH was induced by administering nitrogen gas directly to the ewe for 30 min. Fetal hippocampi were collected at 24 hours after the initial start of hypoxic insult, and cDNA was made from the mRNA using random primers. CASP8, MYD88, and NFKB mRNA expression were measured using real-time PCR and normalized to the abundance of β-actin mRNA. Results (6):R1731), however the relative contributions of intrauterine hypoxia and undernutrition in this process are not understood. Here, we adopted a bioengineering approach to investigate whether fetal chronic hypoxia programmes aortic stiffness and tested whether oxidative stress is an involved mechanism. Methods: Pregnant Wistar rats were divided into normoxic (N: 21% O2) or hypoxic (H: 14% O2) pregnancy +/-vitamin C (5 mg/ml, maternal water) from days 6-20 of gestation. This experimental model does not affect maternal food intake. After birth, offspring were maintained until adulthood. At 4 months, the aorta from 1 male per litter was investigated. Uniaxial tensile tests were performed using a materials testing machine (Instron, Canton, MA). A materials testing software (Bluehill 2, Instron) programmed the stretching rates, extensions and sequences. Fixed aortic sections were subjected to stereology and collagen and elastin was quantified (QuickZyme Biosciences; Fastin elastin assay, Biocolor). Results: Figure 1A shows mechanical relationships in the rat aorta. The ultimate point is maximum stress. The yield is the point at which tissue stops behaving elastically and deforms. The initial stiffness σ1/ε1 is governed by elastin and the maximum stiffness σ2/ε2 by collagen. The elbow point represents the interaction between the initial and maximum stiffness (Wells et al. AJP 1998;274: H1749). Fetal hypoxia programmed an increase in aortic stiffness, partly ameliorated by maternal vitamin C ( Figure 1B & C) . These effects occurred independent of changes in aortic morphology or in elastic or collagen content at adulthood. Conclusions: Increased aortic stiffness is a mechanism underlying the programming of increased cardiovascular risk by fetal chronic hypoxia and oxidative stress. The data offer insight to mechanism and intervention. The goal was to study the differential development of fetal cardiac time intervals (fCTI) in low-risk and high-risk pregnancies. Methods: A total of 441 recordings from fetuses between 27-39 weeks gestational age (GA) were examined. Of these, 232 were classified as highrisk (HR) based on maternal conditions, such as pregnancy-induced/chronic hypertension, that have impact on normal fetal development. The HR group was subcategorized into: Intra-uterine growth restriction (IUGR; 66); and "abnormal tests" (39; biophysical profile (BPP) </= 6, abnormal umbilical artery Doppler values, abnormal fetal middle cerebral artery values and/or nonreactive nonstress test (NST)). The remainder of the HR group was classified as high-risk normal (HR-OK). The 209 studies that were not HR were considered low risk (LR). Magnetocardiogram (MCG) recordings were performed at a sampling frequency of 312.5 Hz. FCTI was calculated with averaged values by oneminute windows. Further we determined the fetal states quiet sleep (1F) and active awake (2F) based on Nihjuis crietira.

The time interval for P, PQ, PR, QRS and RR intervals were computed for all the groups and plotted against GA. We did not observe any statistically significant trend in any of the intervals except an increasing trend in RR intervals (0.992, p<0.05) with gestation. Comparing these variables in the high-risk group to low-risk we found statistically significant differences in the P and PR interval in each of the high-risk sub-groups. The correlation with fetal states did not show any observable effect of state (quite/active sleep) on cardiac time intervals.

to GA between fetuses classified as low-risk and high-risk gives us insight into the electrophysiological trends in fetal cardiac development. First, a subset of severely compromised neonates (pH<7.00 or BE<-12mmol/ mL or APGAR 5'<7; n=5) was identified and compared to a matched healthily delivered babies (pH>7.30, BE>-6mmol/mL, APGAR 5' 9-10; n=5). We analyzed 60 min baseline and the rest of recording until delivery with respect to fHRV measure of root mean square of successive differences of R-R intervals (RMSSD), that we have shown to early reflect onset of acidemia in fetal sheep model of human labor. Next, for all 78 babies, a matrix of 100 fHRV measures was analyzed using multiple linear regression analysis to identify a subset of fHRV measures capable of predicting BE or pH at birth. and -9.4±1.8, respectively. In H/UCO group, FuzEn increased vs. baseline as early as 77±19 min prior to pH<7.00 (p=0.003) and correlated to pH (R=-0.67) and BE (R=0.41). In LPS/UCO group, we observed very early (180±22 min prior to pH<7.00) increases in RMSSD (∼2.7-fold) and Skewness (4.5-fold), likely reflecting the LPS-induced inflammation. Skewness, followed by SampEn (120±22 min prior to p<7.00) continued to significantly differ from baseline at each time point until pH<7.00. Highest correlation to pH and BE showed again FuzEn (R=-0.70 and R=-0.60, respectively). Conclusion: Confirming our hypothesis fHRV monitoring detected incipient acidemia in each fetus regardless the pre-condition. The fHRV dynamics was condition-specific suggesting specific effects of baseline hypoxia or inflammation on specific fHRV measures. Composite fHRV measures need to be developed to capture the spectrum of pre-conditions impacting on fHRV responses to labour. Aim: We previously demonstrated that stimulation or inhibition of nicotinic acetylcholine receptor receptor in the brain, modified brain damage in a wellestablished newborn rat model of hypoxia-ischemia (HI). Our aim is to elucidate whether acetylcholinesterase inhibitor reduces brain brain damage induced by hypoxia-ischemia or not. Study design: 7-day-old Wistar rats were used for this study. Rats were subjected to left carotid artery ligation followed by 2 hours of hypoxia (8% oxygen). We injected galantamine (5mg/kg; n=14, 2.5mg/kg; n=9, 1mg/kg; n=11) or saline to subcutaneously before 2 hours hypoxia to see its neuro-protect effect. After 7 days, we checked for brain damage by eyeball observation. Brain damage in the ligated side was compared with that in the contra-lateral side, which serves as reference.

Results: The group of 5mg/kg injection of the galantamine showed marked reduction of brain damage (p<0.01) compared with saline treated group. 2.5mg/kg injection of galantamine had less protective effect on brain damage compared with saline (p=0.58). Likewise, 1mg/kg injection of galantamine had less protective effect on brain damage compared to saline (p=1.00). Conclusion: We showed that acetylcholinesterase inhibitor reduces brain brain damage induced by hypoxia-ischemia with dose dependent manner in newborn rats.

Kajszczarek, Jacek Marcin Robak, Anna Kwa&#347;niewska. Department of Obstetrics and Pathology of Pregnancy, Medical University in Lublin, Lublin, Poland.

Offspring exposed to prenatal hypoxia has been evidenced to experience behavioral abnormalities as a result of the injury sustained by neuronal cells.

The increased concentrations of neurotransmitters in the mother's circulation induced by hypoxia during gestation imply their role in the regulation of differentiation. This study determined the effects of long-term hypoxia during gestation on the expression of opioid receptors in specific brain regions. The optical density of µ-opioid receptors was determined at E-21 of gestation during long-term exposure to chronic hypoxia. The autoradiography allowed for an assessment of the lesions sustained by fetal brain tissues due to hypoxia.

Overview of results of the influence of chronic hypoxia on the µopioid receptor optical density in different brain structures of the fetal rat brain. The results of A-opioid receptor expression can be detected in specific fetal brain regions that may be attributable to behavioral changes due to hypoxia during gestation. The aim of the study was to develop a novel animal model of premature encephalopathy and asses therapeutic effects of an intracranial mesenchymal stem cell (MSC) transplantation.

The study was conducted in a sham controlled design. Subcutaneous administration of LPS from E. coli and ligation of the left carotid artery followed by 40 minutes of hypoxia (8% O 2 ) on postnatal day 3 induced a ipsilateral perinatal brain injury. On postnatal day 5 pups were anesthetized again and 250'000 human placenta-derived MSCs were injected into the lateral ventricle using a stereotactic frame. A second therapy group additionally received erythropoietin (EPO) (1000 U/kg bw, ip) on postnatal day 6,7, and 8. Evaluation of the brain damage was conducted on postnatal day 12 and 66. Donor cells were detected by immunohistochemistry (HLA class I ABC). Brain injury was evaluated by histology and immunohistochemistry (MBP, GFAP, Caspase-3, NEFH/NF-H, and TUBB-3). Spastic paresis was evaluated by footprint and walking pattern. RESULTS: Donor cells were detected in the periventricular white matter post transplantation. Assessment of the damage by immunohistchemistry indicates a perinatal brain injury in this novel animal model. Application of MSC's +/-EPO attenuates the extend of the perinatal brain damage and resulted in alleviation of spastical paresis. CONCLUSIONS: Transplantation of human placenta-derived MSC into the lateral ventricle of neonatal rats +/-EPO is possible. This complex animal model induces brain damage primary in the white matter. Application of donor cells results in incorporation of MSCs in the lesioned brain area and reduction of cerebral palsy. Conclusions: Elovl6, Scd-1 and Fasn catalyze synthesis and conversion of saturated fatty acids (FAs) into mono-unsaturated FAs (MUFA). The accumulation of MUFAs is associated with adiposity. The upregulation of these hepatic enzymes coupled with increased expression of pro-adipogenic genes may prime offspring from hypoxic pregnancies to early obesity risk. (Supported by NIH grants HD31226 and HD51951)

Effect of Long Term Hypoxia on eNOS Phosphorylation and Cortisol Biosynthesis in the Ovine Fetal Adrenal. E Newby, 1 K Kaushal, 1 D Myers, 2 C Ducsay. 1 1 Ctr. for Perinatal Biology, Loma Linda Univ. SOM, Loma Linda, CA, USA; 2 Dept. of Ob/Gyn, Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. Background: We previously showed that nitric oxide (NO) plays a role in regulating cortisol synthesis in the long term hypoxic (LTH) ovine fetal adrenal. We have also shown that the MEK/ERK 1/2 inhibitor UO126 (UO) inhibits cortisol production in normoxic adrenals, while stimulation with ACTH has no effect on eNOS phosphorylation (peNOS). The present study was designed to determine the effects of LTH on the relationship between the MEK/ERK pathway and peNOS and cortisol production in fetal adrenal cortical cells (FACs). Materials and Methods: Pregnant ewes were maintained at high altitude (3820m, LTH, n=4-7) or near sea level (control, normoxic, n=7) for 100 days. Adrenal glands were collected from near term (139-141 days gestation) fetuses. Dispersed FACs were untreated (control), or stimulated with either 100pM ACTH or 10mM 8Br, with or without 10µM UO pretreatment. Values for cortisol (ng/2.5x10 5 cells) and peNOS (SER1177/79) by Western blot (relative optical density, ROD) at 60min post-stimulation are shown in Table 1 . Results: Both ACTH and 8Br significantly increased cortisol production that was blocked by UO pretreatment in both normoxic and LTH FACs. Treatment with either ACTH or 8Br did not affect levels of peNOS in normoxic controls. In contrast, ACTH treatment significantly reduced levels of peNOS in LTH FACs. 8Br treatment resulted in a similar trend. 

on eNOS phosphorylation appears to be independent of MEK/ERK inhibition in LTH FACs compared to normoxic controls. Together these data suggest that peNOS is closely linked to cortisol synthesis in LTH fetuses while other mechanisms play a more dominant role in normoxic adrenals. NIH HD31226, HD51951

Neuronal Nitric Oxide Synthase (nNOS) Is Increased in Serum of Encephalopathic Neonates. Elisabeth Nigrini, 1 Ernest Graham, 1 Sarahn Wheeler, 1 Talaibek Borbiev, 2 Michael Johnston, 2 Irina Burd. 3 Objective: Currently, neonatal encephalopathy is a clinical diagnosis, and there are no markers to diagnose or prognosticate the injury. The purpose of this study was to determine: 1) whether neuronal nitric oxide synthase (nNOS), a neuron-specific marker of oxidative stress, is present in serum of neonates within 6h of birth; and 2) whether the level of nNOS differs in neonates with a clinical diagnosis of neonatal encephalopathy (NE) compared with neurologically normal neonates. Study Design: This was a case-control study of neonates greater than 36wks of gestation. Cases were neonates clinically diagnosed with moderate to severe NE who underwent whole body cooling per institutional protocol. Controls were age-matched neurologically normal neonates. Enzyme-linked immunosorbent assays (ELISA) for nNOS in serum of neonates within 6h of birth (prior to cooling in NE) were performed. Standard statistics were utilized including t-tests and receiver operating characteristic (ROC) curves. Sensitivity and specificity were calculated. Results: ELISA for nNOS was performed on 41 neonates (n=17 cases and n=24 controls). nNOS was present in neonatal serum within 6h of birth. nNOS was significantly increased in NE cases (mean= 1.19+/-0.86) compared with controls (mean=0.71+/-0.39) within 6h of birth (p<0.01). ROC curve yielded an area under the curve (AUC) of 0.734. We applied stringent criteria to these clinically encephalopathic neonates, and isolated only those that had umbilical artery cord pH<7.0 and base deficit of >12 to compare with neurologically normal Conclusions. Pf is a crucial part of the neuronal substrate that mediates HV roll-off in lambs. Thus, the same thalamic sector involved in H inhibition in the fetus is critically involved in postnatal HV depression. Pf (fetal and postnatal) has a moderate density of A 2A R and hence is a putative locus for O 2 sensing related to hypoxic respiratory depression in fetal and postnatal life (Koos, 2011 Background: Hemodynamic forces such as wall shear stress are critical environmental factors that regulate the genetic program for heart development and cardiac remodeling. Alterations in blood flow at early embryonic stages can lead to detrimental remodeling and heart defects. However, the structural adaptations of the heart to altered hemodynamic conditions are not well understood. We hypothesize that deposition of collagens will be increased as shear and wall stress is increased --leading to a stiffer heart wall. Methods: To test this hypothesis a suture (OTB) was tightened around the outflow tract (OFT) of stage HH18 (day 3) chick embryos to reduce cross sectional area of the lumen, thereby increasing blood velocities and shear stress at the wall. After 24 hours sham and OTB embryos were collected, immunostained for collagen I, VI and XIV, and imaged with confocal microscopy. Staining was quantified by grayscale image analysis according to anatomic region (n=5-6/group). Banded hearts were compared to the sham controls. Results: Collagen I was not significantly changed in the OTB group. However, the deposition of collagen XIV, normally minimal or absent at this stage of development, was increased by 50% throughout the banded OFT (p=0.04) in a degree-of-constriction dependent manner. Collagens VI and XIV were significantly elevated in the endocardium, cardiac jelly, and myocardium of the OFT's outer curvature, distal to the band site (p<0.02). In the proximal OFT, where shear stresses are usually lower, deposition was increased only in endocardial cells (p<0.01). Conclusions: Hemodynamic overload alters the collagen composition of the OFT, which may lead to a detrimental increase in wall stiffness. The observed increase in collagen VI and XIV deposition, with increasing degree of constriction, suggests they play a key role in structural adaptation to increased hemodynamic pressure. The location-specific increased deposition likely reflects local shear and wall stress patterns in the constricted OFT. In future, these data will lead to a better understanding of early changes in cardiac wall adaptation to hemodynamic conditions and detrimental cardiac remodeling that underlie heart defects and adult onset cardiovascular disease. Introduction Despite of the huge development associated with the progress for diagnosis and treatments in the obstetrics in the last few decades, there are still no effective treatments for premature low-birth-weight infants with cardiopulmonary abnormalities. And also, the compulsory artificial respiration is the only method for premature infants. The aim of this study was to develop a new high-performance membranous oxygenator as an artificial placenta and to prolong the survival time compared with the previous studies using premature lambs. Methods We used Suffolk sheep. Fetuses surgically delivered by caesarean section at 125 -135 d (term = 147 d). After C-section, the fetuses were connected to the pumpless artificial placenta with placement of the catheters into the umbilical vessels. Both the fetuses and the circuit of the artificial placenta were submerged in a warm saline bath. Results We could conclusively success to keep five fetuses alive for 18.2 ± 3.2 hours (mean ± SEM) after connecting to the artificial placenta. Cause of death was cardiac decompensation because of hyperlactacidemia from peripheral circulation insufficiency, not infection or performance degradation of the artificial placenta. 

We could success to keep fetuses alive for a longer time compared with previous reports using new type of pumpless artificial placenta. These data suggest a huge prospect for new treatment for premature low-birth-weight infants.

Occlusions with Severe Acidemia in the Ovine Fetus. Objective: Variable fetal heart rate decelerations due to umbilical cord occlusion (UCO) when frequent and/or severe during human labour can lead to acidemia at birth with increased risk for newborn encephalopathy and longer term neurologic sequelae. We have previously shown in the ovine fetus that repetitive UCOs leading to severe acidemia will result in a fetal inflammatory response, both systemically and locally within the brain at 24 hours, as potential mechanisms contributing to brain injury. We have therefore hypothesized that repetitive UCOs with severe fetal acidemia will result in inflammation within the brain which continues to be evident at 48 hours and relates to measures of brain injury. Methods: Near term fetal sheep were chronically instrumented with vascular catheters and an inflatable cord occluder (Control n=10; UCO n =14). All UCO animals underwent repetitive UCOs for up to 4 hours or until fetal arterial pH decreased to < 7.00, with occlusions occurring for 1 min every 2.5 mins.

After 48 hours, fetal brains were perfusion-fixed then sectioned for histological analysis of microglia and mast cell counts, as measure of inflammation; as well as cleaved caspase-3 and TUNEL positive cells, as indicators of apoptotic brain injury. Results: Repetitive UCO resulted in worsening acidosis over 3 to 4 hours with arterial fetal pH decreasing in the UCO group to 7.00±0.03 (mean±SEM), but with most animals recovering to baseline by 24 hours. For all brain areas studied, activated microglia and mast cell counts averaged 9.0±0.3 and 0.4±0.1 cells per high power field (HPF) (400x), respectively, with no significant differences between groups. TUNEL and cleaved caspse-3 positive cells for all brain areas averaged 0.02±0.01 and 7.8±0.8 cells/HPF, respectively, again with no differences between groups. Conclusion: While repetitive UCOs over 3-4 hours leading to severe acidemia in the ovine fetus may result in a local inflammatory response within the brain, this appears to be dissipated by 48 hours and is not associated with an increase in apoptotic cell injury, presumably due to adequacy of neuroprotective mechanisms with these insults. Introduction: Impaired spiral artery remodeling leads to placental ischemia and hypoxia that has been recognized as a leading placental pathophysiology, rendering hypoxia as a key physiological/pathological factor for regulating placental gene expression and development. However, the effects of hypoxia on global trophoblast protein expression have not been reported. Objectives: to establish a quantitative proteomics approach for analyzing trophoblast proteome in response to hypoxia and to analyze the functional links of hypoxia-responsive trophoblast proteome. Methods: Human choriocarcinoma BeWo cell line was used as a trophoblast model. A quantitative proteomics approach was developed based on Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for analyzing hypoxia-responsive proteome. The cells were labeled with "light" (L-12 C 6 14 N 4 -Arg and L-12 C 6 14 N 2 -Lys) or "heavy" (L-13 C 6 15 N 4 -Arg and L-13 C 6 15 N 2 -Lys) amino acids in RPMI-1640 medium. When reaching 70% confluence, the "light" cells were switched to hypoxia condition (2% O2, 5% CO2, and 93% N2) and the "heavy" cells were cultured under normoxia. The cells were harvested at 48 hr post-treatment and equal amounts of cellular proteins from the "light" and "heavy" cells were mixed and trypsin digested, followed by mass spectrometric LC MS/MS for protein identification. The "light"/"heavy" ratios of all peptides identified were calculated for determining the hypoxiaresponsive trophoblast-proteome. Ingenuity pathways analysis was used to analyze the functions of the trophoblast proteins identified. Results: In the trophoblast proteome, 52 proteins were identified to be constitutively expressed and 279 proteins were altered by hypoxia treatment. In the hypoxia-responsible trophoblast proteome, 22 proteins were significantly down-regulated and 257 proteins significantly up-regulated by hypoxia. Pathway analysis suggested that the hypoxia-responsible trophoblast proteins participate into the regulation of various cellular functions, including apoptosis, cell structure, migration, metabolism, etc. Conclusion: Hypoxia regulates various functions of human trophoblast cells by altering global protein expression (proteome). The hypoxia-responsible trophoblast proteome provides an important database for further investigations of the role of hypoxia in placental physiology and pathophysiology (supported by RO1 HL 70562 & R21 HL98746).

Intermedin: A Novel Regulator of MUC1 at the Maternal Fetal Interface. Rexanna Chan, Meena Balakrishnan, Chandra Yallampalli, Madhu Chauhan. Ob/Gyn, University of Texas Medical Branch, Galveston, TX, USA. Introduction: Implantation of embryo depends on direct interaction of the blastocyst with the luminal epithelium of the receptive uterus. The apical surface of uterine epithelia expresses a highly glycosylated transmembrane mucin, MUC1 that acts as a barrier to microbial infection and enzymatic attack. In mammals, expression of MUC1 is increased in the receptive epithelium and is lost only at the implantation site to facilitate embryo attachment. The specific role of MUC1 in human embryo implantation is unclear. However, MUC1 decreases the invasion of first trimester trophoblast (HTR-8sv/neo) cells in human pregnancy and its expression is elevated in severe pre-eclamptic placentas. Intermedin is a novel petide expressed in rat implantation site and placenta and increases the invasive capacity of HTR-8sv/neo cells. In addition, infusion of IMD antagonist in pregnant rat inhibits embryo implantation and feto-placental growth. Hypothesis: IMD regulates expression of MUC 1 in trophoblast cells and uterine epithelium to facilitate embryo implantation. Objective: 1) assess the effect of IMD on the expression of MUC1 mRNA in RL-95 cells, 2) assess the effect of IMD on the immunoreactivity of MUC1 in RL-95 monolayer, 3) assess the effect of IMD on MUC1 immunoreactivity in HTR-8sv/neo monolayer and 4) assess the effect of IMD on the expression of MUC1 immunoreactivity in HTR spheroids. Methods: RL-95 and HTR cells were cultured in MEM and RPMI respectively, supplemented with 10%FBS. For generating spheroids, 750 cells were resuspended in 7ul of RPMI containing 2%FBS and 1% methylcellulose and cultured as hanging drop on the lid of petri dish and incubated in a humidified atmosphere of 95% (vol/vol) air-5% (vol/vol) CO 2 at 37°C for 24 hrs. Two-four spheroids were transfered to each well of lab-tek chambers in presence of IMD 10-8 M and cultured as above for 24 hrs. The spheroids were then fixed for immunofluorescent staining with MUC1 antibody. Results: IMD treatment 1)elevates the expression of MUC1 mRNA in RL-95 cells (P<0.05), 2) increases MUC1 immunoreactivity in RL-95 cells (P<0.05), 3) decreases the expression of MUC1immunoreactivity in HTR spheroids (P<0.05) and 4) decreases MUC1 immunoreactivity in HTR cells (P<0.05). Conclusion: These studies suggest an involvement of IMD as a potential regulator of MUC1 in fetal and maternal compartments that may play an imoportant role during impalntation and early placental development. Background: During early implantation, signaling at th trophectoderm-uteirne epithelal interface is mediated by factors, many of which are glycosylated. Abnormal glycosylarion of selectin and Notch1 result in implantation defects. Successful protein glycosylation requires fucose transportation by Solute carrier family 35, member C2 (SLC35C2), a GDP-fucose transporter. Although SLC35C2 was initially discovered in mouse trophoblast cells, its role in embryo implantation is largely unknown.

We established SLC35C2 knockout mouse model in which SLC35C2-/-mouse exhibited embryo lethality in peri-implantation stage (8.5 dpc) and SLC35C2+/females showed subfertility determined by pup number per litter, suggesting that deletion of SLC35C2 causes less effecient embryo implantation. To further define the role of SLC35C2 in the implanation stage, we assessed SLC35C2 expression in human endometrium by immunohistochemistry found SLC35C2 expressed in both luminal epithelial cells and stromal cells at middle secretory phase. Furthermore, we knocked down SLC35C2 in human endometrial epithelial cells in vitro by siRNA. By measuring Aleuria aurantia lectin (AAL), a lectin that binds fucose, we found a significant reduction in global fucosylation in human endometrial epithelial cells. Further, after adding 10mM fucose in culture for 24hrs in the SLC35C2 knock-down cells, we found that fucosylation level was partially recovered. Finally, we knocked down SLC35C2 in human extravillous trophoblast line HTR8/SVneo, and found a significan reduction in trophoblast proliferation. Conclusion: SLC35C2 plays important role in the glycoprotein mediated cross talk between embryo and uterine epithelia during implantation by regulating protein fucosylation in both trophoblast and uterine epithelia. Rescue trophoblast function in vivo by tetraploid aggregation and generate uterine conditional SLC35C2 mouse are the focus of ongoing investigation. Background: The human endometrium is a dynamic sex steroid-dependent organ subject to cyclical episodes of breakdown (menses), repair, regeneration & angiogenesis. Studies in mice suggest repair at the time of menses is estrogen independent, a finding consistent with low circulating concentrations of estrogen during the luteo-follicular transition in women. In normal women the circulating level of androgen is higher than estrogen, however a role for androgens in modulating recovery of tissue homeostasis following menses (scar-less repair) has not been previously investigated. In the present study we used a modified mouse model of menstruation to explore the relationship between androgens & angiogenesis during endometrial breakdown, repair & the initial stages of regeneration. Methods: Artificial 'menstrual' cycles were induced in mice using the following protocol: ovariectomized mice were treated with estradiol (E2) & progesterone (P)-3xE2 (100ng/100ul in oil), P-secreting pellet (100mg/ml, s.c day 6), E2 (5ng/100ul, days 6-8). Decidualization was induced on day 8 in one uterine horn using oil whilst the contra-lateral horn acted as a control. Endometrial breakdown was initiated by removing the P pellet, 96hrs after oil injection. Each mouse received either dihydrotestosterone (DHT) (2mg/ml) or vehicle at the time of P withdrawal. Uterine horns (control vs decidualized) were recovered for RNA extraction or fixed for immunohistochemistry. Results: In control animals, endometrial breakdown was associated with detectable blood in vaginal smears & in the uterine lumen 4hrs after removal of the P pellet (n=7). Evidence for delayed onset of 'menses' was observed in DHT-treated animals with blood only detectable 8hrs after P withdrawal (n=4). Preliminary data indicate that treatment with DHT significantly up-regulated expression of VEGF-A mRNA at 4hrs & WT-1 mRNA 8hrs after P withdrawal (P<0.0001). Expression of the angiogenic chemokine CXCL12 mRNA was down-regulated at 4hrs & 8hrs after P withdrawal (P<0.05). Discussion: Studies in mice implicate androgens in regulating expression of genes associated with regulation of angiogenesis at the time of endometrial breakdown. These results highlight the potential that disturbances in peripheral/ local androgens may contribute to disorders including heavy menstrual bleeding.

Interleukin-8 (IL-8) is a proinflammatory cytokine with important functions in the endometrium including chemotaxis of neutrophils and lymphocytes, and angiogenesis. Physiologic regulation of its production is not well understood. There is evidence that endometrial expression of IL-8 is regulated by prostaglandins and relaxin; both bind to G-protein coupled receptors and increase intracellular levels of cAMP. However, whether cAMP mediates regulation of IL-8 has not been determined. We tested the hypothesis that increased intracellular cAMP stimulates IL-8 expression in a human endometrial epithelial (HEE) cell line. Studies were performed to determine the relationship between intracellular cAMP and IL-8 protein expression in HEE cells. Cells were incubated in Medium 199 with 0mM, 0.1mM, 0.25mM, 0.5mM, or 1mM 8-br-cAMP. Cells were also incubated with PMA as a positive control. Conditioned medium was collected after 24 hours of treatment, and IL-8 protein levels were assessed by a human IL-8 specific enzyme immunoassay. Three independent experiments, each in triplicate, were performed. IL-8 levels in medium from control cells were set at 100%; values from treated cells were expressed as percent of control. Data were analyzed using nonparametric Kruskal-Wallis (ANOVA) and Mann-Whitney tests. A p-value <0.05 was considered significant. As shown in Figure 1 , no significant effects of any concentration of 8-br-cAMP upon IL-8 protein levels were detected (p = 0.4473). This is in contrast to the generally marked elevated levels of IL-8 protein detected in medium of PMA treated cells in these experiments (data not shown), indicating substantial cellular activity, as well as the significant dose-related stimulation of other secreted products from these cells by increased intracellular cAMP that we have shown previously. These data suggest IL-8 expression is regulated in a cAMP-independent manner in HEE cells. Further studies are needed to elucidate which other signaling mediators, such as MAP-kinase or PI-3-kinase, regulate IL-8 expression in human endometrium.

Bone Morphogenetic Protein 2 (BMP-2) Signaling. Leo F Doherty, Hugh S Taylor. Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA. Objective: Endometrial BMP-2 signaling is necessary for embryo implantation. We have previously shown that TGF-β3, secreted by fibroids, impairs endometrial receptivity by repression of BMP-2 mediated HOXA10 expression.

Here we examined the effect of fibroid-conditioned media (F-CM) on ESC expression of BMP receptors and assessed whether TGF-β blockade prevents endometrial changes associated with diminished receptivity. Methods: Human fibroid cells and ESC were isolated from surgical specimens. ELISA was used to quantify TGF-β3 concentrations in conditioned media. ESC were pre-treated with TGF-β neutralizing antibody or non-specific rabbit IgG (control), and then exposed to F-CM. qRT-PCR was used to quantify BMP receptor types 1A (BMPR1A), 1B (BMPR1B), and 2 (BMPR2) in a subset of ESC. To employ a second approach to TGF-β blockade, additional ESC were transfected with a TGF-β receptor type II mutant (K227R mutation) expression vector or empty vector (control), and then exposed to FCM. After F-CM treatment, ESC were treated with recombinant human BMP-2 (rhBMP-2). qRT-PCR was used to quantify LIF expression. ELISA and PCR data were compared using Student's t-test. Results: Mean TGF-β3 concentrations were more than 5-fold greater in F-CM compared to conditioned media from ESC (52000 pg/ml vs 9200 pg/ml, p<0.05). F-CM treatment of ESC significantly reduced expression of BMPR1B and BMPR2 (0.61 fold and 0.59 fold, respectively, p<0.05). Pre-treatment with TGF-β neutralizing antibody prevented repression of BMP receptors (p>0.05). LIF expression was repressed in ESC treated with rhBMP-2, subsequent to F-CM exposure (0.29 fold, p<0.05). Pre-treatment with TGF-β antibody prevented repression of BMP-2 mediated LIF expression (0.78 fold, p>0.05). LIF expression was also repressed in ESC transfected with empty vector prior to F-CM and rh-BMP2 treatments (0.64-fold, p<0.05). However, LIF expression was increased in ESC transfected with K227 mutant TGF-β receptor prior to FCM and rh-BMP2 exposure (1.65-fold, p<0.05). Conclusion: Exposure of ESC to F-CM, shown to have high concentrations of TGF-β, reduces the expression of BMP receptors 1B and 2. Altered BMP-2 receptor expression prevents BMP-2 induction of LIF, a critical regulator of Introduction Endocrine disruptors (EDs) pose a potential threat to human reproductive health, being able to interfere with the endocrine system function, particularly with sexual and thyroid hormones homeostasis. Among EDs the mainly spread is Bisphenol A (BPA), well known xenoestrogen compound being able to bind both alpha and beta oestrogen receptors. BPA is associated with earlier puberty in females as well as long-term adverse effects on reproductive tract such as adenomyosis, leiomyomas, endometrial atypical hyperplasia and others. Recently, it has been demonstrated that treatment with BPA 100mM on isolated human endometrial endothelial cells was able to decrease their proliferation and viability. In the present in vitro study we wanted to further investigate the possible influence of BPA on endometrial function studying the effect of this compound on stromal decidualization of secretive human endometrial cells in vitro. Actually, some previous studies demonstrated that BPA exposition negatively affected endometrial receptivity and the functional differentiation of the endometrium during the window of implantation, being able to alter some of the gene expression pathway involved in this delicate and finely regulated process.

Secretory endometrial specimens were obtained by uterine curetting from women -without hormonal or endometrial pathology-undergoing laparoscopy. The separation of endometrial stromal cells (SC) from epithelial cells was performed. Secretive SC were incubated for 16 days in presence of 10 -8 M E2 and 2x10 -7 M MAP with or without BPA (1-10-100-1000 nM). In vitro decidualization and in the SC culture medium the levels of prolactin (PRL) and Insulin-like growth factor binding protein (IGFBP) -markers of stromal differentiation-were assayed by ELISA.

After 16 days incubation we observed that BPA was able to negatively influence the decidualization process of SC being both PRL and IGFBP significantly reduced in the culture medium.

In the present study we observed that the decidualization process was negatively affected by the incubation with BPA. Our in vitro results may offer important clues to the mechanism of action of BPA being able to interfere at several levels in human reproduction.

High Levels of Insulin Promote AKT and mTOR Specific Insulin Resistance in Primary Stromal Endometrial Cells. Clare A Flannery, Emily C Turner, Elizabeth Oliver, Hugh S Taylor. Ob Gyn, Yale University. Women with PCOS have higher rates of early miscarriage, which is in part a result of impaired decidualization and implantation. PCOS is associated with elevated fasting and post-prandial levels of insulin. The effect of chronic hyperinsulinemia on decidualization is unknown. Our prior studies show that insulin activates the AKT pathway through the insulin receptor in endometrial stromal cells. AKT activation is necessary for insulin stimulated glucose uptake, although the role of insulin dependent glucose uptake in decidualization is unknown. Other investigators have shown successful decidualization requires increased glucose uptake for the storage of glycogen and synthesis of cholesterol and nucleotides. We sought to determine whether sustained, high levels of insulin induce resistance to insulin along the AKT, MAPK, or mTOR pathways in endometrial cells. Primary stromal cells were isolated from reproductive age women without endometrial pathology, and treated with estradiol (E 2 ) 10nM and medroxyprogesterone (MPA) 0.1 µM with or without insulin 100nM for 21 days in physiological levels of glucose (5.6mM). After 24 hour starvation, cells were acutely stimulated with insulin, and the activated state of AKT, MAPK, and mTOR pathways was assessed. IGFBP1 secretion was measured as a physiological marker indicating insulin action in endometrial cells. We found that chronic insulin exposure obliterated acute insulin induced AKT activation, while E2 and MPA alone did not alter acute insulin action. In cells treated with chronically high levels of insulin, phosphorylation of S6K and 4-EBP were also reduced at baseline and upon acute insulin stimulation, consistent with inhibition of the mTOR pathway. Further, reductions in TSC2 ser939 phosphorylation indicate mTOR was inhibited as a result of decreased AKT activation. In contrast, MAPK activation remained unaltered from baseline. IGFBP1 secretion was suppressed by insulin. These findings indicate that chronic hyperinsulinemia, as seen in PCOS, may promote selective AKT insulin resistance in endometrial cells. While this impairment in the AKT pathway is protective against the effects of high levels of insulin, it may impact the ability of stromal cells to obtain sufficient glucose uptake to complete decidualization successfully.

Are Insufficient for Decidualization. Clare A Flannery, Elizabeth Oliver, Emily C Turner, Hugh S Taylor. Ob Gyn, Yale University. Decidualization is critical for successful embryo implantation. Different in vitro models of decidualization are published, but without direct comparison. We sought to observe the time course of morphological change, protein secretion, and gene alteration in cells treated with established in vitro decidualization protocols. Primary stromal cells were isolated, and cultured in DMEM with 5.6mM glucose. At pass 3, cells were treated with 10% calf serum or 1 of 3 protocols: estradiol (E2) 10nM and medroxyprogesterone (MPA) 0.1µM, E2 and progesterone (P4) 1µM and epithelial growth factor (EGF) 20ng/ml, or 8-bromo-cyclic AMP 0.5mM and MPA 1µM. Secreted IGFBP-1 and prolactin were collected every 3 days and quantified by ELISA. 

Hormonal stimulation with E2+MPA did not produce significant morphological changes or increased prolactin secretion at any time point over 17 days, similar to cells treated with 10% serum alone. Whereas, E2+P4+EGF and cAMP+MPA treated cells demonstrated transition to cuboidal cells, and secreted higher levels of prolactin peaking at days 6 and 3, respectively. Neither group showed sustained prolactin secretion over time. The time course of IGFBP1 secretion was indistinguishable for E2 + MPA and serum treated cells. E2+P+EGF and cAMP+MPA treated cells secreted peak levels of IGFBP1 protein at days 6 and 3, respectively, but were unable to sustain production over 17 days.

These results indicate that estradiol and a progestin are insufficient for transformation of stromal cells to decidualized secretory units. Two established in vitro decidualization protocols using high levels of epithelial growth factor or cAMP promote classical morphological changes and increases in protein markers. However, in the presence of physiological levels of glucose, the cells were unable to sustain prolactin and IGFBP1 synthesis. Women often present at emergency departments in early pregnancy with a 'pregnancy of unknown location' (PUL) and diagnosis/exclusion of EP is challenging due to a lack of reliable biomarkers. Recent studies suggest that serum levels of a disintegrin and metalloprotease protein-12 (ADAM-12), activin B, placental growth factor (PIGF), and fibronectin (FN1) can be used to differentiate EP from viable intrauterine pregnancy (VIUP). However, none of these markers have sufficient specificity and sensitivity in isolation. Herein we describe a study evaluating the performance of these biomarkers in combination with markers of the major risk factors for EP (PgP3 protein for Chlamydia trachomatis; cotinine for smoking) in differentiating EP from the full spectrum of alternative PUL outcomes. Sera were collected from 120 patients at their first clinical presentation with a PUL and assayed for the above markers by ELISA. 

We emailed an electronic survey to the medical directors of all fertility centers in the US. After the initial invitation, a reminder email was sent to non-respondents. The survey included demographic and non-demographic questions. Non-demographic questions asked respondents about the frequency of antibiotic and steroid use for ART in their practice. Data are presented as proportions.

The survey was sent out to 292 US fertility centers. A total of 95 respondents completed the survey (33% response rate). Respondents were from 32 states and the District of Columbia and the largest representation was from California, with 14 (14.7%) of the respondents. 75.8% of respondents worked in a private practice and the majority (80.0%) worked in a practice with fewer than five fellowship-trained physicians. 80.0% of respondents conducted less than 500 IVF cycles in 2010. 63.2% of respondents use antibiotics with IVF and of those, 98.3% use them routinely. 60.0% prescribe them before oocyte retrieval, 63.3% prescribe them in between oocyte retrieval and embryo transfer and 6.7% prescribed them after transfer. 56.8% of respondents use steroids with IVF and of those, 88.9% use them routinely. 14.8% of respondents prescribed them before oocyte retrieval, 87.0% prescribe them between retrieval and embryo transfer and 8.0% prescribe them after transfer.

53.7% of respondents reported that their practice is influenced by training, 46.3% reported that their practice is influenced by personal choice and 43.2% reported that their practice pattern is influenced by the medical literature. CONCLUSIONS This survey of 95 US fertility centers indicates that more than 50% of practitioners prescribe steroids and antibiotics routinely during IVF treatment, and this decision is mostly influenced by their training. This is an interesting finding given the lack of evidence of any significant effect of routine steroid and/or antibiotic use in IVF.

Oral Methods: A prospective observational cohort study was performed on women with regular menses; 6 naturally cycling (NC) and 6 taking OCPs. Uterine pathology was ruled out. Endometrial biopsies (EMB) and P levels (ELISA, ng/mL) were taken on CD 10 in both cohorts (NC10 & OCP10) and on LH+6-7/CD20 (NC20 & OCP20). Immunostaining was performed to quantify the percent of EECs with NCSs. Results: NC10 biopsies had no NCSs, whereas OCP10 biopsies had no or few NCSs. There was a bimodal distribution of %EECs with NCSs in the NC20 and OCP20 biopsies ( Fig. 1 ; Mann-Whitney test [MWT] p=0.063). The four NC20 biopsies with robust NCS presence had corresponding P levels of 4.2-9.7, whereas those with minimal NCS presence had P levels of 2.5 and 3.5. OCP20 biopsies with none or few NCSs had P levels of ≤ 1.9. However, the two OCP20 biopsies with abundant NCS presence had P levels of 4.4 and 7.9, indicating escape ovulatory events. Spearman correlation (SC) of NC20 and OCP20 biopsies for %EECs with NCSs and P level was strong (r=0.83, p=0.002). In a sensitivity analysis, the two OCP20 biopsies with escape ovulatory events and the NC20 biopsy with a P level of 2.5 (less than the established 3.0 threshold for ovulation) were excluded. MWT of the remaining NC20 vs. OCP20 biopsies for %EECs with NCSs yielded p=0.016. SC remained strong (r=0.85, p=0.007). Discussion: OCPs interfere with NCS formation, likely by inhibiting ovulation and minimizing circulating levels of P. Synthetic progestins can lead to some NCS formation on CD 10, however, all three progestins used appeared equally likely to lead to NCS formation. The NC20 biopsies with minimal NCS presence and P levels of only 2.5 and 3.5 are consistent with a threshold of P (∼4) for robust NCS formation. 

Decidualization of endometrial stromal cells is a prerequisite for implantation of human embryo. Previously, we reported that the a2 isoform of vacuolar ATPase (ATP6V0A2 referred to as a2V) plays a role in successful pregnancy and maintains the delicate immunological balance at the feto-maternal microenvironment. The objective of this study is to determine whether a2V influences decidualization.

To study this phenomenon, we used Ishikawa endometrial carcinoma cell line. Treatment with ovarian steroid hormones resulted in increased expression of decidualization markers such as LIF, Wnt4 and BMP2 as well as a2V expression. In addition, the treatment showed enhanced cell migration and proliferation. a2V small interfering RNA (Si RNA) treatment resulted in significant reduction of ovarian steroid mediated decidualization markers expression. Furthermore, depletion of a2V induced apoptotic morphology and increased apoptotic gene expression in Ishikawa cells. Our results collectively indicate that a2V positively regulates Ishikawa cells for endometrial cell migration and proliferation. This study demonstrates that a2V could play a pivotal role in endometrium during implantation to regulate decidualizaiton and differentiation. a2V may also be utilized in diagnostic screens and may be used as a target for drug discovery in women with endometrial-based infertility. Introduction: Leptin, an adipocytokine produced by adipose tissue and, in some species the placenta, regulates both energy homeostasis and reproduction. Undernourished women lack serum leptin, while obese women are often leptin resistant; these women also share elevated rates of infertility. LEPR is found in the uterus and placenta in mouse and human, implying a local function of leptin. Mice and humans lacking expression of leptin (Lep ob/ob ) or Lepr are obese, diabetic, and infertile. Leptin replacement through pregnancy day 6.5, but not 3.5, in Lep ob/ob mice reverses infertility, indicating leptin signaling has an essential role in establishment of pregnancy, but it does not indicate which signal -that derived from the hypothalamus, ovary, uterus or fetus -is required. Evidence is mixed, as mice with Lepr expressed only in neurons are at least partially fertile, yet injection of a leptin antagonist into one uterine horn completely blocks implantation in that horn in normal mice. We hypothesize that leptin signaling in the uterus is essential for optimal fertility. Methods: Lepr uterine null mice were generated by crossing mice expressing cre recombinase driven by the progesterone receptor (Pgr) promoter with Lepr flox mice. Immunohistochemistry (IHC) for Lepr was performed in non-pregnant and pregnant uteri and ovaries from control (Pgr cre/+ Lepr flox/+ ) and null (Pgr cre/+ Lepr flox/flox ) mice. Fertility and fecundity is being assessed in knockouts and controls. groups, were used for statistical analysis. Significance was defined as a P value of <0.05. Accumulation of lipid droplets, resulting from inhibition of fatty acid oxidation, was assayed by Oil Red staining and bodipy staining. MTT assay was performed to determine cell viability after etomoxir exposure. Results: Low concentrations (below 50µM) of etomoxir are tolerated by human immortalized ESCs. Exposure of human primary ESCs and ESC-Ts to etomoxir during decidualization in vitro decreased the expression levels of PRL, IGFBP1, IL-15 and SMST, starting as early as day 1 and continuing through day 9. Specifically, the expression of these decidual markers with 50µM etomoxir exposure are similar to those in non-decidualized ESC-T. Accumulation of lipid droplets in hESC-Ts was induced by etomoxir after 9 days of culture, indicative of blockage of beta-oxidation. Conclusions: Etomoxir, a known inhibitor of the free fatty acid oxidation pathway via inhibition of the carnitine palmitoyl-transferase I step, inhibits decidualization. Etomoxir prevents decidualization in a dose-dependent manner in both immortalized and primary endometrial stromal cells in vitro. These findings suggest that beta-oxidation is critical for the process of ESC decidualization. Support: R01 HD065435 The human endometrium is a dynamic tissue which undergoes rapid remodeling, proliferation and differentiation in response to ovarian steroids. Despite its importance in human biology and to the pathology of common reproductive diseases a comprehensive analysis of the endometrium is far from complete. The growth and differentiation of the endometrium is regulated by the dynamic interplay of the epithelial and stromal cell components confounding the ability to analyze biomolecules which are differentially regulated between the two cellular compartments. We isolated endometrial epithelial cells by laser microdissection (LMD) from 15 proliferative and 15 secretory premenopausal samples. These were subjected to tryptic digestion and mass spectrometrybased quantitative proteomics analysis resulting in the identification of 1318 total proteins, amongst which 230 were found to be differentially expressed between the two phases (p<0.05). These included the progesterone receptor B and PCDNA (proliferative phase proteins) and PAEP (gycodelin A, secretory phase protein), substantiating our methodologies. Similarly, proteomic analysis of LMD isolated endometrial stromal cells (proliferative n=6, secretory n=6) resulted in the identification of 1182 proteins of which 99 were differentially expressed between the proliferative and secretory phases (p<0.05). Importantly we validated several of these proteins using immunohistochemistry in an independent set of samples. Specifically, we confirmed the differential abundance of periplakin (elevated in secretory epithelium), MME (CD10) (elevated in secretory stroma) and Tenascin C (elevated in proliferative stroma). This study is the first large scale proteomic analysis of the individual epithelial and stromal cell compartments of pre-menopausal endometrium. Furthermore, the defined proteomic alterations during endometrial remodeling provide a basis for numerous follow-up investigations on the function of these differentially regulated proteins.

Uterine , an inhibitor of Activin A activity, has fundamental roles in female reproduction. FST has two splice variants: FST288 is mainly associated with cell surfaces; FST315 is the main circulating form. Female mice with both a targeted deletion of the mouse FST gene and expressing a human FST315 transgene (tghFST315) have severe reproductive abnormalities (reduced ovarian follicular population, no corpora lutea, and increased ovarian and uterine inflammation). This study characterised the morphology of tghFST315 oviducts and uteri. We postulated that tract defects reflected abnormalities of ovarian function. The reproductive tracts (oviduct, uterus) of WT and tghFST315 mice were examined in neonatal (day 0) and adult mice (8w) using histology and immunohistochemistry (αSMA, CD31, CD45). To remove the influence of abnormal ovaries, adult WT and tghFST315 mice were ovariectomised and treated with vehicle, exogenous oestradiol-17ß (100ng s.c. injection, dissection after 24h) or progesterone (1mg x 3 daily s.c. injections, dissection 24h after last injection) (n=6 per group). The oviduct and uterus of tghFST315 mice at birth were not different to WTs, but abnormalities had developed by adulthood. In contrast to WT mice, the oviducts of tghFST315 mice failed to coil and the myometrial muscle layers were disorganised. Uteri/ oviducts contained abundant CD45 positive leucocytes. Preliminary analysis suggests that endometrial cell proliferation in tghFST315 mice in response to exogenous E or P is consistent with that of WT mice, but the abundant CD45 leucocyte infiltration was not resolved by ovariectomy. We conclude that absent oviductal coiling and disorganisation of the myometrial layers represent a developmental defect of the Mullerian duct resulting from FST288 absence. The mechanism responsible for the oviductal/uterine inflammation, which develops after sexual maturity (suggesting oestradiol involvement) but is not resolved by ovariectomy (absence of ovarian steroids), has yet to be determined. Through a hormonally coordinated program of epithelial and stromal cell proliferation, angiogenesis, differentiation, leukocyte recruitment, remodeling, vasoconstriction, apoptosis and desquamation, the human uterine lining is reconstructed cyclically, unless pregnancy is established. Errors in these steps could potentially lead to uterine dysfunction, miscarriage, failed embryonic implantation and infertility or alternatively, endometrial hyperplasia and cancer. Recent studies in our laboratories and others indicate that endometrial stromal differentiation in mouse and primate uteri is critically regulated by gap junctions comprised of connexin (Cx) 43 subunits. In the current studies, isolated human endometrial stromal cell (ESC) cultures were exposed to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP (E2+P4+cAMP) for 7 days, resulting in morphological and biochemical decidualization, including characteristic epithelioid changes in cell shape and secretion of prolactin. Pharmacological blockade of gap junction intercellular communications (GJIC) with 18α-glycyrrhetinic acid (AGA) inhibited decidualization and induced ESC apoptosis, as manifested by reduced cell viability, increased TUNEL staining, caspase 3 activation, and sub-G 1 chromosomal DNA complement, as well as shortened telomere length. Moreover, in contrast to the reduction in prolactin secretion by ESC exposed to AGA, gap junction interference led to an increase in production of the chemokine RANTES. The findings indicate that GJIC play a role in the maintenance of ESC decidualization. Disruption or blockade of GJIC induces ESC apoptosis and stimulates the secretion of RANTES, which in turn can mediate the chemotaxis of monocytes and T cells to the degenerating decidua. Our data would predict that apoptotic ESC, through a process of efferocytosis, are removed before they lose membrane integrity and leak toxic enzymes and pro-oxidants into the surrounding endometrium basalis, protecting the tissue for subsequent reproductive cycles.

Role of COX-2 in Decidualization of Endometrial Stromal Cells. Joelle Taylor, Terry Jacot, David Archer. Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Norfolk, VA, USA.

Decidualization is the differentiation of endometrial stromal cells into decidual cells. Although several molecules and pathways have been identified, the sequences of these pathways and direct relationships in this process remain unknown. The importance of prostaglandins (PGs) in initiation and maintenance of decidualization and implantation has been previously identified.

Poster Session: Andrology/Testis Biology (Friday, 3/22/2013, 9: 00 AM -11:00 AM) Scientific Abstracts Reproductive Sciences Vol. 20, No. 3 (Supplement) , March 2013 229A Friday Cyclooxygenase (COX), the rate limiting enzyme in PG synthesis exists in two isoforms, COX-1 and COX-2. These enzymes catalyze the conversion of arachidonic acid into a variety of PGs. Previous studies have demonstrated that COX-2 expression increases during the window of implantation, and most observations point to endometrial epithelial cells as the main source of PGs. The aim of the present study was to investigate whether COX-2 and PGE2 are increased during in vitro decidualization suggesting the decidual cell itself is another source of PGs mediating downstream events. Immortalized human endometrial stromal cells (T-HESCs) were cultured for 8 days in Dulbecco's Modified Eagles Medium -0.5% charcoal stripped fetal bovine serum with 1 µM medroxyprogesterone acetate (MPA), 10 nM estradiol and .25mM cyclic AMP (cAMP). Markers of decidualization, prolactin (Prl), insulin growth factor binding protein 1 (IGFBP-1) and bone morphogenic protein 2 (BMP-2), were measured using qPCR (quantitative or real-time PCR). COX-2 was measured by RT-PCR (reverse-transcription-PCR). PGE2 levels in culture media were measured by enzyme immunoassay (EIA).

To determine that endometrial stromal cell decidualization occurred, qPCR was used to measure known markers of decidualization. Prl, IGFBP-1 and BMP-2 levels were significantly increased in treated cells compared with controls (untreated). COX-2 mRNA levels also increased in cells undergoing the in vitro decidualization process. Since PGE2 is a product of COX-2 enzyme activity, levels were determined in culture media from decidualized (treated) versus undecidualized (untreated) cells. EIA quantitation confirmed that PGE2 levels are increased in treated cells.

These results indicate that COX2 and PGE2 are produced by decidualized endometrial stromal cells. The exact role of PGE2 in the decidualization process remains unknown but further studies may elicit their primary purpose and subsequent downstream effects. Continual discovery of biomarkers of endometrial function may become the basis for diagnosing causes of infertility.

MFG-E8 is a protein recently discovered by us in the human endometrium, with predominant localization in the epithelium during the window of implantation. MFG-E8 has multiple functions in many extra-uterine tissues related to apoptosis and cell adhesion. Little is known about the regulation of its secretion by the endometrium and the underlying mechanisms. Since TNFα is synthesized by both decidual and trophoblast cells, we hypothesized that MFG-E8 secretion may be regulated by TNFα via p42/44 MAPK and NF-κB pathways, and that MFG-E8 may also be involved in TNFα-mediated apoptosis in endometrial epithelial cells.

Ishikawa cells were used as surrogate for human epithelial cells and exposed to time-and concentration-dependent effects of TNFα. Cell viability and apoptosis were determined by MTT cell proliferation assay and TUNEL fluorescence staining. MFG-E8 mRNA and protein levels were measured by real-time PCR and ELISA, respectively. Signaling pathway activation was analyzed using inhibitors of MEK1/2 (U0126) and NF-κB (Bay 11-7082) by western blotting. Immunofluorescence staining was performed to identify NF-κB translocation and degradation of IκB-α.

Exposure to TNFα induced a dose-dependent apoptosis in Ishikawa cells after 24 hours. TNFα at 25ng/ml or higher significantly up-regulated MFG-E8 mRNA (p <0.05) and protein (p< 0.05) expression levels. Moreover, TNFα caused phosphorylation of p42/44 MAPK, translocation of NF-κB into the nucleus, and degradation of IκB-α. After stimulation with 50ng/ml TNFα for 24hours, MFG-E8 protein level was significantly attenuated by pretreatment with U0126 and Bay 11-7082 (both p< 0.05). These results suggest that, phosporylation of p42/44 MAPK and transactivation of NF-κB, are involved in TNFα-induced MFG-E8 expression in Ishikawa cells.

These data demonstrate that MFG-E8 is regulated by TNFα and is involved in TNFα-induced apoptosis. We also found that p42/44 MAPK and NF-κB may be the critical pathways that regulate MFG-E8 expression. These results strongly support a modulatory role of MFG-E8 in trophoblast implantation, and reveal the mechanisms of TNFα action in human endometrial epithelial cells. To improve understanding of the morphological changes that occur during decidualization, a valid method to quantitate these changes was developed. We previously showed involvement of MAP kinase (MAPK) in secretion of IL-11, VEGF from cAMP stimulated decidualized human endometrial stromal cells (HESC). Our aim was to quantitatively assess the role of MAPK in the morphological changes seen in decidualization. A well characterized HESC line, which undergo decidualization in response to increased intracellular cAMP was used. Cells were incubated with phenol red-free medium in the absence or presence of 0.5mM 8-br-cAMP for 9 days. For studies of the role of MAPK in decidualization, cells were incubated with cAMP in the presence of UO126, a specific inhibitor of MAPK activity. Cells were imaged at 200X using bright field microscopy after being stained with hematoxylin. CellProfiler image analysis software, previously used to evaluate morphology in other cell types, was validated and used to assess morphology using an evaluation of cell shape named FormFactor (FF), calculated as 4Xπ XArea/Perimeter2. Assessment of at least 2500 cells demonstrated a FF value for control cells of 0.177+/-0.001, for cAMP stimulated cells of 0.252+/-0.002 and for cAMP+UO126 treated cells of 0.196+/-0.002 (mean+/-SEM). The mean FF of control cells was significantly lower than that of cAMP treated cells (P<0.001); the mean FF of cAMP+UO126 treated cells was significantly lower than that of decidualized cAMP treated controls (P<0.001)(T-test). Decidualized cells had a rightward shift in FF distribution consistent with their cobble-like cell shape, compared to elongated fibroblast-like controls. In cells treated with cAMP+UO126, the right shift in FF distribution was almost completely inhibited, consistent with the morphological changes observed by microscopy.

These data demonstrate the usefulness of this quantitative method of cell analysis in decidualized cells and support the novel role of MAPK in the morphological changes during decidualization. In double IUI, the first insemination is performed 12-18 hours after human chorionic gonadotropin (hCG) injection, and then the second IUI 34-60 hours later. The number of total motile sperm (TMS) is an important parameter in achieving clinical pregnancy. Longer periods of abstinence lead to increases in TMS. Total motile sperm recovery of less than 5 million is known to negatively impact pregnancy rates. The objective of the study was to determine if the shorter abstinence period in double IUI would negatively affect motile sperm recovery after processing. Data from 57 couples who underwent a total of 120 Double IUI cycles between January 1, 2009 and December 31, 2011 were reviewed. The data were analyzed using Mann-Whitney rank sum test. The median TMS at IUI#1 was 12.7 million (Interquartile range (IQR) 5.7-21.2 million), and 10.3 million (IQR 4.0-17.5 million) at IUI #2. There was no statistically significant difference between the TMS at IUI#1 or IUI #2 (p=0.075). Of 120 cycles, the TMS was higher at IUI#1 than IUI#2 in 76 cycles(median IUI #1=23.1 million, median IUI #2=12.6 million), the same in 7 cycles, and lower in IUI#1 than IUI #2 in 37 cycles(median IUI #1= 6.7 million, median IUI #2=12.2 million). The TMS was ≥ 5 million in 92/120 cycles and < 5 million in 28/120 cycles. Of the 92 cycles with TMS ≥ 5 million at IUI #1, 10.8% (n=10) had a TMS < 5 million at IUI #2. Of those 28 with TMS <5 million at IUI #1, 14.2% (n=4) had a TMS ≥ 5 million at IUI #2.

There is no significant difference between the TMS at IUI #1 vs. at IUI#2 regardless of initial TMS. For the majority of men, TMS will not significantly change after one day of abstinence. Double IUI can be performed without concern for a significant reduction in TMS at the time of the second IUI.

Comparative Objective: To determine the live birth potential in women undergoing IVF with very low AMH (<0.4 ng/ml) as compared to age matched women with normal AMH (1.0-10.0 ng/ml). To determine if a difference in live birth rates exists after matching for embryo number and quality. Design: Retrospective analysis Materials and Methods: All women presenting for their first cycle of IVF between 1/2009-12/2011 with an AMH of <0.4ng/ml or 1.0-10.0ng/ml were included. Patients with severe male factor or abnormal uterine cavity were excluded. The primary outcome was live birth rate. Mann U Whitney and Fisher's exact tests were used in analysis. P<0.05 was considered statistically significant. Results: 712 patients, 473 with normal AMH and 239 with very low AMH were included. Patients with very low AMH (mean age 39.13+4.21) achieved a live birth rate of 12.23% per cycle start; this was lower than their normal AMH counterparts (mean age 34.64+4.9), reaching a live birth rate of 40.32% (P=0.0001). Number of oocytes retrieved, mature and fertilized were also higher in the normal AMH group. These differences remained significant after matching for age. 67.58% of patients with normal AMH progressed to transfer of ≥ 2 grade II or better embryos versus 38.42% in an age matched group with very low AMH (P=0.0001). In patients who progressed to transfer of ≥ 2 grade II embryos, pregnancy rates were not statistically different between the two AMH groups. These results are shown in table 1. Objective: Assisted reproductive technology success rates largely depend on ovarian reserve. Women with low anti-mullerian hormone (AMH) are usually advised to undergo in vitro fertilization (IVF) early because "they are running out of eggs". However, the benefit of this approach is unclear as poor responders consistently have low pregnancy rates. Therefore, our goal was to compare the outcomes of intrauterine insemination (IUI) and IVF, in women with low levels of AMH. Methods: Retrospective chart review from January 1st, 2008 until October 4 th , 2012 was performed. All patients with known AMH levels were included, and egg donor cycles were excluded. Canceled cycles were not excluded, but they were specifically annotated. Pregnancy was defined as a positive serum β-HCG. Patients were managed according to their physician's practice preferences. Data were analyzed by t test, Mann-Whitney U test for continuous variables and Chi-square test for proportions where appropriate. Results: A total of 206 patients underwent IUI, whereas 167 patients underwent IVF. There was no significant difference in mean age, BMI, gravidity and parity between the two groups. Cumulative pregnancy rates (PR) between IUI and IVF cycles across all AMH levels were 61 (29.6%) and 82 (49.1%) respectively (P<0.001, Table 1 ). A total of 67 women with AMH <1 underwent IUI, whereas 43 women with AMH <1 underwent IVF. Interestingly, there was no statistical difference in cumulative PR between IUI vs. IVF in women with an AMH of <1.0 (25.3% vs. 27.9%, respectively). The mean age for these two groups was also similar (37.8 ± 4.5 vs. 37.4 ± 4.5, respectively, Table 2 ). The mean number of IUI and IVF cycles in women with an AMH <1 was 2.7 ± 2.0 and 1.5 ± 0.8, respectively. 

Pregnancy rates with IVF decrease with decreasing AMH as expected. Importantly and surprisingly, pregnancy rates between IUI and IVF at low AMH levels (<1) were not found to be significantly different. This preliminary study, which to our knowledge is the first to look at this relationship, suggests that IUI--which is less invasive and less costly--may be a preferred treatment in women with low AMH levels. 1.14) . There was no significant association between ovarian tumors and fertility drug use among infertile women ( Table  1) . Findings were similar when stratified by gravidity and when analyzed separately for borderline vs invasive tumors. 

The choice of gonadotropin is often dependent on availability, cost and ease of administration. Donors stimulated with rFSH required a significantly lower total dose of gonadotropins and had more oocytes retrieved. Also, there was a trend towards improved IR and LBR/OPR with rFSH. The increased oocyte yield with rFSH could potentially increase the availability of embryos for preimplantation genetic diagnosis and proportion of oocytes available for shared cycles, alleviating the financial burden and allowing more women the opportunity for donor IVF cycles. These conclusions confirm that treatment given to these donors with excellent ovarian reserve can be based on drug availability without concern for adverse cycle outcome.

How Conclusions Optimal donor oocyte cycles are measured by donor response to treatment, the amount of viable oocytes retrieved, and ultimately, pregnancy outcome. The recruitment location of donors could contribute to differences in donor response/pregnancy rate depending on the recruitment methods of outside agencies compared to those of the NYUFC. This study demonstrated that agency donors required a lower total dose of gonadotropins and had significantly more oocytes retrieved than those recruited by the NYUFC, which may be age-related as these donors were significantly younger. However, this did not translate to differences in clinical outcomes with similar pregnancy rates achieved in both groups. Centers with donor programs can be reassured that recruitment method does not appear to significantly alter pregnancy outcomes. As expected D3 embryo pregnancy rates were lower, but acceptable. The demographics demonstrated that the D3 women were older with lower AMH levels. The multiple pregnancy rates were higher with D5 ET, but not significantly so, as well as showing higher biochemical/SAB rates. Discussion: Applying selective ET guidelines to ET decision making in an effort to avoid prolonged culture in less robust embryos is associated with lower but acceptable pregnancy rates while selecting out older women with lowered ovarian reserve.

Can Nitric Oxide Explain the Poor Follicle Quality of Women with Diminished Ovarian Reserve? Manuel A Doblado, K Joseph Hurt, Nanette Santoro. Obstetrics and Gynecology, University of Colorado, Aurora, CO, USA. Background Vascularization is a critical process in ovarian follicle maturation. Angiogenesis is enhanced by nitric oxide (NO) production. A role for NO in follicle development and ovulation has been suggested. NO degrades to NOx within minutes in blood, but NOx is rapidly excreted and concentrated as a stable metabolite in urine. Urinary NOx may be a more reliable indicator of NO production over the menstrual cycle. To our knowledge, NOx has not been investigated in urine over the course of the menstrual cycle. We hypothesized that women with diminished ovarian reserve (DOR) would have decreased overall NOx or would lack a midcycle spike in NOx when compared to women with normal ovarian reserve. Method We performed a secondary analysis of NOx in stored urine from eight women with diminished ovarian reserve (DOR). Participants collected daily, first morning urine samples that were preserved in 7% glycerol and then frozen and stored at -20C. Specimens were collected for an entire menstrual cycle on each participant and urinary hormones were profiled for LH, FSH, estrone conjugates (E1c) and pregnanediol 3-glucoronide (PDG) levels, centered on ovulation as day 0, and indexed to creatinine [Fertil Steril. 2010; 93(4) :1074-9]. NOx was measured in triplicate using the Griess colorimetric assay. We indexed urinary NOx to urinary creatinine. Average follicular (10 days prior to ovulation), ovulatory (day of ovulation), and luteal phase (10 days post ovulation) NOx levels were compared by ANOVA. We also calculated daily deviation from average urinary NOx for each patient. For each cycle day, linear regression was performed on average daily deviation to identify significant changes in NOx.

Mean NOx +/-SEM was similar (p=0.96) for follicular (255 +/-29.6 µmol/ mg Cr), ovulatory (264.3 +/-30.8 µmol/mg Cr), and luteal phases (264.7 +/-29.3 µmol/mg Cr). Deviations from average urinary NOx levels were not significantly correlated with menstrual cycle day (mean NOx 238.9 µmol/ mg Cr +/-24.3). Conclusions These results suggest that systemic NO levels do not vary across the menstrual cycle in women with DOR. A control group of women with normal ovarian reserve is currently being assessed to determine whether this absence of a midcycle spike in NOx is a characteristic feature of DOR. Background: There is a paucity of data describing the role that racial, socioeconomic, and cultural factors play in influencing a woman to seek, initiate and continue infertility treatment. Objective: To identify and characterize socioeconomic/cultural factors that influence attitudes toward infertility treatment. Design: Demographic surveys and in-depth semi-structured interviews. Methods: 44 women undergoing infertility treatment at a university medical center participated. Transcribed interviews were analyzed using a grounded theory approach, in which coders identified major themes and sub-themes that emerged from interview texts. Three investigators coded the interviews and consensus around interview codes was confirmed. Results: 50.0% of the subjects identified as White (CW), 29.5% as Black (AAW), 11.4% Hispanic (HW), and 9.1% Asian (ASW). The mean age was Poster Session: Infertility/ART (Friday, 3/22/2013, 9:00 AM -11:00 AM) Scientific Abstracts Reproductive Sciences Vol. 20, No. 3 (Supplement) , March 2013 233A Friday 35.7 ± 0.65 years. 81.8% of CW, 53.8% of AAW, 50.0% of ASW, and 40.0% of HW had an annual household income of >$100,000. 100% of CW, 76.9% of AAW, 100% of ASW and 60.0% of HW had at least a bachelor's degree. Only 13.6% of women had no infertility coverage. White and Black women had been trying to become pregnant for 21 and 28 months on average, respectively. Major themes that emerged included: women's emotional reactions to being infertile or receiving infertility treatment; barriers faced during the treatment process, such as financial and insurance problems, logistical issues, and religious beliefs; and the impact of treatment on relationships. Preliminary results of thematic analysis indicate that AAW were more likely to have religious support, consider the use of alternative treatments, and to consider terminating treatment. CW were more likely to have shared experiences with others undergoing infertility care.

There are universal themes that impact patients' continuation in and experience of infertility treatment. There are, however, specific themes that appear to cluster by race. These findings have implications for improving patient recruitment and retention amongst a diverse patient base. After a SIS for cavity evaluation, a FemVue Saline-Air device was connected to the balloon-catheter. Slow instillation of air-saline was used to sequentially assess right and left tubal patency. This was followed by an HSG performed the same day. Secondary measures included pain scale using a Likert Scale and time of procedure, prior and following each procedure. Cohen's kappa coefficient was used to assess agreement between each procedure. Results: Mean age ± SD was 33.1 ± 3.2 yrs. Overall, uterine cavity findings (K=0.649, p=0.01) and right (R) fallopian tube patency (K=0.58, p=0.02) showed significant agreement, but not the left (L) (K=0.49, p=0.07). All (8/8) normal SIS were identified as normal by HSG, while only 60% (3/5) abnormal SIS were seen on HSG. Air contrast and HSG similarly identified R and L tubal patency 100% (9/9) and 88% (7/8), respectively. However, only 50% (2/4) and 60% (3/5) of R and L tubal occlusions were in agreement with HSG. No significant differences were found between Air Contrast HyCoSy and HSG with respect to pain or procedure time. Conclusions: Our preliminary findings reveal a high agreement between SIS-HyCoSy and HSG for uterine cavity and tubal patency assessment. However in the absence of air bubble contrast in one or both tubes, further evaluation may be indicated. Conclusions: In patients who have 1-2 euploid embryos within a given cohort, the transfer of 2 embryos still confers an improved CPR, albeit with a significant increase in multiple gestations. However, in patients with a greater number of euploid embryos, strong consideration should be given to SET given that the CPR remains high while obtaining a ten-fold decrease in multiple gestations. This is likely because a greater cohort of euploid embryos allows clinicians to more stringently select blastocysts on morphology in addition to chromosomal competence. Identification and education of these ideal candidates for SET should be an inherent part of any ART program that is performing PGS. 43.9 0.0206 *ES cutoff of 5.7mm determined by the median ES in the lowest quartile of study population CONCLUSIONS: The change in thickness of the endometrial lining during an IVF cycle can be used as a predictor of pregnancy outcomes in that cycle. Patients with an endometrial lining that increases by <5.7mm throughout their IVF cycle have significantly worse pregnancy outcomes than those with an endometrial lining that increases by >5.7mm.

Endometrial Expression of Urocortin 1 in Infertile Women Preparing for In Vitro Fertilization: A Potential Indicator of Treatment Outcome? Carolina P Rezende, 1 Helen L Del Puerto, 1 Pasquale Florio, 2 Fernando M Reis. 1 1 Ob/ Gyn, UFMG, Belo Horizonte, Brazil; 2 Ob/Gyn, University of Siena, Siena, Italy. Urocortin 1 is a peptide originally found in the brain that is also expressed in human endometrium, especially during the secretory phase of menstrual cycle, where it promotes stromal cell decidualization and putatively favors embryo implantation. We have previously shown that urocortin 1 is measurable in endometrial washing fluid and predicts outcome of intrauterine insemination. In the present study, we sought to investigate whether the endometrial washing fluid urocortin 1 concentrations and the mRNA levels of urocortin 1 and its receptors in secretory phase endometrial biopsies of women undergoing in vitro fertilization (IVF) change according to the treatment outcome (clinical pregnancy). Methods: This was a prospective cohort study of 135 consecutive women undergoing IVF due to isolated male factor or previous tubal sterilization, and was conducted at the reproductive medicine center of an academic hospital in Brazil. Endometrial washing fluid (2 ml sterile saline gently flushed into the uterine cavity and recovered by slow aspiration) and endometrial biopsy were collected at day 18-20 of the spontaneous menstrual cycle immediately before the IVF cycle. Urocortin 1 protein concentration was measured by enzyme immunoassay, whereas mRNA expression was detected by real time PCR. Results: Embryo transfer occurred in 102 cases, of whom 29 resulted in clinical pregnancy, confirmed by detection of fetal heart beat at ultrasound. The comparison between pregnant vs. non-pregnant women revealed that those who achieved pregnancy had higher endometrial urocortin 1 mRNA levels (fold change = 1.2, p<0.05), whereas the transcripts for receptors type 1 and type 2 were expressed similarly in both patient groups. Urocortin 1 was detectable in endometrial washing fluid at very low concentrations (median = 0.04 ng/ml) and did not differ significantly between the two outcome groups. ROC curve analysis did not identify optimal cut-off points of urocortin 1 concentration for pregnancy prediction in this cohort. Conclusion: Urocortin 1 and its receptors are expressed in secretory phase endometrium of women preparing for IVF, and urocortin 1 mRNA expression is slightly higher in endometrial samples of women who achieve clinical pregnancy. However, urocortin 1 measurement in endometrial fluid is unlikely to provide accurate information for pregnancy prediction in this setting. 

To investigate national practices of ART for same sex couples (SSC) seeking parenthood and report areas of needed improvement.

Questionnaire-based observational study.

An IRB approved Society for Assisted Reproductive Technologies (SART) questionnaire designed to investigate national practices of ART for SSC was distributed electronically to practice directors of all SART-registered in vitro fertilization (IVF) practices (n=392). Participation was voluntary and responses remained anonymous.

Of the 392 questionnaires distributed, 99 (25%) were completed. The majority are in private practice (71%), and perform between 0 to 5000 IVF cycles per year (mean 434.0±711.2). Nearly all respondents offer egg donation (95%), autologous egg/gestational carrier (GC) (91%) and egg donation/GC (85%), but only 23% offer traditional surrogacy. While most practices always inquire about a patient's marital status (72%), only 57% consistently evaluate a patient's sexual orientation and 17% never do. Approximately half have policies regarding treatment of female (57%) and male (48%) SSC. Very few (6%) do not offer IUI or IVF to female SSC, while 29% do not offer IVF to male SSC. Reasons cited for not offering these services include lack of inquiries, hospital policy, state laws/policy against surrogacy, religious objections and lack of expertise. Forty-three percent have protocols with specific criteria to deny access to ART treatment. Practices that perform at least 300 IVF cycles per year are more likely to have such a protocol (OR 5.2, 95% CI 2.1-13.3, p<0.01) and to offer IVF to male SSC (OR 16.7, 95% CI 2.1-130.7, p<0.01). More practices require male SSC (53%) to have psychiatric evaluations prior to treatment compared to females (40%). There were large discrepancies among practices regarding policies on consent forms and legal contracts. Forty-six percent and 33% disagree with their states' laws regarding gay marriage and gestational surrogacy, respectively.

Vastly different state laws, hospital policies, provider beliefs and expertise contribute to inconsistent availability and approaches for SSC seeking parenthood. There is a need for uniform standards of care and practice patterns among providers when caring for SSC.

What Background: Numerous professional societies advocate for weight loss among subfertile obese women prior to initiating fertility treatment and some centers use BMI cutoffs to restrict treatment, but data is lacking from overweight and obese women regarding their preferences for weight management and fertility treatment. Methods: We performed a cross-sectional survey of overweight and obese women presenting for their initial fertility consultation at a university-based fertility clinic. Standard univariate statistics were used to assess patient characteristics. Bivariate statistics were used to determine associations between patient characteristics and responses. Further multivariable regression analyses were performed to control for potential confounding. Results: 76 women participated. Women who agreed that obesity affected fertility were more likely to have been treated for infertility in the past (RR 1.4, 95% CI 1.04-1.7), and trended towards active weight loss efforts (RR 1.3, 95% CI 0.98-1.9). There was a trend among women older than 34 to state they would not defer fertility treatment for weight loss (OR 0.5, 95% CI 0.25-1.02). 4 months was the longest time period anyone over age 34 stated they would defer treatment for weight loss. Caucasian women were much more likely than non-Caucasian women to be engaged in a program that included both diet and exercise to lose weight (RR 3.2, 95% CI 1.1-9.1). Only women whose partners were willing to participate in a weight loss program with them would be willing to participate in a randomized trial of a formal weight loss intervention program (22/45 vs. 0/8). Conclusions: Overall, overweight and obese women agree that BMI impacts fertility, but many do not want to defer fertility treatment for weight loss. Among women willing to defer treatment, the period of time they are willing to defer is not conducive to meaningful weight loss. Non-Caucasian women are less likely to engage in weight loss strategies that include both diet and exercise. Whether disparities exist among non-Caucasian women to engage weight loss strategies that include exercise is unknown. Women may be more likely to adhere to stipulations of weight loss programs if their partner participates with them. Our findings may be helpful in designing successful intervention programs for weight loss among overweight and obese women seeking fertility treatment.

Single-Center Cohort. W van Dorp, 1 J Laven, 1 C den Bakker, 1 J Rietveld, 2 C Hukkelhoven, 3 I Schipper. 1 1 Div Reproductive Medicine, Dept Obstetrics&Gynecology, Netherlands; 2 Dep Obstetrics&Gynecology, Netherlands; 3 NPR, The Netherlands Perinatal Registry, Utrecht, Netherlands. INTRODUCTION: Egg donation (ED) has been used as a treatment option for infertility over the last decades, often as a final resort. Pregnancy rates range considerably, i.e. 22%-67%. Although pregnancy outcomes have been evaluated, they usually have not been adjusted for potential confounders. AIM: To evaluate both treatment and pregnancy outcomes in a Dutch singlecenter ED cohort and compare with a matched IVF cohort after adjustment for potential confounders. METHODS: A single-center study was performed including all women who underwent ED treatment between 1992 and 2009. Treatment outcomes were retrieved from medical records. Both maternal (e.g. pregnancy-induced hypertension (PIH), preeclampsia, postpartum haemorrhage) and neonatal outcomes (e.g. Apgar-score, perinatal death) were retrieved from a nationwide perinatal registry. ED and IVF were matched for maternal age, center, ZIP code and date of embryo transfer.

A total of 277 women underwent 541 ED cycles. The median acceptor age was 34.9 years (20. 6-45.2) , while the mean donor age was 34.4 years (23.8-45.5) . A total of 144 clinical pregnancies was observed, the overall clinical pregnancy rate was 26.6%. 89 women had 96 deliveries, resulting in the birth of 128 life born children. Multivariate analysis showed that donor age in years (OR 0.93, 95% CI 0.88-0.99, P=0.02) and a previous clinical pregnancy (OR 1.69, 95% CI 1.02-2.78, P=0.04) of the acceptor, were significantly associated with pregnancy rate. Regarding the outcome of singleton pregnancies, ED was associated with an increased risk of PIH as compared to IVF (OR 1.99, 95%CI 1.02-3.89, P=0.04). Regarding multiple pregnancies, ED was associated with preeclampsia (OR 6.43, 95% CI 1.67-24.7, P=0.007), PIH (OR 3.54, 95% CI 1.03-12.14, P=0.04) and postpartum haemorrhage (OR 4.91, P=0.03) . No differences in neonatal outcome parameters of ED and IVF pregnancies were observed. CONCLUSIONS: Younger donor's age and multiparity of the acceptor are associated with a higher pregnancy rate. Furthermore, ED is associated with an increased risk for PIH, independent of confounders. However, egg donation has no influence on the overall neonatal outcome. Objective: Several different pathways for preterm birth (PTB) have been proposed. Our objective was to investigate whether potential biomarkers of PTB along with transvaginal cervical length (CL) were more predictive of PTB than CL alone. Study Design: We performed a prospective cohort study of asymptomatic women with a prior spontaneous PTB or cervical surgery (8/11-8/12). Biomarker, fetal fibronectin (fFN) and CL data were obtained at both 20-24 (V1) and 24-28 (V2) wks. Samples were analyzed for soluble E cadherin (sE-CAD) and surfactant protein D (sp-D) using standard ELISAs. The association between PTB and each screening tool was determined. Areas under receiver operator curves (AUC) to predict PTB were calculated and compared. PTB was defined as delivery <37 weeks. Results: CL, fFN, and delivery information was studied (n=105). The PTB rate was 25.5%. Median CL was shorter in those with PTB at V1 (3.02 vs 3.38, p=0.002) and V2 (2.80 vs 3.32, p=0.08) and the odds of PTB decreased with each unit increase in CL (V1: OR 0.38, 95%CI 0.21-0.72; V2: 0.41, 95%CI 0.15-1.14). There was no association between change in CL (V1-V2) and PTB (p=0.80). Using 2cm as a cut-point, short CL was associated with specificities of 96% (V1) and 95% (V2) to predict PTB. Characteristics for fFN were similar to published findings (V1: sens 32%, spec 75.6%; V2: sens 28.6%, spec 86.4%). CL was superior to fFN in predicting PTB at V1 (0.72 vs 0.53, p=0.02) and V2 (0.72 vs 0.55, p=0.06). There was an association between PTB and OBHx, but not between PTB and median V1 or V2 biomarker values ( BACKGROUND Human labor is associated to intrauterine increase of different mediators including chemokines, pro-inflammatory cytokines, prostaglandins and matrix metalloproteinases. The sudden increase in many of these compounds has been linked to induction of some events of human labor such as myometrium contractions, cervix ripening and the rupture of the fetal membranes. Cellular sources of these mediators have not been clearly identified. OBJECTIVE. To evaluate in vitro production of chemokines, cytokines and matrix metalloproteinases in choriodecidual leukocytes (ChDL) obtained at term of gestation and compare these secretions with placental blood leukocytes in the same women. METHODS. A method was developed to obtain ChDL from term fetal membranes. Subsets of ChDL were characterized by flow cytometry and isolated ChDL were culture up to 72 h. Media were collected and analyzed by gelatin zymography. In addition MMP-9 and 20 cytokines/ chemokines were measured using a Multiplex assay. Placental blood leukocytes were analyzed and compared with ChDL. RESULTS. Major subsets of choriodecidual, and placental blood leukocytes were T lymphocytes and NK cells. Monocytes are the only subset significantly enriched in choriodecidua.

ChDL differentially secrete IL-2, INFγ, IL-6, TNFα, IL-1β, IL-8, IL-10, IP-10, MCP-1, MIP-1α y MIP-1β, compared to placental blood leukocytes. These cells also secreted large amounts of proMMP-9; active forms were observed after 24 h of culture. We identified MMP-3 as an endogenous activator of MMP-9. DISCUSSION. Leukocytes located in the choriodecidua at term of gestation exhibit a differential secretion pattern of cytokines, chemokines and MMPs, compared with similar cells located in placental circulation. These compounds may be involved as primary messengers or secondary mediators in the signaling pathway for induction of labor. Amount of secreted MMP-9 make leukocytes the main potential source for extracellular matrix degradation ocurring during rupture of the fetal membranes. Findings suggest that the choriodecidual microenvironment is enriched in specific subsets of leukocytes specialized in secretion of some of the mediators of human labor. Objective: Mechanisms underlying the initiation of preterm labor (PTL) are not fully elucidated. Cervical ripening plays an important role. We hypothesize that preterm cervical ripening presents different pathways compared with term. We aimed to investigate possible differences in gene expression between PTL and term labor (TL) and between PTL and preterm premature rupture of membranes (PPROM). Methods: Cervical biopsies were obtained from 28 women, PTL (N=9), PPROM (N=7) and TL (N=12). Affymetrix GeneChip Human Gene 1.0 ST Array was utilized. Data analysis was performed in Plier using the ANOVA applying fold change 1.7 and p<0.01 as cut-off. Gene ontology was investigated using IPA (Ingenuity Pathway analysis). Real time RT-PCR was performed to confirm expression of selected genes. Immunohistochemistry was used to localize selected proteins. The groups were compared using Mann-Whitney U test. Results: 107 genes were differentially expressed between PTL and TL. Pathways of inflammatory and immunological diseases were over expressed in preterm labor. 7 genes were selected for further analysis. According to the microarray analysis, aquaporin-9 (AQP9) and prokineticin 2 (PROK2) had 4-fold higher expression in PTL compared with TL. However, when analyzed by Real time RT-PCR, an under expression of cervical AQP9 (-12 fold, p=0.001) and PROK2 (-9 fold, p=0.01) was found. In the PPROM group, expression of AQP9 and PROK2 was at the same level as TL with significant difference (p=0.01) compared to PTL. AQP9 and PROK2 were localized both in epithelium and stroma with no differences between the groups. Four olfactory receptor genes and gonadotropin releasing hormone receptor (GnRHR) gene were significantly under expressed in PTL in Microarray results, but had very low expression when analyzed by Real time RT-PCR with no differences between the groups.

Conclusion: Gene expression pattern in preterm cervical ripening was mainly dominated by inflammatory and immunological pathways. AQP9 and PROK2 were identified in the human cervix and significantly under expressed in PTL. Differences in gene expression were found between PTL and PPROM, suggesting that preterm cervical ripening is a multifactorial disorder with different pathways involved for PPROM. Takashima in 2004 suggested that the amnion protects itself from digestive enzymes by the secretion of Alfa1-antitrypsin (AAT). This hypothesis suggested that an increase of AAT production in the amnion may prevent premature rupture of membranes. The aim of this study was to define the best method to isolate cells producing AAT from the amniotic membrane. Amnion derived cells were obtained from caesarian section births in healthy women. The phenotype of cells were elucidated in histologic sections of AM by antibodies directed to epithelial and mesenchymal cells: cytokeratin 7 and AE1/AE3, vimentin, desmin and AAT. We applied two different protocols: digestion of tissues with trypsin (T) or collagenase (C). Cells were characterized by immunohistochemistry using the specific panel of above described antibodies.

Mesenchymal cells (MC) on the histologic sections were positive for desmin and vimentin (specific for MC), while AECs showed a co-expression of epithelial and mesenchymal markers (cytokeratin 7 and AE1/AE3 and vimentin). Some AECs stained for AAT, whilst others were negative. All cells expressed both cytokeratin and vimentin but only cells treated with C were positive for AAT. Cells treated with T showed a tendency to form clones, whereas, cells treated with C tended to form a monolayer. This is probably due to the different grade of functional differentiation. Protocol including digestion with collagenase allows for the selection of AECs which are capable of expressing AAT. This is the starting point to characterize the role and function of these cells and opens the road for future research and clinical application of these powerful cells in human cell therapy. 

Conclusion: These findings demonstrate that fetal membranes can respond to different bacterial TLR and NLR agonists by generating specific and distinct inflammatory cytokine profiles, thus highlighting the heterogeneity of fetal membrane responses to different types of bacterial infection. However, the localisation of Calgranulin to the fetal membranes and placenta, and correlation of DNA within fetal membranes and calgranulin has not been previously studied. Methods 15 placentae from preterm deliveries (Preterm labour, Preterm SROM and maternal hypertension) and 15 from term elective caesarean deliveries were studied. Immunohistochemistry was performed for the Calgranulin A subunit.

Qualitative PCR was performed for 16S Bacterial DNA from DNA extracts of placental tissue.

Calgranulin A was present in the amnion, chorion and decidua of all membranes regardless of gestation. Placental tissue did not express calgranulin A, except in a neutrophilic infiltrate most commonly found in placentae collected after pPROM. The prevalence of bacterial DNA from preterm placentae was 56.3% compared with 0% of term controls. Densitometry was performed to compare preterm with term controls and study the association between calgranulin A and bacterial DNA. Intensity of DAB stain for Calgranulin was higher in all layers of the fetal membranes in the samples collected after preterm delivery. Intensity of DAB stain in the amnion epithelium was statistically stronger in the presence of bacterial DNA within fetal membranes.

Calgranulin expression is increased in placental tissue where bacterial DNA was detected. Calgranulin was also expressed by neutrophils in placental tissue affected by chorioamionitis. This makes it an excellent candidate for a biomarker of infection in women affected by subclinical infection. (n=31) were used. On day 15 of gestation, a FUS system was placed on the abdominal surface of anesthetized rats at the level of the internal cervix. In control rats, the FUS system was placed but no energy applied. In treated rats, 680 kHz FUS at 25 Hz, 2-4 msec pulse duration was directed to the cervix for 0.5 to 1 hour. I SPPA.3 was 40W/cm 2 , which is considered safe (FDA limit of 190W/cm 2 ) and the mechanical index of FUS was calculated to be 0.2 and thus lower than safe limits of 1.9. Measurements of cervical light-induced florescence (LIF, photon counts of collagen x-bridge fluorescence) were made before and after FUS treatment and daily until spontaneous delivery (day 22 of gestation) to estimate changes in cervical collagen. After FUS, the cervix was examined for mechanical changes and visually with an endoscopic camera. Delivery times, fetal weights and fetal viability were recorded after delivery of all rats. Results: In controls the LIF values (mean photon counts ± SD) slowly and significantly decline from day 15 (2027 ± 629) and are lowest during delivery (637 ± 133) at term (day 22). However, LIF values are significantly (P<0.05) lower in FUS groups (treated 1 hour on day15) on days 16 (700 ± 237 vs control 1319 ± 241) and 17 (503 ±231 vs control 1000 ± 178) and remain low until delivery (day 22, 637 ± 66). Also ultrasound application for as little as 0.5 hour reduces LIF levels (from 2339 ± 145 to 1119 ± 89) and so ripening is accelerated and comparable to delivery on day 22. Delivery times, fetal weights (g ± SD, FUS = 5.41 ± 0.25 vs control = 5.48 ± 0.53) and viability are not significantly different (P>0.05) in control vs FUS-treated animals. Cervical stretch resistance (g/mm ± SD) after FUS is significantly lower (P<0.05) than controls (FUS, 3.42 ± 0.25 vs control, 5.48 ± 0.53). Conclusions: 1) FUS of the cervix, with ultrasound effect indices considered safe for humans, offer a unique method to ripen the cervix without pain, damage or early birth; 2) FUS will rapidly ripen the cervix; 3) Optimal FUS parameters remain to be defined; 4) The mechanism for FUS ripening may include neural activation or fragmentation of collagen X-bridges.

Magnesium Sulfate and Dexamethasone Inhibit Matrix Metalloproteinase-9 Induced Blood Brain Barrier Degradation. Introduction: Magnesium decreases the risk of cerebral palsy in preterm infants, possibly by preventing inflammatory degredation of the fetal bloodbrain-barrier. Dexamethasone is given in preterm labor to induce fetal lung maturation. An in vitro blood-brain-barrier model was used to test whether steroids and/or magnesium inhibited blood-brain-barrier degradation by inflammation. Methods: An in vitro blood-brain-barrier model was created with a two-chamber cell culture apparatus. Human umbilical vein endothelial cells (HUVEC) were seeded in the apical well and incubated with conditioned media from astroglial cells to induce tight junctions. Monolayers were treated with dexamethasone, magnesium, and MMP-9. Membrane permeability was assessed by measuring transendothelial electrical resistance (TEER) and diffusion of fluorescentlyconjugated dextran across the apical (blood) and basal (brain) compartments.

Tight junctions were assessed with immunocytochemistry using epifluorescence microscopy with antibodies to zona occludens-1 (ZO-1).

Overnight incubation with astroglial conditioned media decreased dextran transit across the HUVEC monolayer (from permeability scores of 2.9±2.1 to 1.9±0.4; n=4 replicates). MMP-9 treatment increased dextran transit (permeability score 11.6±1.2 in vehicle, 34.4±7.9 with MMP-9, (n=4)). Magnesium attenuated increased permeability at 0.5mM, but not at 5mM. Addition of 100nM dexamethasone to 0.5mM magnesium further decreased dextran transit.

TEER measurements confirmed these results (p<0.05). One-way analysis of the variance and post-hoc multiple comparisons test with Bonferroni correction were used. ZO-1 immunoreactivity was evident at HUVEC junctions. Conclusions: Dexamethasone and magnesium preserved vascular integrity after inflammatory insult by MMP-9 in an in vitro model of blood-brainbarrier degradation. These results suggest a mechanistic role for steroids and magnesium in fetal neuroprotection. 

LPS and compared to d15 and d18 controls. Second harmonic generation (SHG) and electron microscopy (TEM) were utilized for visualization of collagen morphology and ultrastucture. Cervical compliance of term and PTB models was compared by biomechanical measurements. The hypothesis that discrete areas of collagen near MMP8-secreting neutrophils may be remodeled to allow changes in tissue compliance was tested by co-immunofluorescence staining for neutrophils and MMP8 along with SHG imaging of collagen. Quantitative assessment of SHG signal and collagen morphology will be used to decipher local areas of collagen remodeling. SHG and TEM image quantification confirm an absence of collagen disorganization in LPS-mediated ripening.

Despite the absence of disorganization, LPS treated cervices have similar compliance to both RU486 and term cervices. Dual neutrophil and MMP8 staining along with collagen SHG revealed diminished SHG signal in regions surrounding neutrophils as compared to regions devoid of neutrophils. While LPS-mediated cervical ripening does not result in appreciable changes to the collagen ultrastructure, the increased tissue compliance was similar to term ripening. Our data suggests that localized "pockets" of collagen remodeling are occurring in the vicinity of collagenase expressing neutrophils. We conclude the regionalized restructuring of collagen architecture may in part contribute to the unique pathway of LPS-mediated cervical ripening. Prostaglandins produced by the intrauterine tissues of both mother and fetus (myometrium, decidua, placenta, chorion and amnion) are involved with all the physiologies of parturition. On the other hand, progesterone and IL-10 are thought to be key factors for the maintenance of pregnancy. Reports of IL-10 production in the fetal membranes have been inconsisitent.Recently, progesterone is the most popular treatment of premature delivery. Thus, we investigated treating decidual cells with IL-1β(5ng/ml)in vitro. Microarray analysis showed that NFκ-B was a key factor in decidua induced with IL-1β in our preterm model. In the decidual primary cultured cells treated with IL-1β, the levels of PTGS-2 (COX-2), PTGFR (FP), NFκ-B and IL-17 mRNA were increased comparing with the cells treated with vehicle. In contrast, progesterone (P4)+ IL-1β inhibited the levels of all of the genes compared with treatment with IL-1β alone(p<0.05).Up-regurated genes treated with IL-1β were associated with immune response IL-17 signaling pathway and NFκ-B signaling. On the other hand, up-regurated genes treated with IL-1β+progesterone were not related to inflammation pathway. Hence, progesterone may have an important role anti-inflammation effect. For example, the expression levels of IL-8, inflammatory gene, down-regulated in IL-1β+progesterone,which is an up-regulated gene by stimulation of IL-1β alone. We suggest that progesterone makes changes in the expression of genes toward anti-inflammation.

Reactive OBJECTIVE: Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however the mechanistic role of ROS in inducing these complications is unclear. We have reported higher concentration of ROS marker F2-Isoprostanes in the amniotic fluid of pPROM subjects and even higher in those who reported cigarette smoking. Similarly, aging and ROS marker, telomere length, was reduced in the membranes and fetal leukocyte DNA suggesting 'senescence' associated with pPROM. Using cigarette smoke, a risk factor for PTB and pPROM, we examined ROS induction, ROS associated DNA damage and senescence in human fetal membranes and amnion cells to further characterize ROS pathway. METHODS: Normal term, not in labor, fetal membranes collected from Cesarean sections and amnion cells isolated from these membranes were cultured in vitro. Tissue and amnion cells were exposed to cigarette smoke extract (CSE) for 24 hrs. The kinetics of CSE-induced ROS levels in amnion cells were monitored using 2'7'-dichlorodihydro-fluorescein diacetate. Oxidative DNA damage in amnion cells was documented by assays using Fragment Length Analysis using Repair Enzymes (FLARE assay). Western blot analysis was done to examine levels of PPAR-γ and p38 MAPKs. Objective: The biomechanical integrity of the cervix is critical during pregnancy. Collagen crosslink density in cervical tissue is thought to influence its mechanical properties, and the role of crosslink remodeling during cervical ripening in pregnancy remains unclear. Our aim was to determine the relationship between collagen crosslink density and cervical mechanical properties in human tissue. To this end, we quantified cervical material stiffness and pyridinoline (PYD) density in nonpregnant (NP) and pregnant (PG) tissue. Methods: Cervical tissue was obtained from NP (n=4) and PG (n=4) total hysterectomies. 4mm axial slices were obtained at the internal os, and multiple stroma locations were indented with a 6mm diameter steel sphere in a PBS bath. Indentation was done to depth of 0.5mm, with a 20s ramp time and a 120s hold time. A viscoelastic material model was fit to the load-relaxation data, where the material stiffness parameters Go and Geq represent the instantaneous and equilibrium shear moduli, respectively. 4mm biopsies were then taken adjacent to the mechanically tested sites and evaluated for PYD and collagen using colormetric and HPLC methods. Crosslink density was calculated as PYD mole per collagen mole. Results: NP tissue was significantly more stiff than PG tissue (Student's t-test,p<0.05), where Go=7.1±5.4kPa and Geq=2.7±1.6kPa for NP tissue and Go=1.6±0.9kPa and Geq=0.8±0.4kPa for PG tissue. The crosslink density was proportionality related to tissue material stiffness, where tissue with the highest stiffness, Go and Geq, had the greatest crosslink density (statistics in Fig 1) .

Conclusion: Cervical mechanical properties were correlated to collagen crosslink density. These findings suggest that cervical tissue with a higher crosslinking density are more stiff and have an increased resistant to mechanical deformation. These data are essential to developing mechanical models that characterize normal and abnormal cervical function in pregnancy.

In Vivo Detection of Biochemical Change in the Pregnant Cervix in Humans and Mouse Models. C O'Brien, 1 E Vargis, 1 N Borwn, 2 J Reese, 2 BC Paria, 2 A Mahadevan-Jansen. 1 1 Biomedical Engineering, Vanderbilt University; 2 Pediatrics, Vanderbilt University. Objective: Preterm labor is the leading cause of infant mortality and its causes and mechanisms are poorly understood. Raman spectroscopy (RS) is a noninvasive optical technique that can longitudinally probe the biochemistry of a substance based on its inherent vibrational energy levels, and has been used by our group to study the pregnant cervix in women. To better understand Raman spectra from humans, a similar study was established using wild type (WT) mice and abnormal labor mouse models. Spectra from WT mice will be compared to mouse models to identify essential pathways necessary for labor to occur. Methods: RS was acquired using a portable, in vivo Raman system with fiber optic probe from the cervix of 20 pregnant women and four mouse models [n=20 for each model] including WT, preterm models RU-486 and Lipopolysaccharide, and delayed labor model COX-1 KO. Biomechanical tests were performed on excised mouse cervix via creep testing and compared to Raman spectra in order to correlate biochemical changes to structural properties of the cervix. Results and Discussion: WT mice had significantly higher length to tear measurements compared to COX-1 KO mice at different ages of gestation, as seen in Figure 1 . Statistically significant Raman spectral changes were observed between WT and COX-1 day 19 measurements in regions attributed to differences in amide bonds and proteins, potentially correlating with collagen cross linking and concentration, as shown in Figure 2 .

Conclusion: RS generated comparable information as the biomechanical tests, demonstrating that RS can be used in humans to non-invasively measure biomechanical properties of the cervix during pregnancy.

Strength of the Human Amnion near Term. M Puthiyachirakkal, 1 K Lemerand, 1 B Chung, 1 R Moore, 1 D Kumar, 1 B Mercer, 2 JJ Moore. 1,2 1 Pediatrics, CWRU; 2 Reproductive Biology, Metrohealth/CWRU. Forty percent of preterm birth is due to preterm premature rupture of the fetal membranes (pPROM). The mechanisms by which fetal membranes (FM) weaken and rupture at term and preterm gestation are not understood. Characterization of full thickness FM has shown that tensile strength increases until 20 wks, is level until near term (37 weeks), then falls dramatically. We have shown that there is a weak zone in full thickness FM overlying the cervix that develops independent of labor near term. Here, we characterize the amnion (strength component of FM) very near-term (36-40 wks) to determine changes in biomechanics and topographical relationships of weakening during late gestation. Methods: Using our published mapping and biomechanical testing procedures, placentas from well-timed vaginal and cesarean section (36-40 wks) deliveries were collected, cut, and biomechanically tested. The results were transposed onto tracing paper ensuring that orientation of each test site was maintained. Prior to mechanical testing, the amnion was carefully separated from the chorion (reflected amnion). The same procedures were done with amnion overlying the placental disc (placental amnion). Results: Topographic mapping of amnion strength showed a weak paracervical area with rupture strength 10-30% of the mean. As gestation advances from 36 to 40 wks, there is less change in the strength of the weakest area but there is patchy general weakening of the reflected amnion membrane. In very late gestation reflected amnion, the weakest areas (lowest 10%) are clustered in a region previously described as the paracervical weak zone. The second-weakest areas (10%-20%) are distributed near the weakest areas or in another cluster located away from the first. By 40 wks gestation, the reflected amnion shows a deceased mean strength but the placental amnion does not. There is no difference between the mean amnion strength of cesarean (unlabored) deliveries (7.02+/-2.70N) vs. vaginal (labored) deliveries 6.73+/-2.50N; p=0.35). Conclusions: As gestation progresses near term, there is initially 1 area of amnion weakness. This area develops independent of labor and is present by 37 weeks gestation. The amnion then weakens as a whole in a patchy manner; it does not appear that the weak zone weakens faster than the remainder of the tissue. Understanding the mechanisms of amnion weakening may identify a target for pharmacologic intervention allowing a reduction in pPROM and thus preterm birth.

Caspase - Preterm and term labor are both associated with cervical ripening and shortening mediated in part through increased proteolyic activity. We previously identified a non-apoptotic, proteolytic role for active caspase-3 in the pregnant uterus.

In this study we have identified a similar role for caspase-3 in the pregnant cervix. Western blot analysis revealed abundant caspase-3 in the pregnant mouse cervix from mid to late gestation. Immunohistochemical analysis isolated active caspase-3 to the stromal compartment of the pregnant mouse cervix. Dual immunoflourescence established robust caspase-3 activity segregated to the peripheral nerves infiltrating the stromal compartment of the pregnant cervix. Cervical nerve caspase-3 activity is associated with elevated anti-apoptotic signaling and a lack of TUNEL staining confirming cervical caspase-3 activation is non-apoptotic. To determine the role of caspase-3 in the human cervical stromal compartment we utilized a previously described 3D model of primary human cervical stromal cell culture. Employing the 3D culture model allowed us to identify that caspase-3 positive cells embedded in the cervical stromal compartment have the capacity to initiate extracellular matrix remodeling. Caspase-3 positive cells are associated with areas of intense collagen cleavage, a critical step in both extra cellular matrix remodeling and cervical ripening. Previous work has shown a role for activated caspase-3 in collagen breakdown in other tissues such as hyaline cartilage and vascular endothelium. Given the importance of collagen to the anatomical integrity of the cervix preterm, stromal caspases-3 may alter the mechanical strength of the cervix thereby contributing to loss of integrity at term. Yellon et al have also previously shown that cervical nerves play a role in determining gestational length by modifying the timing of cervical ripening. Therefore we speculate that progressive infiltration of the pregnant cervix by peripheral nerves during pregnancy provides a source of active caspase-3 to the stromal compartment of pregnant cervix allowing for increased collagen breakdown aiding in cervical remodeling and ripening ultimately regulating gestational length. Amnioreduction prior to the cerclage did not decrease the likelihood of the need for manipulation of membranes during the procedure or decreased PTB <24wks, <28wks or <32wks. Conclusion: Amnioreduction did not decrease the need for membrane manipulation at the time of rescue cerclage placement or reduce the risk for preterm birth less than 32 weeks. This provides additional information for management of women with dilated cervix before 24 weeks.

Thrombin-Induced Tissue Factor Expression in Human Decidual Cells Is Not Affected by Enoxaparin. Michael P Smrtka, Chad A Grotegut, Liping Feng, Amy P Murtha. Obstetrics and Gynecology, Duke University, Durham, NC, USA. OBJECTIVE: Vaginal bleeding during pregnancy is a common complication and is a source of significant pregnancy morbidity. Thrombin generation at the uteroplacental interface induces inflammation and weakens fetal membranes. Tissue factor (TF) is a powerful procoagulant and thrombin exposure dramatically increases TF expression in human term decidual cells. Thus, TF expression may play an important role in modulating thrombin-induced inflammation. The anti-inflammatory properties of heparin are thought to contribute to their ability to reduce adverse pregnancy outcomes. The purpose of this study was to assess the effect of enoxaparin on basal and thrombininduced TF expression in human term decidual cells. STUDY DESIGN: Human fetal membranes were collected from uncomplicated term pregnancies undergoing cesarean delivery before the onset of labor. Decidual cells were collected and cultured after mechanical and enzymatic purification. Third passage decidual cell cultures were conditioned in defined media (Phenol red-free DMEM/F12 with 10% stripped calf serum, 10-7 M MPA and 10-8 M ethinyl estradiol) for 1 week. Conditioned cells were then treated with enoxaparin (10, 50, or 100 mcg/ml) with or without thrombin (2.5 units/ mL) for 24 hours in serum-free defined media. Western blots were performed on cell lysates from 8 independent experiments and TF expression was measured semi-quantitatively by optical densitometry normalized to GAPDH. ANOVA was used to compare normalized TF expression among treatment groups. RESULTS: Treatment of decidual cells with thrombin (Thr) alone (2.5 U/ml) resulted in a 2-fold increase in TF expression compared to basal (control) levels (p<0.0001). Treatment with enoxaparin did not affect TF expression and the addition of enoxaparin to thrombin also did not affect TF expression. (Figure) CONCLUSION: Enoxaparin does not affect basal TF expression in human term decidual cells and does not decrease thrombin-induced TF expression. The potential mechanism for the anti-inflammatory effects of low-molecular weight heparins in pregnancy may not be mediated through tissue factor.

Distribution of Toll-Like Receptor 4 in Preterm Fetal Membranes. Jennifer L Thompson, 1 Brian C Antczak, 1 Liping Feng, 1 Chad A Grotegut, 1 Patrick C Seed, 2 Amy P Murtha. 1 1 Obstetrics and Gynecology, Duke University, Durham, NC, USA; 2 Pediatrics, Duke University, Durham, NC, USA. Objective-Toll-like receptor (TLR) 4 is responsible for recognition of gramnegative bacteria. An association between TLR4 expression in fetal membranes and preterm birth has been reported. The objective of this study was to determine if TLR4 expression varied by histological layer, proximity to rupture site and clinical phenotype in subjects delivered preterm. Methods-Fetal membrane samples were prospectively collected from the rupture site and a site distant from rupture in subjects with preterm premature rupture of membranes (PPROM; n=10), preterm labor (PTL; n=10), and preterm-no labor (PTNL; n=10). Samples were formalin fixed, paraffin embedded, and probed for TLR4 using primary antibody to TLR4 (Abcam). Slides were imaged and scored on a 4 point scale for degree of staining. Ten images were obtained per slide and scored by three independent blinded raters. Median values were obtained for each scorer for each slide. Mann-Whitney and Kruskal-Wallis tests were used for analysis (AnalyseIt; UK) Results-TLR4 expression was significantly lower at the rupture site in all cell layers. At the rupture site, TLR4 expression was significantly lower in the chorion of PPROM subjects compared to PTL and PTNL (1.3, 1.5, 1.9, P=.02). Similar trends were seen in decidua (P=.08). At sites distant from rupture, TLR4 expression was significantly higher in the amnion of PPROM subjects compared to PTL and PTNL (1.8, 1.2, 1.3, p=.007) . When subjects with chorioamnionitis were compared to those without, TLR4 expression was significantly lower at the rupture site in the chorion and decidua but not amnion (figure). Discussion-Diminished expression of TLR4 at the rupture site may represent consumption of TLR once activated by bacterial products. TLR4 expression is reduced in subjects with chorioamnionitis especially at the rupture site suggesting that infection leads to increased activation of receptors and therefore increased consumption. Additional work is required to better understand the relationship of specific TLR signaling, bacterial invasion and PPROM. The innate immune system uses toll-like receptors(TLRs) to recognise microorganisms. This study profiles TLR signalling in fetal membranes from preterm births with and without CA. Methods Fetal membrane explants were collected from 3 groups of women; term spontaneous labour without CA(TSL -CA )(n=10), PTB <34 weeks without CA(PTL -CA )(n=8), PTB <34 weeks with CA(PTL +CA )(n=13). Presence or absence of CA was determined by Redline criteria (maternal inflammatory response stage 2 and above). Membranes were separated into amnion and chorion and RNA extracted. TLR signalling arrays were used for all 3 groups to determine expression of 84 genes. Individual genes shown to be significantly up or down regulated were then selected for validation by qPCR.

In the amnion 6 genes were differentially expressed between PTL +CA and TSL -CA . 5 genes were differentially expressed between PTL +CA and PTL -CA (P<0.1; fold change >2). In the chorion 6 genes were differentially expressed between PTL +CA and TSL -CA . 10 genes were differentially expressed between PTL +CA and PTL -CA (P<0.1; fold change >2). Increased expression of TLR1, TLR2, LY96, IRAK2 and IL8 mRNA was confirmed in both amnion and chorion in PTL +CA and decreased expression of HMGB1 and SIGIRR was confirmed in the amnion in PTL +CA when compared to both groups. Conclusion Collectively, these data show a correlation between the presence of CA and up-regulation of TLR1 andTLR2, together with decreased expression of two regulators of pathological inflammation the alarmin HMGB1 and negative regulator SIGIRR.

Collagen V, Important in Assembly of Fibril Collagens, Is Regulated in the Cervix during Pregnancy. Karen Wilson, Mala Mahendroo. Obstetrics & Gynecology, University of Texas Southwestern, Dallas, TX, USA. Fibrillar collagen I and III are main structural proteins of the human and mouse cervix. During normal pregnancy, regulated changes in processing and assembly of fibrillar collagens allow for the progressive decline in tissue compliance that reaches a maximal loss at the time of birth. We previously showed, via electron microscopy, that collagen fibril morphology is altered during pregnancy. Collagen fibrils exhibit a progressive increase in diameter that correlates to the progressive decline in tensile strength. Additionally, there is a reduction in collagen cross-linking when there is a decline in proteins that regulate collagen fibrillogenisis such as, thrombospondin 2 and tenascin C. These alterations contribute to formation of collagen fibrils with reduced strength and altered collagen morphology. Collagen V is classified as a nucleator of collagen and functions as an initiating point for fibrillogenesis to occur. Loss of collagen V is embryonic lethal due to lack of collagen fibril formation, despite normal levels of fibrillar collagens. This highlights its importance in normal collagen fibrillogenesis. Deficiency in collagen V results in a decline in fibril number and alterations of fibril diameter. This current study is to evaluate gene and protein expression of collagen V in the mouse cervix during pregnancy in order to determine if collagen V may regulate fibril assembly during stages of cervical remodeling. Collagen Va1 gene and protein expression was assessed in the non-pregnant state and during pregnancy (days 6-18) and postpartum (approx. 2 & 12hrs PP) in the mouse cervix by quantitative real time PCR (qPCR) and western blotting. An antibody specific to the alpha 1 subunit of collagen V was obtained from Dr. David Birk (USF, Tampa) Compared to the NP cervix, Collagen V expression was increased from gestation days 9-16 and then gradually declined back to NP levels at term (gestation d18) and postpartum. This expression pattern was consistent at the level of both mRNA as well as protein.

Similar to fibril collagen I, collagen V gene and protein expression is increased during pregnancy. This finding suggests that increased collagen turnover is

Density in Normal and Disrupted Parturition Mouse Models. K Yoshida, 1 C Reeves, 2 J Kitajewski, 2 N Zork, 2 J Vink, 2 R Wapner, 2 M Kim, 3 D Paik, 3 K Myers. 1 1 Mechanical Engineering, Columbia University; 2 Obstetrics and Gynecology, Columbia University Medical Center; 3 Opthalmology, Columbia University Medical Center. Objective: The pregnant cervix ripens in part due to the remodeling of collagen crosslinks in the tissue. The correlation between alterations in collagen crosslinks and cervical mechanical stiffness in pregnant mice are unknown. We sought to establish this correlation by measuring collagen crosslink density and quantifying cervical mechanical properties through osmotic swelling using mouse models of normal and defective cervical ripening. Methods: Cervical tissue was harvested from 3-4 month old wild-type (WT) and Anthrax Toxin Receptor 2 (Antxr2-/-KO) female mice that were nonpregnant (NP) or pregnant (PG) on gestational day 18.5. The KO mice exhibit defective cervical ripening and a block in parturition. Cervices were equilibrated in hypertonic 2M NaCl and swelled in isotonic phosphate buffered saline (PBS), while tissue volume was continuously measured (Fig.1A) . The collagen network strength Ec, a tissue mechanical property, was determined by fitting a Donnan equilibrium relationship to the swelling response using finite element analysis. In corresponding littermates, pyridinoline (PYD) and collagen content of whole cervical tissue was measured by HPLC. Crosslink density was calculated as PYD mole per collagen mole.

Results: WTPG tissue swelled significantly more than the WTNP and KOPG, corresponding to a statistically lower Ec (Fig.1B&C ) (Student's t-test, p≤0.05). For KOs, there was no statistical difference for Ec and PYD density with gestation. WTPG tissue had a statistically lower Ec and a lower PYD density compared to NP tissue. In general, Ec increased with PYD density, but not with collagen content (data not shown). Conclusion: Through osmotic swelling, we measured the mechanical properties of the collagen network for normal and disrupted remodeling in the cervix. We found that PYD density influences mechanical function of the cervix during pregnancy, where the absence of PYD turnover results in mechanically stiff cervix in full term KO mice.

Pregnancy. Chander P Arora, 1 Brian Yadegari, 1 Payush Chatta, 1 Calvin J Hobel. Los Angeles, CA, USA; 2 Ob-Gyn, David Geffen School of Medicine, UCLA. OBJECTIVE: To identify the association between BMI and circulating levels of vitamin D during pregnancy. HYPOTHESIS: Obesity moderates the bioavailability of vit D during pregnancy. STUDY DESIGN: In a behavior in pregnancy study, 524 ethnically diverse women were followed up at T1 (18-20 weeks), T2 (28-30 weeks) and T3 (34-36weeks), maternal samples collected at each stage. Caucasian subjects (N=99) were categorized into normal weight (BMI 18-24.9), overweight group ) and obese group (BMI 30 and above) and plasma samples analyzed for circulating levels of 25(OH) D. RESULTS: Out of the Caucasian population with complete sample sets for three visits, subjects were grouped as normal weight, overweight and obese and their circulating levels of vitamin D were measured at each visit. Normal weight group (Mean BMI 21.31 ± 2.23) had the highest and sufficient levels (40 nmol/l) of vitamin D at all three visits (T1: 58.89 ± 17.34 nmol/l; T2: 32.85 ± 5.59 nmol/l; T3: 41.55 ± 7.99 nmol/l) followed by the overweight (Mean BMI 26.76 ± 1.58) group (T1: 53.85 ± 16.58 nmol/l; T2: 30.29 ± 4.35 nmol/l; T3: 47.19 ± 6.77 nmol/l) whereas the obese (Mean BMI ± 2.04) group had the minimum and insufficient levels (25-40 nmol/l) of vitamin D (T1: 46.24 ± 17.47 nmol/l; T2: 27.1 ± 4.48 nmol/l; T3: 34.88 ± 8.07 nmol/l) and significantly different from the normal weight group (p<.001). Vitamin D levels at T1, T2 and T3 followed the same pattern in all three groups, being maximum at T1 and minimum at T2 visit. Most of the obese group indicated vit D deficient levels (25 nmol/l) at T2. CONCLUSIONS: The prevalence of hypovitaminosis D is high among obese subjects. Vitamin D deficiency or even insufficiency may cause of an exagerated inflammatory response and a counter regulatory stress response towards the end of pregnancy. There appears to be a resilience response during late pregnancy (34-36 weeks) and less so in the obese group. This longitudinal relationship has not been previously recognized and may be associated with poor pregnancy outcome.

First Trimester Maternal Serum C-Reactive Protein as a Predictor of Third Trimester Impaired Glucose Tolerance. Erica Berggren, 1 Hilary Roeder, 2 Emilia Campbell, 3 Kevin Moss, 4 Stephen Offenbacher, 4 Kim Boggess, 5 Chad Grotegut. 3 1 Ob/Gyn, Thomas Jefferson University; 2 Ob/Gyn, UC San Diego; 3 Ob/Gyn, Duke University; 4 Dentistry, UNC at Chapel Hill; 5 Ob/Gyn, UNC at Chapel Hill. Objective: Impaired glucose tolerance (IGT) in pregnancy is associated with adverse perinatal outcomes. High sensitivity C-reactive protein (hsCRP) is one serum marker associated with glucose when measured at the time of standard third trimester gestational diabetes (GDM) screening. We evaluated whether a first trimester hsCRP is also predictive of third trimester GDM screening result among women not diagnosed with GDM. Methods: We performed a secondary analysis of the prospective Oral Conditions and Pregnancy cohort of non-diabetic singletons enrolled at <26 weeks gestation. Our analysis included women with hsCRP (mg/L) collected at <14 weeks and GDM screening (50g 1-hr oral glucose load) at 24-28 weeks gestation. Women with GDM screening results 135 to <200 mg/dL but without GDM were classified as IGT.

We assessed the linear relationship of log hsCRP and glucose, and evaluated test-for-trend of mean hsCRP result for each one standard deviation (sd) increase in glucose. Mean log hsCRP was compared between normal women and women with IGT. Multivariable modeling estimated the association of log hsCRP and IGT. Models adjusted for maternal BMI. Results: Among 300 women, 13% (39/300) had IGT. Mean glucose result was 107±26 mg/dL. hsCRP was positively associated with glucose result (p=0.005; r 2 =0.03). The test-for-trend was significant in unadjusted (p=0.01) but not adjusted (p=0.31) analyses. Compared with normal women, those with IGT had higher log hsCRP (0.87±0.66 vs. 0.67±0.60, p=0.04). Log hsCRP was associated with IGT (OR 1.70 95%CI 1.01,2.99) but was not significant in adjusted models (aOR 1.20 95%CI 0.65,2.21). Conclusion: Data have suggested hsCRP, a marker of inflammation, is associated with IGT and GDM. In our data, early hsCRP is not a stronger predictor of later pregnancy IGT than BMI. Early identification of women at risk for later IGT and GDM remains a priority. Other promising candidate serum markers may be found to better predict these diagnoses in future analyses. ), obese (BMI 30.0-39.9), and extremely obese (BMI ≥ 40.0). Each BMI group was further classified as gaining less-than-recommended, recommended, or more-than-recommended weight as defined by the 2009 Institute of Medicine (IOM) guidelines. The primary outcome was neonatal birth weight: small for gestational age (SGA) (<2,500 grams), normal weight (2, 999 grams) and large for gestational age (LGA) (≥4,000 grams). χ 2 and Fisher's exact tests were used with a p-value of <0.05 considered significant. Results: Among women of normal weight, less-than-recommended weight gain was associated with an increased risk in SGA but a decreased risk of LGA; more-than-recommended weight gain was associated with an increased risk of LGA. Among overweight women, less-than-recommended weight gain was associated with an increased risk of SGA; more-than-recommended weight gain decreased the risk of SGA but increased the risk of LGA. Among obese women with more-than-recommended weight gain, there was a decreased risk of SGA but an increased risk of LGA. Conclusion: Less-than-recommended weight gain among obese and extremely obese parturients did not increase rates of SGA neonates. It is reasonable to recommend less than the current 11-20 lbs suggested by the IOM. Background and Aims. Fetal growth patterns may be affected by maternal pre-pregnancy body mass index (ppBMI), gestational weight gain and air pollution exposure. Increased adiposity can influence fetal growth and contribute to adverse perinatal outcomes. The aim is to evaluate the independent contributions of maternal ppBMI and air pollution exposure to fetal growth and to assess the effect modification between air pollutants and ppBMI on fetal growth. Methods. To evaluate the contribution of ambient pollution exposure to fetal growth, we are following a cohort of low-socioeconomic status pregnant women in Mexico City. Nutritional status surveillance was provided monthly to all participants during pregnancy. Repeated ultrasound scans were performed: fetal head circumference (HC), abdominal circumference (AC), femur length (FL), and biparietal diameter (BPD). Repeated fetal biometry data were regressed against gestational age (weeks) to calculate growth rates and attained size of fetal parameters. City-wide averages of PM2.5 and ozone exposure during the first trimester were calculated for each woman living in Mexico City. Results. 274 mother-fetal pairs were included. Pre-pregnancy BMI distribution showed 4.7% initiated pregnancy underweight, 41.9% normal, 33.5% overweight and 19.7% obesity. Growth rates were not significantly impacted by maternal ppBMI. For attained growth, only femur length and abdominal circumference were lower among the underweight compared to normal weight,abdominal circumference and head circumference were lower among obese compared to normal. Each increase of an inter-quartile range of PM2.5 air pollution exposure was associated with a decrease in attained growth in femur length only among underweight. Ozone air pollution tended to be associated with attained growth among obese and underweight women. Conclusions. Our results support that air pollution may affect differentially fetal growth. The additive effect of abnormal ppBMI and high air pollution exposure can contribute to impair fetal growth. Future analysis will evaluate diet and weight gain during pregnancy. The supplemented group shows significantly higher abdominal and head circumferences at 36 weeks (75 vs 50 centile), while lower fat mass in subscapular areas (p<0.01) and mid upper leg (p<]0.05) is detected at 32 and 36 weeks. Conclusion. DHA supplementation seems to influence fetal body composition, with lower fetal fat mass but no differences in birthweight in normal weight mothers. Background: There is growing interest of whether dietary factors can affect the risk of preterm delivery. Probiotic food is associated to an overall protective effect, while sugar and artificially sweetened beverages is associated to an increased risk of preterm delivery. No one has investigated dietary patterns in women with preterm delivery and it is not clear if the dietary pattern, among these women could be responsible for the findings. Objective: To identify overall dietary patterns in a large sample of pregnant women and examine whether dietary patterns were associated with preterm delivery. Design: This is a prospective study of 60 761 pregnant women in the Norwegian Mother and Child Cohort Study. Diet was assessed by a semi-quantitative food frequency question and explorative dietary patterns were identified using principle component analysis. Preterm delivery was the primary outcome and data was obtained from the Medical Birth Registry of Norway.

Impaired Lipid Transport in Gestational Diabetes. Shannon K Flood-Nichols, 2 Monica A Lutgendorf, 2 Peter G Napolitano, 2 Danielle L Ippolito. Introduction: Gestational diabetes (GDM) complicates 5-6% of pregnancies, which places affected individuals at higher risk for pregnancy related complications, in addition to the risk for persistent metabolic and cardiovascular disorders later in life. In this study, we evaluated the correlation between apolipoproteins and plasma cholesterol levels in GDM subjects relative to age-matched control subjects over pregnancy. Study Design: Plasma was prospectively collected from 311 nulligravid women during 3 gestational age ranges (4-12, 16-22, and 26-28 weeks) . Six GDM cases were identified (mean 1-hour blood glucose concentration = 158+/-8.2mg/dL). Mass spectrometry and enzyme-linked immunosorbant assays (ELISA) were used to measure the relative and absolute changes in apolipoprotein abundance. High density lipoprotein (HDL) and low density lipoprotein (LDL/VLDL) cholesterol levels were quantified by enzymatic assay. Mann Whitney U tests were used to determine statistical significance (p<0.05).

The rate of change of both apolipoproteins and VLDL/LDL cholesterol over gestational time was significantly lower in GDM patients relative to age-matched controls. In the control subjects, plasma concentrations of ApoAII measured by ELISA increased from 4-12 weeks to 26-28 weeks, while concentrations did not significantly increase in GDM subjects (p=0.04).

Gestational Diabetes Control VLDL/LDL 111 +/-12% 134 +/-17% Apolipoprotein AII 100 +/-8% 135 +/-12% 24-28 weeks as a percent of trimester 1 Likewise, VLDL/LDL cholesterol levels increased in the controls but not in the GDM cohort (p=0.05). HDL cholesterol was not significantly different between cohorts. Mass spectrometry analysis indicated a lower relative abundance of ApoAII at 4-12 weeks and a lower abundance of ApoCIII at 16-22 weeks in the GDM cohort relative to age-matched controls (p=0.02-0.03). Conclusion: Both apolipoproteins and VLDL/LDL cholesterol showed (a) lower abundance and (b) a decreased rate of change over gestational time in the GDM cohort relative to controls. These results indicate that the normal hyperlipidemia of pregnancy is significantly lower in GDM cases than in control subjects, supporting support a role for impaired lipid transport and homeostasis in GDM. Offspring of obese mothers are at increased risk for developing insulin resistance and obesity. We have developed an animal model to study the effects of obesity on the maternal adaptations to pregnancy. We are particularly interested on the effects obesity has on the maternal metabolic and inflammatory adaptive responses. Thus the aim of the present study was to determine the effect of maternal obesity on adiponectin expression in the adult offspring. METHODS: Sixteen non-pregnant sheep were allocated to be fed at either 100% of recommended nutritional allowance or ad libitum for three months.

Sheep were mated to the same ram following induced estrus and were maintained in the same feeding regime until weaning of the lamb. Offspring were studied at 9-12 mo of age. Sheep were chronically instrumented under general anesthesia to place vascular catheters. Plasma samples were obtained prior to necropsy. At necropsy perirenal white adipose tissue (WAT) was harvested and. Perirenal WAT protein and mRNA were extracted for determining expression levels of ADP. Plasma levels of high (HMW) and low molecular (MMW) ADP multimers were measured using western blot. Data are expressed as Mean±SEM and were analyzed by ANOVA.

[figuere1] RESULTS: Ad lib fed sheep gain > 50% of the original weight prior to mating and maintained a significant weight difference during pregnancy. As expected, leptin was significantly higher in the obese group. While adipose tissue expression of HMW was significantly decreased in offspring of obese mothers (*, p<0.05), no changes were observed in plasma. In contrast, MMW ADP was increased in tissue, but decreased in plasma in offspring of obese mothers.

Our data show that maternal pre-pregnancy obesity alters ADP in offspring. This observation may explain in part the increased risk for developing insulin resistance in offspring of obese mothers. Further studies are needed to determine the impact the changes in ADP expression have on glucose handling. HL 89840 and 68728. Intrauterine growth restricted (IUGR) offspring are at increased risk of adult obesity, as a result of changes in energy balance mechanisms. We hypothesized that impairment of hypothalamic insulin signaling contributes to hyperphagia in IUGR offspring. Studies were approved by the Animal Research Committee of the Institute and were in accordance with the Laboratory Animal Care guidelines. Pregnant dams were 50% food restricted from days 10 to 21 to create IUGR newborns. At 5 weeks of age, food intake was measured following intracerebroventricular injection (icv) of vehicle or insulin (10 mU) in control and IUGR pups. At 6 weeks of age, with pups in fed or fasted (48 hours) states, pups received icv vehicle or insulin after which they were decapitated, and hypothalamic arcuate nucleus (ARC) dissected for RNA and protein expression. At birth, the pups from food-restricted dams (IUGR) had approximately 19% lower body weights compared with Control pups (6.12 ± 0.14 vs 7.57 ± 0.08 g). Body weights of IUGR rats caught up to Control rats by 6 weeks of age. IUGR rats consumed more food than controls under basal conditions (1.2-folds), consistent with upregulated ARC NPY (orexigenic peptide) mRNA expression (1.8-folds) . Insulin acutely reduced food intake in both control and IUGR rats. Consistent with anorexigenic stimulation, central insulin decreased NPY and increased POMC (anorexigenic peptide) mRNA expression and pAkt/Akt (insulin signaling) protein ratio, with significantly reduced responses in IUGR as compared to controls. IUGR offspring exhibit a persistent state of orexigenic stimulation in the ARC and relative resistance to the anorexigenic effects of icv insulin. These results suggest that impaired insulin signaling contributes to hyperphagia and obesity in IUGR offspring.

Can First Trimester PAPP-A, beta-hCG, NT and Maternal Characteristics Improve the Prediction of Gestational Diabetes Mellitus? Padmalatha Gurram, 1 Kisti Fuller, 1 Peter Benn, 2 Christine Crawford, 1 Garry Turner, 1 Winston Campbell. 1 1 Maternal Fetal Medicine, University of Connecticut, Farmington, CT, USA; 2 Genetics and Developmental Biology, University of Connecticut, Farmington, CT, USA. Objective:To investigate if combining first trimester PAPP-A (pregnancy associated plasma protein), total beta hCG, NT (nuchal translucency) and maternal characteristics can improve prediction of development of gestational diabetes mellitus (GDM) compared to maternal factors alone. Methods:We performed retrospective analysis of all women who received first trimester screening for fetal aneuploidy (11 to 13+ 6 wks) and delivered at our facility between 1/2005-12/2011. All patients were screened at 24-28 wks for GDM with 50 g oral glucose challenge. Positives (>140mg/dl), were confirmed with 3 hour glucose tolerance test. Known aneuploidy, multifetal gestations and pregestational diabetes were excluded. Maternal factors (age, weight, race, parity), gestational age (GA) at the time of screening, crown-rump length, NT, PAPP-A and hCG were compared between the patients who developed GDM vs normal controls (NGDM). PAPP-A, hCG and NT expressed as multiples of the median (MoM corrected for maternal weight, race, GA) were compared using Mann-Whitney test. Logistic regression models were developed for significant variables (p<0.05). The predictive ability was assessed by ROC curves. Different models were compared by areas under ROC curves (AUROC). Results: Of 1327 eligible patients, 64 (5.07%) developed GDM. Maternal age, weight and race were significantly different between GDM and NGDM (Table) . There was 9% increase in hCG in GDM patients. PAPP-A and NT did not differ between groups. The regression model using only maternal factors age, weight, race yielded an AUROC of 0.768 (95% CI 0.704-0.831, p<0.05), a sensitivity (Sn) of 56.9% for 80% specificity (Sp). The combined model (hCG and maternal factors) had an AUROC of 0.772 (95% CI 0.710-0.834, p<0.05), a Sn of 56.6% for 80% Sp. Conclusion: Combining maternal factors and first trimester screening markers did not improve clinical prediction of gestational diabetes compared to only maternal factors. Objective: The one step 75g 2 hour oral glucose tolerance test (GTT) is used for screening /diagnosis of gestational diabetes (GDM). It is inclusive of ≥1/ 3 abnormal glucose values for diagnosis. We hypothesize that patients with 1 abnormal glucose value have less significant disease than those with ≥2 abnormal values. Methods: In 2011, women were screened for GDM using a 2 hour 75g GTT. In a retrospective chart review, patients with 1 abnormal value on the test were compared to those with ≥2 abnormal values. Patients were divided into 4 groups: Group 1, isolated abnormal fasting blood glucose (BG); Group 2, isolated abnormal 1 hour BG; Group 3, isolated abnormal 2 hour BG; and Group 4, ≥2 abnormal BGs. Body mass index (BMI) was defined as kg/m 2 . Macrosomia was defined as >4000g. Results: 270 women had GDM. 177 (66%) patients were diagnosed with 1 abnormal value; Group 1 had 19 (7%) patients, Group 2 had 99 (37%), and Group 3 had 59 (22%). Group 4 had 93 (34%) patients. Group 1 had a significantly higher BMI and had more Black patients than the other groups; no other pairwise comparisons were significant. 89% of the people in Group 1 were on hypoglycemic agents, compared to 61% in Group 4; where as Groups 2 and 3 similarly had only 38% patients on agents (P value <0.0001). There was no difference among the groups in maternal age and macrosomia (see chart below). Background: LMWH prophylaxis has been recommended for morbidly obese pregnant women (>40 kg/m 2 ). However, no data exists on the anticoagulant effects of LMWH in this group. Aim: We investigated different dosing regimens; fixed dose versus weightadjusted dose on the anticoagulant effects of the LMWH, tinzaparin used for thromboprophylaxis in obese pregnant women. Method: Twenty morbidly obese pregnant women were started on a fixed dose of tinzaparin (4,500iu/day) at 30 weeks gestation and then changed to a weight-adjusted dose (75iu/kg/day) for the remainder of their pregnancy. Four hour post-dose venous blood were taken after each initial dose and repeated every 2 weeks until delivery. Endogenous thrombin potential (ETP), tissue factor pathway inhibitor (TFPI) and anti-Xa were measured and compared with levels in twenty normal weight women at the same gestation. Results: Prior to LMWH prophylaxis, TFPI levels in the obese group at 30 weeks were significantly lower (p<0.001) and ETP and peak thrombin levels in obese group were significantly higher compared with controls (P<0.0001; P<0.001).

Within the obese group, there was no significant difference between ETP levels before and after fixed LMWH dose. However, ETP levels were significantly lower post weight-adjusted dose (75iu/kg tinzaparin) compared with post fixed dose. There was a significant effect of LMWH on TFPI levels, (p<0.0001). Peak anti-Xa levels correlated significantly with total body weight at 75iu/kg tinzaparin (r=0.777) (p<0.01) but not at fixed dose. ETP correlated positively with total body weight at fixed dose (r=0.578)(p<0.05). At weight-adjusted dose, ETP levels demonstrated a weak negative correlation (r= -0.430) but did not reach significance (p=0.059). Conclusion: Morbidly obese pregnant women have increased thrombin generation and reduced natural anticoagulant in third trimester. ETP is sensitive to the anticoagulant effects of LMWH at different dosages and is a potential tool for monitoring LMWH in the morbidly obese. The prothrombotic state in pregnant morbidly obese women was substantially attenuated by weightadjusted LMWH doses. Objective: To evaluate the association between leptin levels and body mass index (BMI) in preterm (PTD) and term deliveries (TD). Methods: 78 women at risk for PTD defined by preterm labor in the current pregnancy or a prior PTD were evaluated in a prospective case-control study. Leptin samples were collected between 23-34 weeks, measured with ELISA assays, and compared between PTD and TD, stratified by pre-pregnancy BMI (kg/m 2 ). Pearson correlations were calculated for the relationship between Poster Session: Nutrition, Obesity, Diabetes I (Friday, 3/22/2013, 9:00 AM -11:00 AM) Scientific Abstracts Reproductive Sciences Vol. 20, No. 3 (Supplement) , March 2013 247A Friday leptin and BMI, for both PTD and TD. Linear and multiple logistic regression was used report the association between PTD (<37weeks) and leptin levels, BMI, and other covariates (race, parity, tobacco use).

Results: There were no differences in maternal characteristics and treatment (age, race, BMI, parity, prior PTD, diabetes, asthma, tobacco use, cerclage, 17-hydroxyprogesterone use, corticosteroids for fetal lung maturity; p>0.05) except for chronic hypertension (18.8% vs. 2.2%, p=0.02) between PTD and TD. There were no differences in leptin levels (ng/ml) between PTD and TD (56±38 vs. 39±32 for BMI<30, p=0.14; 66±38 vs. 55±24 for BMI≥30, p=0.31). The correlation between leptin levels and BMI was low (r=0.18, p=0.34) for PTD, but higher for TD (r=0.33, p=0.03).

For every 1kg/m 2 rise in BMI, leptin increased by 0.03ng/ml (p=0.34) in PTD and by 0.28ng/ml (p=0.03) in TD. Leptin levels >60ng/ml (OR 2.2, 95%CI 0.77-6.4) and BMI≥30 (OR 1.2, 95%CI 0.45-3.4) were not significant predictors of PTD, max-rescaled R 2 =0.09. Conclusion: There is a higher correlation between leptin levels at 23-34 weeks and BMI in TD, but a greater sample size is needed to investigate the role of leptin and BMI in PTD.

Waist Objective: The waist to hip ratio (WHR), as a measure of central adiposity, is a better predictor of obesity related outcomes than BMI in nonpregnant populations. Our objective was to determine the relation between these measures of obesity and LGA and cesarean delivery (CD). Methods: This is a secondary analysis of data from the Combined Antioxidant and Preeclampsia Prediction Study (CAPPS), a multicenter RCT of vitamin C and E to prevent preelampsia. Women who were enrolled at 9-16 weeks gestational age with data available for WHR and BMI were included. Those with a WHR of ≥0.85 and 0.80-0.84, standard ratios identified as high and moderate risk for adverse outcomes, were compared to those with a WHR <0.80. Women with early pregnancy BMI ≥ 30 kg/m 2 (obese) and 25-29 kg/m 2 (overweight) were compared to those <25 kg/m 2 .

LGA was defined as >90% by Alexander nomogram. Univariable analysis, logistic regression (adjusting for maternal age, gestational age at enrollment, years of schooling, race, alcohol, and smoking status), and ROC curves were used. Results: 2,276 women were analyzed. After correcting for potential confounders, only BMI ≥ 30 was significantly associated with LGA (aOR 2.07, 1.35-3.15) while BMI 25-29 (aOR 1.5, 0.98-2.28), WHR 0.8-0.84 (aOR 1.33, 0.83-2.13) and WHR≥0.85, (aOR 1.05, 0.67-1.64) were not. Area under the ROC curve (AUC) for LGA were higher for BMI compared to WHR (0.62 vs 0.51; p<.001). Risk for CD was increased for women with elevated WHR and with higher BMI compared to normal (Table 1) . AUCs for CD were similar for WHR and BMI (0.58 vs 0.61; p=.07). The ROC curves remained similar when only CD performed for cepahopelvic disproportion was evaluated. Conclusion: Although a useful measure of central obesity, WHR is not associated with LGA. While BMI performed better than WHR, neither was a strong predictor of LGA or need for CD in low risk nulliparous women. Objective: To investigate histologic architecture of subcutaneous (SQAT) and visceral adipose tissue (VAT) growth in relationship to gestational weight gain (GWG). Epidemiological data suggest that SQAT expansion may be protective of obesity related co-morbidities, whereas VAT expansion is associated with Type-2 diabetes risk. We hypothesized that in normal gravidas, GWG would be associated with hypertrophy of SQAT and not VAT.

Observational Dietary Study (PPODS) and undergoing Cesarean delivery had SQAT (midline superior edge Pfannenstiel incision) and VAT (inferior omental periphery) biopsies after neonatal delivery, uterine closure and hemostasis achievement. Excised tissues were fixed and stained. Average adipocyte size and capillary density were assessed in 10 independent sections per AT depot per subject. GWG determined by the difference of weight at first visit and immediately postpartum (1-4 days post-op). GWG plotted vs mean SQAT or VAT adipocyte size. Results: Table illustrates general clinical characteristics of the 5 subjects. Figure A demonstrates SQAT and VAT mean adipocyte size with representative sections from patient E depicted above bar graphs representing means and SEM from 5 patients. SQ adipocytes were significantly larger than those from VAT. Significant positive correlation was noted between GWG and SQAT adipocyte size ( Figure B ), but not VAT adipocyte size ( Figure C ). Discussion: Preliminary results reveal that in normal pregnancies, GWG is associated with changes in SQAT but not VAT architecture, which reflects lipid accumulation. These results are consistent with the model that SQAT is specifically adapted for healthy lipid storage, and provides a basis for comparison between normal gravidas and those with GDM. Turretfield Research Centre, SARDI, Australia. Exposure to maternal obesity (MO) is associated with increased risk of obesity & insulin resistance in her offspring. Dietary restriction (DR) regimes have, therefore been proposed for obese women seeking to become pregnant to limit these effects. We have previously shown that MO during the periconceptional (PC) period results in increased body fat mass in female lambs, which is ablated by maternal DR. The impact of MO & of DR in the PC period on insulin signalling in different fat depots in the offspring is unknown. We hypothesised that MO in the PC period would result in increased abundance of insulin signalling molecules in fat of the offspring & DR in obese ewes would abolish these effects. Donor ewes were allocated to 1 of 4 groups & were fed the following diets in the PC period: 100% metabolisable energy requirements (MER) for ≥ 20wks (CC); 100% MER for ≥ 16wks & then 70% MER for 4wks (CR); ∼180% MER for ≥ 20wks (HH); ∼180% MER for ≥ 16wks & then 70% MER for 4wks (HR). This continued for 1wk postconception before single embryos were transferred into recipient ewes of normal weight. At 16wks after birth, omental (OF), perirenal (PF) & subcutaneous (SF) fat samples was collected for determination of insulin signalling molecules abundance by Western blotting. There was decreased insulin receptor, p85α, Akt2, PKCζ & GLUT4 abundance in OF of HH lambs. Maternal DR did not ablate these effects. The abundance of p85α, PKCζ & GLUT4 was also decreased in OF of CR lambs. There was decreased phospho-Akt abundance in PF of HH lambs; an effect that was abolished in HR lambs. There was no effect of MO on the abundance of insulin signalling molecules in SF of lambs. There was, however, increased Akt2 & GLUT4 abundance in SF of CR & HR lambs.

In conclusion, MO in the PC period appears to preferentially program decreased insulin signalling in metabolically active visceral fat but not SF of the offspring, which may contribute to the emergence of insulin resistance in later life.

Maternal DR was unable to abolish these effects in visceral fat. Furthermore, DR in both normal weight & obese ewes also resulted in increased abundance of key insulin signalling molecules in SF of lambs. In addition, a good glycemic control is difficult to achieve with diet therapy alone. The autoimmune mechanism, underlying thyroid dysfunction, could be an important factor involved in the development of GDM, as previously demonstrated for type 1 DM. Thus, the screening for thyroid dysfunction should be performed also in women with a previous GDM or other autoimmune pathologies.

Does Objective: To determine if cord blood concentrations of glyburide (GLY) are associated with neonatal blood glucose levels. Study Design: Women with GDM, who were treated with GLY, and signed informed consent were included in this prospective, observational study. Upon admission for delivery, the GLY dose and time of last dose were recorded.

Maternal fingerstick blood glucose (BG) measurements were obtained every 2-4 hrs until active labor and then every hour until delivery in laboring patients, and once upon admission in patients undergoing elective C-section. Immediately post-delivery, a maternal and an umbilical venous serum sample were obtained, centrifuged, and the plasma was frozen for future evaluation. Neonatal BG was measured via heal-stick as per pediatric protocol. GLY concentrations in maternal and cord blood samples were determined using high performance liquid chromatography-mass spectrometry, as previously described and validated. The limit of detection was 0.5 ng/mL. Results: Twenty-two mother-newborn sets were analyzed. The mean maternal GLY dose was 7.05 ± 6.61 mg/day. Maternal GLY concentrations ranged from 0.93-70.7 ng/mL, with a mean and median of 13.6 ± 19.8 ng/mL and 5.26 ng/ mL, respectively. Umbilical GLY concentrations ranged from undetectable-32.4 ng/mL, with a mean and median of 6.6 ± 7.9 ng/mL and 3.54 ng/mL, respectively. Maternal BG concentrations averaged 86.7 ± 12.2 mg/dL and the mean final BG value was 84.8 ± 7.9 mg/dL. Mean EGA at delivery was 38.5 ± 1.5 wks and mean birth weight was 3237 ± 325g. Mean neonatal BG was 65.1 ± 15.9 mg/dL, obtained at a mean of 28 ±18 min of life. Figure 1 depicts the relationship between umbilical GLY concentrations and neonatal glucose values. An increase of 1 ng/ml in umbilical GLY resulted in a decrease of 1.21 mg/dl in neonatal glucose (p <0.01) Conclusion: Neonatal blood glucose concentrations appear to be inversely correlated with umbilical vein glyburide concentrations at birth. Materials and Methods: Thirty one GDM patients who failed to achieve desired level of glycemic control during pregnancy were evaluated during their induction of labor until 24 hours post-partum using a continuous glucose monitoring system. All subjects were given Lactate Ringer intravenous fluid during labor. The continuous glucose monitoring system measured in subcutaneous tissue interstitial glucose levels within a range of 40-400 mg/ dL every 5 minutes for a total of 288 measurements per day. The CGM data were downloaded into our computerized database and statistical analyses were performed using SPSS 15.0 software. Quantitative variables were compared by ANOVA. The level of significance was set at 5% in all analyses. Results: 70% of the patients were treated with glyburide, 30% were managed by diet alone. Average BMI was 30kg/m2. Asymptomatic hypoglycemic events were 2-fold higher in the diet-controlled women compared to the glyburide treated subjects (100% vs. 57% respectively, p<0.05). Hyperglycemic events were found in 66% of the diet and 86% of the glyburide treated patients. Further analysis of the different stages of labor resulted in a mean recorded blood glucose of 90 mg/dL -latent phase, 79.5 mg/dL -active phase, 88.6 mg/dLsecond stage and 130.5 mg/dL within the 24 hour post-partum period (p=0.008). Conclusions: Our data suggest that pharmacological therapy is not indicated in GDM patients treated with diet or glyburide during IOL in order to obtain desired levels of glycemic control and stability. However during the postpartum period, more assertive glycemic control is needed to maintain desired levels of blood glucose.

Fetal HDL-Associated ApoM-S1P Complex Mediates Vasoprotective Action on the Feto-Placental Endothelium. In Gestational Diabetes Mellitus (GDM) Endothelial Barrier Integrity Is Impaired. 

Fetal high density lipoprotein (fHDL) has a unique composition when compared to maternal HDL, GDM causes quantitative and qualitative alterations of maternal and fetal HDL proteome (Sreckovic, 2012) . In adult, HDL-associated sphingosine-1-phosphate (S1P) is bound mainly to apolipoprotein M (apoM) and maintains vascular integrity. The aim of this study was to identify whether fHDL carries S1P and its regulatory effect on feto-placental endothelium in GDM.

HDL was isolated by ultracentrifugation from control and GDM maternal/fetal donors (n=11). ApoM, S1P were quantified by ELISA. S1P receptor (S1PR1) expression was determined by qRT-PCR and immunoblotting. Transendothelial electrical resistance of human placental endothelial cells (HPEC) was measured by an impedance sensor (ECIS). Cells were treated with S1P, HDL, S1PR1-, Rho-and Rac-inhibitors. Rearrangement of cytoskeleton by S1P on HPEC was visualized by phalloidin and vinculin staining.

ApoM-S1P levels are equal in healthy, maternal and fHDL. In GDM, circulating S1P bound to fHDL is decreased to 20% (p<0.01), due to the 40% lower association of apoM to fHDL (p<0.01). S1PR1 is highly expressed on HPEC. Healthy, fHDL increases barrier integrity in HPEC, while GDM fHDL attenuated permeability to 50-60% (p<0.01). fHDL and S1P protective mechanism in HPEC is induced by S1PR1/Rac pathway. S1P cytoskeletal rearrangement was confirmed by immunofluorescence staining. Conclusion:fHDL exhibits S1P and mediates protective actions on the fetoplacental endothelium. S1P metabolism is affected by GDM and corroborated with endothelial barrier dysfunction resulting in marked increases in vascular permeability that is one of the central features of inflammation. Objective: Insulin Detemir (ID) is a long-acting insulin analogue widely used in the treatment of diabetes, but there are almost no published data on its use in pregnant women. We previously observed that women treated with ID had a higher rate of large infants (>90th% BW) as compared to pregnant women treated with insulin NPH (16.5% vs. 2.5%). We sought to determine the risk factors associated with large infants in women treated with ID. ]Methods: This is a retrospective observational study of women with GDM or type 2 diabetes who were treated in our Diabetes in Pregnancy Program and required insulin therapy. Women using ID for at least 6 weeks were included in the study. ID was administered twice daily together with rapid acting insulin aspart, given before meals. Patients were instructed to monitor fingerstick glucose levels 4-7 times a day. Fasting glucose target levels were 60-90mg/ dL and <120mg/dL 2-hours post-prandial. Variables associated with glucose control, insulin treatment, and maternal characteristics were compared between women who delivered large infants (LGA: BW>90th%) and appropriate weight infants (AGA: BW ≤ 90th%). Chi-square, Fisher's exact, and Student's t-test were used, as appropriate. ]Results: 37 women were treated with ID (21 with type 2 DM, and 16 with GDM) and 6 of these delivered LGA infants. Patient characteristics are presented in the table.

LGA Conclusion: Similar to other pregnant women with diabetes, LGA in pregnant women treated with ID appears to be associated with glucose control and weight gain during pregnancy, and not with ID per se. (<11lb), 321 (17%) gained the recommended amount of weight (11-20lb), and 1134 (61%) gained more than the recommended amount (>20lbs). The rate of hypertensive disorders rose as the amount of weight gained during pregnancy increased, but only those gaining >20lbs had statistically significant difference due to higher rate of pre-eclampsia. Neonates born to obese women with less than recommended amount of weight were born earlier and had lower, but appropriate birth weight for gestational age. Conclusion: Obese women who gain less than the recommended amount of weight have similar rate of hypertensive disorders in pregnancy, but deliver earlier. Obese women gaining more than the recommended amount of weight are at higher risk of pre-eclampsia. Introduction The effect of physical activity (PA) in pregnancy on maternal glucose metabolism seems to be dependent on the timing. PA before and in early pregnancy has been reported to reduce risk of gestational diabetes, whereas PA in the second half of pregnancy was not found to have an effect in normalweight women. It might be that, in later pregnancy, maternal insulin sensitivity is regulated to achieve optimal fetal growth and is not sensitive to increases in PA. In this study, the effects of objectively measured PA in both early and late pregnancy on maternal glucose and insulin were assessed in overweight and obese women at risk for gestational diabetes. Methods A total of 55 women were prospectively followed throughout pregnancy. At 15, 24 and 32 weeks of gestation, PA and glucose, insulin, HbA1c were assessed in the fasting state. At 24 and 32 weeks, an 100 g oral glucose tolerance test was performed in addition, with samples taken at 30, 60, 90, 120 and 180 mins. The number of minutes in moderate-to-vigorous PA per week were calculated based on data derived from the ActiTrainer accelerometer (ActiGraph™) and from a questionnaire. Effects of PA on fasting glucose, insulin, HbA1c, insulin sensitivity, first-phase and second-phase insulin response and beta cell function were assessed. Results Objectively measured PA at 15 weeks was related to a reduction in first and second phase insulin response at 32 weeks. PA at 32 weeks of gestation was related to reduced fasting insulin, increased insulin sensitivity, and reduced first-and second-phase phase insulin response. No relations between PA and glucose levels or HbA1c were observed at any time point. PA measured with questionnaires was not related to metabolic outcomes. Discussion In contrast to earlier studies in pregnancy, we found that in overweight and obese women, insulin sensitivity can apparently be improved with PA. Therefore, for this group of pregnant women, PA throughout pregnancy will have a positive effect on maternal metabolism, and could reduce the risk of infants born large-for-gestation.

Peroxynitrite Affects the Metaphase-II Mouse Oocyte Spindle Structure In Vitro in Presence and Absence of Cumulus Cells. Jashoman Banerjee, Faten Shaeib, Dhiman Maitra, Michael P Diamond, Husam M Abu-Soud. Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA. Objective: To demonstrate the effects of peroxynitrite (ONOO¯) on metaphase-II mouse oocyte spindle structure and chromosomal alignment with and without the presence of cumulus cells in vitro. Materials and Methods: Metaphase-II mouse oocytes, both with and without cumulus cells, were incubated in human tubular fluid (HTF) media for 60 minutes (5% CO2 at 37° C). Each group was treated with (25, 50 µM/mL) of peroxynitrite (ONOO¯) for 30 minutes. Unexposed oocytes or oocytes treated with nitrite/nitrate, the final product of ONOO¯, served as controls. Oocytes treated with ONOO¯, with or without cumulus cells, also underwent viability staining using the Trypan blue dye exclusion method and were compared with unexposed controls. The oocytes were fixed using the microtubule stabilizing (MTSB-XF) protocol and subjected to indirect immunofluorescence for detection of changes in the spindle and chromosomal alignment. The alterations were then scored by two independent observers utilizing both immunofluorescent and confocal microscopy based on previously published scoring system. Results: Our results clearly showed that cumulus cells failed to provide protection against peroxynitrite induced deterioration of metaphase-II mouse oocyte spindle and chromosomal structure in vitro. Most oocytes had poor scores for the spindle and chromosomal alignment on exposure to 50 µM/mL of ONOO¯. Trypan blue staining revealed that most of the cumulus cells failed to survive treatment with ONOO¯. Conclusions: Collectively, ROS not only affects the total antioxidant machinery in cumulus cells, but it also affects their viability which compromises oocyte quality and may lead to female infertility.

Hydroxyl Radical and Mouse Oocyte Quality. Jashoman Banerjee, Faten Shaeib, Dhiman Maitra, Michael P Diamond, Husam M Abu-Soud. Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.

Here we demonstrate that hydroxyl radical (•OH) generated through the Fenton reaction alters metaphase-II mouse oocyte microtubule (MT) and chromosomal alignment (CH). Metaphase-II mouse oocytes, obtained commercially, were grouped as: control, hydrogen peroxide (H2O2) (5, 10, and 20 µM), Fe (II) (100 µM), and Fe (II) (100 µM) pretreated with 10 µM H2O2. After 7-10 minutes of incubation, at 37 °C, MT and CH were evaluated on fixed and stained oocytes and scored by two blinded observers. Pearson Chi-square test, Fisher's Exact test were used to compare outcomes between controls and treated groups, and also amongst each group. Our results showed that poor scores for MT and CH increased significantly in oocytes treated with combination of H2O2 and Fe(II) (p <0.001); oocytes treated with H2O2 alone or Fe(II) alone showed no or little changes compared to control. Comparison of oocyte groups that received increasing concentrations of H2O2 and fixed amount of Fe (II) showed that 70 -80 % demonstrated poor scores both in MT and CH when pretreated with 5 µM H2O2, and increased up to 100% when treated with 10-20 µM of H2O2. Hydroxyl radical alters the metaphase-II mouse oocyte spindle and CH alignment, which is thought to be a potential cause of poor oocyte quality. Thus, free iron and/or ROS scavengers could attenuate the •OH-mediated spindle and chromosomal damage, thereby serving as a possible approach for further examination as a therapeutic option in inflammatory states. INTRODUCTION Increasing age correlates with a low oocyte recovery rate and low egg quality. However, poor ovarian response in young patient is not rare. We endeavored to examine human granulose cell gene expression in elderly women vs. young poor-response patients.

Mural and cumulus granulosa cells (mGCs and CCs, respectively) were obtained at the time of oocyte retrieval for IVF procedures. Poor ovarian response (POR) was defined according to the Bologna criteria as a low oocyte retrieval rate (<4) in response to stimulation with FSH (150 IU). Young POR were defined as patients younger than 38 with POR. The elderly group was defined as patients who were older than 40 with POR. The control group was defined as patients younger than 38 with a normal oocyte retrieval rate (>5). Each group consisted of 9-12 women assembled into 3-4 pools of 3 patients each. Gene expression was analyzed using quantitative real time PCR.

In mGCs, expression of AMH was significantly lower in the elderly group compared to the control group (expression ratio 0.35, P=0.01) while expression in young poor-response was not significantly different than control. FSH receptor and amphiregulin appeared to be expressed equally in the three groups. LH receptor, epiregulin and aromatase expression seemed higher in both the young and elderly POR compared to control albeit the difference in expression did not reach statistical significance. In CCs, STAR expression was significantly higher in the elderly group (expression ratio 2.75, P=0.04) while the expression in young POR was similar to control. GPX3 expression in CCs was significantly lower in elderly POR (expression ratio 0.35, P=0.04) while expression in young POR appeared to be more moderately reduced compared to control without reaching statistical significance (expression ratio 0.65, P=0.18). We did not find significant differences of expression in other genes examined in CCs (LH receptor, EGFR, GREMLIN, HAS2, ADAMTS1, UGP2 and PTGS2). CONCLUSIONS Gene expression pattern in human granulose cells of young poor-response patient is different than that of elderly patients with similar ovarian response and more closely resembles that of young patient with a normal ovarian response. negatively impacts oocyte quality in mice. DHEA is a known uncompetitive inhibitor of the pentose phosphate pathway (PPP). NADPH, a product of the PPP, is necessary for many metabolic pathways in the oocyte. The purpose of this study is to determine whether the effects of DHEA on the PPP contribute to the poor oocyte quality and are reversible by adding an NADPH precursor. Design: Experimental animal study Methods: Three-week-old female mice were fed rodent chow or chow supplemented with 0.01% or 0.1% DHEA for 2 weeks. Another cohort of mice was placed on a 0.1% DHEA plus 0.01% nicotinic acid (NA) diet. MRIs were performed to assess changes in body composition. Oocytes were collected after superovulation and (1) citrate levels were measured or (2) the oocytes were stained for intracellular lipid content with BODIPY. Images were captured by confocal microscopy & analyzed with Image J software. Results: Mice fed the 0.01% and 0.1% DHEA diets are slightly leaner than controls, and mice supplemented with DHEA plus NA have increased body fat compared to the DHEA-fed mice, suggesting a reversal of the metabolic effect. Similarly, oocytes from mice exposed to DHEA have a decrease in lipid content and citrate levels compared to controls. NA, and NADPH precursor, reverses this effect and increases both lipid and citrate levels in oocytes. Conclusion: Inhibition of the PPP with DHEA leads to a decrease in body fat and a decrease in lipid storage within the oocyte. Providing products of the PPP in the form of NA reverses this defect, allowing improved lipid metabolism in the oocyte. Citrate, a second source of NADPH, may partially compensate for inhibition of the PPP. Future studies will evaluate the competence of oocytes exposed to DHEA and NA. We will also use other inhibitors of the PPP to recapitulate our findings. These studies may lead to therapeutic targets of the PPP to improve oocyte quality in women with elevated DHEA. Objective: microRNAs (miR) are small noncoding RNAs that regulate gene expression at post-transcriptional level. A special feature of the microRNAs is their stability against ribonuclease enzymatic activity which make them stable in body fluids. Our preliminary work showed differential expression of microRNAs in the Granulosa Lutein Cells (GLC) of anovulatory and ovulatory women. We aim to show that this differential microRNAs expression is being expressed also in the follicular fluids and the serum of the women. This miRNA expression profile in the serum can be used as a biomarker for the ovulation process. Materials & Methods: Follicular fluids (FF) and serum were obtained from anovulatory women (Poly Cystic Ovary Syndrome) and normally ovulating women undergoing oocyte retrieval during the IVF cycle. microRNA was extracted using the miRVANA paris kit. The FF and the serum of all women from the each group were pooled. miRNAs were reverse transcribed using stem loop primers specific to the known human miRNAs. As serum and follicular fluids contains minute amounts of miRs the sample was pre-amplified using 12 cycles before qRT-PCR was performed using the TaqMan® Array Human microRNA Card Set v3.0. Specific card results were validated using primers for the specific miRs by single qRT-PCR. Results: Follicular fluids and serum were collected from a total of 8 patients with PCOS and 8 normal ovulating patients at the time of ovum pick up during an IVF cycle. Human microRNA array cards identified 27miRs that were up regulated and 19 miRs that were down regulated in the FF of the PCOS group (more than 2 fold change, p<0.05), all with a Ct value of less than 35. Interestingly, serum card analysis identified 266 down regulated miRs and only 4 up regulated miRs in the PCOS group. Using microRNA card analysis for the GLC from the same groups we identified a group of 6 different miRs, which show the same expression pattern in all 3 biological materials. Conclusion: microRNAs play a role in the human ovulation process and the formation and function of the corpus luteum. The miRNA expression profile in the serum might be a reflection of their ovarian expression. These can be used as a novel serum-based biomarker, potentially offering insight for the diagnosis and follow up of the ovulation process. Objective: microRNAs (miR) are small noncoding RNAs that regulate gene expression at post-transcriptional level. microRNAs play important roles in essential processes, such as differentiation, cell growth, and cell death. The ovulating follicle, besides from producing a mature oocyte, forms a functional corpus luteum that plays a critical role in the secretion of gonadal hormones and the maintenance of the early pregnancy. We aim to identify and characterize differential microRNAs expression in the human periovulatory granulosa lutein cells (GLC) of anovulatory women and normal ovulating women. Material and Methods: Follicular aspirates containing GLCs were obtained from anovulatory women (Poly Cystic Ovary Syndrome (PCOS)) and normally ovulating patients (Male factor infertility) undergoing oocyte retrieval during the IVF cycle. microRNA was extracted using standard protocol. The GLC of all women from the same group were pooled. miRNAs were reverse transcribed using stem loop primers specific to the known human miRNAs. Quantitative real-time PCR was performed using the TaqMan Array Human MicroRNA Card Set v3.0. specific card results were validated using primers for the specific miRs by single qRT-PCR.

Results: GLC was collected from a total of 8 PCOS patients and 8 normal ovulating patients at the time of ovum pick up during an IVF cycle. The cells from each group were pooled to one sample. Human microRNA cards identified 101miRs that were up regulated and 47 miRs that were down regulated in the PCOS group (more than 2 fold change, p<0.05), all with a Ct value of less than 35. Cluster analysis revealed their position in multiple large clusters across the genome. Pathway analysis suggest the involvement of the differentially expressed miRs in ovarian steroid synthesis and function. Six differentially expressed miRs were selected for further analysis in order to unravel their role in the ovulation process. Conclusion: microRNAs play a role in the formation and function of the human corpus luteum. There is a different microRNA expression pattern in human GLCs from anovulatory women (PCOS). This different expression pattern might play a role either in the pathogenesis or in the ovarian response to the deviation in the ovulation process in women who failed to ovulate. Adiposity is associated with impaired reproductive capacity in ovulatory women. Both ovarian and pituitary hormones are affected, however, the deficit in urinary progesterone metabolite excretion exceeds the deficit in gonadotropin secretion. Therefore, we hypothesize that adiposity may directly affect corpus luteum (CL) function. Objective: To investigate the effect of weight fluctuations on luteal gene expression. Methods: Two regularly cycling female vervet monkeys (16 yo) were followed for 10 months: 1) Ad libitum intake of 120 calories per kg body weight (monkey M1030), 2) Caloric restriction, ∼25% (monkey M1031). P4 was measured during 2 cycles (at baseline CL collection and at 10 months). The mid-luteal CL tissue was collected by laparotomy at baseline and 10 months. The CL gene expression profiles were established using Affymetrix arrays. The primers for qrtPCR validation were designed using the first generation vervet genomic assembly and the expression changes were calculated by the 2 -∆∆Ct method after normalization to GAPDH. Results: Over 10 months, M1030 gained 15.9% (4.4→5.1 kg) and M1031 lost 17% of the baseline body weight (6.9→5.7 kg). The menstrual cycle length remained unchanged. Mid-luteal serum P4 decreased with weight gain (8.6 ng/mL vs. 2.4 ng/mL) but was unchanged with weight loss. Mid-luteal gene expression changes were noted for CL at baseline and after the weight change: the expression of 174 transcripts for M1030 and 100 transcripts for M1030 (≥2-fold) were altered. Weight gain induced expression changes in a group of CL genes involved in lipid metabolism. APOA1, a gene associated with lipid transport and luteolysis was downregulated in CL of the weight-gain monkey, implicating premature luteolysis. While the transcript abundance of integrin1b was increased in CL after weight gain, it decreased after weight loss, suggesting opposing transcriptional regulation. Conclusions: Human gene expression arrays can be utilized to examine transcript levels in vervet monkey CL. The altered gene expression with weight fluctuations of the luteal genes involved in lipid metabolism is intriguing. If confirmed in larger studies, our results imply that adiposity potentiates luteal dysfunction.

Ettie Maman, 1,2 Nirit Rubenstein, 1,2 Yuval Yung, 1,2 Libby Shalev, 1,2 Ariel Hourvitz. 1, 2 1 Obstetric and Gynecology, Sheba Medical Center, Ramat Gan, Israel; 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Introduction: Anti Mullerian hormone (AMH) is a member of the transforming growth factor beta family and its action is mediated through the AMH receptor type II (AMHRII). AMH role in the ovarian physiology was studied extensively however; the role of AMHRII was poorly investigated. Objective: We aimed to elucidate the AMHRII expression patterns in human granulosa cells (GC) from antral to preovualtory stages, and to investigate the correlation between AMHRII expression and oocyte function. Setting: Referral center Design: Luteinized preovulatory GC were obtained from preovulatory follicles aspirated during in vitro fertilization (IVF) (>17 mm). Luteinized and nonluteinized GC from small-(<10 mm) follicles were obtained during in vitro maturation (IVM) procedures. Cumulus GC were obtained during oocyte denudation for intracytoplasmatic sperm injection procedures (IVF). Main Outcome Measures: AMHRII expression levels in mural and cumulus GC of different follicular sizes and their correlation to oocyte outcome. Results: AMHRII expression was highest in small antral follicles and decreased with the increase in follicle size. Expression levels were higher in cumulus GC compared to mural cells with the highest in cumulus GC denuded from germinal vesicle (GV) oocytes. Interstingly, higher expression of AMHRII was found in cumulus GC of MII oocytes that failed to be fertilized when compared to cumulus of GC form MII oocytes that were fertilized. AMHRII expression was higher in non-luteinized GC from small antral follicles compared to luteinized GC from follicles in the same size. In cultured GC, AMHRII expression was decreased after hCG. Conclusions: The highest expression of AMHRII is found in small antral follicles, and cumulus of immature (GV) oocytes. This expression pattern in human GC correlates to previously reported expression levels of its ligand. High expression levels in preovulatory cumulus cells may reflect an oocyte that is not maturing properly and may fail to react to the LH surge signal. The role of AMHRII as biomarker for oocytes and embryos quality should be further studied.

Oxygen Consumption of Human Granulosa Cells and Impact on Follicular Oxygen Levels. Gabe P Redding, John E Bronlund. School of Engineering and Advanced Technology, Massey Univeristy, Palmerston North, New Zealand. The follicular oxygen environment is crucial to follicular development and has been shown to impact on the developmental competence of the oocyte. Mathematical models have been used to predict the oxygen levels in developing follicles, largely due to the difficulties associated with direct measurement. These models clearly show that the oxygen levels experienced by the oocyte are largely dictated by the oxygen consumption rates of the granulosa cells. Unfortunately, this parameter has not been measured for humans and hence limits the utility of any such models. The objective of this work was to measure the oxygen consumption rates of human granulosa cells and to subsequently use these results to predict the oxygen levels experienced by the oocyte throughout follicular development. Granulosa cells were isolated from the follicular aspirates of patients undergoing IVF oocyte pickup. These cells were subsequently transferred to a custom built respirometer consisting of a 2 ml glass chamber and a fluorescence based oxygen probe inserted through a septum. The respirometer was controlled at 37 °C. Oxygen partial pressure profiles were collected for each sample and subsequently used to determine consumption rates based on the cell concentration. The mean oxygen consumption rate of human granulosa cells was 0.032 mol.m -3 .s -1 (std. dev. = 0.008, range of 0.018 to 0.042, n = 14). This result is similar that reported for ovine granulosa cells (0.036 mol.m -3 .s -1 ) and 4.5 times higher than that reported for human oocytes (0.007 mol.m -3 .s -1 ). The measured consumption rates were used in conjunction with existing mathematical models and data describing human follicle growth to predict the oxygen levels experienced by the oocyte throughout follicle development. The results showed that oocytes in preantral follicles experience reduced oxygen levels as the follicle grows. The oocyte first experiences hypoxia around the point of antrum formation. Antrum formation was shown to increase oocyte oxygenation, a result consistent with hypotheses that suggest antrum formation may be a mechanism for the avoidance of hypoxia. However, oxygen levels in antral follicles were still low. Predictions based on geometric data for individual preovulatory follicles of diameter greater than 16 mm showed the mean oxygen levels to be 3.2 vol% (std. dev. = 2.0, n = 14). This result supports the clinical practice of culturing oocytes and embryos at low oxygen levels.

Endothelial Growth Factor (VEGF) Gene Expression in Human Luteinizing Granulosa Cells. Li Hou, Antonio CM Francisco, Robert N Taylor, Tamer M Yalcinkaya. Ob/Gyn, Wake Forest SOM, Winston-Salem, NC, USA. Background: Luteinization of ovarian follicles is associated with a dramatic increase in neo-vascularization and vascular permeability across the basal lamina, since hematogenous route is critical for corpus luteum (CL) endocrine function. Excessive angiogenesis is associated with ovarian hyperstimulation syndrome (OHS). Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) belong to the same cysteine knot family of angiogenic growth factors. VEGF binds with high affinity to VEGF-R 1 and 2, whereas PlGF binds preferentially to VEGF-R 1, a relatively inert tyrosine kinase receptor. Objective: To examine the regulation of VEGF and PlGF expression in human GC by HCG and the cAMP pathway. Design: Analysis of VEGF and PlGF mRNA and protein expression in cultured GC by real-time RT-PCR and ELISA. Methods: Human mural GC and cumulus cells (CC), isolated from follicular aspirates of patients undergoing IVF for tubal or male factor infertility, were cultured with FSK (10 uM), HCG (50 ng/ml), and with +/-H89, PKA inhibitor (10 uM) for 48 h. For the combination treatment, cells were preincubated with H89 (10 uM) or MAPK inhibitor (SB202190) for 1h, followed by FSK or HCG for 24h-48h. Then, RT and real-time PCR was performed to measure mRNA for PlGF and VEGF in GC. PlGF and VEGF were measured with ELISA. Results: We demonstrate that HCG and FSK induce VEGF gene and protein expression in GC. By contrast, PlGF mRNA and protein were decreased by HCG and FSK. VEGF mRNA was increased 175 ± 28% and protein secretion was 168 ± 24% higher after HCG or FSK treatment (P<0.05). PlGF gene expression was decreased to 49 + 21 % (n=8) and 68% + 49 % (n=8) of the basal level by FSK and HCG in human CC, respectively, and decreased to 31% + 15 % (n=8) and 39 + 24 % (n=8) in GC. The expression of PlGF at the mRNA and protein levels was substantially lower (almost 100-fold) than that of VEGF. Conclusion: We demonstrated that VEGF and PlGF genes are expressed in GC and differentially regulated by HCG. As reported previously, VEGF is increased, whereas PlGF is inhibited by HCG. We postulate that counter-regulatory control of these two angiogenic proteins by HCG allows compensatory modulation of CL angiogenesis through combinatorial VEGF-R 1 and 2 signaling. We speculate that dysregulation of the balance of VEGF and PlGF expression might result in abnormal CL vascular permeability, which can present with severe manifestations in OHS.

Kinase A (PKA) Independent Pathways. Li Hou, Antonio CM Francisco, Robert N Taylor, Tamer M Yalcinkaya. Ob/Gyn, Wake Forest SOM, Winston-Salem, NC, USA. Background: Luteinization of ovarian follicles is associated with a dramatic increase in neovascularization across the basal lamina. VEGF, produced by GC, is known to contribute to angiogenesis and vascular permeability in this compartment. Hematogenous route is critical for corpus luteum function. Furthermore, excessive VEGF increases the risk of ovarian hyperstimulation syndrome (OHS) during in vitro fertilization (IVF) cycles. Objective: To elucidate the regulation of VEGF and ovarian steroid hormone production following gonadotropin stimulation in GC. Methods: GC were isolated from follicular fluid during IVF in 21 patients and cultured. Then GC were incubated in the presence of FSH (50 ng/ml), HCG (50 ng/ml) or forskolin (FSK,10 uM) +/-inhibitors of PKA (H89, 10 uM) or of MAPK (SB 202190, 10 uM). After 24h-48 h, total RNA was isolated for CYP11A1, StAR and VEGF gene expression analysis by real time PCR. VEGF in the medium were measured by ELISA, and P 4 and E 2 by RIA. Results: FSH, HCG and FSK treatments all increased VEGF, CYP11A1 (P450scc) mRNA, and StAR mRNA in GC. The increment appeared more robust in primary cumulus cells than in mural GC. These increases ranged from 1.5 to 3 fold (p<0.05). To assess whether the PKA or p38 MAPK pathways were involved, specific pharmacological inhibitors were studied. PKA inhibitor H89 failed to block FSH-, HCG-or FSK-mediated VEGF gene expression. However a specific P38 MAPK inhibitor decreased VEGF suggesting that P38 activation may be linked to VEGF synthesis. FSK and HCG also increased CYP11A1 and StAR gene expression. P4 and E2 secretion increased during FSH, HCG and FSK treatments, but such increases were not affected by addition of either H89 or SB 202190 inhibitors. In these experiments, P4 increased with the upregulation of StAR and CYP11 in the HCG-stimulated human GC. Conclusion: VEGF gene expression in human GC is under the control of FSH and HCG, likely mediated by the generation of cAMP. However, PKA does not appear to be the operative signaling pathway, as the selective inhibitor H89 did not significantly suppress VEGF protein secretion. In contrast, SB 202190, an inhibitor of MAPK blocked the stimulation of VEGF gene expression by gonadotropins and FSK. Ongoing experiments are designed to define MAPK signaling in this critical reproductive axis. Retrospective chart review from 2002 to 2012. Two comparison groups: abnormal cord insertion (ACI) and normal cord insertion (NCI) singletons. NCI subjects were randomly selected from the entire sample of NCI singletons. ACI included VCI and MCI cases that were pathology confirmed. MCI was defined as cord insertion at the placental edge. Exclusion criteria: < 20 weeks of gestation, iatrogenic terminations, spontaneous abortions and congenital fetal anomalies. The primary outcomes were maternal serum markers in the first (PAPP-A, free B-HCG) and second trimesters (AFP, free B-HCG). All serum markers were evaluated using multiple of the median (MoM) values reported as part of routine aneuploidy screening. Each serum marker was compared between the 2 groups using the Mann-Whitney test. Medians and interquartile ranges (IQR) (non-parametric measure of dispersion 75 th -25 th percentile) were reported. Results: 366 singletons were included: 246 NCI, 120 ACI (pathology confirmed as 51 MCI and 69 VCI). There were significant associations between cord insertion group and PAPP-A (P < 0.03), AFP (P < 0.005) and second trimester free B-HCG (P < 0.005). In the ACI group there was an increase of 21% and 50% in median MoM of AFP and second trimester free B-HCG respectively, but a decrease of 11% in median MoM of PAPP-A compared to the NCI group. Conclusion: Singletons with ACI had higher levels of AFP and second trimester free B-HCG and lower levels of PAPP-A than NCI. Prospective studies are needed to confirm these results and determine how it may relate to outcomes. . Exclusion criteria: < 20 weeks of gestation, iatrogenic terminations, spontaneous abortions and congenital fetal anomalies. Maternal serum markers in the first (PAPP-A, free B-HCG) and second trimesters (AFP, free B-HCG, Estriol, Inhibin) were evaluated using multiple of the median (MOM) values reported as part of routine aneuploidy screening. Mann-Whitney tests were used to compare each serum marker between the two groups of interest. Medians and interquartile ranges (IQR) (non-parametric measure of dispersion 75 th -25 th percentile) were reported.

The study included 1,400 sets of twins; 80 in Group A and 1,320 in Group B. However, serum markers were not always available. No significant difference in free B-HCG (first trimester), AFP, Inhibin, Estriol and free B-HCG (second trimester). PAPP-A (MoM) was lower in Group A as compared to subjects in Group B. Although not statistically significant, it represented a trend toward significance (P < 0.0525).

Our study shows that there is no statistically significant association between serum markers and abnormal cord insertion in twins. Future prospective studies are needed for further evaluation. The protein C pathway regulates inflammation and coagulation in the endothelium. So far the presence and/or function of this pathway in human trophoblasts studied in vivo has never been investigated even though studies in mice suggest its importance for a successfull pregnancy. Objective: To investigate the protein C pathway in human trophoblast cells.

The protein and mRNA expression of the endothelial protein C receptor (EPCR) and thrombomodulin (TM) were studied on isolated, cytokeratine positive purified trophoblasts of human placentas from the first trimester of pregnancy. Trophoblasts expressed both EPCR and TM and were able to activated human protein C with greater efficiency than human endothelial cells (1,1 x 10 -7 nmol/L of activated protein C produced per cell per minute). Both receptors were present on villous and extra-villous trophoblasts.

When treated with the pro-inflammatory cytokine TNF-α (1 nmol/L for 24 hours), trophoblast EPCR expression decreased by 20% and was totally restored by pre-treatment with unfractionated heparin (1 U/mL), but only partially by low molecular weight heparin (enoxaparin, 1 U/mL), while TM mRNA was not affected by either treatment.

First trimester human trophoblasts activate efficiently protein C thus showing anticoagulant properties, which are important to maintain blood fluidity at the maternal-fetal interface. Inflammatory mediators affect expression of EPCR, a receptor important for both protein C activation and for regulation of inflammation. Low molecular weight heparin, a drug often (mis)used in obstetric complications, does not protect, at least in vitro, the trophoblast from the deleterious effects of inflammation on the protein C pathway. Objective: Trisomy 21, 18 and 13 (T21, T18, T13) are the most common aneuploidies, and therefore, the focus of pregnancy screening. We had previously found that DNA methylome of placenta changes during normal gestation. Herein, we sought to investigate whether there are differences between DNA methylation profiles of euploid vs. aneuploid placenta. These differences may lead to better understandings of the developmental abnormalities associated with these aneuploidies.

Methods: We collected samples of the maternal-fetal (decidua-placental) interface: 20 trisomy cases (T21, n=10; T18, n=6; T13, n=4) and four gestational age-matched, euploid controls (14-22 weeks). Interphase FISH was used to confirm the ploidy of cases and controls. DNA methylation profiles were assessed using 450K Ilumina Infinium platform that interrogates >485, 000 methylation sites in human genome. Differential DNA methylation was calculated by changes in median beta-values above 0.2 for each locus for every trisomy group in comparison to control. We used R package for our analysis.

Results: Comparing DNA methylome of each trisomy to control, we observed T18 to have the highest number of differentially methylated CpG sites (5335), followed by T21 (3579) and T13 (2820). Next, we assessed whether these changes are gain or loss of methylation compared to control. We observed that T21 shows six times more gain (3084) vs. loss (495) of methylation; T18 has almost the same number of gain (2729) vs. loss (2606), and T13 shows 0.5 times gain (870) vs. loss (1949) of methylation compared to control. This means that T21 placenta is hypermethylated compared to control, T13 is hypomethylated compared to control, and T18 global methylation level is comparable to that of control but has a different profile. Our results indicate that both the extent and the patterns of DNA methylomes are different in decidua-placental interface of each of these aneuploidies in comparison to control. Conclusion: Significant differences observed in epigenetic profiles of maternalfetal interphase in Trisomy 21, 18, and 13 underscore the importance of better understanding the molecular consequences of the most common aneuploidies on the development of the fetal/maternal unit with the potential to yield better diagnostic and therapeutic approaches.

Effects Background: Perturbation of the uteroplacental haemostasis has been implicated in placenta mediated pregnancy complications in thrombophilic women. LMWH may be effective in altering local thrombin production in the uteroplacental compartment. Aim: We determined the effects of LMWH (tinzaparin) on the peripheral, uteroplacental and fetal circulation and on haemostatic gene and antigen expression in placental tissue. Method: Eight women on antenatal LMWH prophylaxis (tinzaparin 75 iu/ kg) due to moderate risk of VTE undergoing caesarean section (CS) and a control group of 15 healthy pregnant women undergoing CS had venous blood taken from the peripheral and uterine vein before delivery of placenta. Simultaneously, cord venous blood and placental biopsy was collected. Tissue factor pathway inhibitor (TFPI), thrombin antithrombin (TAT) and endogenous thrombin potential (ETP) were measured. Real-time PCR and ELISA were used to quantify mRNA and protein expression of TFPI and TF in placental tissue. Results: TAT levels within uterine vein are significantly higher compared to maternal peripheral circulation in both the control group (P<0.0001) and LMWH group (P<0.02). In the LMWH group, TAT is reduced compared with controls in the uterine vein (P<0.001). ETP and TFPI within uterine circulation is reduced significantly in the LMWH group (P<0.05) and (P<0.02) respectively. Down-regulation of placental TFPI and TFPI 2 mRNA expression was also found (p<0.05). Placental TF mRNA expression in LMWH group showed a non significant increase compared to control and this is replicated in placental TF antigen expression. Conclusion: TAT is reduced in uteroplacental circulation in thrombophilic women on LMWH prophylaxis and this is mirrored by decreased ETP in uteroplacental circulation. LMWH may be effective in reducing in-vivo thrombin production in the uteroplacental circulation of thrombophilic women.

Smoking-Related Changes to Placental DNA Methylation Are Associated with Gestational Age. Jennifer Z Joukhadar, 1 Devin C Koestler, 2 Karl T Kelsey, 3 Carmen J Marsit. 4 Friday detrimental effects on the quality of the in utero environment and is associated with low birth weight, pre-term birth and other poor reproductive outcomes. Tobacco smoking during pregnancy is also associated with alterations to normal DNA methylation patterns in the placenta. We hypothesize that maternal tobacco smoking during pregnancy is associated with DNA methylation changes in the placenta and that because these changes are functional, they will be related to infant clinical characteristics. Methods: In a birth cohort of 206 mother-infant pairs, DNA methylation data on more than 27,000 CpG loci, assayed using Illumina's Human Methylation27 BeadChip technology, were analyzed using a locus-by-locus approach for associations between methylation extent and maternal tobacco smoking during pregnancy.

Results: Seven of the loci with methylation changes significantly associated (p<0.05) with smoking during pregnancy were located within the introns, exons and promoter regions of the RUNX3 gene. One of these seven loci was significantly associated with decreased gestational age as well as maternal tobacco smoking during pregnancy (p=0.04). Conclusions: Methylation changes within RUNX3 could provide a potential mechanism for altered gestational age associated with maternal tobacco smoking during pregnancy. These findings contribute to a greater understanding of the effects of maternal tobacco smoking during pregnancy on the placenta and on the developmental origins of health and disease. We hypothesize that while placental anastomotic sharing is important in determining birthweight (BW) discordance, we predict specific differences in placental surface vascular network characteristics are correlated with BW discordance. Materials and Methods: Forty-two cases of TTTS monochorionic twins delivered without intrauterine laser therapy had their placentas, post delivery, injected with colored dyes to distinguish each arterial (A) and venous (V) circulation. The placentas were photographed and the four circulations traced according to protocol by one observer (MK, example. lasered twin); from the tracings were extracted 30 measures of A and V network structures.

Results: Being the larger twin was significantly correlated with a greater vessel/ surface area ratio, numbers of vessel branch points. And branch generations, the arc length of the vessels, differences in the extension of vessels to the disk perimeter, and the degree to which arteries and vein ran in parallel on the surface (p=0.003 top<0.0001). Conclusions: We have identified structural network features that are correlated with twin size in TTTS monochorionic infants delivered without laser intervention. The extent to which A-and V network features, reflecting differences in branching angiogenesis before the mid-trimester, are influenced by or independent of TTTS anastomotic communication is our current subject of analysis. The impact of such differences in branching angiogenesis on later childhood or adult outcomes is unknown.

Signaling. Susanne Lager, 1 Anne-Maj Samuelsson, 2 Paul D Taylor, 2 Lucilla Poston, 2 Theresa L Powell, 1 Thomas Jansson. 1 Background Children born to obese mothers are at increased risk for developing metabolic and cardiovascular disease. We have previously shown that diet-induced obesity in mice results in elevated maternal circulating levels of insulin in late pregnancy, and metabolic programming of the adult offspring, including elevated body weight, fat mass and hypertrophy of adipocytes. mTOR signaling has been proposed to function as a key nutrient sensor in the placenta, matching fetal growth to maternal nutrient availability by altering placental nutrient transport. Activity of placental mTOR signaling is positively correlated to fetal growth in many animal models and in humans. We hypothesized that diet-induced obesity activates placental insulin-and mTOR signaling resulting in accelerated fetal growth. Methods Female C57BL/6J mice were fed an obesogenic diet (16% fat, 33% sugar) or standard chow (3% fat, 7% sugar) for 6 weeks prior to mating and throughout pregnancy. At gestational day 18 the dams were euthanized, and fetuses and placentas were collected. Fetal and placental weights were recorded (n=9 dams/group). Placental tissue from each litter was pooled and homogenized, protein expression/phosphorylation for mTOR/insulin and inflammatory pathways were analyzed by Western blot (n=placentas from 5-7 dams/group). Results Fetal/placental weight ratio was significantly lower in obese dams compared to controls (p<0.01) although fetal and placental weights were not significantly different between the groups. Maternal obesogenic diet reduced placental mTOR signaling, as measured by phosphorylation of ribosomal protein S6 (p<0.05) and did not affect 4EBP1 phosphorylation. Expression and activation of inflammatory signaling pathways (IκB, JNK, p38) and insulin signaling (AKT, IRS1, ERK, PI3K) were not affected. Discussion In contrast to our hypothesis, diet-induced obesity in mice was associated with an inhibition of placental mTOR signaling. This is, however, consistent with lower fetal/placental weight ratio, which indicates reduced placental efficiency. mTOR activity is regulated by a large number of upstream signals and we speculate that inhibitory signals predominate over stimulatory signals in the placenta in this mouse model of diet-induced obesity in pregnancy. Sponsored by: British Heart Foundation and Tommy's the baby charity

Pregnancies. Background: Preeclampsia is a major cause of maternal and neonatal morbidity and mortality; however, the etiology of which remains poorly understood. Endothelial dysfunction in maternal vasculature, especially increased endothelial permeability caused by dissembled endothelial junction proteins, is intimately associated with preeclampsia. Nonetheless, it is still unclear if vascular growth and endothelial dysfunction in placentas significantly impact on preeclampsia. Hypothesis: Vascular growth and expression of endothelial junction proteins in placentas are involved in the pathophysiological event of preeclampsia. Methods: Expression of platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and vascular endothelial-cadherin (VE-Cad), two functionally critical proteins in endothelia, was evaluated using immunohistochemistry and Western blotting in placentas from the first trimester (FT), normal (N) and preeclamptic (PE) pregnancies. Using CD31 as an endothelial marker, capillary number density (CND) and capillary area density (CAD) in N and PE placentas were also analyzed. Results: Both CD31 and VE-Cad were immunolocalized primarily in endothelia of FT, N, and PE placentas. However, no differences in CND and CAD were found between N and PE placentas. Using the same antibodies as those used in immunohistochemistry, Western blotting revealed the levels of CD31 and VE-Cad in N placentas were 3.7 and 8.0 fold higher (P ≤ 0.05) than those in FT placentas, respectively. VE-Cad levels were similar between N and PE placentas. CD31 was undetectable in PE placentas, while CD31 was easily detected in N placentas. These data confirm the robust vascular growth in placentas during pregnancy and indicate similar vascular growth between N and PE placentas. However, the failure to detect CD31 using Western blotting suggests abnormal formation of CD31 in PE placentas, which may potentially adversely impact placental endothelial communication and function. Conclusion: Dysfunction of placental endothelial junction likely plays a significant role in preeclampsia, although we were unable to find major differences in vascular growth between N and PE placentas (Supported by NIH HD38843).

Effect Results: While placental MtDNA copy number significantly increased with increasing adiposity in female (p<0.05), a similar but nonsignificant trend was seen in male. Expression of PGC1α, PPRC1, NRF2 and TFAM in the LN female placenta was higher than in the corresponding male placenta, increased significantly in the female OW group and was significantly greater (p<0.05) than the male OW group. Whereas expression of PGC1α, PPRC1, NRF2 and TFAM in male placentae continued to increase further in the OB group (3) (4) (5) (6) (7) fold in OB, p<0.001), expression of these genes in female OB placentae fell markedly compared to the female OW group and were all significantly lower than the male OB group. NRF1 expression was altered only in the OB group for both genders; whereas NRF1 increased 3 fold in males, it declined 43% below LN levels for females. Expression of mTFb, increased nearly 3 fold (p<0.001) with increasing maternal adiposity in both genders. Conclusions: Maternal adiposity and fetal gender affected the expression of placental mitochondrial biogenesis genes, but patterns of change were gene specific. MtDNA copy number increased in villous tissue from a female fetus consistent with enhanced responsiveness in several biogenesis genes with increasing maternal adiposity. Differences in response of biogenesis genes and failure to increase mtDNA copy number with maternal adiposity in placenta from a male fetus may be relevant to poorer perinatal outcomes.

Autophagy The placenta serves as a formidable barrier to protect the fetus from maternalfetal transmission of pathogens. Fetal trophoblasts in the placental villi, which are in direct contact with the maternal blood supply are known to facilitate the protection from pathogens. In particular, the syncytiotrophoblasts (STB), which cover the villous surface and are derived from highly proliferative cytotrophoblasts (CTBs), have been shown to be more resistant to infection by diverse pathogens than CTBs. However, the mechanism(s) are still poorly understood. Our central hypothesis is that, autophagy, a conserved cellular degradative pathway and a vital part of immune response to microbial infection, plays an anti-pathogenic role in placental infection. Autophagy has been shown to occur in both CTBs and STBs. Human CTBs (BeWo cell line) were induced to differentiate into STBs. Both CTB and STB populations were infected with the gram-negative bacterial pathogen, uropathogenic E. coli (UPEC) and intracellular bacterial colony forming units (CFU) were measured 2 hours post infection. First, we found that autophagic activity, as measured by light chain 3-II (LC3-II, an autophagic marker) levels and conversion of LC3-I to LC3-II is significantly higher in STBs than CTBs (p <0.05) at baseline. Second, we show that treatment with rapamycin, an inducer of autophagy, results in a highly significant decrease in intracellular levels of UPEC in CTBs (p<0.01). Conversely, treatment with 3-methyladenine (3-MA), a potent inhibitor of autophagy, elicits a significant increase in intracellular infection levels. Finally, we demonstrate that intracellular bacterial colonization in STBs is overall significantly lower than in CTBs (p <0.05). Together, our findings suggest that autophagy is a key mechanism governing susceptibility to bacterial infection in the placenta; that the basally higher level of autophagic activity in STBs may explain the differential susceptibility to infection in CTBs and STBs and underlie STB infection-resistance. This is the first study to reveal a role for autophagy in placental infection and opens new avenues for further investigation.

Placental Objective: There is sustained delivery of oxygen and decreased delivery of glucose to the fetus in chronic (altitude-induced) hypoxia in vivo. We have previously hypothesized that placental metabolic reprogramming, a cellular shift to a hypometabolic state, might be responsible for this effect. Confirming this, in vitro studies have shown a decrease in trophoblast oxygen consumption following stimulation of the hypoxia response. In an attempt to discover the mechanisms we tested one candidate, pyruvate dehydrogenase (PDH) kinase-1 (PDK1) which regulates the activity of PDH, the bridge between glycolysis and the tricarboxylic acid cycle, converting pyruvate to acetylCoA. We chose to examine PDK1 since hypoxia-induced increases in PDK1 expression have been observed previously in concert with reduced PDH activity in lymphoma, renal carcinoma and embryonic fibroblast cells. We hypothesized that PDK1 expression would be increased and PDH activity decreased by hypoxia in BeWo choriocarcinoma cells, a trophoblast cell model. Methods: BeWo choriocarcinoma cells were incubated for 24 hr. in dimethyloxalylglycine (DMOG, 1 mM), a hypoxia mimetic, then extracted for assay. Extracts were blotted for PDK1, transferrin receptor (TfR, positive control) and beta-actin. PDK1 and TfR expression were normalized to betaactin. PDH activity was measured using an immunocapture ELISA. Data are presented as percent of control (mean ± SEM). Results: TfR expression was up-regulated (150 ± 21 %; p < 0.05, n = 5, t test) following DMOG treatment however PDK1 expression was unaltered (104 ± 7%; NS, n=5). PDH activity increased to 223 ± 17% of control following DMOG treatment (p < 0.01; n = 5).

The up-regulation of TfR by DMOG demonstrates that the BeWo cells are responding to the hypoxic stimulus. The absence of a change in PDK1 expression suggests that hypoxia does not affect PDK1 in these cells, contrary to published reports in other cell types. Moreover the increase in PDH activity is opposite to the effect that might be expected as a result of hypoxia-induced PDK1 inhibition of PDH. One possible explanation is delayed activation of the hypoxia response program in these cells. Alternatively PDK1 may not be a part of the oxygen-sparing, placental metabolic reprogramming mechanism. (Supported by NIH HD046982, HD068954, HD073408 ). Objective: Abnormal serum analytes in pregnancy and uterine artery (UtA) Doppler indices are predictive of adverse outcomes, such as preterm preeclampsia (pPET) and birthweight (BW) <3rd percentile. We sought to determine if, in the context of abnormal analytes, UtA Doppler abnormalities correlated with specific findings on placental histopathology. Study Design: We retrospectively reviewed cases referred to our institution between Jan 2010 and Mar 2012 with at least one abnormal analyte on Friday prenatal genetic screening (PAPP-A<0. 3, hCG>3.0, AFP>2.5, inhibin>2.0, or unconjugated estriol<0.3 MoM) . Pulsatility index (PI) and notching were assessed by UtA velocimetry at approximately 24 weeks gestational age (GA). Placental pathology was performed by a single pathologist and assessed for placental weight (adjusted for GA), maternal vascular pathology (decidual vasculopathy, villous hypermaturity, and infarction), fetal thrombotic vasculopathy (FTV) and related lesions, and normoblastemia. Results: Eighty patients with ≥ one abnormal analyte who received UtA Doppler screening and histopathological evaluation of the placenta were identified. The percentage of patients with 1, 2, or 3 abnormal analytes was 75%, 23%, and 2% respectively. Twenty-four (30%) had an abnormal mean UtA PI (PI>1.6) and/or UtA notching. Of those with abnormal UtA Doppler imaging, 22 (92%) demonstrated evidence of maternal vascular pathology (p=0.015), and 8 (33%) had FTV-related lesions (p=0.02); however, abnormal UtA Dopplers were not significantly associated with either small placenta or normoblastemia.

In patients with abnormal analytes, there is a strong association between abnormal UtA Doppler imaging and 1) placental insufficiency, characterized by maternal vascular pathology, and 2) FTV-related lesions. Given that published studies have shown an association between both abnormal analytes and abnormal UtA Doppler findings and pPET and low birth weight percentile, our data suggest that underlying placental pathology may mediate these pregnancy complications and highlight the importance of a full placental examination in these clinical settings.

Reproductive and Nonreproductive Arteries during Pregnancy. Charles R Rosenfeld, Xiao-tie Liu. Pediatrics, UT Southwestern Medical Center, Dallas, TX, USA. Background: K + channels contribute to cardiovascular adaptation and down-stream regulation of vasodilation, myogenic responses and modulation of vasoconstrictor effects. Large-conductance Ca 2+ -activated K + channels contribute to uterine vasodilation during pregnancy, the ovarian cycle and after exposure to estrogens (E). K V also contribute to uterine responses to E and apparent blood pressure regulation after E (Endocrinology, in press); but it is unclear which K V isoform is involved and if this is modified by pregnancy. Objective: To screen ovine reproductive and systemic arteries for K V α-subunit expression and determine if pregnancy differentially modifies their vascular smooth muscle (VSM) expression in these arteries. Methods: Mammary arteries, 1 st to 5 th generation uterine arteries, myometrium and select systemic arteries (carotid, mesenteric, popliteal) were collected from nonpregnant ovariectomized (NP) ewes without E2β for 5d and term pregnant (P) sheep. Western analysis was performed with 20ug soluble VSM protein from each artery and antisera for K V1.1, 1.2, 1.5, 3.1 (chosen for observed vascular sensitivity to 4-aminopyridine and tetraethylammonium); loading protein was α-actin. Comparisons were made on single immunoblots. Results: K V3.1 was barely detected in any artery from NP or P ewes, while K V1.1, 1.2, 1.5 , all sensitive to 4-aminopyridine, were easily detected in all arteries; especially K V1.5 . Since K V1.5 predominated, we compared expression in uterine and systemic VSM in a term P ewe. Expression was greatest in 3 rd generation uterine, myoendometrial and carotid arteries and myometrium. When NP and P arteries were compared, K V1.5 was >5-fold higher in P myoendometrial artery, while myometrium was ∼2-fold higher in P. Differences in systemic arteries were not apparent. Conclusions: These are the first data characterizing the expression of several K V α-subunits within the uterine and systemic vasculature of NP and term P ewes. The apparent over-expression of K V1.5 throughout the vasculature suggests it is an important modulator of vascular function. Moreover, its fold increases in P myoendometrial VSM and myometrium suggests a role in modulating myogenic and vasoconstrictor responses in pregnancy and possibly myometrial function. More detailed studies of expression are now underway. Little is known about how placental characteristics vary across populations in normal pregnancies. Investigation of the functional-morphological properties of the placenta can help us better understand not only what drives birth weight, but also how the intrauterine environment sets lifecourse health trajectories in motion across varying socioecological settings. Here we present preliminary data on 1) the influence of placental size and morphology on birth weight of babies born to women who have been lifelong enrollees in the Cebu Longitudinal Health and Nutrition Survey in Cebu City, Philippines and 2) evidence for population differences in placental characteristics. In contrast to findings in several populations, placental and birth weights were not correlated in our preliminary sample (n=9, r=0.42, p=0.27), although lower birth weight neonates tended to have lower-weight placentas (418 g vs. 638.75 g; t=-2.30, p=0.06). We compared the Cebu sample to an identically-sized sample of control pregnancies (i.e. not growth-restricted; n=9) from Nottingham, UK. Cebu birth and placental weights were lower (though not significantly so) than the Nottingham sample, but Cebu placental villous surface area was significantly lower (6.29 m2 vs. 11.0 m2, p<0.01). The Cebu placental villous surface area was similar to the Nottingham values for growth-restricted fetuses (n=5, 6.47 m2). Since the surface of the placental villi is responsible for maternal-fetal nutrient transport, variation in this compartment has important implications for understanding population variation in fetal growth patterns, disparities, and developmental programming. Baseline differences also have implications for classifying and treating "pathology." Objective: Normal development of the placenta during gestation is well understood at the anatomic and histologic levels but less well understood at the physiologic and molecular levels. Nowhere is this more true than with regard to microRNAs (miRNAs), small (21-23nt long) RNA species that are powerful regulators of gene expression. While there have been some miRNA expression studies in abnormal events such as preeclampsia, IUGR, and chorioamnionitis, no study of miRNA expression in normal placentae has been carried out except as controls against which abnormal placentae are compared. Methods: De-identified placental tissues were obtained through Department of OB/Gyn banking efforts at the University of Iowa. Tissues were stored in RNAlater at -80 o . Total RNA was extracted from 0.1g of placental tissue using the miRvana miRNA Isolation Kit (Ambion, Life Technologies). RNA yield and quality was assessed on an Agilent Model 2100 Bioanalyzer. MicroRNA expression profiling was carried out on TaqMan Low Density Arrays (TLDA, Applied Biosystems, Life Technologies). For this study, the A-set array composed of 377 human microRNAs was used for normal placental tissues both at later stages of gestation (26 to 40 weeks) and, for the first time, at a very early stage of gestation (9 weeks). Results: In 357 of 377 miRNAs, expression does not change between early and late gestation. Of note, the 36 miRNAs of the primate specific, placenta specific Chromosome 19 cluster on our TLDAs also demonstrated no change.

Our results have identified a small number of miRNAs (20 of 377) whose expression changes significantly between early and later gestation. Genes targeted by these miRNAs are generally involved in gestationally relevant processes such as angiogenesis, cell growth and immune responses. Conclusion: The vast majority of miRNAs expressed in placentae during gestation do NOT change expression suggesting that to whatever degree they are involved in establishment and maintenance of the human placenta, those roles are established early and do not change substantially. Thus, among these miRNAs, significant changes in expression are likely to be associated with pathogenic processes.

Sildenafil There is no curative treatment available. Catechol-Omethyltransferase knockout mice (COMT-/-) exhibit many signs of PE/FGR (including aberrant uteroplacental blood flow) and deliver growth-restricted pups in comparison to wild type C57Bl6/J (WT) mice. We have reported that treatment of COMT-/-mice with Sildenafil Citrate (Viagra) during pregnancy ameliorates the PE/FGR phenotype and rescues fetal growth. We hypothesized that these effects would be reflected in differences in the mouse metabolome between WT and COMT-/-mice, with resolution of differences consequent on Sildenafil.

Methods WT and COMT-/-mice were randomly assigned to either Sildenafil (0.2 mg/ ml) or placebo from gestational day 12.5 -18.5 (n = 10-23 in each group). Dams were sacrificed on d18.5 and cardiac puncture performed. We applied a targeted quantitative metabolomics approach to analyze serum samples using a combination of direct injection mass spectrometry (AbsoluteIDQ™ Kit) with a reverse-phase LC-MS/MS Kit (BioCrates Life Sciences AG) on an ABI Qtrap 4000 mass spectrometer. A targeted profiling scheme quantitatively screened for known small molecule metabolites using multiple reaction monitoring, neutral loss and precursor ion scans.

In untreated animals, the PE/FGR phenotype was reflected in significant differences in 24 metabolites (p<0.05, WT vs COMT-/-); predominantly glycerophospholipids, sphingolipids, acylcarnitines and biogenic amines. Sildenafil therapy resulted in altered levels of 12 metabolites (P<0.05): nine aminoacids, two glycerophospholipids and hexose. In treated animals, significant differences in 9/24 metabolites (p<0.05, WT vs COMT-/-) persisted.

Metabolomics profiling can provide insights into the pathogenesis of PE/ FGR and also to the mechanism of therapeutic effects. For example Sildenafil elicited marked changes in Kynurenine; kynurenic acid concentrations in early pregnancy have recently been found to convey increased risk of PE in obese women.

VHL and subsequent degradation. As well, oxidative stress conditions trigger HIF-1α activity by inducing de-SUMOylation of the HIF-1α co-activator p300 via SENP3.We previously reported elevated HIF-1α levels in early placental development and in preeclampsia. However, no information is available on HIF-1α SUMOylation in the human placenta. Herein, we investigated the expression of SUMO2/3, SENP3 and HIF-1α SUMOylation in normal placental development and preeclampsia. Methods SUMO2/3 and SENP3 protein levels were assessed in placental tissues from early gestation (7-17 weeks, n=21) and from preeclamptic (PE, n=14) and normotensive preterm age-matched controls (AMC, n=12) by immunoblotting. HIF-1α SUMOylation and HIF-1α/SENP3 associations were assessed by immunoprecipitation. The effect of oxidative stress was evaluated by treating choriocarcinoma JEG3 cells with sodium nitroprosside (SNP; 2.5 mM and 5.0 mM), a nitric oxide donor or by maintaining JEG3 cells at 3% and 20% O 2 . Results SUMO2/3 expression was significantly increased at 7-9 weeks gestation and this inversely correlated with that of SENP3. Interestingly, SUMO2/3 and to a greater extent SENP3 expression levels were increased in PE compared to AMC. Immunoprecipitation experiments revealed increased HIF-1α SUMOylation during early placenal development (7-9 weeks) and in PE. Similar to PE, SENP3 expression was increased in JEG3 cells following SNP and 3% O 2 treatments. Immunofluorescence analysis showed increased nucleoplasmic redistribution of SENP3 and its association with HIF1α in JEG3 cells after SNP and 3% oxygen treatments, indicating an involvement of this protease in HIF-1α de-SUMOylation.

Oxidative stress-induced expressions of SENP3 in preeclampsia may in part contribute to increased HIF-1α stability and activity found in this pathology. Obstetrics and Gynecology, MedStar Harbor Hospital. Objective: Evaluate impacts of prior pre-eclampsia (Hx PE) on first-trimester (T1) maternal physical parameters, uterine artery Dopplers, pregnancyassociated plasma protein A (PAPP-A) and free ß human chorionic gonadotropin (ßhCG) levels in subsequent pregnancies. Methods: Parous women with singleton pregnancies were prospectively enrolled at 9-14 weeks. History, demographics, blood pressure (BP), body mass index (BMI), uterine artery pulsatility index (UtA-PI), PAPP-A multiples of median (MoM) and ßhCG MoM were compared between women with uncomplicated obstetric history and those with Hx PE. Non-parametric, Chi-square, and logistic regression analyses were used considering p <0.05 as significant. Results: 52 (4.1%) of 1283 women had Hx PE and were more likely to have underlying hypertension (HTN) and diabetes (DM). They had higher T1 mean arterial pressure (MAP) ( Table 1) , while Doppler parameters and serum analytes were unaffected. Increases in MAP in women with Hx PE became more exaggerated with increasing parity (Figure 1 ). Prior PE was an independent contributor to elevated T1 MAP (R 2 =0.22, p=0.03). 6/52 (11.5%) women with Hx PE developed recurrent PE and had higher T1 BP than those who did not develop PE. Conclusions: Hx PE increases T1 BP in subsequent pregnancies independent of other cardiovascular risk factors. However, there is no residual impact on placenta derived serum analytes or ultrasound variables, suggesting a potential for normal placentation in patients with normal BP. The amylome represents the sum of proteins with propensity to aggregate into amyloid-like fibrils. Amyloid precursor protein (APP), prion protein, IgG free light chains (FLC) and alpha-1 antitrypsin are amylome proteins involved in pathogenesis of Alzheimer's, prion disease, light chain amyloidosis and serpinopathies. Recent evidence suggests preeclampsia (PE) shares similar pathology. Our objective was to identify, characterize and provide insight into the origin of the PE amylome. Blood and urine samples were collected from 91 pregnant women as follows: healthy pregnant controls (CRL, n=13), chronic hypertension (crHTN, n=5), mild PE (mPE, n=5), severe PE (sPE, n= 51), superimposed preeclampsia (spPE, n=11) and non-PE proteinuric conditions (n=6). Amylome proteins were enriched by Congo Red-assisted precipitation and subjected to tandem mass spectrometry and gel electrophoresis. Protein identity was confirmed by Western blotting. Aggregation propensity was modeled using AGGRESCAN software. Kappa and lambda FLC were measured in serum and urine of sPE and GA-matched CRLs with the Bence Jones protein immunoassay. Expression of kappa and lambda FLC in placenta and fetal membranes was sought by immunohistochemistry and RNA-in situ hybridization (RISH). Urine Congo Red precipitates were obtained in 90% of PE samples but not in CRL, crHTN or non-PE proteinuric subjects. The amount of isolated precipitate/volume of urine was proportional to PE severity. Alpha-1 antitrypsin, ceruloplasmin, kappa FLC and non-random albumin fragments were represented in all precipitates while fragments matching to APP, and the interferon-inducible protein G1P3 displayed a more selective pattern among sPE and spPE. AGGRESCAN identified G1P3 as having similar aggregation propensity to beta-amyloid. Kappa and lambda-FLC were identified in blood and urine of pregnant women with increased fractional excretion in sPE for both. Kappa/lambda FLC ratio was decreased in PE compared to GA-matched CRLs (P=.016). Placenta and fetal membranes of sPE women express high levels of kappa and lambda FLC at protein level but only kappa mRNA specifically in trophoblasts. In conclusion, the PE amylome displays a characteristic profile of both shared and unique identities which may explain the heterogeneity in clinical manifestation of the disease. This is the first evidence that human trophoblasts synthesize FLC and may have relevance for PE pathogenesis.

Developing Novel Therapeutics for Preeclampsia. Relaxin (RLX) emanates from the corpus luteum during pregnancy contributing to the marked vasodilation of maternal circulation. RLX vasodilatory action is in part mediated through activation of the well-known anti-apoptotic PI3K/ Akt pathway and nitric oxide production in endothelial cells. In the same vein, human Tr cells express erythropoietin (EPO)/ EPO receptor mRNA, protein and activity, as well as β-common receptor mRNA. EPO has been shown to protect cells from apoptosis caused by ischemia-reperfusion injury through activating the β-CR/EPO-R heterodimer and PI3K/Akt. The peptide mimetic, Brines' helix B surface peptide (HBSP), acts as an EPO-βCR agonist without stimulating the classic EPO-R and erythropoiesis. We hypothesized that RLX, HBSP or a combination would attenuate apoptosis in HTR-8/SVneo first trimester extravillous Tr cells subjected to hypoxia-reoxygenation injury. HTR-8/SVneo cells were cultured under standard conditions or in serumfree media and 1% O 2 . Pre-treatment with rhRLX (300ng/mL) was done 8 hrs prior to serum-starvation and again after changing to serum-free media. HBSP (25ng/mL) was only added after changing to serum-free media. Cells were serum-starved for 17 hrs, then placed in 1% O 2 for another 8 hrs. After the 8 hr hypoxic period the media and treatments were replenished. Fixing and staining for apoptosis and nuclei using TUNEL and Hoechst, respectively, was done 1 hr later. Percent apoptosis was calculated as a ratio of TUNEL-stained nuclei to Hoechst-stained nuclei. The preliminary data show that cells cultured under standard conditions (n=7 wells) yielded 0.98 ± 0.13% (SEM) and serum starvation with hypoxia (n=6) 5.54 ± 0.8% apoptosis. The rhRLX treatment group (n=6) reduced apoptosis to 3.59 ± 0.22%. The combined rhRLX+HBSP (n=7) and HBSP-alone (n=4) treatment groups slightly decreased apoptosis to 5.13 ± 0.59%, and 5.05 ± 1.13% respectively. On balance, the results so far suggest that RLX attenuates Tr apoptosis in response to hypoxia-reoxygenation in vitro. However, additional experiments are clearly needed, in order to increase the N number and substantiate this finding. In view of RLX's vasodilatory effects, as well as the emerging evidence that it may stimulate invasion and reduce apoptosis of Tr cells, the hormone or a molecule mimetic may be a promising therapeutic in PE.

Objective: We estimated the frequency of abnormal labs in pregnancyassociated hypertension (PAH) and assessed the relationship with pregnancy outcomes. Methods: Secondary analysis of a multi-center trial of vitamin C/E for prevention of PAH in low-risk nulliparas. Women with new-onset hypertension were analyzed. Those without PAH or with major fetal anomalies were excluded. Lab abnormalities included: platelet count <100K, AST ≥100 u/L, Cr ≥ 1.5 mg/dL, LDH ≥ 600 u/L, total bilirubin ≥ 1.2 mg/dL, or hemolysis on peripheral smear. Pregnancy outcomes were compared by 4 groups: I. mild PAH alone (BP140-159/90-109)-referent; II. mild PAH + abnormal labs; III. severe PAH (BP≥160/110) or severe clinical signs alone IV. severe PAH/signs + abnormal labs. Severe clinical signs included headache, epigastric pain, blurred vision, pulmonary edema, eclampsia, or oliguria. Results: Of 9,969 women, 2,779 (28%) developed PAH and of these 2,752 were analyzed. Women with lab abnormalities (7.3%) were more likely to be older, Hispanic or Caucasian, non-smokers, with higher education level and lower BMI. The frequency of lab abnormalities increased with severity of PAH: mild PAH alone (4.9%), severe PAH alone (8.9%), mild or severe PAH with severe clinical signs (12.2%); p-value for trend <0.001. The incidence of adverse outcomes in each PAH category are presented in Table 1 . In general, compared to women with mild PAH alone, selected outcomes are higher when labs are abnormal.

The frequency of abnormal labs in women with PAH is low but increases with the severity of disease. Selected adverse perinatal outcomes are increased in the presence of abnormal labs. 

The impact of a prior diagnosis of preeclampsia (PRE) on subsequent pregnancy outcomes remains unclear. Due to limited data, our ability to counsel patients about the risk of future adverse pregnancy outcomes (APO) is poor. The objective of this study was to assess if prior PRE impacts the risk for APO in a subsequent pregnancy.

A retrospective cohort study (SOAP) was performed. Women previously enrolled in our Preeclampsia: Mechanisms and Consequences (PMC) study with investigator confirmed preeclampsia, comprised our cohort. For SOAP, women were considered exposed or unexposed based on the presence of preeclampsia in the index pregnancy (PMC), as determined prospectively by the study investigators. Women having a subsequent pregnancy beyond 16 wks at the same institution were eligible (N=338 The increased risk for APO and PRE remained whether PRE was diagnosed preterm (AOR=2.9 [1.5-5.67 ], p=0.00) or term (AOR=2.0 [1.17-3.6 p=0.01) .

Prior exposure to PRE did not significantly increase the risk of subsequent SPTB.

Using a prospectively identified cohort, in patients with prior PRE, we have demonstrated a 46% and 28% recurrence rate for PRE and APO respectively. More importantly, prior PRE, regardless of gestational age, also increases the risk for other APOs. These data provide new information to effectively counsel patients with prior preterm AND term PRE about future pregnancy risks. These data also underscore the need for more research regarding the pathogenesis of PRE and the discovery of new therapeutic targets to reduce APOs. Background: Early detection/diagnosis of pre-eclampsia allows appropriate monitoring and targeting of therapeutic strategies. Urinary proteomics is a rapidly developing field that allows detection and identification of individual proteins. Our hypothesis is that a distinctive pre-eclampsia urinary proteome profile can be identified. Methods: At time-of-disease, urine samples were collected from twelve preeclamptic women and twelve gestation-matched controls following informed written consent. A proteome profile was established using a validated workflow, involving selective immunodepletion of albumin and immunoglobulins, 1D SDS-PAGE gel fractionation, in-gel trypsin digestion of gel sections, LC-MS/ MS analysis, spectral analysis and selection of candidate proteins for Multiple Reaction Monitoring (MRM) verification and quantification. Results: Nine hundred and eighty one proteins were identified using minimal stringency in Scaffold, from these, 8 proteins were differentially expressed between cases and controls. Subsequent MRM area-under-curve peak analysis revealed 7 proteins which were present in significantly higher levels in preeclamptic urine samples compared to controls and 1 protein was significantly lower in pre-eclamptic samples.

Preeclampsia Is Associated with Decreased Expression of Intermedin: Potential Angiogenic Role of Intermedin. Madhu Chauhan, Rexanna Chan, Meena Balakrishnan, Chandra Yallampalli. Ob/Gyn, University of Texas Medical Branch, Galveston, TX, USA. Introduction: Pathogenesis of preeclampsia involves abnormal remodeling of the placental vasculature, utero-placental ischaemia, and endothelial cell dysfunction. One hypothesis of inadequate trophoblast invasion leading to focal regions of placental ischaemia/hypoxia correlates a localized overproduction of pro-inflammatory cytokines with a corresponding loss in endothelial function resulting in abnormal vascular remodeling. Intermedin (IMD) is a novel placental peptide that supports rat feto-placental vasculature with a potential to increases the trophoblast cell invasion in early pregnancy. Hypothesis: IMD regulates normal endothelial function in human pregnancy and, a reduced expression of placental IMD in PE could result in endothelial dysfunction. Objective: To assess, 1) Expression of IMD mRNA in placentas from preeclamptic pregnancies and age matched controls, 2) Effect of PE on IMD immunoreactivity in villous tissue, 3) effect of IMD on tube formation in HUVEC cells, and 4) Effect of IMD on TNF-α induced inhibition of tube formation in HUVEC cells. Methods: Preeclampsia was defined as blood pressure > 140/90 mm Hg on at least two consecutive measurements and protein urea of at least 300 mg per 24 hours. Consents were obtained from patients. Placental tissues were collected from preeclamptic and gestational age matched normal pregnancies for gene expression and immuno-histochemical staining studies. Effect of IMD on tube formation was assessed in HUVECs (ATCC).

Tube formation was visualized with phase contrast microscope and quantified by measuring the total tube length in 4 random 4x power fields per well with ImageJ software. Results: 1)Preeclampsia is associated with a decline in the expression of IMD mRNA expression in Placenta compared to the age matched controls and this effect is more prominent in pregnancies with male than female fetus (P<0.05), 2) IMD immunoreactivity is significantly lower in preeclamptic placentas compared to age matched controls, 3) IMD increases formation of tubes of HUVEC cells and these effects are mediated through ERK signaling pathway (P<0.05), 4) IMD reverses the inhibitory effect of TNF-α on the tube formation in HUVEC cells (P<0.05). Conclusion: These studies demonstrate that lower levels of IMD in placenta are associated with preeclampsia and play a role in preserving normal endothelial function.

Potential Regulation by microRNA-21. T Cindrova-Davies, 1 EA Herrera, 1,2 J Kingdom, 3 DA Giussani, 1 GJ Burton. 1 Department of Obstetrics and Gynaecology, University of Toronto, Canada. Introduction: Hydrogen sulphide (H 2 S) is a gaseous vasodilator produced endogenously by cystathionineβ-synthase (CBS) and cystathionineγ-lyase (CSE) 1 . The haemodynamic role of H 2 S in the fetoplacental circulation is unknown. The aims were to study the expression of H 2 S-producing enzymes in placentas from abnormal pregnancies associated with elevated umbilical arterial resistance and to explore its regulation. Methods: We analysed placentas from intrauterine growth restricted pregnancies (IUGR, n=6), and growth restricted pre-eclamptic pregnancies with abnormal (PE-AD, n=6) and normal umbilical artery Doppler waveforms (PE-ND, n=7) for protein and mRNA expression of CSE, CBS. We also measured microRNA-21 (miR-21), which is known to downregulate CSE 2 . Placental explants (n=4) were subjected to normoxia (10%O 2 ) or hypoxia-reoxygenation (0.5%O 2 /10%O 2 ) for 20hr and above parameters analysed. Results: CSE protein levels were down-regulated in IUGR and PE-AD, but not in PE-ND placentas. Reduced CSE expression was associated with increased expression of miR-21. There were no changes in CBS expression between the groups (Fig.1) . We mimicked the changes in CSE and miR-21 expression in placental explants subjected to hypoxia-reoxygenation, suggesting oxidative stress is a mechanism for this effect.

We demonstrate differential CSE expression in placental pathological samples with respect to umbilical vascular resistance and elude to the mechanism of such pathological downregulation via miR-21 upregulation. The changes can be mimicked in placental explants subjected to hypoxia-reoxygenation. Reduced CSE expression and consequent reduced H 2 S bioavailability could contribute to the IUGR pathology by increasing resistance in the umbilical circulation. In an effort to clarify the relationship between Vitamin D (VitD) deficiency/ supplementation and human hypertensive pregnancy, we sought to determine the effects of hypoxia on placenta explants from patients with chronic hypertension (CHTN), and to determine if VitD supplementation would blunt the effects of hypoxia induced inflammation in CHTN.

Placentas from CHTN women were collected after cesarean delivery. Uniform segments of placental tissue were placed in wells containing matrigel media as well as media supplemented with VitD at concentrations of 50µM D2, 100µM D2, 50µM D3, and 100µM D3. The wells were exposed to hypoxic (1% O 2 ) or normoxic (6% O 2 ) conditions for 24 hours. ELISAs were performed on cell culture media and normalized to total protein to determine variations in cytokine secretion.

Hypoxia stimulated pro-inflammatory cytokines/antiangiogenic factors while decreasing anti-inflammatory cytokines in the media of CHTN placentas. IL-6 increased from 8.5 to 73.3pg/mg, TNFα increased from 0.12 to 0.75pg/ mg, and sFlt increased from 14.2 to 21.1pg/mg while IL-10 fell from 0.59 to 0.32pg/mg in response to hypoxia. When treated with 50µM D2, 100µM D2, 50µM D3, or 100µM D3 under hypoxic conditions, IL-6 decreased from 73.4 to 28.9, 34.5, 20.65, and 23.7pg/mg, respectively; TNFα decreased from 0.76 to 0.19, 0.08, 0.076, and 0.19pg/mg; and sFlt fell from 21.08 to 21.69, 13.73, 16.22, and 22.87pg/mg. IL-10 rose from 0.33 to 0.63 pg/mg with only the highest dose of VitD3.

VitD supplementation has a more consistent and profound effect on CHTN placentas in suppression of IL-6, TNFα, and sFlt. These data support the hypothesis that VitD deficiency is associated with CHTN and immune activation in pregnant patients and that VitD supplementation could prove to be beneficial therapy for gestational hypertension caused by placental ischemia/insufficiency. Objective: We investigated mechanism of placental injury and effect on maternal and fetal wellbeing in a mouse model for preeclampsia. Mating a female mouse transgenic for human angiotensinogen (hAGT) with a male mouse transgenic for human renin (hREN) results in hypertension (HTN), proteinuria, and seizures during pregnancy. Study Design: Mice were genotyped as hAGT, hREN, or wild type (WT). Telemetric transmitters were implanted preconception to continuously assess blood pressure (BP). Mice were cross-bred: hAGT x hRen for preeclamptics and controls of hAGT x WT or WT x WT. Blood was collected gestational day (GD) 12, 15, and 18. ELISA was performed for plasma sFlt-1 and VEGF. Necropsies were performed GD 19. Conceptuses were sectioned and stained. Univariate analysis was performed where appropriate. Results: Ten preeclamptic cases and nine controls were evaluated. Systolic BP density demonstrated a shift toward higher values (more HTN) in preeclamptic mice compared to controls; see Figure 1 . HTN by telemetric method is less severe than previously reported tail cuff data. Preeclamptic mouse placentas demonstrated cystic endometrial hyperplasia, increased focal necrosis, increased fibrin deposition, and pregnancy resorption. Plasma sFlt-1 declined in controls (β=-143.66) but increased in preeclamptic mice (β=2539.94; p=0.017). Plasma VEGF did not differ. Preeclamptic mice had higher incidence of placental necrosis (60% vs. 0% wild type controls; p=0.018) and a significantly higher spontaneous maternal death rate (30% vs. 0%; p=0.028).

Increased RAS activity and associated HTN in this preeclamptic mouse model are detrimental to placenta and maternal wellbeing. Increased sFlt-1 may be a surrogate for placental injury. RAS activity plays a key role in both maternal wellbeing and placental perfusion to support carrying a viable gestation.

Meta-Analysis of Pre-Eclampsia Microarray Data. Justyna A Dopierala, Gordon C Smith, Stephen D Charnock-Jones. Obstetrics and Gynaecology, University of Cambridge, Cambridge, United Kingdom. Background: Factors released by placenta into maternal circulation cause the maternal symptoms of pre-eclampsia (PE) and may be useful as PE biomarkers. Microarray studies of the placenta in PE show little consistency due to the small sample size and variability in the choice of the platform and the tissue used. The aim of this study was to perform meta-analysis using raw data to obtain a more accurate and reliable list of differentially regulated transcripts.

We searched PubMed, GEO and ExpressArray for PE array datasets (DS) and we contacted authors whose data were not publically available. The scripting language Python was used for data extraction and management. We mapped probes to Ensembl gene IDs (ENSG) using biomaRt R package. The RankProd R package was used for the meta-analysis.

We identified 39 microarray studies of PE placentas, which generated a list of 1005 transcripts described by the authors as differentially regulated at a "significant level". Of the 39 studies, 8 DS were publically available and 4 were directly provided by the authors. Eleven different platforms were used among the 12 available DS and generated a list of 368,124 probe IDs. These were mapped to approx. 40,000 ENSG. We collected a total of 263 array data files, of which 122 were generated from PE samples. Through the meta-analysis approach we identified 348 up-regulated and 517 down-regulated transcripts with p-value <10E-6 and percent false positive (pfp) <10E-5. Of these 229 up-regulated and 437 down-regulated transcripts have not been previously described in association with PE. The lists contain protein-coding mRNAs as well as pseudogenes, lincRNAs and microRNAs. Thirty-nine and 74 of the top 100 transcripts in the up-and down-regulated list, respectively, were not previously described in the PE microarray literature, including 4 of the top 5 positions in the down-regulated list. Well-known transcripts: LEP, FSTL3, CGB5, PAPPA2 and HTRA4 occupied the top 5 positions in the up-regulated list, confirming the utility of the meta-analysis approach.

Through the use of the meta-analysis technique, we were able to confirm the importance of some previously reported transcripts, which are likely to play a role in PE. However, several of our top-scoring transcripts were not found in any of the individual studies. Our results suggest that the meta-analysis approach is an effective method to identify the consistently regulated transcripts. Effective identification of women at risk of severe early-onset preeclampsia (sPE) is a key objective in obstetrics. We followed 20 normotensive women at high-risk for sPE based on 2 + of: complex medical/OB history, abnormal IPS testing, abnormal uterine artery Doppler/placental size/shape at 16-20 weeks. All had serial non-invasive cardiac output monitoring (NICOM) by thoracic bioreactance and measurement of uric acid and sFlt1/PlGF ratio. 6/20 (30%) delivered <33 weeks with sPE. Results in Table. Despite normal blood pressure, SVR was significantly elevated in those developing sPE. Striking differences for sFlt-1 and PlGF were found. Similar significant data was found for uric acid. The AUC and cut-offs, sensitivity and specificity to predict sPE at 24 weeks were: SVR: 0.84, 1250, 80%, 93%, sFlt1/PlGF ratio: 0.94, 55, 100%, 93% and for uric acid: 0.99, 255, 100%, 93%. Uric acid highly correlated with both sFlt1(p=0.003) and sFlt1/PplGF ratio (p=0.02). sFlt1 was highly-correlated with SVR in the pre-hypertensive phase (R 2 0.38), but not in women remaining normotensive. SVR was not associated with uric acid (p=0.13). Our novel prospective data demonstrate that women at high risk of sPE are in a compensated normotensive state characterized by high SVR that is accompanied by substantial elevations in both uric acid and the sFlt1/PlGF ratio. Obesity increases the risk of pregnancy complications including preeclampsia. SDF-1 is a cytokine involved in the recruitment of stem cells and hematopoietic cells, and is induced by hypoxia and pro-inflammatory stimuli. We have shown previously that circulating SDF1 is significantly elevated in preeclampsia. We hypothesized that adipose tissue may be dysfunctional in obese subjects and a source of SDF-1 during pregnancy. Objective: We measured the expression of SDF-1, HIF1α (as a measure of hypoxia), c-KIT (a stem cell cytokine receptor), TGFβ and collagen 5a3 (Col5a3) (as indicators of inflammation and fibrosis). We investigated associations between these five factors, maternal adiposity and pregnancy outcome. Study Design: Subcutaneous maternal adipose tissue was collected at C-section. Total RNA was extracted and quantified with real time RT-PCR. Statistical analysis was by t-test, Wilcoxin rank sum, and correlation analysis was performed using Pearson product moment correlation coefficient. Results: There were no differences in SDF-1 expression between women with preeclampsia (n=11) and controls (n=13). Samples were combined and investigated for lean vs. obese differences. The expression of SDF-1, HIF1α and c-KIT were significantly elevated in adipose tissue of obese pregnant women compared to lean pregnant women. The expression of TGFβ and Col 5a3 were also elevated in obese women, but not significantly. There were significant correlations between SDF-1 and HIF-1α (r2=0.48, p<0.01) and TGFβ and Col5a3 (r2=0.54, p<0.01) expression. None of the tested analytes correlated with BMI.

Conclusion: Adipose tissue in obese pregnant women evidences hypoxia and fibrosis and overexpresses SDF-1, HIF1α and c-KIT. These data suggest adipose tissue dysfunction and decompensation in obese pregnant women. BMI alone is not sufficient to indicate adipose tissue dysfunction. Endothelial progenitor cells (EPCs) are bone marrow-derived cells that enter the systemic circulation to replace defective or injured mature endothelial cells. We have previously reported significantly elevated circulating EPCs in human and mouse pregnancy. Patients with reduced or undetectable EPCs are at increased risk of endothelial dysfunction and cardiovascular disease. Obesity is associated with an increased risk of cardiovascular disease. We hypothesized that EPCs would be significantly lower in obese pregnant women. Objective: The objective of this study was to quantify circulating EPCs in lean and obese pregnant women and mice. Study Design: EPCs were quantified from whole blood by flow cytometry from 6 lean (BMI= 23.1±1.9 kg/m2) and 8 obese (BMI= 34.7±5.7 kg/m2) pregnant women sampled in the first trimester (average 11 weeks gestation). EPCs were also quantified in whole blood from 4 lean and 9 high fat diet-induced obese pregnant mice. EPCs in human samples were identified as circulating angiogenic cells (CACs) 1. CD117+/CD34+, 2. CD133+/CD34+, and 3. CD45dim/CD34+/CD31+/CD133+. EPCs in mouse samples were identified CD45-/ sca1+/ Flk1+ and CD45-/CD34+/Flk1+, and were quantified per blood volume. Statistical analysis was by Wilcoxon rank sum. Results: Circulating EPCs were significantly elevated in obese pregnant women compared to lean pregnant women (Table) , and elevated in obese pregnant mice compared to lean mice (p<0.01). Non-pregnant obese mice also had elevated EPCs in compared to the lean non-pregnant controls. Conclusion: Contrary to our hypothesis, circulating EPCs were significantly elevated in obese pregnant women and high fat diet-induced obese pregnant mice. While pregnancy increases circulating EPCs, obesity appears to further increase circulating EPCs. It remains to be determined, however, if these cells are functioning appropriately. This project supported by National Institutes of Health grants P01-HD30367 and R01-HL091094. in patients with PE at 11-14 weeks of gestation. 2-ME is synthesized from 17-ß-estradiol, which is produced from testosterone by aromatase. Hypoxia has been seen to regulate aromatase transcription. Given that placentas from PE patients are hypoxic, we evaluated if aromatase expression was altered in placentas of PE patients. Aim: Our aim was to determine if aromatase expression (RNA and protein levels) is altered in women who develop PE compared to women with a normal pregnancy. Methods: Pregnant women (control=32 and PE=16) were recruited from hospital Parroquial de San Bernardo with informed consent. Aromatase RNA and proteins were assessed from term placenta samples. Total RNA was obtained with RNA purification kit (Master Pure, Epicentre) and was quantified observations in adjacent trophoblasts implicated in promoting a PE-related local pro-inflammatory cytokine milieu. The current observations that activation of TLR-4 by LPS in the presence of PE-associated TNFα in first trimester human DCs promotes PE by inhibiting angiogenesis-mediated spiral vascular transformation via enhanced sFLT-1 expression.

Louise C Kenny, 1 David I Broadhurst, 2 Wen Hong, 3, 4 Joanna Stanley, 4 Irene Andersson, 4 Christian Rueda-Clausen, 4 Ruparsi Mandal, 6 David Wishart, 6 Philip N Baker. 5 1 Obstetrics and Gynaecology, University College Cork, Cork, Ireland; 2 Department of Medicine, University of Alberta; 3 Obstetrics and Gynaecology, Zhejiang University; 4 Obstetrics and Gynecology, University of Alberta; 5 Liggins Institute, University of Auckland; 6 TMIC, University of Alberta. Background: Preeclampsia (PE) complicates 3-4% of human pregnancies. There is currently no widely available sensitive early pregnancy predictive test. We have previously presented biomarkers, discovered using GC-MS and LC-MS metabolomic profiling of plasma that predicted the subsequent development of later preeclampsia. Here we present the results of NMR profiling of early pregnancy serum samples from women who subsequently developed preeclampsia compared with matched controls. Methods: Nulliparous healthy women with a singleton pregnancy were invited to participate in the SCOPE (Screening for Pregnancy Endpoints) Ireland study. Blood samples were obtained at 15 weeks' gestation. 49 women who subsequently developed preeclampsia were matched with 49 women with uncomplicated pregnancies. Serum samples were deproteinized and transferred to a standard NMR tube for subsequent spectral analysis. 1H-NMR spectra were processed using the Chenomx NMR Suite Professional Software package version 7.1 (Chenomx Inc, Edmonton, AB). 43 unique endogenous metabolites were reproducibly identified and subsequently quantified. After data normalization Wilcoxon signed-rank test was performed on each metabolite to assess whether the population mean ranks differed significantly. Correction for multiple testing was performed using the Benjamini and Hochberg procedure (q-values).

Amines and Amino Acids were observed. Background: Maternal vitamin D deficiency is associated with adverse pregnancy outcome; however, the evidence is inconsistent. We examined the effect of circulating serum 25-hydroxyvitamin D (S25OHD) concentrations in early pregnancy on the risk of uteroplacental dysfunction as a composite outcome of small for gestational age (SGA) and preeclampsia. Methods: Nulliparous healthy women with singleton pregnancy were invited to participate in the SCOPE Ireland (Screening for Pregnancy Endpoints) study. Women were interviewed at 15 and 20 weeks? gestation and provided blood samples. S25(OH)D concentration (calculated as the sum of 25(OH)D2 and 25(OH)D3) was measured by liquid chromatography-mass spectrometry (LC-MS/MS) at 15 weeks gestation. Women were grouped into 4 categories of S25OHD concentrations; <30; 30-49.9; 50-74.9 and ≥75nmol/L (Institutes of Medicine, 2011). SGA was defined as birthweight ≤ 10th customized centile. Preeclampsia was defined as ≥140 mm Hg or diastolic blood pressure ≥90

Poster Session: Preeclampsia and Related Disorders (Friday, 3/22/2013, 9:00 AM -11:00 AM) Scientific Abstracts Reproductive Sciences Vol. 20, No. 3 (Supplement) , March 2013 265A Friday mm Hg, or both, on at least two occasions four hours apart after 20 weeks? gestation but before the onset of labour, or postpartum, with either proteinuria or any multisystem complication. Logistic regression was used for data analysis adjusting for season, supplement use, recreational walking and maternal characteristics such as age, smoking and BMI. Results: Among 1769 women, 190(10.8%) had SGA infants and 68(3.8%) had pre-eclampsia. Mean (SD) age was 29.9 (4.5) and 98% were Caucasian. Mean (SD) S25OHD was 56.7(25.9) nmol/L; 295 (17%) had S25(OH)D < 30 nmol/L and 44% were below 50nmol/L all year round. In winter, these prevalence figures increased to 25% < 30 and 57% < 50 nmol/L. The adjusted odds ratios are presented in the Table 1 . Conclusions: There was no evidence that vitamin D deficiency (<30nmol/L) is associated with increased risk of SGA and/or pre-eclampsia in this cohort. These findings suggest that vitamin D levels above 75 nmol/L may be protective against SGA and preeclampsia. Severe pre-eclampsia (sPE) is a hypertensive pregnancy disorder characterized by systemic maternal vasculopathy and impaired fetal growth. The antiangiogenic state is in part mediated by excess release of sFLT-1 largely originating from pathologic transcriptionally-active syncytial knots in the syncytiotrophoblast (SCT) layer. The peroxisome proliferator-activated receptor-gamma (PPAR-γ), a nuclear receptor and transcription factor, plays a key role in physiologic SCT differentiation in mice and humans. We demonstrated that activation of PPAR-γ with a selective agonist rosiglitazone ameliorated, while inhibition of PPAR-γ promoted, features of sPE in disease rat model. PPAR-γ targets the expression of heme oxygenase-1 (HO-1), a molecule that possesses anti-inflammatory and cytoprotective properties. We tested the hypothesis that PPAR-γ promotes HO-1 transcription in the villous trophoblast using the human chorio-carcinoma-derived cell line BeWo that mimics SCT formation in-vitro. BeWo cells were cultured for 3, 6, 24, and 48 hrs at 37 o C under 20% O 2 tension with treatments and their appropriate controls. PPAR-γ and HO-1 mRNA levels were assessed by RT-qPCR. Cellular HO-1 protein content and release were assessed by ELISA. Following rosiglitazoneinduced activation of PPAR-γ, a rapid rise in HO-1 was observed as early as 3hrs of culture (2.4 fold increase, n=5, p<0.005), along with a decrease in PPAR-γ expression by 33% (n=5, p<0.05), indicating a novel negative feedback mechanism of PPAR-γ autoregulation. Furthermore, the PPARγ-specific antagonist inhibited HO-1 induction when administered either alone or with rosiglitazone. PPAR-γ stimulation also increased cellular HO-1 protein expression (2-fold, n=5, p<0.05) and release (1.9-fold, n=5, p<0.05) after 48hrs of treatment. These data support our initial hypothesis and may provide further insight into the involvement of the PPAR-γ/HO-1 pathway in villous trophoblast differentiation. Lower HO-1 expression has been reported in sPE placentas, thus, pharmacologic upregulation of HO-1 offers insights into a pathway relevant to the secondary prevention and/or treatment of sPE. Funding: CIHR grants to JK; Bernard Ludwig Studentship, MSH (2011-12) and Dr. Samuel and Evelyn Librach Award, UofT (2012-13) to KL.

The Influence of Vaccinium angustifolium Var. laevifolium House (Ericaceae) Extracts on Trophoblast Biology. Christina Ly, 1 Julien Yockell-Lelievre, 2 Jonathan Ferrier, 3 Lana Saciragic, 4 Ammar Saleem, 3 John T Arnason, 3 Andree Gruslin. 1,2,4 1 Cellular and Molecular Medicine, University of Ottawa; 2 Reproductive Medicine, Ottawa Hospital Research Institute; 3 Biology, University of Ottawa; 4 Obstetrics and Gynecology, The Ottawa Hospital. BACKGROUND Vaccinium leaf extracts have been described as important anti-diabetic therapies and highly active in preventing the formation of advanced glycation endproducts (AGEs). Mean serum levels of AGEs were found to be increased in preeclamptic women. Immunohistochemical analysis of preeclamptic placenta indicated enhanced oxidative modifications of lipids and DNA resulting from upregulated binding between AGE and its receptor (RAGE). Upregulation of the AGE-RAGE system triggers oxidative stress, a characteristic of preeclampsia. Preventing oxidative stress with anti-AGE therapy might therefore contribute to the prevention of preeclampsia and could lead to improved trophoblast function. OBJECTIVE We aimed to assess the influence of V. angustifolium extract on trophoblast migration, apoptosis and proliferation. We hypothesized that this extract will promote trophoblast migration without influencing cell death or proliferation. METHODS Trophoblast biology was evaluated using a human first-trimester extravillous trophoblast cell line (HTR-8/SVneo). Cells were treated with 0 ng/mL to 2x10 5 ng/mL of V. angustifolium for 24 hours. Cell migration was assessed using a standard migration assay with 8µm Boyden chamber inserts. Cell apoptosis was assessed by TUNEL assay and trypan blue exclusion test. Cell proliferation was determined using 5-bromo-2'-deoxyuridine (BrdU) and anti-BrdU antibody. Data were analyzed using one-way ANOVA and Bonferroni post hoc test. RESULTS Trophoblasts treated with 20, 2x10 2 , and 2x10 3 ng/mL of V. angustifolium displayed a significant increase in migration compared to the control group (p < 0.01). Based on previous bioavailability studies, we decided to pursue 20 ng/ mL in our apoptosis and proliferation assays. At 20 ng/mL, V. angustifolium had no effect on trophoblast apoptosis or proliferation. However, cells treated with 2x10 5 ng/mL of the extract experienced a significant increase in cell death (p < 0.01). CONCLUSIONS Our data suggest that at 20 ng/mL, V. angustifolium improves trophoblast migration with no effect on apoptosis or proliferation. Further investigations are needed to assess the potential preventative benefits of this extract in the context of preeclampsia.

Pregnancy-Induced Hypertension Model Mouse Using CD40L Gene Delivery. Yuko Matsubara, Keiichi Matsubara, Miki Mori, Yuka Uchikura. Obstetrics and Gynecology, Ehime University, Toon, Ehime, Japan. Objectives: It has been reported some kinds of pregnancy-induced hypertension (PIH) model mice using gene delivery and cytokine injection after conception. However, these model mice are not fitted to investigate the beginning of the pathogenesis of PIH since early pregnancy is a critical period for the pathogenesis of PIH. It is known that CD40 ligand (CD40L) is expressed on T cell and regulates immune system including helper T call. CD40L also plays an important role in the antigen-antibody interaction. We succeeded to develop a new breed of PIH model mouse with the early pregnancy period using CD40L gene-transfected blastocysts. Material and Methods: Blastocysts were obtained from uterine horns of ICR female pregnant mice (10 -12 weeks old) after PMSG and hCG peritoneal injection. The blastocysts were incubated with adenovirus including CD40L gene and transferred into the uterine horns of pseudopregnant ICR mice. We measured blood pressure using tail cuff method and collected urine samples using metabolism cage from the 5th day after the embryo transfer (the 5th day). The mice were killed on the 14th day and the weight of fetuses and placentae was measured. Also, the placenta was stained using hematoxyline-eosin staining and the kidney was stained by PAS staining method. Results: Blood pressure was significantly increased on the 12th day. Fetal weight was significantly decreased in the CD40L-transfected mice; however, the placental weight was not different. PAS stain demonstrated that mesangial matrix was proliferated in the renal glomeruli from CD40L-transfected mice. We couldn't find any change in the liver. Discussion: It is thought that CD40L plays a critical role in the pathogenesis of PIH through the immune system activation since the CD40-CD40L interaction is important for cellular immune system. Our PIH model mouse might supply a good solution to investigate the pathogenesis of PIH. Introduction Pregnancies complicated by preeclampsia have shown signs of concentric cardiac hypertrophy which may explain the increased risk of cardiovascular disease that occurs later in life. Peroxisome proliferator activated receptor gamma (PPAR-γ) has an anti-hypertrophic effect in the setting of maladaptive ventricular remodelling. The reduced uterine perfusion pressure (RUPP) rat model of preeclampsia is characterised by cardiac fibrosis and heart hypertrophy. Our study aimed to determine the type of cardiac hypertrophy present in the RUPP rat and whether administration of a PPAR-γ agonist, rosiglitazone, affected any observed changes in ventricular structure. Methods Surgical reduction of uteroplacental perfusion was performed in normal pregnant (NP) rats. RUPP rats were treated with either vehicle or the PPAR-γ agonist, rosiglitazone, post (D16-18) RUPP surgery. Vehicle treated NP and sham-operated NP rats were also included in the study. Post the in vivo study, rats were culled and the hearts harvested. Cardiac tissue was processed for haemotoxylin and eosin staining and morphological analysis. Left ventricular (LV) and right ventricular (RV) wall thicknesses, LV chamber area (% of total LV area) and cardiomyocyte diameter were measured using computer planimetry.

Heart weight to bodyweight ratio (HW:BW) was increased in the RUPP model (3.49±0.1 vs. 2.88±0.1mg/g; P<0.01; n=9-11) indicative of cardiac hypertrophy and this was unaffected by rosiglitazone treatment. No significant differences were observed in LV (2.4±0.2 vs. 2.3±0.1mm; P>0.05; n=4-6) , RV (1.2±0.3 vs. 1.1±0.1mm; P>0.05; or interventricular (2.2±0.1 vs. 1.7±0.2mm; P>0.05; n=4-6) wall thicknesses in the RUPP rat compared with the NP. Neither cardiomyocyte diameter (13.1±2.2 vs. 12.4±2.6µm; P>0.05; n=4-6) nor LV chamber area (22±2 vs. 23±2%; P>0.05; n=4-6) were significantly altered in RUPP rats compared with NP rats.

While HW:BW was significantly increased in the RUPP rat, indicative of cardiac hypertrophy, no changes were detected in ventricular dimensions, suggesting that any hypertrophic changes associated with this model are subtle and unlikely to be of the concentric variety. As changes in cardiac fibrosis have been shown to underlie cardiac dysfunction the RUPP model may still be associated with comprised cardiac contractility. Compelling evidence suggests that cerebral edema underlies the neurologic complications of preeclampsia, including seizure risk. Our prior work using MRI quantification of brain water diffusion suggests that normal pregnancy leads to increased diffusivity within posterior cerebral subcortical white matter.

The objective of the current study is to use high resolution MRI to quantify pre-pregnancy cerebral water characteristics of women with a history of preterm preeclampsia as compared to nulliparous control patients. METHODS: Twenty-seven healthy non-pregnant nulliparous women (NP) and fifteen non-pregnant women with a history of preterm preeclampsia (PP) were examined using a Philips Achiva TX 3T MRI. Subjects were provided a controlled diet for 3 days prior to testing. PP subjects were an average of 2.65 years postpartum. A diffusion tensor pulse sequence was employed to measure cerebral water diffusion. Histograms of whole brain mean diffusivity values were fitted using a dual Gaussian model corresponding to brain parenchyma, CSF, and a mixed volume compartment. Data is presented as mean ± SD. RESULTS: Subjects in the two groups demonstrated no significant difference in age (PP=32.8 ± 5.4, NP= 30.7 ± 4.6) , BMI (PP= 27.3 ± 6.5, NP= 24.8 ± 6.1) , and blood pressure (MAP: PP=89.8 ± 10. 6, NP= 86.75 ± 5.72 ). The overall mean diffusivity of brain parenchyma is lower in the women with a history of preeclampsia compared to the controls (PP= 0.73 ± 0.013, NP= 0.74 ± 0.011) x 10 -3 mm 2 /s. Regional analysis of mean diffusivity values using the JHU white matter atlas showed decreased mean diffusivity in 18 of 20 tracts assessed, with the bilateral anterior thalamic radiations (p<0.002) and fornix minor (p=0.014) showing the most significant differences. CONCLUSIONS: Patients with a history of preterm preeclampsia (PP) demonstrate significantly lower values of mean diffusivity of brain parenchyma as compared to nulliparous controls (NP). The association of these differences in with underlying physiologic parameters including plasma volume and cardiac output is under investigation.

Identification of Different Phenotypes of Preeclampsia by Clustering Analysis of Biomarker Profiles. Leslie Myatt. for the Eunice Kennedy Shriver National Institute of Child Health and Human Development, Maternal-Fetal Medicine Units Network, Bethesda, MD, USA. Background: Preeclampsia (PE) is a maternal syndrome defined by development of gestational hypertension and proteinuria. The pathophysiology includes variable involvement of different organ systems and pathways suggesting distinct pathologic phenotypes of PE may exist. Objective: To determine whether different phenotypes of preeclampsia may be identified based on biomarker profiles. Methods: Women with clinically diagnosed preeclampsia (n=176) in the prediction arm of a multicenter randomized control trial of antioxidants to prevent preeclampsia in nulliparous singleton pregnancies were included. The biomarkers ADAM-12, PAPP-A, PP13 (trophoblast invasion), IGFBP1 (invasion and metabolic syndrome/insulin resistance), PlGF, sFlt-1, endoglin (pro and anti-angiogenic factors), CRP, leptin (inflammation), cellular fibronectin (vascular condition), platelet number and volume (coagulation cascade) were measured within 4 wks of delivery. Uterine artery Doppler notch, bilateral notch PI and RI (uteroplacental adaptation) were measured at 16.9+1.1wks. Correlation among markers was analyzed by Spearman's correlation coefficient and a hierarchical clustering procedure was used to group patients into different clusters. Exact chi-square test was used to evaluate whether any two clustering outcomes were associated. Results: Of 28 pairwise comparisons examined, 9 significantly associated clustering outcomes were identified. These were IGFBP1 with mean RI and PI (p=0.031), IGFBP1 with sFlt-1 and endoglin (p<0.0001), IGFBP1 with CRP and leptin (p=0.040), ADAM12, PAPP-A, PP13 with PlGF (p=0.014), and with sFlt-1 and endoglin (p=0.035), mean PI and RI with sFLT-1 and endoglin (p=0.042), PLGF with sFlt-1 and endoglin (p=0.003) and mean platelet volume and platelet count with cellular fibronectin (p=0.039) or with CRP and leptin (p=0.010). A correlation strength based heatmap showed a similar pattern. Conclusion: Identified clustering associations link IGFBP-1, a marker of trophoblast invasion but also metabolic adaptation, with uteroplacental adaptation, angiogenic and inflammatory pathways. Other invasion markers and uteroplacental adaptation associated with pro and anti-angiogenic pathways, and coagulation cascade with vascular condition and inflammation. A lack of associated clustering outcomes among 19 other pairwise comparisons suggests other pathways and illustrates the heterogeneity of preeclampsia. Human Development, Maternal-Fetal Medicine Units Network, Bethesda, MD, USA. Background: Normal pregnancy is an inflammatory state and inflammation is increased in preeclampsia (PE). Obesity is also a state of chronic low grade inflammation and women of increased BMI are at increased risk of developing PE, presumably due to increased inflammation. In non-pregnant individuals, levels of C reactive protein, produced by the liver, and leptin, produced by adipose tissue and trophoblast, are related to BMI. Objective: To evaluate the relationship between BMI and maternal 1 st trimester CRP and leptin levels, and determine if these markers are associated with PE independent of BMI. Methods: A secondary analysis of 750 women, in whom prepregnancy BMI was available, from a multicenter randomized control trial of antioxidants to prevent preeclampsia in nulliparous singleton women. Serum CRP and leptin at enrollment (11.4+1.1wks, mean+SD) were measured by Luminex assay. Results: 171 women subsequently developed PE. There was a significant association (p<0.0001) of the development of PE with increasing adiposity. First trimester concentrations of both leptin and CRP were positively associated univariately with increasing BMI (both p<0.0001). There was a significant association of leptin with development of preeclampsia (p=0.0163) after adjusting for prepregnancy BMI. For a 10 unit (ng/ml) increase in leptin there is a 1.17 fold increase (95%CL 1.03, 1.32) in the odds of developing preeclampsia. There was no association of leptin with development of severe preeclampsia or with late onset preeclampsia (>37wks). However the influence of leptin on the odds of developing early onset PE (<37 wks) were 1.65 (95%CL 1.15, 2.37, p<0 .0062) in lean women, not significantly different in overweight women, and 2.19 (95%CL 1.37, 3.49, p<0 .0011) in obese women. No significant association of CRP levels with development of preeclampsia was found. Conclusion: We confirm that increased BMI is associated with PE, and that BMI correlates with the inflammatory markers leptin and CRP in the 1 st trimester. 1st trimester leptin is associated with development of preeclampsia but the effect on early onset PE is dependent on maternal adiposity suggesting differing influences in lean vs obese women that may reflect placental vs adipose sources of leptin. CRP as a general marker of inflammation was related to BMI but does not appear to be associated with preeclampsia.

Epigenetic Regulation of Interleukin-17 Cytokines and Their Role in Neutrophil Infiltration in Preeclampsia. William H Nugent, 1 Sonya L Washington, 1 Jerome F Strauss III, 1 Scott W Walsh. 1, 2 1 Ob/Gyn, Virginia Commonwealth University, Richmond, VA, USA; 2 Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, producing T-cells are elevated in women with preeclampsia. IL-17 cytokines are potent inflammatory agents and implicated in hypertension. DNA methylation is a major epigenetic mechanism controlling gene expression and reduced methylation is associated with increased gene expression. Hypothesis: IL-17 cytokines have reduced methylation in preeclampsia. Methods & Results: We used the Illumina platform to conduct a global assessment of DNA methylation in omental arteries and leukocytes of normal pregnant and preeclamptic (PE) women. We found significantly reduced methylation for IL-17A, IL-17D, IL-17E, IL-17F in PE. Significantly reduced methylation was also present for IL-2, which regulates T-cells, for Th2 cytokines, IL-4, IL-5, IL-10, and for TNFα. To test if methylation regulates IL-17 cytokines, a lymphocyte cell line (Jurkat), was cultured with 5-Aza (5 µM), a hypomethylation agent, for 48 h (n=8). Specific primers and qRT-PCR were used to assess gene expression. Compared to control, 5-Aza significantly increased IL-17D, 8±3-fold, IL-17E, 18± 6-fold and IL-17F, 19±6-fold (P<0.001). IL-17A was not expressed by Jurkat cells. 5-Aza also increased IL-2, 82±26-fold and TNFα, 34±3-fold (P<0.001). To test if IL-17 cytokines could be responsible for infiltration of neutrophils, human vascular smooth muscle cells were cultured with IL-17 and neutrophil chemokines were measured. IL-17 significantly increased IL-8, 2. OBJECTIVE: It is widely accepted that aberrant cell cycle progression is responsible for the inadequate placentation processes typical of Preeclamptic (PE) placentae, characterized by increased trophoblast cell death and turnover. The Activator Protein 1 family member JunB inhibits cell cycle progression by regulating Cyclin-D1, key promoter of G1/S phase transition. We previously reported JunB over-expression in PE Placental Mesenchymal Stromal Cells (PDMSCs). a pivotal structural component of placental villi. Since normal PDMSCs possess the ability to regulate proliferation in neighbouring cells, herein we investigated JunB-mediated regulation of Cyclin D1 pathway in normal and PE PDMSCs in order to clarify their role in normal and pathological placentation. METHODS: PDMSCs were isolated from control (n=12) and PE (n=12) placentae. At passage 5, control and PE PDMSCs were plated (1x105 cells/ ml) and JunB siRNA was performed. Normal control villous explants (n=48) were treated for 72h with media conditioned (CM) for 48h by control or PE PDMSCs. Cells and explants were processed for mRNA and protein isolation. Objective: Increasing evidence seems to suggest that cases of pre-eclampsia with different onset may represent different pathologic entities. This may also apply to eclampsia; therefore, in this study we aimed to describe characteristic and maternal outcomes based on eclampsia onset. Study Design: We reviewed medical records of all women diagnosed with eclampsia in our hospital from August 1998 through April 2011 and abstracted clinical and socio-demographic data. Characteristics and outcomes were compared among cases of antenatal and postnatal eclampsia, and early (diagnosed before 32 weeks of gestation) versus late antenatal cases (diagnosed at or after 32 weeks of gestation). Data are presented in absolute numbers and percentages, and comparisons are made between groups using Chi square, Fisher's exact test, independent T test and Mann Whitney U test. A two-sided p-value of less than 0.05 was considered statistically significant. Results: We identified 87 eclampsia cases out of 59,388 deliveries (0.15%). 62 (71.3%) cases were diagnosed during the antenatal period and 25 (28.7%) had a postnatal onset. Among the 62 cases diagnosed before delivery, there were 41 (66%) cases of early eclampsia and 21 (34%) cases of late eclampsia. As shown in the table antenatal eclampsia cases had higher blood pressures (p=0.02), more abnormal proteinuria (p=0.002) and lower platelet counts (p<0.001) than postnatal cases. Early eclampsia outcomes were similar to those of late eclampsia with the exception that early cases were more often complicated with HELLP syndrome (p=0.007).

Conclusion: Eclampsia continues to be a rare complication of pregnancy. Our data indicate that antenatal eclampsia may be a different and more severe disease than postnatal eclampsia. Early and late eclampsia seem to have similar characteristics and outcomes with the exception that HELLP syndrome is more often present in patients who develop early eclampsia than in patients with late eclampsia.

Role of Matrix Metalloproteinase-14 (MMP-14) in Trophoblast Invasion and Preeclampsia. Serkalem Tadesse, 1 Tianmeng Luo, 1 Vikki Abrahams, 1 Seth Guller, 2 Paolo Toti, 3 Felice Arcuri, 3 Errol Norwitz, 1 Anika Agarwal. 1 1 Ob/Gyn, MIRI and MORI, Tufts University, Boston, MA, USA; 2 Ob/Gyn, Yale University, New Haven, CT, USA; 3 Human Pathology & Oncology, Univ. of Siena, Siena, Italy. OBJECTIVE: Failure of extravillous cytotrophoblast (EVCT) cells to invade and remodel the maternal vasculature leads to suboptimal placentation that manifests clinically as preeclampsia (PE). MMP-14 is an upstream regulator of MMP-9, MMP-1, and G protein-coupled protease-activated receptor, PAR1, all of which have been shown to regulate angiogenesis and invasion in a variety of human cancers (Agarwal, Mol Cancer Ther 2008; 7:2746) . Although expressed in the placenta, the role of MMP-14 in PE has not been examined. This study investigates the expression of MMP-14 in preeclamptic placentas and in EVCTs in vitro under hypoxic and normoxic conditions. METHODS: Expression of MMP-14 in placentas from patients with PE vs gestational age-matched normotensive controls (n=5 for each) was examined by immunohistochemistry (IHC) and quantified by H-scoring. EVCTs were isolated and purified from placentas collected from elective cesarean at term Introduction: Limited information exists regarding post partum persistence of pregnancy induced hypertensive (PIH) disorders. We aimed to identify risk factors that may serve as early indicators of persistent or worsening disease. Materials and Methods: A case-control review of women admitted postpartum (<6 weeks after delivery) with persistent severe or worsening PIH diagnosed antenatally from January 2008 to June 2012 was performed. Mann-Whitney U and chi-square tests were used; a P value <0.05 was considered significant. Results: Among 46 cases and 92 controls, hematocrit < 30% (OR 8, 95% Cl 1.79-35.74), body mass index (BMI) > 30 lbs./in2, lactate dehydrogenase (LDH) > 450 IU/L (OR 4.5, 95% Cl 1.09-20.1), and tobacco use (OR 7.5, were identified as significant risk factors for readmission with worsening or persistence severe PIH. Similar use of antihypertensive medications and magnesium sulfate was observed among cases and controls. Conclusion: In a subset of women, delivery may not be the culminating event of PIH as traditionally considered. As expected, obesity and increased LDH are risk factors for readmission. Contrary to what occurs antenatally, hemoconcentration was not associated with worsening disease while tobacco use was not protective for worsening or persistent severe PIH. The use of antenatal or intrapartum antihypertensives had no significant impact on the risk of readmission. Epidemiology and Clinical and Translational Research, University of Pittsburgh, USA; 4 Centre for Public Health, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast; 5 School of Nursing and Midwifery, Queen's University, Belfast; 6 Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, United Kingdom. Objective: Haptoglobin is a powerful antioxidant and proangiogenic factor, however the proangiogenic and antioxidant capacities of haptolgobin differ between the 1-1, 2-1 and 2-2 phenotypes. Haptoglobin phenotype predicts cardiovascular risk and responsiveness to vitamin E, or vitamin C and E, supplementation in non-pregnant individuals with diabetes. This study determines whether haptoglobin phenotype influences preeclampsia risk, or the effectiveness of Vitamin C and E in preventing preeclampsia, in women with Type 1 diabetes. Methods: This is a secondary analysis of a randomized controlled trial in which 762 women with Type 1 diabetes received daily vitamins C and E, or placebo, from 8-22 weeks gestation until delivery. Haptoglobin phenotype was determined in white women who completed the study, and had samples available (n=685). Results: Compared to haptoglobin 2-2, haptoglobin 1-1 (odds ratio: 0.67 (95% confidence interval: 0.34-1.34)) and 2-1 (1.07 (0.69-1.65)) were not associated with significantly decreased preeclampsia risk. Vitamins C and E did not prevent preeclampsia in women of any haptoglobin phenotype. 

Haptoglobin phenotype did not significantly affect preeclampsia risk, or modify the effectiveness of Vitamins C and E in preventing preeclampsia, in women with Type 1 diabetes. The peptides of calcitonin gene-related peptide (CGRP) family, CGRP, adrenomedullin (AM) and intermedin (IMD) are potent vasodilators and we have shown mesenteric vasorelaxation responses to all 3 peptides are amplified during pregnancy. All 3 peptides use a common 7-TM receptor, calcitonin receptor like receptor (CRLR) and one of the receptor activity modifies protein (RAMP) and the specificity appear to be related to RAMP type: RAMP 1 for CGRP; RAMP 2 or 3 for AM; and any RAMP for IMD. Because all 3 RAMPs are expressed in vascular smooth muscle, we examined the specificity of RAMP involvement in each of the peptides action. Methods: We assessed generation of cAMP by mesenteric artery (MA) vascular smooth muscle cells (VSMC) in vitro upon CGRP, AM and IMD stimulation, in the presence of their antagonists or in cells with specific RAMP inhibition using shRNA methods. VSMC from MA of nonpregnant rat at passages 2-4 are used and plated into 6 well plates. Following 24h in serum free DMEM, cells are treated for 1h with 1µM of CGRP 8-37 , AM 22-52 or IMD 17-47 and then challenged with 1, 10 or 100 nM of CGRP, AM or IMD for 5 min in the presence of 1BMX (0.1mM). cAMP in cell lysates were measured by RIA kit. In the 2 nd set of studies, cAMP responses to all 3 peptides were assessed after RAMP 2 or 3 mRNA was inhibited (shRNA: more than 80% inhibition). Results Objective: Emerging evidence has shown that podocyturia is an indicator of podocyte injury in preeclampsia (PE). Recent studies also found podocyte protein nephrin and podocalyxin in urinary specimen in PE. This study was undertaken to test our hypothesis if urinary podocyte protein can be used as a biomarker of postpartum podocyte function recovery in PE. Methods: Urinary specimen was collected from 50 pregnant women, 17 from normal and 33 from PE pregnancies before delivery. Among them, urinary specimen was also collected 2-3 months after delivery from 8 normal and 10 PE pregnancies. Urine levels of nephrin, podocalyxin, creatinine, and protein were measured by ELISA. Concentrations of nephrin and podocalyxin were normalized by creatinine levels. Data are expressed as mean ± SE and p<0.05 was considered statistically different. Results: 1) Prenatal urine nephrin and podocalyxin levels were significantly higher in PE than in normal pregnancies, nephrin: 4.64±1.2 vs. 1.07±0.14 µg/mg creatinine, p<0.05; and podocalyxin: 196±15.4 vs. 12.1± 2.7 ng/mg creatinine, p<0.01, respectively; 2) Both nephrin and podocalyxin levels were significantly reduced after delivery in PE, nephrin: 1.48±0.22 µg/mg creatinine, p<0.01 and podocalyxin: 5.9 ± 1.6ng/mg creatinine, p<0.01. There was no significant difference in nephrin and podocalyxin levels before and after delivery in normal pregnancies; 3) Prenatal urine nephrin and podocalyxin levels are highly correlated in PE, r2=0.88, but not in normal pregnancies, r2=0.006; and 4) Concentrations of prenatal nephrin and proteinuria were highly correlated in PE, r2=0.388, but postpartum concentrations were not, r2=0.041. Conclusions: Urine nephrin and podocalyxin levels are significantly higher before delivery but significantly reduced after delivery in women with PE. Reduced urinary nephrin and protein levels postpartum in PE indicate that urinary nephrin levels could serve as a sensitive biomarker of podocyte function. Nephrin is a specific podocyte slit diaphragm protein and proteinuria is a signature of glomerular barrier dysfunction; therefore urinary nephrin levels could be an ideal biomarker to monitor ongoing podocyte injury during PE and reduced/absent urinary nephrin level could be an indicator of podocyte function recovery postpartum in PE.

Impact of Subclinical Hypothyroidism in Women with Recurrent Pregnancy Loss. Lia A Bernardi, 1, 2 Early pregnancy impacts thyroid function. The effects of overt hypothyroidism on pregnancy are well documented, but the impact of subclinical hypothyroidism (SCH) is less clear. Studies suggest an association between SCH and sporadic miscarriage. The objective of this study was to assess SCH in women with recurrent pregnancy loss (RPL).

Observational cohort study using prospectively collected data in an academic RPL Program. Methods Entry criteria included women: 1. Seen in consultation by MDS from July 2004 to December 2011, 2. With a history of RPL, defined as two or more documented pregnancy losses under 10 weeks of gestation, excluding "explained" miscarriages due to numeric chromosome errors, 3. With an RPL evaluation, which included a serum thyrotropin (TSH) 4. Who gave written consent. From 2004-2007, women with SCH, defined as a TSH >2.5 mIU/L with a normal free thyroxine, were not treated. From 2008, levothyroxine was prescribed prepregnancy, with dosing adjusted to maintain the TSH ≤2.5mIU/L. Chi-squared or Fisher's exact tests were used for categorical variables. A generalized estimating equations (GEE) approach was used to account for multiple subsequent pregnancies in the same woman, which ranged from one to five. P<0.05 was accepted as statistically significant. Results 286 women met criteria. The prevalence of SCH was 19% (55/286). 68% (193/286) of the women were euthyroid and 13% (38/286) had overt thyroid disease. 180 of the women had 290 subsequent monitored pregnancies. The cumulative live birth rate (LBR), defined as a live birth or ongoing pregnancy, was 69% (27/39) for RPL/SCH vs. 74% (104/141) for RPL/euthyroid (p= 0.57). The per pregnancy LBR was 49% (34/69) for RPL/SCH vs. 58% (129/221) for RPL/euthyroid (p=0.28). The cumulative LBR was 71% (17/24) for RPL/treated SCH vs. 67% (10/15) for RPL/untreated SCH (p=1.00). The per pregnancy LBR was 48% (22/46) for RPL/treated SCH vs. 52% (12/23) for RPL/untreated SCH (p=0.83).

The prevalence of SCH in this cohort of RPL patients was higher than expected. There was no significant difference between groups for cumulative or per pregnancy LBR. To confirm that SCH is associated with a 5% lower LBR in RPL, a sample size of 2,558 would be required.

Modulation of Cx43 by Retinoic Acid in Human Granulosa Cells. Monica W Best, Neil Sidell. Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, GA, USA. Oocyte growth and development in the ovarian follicle are dependent on gap junction communication (GJC) between granulosa cells and the oocyte. Gap junctions (GJs) are transmembrane proteins consisting of hydrophilic pores which allow trans-cellular flow of ions and small molecules. Connexin (Cx) proteins are the major components of GJs with Cx43 being most widely expressed in granulosa cells. The channel gating of Cx43 is modulated by phosphorylation of various amino acid residues and is regulated by gonadotropins, steroid hormones, and cytokines. Previous animal studies have highlighted the modulation of Cx43 by luteinizing hormone (LH) leading to resumption of meiosis by the oocyte. Our experiments focused on exploring modulation of Cx43 in human granulosa cells by retinoic acid (RA) because of its essential role in reproduction. RA is known to exert effects on folliculogenesis, steroid production, oocyte maturation, and embryogenesis. We found that treatment of primary human cumulus granulosa cells with RA enhanced expression of the non-phosphorylated (P0) form of Cx43 in a dose dependent fashion while decreasing the phosphorylated (P1 and P2) Cx43 species.

This finding has also been reproduced in KGN cells, a human granulosa cell line. RA-mediated dephosphorylation was shown to occur at the serine-262 residue of Cx43. Dephosphorylation of Cx43 at serine-262 has been associated with upregulation of GJC in other tissues. Additionally, human chorionic gonadotropin (HCG) enhanced the overall protein levels of Cx43 without changing its phosphorylation status while addition of RA along with HCG caused a similar shift in phosphorylation species of Cx as seen with RA alone. Our studies have also determined that human cumulus granulosa cells are capable of producing retinoic acid from retinol and that high follicular fluid RA levels were associated with oocytes yielding higher quality embryos with in vitro fertilization. Together, these findings suggest that RA plays a beneficial role in oocyte development and folliculogenesis through modulation of GJC, perhaps in concert with LH/HCG signaling, through alteration of the phosphorylation status of Cx43. , Chandra Yallampalli, Kunju Sathishkumar. Ob/Gyn, University of Texas Medical Branch, Galveston, TX, USA. Animal and human studies have shown sex differences in the development of hypertension. Although most studies focused on estrogen, several observations suggest that testosterone may contribute for development of hypertension. Studies show that castration reduced hypertension and testosterone replacement reversed this antihypertensive effect of castration. Further, treatment with androgen antagonists mimicked the effects of castration. Testosterone treatment increased vascular resistance and responsiveness to vasoconstrictor agents in animals and humans. Thus, androgens appear to amplify the hypertensive process. Our previous studies show that long-term exposure to elevated levels of testosterone increased contractile responses of angiotensin II (Ang II) in resistance sized mesenteric arteries. The molecular mechanisms contributing to androgen amplification of vascular reactivity remain to be fully elucidated. We hypothesized that androgens may potentiate mesenteric vascular responses to Ang II by directly upregulating the expression of key regulators of vascular tone, Ang II receptors (AT 1 R and AT 2 R) and/or protein kinase C (PKC). Methods: Mesenteric arterial smooth muscle cells were isolated from rats. These cells were cultured with testosterone (5nM-100nM) for five days with fresh medium containing testosterone replaced every day. The expression of AT 1 R and AT 2 R and PKCα and δ isoforms were assessed by qPCR and Western blot. We used bioinformatics to identify and ChIP assay to validate novel androgen response elements (ARE) in the PKCδ gene. Results: Testosterone treatment did not change the expression of AT 1 R and AT 2 R. However, PKCα and PKCδ levels were upregulated with PKCδ showing a dramatic dose dependent increase.

Analysis of the PKCδ promoter demonstrated presence of five putative androgen responsive elements (ARE) located between -4716kb to -1350kb. Furthermore, ChIP assay showed that the androgen receptor binds to the PKCδ ARE in response to testosterone stimulation. Conclusion: We demonstrate for the first time that PKCδ is a physiological target for testosterone, and androgens can directly activate the transcription of PKCδ via novel AREs present upstream to the PKCδ gene. Such regulation may have considerable effect on PKCδmediated contractile responses.

Effect of Biologic pH Buffers on the Rate of Embryo Development and Blastocyst Size. Natalie A Clark, 1 Matthew Will, 2 Jason E Swain. 3 1 OB/GYN, University of Michigan; 2 REI, Midwest Fertility Specialists; 3 REI, University of Michigan. INTRODUCTION: pH buffers utilized in culture media play a crucial role in ART, stabilizing the environment for cells. Prior studies indicate cellspecific sensitivity to individual buffers, with some buffers compromising cell development and function. This may be true for embryos. It has been suggested that buffers may impair Cl-channel function and blastocoel formation. This study sought to examine various biologic pH buffers and determine their impact on embryo development. METHODS: HTF media was formulated using 5mM bicarbonate supplemented with 21mM of either HEPES, MOPS, TES, . Media were compared in their ability to support mouse embryo development over 96h. Groups of ten 1-cell embryos were cultured in 500µl of media under 300µl oil at 37ºC and gassed with 5% CO2 for 45min tid. Embryo development was examined using Cronus imaging software. Blastocyst cell numbers were counted following Hoescht staining. Data were collected over 3 replicates and analyzed using ANOVA with Tukey analysis RESULTS: No difference in cleavage past the 1-cell stage at 6h was apparent between any of the 5 buffers studied. Use of TES yielded higher rates of embryo development >2-cell at 30h (45%±6.5) compared to PBS (22.5%±10.3; p<0 .02), similar to rates from HEPES (37.5%±7.5), MOPS (25%±8.7) and DIPSO (27.5%±4.8) . No differences in rates of 8-cell development or early compaction were apparent at 48h, nor were rates of early blastocyst formation at 72h. Use of PBS yielded lower rates of total blastocyst formation at 96h (50%±4.1) compared to HEPES (90%±5.8), MOPS (75%±2.9), TES (90%±0) and DIPSO (80%±7.1) (p<0.01). There were no differences in rate of blastocyst hatching at 96h between buffers, though TES tended to yield higher rates compared to PBS (p<0.08). No differences in total blastocyst cell number were apparent. Comparison of blastocyst perimeter demonstrated that TES yielded significantly larger blastocysts (12815µm) compared to PBS (10822µm; p<0.03), similar to HEPES (11114µm), MOPS (11961µm) and DIPSO (11539µm) . CONCLUSION: This is the first report examining the impact of pH buffers on blastocyst size. Commonly used buffers, HEPES and MOPS, yielded similar results for all endpoints, similar to DIPSO. PBS is an inappropriate buffer for embryo culture. TES may be an alternative and potentially superior buffer, yielding the highest rates of blastocyst development and size. Our previous studies have shown that the downregulation is the result of a post-transcriptional mechanism mediated by an LHR mRNA binding protein (LRBP). Further studies revealed that LRBP causes accelerated degradation of LHR mRNA. Since LH/hCG has been shown to regulate VEGF-A expression in the ovary, the preset study focused on the consequence of LHR mRNA downregulation on VEGF-A, one of the most important regulators of angiogenesis in the ovary. 23 day old female rats were treated with a single dose of PMSG (50 I U) to induce follicle growth followed 56 h later by varying doses of hCG (6.25 IU, 12.5 IU, 25 IU, or 50 IU) to induce ovulation. At 12 h, when the expression of LHR mRNA has been shown to undergo downregulation, ovaries were harvested and, LHR mRNA and VEGF-A mRNA expressions were determined by real-time PCR. A second group of ovaries were processed for determination of LRBP activity, by RNA electrophoretic mobility gel shift assay. The results showed that LHR mRNA downregulation occurs following the administration of hCG in a dose-dependent manner with a fold change of 0. 85, 0.36, and 0.28 in response to 12.5, 25, and 50 IU of hCG, respectively (p<0.05, n=4) , compared to saline treated controls. A parallel decrease in VEGF-A mRNA expression was also seen with a fold change of 0.88, 0.79, 0.73, and 0.14 for rats receiving 6.25, 12.5, 25, and 50 IU of hCG respectively (p<0.05, n=4) , compared to the controls. LRBP activity showed an expected increase during LHR mRNA downregulation, consistent with the notion that LHR mRNA expression undergoes downregulation with a corresponding increase in LHR mRNA binding activity. What was most striking was the finding that VEGF-A mRNA expression showed a parallel, dose dependent decline when LHR mRNA expression was downregulated in response to treatment with ovulatory doses of hCG. Given the known role of VEGF-A in ovarian hyperstimulation, these results suggest that LHR mRNA downregulation in response to an ovulatory bolus of LH/hCG might serve as a mechanism to prevent ovarian hyperstimulation by transiently downregulating the expression of VEGF-A expression.

Effect of the Anti-Androgenic Endocrine Disruptor Vinclozolin on Human CYP3A4 Expression In Vitro. Oumar Kuzbari, 1 John G Lamb, 2 Ahmad Hammoud, 1 Erica B Johnstone, 1 Michael R Franklin, 2 C Matthew Peterson. 1 1 Department of Obstetrics and Gynecology, University of Utah, Salt Lake City, UT, USA; 2 Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, USA. Objective: Vinclozolin is a common fungicide with antiandrogenic properties that has been reported to interact with the pregnane X receptor (PXR). Human CYP3A4 enzyme has an essential role in hydroxylation of steroid hormones and is regulated at the molecular level by the PXR. The purpose of our study is to determine the effect of vinclozolin on CYP3A4 transcriptional expression in the engineered human hepatoma cell line, DPX2. Design: Laboratory-based study. Materials and Methods: DPX2 cells, human hepatoma cells that are stably integrated with human PXR and a luciferase construct containing the CYP3A4 promoter, were exposed to vinclozolin at relevant concentrations in a 96 well plate. Activation of human PXR regulated gene expression was determined by luciferase activity (normalized to cell viability). All assays were measured with a BioTek Synergy2 plate reader. DMSO vehicle served as control. Results: Vinclozolin (10-50µM) caused a significant activation of the CYP3A4 promoter, via the PXR compared to control (p<0.05). The calculated EC50 was 10µM, and the calculated maximum induction was 4 fold Effect of Vinclozolin on CYP3A4 Promoter Expression as Indicated by the Luciferase Luminescence Activity Assay in Human DPX2 Cells

Mean fold induction in CYP3A4 promotor expression and standard deviation DMSO 1 +-0.5 Vinclozolin 1µM

1.8 +-0.2 Vinclozolin 5µM 1.8 +-0.5 Vinclozolin 10µM

2.6 +-0.7* Vinclozolin 25µM 3 +-0.5* Vinclozolin 50µM 3.5 +-0.8* *statistically significant at p<0.05 compared with DMSO-treated control Conclusions: Vinclozolin, in relevant exposure ranges, demonstrates induction of CYP3A4 expression in human hepatoma cells in vitro which may affect endocrine function by altering steroid hormone metabolism/action through a PXR mediated mechanism, in vivo. Objective: Recurrent pregnancy loss (RPL) and fetal miscarriage (FM) are devastating conditions for couples trying to have a child. While chronic endometritis has been implicated in RPL and FM, the prevalence and outcomes following treatment have not been reported. The objectives of this study were to ascertain the prevalence of chronic endometritis in RPL and/or FM, to determine the cure rate following a course of antibiotics, and to report subsequent pregnancy outcomes. Design: Observational cohort of prospectively collected data in an academic RPL Program. Methods: IRB approval was obtained. Chronic endometritis was defined by the presence of plasma cells within the endometrium.

Entry criteria: 1. Seen in consultation by MDS from 2004 to 2012, 2. Had a history of RPL, defined as ≥2 pregnancy losses of <10 wks size, and/ or FM, defined as an intrauterine demise of 10-20 wk size, 3. Had an endometrial biopsy (EB), and 4. Gave written consent Potential subjects were identified with ICD-9 codes and RPL Database (Microsoft ACCESS 2007) . Data was transferred to EXCEL 2007 for analysis. Results : 396 women met criteria. Overall, the prevalence of chronic endometritis was 8.8% (35/396); 7.5% (21/282) in RPL, 14.0% (8/57) in FM, and 10.3% (6/58) in women with both RPL and FM. Objective: Ulipristal acetate (UPA), a selective progesterone receptor modulator (SPRM), is currently approved in the United States for use as emergency contraception. In has recently been approved in the European Union as a preoperative treatment for uterine fibroids due to its ability to decrease fibroid size. Prior concerns regarding a theoretical increase in endometrial neoplasia have not been substantiated and studies have concluded there is no increase risk of endometrial hyperplasia with use of UPA for 3-6 months. To date, no studies have evaluated the impact of UPA therapy on the histology of fallopian tube. In this case series, we evaluated the impact of UPA use on fallopian tube pathology. We hypothesized that exposure to UPA for 3-6 months will not detrimentally affect the epithelium of the fallopian tube. Design: Retrospective Case Series Materials and Methods: This is a sub-analysis of a randomized-controlled trial evaluating the effect of UPA on leiomyoma size. Patients that underwent total abdominal hysterectomy and bilateral salpingo-ophorectomy at the end of treatment were retrospectively identified. Two pathologists reviewed the hysterectomy specimens for evidence of hyperplasia and carcinoma. Immunohistochemical (IHC) staining was preformed for p53, MiB-1, estrogen receptors, and progesterone receptors if any aytpia was discovered.

Results: Four patients that underwent surgical management at the end of the trial were identified. The duration of treatment with UPA was 3-6 months. No occurrences of fallopian tube cytological atypia were identified in this case series. Immunohistochemical staining revealed no evidence cellular markers concerning for future development of fallopian tube malignancy Conclusions: The fallopian tube pathology from hysterectomy specimens of four patients was normal. This small case series demonstrates that short-term exposure to UPA does not predispose to fallopian tube neoplasia. Larger studies are needed to confirm these findings. Support: Program in Reproductive and Adult Endocrinology, NICHD, NIH

The As a tumor expands from a functional blood supply, the metabolic needs of the cancer cells deplete the environment of oxygen and nutrients, resulting in hypoxia. Master regulators of the hypoxic response are the Hypoxia-Inducible Factors (HIFs). HIFs are transcription factors stabilized under hypoxic conditions that induce genes promoting cell survival, including glycolytic enzymes, migration factors, and angiogenic factors. The histone demethylase JMJD2B is also a HIF target, linking the hypoxic tumor microenvironment to epigenetic regulatory mechanisms. In a microarray analysis using siRNA to JMJD2B in HCT116 colon carcinoma, RCC4 clear cell renal cell carcinoma, and SKOV3ip.1 ovarian serous adenocarcinoma cell lines, we identified sets of potential JMJD2B targets with clear associations to cancer cell growth, migration, and metastasis. While hundreds of genes were specifically regulated in each cel line by JMJD2B, 17 genes were commonly regulated in all three lines, and their expression confirmed by quantitative real-time PCR. More than half of the 17 commonly regulated genes have documented contribution to tumor progression, identifying some general mechanisms regulated by JMJD2B in hypoxia. Ingenuity Pathway Analysis identified oxygen dependent regulation of cellular mechanisms by JMJD2B, with a greater proportion of genes involved with cell proliferation in atmospheric conditions and more genes involved with inflammation and cellular movement in hypoxia. In vitro functional analysis using siRNA and stable shRNA constructs against JMJD2B confirmed the contribution of JMJD2B to SKOV3ip.1 proliferation in 21% oxygen, while invasion was affected in 21%, 2%, and 0.5% oxygen. Immunohistochemical analysis of serous adenocarcinoma biopsies demonstrated robust expression of JMJD2B in cancer compared to benign masses and normal ovary. Combined, our results suggest that JMJD2B may contribute to ovarian cancer progression, making JMJD2B or its target genes potential candidates for improving existing therapies. We previously showed that offspring exposed to chronic maternal low protein diet (MLP) in early development have significantly lower body weights up to 1 year of age (1y). Gene expression profiling revealed significant overexpression at 1y of genes in the liver that encode cohesins, which are important for chromatin organization and long-range gene expression regulation. To determine whether there are potential metabolic ramifications of these changes, we characterized the metabolic phenotype of the adult male offspring. C57BL/6J dams were fed 8% (MLP) or 20% (C) protein diets from four weeks prior to mating throughout lactation. Male pups were weaned to a standard lab rodent diet and single-housed at P21 until 8-16 weeks of age [n=7/treatment]. Body composition was then measured by Quantitative Magnetic Resonance (QMR), followed by a 5d assessment of energy expenditure (EE), food intake and cage activity using a Comprehensive Laboratory Animal Monitoring System (Columbus Instruments). Effects of treatments were compared using ANOVA. For energy expenditure and food intake, individual values for lean and fat were used as covariates.

There was no difference in body weight, but MLP offspring had significantly increased % body fat compared to controls (P=0.027) even though their food intake was significantly lower than for control offspring (P<0.05); the difference in intake was manifested only at night (active phase; P<0.05). Consistent with these differences in intake, respiratory quotient as higher for controls at night (P<0.001) and similar for both groups in the daytime (P>0.9). Total EE was significantly lower in MLP offspring (P<0.05) and this also was evident only at night (P<0.01). There was no difference between treatments in daytime (resting phase) EE (P>0.1) or resting EE (P>0.2). There were no differences in spontaneous cage activity. The differences in the daily patterns of energy intake and expenditure suggest that the MLP diet influenced the diurnal pattern of feeding behavior in the offspring. This had consequences for EE, and because activity and resting EE were similar, it suggests that nonshivering thermogenesis and/or the thermic effect of food most likely were responsible. Ultimately, these differences in EE were sufficient to produce differences in energy balance that were manifested by the difference in adiposity of the two groups. Studies have shown that prenatal maternal protein restriction leads to altered behavior in progeny, but this has not been studied in chronic maternal protein restriction and the underlying mechanisms are not well understood. We previously reported that in mice, offspring exposed to chronic maternal low protein diet (MLP) have significantly lower body weights up to 1 year of age (1y), lower hind-leg muscles weights at 21 days (P21) and 1y, decreased serum levels of ALT and LDH, and significant gene expression differences in 1y liver, but not in muscle. To study if these phenotypes are also accompanied by behavioral abnormalities, we performed behavioral assessment of adult male offspring exposed to chronic MLP during pregnancy and lactation.

We fed adult C57BL/6J dams an 8% protein diet (MLP) or 20% protein control diet (C) from four weeks prior to mating throughout lactation. Male pups were weaned to standard lab rodent diet and single-housed at P21 (N=9/each group). Adult male offspring (8-16 weeks of age) underwent detailed behavioral analysis (3-Chamber Test for social interactions, Elevated-Plus Maze, Open-Field Activity, Automated Hole Board and Marble Burying). Male offspring exposed to chronic MLP diet during pregnancy and lactation demonstrated anxiety-like behavior. On the Elevated-Plus Maze, MLP-exposed offspring spent less time in the open arm than C-exposed offspring (p=0.018), more time in the center (p=0.005) and decreased latency to first entry in closed arm (p=0.01). MLP-exposed offspring also had less digging behavior on the Marble Burying test (p=0.043). In the 3-chamber test, both C-exposed (p=0.003) and MLP-exposed mice (p=0.014) interacted more (sniffing behavior) with a novel mouse than a novel object. However, both spent equal times in chambers with a novel mouse or a novel object [p=0.17 (MLP); p=0.8 (C)]. Open-Field Activity and Automated Hole Board test did not reveal any significant differences between the groups. Our results demonstrate that chronic maternal protein malnutrition during early development may lead to anxiety in male offspring. The changes in social behavior in both MLP and C-exposed mice were unexpected. We propose that this may be related to post-weaning single-housing of animals. Future experiments will focus on studying the molecular mechanisms that lead to the altered developmental programming of behaviors caused by chronic maternal protein deprivation.

Untreated out of these regressed spontaneously back to normal or low-grade lesions. Nineteen (18%) defaulted one or more follow-up visits. The likelihood of regression increased with young age, small lesion size, low-grade cytology or colposcopy and HPV subtypes other than 16 (p<0.05). Results on HPV-related biomarkers are available in 40% of the included cases and will be presented. High-risk HPV DNA test had the highest sensitivity (94%) but relatively lower specificity. The combination of tests improved specificity to 96%. There were no progressive invasive lesions. Conclusion: Some of the combinations of HPV-related biomarkers may have significant accuracy in predicting lesions likely to regress. This could allow conservative management for women at low risk and avoidance of unnecessary intervention and/or treatment. The estimated proportion of excision varied significantly between 5% and 41%(median 13%). Multivariate linear regression revealed that the proportional deficit at 6 months was determined mainly by the proportion of the excised volume. Subgroup analysis revealed similar findings for each imaging technique. Fourteen women have conceived following treatment. Nine have already delivered, 6 at term, two at 35-36 and one at 33 weeks of gestation. The risk of prematurity seems to correlate to the proportion of excision.

Conclusions: Careful assessment of risks and benefits of treatment is essential when deciding to treat women who wish to have future pregnancies. Assessment of the proportion of the volume/length excised might identify those that need further surveillance during future pregnancy. We previously showed that offspring exposed to chronic maternal low protein diet (MLP) in early development have significantly lower body weights up to 1 year of age (1y), lower hind-leg muscle weights at 21 days (P21) and 1y, and decreased serum levels of liver enzymes (ALT, LDH) at 1y. Gene expression profiling revealed no differences in skeletal muscle at P21 and 1y, but significant overexpression in liver at 1y of genes encoding cohesins, which are important for chromatin organization and long-range gene expression regulation. To further explore why mice have lower body-weights and what the role for cohesin gene expression changes is in other organs after MLP, we performed gene expression profiling in the hypothalamus of offspring, as well as targeted analysis of candidate genes with a known role in satiety control. We fed adult C57BL/6J dams an 8% protein diet (MLP) or 20% protein diet (C) from 4 weeks prior to mating throughout lactation. Male pups were weaned to standard lab rodent diet and single-housed at P21. Mice were sacrificed at P21 and 1y and RNA from 3 MLP and 3 C hypothalami at each age were hybridized to Affymetrix Gene 1.0 ST arrays. The microarray data were array and gene centered and two-sample T-statistics were calculated for each gene with variance inflation factor of 0.05, to correct for small sample sizes. T-statistics were further adjusted via central-matching and corrected for multiplicity by the Benjamini-Hochberg step-up procedure. RT-qPCR was used for confirmation as well as for the study of gene expression of the orexigenic-anorexigenic pathway.

After controlling the FDR at 10%, we found 316 genes at P21 and 141 at 1y with significantly altered expression in hypothalamus after MLP exposure, but we could not confirm any by RT-qPCR analysis at P21, and only one gene, Actr10 (P=0.01; n=3/group) at 1y. We did not find any significant differences in the genes from the orexigenic-anorexigenic pathway.

The absence of differences in gene expression at P21 and the minor differences at 1y indicates that observed phenotypes are not associated with long-term detectable gene expression changes in total hypothalamus. Future experiments will focus on regional differences in selected hypothalamic nuclei.

Differences (Fig. 1 ). In contrast, postnatal GC exposure at P4/5 induced a hyporesponsive HPAA. Prenatal GC at E15/E16 led to longer floating times in the FST suggesting depressive behavior (p<0.05). Prenatal GC at E19/20 worsened motor stroke outcome (Fig. 1 ). GC exposure on other days had less pronounced effects on behavior and stroke outcome (Fig. 1 There were no differences between groups in vascular responses to the alpha agonist phenylephrine, the endothelium-dependent vasodilator methacholine or the endothelium-independent-vasodilator sodium nitroprusside. However, big endothelin-1 (bET-1) mediated constriction was reduced in resveratrol-treated offspring (28% reduction in the area under the curve of the bET-1 cumulative concentration curve; P<0.05). L-NAME potentiated the bET-1 constriction in both groups (P<0.001), and normalized the differences between control and resveratrol-treated offspring. Prenatal resveratrol treatment also lowered fasting glucose levels (-9%; P<0.05), as well as fat mass (-13%; P<0.05) which corresponded with an increase in lean body mass (+1.7%; P<0.05).

Conclusions: Resveratrol treatment improved vascular and metabolic outcomes in the SHR, despite no changes in BP. While these effects may be subtle, the therapeutic benefits may be appreciable in conditions associated with reduced physiological reserve, such as with advanced age. Interestingly, resveratrol appears to be tolerated during pregnancy, but was detrimental to neonatal growth during lactation.

Negative Effects of Antenatal Betamethasone Treatment in Twin Pregnancies. Thorsten Braun, 1, 4 Hanna Gil, 1 Boris Tutschek, 3 Deborah Sloboda, 4 Thomas Harder, 1, 4 Conclusion: Antenatal BET therapy in twin pregnancies led to a reduction in fetal growth in twins with potential consequences for the long-term morbidity.

Higher BET doses resulted in the greatest growth reduction in male twins, without further improvement in Apgar scores. These data suggest that male twins appear more sensitive to the growth restricting effects of antenatal BET therapy, compared to twins that have one or more females. 

It is reported that male infants with prenatal betamethasone exposure exhibit higher systolic blood pressure than females in their early adolescent. The mechanism is not well known. Previous study showed that alterations in the ACE and ACE2 pathways might be involved in developing hypertension, but the data is controversial. For example, in isolated proximal tubules, 50% lower ACE2 activity and no change in ACE activity was noted in adult male sheep with prenatal betamethasone exposure. Current study was designed to compare the effect of prenatal steroid exposure on renal cortex ACE and ACE2 activity in male and female sheep.

Date-mated sheep received saline or betamethasone (2 doses of 0.17mg/kg, 24 hour apart) at the 8oth day of gestation and delivered at term. At 1.8 years, sheep were anesthetized and nephrectomy was performed. Renal cortex was dissected on ice and frozen at -80 ºC for further study. ACE and ACE2 activities (expressed as fmol/min/mg protein) were assayed by radioactivity assay. All data were expressed as the means±SEMs. t test was used for data analysis. P<0.05 was used as statistical significance.

In male sheep, compared to saline group (C), prenatal steroid exposure (S) had no effect on ACE activity (1247±77.1 (C) vs. 1204±68.7 (S)), but decreased ACE2 activity (1580±129.4 (C) vs. 1101±105.6* (S), *p<0.05 vs. control) and consequently increased ACE/ACE2 ratio (0.82±0.07 (C) vs. 1.12±0.06* (S), *p<0.05 vs. control).

In female sheep, prenatal steroid exposure had no effect on ACE activity (568±196.3 (C) vs. 521±134.6 (S)), tended to increase renal ACE2 activity (690±163.2 (C) vs. 1173±115.5 (S)) and decreased ACE/ACE2 ratio (0.99±0.33 (C) vs. 0.54±0.09 (S)).

Prenatal betamethasone exposure caused gender specific effects on renal ACE and ACE2 activity. Decreased ACE2 activity and increased ACE/ACE2 ratio may favor elevating blood pressure in male sheep with prenatal betamethosone exposure.

Maternal Malnutrition Impacts Fetoplacental Growth and Placental Fatty Acid Transport in Late Gestation. KL Connor, 1 M Lee, 1 R Maganga, 1 E Bloise, 2 SJ Lye. 1 1 Samuel Lunenfeld Research Institute, Mt Sinai Hospital; 2 Physiology, Univ Toronto. Placental uptake of maternal fatty acids (FA) is required for optimal fetal growth and development. We, and others, have shown that maternal underand overnutrition influence the development of the fetus and its metabolic health long-term. We therefore asked whether changes in maternal nutritional status would adversely impact fetal development via alterations in placental FA transport/metabolism and thus suggest a role for placental FA transport in modifying susceptibility to diet-induced metabolic programming. Female mice were randomised to three groups: mice fed a control diet throughout pregnancy (CON), mice calorically restricted by 30% from embryonic day 5.5 to 18.5 of pregnancy (CR), or mice fed a 60% high fat diet from 8 weeks before mating and throughout pregnancy (HF). At E18.5 (term=19d) maternal, placental and fetal tissues were collected. mRNA expression of genes critical for FA transport/metabolism in the placenta was determined by qPCR and Pfaffl's relative ratio. Data were analysed by one-way ANOVA with Tukey's post-hoc test. Significance p<0.05. Maternal HF diet was associated with maternal hyperleptinaemia, but did not alter maternal weight or fetoplacental weights compared to CON. HF diet increased PPARγ2 expression in male placentae, but did not alter other key FA transport genes. In female placentae HF diet was associated with a trend towards decreased fatty acid translocase (FAT/CD36) and FA transport protein 1 (FATP1) expression compared to CON. Conversely, CR was associated with reduced maternal plasma insulin and leptin concentrations, lower maternal weights and lighter placentae and fetuses in both males and females compared to CON. In male placentae, CR increased PPARγ2, but decreased FAT/CD36 and lipoprotein lipase expression compared to CON. In female placentae, CR did not alter PPARγ2 expression compared to CON but increased its heterodimeric partner retinoid X receptor α. Further, CR increased endothelial lipase and plasma membrane FA binding protein expression, but decreased FATP1 expression in female placentae compared to CON. Fetoplacental growth and placental FA transport gene expression are modified in late gestation in a diet-and sex-dependent manner. The inability of the placenta to increase FA uptake to support fetal growth may be a maladaptive response to nutritional adversity. This may impact both fetal and placental metabolism and programme later metabolic health. Prenatal programming of cardiovascular disease and diabetes may be exacerbated by early postnatal "catch-up" growth, especially in male growth restricted (IUGR) offspring. One underlying mechanism, common to the development of these diseases, is premature aging of the organs involved (heart, kidneys, pancreas). Cellular senescence is a state of permanent and irreversible cell cycle arrest with a reduced capability to respond to stresses. In animal models of IUGR, catch-up growth is associated with reduced lifespan and accelerated aging of the heart and pancreas. Senescence results from activation of the cell cycle regulating P16 pathway, which interacts with the P53-P21 pathway to induce cellular growth arrest. Recently, in a maternal low protein diet-induced rat model, P16 was elevated in male IUGR offspring after a period of catch up growth. However, whether this is a sex-specific effect of postnatal dietary mismatch is unknown. Thus, we postulate that renal markers of senescence are altered via sex-specific mechanisms in a low protein (LP) rat model of IUGR and that maintenance of offspring on a LP diet after birth may be protective. Pregnant Wistar rats were fed control (C) or LP diet throughout pregnancy. After delivery, offspring were either maintained on LP diet (LP1), or switched to control diet (LP2) until postnatal d130. Western Blot was performed for P16, P21 and P53 in kidneys from both male and female offspring (n = 6 per group) at embryonic day 19 (E19), d21 and d130. At E19, P16 protein levels are increased in kidneys from LP-male offspring, while P21 is reduced in LP-females compared with controls (P<0.05). At d21, renal P16 protein is decreased in both LP-male and LP-females (P<0.05). Conversely, P21 levels are increased in both LP-male and LP-females while P53 is increased only in LP-male offspring at d21 (P<0.05). At d130, P16 protein levels are increased only in kidneys from LP2-females, yet P53 is only increased in LP2-males (P<0.05). Thus, renal markers of senescence are altered in kidneys from LP-IUGR offspring in a sex-specific manner, suggesting that premature aging of the kidney may be programmed in utero. Maintaining IUGR offspring on a protein restricted diet throughout life may be protective, since markers of senescence are not increased in male nor female LP1 offspring. Introduction: Amniotic fluid (AF) volume is regulated primarily by intramembranous transport of AF across the amnion. The pathway is a transcellular vesicular process stimulated by VEGF 165 . A VEGF 165 sister isoform, VEGF 165 b, has been identified as a spliced form generated by alternative splicing at a distal site in exon 8 of the VEGF gene. This isoform translates to a protein of the same length but has a different C-terminal sequence and functions as an inhibitor of VEGF 165 . To investigate the role of VEGF in AF transport, we determined the expression pattern of VEGF 165 and VEGF 165 b in human amnion. We hypothesized that 1) VEGF 165 is the primary isoform expressed in the amnion with differential regional distribution and 2) relative levels of VEGF 165 and VEGF 165 b are altered by pregnancy complications. Methods: Human amnion samples were collected at cesarean delivery from term subjects with normal pregnancy (9), gestational diabetes (4) and gestational hypertension (3). Total RNA from placental and reflected amnion was reverse transcribed and amplified by real-time PCR using custom designed Taqman human primers and probes for VEGF 165 (spanning distal exon 7 to 8a) and VEGF 165 b (spanning distal exon 7 to 8b splice site). The relative amount of VEGF mRNA was quantified by the standard curve method and normalized to 18S endogenous control. Data were analyzed by ANOVA.

The relative amount of VEGF 165 was 2 fold higher than VEGF 165 b (P<0.01) in reflected amnion while similar in placental amnion. The levels of both isoforms were higher in reflected amnion (P<0.0001) yielding a VEGF 165 :VEGF 165 b ratio of 3:1 in reflected and 1:1 in placental amnion. There was no fetal gender difference in the amount of VEGF 165 and VEGF 165 b in either reflected or placental amnion. In gestational hypertensives but not diabetics, the level of VEGF 165 b was lower than normal in placental and reflected amnion (P<0.02) raising the ratio by 4 fold and 1 fold, resp. Conclusion: The expression pattern of VEGF 165 and VEGF 165 b in the amnion differs in both relative quantity and regional distribution. A shift in the ratio of VEGF 165 :VEGF 165 b would switch VEGF 165 activity between stimulatory and inhibitory. Thus, changes in the ratio of VEGF 165 :VEGF 165 b in the amnion may alter intramembranous transport resulting in abnormal AF volume particularly in complicated pregnancies.

Effects of an The recognition of adult metabolic diseases such as obesity and non-alcoholic liver disease (NAFLD) in young children is a leading public health concern. The obesity rates in children approach 1 in 5 and it is estimated that 15% of obese children are affected by NAFLD, yet the developmental origins of NAFLD in children remain poorly understood. We previously reported in our Japanese macaque model of maternal obesity that fetal hepatic lipids and oxidative stress were strikingly increased in the early 3 rd trimester. However, there is limited human data in infants exposed to maternal obesity and GDM. Herein we assess the precision of these methods in the neonate and determine the pattern of neonatal fat deposition in offspring of mothers obesity/GDM compared to infants of normal weight women. 25 neonates born to normal weight mothers (n=13, BMI 21.6 kg/m 2 ) and obese mothers with GDM (n=12, BMI 40.1 kg/ m 2 ) underwent MRI to measure sub-cutaneous and intra-abdominal fat and MRS for measurement of intrahepatocellular (IHCL) lipid at 1-3 weeks of age. Mean gestational weight gain (GWG) fell within IOM guidelines in non-obese (14.2 kg), but exceeded IOM guidelines in obese mothers (9.7kg). Infants born to Obese/GDM mothers had increased skinfold thickness compared to infants born to normal weight mothers (p<0.05), however there was no difference in total subcutaneous fat as measured by MRI. Infants born to obese/GDM mothers had a mean 68% increase (P<0.01) in IHCL compared to infants born to normal weight mothers. There was a significant correlation between IHCL and maternal pre-pregnancy BMI (r =0.50, p=0.02), but not between IHCL and GWG (r=0.03, p=0.91), or total adiposity (r=0.09, p=0.71). Intra-abdominal fat did not significantly correlate with MRI subcutaneous fat (r=0.24, p=0.25) or skinfolds (r=0.19, p=0.37). Our results demonstrate that maternal pre-pregnancy BMI is associated with hepatic fat storage at 1-3 weeks of life across a cohort of infants born to both normal weight and obese/GDM mothers, independent of neonatal subcutaneous fat. This finding suggests that fetal storage of fat in the liver is driven in part by excess fuels present in obese mothers throughout pregnancy and may represent an important process for the early onset of fatty liver, distinct from those factors that determine adipose tissue development in subcutaneous tissue. Pregnant rats subjected to a low protein diet have been widely used to explore the mechanisms responsible for fetal programming on hypertension and cardiovascular diseases, with an assumption that dams with or without protein restriction consume comparable amount of diet. Our previous observations did not support this assumption. Appetite is regulated by a well-orchestrated interplay between central system and peripheral organs. In this study, we hypothesized that appetite of pregnant rats is altered by low protein diet. To determine if increases in miR-29 functionally decreased hepatic Igf-1 expression, Clone 9 rat hepatocytes isolated from a 3 week old male hepatoma were transfected with miR-29 mimics and Igf-1 expression was assessed. Over expression of miR-29 resulted in decreased Igf-1 expression by 24 hours. CONCLUSIONS: This study is the first to examine the impact of maternal diet on miRNA expression in the liver long-term. The observed decrease in Igf-1 at day 130 in these LP2 offspring was consistent with the findings of impaired glucose and cholesterol metabolism associated with this model. Furthermore, miRNA microarray and transfection experiments implicate miR-29 as a negative regulator of Igf-1 in these offspring by three weeks of age, but persisting only when the mismatch in the postnatal diet occurred at weaning. Further work is warranted to identify molecular mechanisms regulating the promoter of miR-29 in these IUGR offspring. We acknowledge the Canadian Institutes of Health Research for their funding support.

High Fat Diet and IUGR Alter mRNA Levels of PPARγ and Setd8 in a Sex-Dependent Matter in Newborn Rat Lung. S Ashmore, S Shupe, C Wilson, Y Wang, C Jiang, A Sainz, E Zinkhan, R Lane, L Joss-Moore. Pediatrics, University of Utah. Intrauterine growth restriction (IUGR) increases neonatal lung disease and alters alveolar formation in the developing fetal lung. Alveolar formation depends upon PPARγ expression. PPARγ functions as a transcription factor for the chromatin modifying methyltransferease Setd8. Setd8 methylates histone 4 lysine 20. Subsequently, PPARγ signaling coordinates the expression of genes with PPARγ response elements and those that use H4K20 methylation as a mode of regulating expression. We previously showed that IUGR decreases PPARγ and Setd8 expression in male and female rat lung and that this can be reversed with the addition of a diet high in unsaturated fats. However, the typical Western diet is high in saturated fats and a maternal diet high in saturated fat (HFD) is independently associated with alterations in alveolar formation. We hypothesize that a maternal HFD in combination with IUGR will decrease PPARγ and Setd8 mRNA beyond that of IUGR alone, in newborn rat lung. IUGR was induced by bilateral uterine artery ligation at E19 of gestation. Maternal rats were fed either Standard Rat Chow or HFD chow prior to mating and during gestation. Real-time RT-PCR was used to measure mRNA levels of PPARγ and Setd8 in newborn rat lung. Results are IUGR as % of sex-matched regular diet controls ± SD. In female rat lung, IUGR alone decreased PPARγ (82 ± 7%*) and Setd8 (77 ± 12%*) mRNA. In female lung, HFD alone decreased PPARg (57 ± 15%*) but did not alter Setd8 mRNA levels. The combination of HFD and IUGR decreased PPARγ (62 ± 16%*) and Setd8 mRNA (79 ± 11%*) in female lung, but not more than IUGR alone. In male rat lung, IUGR alone decreased PPARγ (58 ± 25%*) and Setd8 (82 ± 7%*)mRNA levels. HFD alone decreased PPARγ mRNA (42 ± 22%*) in male rat lung. However, HFD alone increased Setd8 mRNA (132 ± 27%*), and HFD with IUGR further increasing Setd8 mRNA (174 ± 40%*). *p≤0.05. Contrary to our hypothesis, we conclude that high fat diet has a sex specific effect on Setd8 mRNA levels.

In female rat lungs, decreased Setd8 with HFD, is consistent with the observed decrease in PPARγ. Interestingly, in male lungs, a similar reduction in PPARγ with HFD is associated with an increase in Setd8 mRNA. Alternative chromatin modifications in the Setd8 promoter in male HFD rat lung may contribute to this dichotomy and warrant further investigation.

IUGR Affects Elastin Alternative Splicing and DNA Exon Methylation in Newborn Rat Lung. L Joss-Moore, Y Wang, C Jiang, A Saniz, J Stiers, K Albertine, R Lane. Pediatrics, University of Utah. Intrauterine growth restriction (IUGR) increases the risk of postnatal lung disease and impairs lung development, with males more severely affected. Lung development depends on programming of elastin expression during a critical developmental window. Programming of gene expression though epigenetics often involves alternative exon usage, which is rarely appreciated. Alternative splicing allows for more specific adaptations to environmental pressures than modifying gene expression alone. Epigenetics contributes to this mechanism of programming through differential methylation of exonic CpG's. We have previously demonstrated that IUGR decreases total elastin mRNA in male and female rat lung. However, it is unknown if IUGR affects alternative splicing of elastin or DNA methylation at alternatively spliced exons. We hypothesize that IUGR alters elastin alternative splicing as well as DNA methylation at alternatively spliced exons in newborn rat lung. IUGR was induced by bilateral uterine artery ligation in rat dams at E19 of gestation (term 21 days). Realtime PCR was used to measure mRNA levels of elastin transcripts containing alternatively splicing of exons 10, 28 and 33 in newborn IUGR and control rat lung. Bisulfite sequencing was used to assess DNA methylation within Exons 10, 28 and 33 of the elastin gene in the same samples. Results are reported as IUGR as % of gender-matched control ±SE. IUGR decreased elastin mRNA levels of alternatively spliced exon 10 in male (73±14%*) and female (65±17%*) lung. IUGR decreased levels of alternatively spliced exon 28 (53±15%*) and 33 (67±23%*) in female lung only. DNA methylation was increased (121±14%*) at exon 28 in female rat lung. *p 0.05. We conclude that IUGR alters alternative splicing of the elastin gene in in a sex-specific manner in newborn rat lung. Given that total elastin expression in the IUGR rat lung is decreased similarly in male and female lungs, sex-specific differences in alternatively spliced elastin transcripts is intriguing. We speculate that sexspecific differences in DNA methylation and alternative splicing of elastin exons may contribute to sex-specific differences lung development in IUGR infants. . Since glucocorticoid (GC) induction of hepatic maturation includes increased PEPCK-2 (mitochondrial isoform) expression we hypothesized 1) fetal PEPCK-2 is up-regulated in fetal liver in IUGR and 2) GC induce PEPCK-2. We measured fetal liver PEPCK-2 and isolated fetal baboon hepatocytes to determine if increased GC exposure and IUGR augments PEPCK expression. Methods: Quantitative RT-PCR (qPCR), immunohistochemistry (IHC), and western blot analysis were performed on liver tissues and hepatocytes from 0.9 gestation (G) CTR and IUGR fetuses obtained at C-Section. To investigate whether increased GC levels in IUGR fetuses augment fetal PEPCK-1 and -2 expression, we cultured 0.9G fetal hepatocytes for 24h with dexamethasone (Dex, 100 nM).

We detected strong PEPCK-2 by IHC in 0.9G baboon liver which increased (5 fold, p<0. For pregnant women, nicotine replacement therapy (NRT) is generally considered to be safer than smoking. Yet, there are still numerous concerns regarding the use of NRT during pregnancy. Animal studies suggest that nicotine exposure during fetal and neonatal life may increase the risk of dyslipidemia and obesity in postnatal life. Indeed, we have recently demonstrated that male rats with in utero and lactational nicotine exposure had elevated hepatic and circulating triglyceride levels (p<0.05) at 26 weeks of age. Given that many hepatic target genes involved in fatty acid synthesis are regulated by the nuclear receptor Liver X Receptor x (LXRα), we hypothesized that LXRα may mediate permanent nicotine-induced changes in liver function. To address this hypothesis, female Wistar rats were randomly assigned to receive daily subcutaneous injections of either saline (vehicle) or nicotine bitartrate (1mg/ kg/day) from two weeks prior to mating until weaning (postnatal day 21). Quantitative PCR analysis revealed that 26-week-old male nicotine-exposed offspring had significantly elevated levels of fatty acid synthase (FAS) and acetyl-coA carboxylase alpha (ACCα) mRNA, both of which are involved in fatty acid synthesis and are targets of LXRα. Other enzymes downstream in the triglyceride synthesis pathway including stearoyl-coA desaturase (SCD-1) and elongase 5 (Elovl5) did not show any change with nicotine exposure. Chromatin immunopreciption revealed that in nicotine-exposed offspring LXRa binding to the FAS promoter at the tentative LXRE element (-667), was elevated with a concomitant increase in hepatic LXRa protein levels.

Collectively, these results suggest that nicotine exposure during pregnancy and lactation contributes to a permanent increase in circulating triglycerides, via an increase in the activation of LXRα. These results provide evidence that perinatal nicotine exposure leads to long-term dysregualtion of fatty acid synthesis in the offspring. Supported by CIHR.

Nurmamat, 1 John F Odhiambo, 1 Peter W Nathanielsz, 2 Stephen P Ford. 1 Female ICR mice were orally gavaged with 1 µmole/kg PCB 126 biweekly such that dams were exposed 48 hours prior to mating, once during gestation and once during lactation. Glucose tolerance testing revealed that male offspring born the PCB exposed dams had impaired glucose disposal at 7 weeks of age, but female offspring were normal at this age. However, at 6 months of age, the female offspring born to PCB exposed dams displayed impaired glucose tolerance while the male offspring were unaffected. Embryo and placenta did not differ in weight when collected at embryonic day 18 from a separate cohort of vehicle and PCB-exposed dams. Taken together, these data demonstrate gender specific affects of maternal PCB exposure on offspring glucose disposal without manifestation of attenuated fetal growth. Future cross-fostering experiments will be carried out to determine whether direct exposure to PCBs in the milk caused the early life glucose disposal impairments in the male offspring since these detrimental effects disappeared over time. In agreement with our hypothesis, perinatal PCB exposure in female offspring led to impaired glucose tolerance when the mice reached 6 months of age. This mouse model provides a powerful in vivo system to explore the mechanisms behind PCBs' detrimental effects and will allow us to establish the critical windows during development when offspring may be most susceptible to PCB toxicity. Obesity, diabetes, and cardiovascular disease are at epidemic levels, and the toxicity of environmental contaminants during development could be a significant contributor to these trends.

Exercise as a Therapeutic Approach for Fetal-Programmed Cardiovascular Dysfunction. Laura M Reyes, 1, 2, 3 (IUGR) has been shown to increase long-term susceptibility to myocardial ischemia/reperfusion injury. Exercise is a practical and effective preventive treatment for cardiovascular diseases, especially in children. Whether exercise can be an effective intervention for hypoxia-induced cardiovascular complications is not known. We hypothesized that the myocardial susceptibility to ischemia insult in offspring born from a hypoxic in utero environment would be ameliorated following exercise training. Methods: Female Sprague Dawley rats were mated at 3 months of age. From gestational day 15 to 21 of pregnancy, rats were exposed to control (room air) or maternal hypoxia (11% oxygen) conditions. At the time of birth (gestational day 22), litters were randomly reduced to 4 males and 4 females. At 10 weeks of age, male and female rats from hypoxic and normoxic pregnancies were randomized to either an exercise-training or sedentary group. Rats were habituated to treadmill running then exercised for 6 weeks; 5 consecutive days/ week, 30 min/day at 20 m/min. After a recovery period of 24 hours, animals were euthanized and their hearts perfused for ∼10 min in retrograde Langendorff mode. After retrograde perfusion, hearts were switched to working heart mode and global, normothermic flow ischemia was induced for 10 min. Following ischemia, hearts were reperfused for 40 min.

Our preliminary data show that, compared to controls, offspring born from hypoxic pregnancies exhibited a decrease in recovery of cardiac performance during the reperfusion period (36.3% controls vs.19.6% IUGR).

Exercise improved the recovery of cardiac performance in both groups (58.2% controls, 36.0% IUGR). These data suggest that while exercise is beneficial, it does not appear to improve the specific complications associated with IUGR. Discussion: Our data suggest that exercise improves cardiac performance after ischemia. Additional studies will, therefore, continue to determine the mechanisms involved in the improved function with exercise in order to develop therapeutic approaches to cardiac complications that may be more severe in offspring born growth restricted. Introduction: Poor fetal nutrition alters islet β-cell development predisposing to adult metabolic problems. Pancreatic development involves establishment of normal relative populations of β and α cells. We reported 1 that female OFF of restricted (R) mothers fed LP in pregnancy had decreased post natal β-cell secretory reserve and premature aging of insulin secretion. We hypothesized that R differentially alters β and α cell populations. We determined effects of R on morphology and cellular composition of pancreatic islets in female OFF from postnatal day (PND) 7 -110. Methods: We studied female OFF. Control (C) mothers ate 20% casein and R ate 10% casein, isocaloric diets in pregnancy with normal diet after delivery in both groups. At PND 7, 14, 21, 36 and 110 pups were euthanized and pancreas obtained. The α and β-cell were analyzed by immunohistochemistry and islet size and α and β-cell area quantified with Image Pro Plus. Results: Islet area was similar in R and C at all ages. At 7 and 110 PND there were no differences in β or α cells between C and R OFF. At 14 and 21 PND β-cell area in R was lower than C and higher at 36 PND. α cell area was preserved in R and C at 7, 36 and 110 PND, at 14 PND it was lower and at 21 PND was higher than C. The β : α cell ratio was lower in R at 7 and 21 PND and higher at PND 36, and unchanged at 14 and 110 PND vs C. 

Goserelin for the Prevention of Cyclophosphamide-Induced Ovarian Damage in Pre-Pubertal Mice. Background: Systemic cyclophosphamide (CTX) has been shown to cause apoptosis of ovarian follicles. We sought to evaluate whether pre-treatment with goserelin, a GnRH-agonist, could prevent CTX's effects in pre-pubertal mice. Materials and Methods: C57BL/6J pre-pubertal mice were randomized to three groups; placebo (group1, n=6), CTX (group 2, n=13), and experimental (group G, n=9). On day 13 of life, groups 1 and 2 received placebo and group G received depot goserelin rods (3.6 mg). On day 18 of life group 1 received placebo and groups 2 and G received 200 mg/kg of CTX, intraperitoneally. All mice were euthanized in late puberty, on day 56 of life. FSH and AMH were measured from serum. Uteri and ovaries were fixed in formalin, embedded in paraffin, and stained with H&E and TUNEL. Follicular apoptosis was graded. Data were analyzed using ANOVA and t-test (SPSS v19).

Results: The variables are reported in the table. BMI, uterine weight and ovarian size did not differ among groups. FSH and AMH were similar in groups 1 and G, however, FSH was higher and AMH was lower in group 2. The number of PMF and PRF/mm2 was smaller in groups 2 and G than group 1. SEF, TEF and corpora lutea/mm2 were similar in groups 1 and 2, but decreased in group G.

Variable measurements in the three groups. Conclusions: Although AMH and FSH measurements would indicate a protective effect of goserelin on ovarian function, histological assessment did not confirm such protection, as the number of PMF and PRF was similarly decreased in mice exposed to CTX whether or not they were pre-treated with goserelin.

Acute Cyclophosphamide-Induced Effects on Pre-Pubertal Mice Ovaries. Background: In adults, cyclophosphamide (CTX) has been shown to cause apoptosis of ovarian follicles. Reduction of the primary follicle reserve in prepubertal cancer patients results in acute or chronic ovarian insufficiency. This has been shown to be associated with early menopause and loss of reproductive capacity. We sought to evaluate the acute effects of CTX on ovarian follicles of pre-pubertal mice. Design: Animal study. Materials and Methods: Pre-pubertal inbred C57BL/6J female mice were randomized to receive placebo (group 1) or 200 mg/kg of CTX (group 2) on day 18 of life. Mice were euthanized 48 hours after CTX administration. Body weight and length, and uterine weight were measured. Uteri and ovaries were fixed in formalin, embedded in paraffin, and stained with H&E and TUNEL to study cellular apoptosis. Five µm-thick sections were serially cut and every 5th section analyzed for follicular counts and apoptosis. Apoptosis was graded in the granulosa cells, the oocyte, or both, in primordial (PMF), primary (PRF), secondary (SEF) and tertiary (TRF) follicles. Data were analyzed using ANOVA and t-test (SPSS v19). Results: Group 2 mice were heavier, but had similar length of group 1. Uterine and ovarian weight were similar. PMF, PRF, SEF, TEF, but not corpora lutea, were present in the pre-pubertal ovaries. PMF and SEF were the most represented follicles in both groups (14 follicles/mm2, CI:10-18, in both groups). PMF and PRF number, but not SEC and TES was decreased after CTX: from 14 to 0.1 PMF/mm2 and 5 to 0.5 PRF/mm2, respectively; p<0.008. Apoptosis was increased in SEF (4%, CI: 2-6, in group 1 to 68%, CI: 62-74 in group 2; p<0.02) and TEF (38%, in group 1 to 89%, in group 2; p<0.02) . Apoptosis was noted only in the granulosa cells in 5 % of the follicles. Conclusions: All follicle categories, except for corpora lutea, were present in the pre-pubertal ovaries. In the acute setting, CTX treatment decreased the number of primordial and PRF, but not of SEF and TEF follicles, albeit increased apoptosis in the latter two. Our results confirmed previous adult outcomes. We also confirmed that apoptosis starts first in the granulosa cells. AP, Hosp. Dr.Peset, Spain; 5 Hospital Clínico Universitario, Valencia, Spain. Breast Cancer (BC) is the most frequent malignancy in women of reproductive age and the main indication for ovarian cortex (OC) cryopreservation and transplantation (OT). Although it was suggested that OC-OT, after non advanced stages of BC has been cured is safe, this has not been properly addressed. The major concern is contamination of OC with cancer cells that could be reintroduced during OT. Although histology is still the gold-standard in the management of BC, new molecular methods will add more safety to the screening of occult metastases in OC from BC patients, before OT is attempted. Women with gynecologic malignancies were significantly older than the women in the other 3 groups. Women with hematologic malignancies were most likely to have been exposed to previous chemotherapy and have the longest stimulations but had a similar number of oocytes retrieved. There was a trend for patients with breast cancer or gynecologic malignancies to have a greater number of oocytes retrieved and gametes frozen compared to patients with other cancer diagnoses. A total of 4 patients were cancelled because of lack of ovarian response. The IVF control patients required fewer stimulation days than patients without a history of chemotherapy. The IVF control also had higher peak estradiol levels, number of oocytes obtained and fertilization rates when compared to cancer patients with and without a history of prior chemotherapy. Conclusions: Factors including age, type of cancer as well as chemotherapy exposure, can influence response to ovarian stimulation. Discussing these findings with patients presenting for FP may aid in setting realistic treatment expectations. Most respondents (80%) prefer a GnRH agonist based stimulation protocol, while 7.5% prefer an antagonist protocol, and 12.5% denied a preference. Variability also existed in preference for triggering agent, as 74.6% of physicians reported using hCG exclusively, and 19% reported using GnRH agonists to trigger. Interesting variability was also noted in responses regarding COH for breast cancer patients. 85.9% reported using letrozole during COH in estrogen receptor (ER) positive breast cancer, and 49.28% also utilize the drug during IVF stimulation for ER negative cancer. Physicians also varied in their preferences regarding which patients they would allow to proceed with oocyte and embryo banking. Of respondents who offer FP to patients undergoing potentially gonadotoxic therapies, 28% have some form of screening criteria (i.e., FSH, age) to determine if they will allow a patient to proceed with oocyte or embryo banking. Interestingly, of the physicians who provide FP for cancer patients, 21.3% were not familiar with the American Society for Clinical Oncology (ASCO) guidelines regarding fertility preservation. Those who were not familiar with ASCO guidelines were less likely to offer ovarian tissue cryopreservation. Conclusion(s): FP practice patterns vary among SREI members in regards to COH protocol preference, letrozole use during COH in breast cancer patients, and agent used for follicle trigger. Although some of this variability may be dictated by local availability of resources, a more standardized, congruent method of managing and counseling patients seeking FP may be helpful.

Characterization of the Gonadotropin Releasing Hormone Response in the Ovary. Objective: Gonadotropin releasing hormone agonists (GnRHa) are increasingly used for fertility preservation in women undergoing gonadotoxic chemotherapy. However, the mechanisms of action for these compounds have not yet been elucidated. In this study, we aimed to determine whether GnRHa have a direct effect on ovarian granulosa/cumulus cells. Design: Experimental study Materials/Methods: Gonadotropin releasing hormone receptor (GnRHR) expression was determined in mouse somatic and gonadal tissues including granulosa/cumulus cells and oocytes using quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Western Blot analysis, and immunohistochemistry. Granulosa cells were isolated from mouse ovaries primed with pregnant mare serum gonadotropin (PMSG). Response to GnRHa in cultured granulosa cells was assessed by determining 1) cAMP response following transfection with a construct containing a luciferase reporter and a cAMP-response element and 2) phosphorylation of downstream mediators of GnRH signaling: ERK, NFΚB, and p36. For all experiments, pituitary tissue and/or AT3-1 mouse pituitary cell line were used as controls.

Results: GnRHR mRNA and protein are expressed in mouse gonadal tissues as well as pituitary, heart and kidney but were absent from lung and spleen. Within the ovary, GnRHR expression is high in granulosa/cumulus cells as well as oocytes. Following GnRHa stimulation at various time intervals, we were unable to detect cAMP increase or activation of the ERK signaling pathway in cultured primary mouse granulosa cells, while activation was detected in the control AT3-1 mouse pituitary cells. Conclusion: We have shown that gonadotropin releasing hormone receptor (GnRHR) is expressed in mouse granulosa/cumulus cells and oocytes. Activation of the canonical GnRH signaling pathways was not detected in our system. Our findings suggest that the mechanism of action of GnRHa in the ovary, is either below the detection level of our experimental design, or is different from that in the pituitary.

Investigating the Role of Apoptosis in the Chemoprotection Effect of GnRH Antagonist. Bo Yu, 1 Snezana Haymes, 2 Yaling Zhou, 2 Gary Levy, 1 James Segars, 1 Alicia Armstrong. 1 1 Program in Reproductive and Adult Endocrinology, NICHD, NIH, Bethesda, MD, USA; 2 Department of Research Programs, Walter Reed National Military Medical Center, Bethesda, MD, USA. Introduction: GnRH antagonist cetrorelix has been shown in some studies to be effective in protecting ovarian function during gonadotoxic chemotherapy. A previous study from our group demonstrated that cetrorelix reduced DNA damage in developing follicles of mice treated with cyclophosphamide (CYC). This study investigated whether genes involved in apoptotic pathway may have been regulated, and therefore contributed to the chemoprotective effect of cetrorelix. Materials and Methods: Six-week-old Balb/c female mice were randomly divided into 4 treatment groups (n=4 in each group): a) cetrorelix 0.5mg/kg/day for 15 days and a single dose of CYC 100mg/kg on day 9; b) a single dose of CYC 100mg/kg on day 9; c) cetrorelix 0.5mg/kg/day for 15 days and a single dose of 0.1ml saline on day 9; and d) a single dose of 0.1ml saline on day 9. After the mice were sacrificed on day 16, ovaries were removed. Total RNA was extracted from one ovary of each mouse and RNA integrity was confirmed. After total RNA was converted to cDNA, mouse apoptosis RT² Profiler™ PCR Array (QIAGEN) was used to analyze the expression levels of 84 key genes involved in apoptosis and anti-apoptosis. Each set of PCR array experiments was repeated in triplicates. The data was analyzed using the software provided by the manufacturer. Results: Compared to the group treated with only CYC (group b), treatment with cetrorelix before and after CYC (group a) did not result in statistically significant difference in expression levels in any of the 84 genes included in the apoptosis PCR array. All 3 sets of experiments generated the same results.

Conclusions: There was no differential expression of any of the 84 genes involved in the apoptotic pathway with cetrorelix treatment before and after CYC. Therefore, the mechanisms contributing to the protective effect of cetrorelix in gonadotoxic chemotherapy remain unclear and deserve futher investigation. 

Perinatal Intra-Cranial Gene Transfer for Permanent Astrocyte Specific Expression in Mice. Rajvinder Karda, Simon N Waddington, Mark R Johnson.

Gene transfer technology has advanced greatly over the past decade. Viral vectors can be prepared at very high titres (∼1x10 14 vector particles/ml) and novel vectors have been developed which have very high tropism for cells of the central nervous system including neurons and astrocytes. By introducing these vectors into the perinatal rodent brain it may be possible to induce a state of cell-specific somatic transgenesis. To test the hypothesis that we would be able to achieve prolonged expression in astrocytes by this approach, we chose a truncated promoter of glial fibrillary acid protein (GFAP). The protein is an intermediate filament which is expressed in mature astrocytes, but not neurons, and is involved in maintaining their structure and motility.

A rodent GFAP (GfaABC1D) was cloned into a lentivirus vector upstream of the genes encoding firefly luciferase and green fluorescent protein (GFP). This vector was injected intra-cranially into wild-type mice at P0 of development and luciferase expression was monitored continually by whole body bioluminescence imaging. We observed long-term, stable expression of firefly luciferase under the control of the truncated GFAP promoter; expression was restricted to the central nervous system including both hemispheres of the brain.

In conclusion, this combination of GFAP promoter and lentivirus vector appears suitable for achieving astrocyte-selective and prolonged somatic transgenesis. We now plan to use this construct for the study, and potential treatment of neonatal hypoxic ischaemic brain injury. Background Preeclampsia (PE) and fetal growth restriction (FGR) complicate ∼ 10% of human pregnancies and contribute significantly to fetal and maternal morbidity and mortality. Although the causes of PE and FGR are not well understood, they are known to be associated with impaired uterine artery blood flow. Resveratrol (Resv), a polyphenol found in many plants, can induce relaxation of uterine arteries in vitro as well as improve many pathological features associated with PE and FGR. Endothelial nitric oxide synthase knockout mice (eNOS-/-) and catechol-O-methyltransferase knockout mice (COMT-/-) exhibit many signs of PE during pregnancy and deliver growth-restricted pups in comparison to wild type (C57Bl6/J) mice. We hypothesize that treatment of eNOS-/-and COMT-/-mice with Resv during pregnancy will improve uterine artery blood flow, ameliorate the PE-like phenotype and rescue fetal growth. Methods Pregnant C57BL/6J, eNOS-/-and COMT-/-mice were randomly assigned to receive either Resv supplemented diet (4 g/kg diet, n=7-11) or control diet (n=7-9) between gestational day (d) 0.5 and d18.5. Uterine artery Doppler waveforms and blood pressure (BP) were measured on d17.5. Dams were euthanized on d18.5 and uteri examined for number of live fetuses and resorptions. Fetuses and placentas were blotted dry, weighed and examined for gross abnormalities. All data are presented as mean±SEM and were compared using a two way ANOVA.

No difference in BP was observed among groups. Resv supplementation increased uterine artery blood flow velocity in the COMT-/-(430.6 ± 35.6 vs 604.5 ± 17.7 mm/s; p<0.01) but not in eNOS-/-(396.8 ± 41.8 vs 500.9 ± 42.2 mm/s) or C57BL/6J (569.1 ± 32.3 vs 554.6 ± 36.7 mm/s) mice. Following Resv administration, a significant increase in pup weight was observed in COMT-/-(1.03 ± 0.02 vs 1.11 ± 0.01g, p<0.05) mice but not in eNOS-/-(0.91 ± 0.02 vs 0.97 ± 0.02g) or C57BL/6J (1.05 ± 0.02 vs 1.08 ± 0.01g) mice. There were no effects of Resv on resorptions, placental weight or fetal weight/placental weight ratio. No evidence of gross abnormalities was observed in fetuses from Resv supplemented dams. Conclusion Resv increased uterine artery blood flow velocity and the fetal weight in COMT-/-mice, suggesting potential as a therapeutic strategy for PE and FGR.

Cortisol Maternal stress during pregnancy reduces fetal growth and programs diseases in later life. It is generally assumed that these effects are mediated by maternal cortisol which crosses the placenta and programs hyperactivity of the fetal hypothalamo pituitary adrenal axis (HPAA). This mechanism does not explain the transfer of maternal stress completely, e.g. early stress has the most pronounced programming effects when fetal central glucocorticoid receptors are not yet expressed.

Objective: We hypothesized, that although maternal catecholamines do not cross the placenta, they have major fetal effects by decreasing uterine blood flow (UBF). Methods: Five pregnant ewes were instrumented at 125d gestation (dGA, term 150d) with maternal and fetal vessel catheters and a uterine ultrasound flow probe. At 130dGA, animals were stressed by isolation for 2h before and after infusion of labetalol, a mixed α and β adrenergic antagonist.

[bold/]Results:[/bold] Ewes responded to isolation with an increase in blood pressure (BP) from 83±2.7 to 89±3.9mmHg (mean±SEM) for 9min (p<0.05), an increase in heart rate (HR) from 98±4.7 to 126.9±6.2bpm for 30min (p<0.05). UBF decreased by 11.8±2.7% for 75min (p<0.05, Fig. 1 ) reflecting uterine vasoconstriction. The fetus responded with a delayed and prolonged pH decrease from 7.39±0.01 to 7.36±0.01, lactate increase from 1.5±0.2 to 1.8±0.2mmol/L and O 2 sat decrease from 75±2.5 to 67±3.6% starting at 60min of isolation (p<0.05). Labetalol prevented the UBF decrease and resulted in an increase by 11±3.4% (p<0.05, Fig. 1 ) reflecting increased cardiac output (BP and HR increase) in the absence of vasoconstriction. Conclusion: Maternal stress induces a catecholamine mediated UBF decrease followed by a prolonged fetal lactate increase and decrease in O 2 sat that may contribute to impaired fetal growth and programming of diseases in later life. 

A definitive diagnosis of CHD is feasible in the first trimester: an early detection of vessel anomalies is more difficult to accomplish then that of cardiac anomalies. As an early diagnosis and follow up of CHD might allow a comprehensive counselling thus avoiding unnecessary pregnancy terminations, a complete cardiac evaluation in the first trimester is advisable. (25), posterior fossa anomalies (24), previous pregnancy with CNS anomalies (23) or non CNS anomaly (2), macrocephaly (19), death of single twin (19), brain cystic lesions (15) and abnormal cavum septum pellucidum (11). 55 body MR scans were performed according to 10 indications. The main indications were gastrointestinal tract (6), renal (6) and gallbladder anomalies (5) as well as abdominal cystic lesions (5) There was no difference in ability to obtain cytogenetics, p =0.4. Reason for termination did affect ability to obtain an autopsy, with fetal anomalies most likely to be successful, p <0.01. Increasing gestational age, > 20 weeks, was associated with higher likelihood of obtaining an autopsy as compared to fetuses < 20 weeks, p < 0.01. There was no difference in the total complication rates between the 2 procedures, p=0.17. Retained placenta was the most common complication with IOL and EBL > 500 mL was most common with D&E. Midtrimester terminations by IOL are more likely to have autopsy and cytogenetics information available, which can provide crucial information for obstetricians when counseling for future pregnancies. OBJECTIVE: Smith-Lemli-Opitz (SLO) is an autosomal recessive syndrome caused by a defect in the biosynthesis of cholesterol related to mutations in the 7-dehydrocholesterol reductase (DHCR7) gene. It is estimated that 3.9 -4.0% of whites carry a DHCR7 mutation, theoretically resulting in a homozygote frequency of 1/2,500. However, it occurs in only about 1/20,000 live births, implying that most homozygotes die before birth and that SLO may account for several percent of stillbirths. Thus, our purpose was to assess the frequency of DHCR7 mutations in stillbirths. METHODS: Prospective, multicenter, population-based case-control study of all stillbirths (fetal deaths >20 weeks) and a representative sample of live births enrolled at delivery in 5 geographic areas at 59 hospitals averaging >80,000 deliveries/year. Cases with stillbirth due to obstetric complications, infection or aneuploidy, as well as those with poor quality DNA were excluded. DNA was extracted from placental tissue stored at -80oC, and exons 3-9 of the DCHR7 gene were amplified, purified, and subjected to bidirectional sequencing to identify mutations. RESULTS: 144 cases met criteria including 73 white, 46 Hispanic 17 black and 8 stillbirths of other race. In the 139 cases with informative assays, 8 (5.7%) had mutations in coding exons, including 4 white (5.4%), 3 Hispanic (6.5%) and 1 black (5.8%). Seven had a single mutation and one Hispanic case was a compound heterozygote. This stillbirth had no clinical features of SLO. Several novel mutations were noted although the frequency of previously described mutations was similar to that noted in the general population. In addition, 15 cases had a benign polymorphism in DHCR7 exon 4 position 240 T77T (C/T) including two heterozygotes. There were no cases of the most prevalent mutation in whites; c.964-1G>C. CONCLUSION: Less than 1% of stillbirths in women without evidence of obstetric complications, infection or aneuploidy had two DHCR7 gene mutations based on sequencing analysis. This is not higher than that anticipated based on the proportion of live births with clinical SLO. Although sporadic stillbirths may be associated with SLO, our data do not support an association between unrecognized DHCR7 mutations and stillbirth.

Effects Endothelium dysfunction was also noted in uterine endothelial cell culture system; binge alcohol decreased levels of major endothelial nitric oxide (NO) synthase (eNOS, rate limiting enzyme for NO vasodilator production) excitatory P635 eNOS/total eNOS (↓23%, P<0.003) compared to the Controls and the related pERK/total ERK signaling (↓25.7%, P<0.001). Discussion: 1) Chronic binge alcohol has detrimental effects on agonist-mediated endothelium-dependent uterine artery relaxation during pregnancy; 2) These data support that uterine vascular dysfunction precedes FASD growth deficits; 3) Chronic binge alcohol has specific effects on uterine eNOS activity and related signaling; 4) Ongoing studies will investigate role of NO, prostacyclin, and endothelium dependent hyperpolarizing factor in alcohol-induced decreases in uterine vascular function; 5) These findings suggest the involvement of the uterine compartment in FASD pathogenesis. NIH AA19446, HL49210, HD38843, HD69750 Conclusion: Our study showed significantly higher amounts of villi for both TC and TA approaches from a single institution compared to previous studies. More information regarding amount of villi and relationship to technique and pregnancy complications is needed. In addition, TC CVS produced more villi than the TA approach. This information may be useful when tests for single gene disorders that require a larger quantity of villi are considered. Endoplasmic reticulum (ER) stress is present in embryos exposed to maternal diabetes. Oxidative stress is the central mechanism in the induction of diabetic embryopathy. However, the relationship between oxidative stress and ER stress has never been explored in diabetic embryopathy. We have previously demonstrated that mitigating oxidative stress, using superoxide dismutase 1 (SOD1) transgenic (Tg) mice, suppresses maternal diabetes-induced malformations. In this study, we designed to using SOD1-Tg mice to examine whether oxidative stress is responsible for ER stress. Methods: Embryonic day 8.75 (E8.75) Wildtype (WT) embryos and SOD1 overexpressing embryos from non-diabetic control (NC) and diabetic mellitus (DM) dams were used for detection of ER stress markers. Results: Protein levels of ER stress markers: C/EBP-homologous (CHOP), calnexin, phosphorylation of eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like ER kinase (PERK) were significantly elevated in WT embryos exposed to maternal diabetes compared to those in WT embryos of the NC group and in SOD1-overexpressing embryos of the DM group. Similarly, mRNA levels of ER chaperones: Bip, calnexin, eIF2α, eIF2αk3 and PDIA3 in WT embryos of the DM group were also significantly increased. WT embryos exposed to maternal diabetes showed distinct RNA splicing of X-box binding protein (XBP1) (205 bp and 179 bp), while WT embryos of the NC group and SOD1-overxpressing embryos of the DM group had only one band (205 bp). Conclusions: Mitigating oxidative stress by SOD1 overexpression blocks maternal diabetes-induced ER stress marker expression, ER chaperone gene expression and XBP1 splicing. Maternal diabetes-induced oxidative stress causes ER stress in diabetic embryopathy. Objective: To compare 2 real-time PCR strategies--Simplex and Duplex methods for Fetal RhD Genotype with maternal dry blood Spot specimens on Guthrie cards. Study Design: Fetal DNA was extracted from both Guthrie card and peripheral blood samples of RhD-negative pregnant in the first trimester. Specific fluorescent probes for real-time PCR were designed to detect fetal gender gene (male -DSY14, female -S05, S06 and S10A), and Exon 4-6 of the RhD gene. Simplex real-time PCR was to detect one target gene (gender genes or RhD exon fragments) per reaction mix while the control gene (β-actin) was processed separately. Duplex real-time PCR was processed as both the target and the reference copy number genes, which were conjugated with distinctive fluorescents, were amplified in the same tube. Fetal sex and RhD genotype were confirmed after delivery. Results: Among 13 patients, fetal RhD genotypes were detected in 10 (76%) at first round using Simplex while fetal RhD genotypes were screened out in all 13 patients using Duplex method. Quantification studies were using the ratio of expressional levels of target genes over the β-actin gene (Simplex) or the reference copy number gene (Duplex). The standard deviation of Simplex realtime PCR was ≤0.01 versus ≤0.03 with Duplex method. One Rh-negative baby was incorrectly diagnosed as positive using either Simplex or Duplex methods. Conclusions: Our study revealed that fetal DNA from maternal dry blood samples placed on Guthrie card during the first trimester screen were reliable sources for fetal sex &fetal RhD determination as well as fetal DNA quantification. Although this method is prospective for convenient testing, fetal DNA trace from previous pregnancy may impair the accuracy of the results. Duplex method, although yielded bigger deviation of ratios, was more sensitive than Simplex method and it also helped to diminish the intergroup errors or loading errors. Background: Amniotic fluid (AF) supernatant cell-free fetal (cff) RNA, obtained at mid-trimester, is a pure source of fetal transcripts from multiple organs, including fetal brain (Hui et al. Obstet Gynecol 2012) . Prior fetal transcriptomic data has been obtained using microarrays. Recent advances in massively parallel sequencing (MPS) technology have made it feasible and affordable to directly analyze fetal genes. Aim: We aimed to compare and contrast biological information obtained from the same AF cffRNA samples by microarray and RNA-Seq analysis. Methods: AF cffRNA was extracted from five euploid mid-trimester fetuses, split and prepared in tandem for either hybridization to the Affymetrix HG-U133 Plus 2.0 GeneChip microarray or for MPS using Illumina HiSeq. Transcriptomes derived by each platform were compared based on the presence of signal, rank-order gene expression, and the enrichment of pathways and networks as performed with Ingenuity Pathways Analysis (IPA) software. Both platforms were also evaluated for dynamic range and the ability to track expression of multiple gene isoforms. Results: For MPS, 32-61% of reads aligned to the genome, commensurate with the degraded nature of AF cffRNA. Fewer genes were observed using MPS than microarray (2,800 versus 8,600) . Within individual samples, correlation of total expression between platforms varied (R=0.39-0.56). Among the top 5% of ranked genes there was 51-54% concordance. MPS data yielded more gene populations with more significant p-values than the microarray data for many pathways in the "physiological systems development and function" and "molecular and cellular functions" categories of IPA (Benjamini-Hochberg corrected p-value <0.05). With MPS, but not the microarray, we were able to detect microRNA host genes (e.g. MIR143HG, MIR181A2HG, MIR205HG) and various transcript isoforms, including those known to be expressed in the developing fetus (e.g. isoforms of IGF2, NRG1, RBPJ). Conclusions: Our analyses show that while the microarray data affords a broader view of gene expression, particularly in low-concentration or degraded samples such as AF cffRNA, the MPS data provide a better focus on alternative splicing and specific biological pathways relevant to the developing fetus.

Placental Glucose Transporter Expression and Activity in Relation to Maternal BMI and Fetal Growth. Ometeotl Acosta, Susanne Lager, Donald Dudley, Theresa L Powell, Thomas Jansson. Dept OB/GYN, University of Texas Health Science Center, San Antonio, TX, USA. Introduction: Obese women are at increased risk to deliver a large infant, however the underlying mechanism remains to be established. Glucose is the primary energy substrate for the fetus and fetoplacental glucose needs are met entirely by placental transfer. Fetal glucose availability is linked to fetal growth by regulating the release of insulin and IGF-I, primary fetal growth hormones. Hypothesis: Fetal blood glucose levels and placental glucose transporter expression and activity are increased in high BMI women giving birth to large babies. Methods: Fasting maternal and umbilical cord glucose and insulin concentrations were determined in 29 non-diabetic women with varying early pregnancy BMI (range 18.8-54.3) giving birth to infants with birth weights ranging from 3025-4133g. Five infants had a birth weight greater than 4000g. We isolated syncytiotrophoblast microvillous (MVM) and basal plasma membranes (BM) from 26 placentas and determined the expression of GLUT1 and 9 (Western blot) and glucose uptake (radiolabeled glucose uptake). We performed linear regression analysis to evaluate significant correlations between glucose, insulin, placental transporter expression and activity with maternal BMI and birth weight. Results: Umbilical cord glucose levels were significantly correlated with maternal BMI (r²= 0.15, n=29, p=0.03), whereas maternal glucose, cord insulin, MVM, and BM GLUT expression were not influenced by maternal BMI. Cord glucose (r²=0.24, n=29, p=0.0075) and insulin (r²=0.20, n=25, p=0.02) and BM GLUT 1 expression (r²=0.21, n=26, p=0 .02) were positively correlated with birth weight. In contrast, maternal glucose, MVM GLUT 1/9 and BM GLUT9 expression were not related to birth weight. In preliminary studies, MVM and BM glucose uptake were not significantly influenced by maternal BMI or birth weight. Conclusion: The observed increase in umbilical glucose levels with increasing BMI and birth weight could not be explained by elevated maternal glucose, suggesting changes in placental handling of glucose with fetal overgrowth in high BMI pregnancies. In addition, GLUT 1 expression in BM and fetal insulin were positively correlated with birth weight in our study population. Because BM is believed to be the rate-limiting step for transplacental glucose transport, enhanced BM glucose transport capacity may be a contributing factor to increased fetal growth in high BMI women. Pre-eclampsia is a pregnancy-specific disease affecting up to 5% of all pregnancies worldwide associated with hypoxia as well as intrauterine growth restriction. It is a major cause of maternal and fetal morbidity and mortality. Transient receptor potential vanilloid 6 (TRPV6) is an epithelial Ca 2+ channel protein expressed in calcium absorbing organs. It is also found in the human placenta. Syncytiotrophoblasts, building a multinuclear epithelial barrier layer between mother and her fetus, carries out the maternal and fetal metabolite as well as ionic exchanges such as calcium transfer. Materno-fetal calcium transport is a crucial process for the fetal calcium homeostasis and skeletal growth. It has been shown that additional calcium supplementation in developing countries drastically reduces the risk of pre-eclampsia. In the aim of this study was to investigate the role of placental TRPV6 in pregnancies compromised by pre-eclampsia. Term placentae following normal pregnancies (control) and pregnancies affected by pre-eclampsia were collected following elective caesarean sections. The gene expression of TRPV6 in whole placental villous tissue was assessed by real-time PCR. mRNA transcripts were normalized to the reference gene GAPDH. Protein expression was analyzed by Western blot analysis of syncytial basal membrane (BM) and apical microvillous-membrane (MVM) fractions, prepared by differential ultra-centrifugation and magnesium precipitation. In normal placenta there was considerably less TRPV6 protein expression in MVM than in BM. in MVM we found less unglycosylated TRPV 6 than the glycosylated isoform, whereas in BM more unglycosylated than glycosylated TRPV6 was detectable. Further, in MVM the unglycosylated isoform was significantly down-regulated in pre-eclampsia when compared to normal. This altered TRPV6 expression pattern might have an impact on placental calcium transport in pre-eclampsia. of 5 AQP genes in human amnion. Because the amnion displays regional differences in gene expression, we compared AQP expression between the placental and reflected amnion. We hypothesized that AQP expression is higher in placental relative to reflected amnion, and AQP1 and 3 are expressed at greater abundance compared to AQP8, 9 and 11, suggesting that they may play a dominant role in intramembranous transport of amniotic fluid. Methods: Subjects at term with normal pregnancies (n=9) and gestational diabetes (n=4) were recruited. Placental and reflected amnion samples were collected immediately after cesarean delivery and quick frozen for gene expression analysis. Amnion total RNA was reverse transcribed and amplified by real-time PCR for AQP1, 3, 8, 9 and 11 using predesigned Taqman human AQP primers and probes (ABI). Target AQP mRNA was normalized to the 18S endogenous reference and quantified by the comparative C T method. Data were analyzed by ANOVA and presented as fold difference relative to placental AQP8. Results: In normal subjects, there were no significant differences in expression of the 5 AQPs between placental and reflected amnion. However, highly significant differences (P<0.0001) existed amongst individual AQPs, with AQP1, 3, 9, and 11 expression at 170, 172, 48 , and 1.8 fold, resp., higher than AQP8. In the reflected but not placental amnion, AQP1, 3, and 9 levels were higher in male than female fetuses (P<0.01). In gestational diabetics, AQP1 expression in placental and reflected amnion was lower than in normal subjects (P<0.01). Conclusion: There is a differential pattern of expression for AQP1, 3, 8, 9 and 11 in human amnion with AQP1, 3 and 9 at greater abundance while regional differences were not observed. Further, AQP1 likely is down regulated in gestational diabetics. We speculate that AQP1, 3 and 9 are the predominant AQPs involved in water transport across the amnion and thus play an important role in regulating amniotic fluid volume and composition particularly in pregnancy complications.

Maternal Obesity during Pregnancy Induces Nitrosative Stress in Mitochondria in the Human Placenta. LaShauna Evans, Leslie Myatt.

Maternal obesity creates an adverse intrauterine environment, is associated with complications during pregnancy such as preeclampsia and gestational diabetes, and programs the offspring for obesity, cardiovascular and metabolic disease. We have previously shown that maternal obesity induces nitrosative stress in the placenta and is associated with inhibition of placental mitochondrial energetics in a sexually dimorphic manner. Nitrosative stress occurs when the cytotoxic oxidant peroxynitrite (ONOO-), generated by the interaction of NO with O2-, covalently modifies tyrosine residues in proteins by nitration. Mitochondria are targets of nitrosative stress which inhibits mitochondrial respiration. We tested the hypothesis that maternal obesity induces nitrosative stress in mitochondrial proteins in the placenta in a sexually dimorphic manner. Methods: Randomly sampled villous tissues were collected at term via cesarean section prior to labor to avoid the confounding effects of hypoxia or ischemiareperfusion from two groups of patients based on prepregnancy body mass index (BMI): normal (BMI 22.3+0.2, n=10) and obese (BMI 36.0+0.3, n=9) with similar gestational ages (38.7+0.1 vs 39.6+0.2 weeks). Tissue was frozen and stored at -80oC. Mitochondria were extracted and solubilized in a sucrose buffer with protease inhibitors. Mitochondrial nitrated proteins were measured by Western Blot and normalized to voltage-dependent anion channel. Results: Significantly (p<0.05) greater amounts of nitrated mitochondrial proteins were found in placentas of obese women compared to normal BMI (1.26+0.22 vs 0.39+0.09 units, obese vs normal, respectively). Although there was a trend to significance we could not demonstrate a sex dependent effect of obesity on nitration of mitochondrial proteins (0.22+0.07 vs 0.93+0.31, normal female vs obese female, 0.48+0.12 vs 1.52+0.30, normal male vs obese male). Conclusions: These data suggest that the increased nitrosative stress of maternal obesity results in increased nitration of mitochondrial proteins. This correlates with the reduced mitochondrial respiration we have previously shown in the obese placenta suggesting that nitration of mitochondrial proteins decreases enzymatic activity in the electron transport chain. This identifies an important effect of maternal obesity on the placenta that could result in mitochondrial and ultimately placental dysfunction.

Maternal Obesity Activates Placental mTOR Signaling and Increases Fetal Growth in Rats. F Gaccioli, 1 V White, 2 A Jawerbaum, 2 TL Powell, 1 T Jansson. 1 BACKGROUND: Obese women have a high dietary fat intake and an increased risk of giving birth to a large baby. The mammalian target of rapamycin (mTOR) pathway has been proposed to function as a placental nutrient sensor and is a positive regulator of placental amino acid transport. We hypothesized that maternal obesity, induced by a high fat diet, up-regulates placental mTOR signaling and nutrient transport resulting in fetal overgrowth. METHODS: Albino Wistar female rats were fed a standard chow +/saturated animal fat for 6 weeks before mating and during pregnancy. Dams were sacrificed at GD21 (N=9 control diet, C; N=10 high fat diet, HF) and maternal and fetal blood was collected. Placental homogenates were prepared for Western blot analysis and trophoblast plasma membranes (TPM) were isolated. Phosphorylation of placental 4EBP1 and rpS6 was determined to evaluate mTOR Complex 1 (mTORC1) activity, and placental expression of P-Akt, P-AMPK and P-ERK1/2 were measured to study signaling upstream of mTOR. Placental inflammation was assessed by P-JNK, P-STAT3, total IkB• and cleaved caspase-1 expression. We measured System A and L amino acid transport activity and expression of SNAT 1, 2 and 4, GLUT 1, 3 and 9, FATP 4-6 transporters and LPL in TPM. Protein expression of FABP 1-3 was determined in placental homogenates. RESULTS: HF-diet significantly increased maternal and fetal serum triglyceride levels (+118% and +35%, respectively) but not maternal fasting glucose levels. Both maternal (+20%, P<0.01) and fetal weights (+6%, P<0.05) were increased by the HF-diet and placental weights trended to increase (+5%, P=0.14). Expression of placental P-4EBP1 (T37/46) and P-rpS6 (S235/236) was increased +39% (P<0.05) and +48% (P=0.07) while P-AMPK (T172) was reduced (-28%, P<0.01) in the HF-group. Phosphorylation of Akt, ERK1/2, JNK, STAT3 and expression of IkB and cleaved caspase-1 was not different. HF-diet did not significantly alter the expression of the FABPs, SNATs, GLUTs, FATPs or LPL. System A and L transport activity in TPM was not different between groups. CONCLUSION: Diet-induced obesity in rats activates placental mTORC1, possibly through the inhibition of AMPK. We speculate that these changes may contribute to the modestly increased fetal growth, however the mechanisms remain to be established.

Transcription Profiling of Autophagy Associated Genes in the Placenta of Preeclampsia. Ronit Haimov-Kochman, 1 Tamar Cesla, 2 Caryn Greenfield, 1 Yoav Smith, 3 Simcha Yagel, 1 Debra Goldman-Wohl. 1 1 Obstetrics and Gynecology, Hadassah Medical Center, Mt. Scopus, Jerusalem; 2 Faculty of Medicine, Hebrew University, Jerusalem; 3 Bioinformatics Unit, Faculty of Medicine Hebrew University, Jerusalem. Macroautophagy, a lysosomal pathway of cellular component degradation, is known to play an essential role in diverse cellular processes, including survival under stress and nutrient deprivation, development, immunity and clearance of defective macromolecules. In addition to its role in normal cellular homeostasis, autophagy is implicated in processes of inflammation, aging and numerous pathologies. Human placental function is subject to variations in maternal nutrition, which impacts the resources accessible to support the developing fetus. We hypothesized that defective autophagy may be involved in the etiology of preeclampsia where placental insufficiency is implicated in the initiation of the maternal syndrome. We used the microarray data publicly available through the NCBI Gene Expression Omnibus (Geo) platform to analyze if 30 autophagy pathway associated genes display differential expression in normal as compared to preeclamptic placental samples. Five data sets were examined for a total of 59 preeclampsia and 87 control samples. Validity of analysis was confirmed by observing genes known to be associated with preeclampsia. Analysis of whole genome expression transcriptional profiling with RMA (quantile based) normalization using the PARTEK GENOMIC SUITE 6.6. PCA (principal component analysis) was performed to remove outlier samples. A t-test performed on the data did not reveal a statistical difference between autophagy associated gene expression of normal and preeclamptic placental samples. We conclude that although preeclampsia displays many of the features that would suggest altered autophagy, including premature aging and placental insufficiency, defective autophagy as a cause of preeclampsia is not supported by whole placental microarray differential expression profiling.

Altered Lipid Accumulation in Mouse Placentas Following Generalized Disruption of Genomic Imprints. Katherine P Himes, 1 Megan Campbell, 3 Erik Koppes, 2 Richard Chaillet. 2 Background: Imprinted gene expression in the placenta is essential for development. To dissect the role of imprinting during development, we examined conceptuses developing in the absence of DNMT1o cytosine methyltransferase. Absence of DNMT1o results in partial loss of methylation on imprinted differentially methylated domains (DMD) in the embryo and placenta. We have previously shown DNMT1o-deficiency leads to a range of fetal and placental growth abnormalities. Objective: We sought to determine if DNMT1o deficiency was associated with accumulation of placental lipids. We also sought to determine if lipid accumulation was related to placental or fetal weight and if a given imprinted gene cluster was associated with lipid accumulation in the placenta. Methods: Placentas were collected at E17.5. Oil red O (ORO) staining was done on cryosections. Gene expression was determined by RT-qPCR. A lipid accumulation score from 1-4 was assigned based on Oil Red O staining. Four wild type (WT) and 12 DNMT1o-deficient placentas were evaluated. Kruskal wallis was used to compare continuous variables. Results: DNMT1o-deficient placentas had greater lipid accumulation compared to WT.

DNMT1o-deficient placentas with lipid scores of 3 or more were heavier than those with a score of 2 or less--median weight 130.4mg versus 94.5mg (p=0.01). Lipid accumulation was not related to fetal weight. There was a trend towards increased Peg1 gene expression in placentas with greater lipid accumulation (p=0.08). Conclusion: Generalized loss of imprinting is associated with accumulation of lipids in the placenta. This is strongly associated with placental weight, but not embryonic weight. Of imprinted genes examined, Peg1 gene expression was higher in placentas with abnormal lipid accumulation. Peg1 gene expression may be important for lipid metabolism in the placenta.

Regulation of Multidrug Resistance in Term Human Placental Explants in Culture. Mohsen Javam, 1 Melanie C Audette, 1 William Gibb, 2 Stephen G Matthews. 1 1 Physiology, University of Toronto, Toronto, ON, Canada; 2 Ob-Gyn, University of Ottawa, Ottawa, ON, Canada. The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), that limit the passage of xenobiotics, certain hormones and nutrients from the maternal to the fetal circulation. While studies have been conducted in animals, there are limited models available to investigate placental multidrug resistance in human tissue. The objective of this study was to investigate P-gp and BCRP expression in the human placenta using an in vitro culture model. As previous studies have demonstrated syncytiotrophoblast regeneration in these culture conditions, we hypothesized that the regenerated syncytiotrophoblast layer would express high levels of P-gp and BCRP. Methods: Term healthy placentae (n=6) were collected and cultured for a 6-day period. Culture medium was collected every 24h to measure human chorionic gonadotropin (hCG; syncytiotrophoblast function). P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) protein and mRNA expression were measured before and after culture in the same placentae using western blot and qRT-PCR. We also measured the protein expression of HIF1α, an established key regulator of P-gp, using western blot. Results: Cultured explants had reduced hCG levels by day 2 and increased levels following day 3, indicative of syncytiotrophoblast shedding and regeneration. There was a very significant decrease in P-gp protein and ABCB1 mRNA expression in cultured compared to pre-cultured tissue, however there was no change in BCRP protein or ABCG2 mRNA expression. There was a significant decrease in HIF1α protein in cultured explants compared to pre-cultured tissue; this was strongly correlated with P-gp expression. Conclusions: We have identified profound differences in the expression of placental P-gp and BCRP transporters following in vitro culture. Interestingly, the regenerated syncytiotrophoblast layer expresses extremely low levels of P-gp, but 'normal' pre-culture levels of BCRP. The reduction in P-gp correlated closely with that of HIF1α, which is known to positively regulate P-gp in other cell types. Understanding the potential role of HIF1α in the regulation of P-gp is important as emerging evidence indicates these proteins are aberrantly expressed in pathologies of pregnancy, including pre-eclampsia and IUGR. Funded by: Canadian Institutes for Health Research.

Effect of Oxygen on Drug Transporters in the First Trimester Human Placenta. Phetcharawan Lye, 1 Enrrico Bloise, 1 Caroline Dunk, 2 Sascha Drewlo, 2 Stephen Lye, 1,2 William Gibb, 3 Stephen G Matthews. 3 Ob/Gyn, Univ Ottawa, Canada. Multidrug resistance P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are the two primary multidrug (xenobiotic) transporters located on the apical surface of the syncytiotrophoblast. Placental P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) efflux a large number of clinically relevant compounds (cancer and HIV drugs, glycosides, pesticides) and endogenous substrates (steroids), limiting their transfer from the maternal to fetal compartments. P-gp and BCRP are present at high levels in first trimester human placenta when the tissue oxygen tension is very low. However, nothing is known as to how oxygen tension influences the expression of these important transporters. In the present study, we hypothesized that the low oxygen tension in early first trimester is responsible for maintaining high levels of ABCB1 (P-gp) and ABCG2 (BCRP) expression in human placenta. METHODS: Placental tissue was collected from first trimester terminations (7-9weeks). Placental villi (n=6-9/treatment group) were dissected and placed into culture wells and equilibrated for 24h at 8% O2. Explants were then incubated for a further 24 or 48h at 3% O2 (mild hypoxia), 8% O2 (physiological) and 20% O2 (hyperoxia). ABCB1/P-gp and ABCG2/BCRP mRNA and protein levels were assessed by RT-PCR and Western blotting. Regional localization of P-gp and BCRP protein was evaluated by IHC in first trimester placenta. RESULTS: ABCG2 mRNA was positively regulated by oxygen; there was a trend to an increase at 24h hours which reached significance by 48 hours exposure to the experimental O2 tension. In contrast, ABCB1 mRNA levels were not significantly affected by altered O2 tension at 24 or 48h. In the first trimester placenta P-gp protein and BCRP were localized to the syncytiotrophoblast membranes. CONCLUSIONS: ABCG2 (BCRP) expression increases with increasing oxygen tension, while ABCB1 (P-gp) is not affected by oxygen in first trimester human placenta. This would indicate that low oxygen tension is not responsible for the high levels of these two important drug transporters in early stages of human pregnancy. However, our data do suggest that changes in placental oxygen levels during pregnancy may play an important role in regulation of BCRP expression, and in doing so alter the transfer of xenobiotics from the maternal to fetal compartment.

with GDM have larger placentas. As the placenta serves as the interface for the transfer of glucose and additional nutrients from the mother to fetus, alterations of placental growth and development may contribute to the increased risk of abnormal fetal growth. We hypothesized that reduced placental apoptosis contributed to enhanced placental growth in GDM pregnancies. Accordingly, we examined the expression of apoptotic genes in GDM and control pregnancies. STUDY DESIGN: Maternal pre-pregnancy and newborn body weights were documented. Placentas were obtained from control pregnancies (no pregnancy complications; N=5) or GDM pregnancies (N=5) in patients at 37-40 weeks gestation. Placentas were weighed and placental villous samples were collected. One specimen was fixed in 4% paraformaldehyde for TUNEL. The other specimen was snap frozen for gene expression analysis using apoptosis qPCR arrays and RT-PCR. Controls and diabetic groups were compared by Student's t-test. RESULTS: Maternal (90±13 vs. 73±9kg, P=0.004), neonatal birth-weight (4017±241 vs. 3359±270g, P=0.05) and placental weight (572±116 vs. 446±42g, P =0.04) were significantly higher in the GDM group, compared to controls. The apoptotic index of placentas from the GDM group was markedly lower than the controls (0.05±0.01 vs. 0.17±0.04, P<0.04). qPCR arrays showed that tumor necrosis factor receptor superfamily 1B (TNFRS1B, death receptor pathway), BCL2L2 and BAX genes (mitochondrial pathway) were significantly increased (2.4, 4.6, and 2.5 fold, respectively) while CASP8P2 was decreased (0.5-fold) in placentas from GDM as compared to controls. RT-PCR confirmed the consistency of the qPCR arrays. CONCLUSION: Increased placental mass was associated with significantly decreased apoptosis in GDM pregnancies. Decreased expression of CASP8P2, a point of convergence of death receptor and mitochondrial pathways, suggests that both apoptotic pathways may be inhibited in GDM placentas. Increased placental growth secondary to reduced apoptosis may contribute to the development of macrosomia in GDM pregnancies.

Metabolites. SO Jobe, JL Austin, GE Lopez, RR Magness. Ob/Gyn, Univ of Wisconsin-Madison. Intrauterine growth restriction (IUGR) accounts for a large incidence of infant mortality and morbidity worldwide. We developed an ovine model of uterine space restriction (USR) which is associated with reduced placental weight and efficiency (Meyer et al, BOR 2010) . The placental is the main source of estrogens and estrogen metabolites which play key roles in regulating pregnancy-related vascular functions that modulate normal fetal growth. We thus investigated if USR associated IUGR demonstrates alterations in the synthesis and metabolism of estrogens and estrogen metabolites. Methods: Using high performance liquid chromatography mass spectrometry (LC/ MS), we analyzed venous plasma levels of estradiol-17β (E 2 β), estrone (E 1 ), estriol (E 3 ), 2-hydroxyestradiol (2-OHE 2 ), 2-hydroxyestrone (2-OHE 1 ), 4-hydroxyesrone (4-OHE 1 ), 2-methoxyestradiol (2-ME 2 ), 2-methoxyestrone (2-ME 1 ), 4-methoxyestradiol (4-ME 2 ), 4-methoxyestrone (4-ME 1 ), 16-ketoestradiol (16-keto-E 2 ), 16-alpha-hydroxyestrone (16-α-OHE 1 ), 16-epiestriol (16-epi-E 3 ) and 17-epiestriol (17-epi-E 3 ) in USR versus control non-space restricted (NSR) late pregnant sheep [LP; gestational day 120-130; n=3]. Results: Total conjugated + unconjugated plasma levels of E 2 β and E 1 were lower (P<0.05) in USR compared to NSR sheep (E 2 β: 858±40.9 vs 1499±16.7 pg/ml, respectively, and E 1 : 475±56.3 vs 1279±17.9 pg/ml, respectively). Plasma levels of 2-OHE 2 were lower (P<0.05) in USR vs NSR sheep (332±77.8 vs 803±77.8 pg/ml respectively). 2-ME 2 was lower (P<0.05) in USR vs NSR sheep (253±100.3 vs 1430±100.3 pg/ml, respectively). 4-ME 2 was also lower (P<0.05) in USR vs NSR sheep (123±23.3 vs 253±23.3 pg/ ml, respectively). 16-keto-E 2 plasma levels were lower (P<0.05) in USR compared to NSR sheep (385 ±97.3 vs 763 ±97.9 pg/ml, respectively). Plasma levels of 16-α-OHE 1 was lower (P<0.05) in USR vs NSR sheep (323±95.3 vs 802±77.8 pg/ml, respectively). Levels of E 3 , 2-OHE 1 , 4-OHE 1 , 2-ME 1 , 4-ME 1 , 16-epi-E 3 and 17-epi-E 3 were all detectable, but not different between groups. Conclusions: We demonstrate that ovine unilateral uterine space restriction leading to placental maladaptations is also associated with specific alterations in synthesis and metabolism of estrogens and estrogen metabolites and thus supports the hypothesis that these steroids are involved in the regulation of mechanisms to enhance support for placental/fetal growth. HL49210, HD38843, HL87144, R25GM083252

Increased Trophoblast Oxygen Consumption in IUGR. C Mando, 1 C De Palma, 2 GM Anelli, 1 M Figus, 1 MI Mazzocco, 1 F Calascibetta, 3 D Trabattoni, 3 T Stampalija, 4 E Ferrazzi, 4 EI Clementi, 2 I Cetin. 1 1 Unit Obst Gyn Dept Biomed Clin Sci Sacco, Univ Milan, Italy; 2 Unit Pharmacol Dept Biomed Clin Sci Sacco, Univ Milan, Italy; 3 Unit Immunol Dept Biomed Clin Sci Sacco, Univ Milan, Italy; 4 Unit Obst Gyn, Children Hosp V. Buzzi, Milan, Italy. BACKGROUND We have previously shown that IntraUterine Growth Restriction (IUGR), is characterized by higher placental mitochondrial (mt) DNA levels than controls (C), and that this increase is negatively related with umbilical venous oxygenation. Here, we evaluate mt oxygen (O2) consumption and respiratory chain complexes (RCC) gene expression, in trophoblast cells isolated from IUGR and preeclamptic (PE) placentas. METHODS Cytotrophoblast cells were isolated from 16 placentas (8 IUGR, 3 PE alone, 5 C) of non-smoking women at elective caesarean section, and characterized by cytofluorimetry using cytokeratin-7 and anti-vimentin antibodies. mRNA levels of NDUFA9 (CI), ETFDH (CII), UQCRC1 (CIII) and COX4I1 (CIV) were quantified by Real Time (RT) PCR. O2 consumption was evaluated by High Resolution Respirometry (HRR), by administration of substrates and inhibitors of different RCC, thus allowing the measure of the global cell and of single complexes activity. Data were normalized by mtDNA content. RESULTS IUGR presented significantly lower UQCRC1 and COX4I1 mRNA levels than C (p<0.05). On the contrary, both raw (figure1A) and normalized data (figure1B) in IUGR with or without PE (but not in PE without IUGR) showed significantly higher O2 consumption levels of RCC, altogether and singularly, vs C (p<0.05 and p<0.01), particularly for CIV. In the entire population most of HRR data negatively related with ga and with placental and fetal weights. DISCUSSION Our data show lower mRNA levels of CIII and CIV subunits in trophoblast cells from insufficient placentas of IUGR fetuses, in contrast with the RCC higher functionality, suggesting a compensatory mechanism to their lower expression. These results shed new light into placental oxygenation in IUGR, suggesting that increased placental oxygen utilization may represent a limiting step in fetal growth restriction. The activity of ATP-binding cassette (ABC) transport proteins can be controlled by a variety of endogenous and environmental stimuli. Prostaglandin E2 (PGE2) is a hormone-like substance that acts as an immunomodulator at the maternalfetal interface. Evidence suggests that PGE2 up-regulates the expression of drug efflux transporters. In the present investigation, we sought to determine the impact of PGE2 on the multidrug resistance protein (MDR) 1 (P-glycoprotein; Gene: ABCB1) and breast cancer resistance protein (BCRP; Gene: ABCG2) in human placental cells. Transporter expression was evaluated by qRT-PCR and Western blot in JAr placental cells before and after PGE2 stimulation. The functional activities of MDR1 and BCRP were measured by the cellular accumulation of fluorescent substrates. Cells were treated with specific prostaglandin receptor (EP) antagonists prior to stimulation with PGE2 to determine the regulatory pathways involved. PGE2 up-regulated BCRP expression in JAr cells, correlating with a decrease in the cellular uptake of the BCRP substrate, Hoechst 33342. PGE2 decreased MDR1 expression, leading to the intracellular accumulation of Calcein-AM (MDR1 substrate). The regulation of BCRP by PGE2 was inhibited by pretreatment of JAr cells with EP3 and EP1 specific receptor antagonists. EP receptor specific antagonists had no affect on MDR1. PGE2 differentially regulates MDR1 and BCRP in placental cells. Alteration of transporter activities may cause a change in drug exposure leading to altered response (safety or efficacy). We find that EP3 and EP1 are key signaling factors in the regulatory pathway by which PGE2 affects placental BCRP. These findings offer new insight into the pathways by which pregnancy can alter drug transport across the placenta. 

Intrauterine growth restriction (IUGR) is a significant cause of fetal mortality and long term morbidity. Current pathology has limited ability to detect the antecedent pathways leading to an individual's low birth weight, in part, because it only considers the two-dimensional domain. This feasibility study set out to reconstruct the three-dimensional structure of the villous tree using innovative digital image analysis technology.

Placentas were obtained from normal and intrauterine growth restricted pregnancies, delivered by elective caesarean section at term. Two hundred consecutive full thickness sections at 5micron intervals were digitized and archived for software analysis. Slice-to-slice image registration was performed to create slide sets for segmentation. Iso-surfacing of the segmented layers yielded three-dimensional reconstructions of the stem villous architecture.

This method successfully enabled the creation of three-dimensional isosurfaced volumes displaying the villous architecture of the placenta. Qualitative assessment of the control specimens revealed a diffusely distributed complex branching network of stem villi. In comparison, the intrauterine growth restricted placenta displayed a sparse and truncated branching pattern.

Three-dimensional reconstruction of placental tissue is a feasible tool for visual analysis of morphology in healthy and pathological placentas. Digital image analysis may improve the understanding of placental architecture and may enable discrete phenotyping of pathological placentas (supported by Cerebra). Traditional histopathologic placental examination relies on expert-led macroand microscopic assessment. However, this method is subjective, and is unable to quantify changes in the placental architecture. Research tools which have improved our understanding of placental structure include stereology and scanning electron microscopy, which are not suitable for routine clinical use. This feasibility study used novel digitised capture and quantitative analysis to quantify vascular parameters within the normal placenta. Methods Placentas were obtained from uncomplicated pregnancies delivered by elective caesarean section at term. Tissue was sampled from the centre and periphery of each placental disk. Standard histological techniques were used and slides were stained with CD-31 to identify endothelial cells. Slides were scanned into an automated system and analysed using a Microvessel Analysis Tool (Aperio ePathology Solutions, Vista, CA, USA).

No significant differences in vessel number were observed within sections obtained from central versus peripheral sites, nor between maternal, central or fetal regions within each section (p=0.2, p=0.13, respectively). Significant differences in vessel number, however, were seen between placentas (p<0.000). Differences in vessel density, lumen area, perimeter and vascular area were also significant (p<0.000).

We observed a trend towards an inverse correlation between vessel number and placental weight.

Through digital image analysis we demonstrated uniformity in vascular parameters within each placenta. This tool could therefore be applied to routine clinical settings. It may be possible to generate vascular reference ranges for the normal placenta through analysing a larger sample size. Further work remains to investigate high levels of inter-individual variation seen across normal placentas and characterise the vascular parameters in pathological placentas (supported by Cerebra). fetal blood. Organized in a fixed orientation, three layers of trophoblast cells followed by a layer of fetal endothelial cells separate the maternal and fetal blood spaces. A mononuclear trophoblast giant cell layer lines the maternal blood spaces, followed by two multi-nucleated syncytiotrophoblast layers (SynT-I and -II respectively). It has been shown previously, that SynT-I and -II cells can be identified by the layer-restricted expression of Syncytin a (Syna) and Gcm1, respectively. In addition, these genes are expressed in a similar, spatially related manner in the chorion, suggesting pre-patterning of labyrinth development. In the present study, we have found that Ly6e, encoding a small molecular weight, GPI-linked glycoprotein of the Ly-6 family of cell surface proteins, is expressed by SynT-I cells within the labyrinth; a pattern reminiscent of Syna expression. Like Syna, genetic deletion of Ly6e is embryonic lethal by mid-gestation, a phenotype originally attributed to heart abnormalities. We show that Ly6e KO placentae display a range of abnormalities, both in the gross organisation of the villous tree and in the ultrastructural organisation of the interhaemal membrane. As evidenced by subtype-specific in situ hybridisation, trophoblast differentiation does not appear to be affected by Ly6e deletion per se, but a reduction in villous branching is evident. Further indicative of a morphogenesis defect, the interhaemal membranes in Ly6e mutant placentae display areas of disrupted syncytiotrophoblast fusion, altered electron density of cell layers and an overall increase in thickness, all profound deficiencies in placental function. Furthermore, these placental phenotypes precede the reported heart abnormalities, and are likely the cause of lethality in these mice. OBJECTIVE: Intrahepatic cholestasis of pregnancy (ICP) is associated with adverse obstetrical outcomes such as preterm birth, meconium passage, and intrauterine fetal demise. The pathophysiology leading to these events is uncertain, and little is known about the effects of cholestasis on the human placenta. The objective of this study was to compare the incidence of histologic markers of oxidative stress and inflammation in placentas from women with ICP versus those without cholestasis. STUDY DESIGN: This was a retrospective case control study. Study subjects were identified using the labor and delivery birth log at our institution. Cases had a diagnosis of cholestasis of pregnancy and no other medical problems that may be associated with placental findings indicative of oxidative stress and inflammation. The control group did not carry a diagnosis of ICP and also did not have any medical problems. The placentas were reviewed in a random order with an expert pathologist who was blinded to the study group. Statistical analyses were performed with the t-test, mann-whitney test, and f-test. A p-value < 0.05 was considered significant. RESULTS: The placentas from a total of 24 cases and 30 controls were reviewed. Seventeen placental characteristics indicative of oxidative stress or inflammation were found (i.e. increased syncytial knots, fibrinous necrosis, necrotizing villitis), but there was no statistically significant difference between cases and controls. Comparing cases with and without cholestasis, there was a difference in gestational week at delivery (36.8 ± 1.3 vs. 38.9 ± 1.2, p<0.0001). After controlling for this, there were no differences in placental weight (513.50 gms (277-675) vs. 584 gms (334-1100), p=0.4), birth weight (3045.20 ± 430.08 gms vs. 3431.70 ± 435.00 gms, p=0.46), or APGAR score at 5 minutes (9 (8-9) vs. 9 (6-10), p=0.74). CONCLUSION: There does not appear to be an increased incidence of placental markers of oxidative stress and inflammation in cases of ICP. Vitamin D (VD) supplementation of pregnant women and infants has engendered international attention and formulation of therapeutic guidelines. Normal in-utero fetal levels and physiologic functions of various VD metabolites have yet to be determined. It is possible that the fetal/placental unit is not dependent on maternal-fetal transfer of VD, instead being able to synthesize these moieties from cholesterol. OBJECTIVE: As the first step in exploring these relationships, this study collected maternal (mat), umbilical vein (UV) and artery (UA) serum samples at delivery of term pregnancies, allowing reliable determination of 25(OH)D3 [D3] and its C-3 epimer [epi-D3]. METHODS: Samples from 26 term deliveries were refrigerated within 1 hour of collection and serum was frozen at -30° within 24 hrs. High Performance Liquid Chromatograph/Tandem Mass Spectroscopy (LC/MS-MS) was used for analyte analysis. RESULTS: Mat D3 (ave ± 1SD)=29.4 ± 11.8 ng/dl. Fetal UA D3 (ave ± 1SD)=17.1 ± 7.7 ng/dl and equaled 59 (±19)% of the mat D3 value. The relationship between mat D3 and fetal D3 was significant (R 2 =0.74; P<0.0001). Mat epi-D3 (ave ± 1SD)=2.3 ± 1.4 ng/dl and accounted for 6.7 (±2.6)% of the total mat D3 (D3+epi-D3). Fetal UA epi-D3 (ave ± 1SD)=1.9 ± 1.2 ng/dl, similar to the mat epi-D3, and accounted for 10.7 (± 2.0)% of the fetal total vitamin D3 (D3+epi-D3). The relationship between mat and fetal epi-D3 was significant (R 2 =0.63; P<0.0001). In reference to total mat and total fetal D3 (D3+epi-D3), the relationship had an R 2 value of 0.784 and was significant (P<0.0001). DISCUSSION: Prior studies have been unable to differentiate the epimer forms of VD. Using LC/MS-MS we have been able to differentiate between D2, D3 and their epimers in samples, which likely reflects the true in-utero values (UA). The fact that epi-D3 levels in both the mother and fetus are similar makes its site of synthesis uncertain; however, since both mat and infant values decrease post-delivery, placental synthesis is probable. Overall, our results show that mat VD levels ultimately affect the VD levels of the fetus. By identifying the different forms of VD (D2, D3, epi-D3) we can trace the physiological mechanisms of each and link deficiency to adverse pregnancy and neonatal outcomes. Placental dysfunction is central to the pathogenesis of conditions such as pre-eclampsia and intrauterine growth restriction, which are associated with increased morbidity and mortality for both the fetus and mother. Descriptive pathology of the placenta in health and in disease is constrained by its twodimensional perspective. This feasibility study investigated the utility of a novel software programme to reconstruct the three-dimensional placental vascular tree from sequential histological sections at microscopic level.

Placentas from an uncomplicated and a pre-eclamptic pregnancy were obtained, with consent, from women undergoing elective cesarean section delivery at term. Placental tissue was processed using a standard protocol, then sectioned sequentially at 5µm thickness over 40 consecutive slices. Once scanned, a slice registration application was utilized to create rigid and non-rigid reconstructions of the terminal villous tree.

Successful registration and reconstruction of terminal villi in both specimens enabled the three-dimensional structure of the placenta in health and disease to be viewed and compared at microscopic level. This initial reconstruction work demonstrated elongation of the terminal villi in the pre-eclamptic placenta. This novel means of assessment may lead to the generation of new hypotheses which may help us understand the mechanisms of impaired feto-maternal blood flow and transfer.

Using software to create three-dimensional reconstructions is a feasible and exciting new method of understanding the placental structure, which may have potential use in both research and in diagnostic pathology services (supported by Cerebra). Introduction: Caveolin-1 (Cav-1) is the principal structure protein of plasma membrane microdomain caveolae that are essential for nitric oxide (NO) signaling and angiogenesis. We have shown that caveolae play a paradoxical role in compartmentalizing angiogenic growth factor signaling to placental angiogenesis in vitro. However, whether Cav-1/caveolae plays a role in placental angiogenesis in vivo is unknown. Targeted Cav-1 disruption causes loss of caveolae and enhances NO production due to eNOS hyperactivation, providing an ideal animal model for investigating Cav-1/caveolae in placental angiogenesis in vivo. Hypothesis: Placental angiogenesis is dysregulated in Cav-1 null mutant mice due to increased nitrosative stress. Methods: Wild-type (wt) and Cav-1 null mutant mice were purchased from Jackson Laboratories. Dated pregnant mice were sacrificed at d13.5 of gestation. The placentas were fixed in formalin and embedded in paraffin. Sections of the placentas were subjected to morphometric analysis of angiogenesis and to immunohistochemical analysis with an anti-nitrotyrosine antibody. Total proteins were extracted from the placentas and used for analyzing eNOS and MnSOD nitrosation by immunoblotting of total nitrosated proteins immunoprecipitated with an anti-nitrotyrosine antibody. Results: Placental weight (mg, n=26) in Cav-1 null mice (97±20) was significantly (p<0.001) greater than that in wt placentas (74±10, n=28), while fetal weight (mg) in Cav-1 null mice (67±15) was significantly (p<0.01) lower than wt controls (83±31). Labyrinth vascular density and total area were significantly (p<0.05) lower in Cav-1 null mice compared to wt controls. Immunostaining with the nitrotyrosine antibody yielded a much stronger signal in Cav-1 null placentas compared to controls. Levels of total nitrosated proteins and nitrosated eNOS and MnSOD were significantly greater in Cav-1 null mice compared to wt controls. Conclusion: Targeted disruption of Cav-1 gene results in intrauterine growth restriction in association with enhanced nitrosative stress and an impaired placental vasculature with decreased angiogenesis, implicating an essential role of Cav-1/caveolae in placental angiogenesis in vivo via regulating NO signaling (supported by RO1 HL74947 & R21HL98746).

and Amniotic Fluid Macrophages. Varkha Agrawal, 1 Emmet Hirsch. 1,2 1 Obstetrics and Gynecology, NorthShore University HealthSystem, Evanston, IL, USA; 2 Pritzker School of Medicine, University of Chicago, Chicago, IL, USA. Objective: Surfactant protein (SP)-A is produced in fetal lungs and may either activate or suppress immune functions. We have shown that SP-A inhibits preterm delivery induced by toll-like receptor (TLR) ligands in mice. Here we examine the effect of SP-A on production of pro-and anti-inflammatory mediators in gestational tissues and amniotic fluid macrophages. Methods: Amniotic fluid was harvested from CD-1 mice on days 14.5 and 16.5 of pregnancy. Adherent cells (confirmed as macrophages using the F4/80 marker) were cultured for 1 hour with or without LPS (5ng/ml) in the presence or absence of SP-A (20µg/ml). Cells were processed for immunofluorescent detection of IL-1β protein. Experiments were repeated twice in triplicate sets. For in vivo experiments, CD-1 mice underwent IU injection on day 14.5 of pregnancy with either peptidoglycan (PGN, a TLR2 ligand, 0.3mg/ mouse)+polyinosinic:cytidylic acid (poly(I:C), (a TLR3 ligand, 1.0 mg/ mouse) or saline in the presence or absence of SP-A (0.105mg/mouse). Animals were euthanized 8 h after surgery and gestational tissues (uteri, fetal membranes, fetuses and placentas) were collected for RNA extraction. RT-PCR was performed in duplicate for IL-1β, CCL5, TNF, CXCL1 and IL-10 with GAPDH as an internal reference. Results: IL-1β protein was not detectable in Day-14.5 macrophages regardless of treatment. SP-A abolished the LPS-induced expression of IL-1β protein in Day-16.5 amniotic fluid macrophages. In vivo treatment with PGN+poly(I:C) resulted in up-regulation of all the assayed transcripts in all of the tissues in which they were detectable. SP-A significantly suppressed the expression of pro-inflammatory markers induced by PGN+poly(I:C) in placentas and fetal bodies but not in uteri and fetal membranes. In a complementary fashion, SP-A significantly increased the expression of the anti-inflammatory factors CXCL1 in placentas, fetal bodies and uteri and IL-10 in uteri (IL-10 was not detectable in placentas and fetal bodies). Conclusion: SP-A suppresses the expression of IL-1β protein in amniotic fluid macrophages. In gestational tissues, SP-A suppresses the expression of pro-inflammatory mediators and enhances the expression of anti-inflammatory. In concert with our prior reports, these findings support a role for exogenous SP-A in inhibiting TLR ligand-induced preterm labor via suppression of inflammation. Objective: Bacterial infection and inflammation of the chorioamnion play a major role in premature rupture of membranes and preterm birth. However, little is known about the impact viral infections have on the inflammatory milieu of the fetal membranes (FM). Infection-associated inflammation at the maternalfetal interface is thought to be mediated by Toll-like receptors (TLR); which signal through the adapter proteins, MyD88 and TRIF. The objective of this study was to evaluate the cytokine profiles of FM towards two viral signatures: viral dsRNA, which activates TLR3, and viral ssRNA, which activates TLR8, and to determine the role of MyD88 and TRIF in these responses. Methods: FM were collected from normal human placentas (n=5) obtained at the time of planned cesarean delivery without labor. FM biopsies were treated with or without Poly(I:C) (a viral dsRNA mimic; 20µg/ml) or viral ssRNA (5µg/ml) in the presence and absence of either a MyD88 inhibitor or a TRIF inhibitor (10µM). After 24hrs, supernatants were collected and measured for cytokines by multiplex analysis (BioRad). Results: When compared to the untreated control, viral ssRNA significantly increased FM secretion of IL-1β by 20-fold; G-CSF by 4.5-fold; MIP-1α by 9.6-fold; MIP-1β by 3.3-fold and RANTES by 5.8-fold (p<0.05). In contrast, Poly(I:C) increased FM secretion of only 3 cytokines: MIP-1α by 5.3-fold; MIP-1β by 3.9-fold and RANTES by 4.2-fold (p<0.05). ssRNA-induced FM upregulation of IL-1β, G-CSF, MIP-1α and RANTES was reduced by the presence of either the MyD88 or TRIF inhibitor (p<0.05). However, neither inhibitor had any effect on ssRNA-induced MIP-1β secretion. Poly(I:C)induced MIP-1α was reduced in the presence of either the MyD88 or TRIF inhibitor (p<0.05). However, Poly(I:C)-induced MIP-1β and RANTES were only reduced by the MyD88 inhibitor (p<0.05). Conclusion: FM secrete different inflammatory cytokine profiles in response viral dsRNA and ssRNA through distinct mechanisms. Viral ssRNA triggers FM responses through both MyD88 and TRIF, or through neither. In contrast, viral dsRNA induces FM inflammation through either MyD88, or both MyD88 and TRIF. These findings suggest that FM inflammatory responses to viral dsRNA are mediated by TLRs, while viral ssRNA responses are TLR-dependent and independent. (HR3,4) . Pharmacologic evidence suggests signals through HR1 and HR2 support uterine contraction and relaxation, respectively. We assert that normal uterus has a regulatory circuit that counters infection-induced inflammation and contractions. We thus hypothesize that during gestation HR1 and HR2 are down regulated in uterine leukocytes while HR2 is up regulated in non-leukocytes. Objective: Examine transcriptional expression of HRs in cell populations of mouse uterus. Methods: C57BL/6 females, non-pregnant (NP, n=9) or at day (D) 7-8 (6), 10-12 (12), 14-16 (5), 18 or post-partum D1 (8) were euthanized. Uteri were separated from the placenta, minced, digested, and made into single cell suspensions. These were either used to isolate RNA directly, or (NP, D10-12, D18+), underwent magnetic cell-sorting (MACS) to separate uterine cells into leukocyte (CD45+) and non leukocyte (CD45-) pools. CD45+ cells from 2-4 uteri were pooled and underwent MACS to obtain CD45+T cell+ (T+) and CD45+T-populations. Cell pools were subjected to RT-QPCR for HR and housekeeping gene (hprt) as control. HR expression was calculated by the ddCt method. Statistical significance (p<0.05) was determined by ANOVA.

Results: Compared to NP, unsorted uterine cells transiently decreased (4.6fold, p=0.0004) HR1 at mid gestation, while HR2 expression increased (8fold, p=0.03). Both CD45-and CD45+ cells expressed HR2 > HR1 (p=0.003, p<0.0001). While CD45-cells did not increase HR2 over gestation (p=0.39), CD45+ cells expressed increasing levels of HR2 reaching a maximum at D18 (p=0.01). While HR1 expression trended downward at mid-gestation in CD45-cells (p=0.13), the expression in CD45+ was stable until D18 when it increased 4 fold over NP (p=0.0034). On D10-12, HR2 was > HR1 in both CD45+T+ (9-fold) and CD45+T-(17-fold, p=0.0003), but the expression of both HR2 and HR1 were higher in T-as compared to T+ subpopulations (6fold and 4-fold, p=0.001). Conclusions: In pregnancy uterine subpopulations show differential HR1 and HR2 transcriptional expression, dominated by HR2. In normal pregnancy, H may participate in a complex system that regulates both inflammation and uterine quiescence.

Role of miR-21 in the Inflammatory Response Elicited by Human Decidual Cells after Streptococcal Infection. Violeta Castro-Leyva, 1,2 Silvia Giono-Cerezo, 2 Francisco Arenas-Huertero, 3 Iyari Morales-Mendez, 1 Aurora Espejel-Nunez, 4 Guadalupe Estrada-Gutierrez. 1 1 Infectology, Instituto Nacional de Perinatologia, Mexico; 2 Bacteriology, Instituto Politecnico Nacional, Mexico; 3 Pathology, Hospital Infantil de Mexico, Mexico; 4 Biochemistry, Instituto Nacional de Perinatologia, Mexico; 5 Labor Room, Instituto Nacional de Perinatologia, Mexico. Background: Decidual cells play a pivotal role in the onset of the inflammation triggered by intrauterine infection. The mechanisms that regulate the production of the inflammatory molecules by these cells have not been fully explored. MicroRNAs (miRs) are small non-coding RNAs that suppress gene expression at the post-transcriptional level, which are implicated in the cellular response to bacteria. Since the inflammatory stimuli induce expression of miR-21 in different cellular models, in this work we address if miR-21 has a role in the modulation of the inflammatory response elicited by decidual cells under experimental infection. Methods: Primary cultures of decidual cells (n=10) were characterized using vimentin as a marker for fibroblasts. Decidualization was induced with 36nM estradiol + 300nm progesterone, and confirmed by prolactin and IGFBP-1 secretion. Cultures were infected overnight with group B streptococci (GBS). Cellular RNA was used to quantify expression of miR-21, IL-1b, and IL-10 by qRT-PCR. In addition RNA extracted from supernatants was used to quantify miR-21 expression. Validation of expression assays was performed using ELISAs for cytokine quantification in the culture supernatants. miR-21 functional analysis was performed using synthetic miR-21 inhibitor in transfected decidual cells. Results: Up to 90% of cells in the primary cultures were identified as decidual cells showing morphological and biochemical markers of decidualization. Expression of miR-21 was significantly up-regulated in both, intra and extracellular compartments. Although expression of IL-1b and IL-10 was significantly up-regulated, confirmation of expression data showed that only IL-10 was secreted. Functional analysis showed that inhibition of miR-21 favors secretion of IL-1b and block secretion of IL-10. Conclusion: GBS infection increases expression of miR-21 by decidual cells favoring an anti-inflammatory response mediated by IL-10 and controlling pro-inflammatory cytokines like IL-1b, highlighting the role of decidual cells as regulators of the immune response triggered by the intrauterine infection.

Degree of Blood Contamination after an Amniocentesis and the Effect on Markers of Amniotic Fluid Inflammation. Marcos Cordoba, 1 Eduardo Aguin, 2 Samet Albayrak, 3 Benjamin Kuritzkes, 1 Laura Meints, 3 Ray O Bahado-Singh. 3 Background Inflammatory markers measured in amniotic fluid (AF) are the best predictors of maternal and perinatal outcomes in preterm labor. White blood cells (WBC) and glucose are the most commonly used markers. However, the impact of blood contamination after a bloody tap on the predictiveness of these markers for perinatal outcomes have not been extensively studied.

A retrospective cohort study was performed. AF glucose and WBC were measured in 70 cases of "mildly bloody tap" (1000-30,000 RBC/mm3) (Group 1), and 17 cases of "grossly bloody tap" (> 30,000RBC/mm3) (Group 2), and compared to 195 "non bloody" controls (<1000 RBC/mm3). Cases and controls were at increased risk for prematurity. Mean (SD) levels of glucose and WBC were compared between groups. The variables used to define AF inflammation were white (WBC) >50 cell/mm3 or glucose <15 mg/dl. The impact of the degree of blood contamination on levels of inflammatory markers and their ability to predict positive AF culture were compared against normal controls.

Overall, WBC was significantly elevated in both Group 1, mean (SD) 619. 2 (244.5) and Group 2, 767.5 (378) compared with controls, 153 (39.2), (p=0.01). There was with no difference in AF glucose among cases and controls (p=0.4). Among cases with a positive culture there was no difference in the frequency of WBC >50 between groups 1 and 2, however in cases with a negative culture, WBC >50 was significantly more frequent both study groups compared to controls (Fisher exact test p<0.005). Glucose <15, OR 0.93 (0.89, 0.97), p<0.001 and WBC >50, 4.81 (2.0, 11.65), p=<0.001 were significant predictors of positive AF culture. Bloody status did not interact with AF glucose or WBC or affect their prediction of these adverse outcomes.

The interpretation of markers of AF inflammation with blood contamination is a clinical challenge. While the frequency of elevated WBC is increased in bloody AF with negative culture, the overall ability of inflammatory markers to predict adverse outcome appears not to be significantly affected by difference in the amount of RBC contamination.

The Effects of LPS on Intermediate Filaments in the Developing Fetal Ovine Lung. Thomas Objective Chorioamnionitis is an important cause of preterm birth. Antenatal infection and inflammation have been associated with improved respiratory outcomes in preterm infants by functionally maturing the lungs. These infants however, remain at risk of lung parenchymal injury caused by the shear stress generated by mechanically ventilating atelectic alveoli. Intermediate Filaments (IF) form a supracellular scaffold within cells that maintains cell and tissue integrity, primarily by providing resistance to external shear stresses. Despite this, there is virtually no information available on the role played by IFs in the biomechanics of the developing and premature lung. Using an ovine model of preterm birth, we aimed to examine the effects of LPS exposure on IF expression in the developing fetal lung. Methods Date mated merino ewes were divided into two groups: i) IU 10mg LPS in 2mL sterile saline (n=8); and ii) IU 2 ml saline (control, n=6). Ewes were anaesthetised and the fetuses surgically delivered 48 hours post intervention at 124d GA (± 2 d; term=150 d). Tissue was collected from the upper lobe of the right lung for quantitative PCR analysis of intermediate filament expression. 

Significant changes in expression (p ≤ 0.05) were seen only in Keratin 1 (p= 0.029). Conclusions These novel data suggest that the functional maturation observed in the fetal lungs after exposure to antenatal inflammation, may not be accompanied by a necessary structural maturation of the tissue. Inappropriate K1 expression has been shown to cause impaired cellular integrity (and associated disease states), and these data suggest a selective maturation of the pulmonary IF complement, that may leave cells susceptible to the mechanotrauma associated with postnatal existence. Further investigation is warranted considering the prevalence of perinatal and long-term respiratory disease in the preterm population. Background: Premature delivery, defined as delivery occurring before 37 weeks of gestation, occurs in 12% of all births, and accounts for nearly half of neurological morbidity, and 60 to 80% of perinatal mortality. The single most common cause of spontaneous preterm birth (PTB) is infection. We have shown that both ET-1 and endothelin-converting enzyme-1(ECE-1) are increased in gestational tissues in E16.5 mice induced to deliver prematurely with lipopolysaccharide (LPS). We have also previously shown that both the ECE-1 inhibitor phosphoramidon and the selective ET A receptor antagonist BQ-123 control PTB in a dose-dependent fashion. In addition, we have found that a novel ET A receptor antagonist synthesized by our group rescues mice from LPS-induced PTB. Finally, we have been able to prevent PTB in LPSstimulated animals, by transfection of E15 mice with ECE-1 RNAi. Objective: Our aim was to test the hypothesis that ET-1 contributes to the cause of preterm birth by up-regulating pro-inflammatory cytokines. Methods: Chorionic villous explants from term placentae obtained from Albert Einstein Hospital were divided into four groups: sham (cultured in media), endotoxin-treated (media plus 500ug/ml LPS), endotoxin and low level ET-1 blockade (media plus 500 ug/ml LPS plus 16 uM BQ-123) and endotoxin and high level ET-1 blockade (media plus 500ug/ml LPS plus 32 uM BQ-123). Explant supernatants from all groups were collected 24 hours after the addition of LPS and evaluated for changes in levels of interleukin 1beta (IL-1B) and tumor necrosis factor alpha (TNF a) by Western blotting analysis. Results: Villous explants treated with LPS released significantly higher amounts of both IL-1B and TNF a into culture media than sham explants. This proinflammatory response was reversed, in a concentration-dependent fashion, with ET-1 blockade by BQ-123. Conclusion: Placental release of IL-1B and TNFa, pro-inflammatory cytokines closely linked to infection-associated PTB, are ET-1 dependent. The mechanism of action of ET-1 blocking agents in tocolysis involves decreased ET-1dependent release of these mediators. Further evaluation of inflammatory mediators affected by ET-1 action may lead to the identification of novel targets for preventive therapy for PTB. Objective: Neutrophils are first responders to infection. They may also play a central role in the processes by which bacterial infection leads to labor. The objective of this study was to investigate the impact of neutrophil depletion on a mouse model of E. coli-induced labor. Methods: Intraperitoneal (IP) injection of rabbit anti-mouse PMN antiserum (0.5 ml of a 1:10 dilution) or PBS (0.5 ml) was performed 29 hrs and 5 hrs prior to laparotomy and intrauterine (IU) injection of either heat-killed E. coli or PBS. Mice were observed after surgery for preterm delivery (delivery of at least one pup within 48 hours). Blood samples were drawn from the retro-orbital sinus of each mouse at the time of surgery and 24 and 48 hrs after surgery. Circulating neutrophil counts were determined manually after nuclear staining with Gentian violet. A separate group of animals was euthanized 8 hours after surgery for tissue collection including whole blood, in which leukocyte typing was conducted by flow cytometry. Results: Pre-surgical IP injection of PMN antiserum significantly decreased the numbers of circulating leukocytes at the time of surgery (1480±1002/µl with antiserum (n=15) vs. 4883±1529/µl with no antiserum (n=14), p<0.0001). Both the absolute number of neutrophils and their proportion among all leukocytes was significantly diminished by antiserum (neutrophil proportion = 5% with antiserum vs. 24% with no antiserum at the time of surgery, p<0.0001; 2.5% with antiserum vs. 48% with no antiserum 8 hours after surgery in animals receiving IU PBS, p=0.0005; and 3.5% with antiserum vs. 48% with no antiserum in animals receiving IU E. coli). IU saline caused no preterm delivery either with (n=4) or without (n=4) antiserum treatment. IU injection of 6 x 10 7 (n=5) or 2 x 10 7 (n=5) E. coli organisms caused preterm delivery in 100% and 80%, respectively, of mice not receiving antiserum and in 100% (n=5) and 67% (n=6) of animals receiving antiserum (p=1.0 for both).

Conclusions: Partial depletion of circulating neutrophils using antiserum does not alter sensitivity to bacterially induced delivery in the mouse model. Preterm birth in this model is not likely to depend on neutrophil function. Objective: To evaluate the perinatal outcomes of pregnant women with a primary CMV infection treated with CMV specific immunoglobulin therapy. Study design: Case series performed at 4 centers in Colombia (COL) and one in the US from 2009 to 2012. The study included 8 women with primary CMV infection. Maternal diagnosis was made by serological testing measuring CMV IgG, IgM and low IgG avidity. If available, fetal infection was confirmed by positive PCR for CMV in amniotic fluid. Detailed ultrasound examinations were done by MFM specialists at entry and serially. All 8 women were treated with IV CMV specific hyperimmune globulin, Megalotect® (Amarey Novamedical SA, COL), or CytoGam ® (CLS Behring) at a dose of 100 U per kilogram of maternal weight monthly until delivery. Neonatal evaluation included testing of urine for CMV by 1 to 5 days of age; physical examination; ophthalmoscopy and brain-stem auditory evoked responses. Fetuses with suspected neurologic injury, had brain computed tomography and MRI. Results: Maternal infection confirmed by serology at 23 weeks gestation on average. Amniocentesis was done in 7/8 patients; 3/7 had a positive PCR for CMV. 3/8 (37.5%) had abnormal findings on ultrasound at entry: IUGR 3/8, placental insufficiency 1/8, and oligohydramnios 2/8. The majority, 5/8 (62.5%) had a normal ultrasound. 5/8 women received hyperimmuneglobulin every 4 weeks until delivery and 3 received a single dose because of indicated preterm delivery. None of the patients had adverse effects with treatment. 2/8neonates had + CMV in urine, and were 2/3 with + CMV in amniotic fluid. One of them had an abnormal hearing test. (Table) Conclusion: In three fetuses confirmed to have CMV in amniotic fluid by PCR, evidence of infection at birth by urine assessment persisted in two out of three neonates. Treatment of pregnant women with CMV-specific hyperimmune globulin appears to be safe. Efficacy and cost effectiveness should be assessed by adequately powered randomized prospective trials, especially in women with + CMV by PCR in amniotic fluid.

NFκB inhibition only blocked LPS-induced MMP9 and Cox-2 expression, NOX inhibition and anti-oxidants fully blocked LPS effects. H2O2 stimulations had no impact on MMP9 and only a slight impact on Cox-2 expression (x1.5 vs. CTL). However, H2O2 induced NFκB phosphorylation, MMP2 and VEGF expression, and Caspase-3 cleavage (x3.1, x3.13, x21, and x2.7 vs. CTL respectively). Moreover LPS induced MMP9, Cox-2 and VEGF expression in Th-P1, but had no such effect on myometrial cells. H2O2, however, induced MMP2 and Cox-2 expression and caspase-3 cleavage in MC. CONCLUSION: These data show that ROS production in macrophage is required for NFκB activation, but also that is activates MC, leading to labor associated features. It suggests a pivotal role for ROS in labor onset, and suggests a potential interest for anti-oxidant supplementation during pregnancy for preterm labor prevention.

LPS Enhances Myometrial Myocyte Contractility In Vitro through TLR4 Stimulation and RhoA/ROCK Pathway Activation. James L Hutchinson, Shalini P Rajagopal, Jane E Norman. MRC CRH, University of Edinburgh, Edinburgh, United Kingdom. Introduction: Infection is thought to be a major cause of spontaneous preterm labor (PTL), and LPS administration is sufficient to induce PTL in animal models. However, the mechanism by which infection-induced inflammation induces myometrial contractions remains uncertain. The aim of this study was to investigate the response of cultured myometrial myocytes to LPS in terms of cytokine production and myocyte contractions. Methods: PHM1-41 pregnant myometrial cells or primary uterine myocytes were cultured in 6-well plates for 24h in the presence of 100ng/ml LPS or appropriate vehicle. Cell supernatants were collected and either analysed by 21-plex ELISA array, or transferred to plates containing fresh cells seeded in collagen gels the previous day (n=6 gels/group). Gel contraction was monitored for 72h followed by assessment of viable cell number. Syntocinon (10nM) was used as a positive control for stimulation of gel contraction. Pharmacological inhibitors were added either 15min before LPS (indomethacin 50µM, LPS-RS 0.01-10µM), or concomitant with conditioned medium to gels (nifedipine 20µg/ ml, Y27632 10µM). Myosin light chain (MLC) phosphorylation following stimulation of PHM1-41 cells with syntocinon or LPS (20s-5m) was assessed by in-cell Western. Results: LPS treatment significantly increased myocyte secretion of 11 of 21 cytokines tested including IL-6 (8.09 vs 0.069ng/ml), GM-CSF (1.62 vs 0.074ng/ml) and TNFα (11.4pg/ml vs undetected, all p<0.001). The response was serumdependent and inhibited by LPS-RS, indicating TLR4 specificity. Syntocinon, but not LPS, induced rapid transient phospho-MLC in PHM1-41 cells (peak 20s, 271% versus vehicle, p<0.001). Syntocinon enhanced PHM1-41 collagen gel contraction by 107% over basal (p<0.01) at 6h. By contrast, LPS did not induce significant contraction until 48h, peaking at 72h post-treatment (108% and 113% respectively, both p<0.01). Neither the L-type Ca2+ channel blocker nifedipine nor indomethacin blocked LPS-mediated contraction, however the ROCK inhibitor Y27632 abrogated LPS-mediated enhancement. Conclusion: Myometrial myocytes synthesise cytokines in response to LPS via TLR4dependent pathways. LPS enhances myocyte contraction with delayed kinetics compared to oxytocin. LPS induced contraction appears to work via RhoA/ ROCK pathways implying LPS-mediated calcium sensitization. Acknowledgements. We are very grateful to Barbara Sanborn for her kind donation of PHM1-41 cells. Objective: Proinflammatory stimuli such as cytokines, bacterial cell wall products and thrombin are proposed to be prominent triggers for preterm parturition. Gene expression networks are fine-tuned by small, non-coding RNAs (microRNA) that recognize mRNA targets in a sequence-specific manner, and inhibit translation into cognate protein or lead to mRNA degradation. This study profiled miRNA (miR) expression in term human decidual cells stimulated with cytokines, endotoxin or thrombin to model sterile, infection-mediated or coagulation-driven inflammation.

Methods: Decidual cells (DCs) cultured from uncomplicated term deliveries were primed in charcoal-stripped, serum-containing medium with 10 -8 M estradiol and 10 -7 M medroxyprogesterone acetate for 14 days. DCs were incubated in serum-free, defined medium for 24 h, & stimulated for 4 h with vehicle (EtOH), IL-1β (1 ng/ml), TNF-α (1 ng/ml), LPS (5 µg/ml) or thrombin (2 U/ml). Small RNAs were isolated using the Qiagen miRNeasy kit and miR expression was profiled using the nCounter platform (Nanostring Technologies, Human v2 miRNA expression assay). Expression data were normalized and analyzed with the nSolver software and downstream bioinformatics tools. Results: Relative to control cells, challenge with IL-1β, TNF-α, LPS, and thrombin resulted in up-regulation (≥ 1.5-fold) of 37, 21, 66, and 30 miRs, respectively, and down-regulation (≥ -1.5-fold) of 227, 274, 178, and 234 miRs, respectively. While 57 miRs were commonly down-regulated among all treatment groups, only 4 miRs (338-3p, 378e, 1912, and 4455) were commonly up-regulated. There were 34 candidate mRNA targets among the commonly upregulated miRs, whereas 2534 mRNA targets were predicted for the commonly down-regulated miRs. Among the latter, miRs targeting several chemokines / receptors, matrix metalloproteinases, transcription factors, and receptors for prostaglandins / growth factors were present. Conclusions: Our data suggest that proinflammatory stimuli trigger large-scale down-regulation of miRs in term DCs. Far fewer miRs are up-regulated by these agents. This is in striking contrast to mRNAs, the majority of which are up-regulated in response to inflammation. This suggests that miR withdrawal could play an important role in unleashing proinflammatory gene expression during preterm parturition. Human beta defensin 1 (HBD1) is an antimicrobial and immunomodulatory peptide present in cervical mucus. Variation in cervical antimicrobial expression is associated with preterm labour. We hypothesized that SNPs in the HBD1 gene may be associated with preterm birth. Methods This is a retrospective case control study. Genomic DNA was extracted from blood collected at 11-13 weeks from women attending King's College Hospital March 2006-September 2010. 50 women with premature prelabour rupture of membranes (PPROM) in the index pregnancy and 50 with spontaneous preterm labour were matched (ethnicity, smoking, BMI) with 300 who delivered at term. The SNPs rs1799946 (5'UTR) and rs1047031 (3'UTR) were genotyped by KASP assay (Kompetitive Allele Specific PCR, KBioscience). Data were analysed using multiplicative (rs1047031) and recessive (rs1799946) models and Chi Square Test, and were assessed for Hardy-Weinberg equilibrium. Results There was no difference in BMI, smoking status or ethnicity between the groups. Genotyping was successful in 98% (n=390) samples for rs1047031 and 97% for rs1799946 (n=386). Allele distribution in controls demonstrated Hardy-Weinberg equilibrium. AA homozygotes (rs1799946) had a significantly increased risk of PPROM; OR 2.24 (95%CI 1.11-4.49), p=0.0.0257. No association was seen with spontaneous preterm labour. Of the 400 women, 117 had at least one prior delivery before 37 weeks and 36 had a history of delivery before 28 weeks. AA homozygotes (rs1799946) had significantly increased risk of delivery before 37 weeks; OR 2.14 (95%CI 1.24-3.68), p=0.005 and an even higher risk of delivery before 28 weeks; OR 4.08 (95%CI 1.93-8.63), p<0.0001. Carriers of the A allele (rs1047031) were less likely to deliver before 28 weeks; OR 0.288 (95%CI 0.121-0.683), p=0.003. In this combined analysis clinical data was not available to differentiate between prior history of PPROM and spontaneous preterm labour. Conclusion rs1799946 is associated with PPROM, a doubled risk of delivery before 37 weeks and a four-fold increase in the risk of delivery before 28 weeks. rs1047031 may be protective. These data suggest that variation in innate immune genotype may contribute to the clinical phenotype of women who deliver preterm. Progesterone plays a key role in pregnancy in all species. In most species labour is heralded by progesterone withdrawal. However the regulation of progesterone function differs from one species to another. Animal models are therefore not ideal to study progesterone function in human pregnancy. Primary cultures of human myometrial cells without passage cannot usually be used to study progesterone function and signalling (or other aspects of myometrial biochemistry) since it is not usually possible to grow sufficient cells at passage 0 for a single experiment and all of its appropriate controls. Immortalized human myometrial cells (eg PHM1 and hTERT-M) have been widely used for this purpose but their expression of progesterone receptors does differ from that of native human myometrium. Otherwise investigators have attempted to extrapolate from data obtained from cancer cell lines. A common criticism of the use of passaged primary human myometrial cells is that they do not retain the characteristics of myocytes and that fibroblasts then dominate. In this study, to determine whether myocytes cultured up to passage 4 retain the original characteristics of myoctyes, the levels of archetypal myocyte proteins α-SMA, OTR, PR-B, and PR-A and the ratio of PR-A to PR-B were examined throughout passage by Western analysis. Levels of α-SMA remained unchanged from passage 0 to passage 4 with minimal variability between individual cultures or from passage to passage. Expression of OTR was also unchanged. Although there was a apparent doubling of expression at passage 2, compared to each other passage number, this was not statistically significant, and expression levels at passage 0 and 4 were the same. Expression of PR-A and PR-B was more varied between individual cultures however passage had no effect upon their expression. The ratio of PR-B to PR-A was consistently >1 and was not affected by passage. Overall, the expression of each of α-SMA, OTR, PR-B, PR-A and the ratio of PR-B to PR-A was the same at passage 4 as at passage 0. We conclude that, in respect of expression of these archetypal myocyte proteins, passaged human myometrial cells up to passage 4 have the same characteristics as at passage 0 and are at least as good a model for progesterone function in human myometrium as immortalised myocytes or cancer cells. Background: Recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) constitutes the first line of defense by the innate immune system. Activation of NOD-like receptors (NLRs) by PAMPs results in the formation of inflammasomes, which function independently of toll-like receptors (TLRs) to activate caspase 1. Caspase 1 in turn catalyzes the maturation of IL-1β and IL-18 and mediates pyroptosis. Previously we showed that activation of TLR2 or TLR4 induces preterm labor in mice. However, the role of NLRs in preterm labor and their interplay with TLRs have not yet been investigated. In this study we determine the effects of inhibition of caspase 1 on production of inflammatory mediators (IL-1β, TNFα and COX2), steroid response elements (membrane progesterone receptor alpha (mPRα), oxytocin receptor (OXTR)) and transcriptional coregulators (steroid receptor coactivator 2 (SRC2) and cAMP response element-binding protein 3 (CREB3)) in macrophages. Methods: RAW264.7 cells (a mouse macrophage cell line) were pre-stimulated with vehicle or 40 µM of YVAD-CMK (caspase 1 inhibitor) for one hour and then challenged with vehicle, lipopolysaccharide (LPS, a ligand for TLR4, 10ng/mL), or LPS+ATP (an inflammasome activator, 5mM) for four hours. Real-time PCR was performed to assess the expression of select transcripts. Results: Both LPS and ATP+LPS significantly increased mRNA levels of IL-1β, TNFα and COX2 compared to controls. The effects of ATP+LPS were attenuated by pre-treatment with the caspase 1 inhibitor YVAD-CMK. Both LPS and ATP+LPS decreased mRNA levels of mPRα, OXTR, and SRC2 compared to controls. These effects were not influenced by pre-treatment with the caspase 1 inhibitor, with the exception of a reversal of ATP+LPS-induced, but not LPS-induced, down-regulation of mPRα expression. Both LPS and ATP+LPS increased CREB3 expression, an effect that was blocked by caspase 1 inhibition only for ATP+LPS treatment, but not LPS-induced treatment alone. Conclusion: These data demonstrate that augmentation of the actions of LPS via specific activation of inflammasomes results in distinct caspase 1-dependent effects, including: suppression of mPRα expression and induction of CREB3. Intrauterine infection and associated inflammation is the most firmly established trigger for preterm labour (PTL). Upon the interaction of bacterial products with toll-like receptors (TLRs) on intrauterine cell surfaces, inflammatory pathways are triggered that culminate in the premature activation of the myometrium. We have previously shown that the transcription factor, activator protein-1 (AP-1), rather than NFkappaB, is predominately activated in the myometrium of a specific LPS (Escherichia coli: serotype 0111:B4)-induced PTL murine model. The objective of this study was to identify key mediators of myometrial AP-1 activity and characterise their role in myometrial activation and inflammation/ infection PTL. Myometrial samples (n≥4 for all experiments) were collected from CD-1 mice subjected to an intrauterine injection of 20µg LPS (0111:B4) or PBS on day 16 of gestation at 1, 2, 4 and 6h post-injection (p.i.) or during active labour (∼7h). Proteins were extracted and Western blotting was used to examine levels of key mitogen-activated protein kinases (MAPKs) known to regulate AP-1 activation. An 3.5-fold increase (P<0.01) in phosphorylated JNK (p-JNK) was detected 1h following LPS-treatment concurrent with significant increases in the activated AP-1 subunits, p-C-Jun (8-fold, P<0.01) and p-C-Fos (3.4-fold, P<0.05). Levels of p-ERK and p-p38 were unchanged. Systemic administration of the specific JNK inhibitor SP600125 (30mg/kg) 2h prior to LPS treatment delayed PTL by 6h (P<0.05), whereas the NFκB inhibitor 15dPGJ2 had no effect. Intrauterine injection of TLR4-/-mice did not lead to preterm labour (delivery 42h ± 9h). Collectively our results show that the effects of LPS (0111:B4) treatment are primarily mediated through the TLR4 receptor, which leads to the activation of JNK and subsequent phosphorylation the AP-1 subunits, c-Jun and c-Fos. The variable effects of different LPS serotypes are a model for human PTL in the context of different micro-organisms. It is unlikely that specific targeting of single transcription factors would prevent most cases of PTL.

fold change). Expression of key mRNA targets in the inflammatory pathway (IL-8, MIP-1α and -2β, CD38, CCL18) predictably correlated with miR-155 expression. Conclusions: miRNA-155 is induced in the pro-inflammatory fetal lung response following a choriodecidual infection and may be an early biomarker or therapeutic target to minimize inflammatory damage of the fetal lung in utero. Background: Ventilation-induced lung injury (VILI) contributes to morbidity associated with preterm birth. Human amnion epithelial cells (hAECs) can reduce inflammation and promote repair. We hypothesised that hAECs would alter the systemic inflammatory response to VILI in preterm lambs. Methods: Preterm lambs were delivered by caesarean section at 126 days of gestation (term 147 days) and 9x10 6 hAECs (in 3ml PBS; n=6) or vehicle (3ml PBS; n=5) were administered into the trachea and intravenously. Lambs were ventilated for 2 hours, with a high tidal volume for the first 15 min. A third group, of unventilated controls (UVC; n=6) was also used. Blood, lymph node and spleen samples were collected at post mortem for isolation of plasma and/or leukocytes. Isolated cells were cultured with T cell stimulants for 5 days. Leukocytes were stained with antibodies (CD1, CD8, CD4, CD21, CD25, CD44, MHC II and γδ T cells) for identification of phenotype using flow cytometry. Plasma cytokine concentrations were measured by ELISA. Results: T cell proliferation tended to increase in the lymph node and blood. Plasma IL-10 cytokine concentrations increased after ventilation (**p<0.01) and tends increased after hAEC administration, but no change was seen in inflammatory cytokines IL-6 and TNF-α in response. Percentage of circulating monocytes increased after hAEC administration but no change was seen in lymphocyte and granulocyte populations. CD21 expression increased after hAECs in the circulating monocyte population (*p<0.05). Expression of other cell markers did not change in the blood or lymph node.

Administration of hAECs increase circulating monocytes and may activate B cells, as indicated by an increase in CD21 expression. Administration of hAECs increases anti-inflammatory (IL-10) plasma cytokines and tends to increase T cell proliferation in the lymph node and blood. In conclusion, hAEC administration alters the systemic inflammatory response to ventilation. Background: Labor requires activation of inflammatory pathways at the maternal-fetal interface. However a dysregulated inflammatory response may lead to adverse maternal and neonatal outcomes. Recent studies have revealed that resolution of the inflammatory response is an active process, involving omega-3 and omega-6 fatty acid derived specialized pro-resolving lipid mediators (SPM). We performed this study to determine if SPM are present in the fetal compartment after labor. Methods: Umbilical cord blood samples were collected at delivery from four infants born to mothers participated in a trial of prenatal EPA-and DHA-rich fish oil supplementation. Specimens were centrifuged within 12 hours of collection, and were stored in 1 ml aliquots at -80 degrees C. Plasma lipid mediator (LM) levels were assessed by LC-MS-MS based LM metabololipidomics targeting DHA, EPA, and arachidonic acid bioactive metabolomes. Results: Using LM metabololipidomcs we identified from the arachidonic acid metabolome lipoxin B 4 and thromboxane B 2 as the most abundant mediators in umbilical cord plasma. Biologically-significant levels of resolvin D1 and maresin R1 were identified in all 4 specimens. Low concentrations of protectin D1 (7-9 pg/ml) were identified in 2 samples collected from babies whose mothers had received DHA-rich fish oil. Resolvin E2, an EPA-derived resolvin, was identified in all specimens. Conclusions: EPA-, DHA-, and arachidonic-acid derived SPM are present in the fetal compartment in biologically-significant levels after labor. Further research is needed to determine whether SPM found in the fetal compartment are of maternal origin or if they result from endogenous fetal biosynthesis.

Mucosal Immunity in Cervicovaginal Infection. Christopher J Nold, Monique Maubert, Lauren Anton, Michal Elovitz. Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. OBJECTIVE: Premature cervical remodeling appears to be an obligatory step in the pathogenesis of spontaneous preterm birth. We posit that the molecular precursor to premature cervical remodeling is activation of mucosal immunity. Specifically, these studies sought to assess if cervico-vaginal infection altered expression of TH17 cytokines and antimicrobial proteins in cervical tissues. STUDY DESIGN: A two prong approach was utilized. In the first approach, day E15 pregnant CD-1 mice were treated with either 500ug of lipopolysaccharide (LPS) or 0.1cc of Replens vaginally. Mice were then sacrificed six hours later (n=4 per treatment group). Cervical tissue was collected and RNA was extracted. Quantitative real-time polymerase chain reaction (qPCR) was performed on the following targets: 1) Th17 cytokines: IL17, IL22, and IL22R; and 2) Anti-microbial proteins: Defensin 1, Defensin 3, Defensin 4, and SLP1. Cervicovaginal fluid (CVF) was also collected and was assessed for IL17, IL22, and IL23. In the 2nd approach, immortalized human ectocervical (ECTO) and endocervical (ENDO) cells were treated with 25ug/ml of LPS or vehicle. RNA was collected at 6 and 24 hours (n=6 per treatment group) and IL17, IL22, and IL23 were analyzed by qPCR. RESULTS: LPS vaginally administered significantly increased the expression of IL22 (p<0.001) and cervical SLP1 (p<0.05) mRNA compared to Replens. LPS did not significantly increase the expression of Defensin 1, and baseline mRNA values of IL17, IL22R, Defensin 3, and Defensin 4 were very low; expression was not altered by any of the treatments. LPS significantly increased the expression of CVF IL22 (p<0.001) but did not significantly increase IL17 or IL23. In vitro, we found that LPS significantly increased the expression of IL23 at 6 and 24 hours (p<0.05) in ECTO cells and at 6 (p<0.01) and 24 (p<0.05) hours in ENDO cells. IL17 and IL 22 expression was very low and not altered by LPS.

CONCLUSION: These studies provide evidence that mucosal immunity is activated in cervico-vaginal infection. Indeed, cervical epithelial cells may be the primary 'immune responders' to cervico-vaginal infection. Activation of mucosal immunity in cervical tissues (and specifically cervical epithelial cells) may be a precursor to cervical remodeling in the setting of infection and, as such, may be an important therapeutic target.

Progestational Agents and Their Effect on the Extracellular Matrix. Christopher Nold, Monique Maubert, Lauren Anton, Michal Elovitz. Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. OBJECTIVE: Alterations in the structure and composition of the extracellular matrix (ECM) occurs during cervical remodeling. The ECM consists of numerous structural components including glycosaminoglycans (GAG). At term, total GAG levels increase due to an increase in hyaluronan (HA). During cervical ripening, increased HA content is due to increased expression of hyaluronan synthase (HAS). Additionally, ripening is associated with a transition from a large to small molecular weight HA, which is regulated by hyalruonidase (Hyal). We hypothesize progestational agents would decrease the expression of hyaluronan synthase (HAS) and hyaluronidase (Hyal). Secondly, we hypothesize progestational agents would increase the expression of collagen binding integrins leading to greater cervical integrity. STUDY DESIGN: Using a mouse model, on days E14-E17 CD-1 pregnant mice were treated with either 0.1cc of 10mg/ml of 17 alpha-hydroxyprogestreone caproate (17P) subcutaneously, 0.1cc of castor oil (CO) subcutaneously, 0.1 cc of 25 mg/ml of progesterone in Replens vaginally (Vag P), or 0.1cc of Replens vaginally, with four dams per treatment group. Mice were sacrificed six hours after treatment on E17.5. Cervices were collected and quantitative real-time polymerase chain reaction (qPCR) was performed on the following targets: A) Enzymes regulating HA: HAS 1, HAS 2, HAS 3, Hyal 1, and Hyal 2; and B) Integrins: Integrin α1β1, Integrin α2β1, Integrin α10β1, Integrin α11β1, and Integrin β6. RESULTS: Exposure to Vag P significantly decreased the expression of Integrin α10β1 (p<0.01) and HAS 3 (p<0.01) compared to Replens. Treatment with 17P significantly increased Hyal 1 (P<0.05) compared to CO. Treatment with either 17P or Vag P did not have a statistically significant effect on the production of HAS 1, HAS 2, Hyal 2, Integrin α1β1, Integrin α2β1, Integrin α11β1, and Integrin β6. CONCLUSION: These studies demonstrate that 17P did not have significant effect on mediators known to modulate the ECM in cervical ripening. However, Vag P decreased the expression of HAS 3 and Integrin α10β1. Whether these molecular changes have any effect on preventing preterm birth requires further study. Objective: Second trimester cervical length (CL) is an important predictor of preterm birth, though the etiology of CL variation has not been fully elucidated. Thus our objective was to evaluate the potential association between first trimester peripheral markers of inflammation and the subsequent occurrence of a decreased cervical length (CL). Methods: Subjects were included if they had undergone both first trimester aneuploidy and second trimester transvaginal ultrasound CL screening. Cases and controls were defined by second trimester CL >/< 2.5 cm and matched 1:2 by date of first trimester screen (FTS) sample collection (+/-30 days) and history of prior preterm birth. Archived residual FTS serum was retrieved and C-reactive protein (CRP) concentrations were measured in all subjects via commercial assay. Subject records were reviewed for FTS results including PAPP-A and hCG. The association between first trimester CRP and decreased second trimester CL was evaluated via Wilcoxon rank test for matched pairs, logistic regression with adjustment for gestational age at FTS, maternal weight, tobacco use and ethnicity and linear regression with cervical length as a continuous variable. Results: A total of 49 cases with CL <2.5 cm were included and matched to 98 controls. No association was demonstrated between second trimester CL and 1st trimester CRP in univariate analysis (p=0.26, Figure 1 ) or when adjusted for potential clinical confounders (Table 1) . Additionally, there was also no association with PAPP-A or hCG concentrations. Recent studies have suggested an association between asymptomatic C. albicans vaginal colonisation and subsequent preterm birth 2,3 , yet little data exists on the fetal inflammatory response to C.albicans intra-amniotic infection.

Ovine models have documented that the fetal skin elicits varying inflammatory responses to Escherichia coli lipopolysaccharide and Ureaplasma parvum 4 that may act as important precursors to the initiation of preterm birth. The aim of this study was to examine changes in inflammatory markers in the ovine fetal skin in response to intra-amniotic C. albicans infection.

Singleton ovine fetuses received an intra-amniotic injection (2mL) of C. albicans (1.25-2.05x10 7 cells/mL) at 122d GA (n=6). Control animals received an identical volume of sterile saline (n=6).

Fetuses were euthanased at 124d GA (term =150d) (2d exposure) and tissues snap frozen prior to microbiological/inflammatory assessment. Data were tested with parametric or non-parametric ANOVA as appropriate.

A p value of <0.05 relative to saline controls was considered significant. Successful infection of the amniotic fluid by C. albicans was indicated by real-time PCR in all 2d-exposed samples. All control samples were negative for the presence of C. albicans. As indicated by real-time PCR analysis for the inflammatory markers: IL-6, IL-8, TNF-α and MCP-2, mRNA expression was significantly higher in the skin of 2d-exposed fetuses compared to saline controls. Average fold change for mRNA expression in 2d-exposed samples was 40, 92, 4 and 59 fold for IL-6, IL-8, TNF-α and MCP-2 respectively. No significant difference was observed in IL-1β mRNA expression. Conclusions C. albicans intra-amniotic infection elicits a vigorous inflammatory response in the ovine fetal skin. These data suggest that intra-amniotic C. albicans colonisation may be an important risk factor for preterm birth. 

Investigating the Anti-Inflammatory Agents Epi-Lipoxin and IL-10 in a labour. However, in the presence of either epi-lipoxin or IL-10, the effect of LPS on pup death appears to be reduced. Our preliminary results suggest that epi-lipoxin and IL-10 deserve further investigation as therapeutic agents for the treatment of PTL.

Investigating Background: Preterm labour (PTL) is commonly associated with the presence of intrauterine infection and/or inflammation. Using antibody-based depletion strategies we aimed to investigate the role of neutrophils in preterm parturition. Methods: On D16 of gestation CD1 mice received an intra-peritoneal injection of either 250µg of anti-GR-1 or 500µg of anti-Ly6g to deplete neutrophils, control mice received the equivalent dose of the appropriate IgG control antibody. Following this, on D17 of gestation mice received an intrauterine injection of 20µg of LPS (from E.Coli 0111:B4) and time to delivery was determined (n=5-8 in each group). In a second cohort, utero-placental tissues were collected 6 hours post-LPS administration for qRT-PCR analysis of inflammatory mediators (n=5-8 in each group). Results: Flow cytometry confirmed that injection of each of anti-GR-1 and anti-Ly6g antibodies on D16 of gestation successfully depleted circulating neutrophils. Mice that received either anti-GR-1 or anti-Ly6g prior to intrauterine LPS injection on D17 of gestation delivered earlier than control mice (9 ± 1.9 hrs in anti-GR-1 group, 19.3 ± 9.4 hrs in anti-Ly6g group vs. 33.1 ± 15.6 hrs, mean ± SE), however this was not statistically significant. Depletion of neutrophils with anti-GR-1 resulted in significantly decreased mRNA expression of IL-1β in the uterus and placenta (3 and 1.5-fold vs. control respectively, p<0.05), and significantly elevated expression of IL-6, COX-2, CXCL1 and CXCL2 in the fetal membranes (3, 2, 3.5 and 3-fold vs. control respectively, p<0.05), 6 hours post-LPS administration. Depletion using anti-Ly6g resulted in a different profile of gene expression changes, with decreased mRNA expression of TNF-α in the uterus (2-fold vs. control, p<0.05), and increased placental CXCL5 expression (2-fold vs. control, p<0.05). Conclusion: Our preliminary data indicate that neutrophils are not required for the induction of PTL in response to intrauterine infection, but suggest that the neutrophils do contribute to the inflammatory response to intrauterine LPS given that the expression of many important inflammatory factors is altered in the utero-placental tissues when neutrophils are depleted. Toll-like receptors (TLR) and nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are types of pattern-recognition receptors (PRR) that allow the innate immune system to recognize and react to pathogens or danger-signals. TLR signaling pathways have been studied widely. However, the role of NLRs (in particular nucleotide-binding oligomerization domain protein 2 (NOD2)) in amplifying inflammatory processes is less understood, especially their interaction with TLRs. The objective of this study was to characterize interactions between TLR and NOD2 signaling pathways.

The RAW 264.7 murine macrophage cell line was cultured in vitro for 4 hours with combinations of specific ligands: lipopolysaccharide (LPS, a TLR4 ligand 5ng/mL), polyinosinic:polycytidylic acid (Poly(I:C), a TLR3 ligand, 10µg/mL), peptidoglycan (PGN, a TLR2 ligand, 1µg/mL), and muramyl dipeptide (MDP, a NOD2 ligand, 20µg/mL). Total cellular RNA was collected. Reverse transcription followed by real-time PCR was used to compare mRNA expression of TNFα and IL-1β, relative to the housekeeping gene GAPDH. Results: As previously shown, the two TLR agonists PGN and poly(I:C) produced synergistic expression of TNFα and IL-1β. In addition, MDP synergized with all tested TLR agonists (LPS, PGN, Poly(I:C), and the combination of PGN+Poly(I:C)) in the induced expression of both IL-1β and TNFα. Figure  1 shows results for IL-1β. undifferentiated cytotrophoblasts. Our objective was to characterize trophoblast cells obtained from TCS using specific protein markers and perform signal cell genetic analysis. Materials and Methods: Cells were collected by TCS at 6-17 weeks of gestation. Fetal trophoblast cells were isolated using HLA-G antibody coupled to magnetic nanoparticles. Isolated HLA-G+ cells were examined by immunofluorescence antibody labeling for the expression of specific trophoblast markers. Isolated single cells underwent multiplex polymerase chain reaction (PCR) for gender determination with amplifying sequences in genes on the X (DMD) and Y (SRY) chromosomes. Results: Immunomagnetically isolated cells from transcervical specimens expressed the general trophoblast markers, β hCG, placental lactogen and cytokeratin 7. Cells were not reactive with antibodies against pregnancyspecific glycoprotein (PSG)-1, integrin α6 or E-cadherin (chorionic villous trophoblast markers). Specific extravillous trophoblast cells markers HLA-G, VE-cadherin, PECAM1, integrin α1 and MMP9 were all expressed. Using primers for X, and Y chromosomes fetal gender was identified from isolated single cells collected by TCS.

Conclusions: TCS paired with immunomagnetic isolation using anti-HLA-G is a unique technique that can be used to isolate extravillous cells that have migrated from the placenta during early pregnancy and permits accurate PCR-based genetic testing at 6 weeks of pregnancy. The TCS can provide a noninvasive platform to obtain trophoblasts for detection of chromosomal, genetic and placental anomalies, and may be a useful approach for monitoring molecular and cellular aspects of the extravillous trophoblast population during gestation. Introduction: Maternal thyroid dysfunction is associated with complications of malplacentation including miscarriages and pre-eclampsia. Thyroid hormones (TH) are known to promote trophoblast proliferation and invasion. We hypothesise that TH also play an important role within human decidua by regulating growth factor and cytokine secretion, which control trophoblast invasion. Methods: Deciduas from human pregnancy were obtained from 1st (8-11 weeks) and 2nd trimester (12-20 weeks) surgical terminations of pregnancy and by uterine curettage at elective caesarean sections at term. Primary cultures of total decidual cells (TDC), and immunomagnetic bead isolated populations of stromal-enriched (CD10+ve) and stromal-depleted (CD10-ve) cells, uterine natural killer cells (uNKs;CD56+ve) and macrophages (CD14+ve) were treated with T3 (0,10,100nM). Assessments were made of cell viability (MTT assay), cytokine and angiogenic growth factor secretion (immunomediated assay) and the effects of decidual cell-conditioned media on extravillous trophoblast (EVT) invasion through Matrigel®.

Results: Immunohistochemistry showed the expression of TH transporters (MCT8, MCT10) and receptors (TRα1, TRß1) required for TH-responsiveness in endometrial glands, uNKs and macrophages from early gestation. The viability of TDC and cell isolates were unaffected by T3. In 1st trimester, T3 reduced IL-10 secretion by TDC and CD10-ve cells (p<0.01), and reduced GM-CSF, IL-10, IL-1ß, IL-6, MCP-1 by macrophages (p<0.01) but increased IL-10 by uNKs (p<0.05). In 2nd trimester, T3 increased IL-10 by TDC (p<0.01) but reduced IL-10 by uNKs (p<0.001). T3 increased VEGF secretion by 1st trimester uNKs (p<0.05), and angiopoietin-2 by 2nd trimester TDC and uNKs (p<0.05). Conditioned media from T3-treated TDC and macrophages did not alter EVT invasion compared to untreated controls. Conclusion: TH regulate decidual cytokine and angiogenic growth factor secretion in a cell-specific and gestation-dependent manner, with the first trimester decidual macrophages being the most responsive to TH treatment. However, the summation of TH effects upon the secretome do not affect EVT invasion in vitro. OBJECTIVE: Placental inflammation is implicated in the pathogenesis of preeclampsia (PEC). Our group has previously shown that excess glucose induces a pro-inflammatory milieu in first trimester trophoblasts (TBs), providing a potential link to the increased risk of PEC in diabetic pregnancies. Recent literature suggests that metformin, an antidiabetes medication, not only acts via its conventional hypoglycemic effects in non-pregnant diabetics, but also exerts anti-inflammatory responses in various cell types. The objective of this study was to evaluate the ability of metformin to modulate the proinflammatory response seen in first trimester TBs exposed to hyperglycemic conditions. METHODS: The human first trimester human extravillous TB cell line (Sw-71) was cultured with media containing 5 mM (normoglycemia, NG) and 50 mM (hyperglycemia, HG) glucose, all in the presence or absence of metformin (0.1 or 0.5 mM). After 72 hrs, supernatants were collected and assayed for the pro-inflammatory cytokine IL-6 by ELISA. L-glucose was used as an osmotic control. Statistical significance was determined using one-way ANOVA. RESULTS: Compared to NG, exposure to HG significantly up-regulated trophoblast secretion of IL-6 (1.72-fold, p<0.001). L-glucose controls showed no difference in IL-6 production. Lower concentration of metformin (0.1 mM) had no effect on the HG-induced pro-inflammatory response, but the higher concentration of metformin (0.5 mM) significantly reduced IL-6 under both the NG (0.79-fold, p<0.001) and HG (0.65-fold, p<0.05) conditions. CONCLUSION: Metformin at the higher concentration was effective in reversing hyperglycemia-induced pro-inflammatory effects on human first trimester TBs in vitro. During the early placentation process, metformin may provide protective benefits in addition to its traditional hypoglycemic effects, thereby decreasing risk of PEC in diabetic pregnancies. OBJECTIVE: Disruption of the anti-angiogenic (sFlt-1 and soluble endoglin, sEng) and pro-angiogenic (VEGF) factor balance is implicated in the pathogenesis of preeclampsia (PEC). We previously demonstrated that excess glucose modulates the angiogenic profile in first trimester trophoblasts (TB), providing a possible link to the increased risk of PEC in diabetics. Metformin has been shown in cancer models to potentially decrease angiogenesis, but little is understood about its effects on TBs. The objective of this study is to elucidate the effects of metformin on angiogenic factor profile in first trimester TBs under different glycemic conditions.

The human first trimester TB cell line (Sw-71) was cultured with media containing 5 mM (normoglycemia, NG) or 50 mM (hyperglycemia, HG) glucose, all in the presence or absence of metformin (0.1 or 0.5 mM). After 72h, supernatants were collected and assayed for sFlt-1, sEng, and VEGF by ELISA. L-glucose was used as osmotic control. Statistical significance was determined using ANOVA. RESULTS: Compared to NG, HG significantly up-regulated TB secretion of sFlt-1 (2.74-fold, p<0.01, Fig.1 ) and sEng (1.61-fold, p<0 .01, Fig.2 ), while down-regulating VEGF (0.17-fold, p<0.01, Fig.3 ). L-glucose controls showed no difference in angiogenic factor secretion. Lower concentration of metformin (0.1 mM) had no effect on the HG-induced up-regulation of sFlt-1 and sEng, but the higher concentration of metformin (0.5 mM) significantly reversed anti-angiogenic factor secretion under both NG and HG conditions. Metformin did not reverse the significant down-regulation in VEGF secretion with HG. CONCLUSION: Metformin at higher concentrations may partially reverse HG-induced anti-angiogenic effects in first trimester TBs in vitro. During the placentation process, metformin may contribute protective benefits beyond to its traditional hypoglycemic effects, thereby decreasing risk of PEC in diabetics.

The Migration Induced by Scratching Regulates the Secretion of sFlt1 in TCL-1 Cells. It is known that sFLT1 acts as a potent endogenous soluble inhibitor of VEGFand PlGF-mediated various biological functions. And the maternal serum levels of sFlt1 increase before the onset of pregnancy-induced hypertension (PIH). However, the mechanism of increase in placental sFlt1 production at the time of placental dysfunction remains unknown. We modeled vascular endothelial dysfunction by scratching and examined the changes in secretion and production of sFlt1. METHODS: TCL-1 cells (immortalized human term pregnant trophoblast cell lines) were starved in serum-free condition for 24hours, and were examined the followings in the presence or absence of scratching. The observation periods were 0, 24, 48, and 72hours.

1) Concentrations of sFlt1 attached on the cell membrane and supernatant were measured by ELISA.

2) The expressions of sFlt1/Flt1/VEGF of cell membrane were examined by Western Blot and real-time PCR.

3) Additional chymotrypsin inhibitor with a concentration of 0, 1, 10, 100µg in culture medium, the concentrations of sFlt1 were measured by ELISA and compared with control (no chymotrypsin inhibitor addition). RESULTS:

1) The concentrations of sFlt1 were undetectable in the supernatant. sFlt1 consecration on the cell membrane at 24hours was increased regardless the presence or absence of serum or scratching. On the other hand, sFlt1 consecration was significantly lower at all observation periods with in presence of serum than no scratching group (P<0.05).

2) The protein levels and mRNA expressions of sFlt1/Flt1/VEGF of cell membrane did not show any differences.

3) The concentrations of sFlt1 on the cell membrane showed significant concentration-dependent increases with additional chymotrypsin inhibitor (P<0.05). CONCLUSIONS: These results suggest that sFlt1 secretions may be regulated by protease like chymotrypsin on the cell membrane, not by the changes in sFlt1/Flt1 production.

Marked The formation of syncytia from mononuclear cells leads to a number of changes in cellular structure. We focus on alterations to the microtubule (MT) cytoskeleton in fused BeWo cells.

[Methods] BeWo cells were used as surrogates for cytotrophoblasts and were induced to fuse by treatment with forskolin (FK). BeWo cells were treated with FK for 48 hours to induce cell fusion; this was followed by treatment with nocodazole to induce MT depolymerization and wash out of the drug to induce subsequent MT re-growth. Antibodies to α-tubulin, and pericentrin were used in immunofluorescence experiments to monitor MT organization. [Results] In both un-fused control and FK-treated fused cells MT depolymerization was observed following 30 minute exposure to ice-cold nocodazole. In both groups of cells, re-growth of MTs was found soon after wash out of nocodazole and return to 37°C. However, MT re-growth was different in the two types of cells.

In controls, re-growth was centered at the centrosome and radiating outward like spokes of a wheel. In fused cells, the re-growth did not originate from single sites but appeared to be nucleated from broad areas that had increased labeling for pericentrin.

We show a dramatic alteration in the amount of and distribution of pericentrin in fused BeWo cells compared to mononuclear cells. Moreover, these expanded areas of pericentrin serve as sites for MT nucleation. (2), ensures cellspecificity. To investigate the use of polymer-DNA nanoparticles to transfect the BeWo Choriocarcinoma cell line we used eGFP as a reporter transgene. Two fragments of the human Cyp19a promoter (-932 and -501 fragments,1) were cloned and a PLAC1 promoter (GeneCopia, Rockville, MD) were used to replace the CMV promoter in the pEGFP-C1 plasmid (Promega, Madison, WI). After sequence confirmation, purified plasmids were incubated with HPMA-DMAEMA polymer at an N:P ratio of 3:1 at room temperature for 1 hour. BeWo cells or human fibroblasts were plated onto chamber slides and incubated with the DNA or nanoparticle overnight in serum-free medium, cells were cultured for a further 72 hours in complete media. Control cells were incubated with either nanoparticles including the CMV-eGFP to compare expression levels with a constitutive promoter or uncomplexed plasmid GFP to demonstrate the benefits of the nanoparticle. Expression was assessed by fluorescent microscopy (Nikon Eclipse 80i). GFP expression was higher in BeWo cells incubated with any of the nanoparticles containing trophoblast-specific promoters than those incubated with the uncomplexed DNA. However, only the PLAC1 or Cyp19a -932 promoters produced GFP expression comparable with the CMV-eGFP expression in BeWo cells. Despite robust GFP expression in fibroblasts following incubation with nanoparticles containing the CMV promoter, no GFP expression was observed in fibroblasts when the trophoblast-specific promoters were used. Gene expression was significantly enhanced by complexing plasmid DNA with an HPMA-DMAEMA polymer. PLAC1 or Cyp19a-932 promoter-driven nanoparticles can be successfully used to drive trophoblast-specific expression of transgenes and are important building blocks towards human placental gene therapies. We examined stem cell function and giant cell differentiation by monitoring the mRNA and protein expression of pluripotency markers Id-1 and Id-2 (inhibitor of DNA binding proteins 1 and 2), and transcription factors PL-1 and 2 (placental lactogen 1 and 2) by quantitative RT-PCR and immunoblotting, respectively. Cell migration was evaluated in wound assays using live-cell imaging.

We detected a sharp decline of Id-1 and 2 mRNA and protein expression and a coordinate up-regulation of PL-1 and 2 (differentiation markers in rodent placenta) mRNA and protein expression during the 10 days differentiation timecourse. Moreover, HtrA1 mRNA and protein expression increased as Rcho-1 cells differentiated into giant cells. When HtrA1 was depleted by shRNA, the expression of Id-1 and 2 was largely unaffected. In contrast, we noted a blunting of PL-1 and 2 expression at the mRNA and protein levels in HtrA1-KD cells compared to LTVc cells. In addition, diminution of HtrA1 decreased the motility of Rcho-1 cells as shown by their inability to close a wound in an in vitro bioassay.

The reduction in the expression of giant cell markers and decreased TSC motility in HtrA1-depleted Rcho-1 cells suggest a role for HtrA1 in both stem cell motility and differentiation. Introduction: Exosomes (40-100 nm vesicles) contain an extensive repertoire of biologically-active proteins and oligionucleotides that are released by many cell type, including MSCs, and exert effects at both proximal and distal sites. Previously, MSC exosomes have been reported to induce endothelial cell proliferation. pMSC display a perivascular localisation in the placenta, however, their role in placental development and vascularisation remains to be established. Aim: To determine the effect of pMSC derived-exosome exposed to different oxygen tensions on hPMEC migration and tube formation. Methods: pMSC were isolated from placental villi obtained at 8-12 weeks of gestation (n=6) by enzymatic digestion (dispase and collagenase) and immunophenotype was confirmed by flow cytometry. pMSC were cultured (DMEM, 10% FCS-exosomes free) under and atmosphere of 8%, 3% or 1% O2 (BioSpherix). Cell-conditioned media was collected and exosomes (exo-pMSC) were isolated by differential and buoyant density centrifugation. Exosome proteins were identified by mass spectrometry (AbSciex 5600). The effect of 5, 10 or 20 µg/ml exo-pMSCs on hPMEC tube formation was established using Matrigel-coated well. The effects of exo-pMSCs on hPMEC cell migration were using an IncucyteTM live-cell imaging system. Results: Exo-pMSC were identified (electron microscopy) as spherical vesicles, with a typical cup-shape and diameters ranging from 50 to 100 nm and the expression exosome markers: CD63, CD81, CD9. Under hypoxic conditions (1% and 3% O2) pMSC exosome released increased by 3.3 and 6.7 fold, respectively, when compared to controls (8% O2; p < 0.05). Exo-pMSC increased hPMEC migration by 1.6 fold compared control (p < 0.05) and increased hPMEC tube formation by 7.2 fold (p < 0.05). MS analysis of exo-pMSC protein identified more than 70 different proteins, including: developmental, immunomodulatory; adhesion; cytoskeletonal; and ion channels proteins and mediators of amino-acid metabolism. Conclusion: pMSC-derived exosomes may participate in hypoxia-associated changes in placental and endothelial function and pathologies during the pregnancy. INTRODUCTION: Placental trophoblast cells release exosomes that may exert paracellular effects and influence the functional activity of resident cells. We propose that one population of cells that may be affected by trophoblast-derived exosomes are pMSCs. We, therefore, hypothesize that cytotrophoblast-derived exosomes (exo-CTs) induce phenotypic and/or functional changes in pMSC. AIM: To establish the effect of cytotrophoblast-derived exosomes on in vitro pMSC migration. METHODS: Cytotrophoblast cells were isolated from first trimester (n=6) using a trypsin-deoxyribonuclease-dispase/Percoll method and characterised by specific markers anticytokeratin-7 and antivimentin. Trophoblast cells were cultured under an atmosphere of 8%, 3% or 1% O2 for 3 days. Cell-conditioned media were collected and exosomes were isolated by differential and buoyant density centrifugation and characterised by Western blot (CD63, CD81 and CD9). Exosome proteins were identified by mass spectrometry. The effects of 5, 10 or 20 µg/ml exo-CTs on pMSC migration were using an IncucyteTM live-cell imaging system. RESULTS: Exo-CTs were identified (electron microscopy) as spherical vesicles, with a typical cup-shape and diameters ranging from 50 to 100 nm and the expression exosome markers: CD63, CD81, CD9 and placental alkaline phosphatase. The release of exo-CTs was inversely correlated with oxygen tension (p < 0.05). MS analysis of exo-pMSC protein identified more than 70 different proteins, including: Vascular Endothelial Growth Factor (VEGF) and Placental growth Factor (PGF). Exo-Cts increased pMSC migration by ∼ 1,8 fold (area under curve = 3844 ± 220) compared to control (area under curve =2163 ± 119; p < 0.05). CONCLUSIONS: The data obtained in this study establishes that the deportation of exosomes from cytotrophoblast cells in vitro increases under low oxygen tensions. Furthermoe, cytotrophoblast-derived exosomes increase placental mesenchymal stem cell migration. These data establish a novel paracellular communication pathway between between cytotrophoblast cells and pMSC that may contribute to stem differentiation and placental development and/or adaptation.

Trophoblast Invasion in Endotoxin Infused Pregnant Rats. Floor Spaans, 2 Gea Kiewiet, 2 Theo Borghuis, 1 Pieter A Klok, 1 Winston W Bakker, 1 Marijke M Faas. 2 1 Pathology, University Medical Center Groningen, Groningen, Netherlands; 2 Medical Biology, University Medical Center Groningen, Groningen, Netherlands. Introduction: Poor placentation (decreased and aberrant trophoblast invasion) is a hallmark of preeclampsia (PE). The cause and mechanism of altered trophoblast invasion is still unknown. The pro-inflammatory agent endotoxin has been shown to induce PE-like signs after a single low dose infusion in pregnant rats. These PE-like characteristics include proteinuria, increased blood pressure and decreased fetal weight. In the present study we evaluated whether trophoblast invasion was decreased in this experimental PE model. Methods: Pregnant rats received a single endotoxin (1.0 µg/kg bw; n=9) or saline (control, n=9) infusion via a permanent jugular vein cannula on day 14 of pregnancy. At the time of maximal trophoblast invasion (day 17 of pregnancy) rats were sacrificed and placentas with mesometrial triangle were collected, fixed in zinc-buffer and embedded in paraffin. 4 µm sections were stained with a monoclonal α-cytokeratin antibody (MNF116). In the mesometrial triangle, the equivalent of the placental bed in humans, the percentage of surface area of trophoblast invasion was evaluated using computerized image analysis. The depth and width of invasion were analyzed by subdividing the mesometrial triangle in three concentric depth levels of equal width and counting to which level the trophoblast cells invaded into the depth and width of the mesometrial triangle.

Results: In the mesometrial triangle a relative decrease of surface area of trophoblast invasion was observed after endotoxin infusion in contrast to saline infused rats (p<0.02). Also, the pattern of trophoblast invasion appeared to be altered in endotoxin infused rats, with a trend towards decreased depth of invasion (p<0.06) but no changes in the width of invasion. Conclusion: In the present experimental PE model, we showed aberrant trophoblast invasion which also occurs in women with PE. Conclusions about the mechanism underlying these alterations cannot be drawn from the present study. However, since trophoblast cells express TLR-4 (an endotoxin binding receptor), a direct endotoxin effect in the present model is not unlikely. Alternatively, endotoxin may also indirectly affect trophoblast invasion via changes in local immune cell populations. Further research into the mechanism of decreased trophoblast invasion and its possible role in the pathophysiology of this model is in progress.

Estrogen Receptor-beta Mediates Impaired Fetoplacental Angiogenesis in Fetal Growth Restriction. Emily J Su, Hong Xin, Diana Monsivais, Serdar E Bulun. Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Objective: Fetal growth restriction with abnormal umbilical artery Doppler velocimetry (FGR UADV) carries significant risks for adverse perinatal outcome beyond that of FGR alone, and histopathologic analyses of these placentas shows evidence of impaired angiogenesis. Hypoxia is one key mediator of angiogenesis, and although physiologic, pO2 within the fetoplacental circulation is significantly more hypoxemic than that of postnatal life (∼12-50 mm Hg). Estrogen receptor-beta (ESR2) is the sole estrogen receptor expressed within fetoplacental endothelium, and expression levels are abnormally high in FGR UADV placentas. We hypothesize that high expression of ESR2 in fetoplacental endothelium results in repression of hypoxia-related transcription factors that promote normal angiogenesis. Methods: Placental villous endothelial cells (PLEC) were isolated and cultured from (1) uncomplicated term; (2) FGR UADV; and (3) gestational age-matched control pregnancies. Cells were subjected to normoxia vs hypoxia, ESR2 RNA interference, transduction of an ESR2 over-expression construct, and treatment with the proteasomal inhibitor MG132. Real-time PCR and western blotting were also performed. Results: Exposure of normal PLEC to hypoxia resulted in up-regulation of hypoxia inducible factor 1-alpha (HIF1A; p<0.01), prostacyclin synthase (PTGIS; p<0.01), and vascular endothelial growth factor A (VEGFA; p<0.05).

In contrast, PLEC from FGR UADV pregnancies demonstrated a present but blunted response to hypoxia. Furthermore, at baseline, FGR UADV PLEC demonstrated increased ESR2 (p<0.05) and decreased hypoxia inducible factor 1-alpha (HIF1A; p<0.01) protein expression. There was also diminished expression of the HIF1A heterodimeric partner aryl hydrocarbon nuclear translocator (ARNT; p<0.001), along with that of PTGIS (p<0.05) and VEGFA (p<0.05). ESR2 antagonism resulted in up-regulation of HIF1A, while ESR2 over-expression repressed HIF1A protein levels. Treatment with MG132 resulted in rescuing of ESR2-mediated repression of HIF1A. Conclusions: Abnormally high ESR2 in FGR UADV endothelium represses the hypoxia-mediated induction of HIF1A via post-translational modifications, which in turn, results in down-regulation of the angiogenic genes VEGFA and PTGIS. This may be one key mechanism by which there is aberrant angiogenesis in FGR UADV pregnancies. The prevalence of maternal obesity (MO) is increasing in women of reproductive age. MO is associated with adverse pregnancy outcomes and also with long term cardiometabolic abnormalities in the offspring. Limited information exists regarding the degree of the inflammatory response in the fetal compartment in pregnancies complicated by extreme maternal obesity. Previous data suggest that Inflammation Markers associated with Obesity (IMO) include adipocyte-secreted hormones that regulate energy and metabolism. AIM: The aim of this study was to evaluate the expression of inflammatory mediators in maternal serum (MS), umbilical arterial (UA) and umbilical vein (UV) cord samples, in pregnancies complicated by maternal obesity. METHODS: MS, UA and UV samples were obtained at the time of elective C-section with maternal consent under an IRB approved protocol. Study groups were (Lean: n= 6; Class II: n=9 and Class III: n=9). IMO were measured in a custom Multi Analyte Platform (MAP) by RBM (Austin Tx), which included 67 known markers associated with inflammation and metabolism. Data are presented as Mean±SEM and analyzed by One-Way ANOVA.

RESULTS: Women were of comparable gestational age, and by design stratified by BMI. Ethnicity was: Hispanic 13, Caucasian 9, Asian 1, and African-American 1. Of all the analytes measured, we present Leptin (LEP), Adiponectin (ADPX), IL18, MMP2, MMP3, Eotaxin (EOTX), Stem Cell Factor (SCF), and Tissue Inhibitor of Metalloproteinases-1 (TIMP), which were significantly altered by MO in MS. In UA only SCF was altered (Table) .

When MS was compared to UA, the analytes presented all exhibited a gradient across the placenta (Figure) . Although not significant, blood pressure was higher in Class II MO.

CONCLUSION: Our data show that as expected LEP↑ and ADPX↓. However, IMO were found to be decreased. Despite a significant concentration gradient across the placenta MO affects only the maternal compartment. ARMC, HL89840

S-120 predicts chronic disease such as hypertension in adulthood. The mother represents an important source of nutrients for the growth of the baby, but maternal characteristics that may influence placental morphology still need to be evaluated. Here we studied placental characteristics in singleton pregnancies at term of normal weight and obese/overweight (OB) women. METHODS: 564 placentas were collected at birth, 432 from normal weight women (18<BMI<25 kg/m 2 ) and 132 from OB (BMI≥ 25 kg/m 2 ). Gestational age, maternal data (age, height, BMI, weight gain, hemoglobin -Hband glucose) and fetal data (weight, length, ponderal index and gas analysis at birth) were collected. After removing fetal membranes and umbilical cord, the larger (D) and the smaller (d) diameters were measured and placental weight obtained. With the hypothesis of an elliptical placental shape the surface area was calculated as: "D" x "d" x π/4. We expressed placental efficiency using the ratios of placental weight (PW), "D" and "d" of surface to birthweight (BW).

Gestational age, maternal age and height, and glucose level were not statistically different in the OB vs normal weight women. Hb levels were significantly lower in OB women (11.32 ± 1.17 vs 11.60 ± 1.15 g/dl, p=0.01) as well as the weight gain during pregnancy ( 

Hemopexin is the scavenger protein for plasma heme and contributes to iron (Fe) homeostasis by recycling the heme Fe released from physiological breakdown of red blood cells (RBCs) in the circulation. During pregnancy, hemopexin may be involved in the distribution/partitioning of plasma heme between maternal and fetal compartments. If so it may be influenced by maternal and fetal Fe demands.

The objective of this study is to longitudinally assess serum hemopexin in relation to Fe status indicators and hepcidin in 66 pregnant adolescents (≤18 years of age). We hypothesized that anemic teens would have lower serum hemopexin because of a greater reliance on RBC breakdown to meet the Fe demands of pregnancy.

Serum hemopexin and Fe status indicators (including: hemoglobin (Hb), serum ferritin, transferrin receptor (TfR), total body Fe, and hepcidin) were assessed at mid-gestation (∼26 weeks), delivery, and in cord blood.

Serum hemopexin decreased significantly by 9% from mid-gestation to delivery (P=0.001) and was 6-times higher in maternal mid-gestation serum than in umbilical cord serum (P<0.0001). Mid-gestation hemopexin was positively associated with maternal Hb at mid-gestation (P=0.04) and predicted both maternal delivery Hb (p=0.01) and cord Hb (P=0.0008) and remained a significant predictor of cord Hb after controlling for maternal mid-gestation Hb. Greater decreases in maternal hemopexin were observed in neonates with poor Fe status evidenced by low cord hepcidin (P=0.005) and high cord TfR (P=0.04).

Our data provide evidence for a role of hemopexin in maternal Fe utilization and suggest plasma heme may be involved maternal/fetal Fe partitioning during pregnancy.

Can Objective: There is an increasing trend of diabetes worldwide. It is estimated that pre-gestational diabetes mellitus (DM) affects 1.8% of all pregnancies. The condition is often first diagnosed during pregnancy. Suspicion of preexisting disease prompts early testing. However, regardless of gestational age at diagnosis, these patients are classified as gestational diabetics. This population provides insight into the treatment of DM as differences exist in the management among gestational and pre-gestational DM. We sought to determine the treatment regimens prescribed to both patients with pregestational and suspected pre-gestational DM. Methods: We retrospectively reviewed the data from patients presenting to RWJUH -High Risk Perinatal Center from 2006-2011 and selected those with DM, both gestational and pre-gestational. Patients diagnosed before 20 weeks EGA were suspected to have pre-existing DM. Treatment regimens for all patients were reviewed. Results: 176 patients with DM were evaluated during our study period. 122 patients were diagnosed with gestational DM. Of these, 38 were suspected to have pre-gestational disease. 55% (n=21/38) were maintained on oral agents. 8% (n=3/38) required conversion to insulin to optimize glycemic control. Prescribed treatment regimens are outlined in Figure 1 .

54 patients had pre-gestational diabetes. 26% (n=14/54) were maintained on oral hypoglycemic agents. 3% (n=2/54) required conversion to insulin in order to optimize glycemic control. Prescribed treatment regimens are outlined in Figure 2 .

Conclusions: Patients with both suspected and confirmed pre-gestational DM can be maintained on oral hypoglycemics agents. A randomized controlled trial is warranted to compare oral hypoglycemics to insulin regimens in this patient population. Objective/Hypothesis: Ultrasound estimates of fetal weight (US-EFW) may be less accurate in obese women. Providers may therefore have a lower threshold to offer a cesarean delivery (CD) for macrosomia to obese women with a borderline large EFW. We sought to evaluate whether this was warranted, by examining the magnitude and direction of estimation error in US-EFW in obese women. Methods: We performed a retrospective cohort study of women delivering at a single tertiary care center with an US-EFW within 1 month of delivery. Women with diabetes, multiple gestations, and deliveries <39 weeks were excluded. We projected US-EFW to the day of delivery by assuming an average fetal growth rate of 30 grams per day. We stratified by delivery BMI category and defined obesity as a delivery BMI ≥ 35. Results: 1109 women met inclusion criteria. Of these, 225 (20.3%) were obese: 133 (12%) with BMI 35-39.9 and 92 (8.3%) with BMI ≥ 40. Obesity significantly decreased the accuracy of US-EFW. The average error in obese women was +/-11.1%, compared to +/-8.9% in non-obese (p < 0.01). For all women, ultrasound was more likely to overestimate fetal growth, with 84% of all estimates being greater than birthweight. Even for the subset of women with a delivery 0-1 days after the EFW, 77% were overestimates. The magnitude of the overestimation was significantly greater for obese women, with 13.3% having an overestimate of > 20%, as compared to 8.3% of non-obese women (p<0.01). However, underestimation was not impacted by maternal obesity, with 1.3% of obese women having an US-EFW > 10% under birthweight, compared to 2.1% of non-obese women.

Conclusion: In our cohort, we found that as maternal BMI increases the accuracy of US-EFW decreases, but only in the direction of overestimation.

Obese women are no more likely than normal weight women to have a birthweight greater than the US-EFW. Providers should not have a lower threshold to offer CD to obese women with nearly-macrosomic estimated fetal weight.

Trimester. Leptin is increased in obesity, and plays an immunoregulatory role. We assessed the first trimester associations between BMI and inflammatory markers including cytokines, CRP, MMP-8 and Leptin as they may help elucidate early pathophysiology related to adverse pregnancy outcomes.

Methods: This is a secondary analysis from a prospective multicenter study. Women over 16 years of age with singleton gestations, and no pre-existing diabetes were recruited in the first trimester. Serum concentrations of Leptin, IL-1a and b, IL-6, IL-8, IL-10, CRP, TNF-alpha, and MMP-8 were determined by Luminex or ELISA. Statistical analysis using paired t-test, and univariate and multivariate regression models were performed.

We analyzed samples from 128 women. Discussion:

Obesity is a pro-inflammatory state. BMI in the first trimester is significantly associated with CRP and Leptin. However, after controlling for BMI the association between CRP and Leptin was only noted at very high BMI levels.

Further studies are needed to relate these biomarkers to adverse outcomes seen with obesity in pregnancy. Objective: There is controversy over the 1 step vs the 2 step method to diagnose gestational diabetes (GDM). In the 2 step, non-fasting patients screen with a 1hour 50g challenge test; if positive a 3 hour 100g glucose tolerance test (GTT) is performed and ≥2 abnormal values define GDM. The 1 step differs in that a fasting patient is screened/diagnosed after a 75g 2 hour GTT which is positive if ≥1 value is abnormal. Using the one step method patients could be diagnosed with GDM having an isolated abnormal fasting blood glucose (FBG). These patients would not be identified on the 2 step method. We sought to investigate the specific characteristics of GDM patients diagnosed by an abnormal FBG on the 1 step.

In 2011, women were tested for GDM using the 1 step method. All patients diagnosed with GDM were managed by the protocol described by Langer et al NEJM 2000. In a retrospective chart review, the patients that were diagnosed with GDM having only an abnormal FBG (Group1)were compared to the all other GDM patients(Group 2, exclusive of the patients with an isolated abnormal FBG). Macrosomia was defined as >4000g.

Results: 270 women were diagnosed with GDM. 19(7%) patients were in Group 1.When these women were compared to Group 2 they had significantly higher BMI, were more likely to be Black and require hypoglycemic agents (84%vs 47%, p<0.001). Mean FBG was significantly higher in group 1 than group 2 (102.21mg/dl vs. 85.71mg/dl, p <0.05). (see chart below). Conclusions: By using the 1 step method, a considerable amount of women are diagnosed with GDM only with an abnormal FBG. These patients were more obese and required hypoglycemic agents, implying severe, extensive disease. These patients would have escaped detection if the 2 step method was used; as the screening component does not require a fasting blood glucose and the diagnostic component requires ≥2 abnormal values.

Characteristics of Recurrent LGA in Obese Women. Arun P Jain, Sarah A Hopkins, Jeffrey A Gavard, Jim J Rice, Rosemary B Catanzaro, Raul Artal. Obstetrics, Gynecology & Women's Health, Saint Louis University, Saint Louis, MO, USA. Introduction: Obesity and prior delivery of a large-for-gestational-age (LGA) infant are risk factors for future LGA births. The aim of this study was to identify the significant characteristics associated with the recurrence of LGA in the second pregnancy in obese women. Methods: A historical cohort analysis of 1,412 obese (BMI ≥ 30 kg/m 2 ) women was performed using the Missouri 1998-2005 longitudinally-linked data. Characteristics of women who had a recurrence of LGA in their second pregnancy were compared to those who did not, using the Kolmogorov Smirnov test. Stepwise logistic regression was performed to estimate adjusted odds ratios (aOR) and 95% confidence intervals (CI) for significant predictors of recurrent LGA. Results: In this cohort, 564 women (39%) delivered a second consecutive LGA infant. Women with a recurrence of LGA delivered a heavier infant in their first pregnancy (p<0.001) and gained more weight in their second pregnancy (p<0.001). In contrast, BMI prior to the second pregnancy was not significantly different between groups (p=0.150). Significant multivariate predictors of recurrent LGA are shown in the Table. The presence of diabetes mellitus was associated with a two-fold increase in odds of recurrent LGA. In contrast, frequent prenatal care (greater than 110% of the expected number of prenatal visits) was associated with significantly reduced odds of a second LGA infant. Conclusion: Obese women with diabetes, and those who had the heaviest babies in their first pregnancy, are at increased risk for recurrent LGA. More frequent prenatal care visits and avoidance of excessive weight gain in the second pregnancy may reduce the risk of delivering a second LGA infant in obese women. To prospectively measure endothelial function by flow mediated vasodilation (FMD) in each trimester in a cohort at risk of developing preeclampsia and to determine if FMD is significantly predictive of obstetric outcomes.

Participants at risk of developing preeclampsia were recruited into this IRB approved, prospective cohort study. FMD was evaluated in each trimester of pregnancy. Demographic data and relevant medical history were obtained from the medical record. Ultrasound measurement of flow mediated dilation (FMD) of the brachial artery in response to reactive hyperemia non-invasively assesses endothelial function. Artery diameter is obtained before and after an occluding forearm cuff placed distal to the antecubital fossa is inflated and then deflated. FMD is the difference in artery diameter after reactive hyperemia has been induced compared to baseline. The data was analyzed by ANOVA and clinical correlates were analyzed by Pearson correlations, multivariable regression and ROC curves. Results 34 participants who did not develop preeclampsia in the index pregnancy are considered in this analysis. The cohort FMD did not significantly change throughout pregnancy. Significant correlations between FMD and pregnancy characteristics included extremes of maternal age, age at time of delivery, operative vaginal delivery, PPROM, abruption and history of preeclampsia. Significant correlations between change in FMD between trimesters and pregnancy characteristics included extremes of maternal age, BMI over 25, type I diabetes mellitus, number of live births and age at the time of delivery. The change in FMD from the first to the second trimester was highly predictive of the subsequent development of gestational diabetes mellitus type A1 (GDMA1) (AUC=0.97, p=0.003).

To our knowledge, this is the first study to prospectively evaluate FMD in a control cohort of patients at high-risk for developing preeclampsia. The change in FMD from the first to second trimesters was predictive of developing GDMA1. This change is observed 8 weeks before routine Glucola testing. This finding suggests a predictive modality and an early physiologic pathway for analysis in the development of poor outcomes in women at risk for developing preeclampsia. Pregnant adolescents are at risk of iron deficiency and anemia however few data are available among this group and their newborns, and the impact of maternal Fe status on neonatal Fe stores at birth remains unclear. We assessed the Fe status among 238 pregnant teens (≤18 y) and their babies. Teens were over-nourished as a whole as evidenced by their mean pre-pregnancy BMI (ppBMI; 24.6±5.5); with 17% overweight or obese at entry into pregnancy; and 63.2% of teens with excessive weight gain (16.4 ± 7.2 kg). Maternal Fe status (Hb, transferrin receptor; TfR, serum ferritin; SF, total body iron;TBI) was assessed at mid-gestation (MG, ∼26 weeks) in a subsample of the cohort (n=143, 60%), and at delivery (39.3±2.7 weeks) in both teens and in neonates (umbilical cord blood). Maternal Fe status at MG were highly correlated with values obtained at delivery (r=0. 4-0.6, p <0.0001) . Sixteen percent of teens were anemic at MG, this increased to 34% at delivery. On the basis of Hb and SF levels, 5.6% and 8.5% of teens were classified as having Fe deficiency anemia (IDA). Serum folate and vitamin B12 were within normal ranges. A quarter of teens had depleted Fe stores (SF ≤12 µg/L) and half of the teens had tissue Fe deficiency (TfR >4.4 mg/L) at MG. Twenty three percent of newborns were anemic (Hb <13.5 g/dL) at birth, and 23% of neonates had SF <76 µg/L, a cutoff that has previously been associated with impaired language ability, tractability and fine motor skills in children at 5 y of age. In this group, maternal Fe status had a significant impact on neonatal Fe stores. Neonates born to Fe-deplete teens at MG had significantly lower Fe stores than those born to Fe-replete teens at MG [99.5±53 vs 154.7±81.1 µg/L, p <0.0001]. Likewise, neonates born to teens with tissue Fe deficiency at MG had significantly higher sTfR concentrations at birth [9.1±3.5 mg/L vs 7.5±2.7 mg/L, p <0.0084]. Among a group of otherwise healthy pregnant adolescents, anemia and depleted Fe stores were prevalent and maternal Fe insufficiency was linked to low Fe status in neonates at birth. Because of the growing awareness of the impact of neonatal anemia on long-term, irreversible neurophysiological and cognitive outcomes, increased attention on optimizing Fe status among pregnant teens and their newborns is warranted.

Activity and Expression of Placental Glutathione Peroxidase in Pregnancies Affected by IUGR and Macrosomia. Margarida Y Lei, Suvajit Sen, Gautam Chaudhuri, Carla Janzen. Obstetrics and Gynecology, UCLA David Geffen School of Medicine, Los Angeles, CA, USA. Background: Increased placental mitochondrial activity during pregnancy generates reactive oxygen species (ROS) including superoxide. Free radicals contribute to increased oxidative stress (OS) documented in pregnancies complicated by preeclampsia, IUGR and diabetes. Glutathione peroxidases (GPx) are antioxidant enzymes able to metabolize H2O2 and lipid peroxides to maintain a stable redox balance despite increased OS. GPx is the only antioxidant enzyme known to reduce lipid peroxidation. Abnormally reduced placental GPx activity has been found in pregnancy affected by preeclampsia, which may explain excessive OS contributing to the complications of this disease. Few studies have analyzed placental GPx activity in pregnancies complicated by idiopathic fetal growth disorders (IUGR and macrosomia in the absence of known maternal risk factors). Objective: To determine whether there are changes in placental GPx expression and GPx activity in pregnancies affected by idiopathic IUGR and macrosomia. Methods: Under IRB approval, placentas were obtained from women with IUGR (n=7), macrosomia (n=7), and gestational age-matched controls (n=7). Placentas were dissected to separate the maternal from the fetal side. Paraffinembedded sections of placental biopsies were stained for against GPx. GPx activity was analyzed by a spectrophotometric assay (BioVision, Inc). Real time-PCR using primers specific to GPx1 was performed using SYBR Green Dye. Results: We found a significant increase in GPx activity in both the maternal and fetal aspects of placenta in pregnancies affected by both idiopathic IUGR and macrosomia (Fig 1) . Increased GPx activity correlated to a trend toward increased GPx1 mRNA expression. Conclusions: Our results suggest that placental GPx activity is increased in idiopathic IUGR and fetal macrosomia. Increased GPx activity may represent an adaptive response to OS in order to resist oxidative injury in the fetal compartment. Additional studies are planned to assess the mechanism by which GPx activity is regulated in pregnancy complicated by abnormal fetal growth. Objective: To examine maternal and neonatal outcomes in obese, nulliparous women with an unfavorable cervix, undergoing elective induction of labor as compared to expectant management after 39 weeks gestation. Study Design: This is a retrospective analysis of a cohort of nulliparous women with a singleton gestation in vertex presentation who delivered at MedStar Washington Hospital Center from 2007-2012. Patients with unfavorable cervix (defined as a modified Bishop score <5) and a body mass index (BMI) >30.0 kg/m 2 were included in analysis. Patients undergoing elective induction of labor between 39 0/7 and 40 6/7 weeks' gestation were compared with patients who were expectantly managed beyond 39 0/7 weeks'. Maternal and neonatal outcomes were compared using χ 2 , student t or Wilcoxon rank sum tests as appropriate with significance set at p<0.05. Results: Sixty patients meeting inclusion criteria underwent elective induction of labor and were compared to 460 patients who were expectantly managed beyond 39 0/7 weeks. The rate of cesarean delivery was significantly higher in the electively induced group vs. those managed expectantly (40.0% vs. 26.3% respectively, p=0.026). Other maternal outcomes, including operative vaginal delivery, rate of 3 rd or 4 th degree lacerations, chorioamnionitis, postpartum hemorrhage and need for blood transfusion were similar. Birth weight was lower in the expectantly managed group (3509±445g vs. 3378±400g, p=0.019) and NICU admission was higher in the electively induced group (18.3% vs. 7.2%, p=0.004). Umbilical artery pH<7.0 and Apgar < 7 at 5 minutes were similar between the two groups. Conclusion: Elective labor induction in obese nulliparous parturients carries an increased risk of cesarean delivery and higher NICU admission rate as compared to expectant management. Thomas' Hospital Foundation Trust, London, United Kingdom. Background. Obesity is associated with heightened risk of gestational diabetes (GDM). A test in early pregnancy which identified obese women who later developed GDM would enable stratification of care and targeted intervention. Design. We evaluated the role of clinical, demographic and anthropometric variables and routine and novel plasma biomarkers (total, HDL-, LDLcholesterol, triglyceride, CRP, insulin, AST, ALT, ferritin, t-PA, adiponectin, IL-6, fructosamine, visfatin and leptin) in non-fasted samples obtained at 15 +0 to 17 +6 weeks' gestation, for prediction of GDM as assessed by an OGTT (IADPSG criteria).

Subjects. 106 women participating in the pilot study of the UPBEAT trial, a complex intervention of a low glycemic index diet and increased physical activity in obese pregnant women. Results. Women who developed GDM (n=29) were older (mean age 33.48 v 30.19 yrs, p=0.002), had higher parity, higher blood pressure, greater total sum of skinfolds (93.88mm v 86.06mm p=0.031) and were more likely to be black (55.2% of women with GDM were black p=0.009) than non-GDM women. Women with GDM had significantly higher early 2 nd trimester AST (mean 30. Conclusion. An algorithm comprising clinical, demographic and anthropometric variables with addition of plasma adiponectin and AST may prove valuable in prediction of GDM in obese women. Validation is required in a larger population. OBJECTIVE: The aim of this study was to determine levels of early (less than 20 weeks) HgA1C (A1C) testing less than the cut off for overt DM, but predictive of the development of gestational diabetes (GDM). STUDY DESIGN: This is a retrospective review of 281 patients delivered at a public hospital in Southern California, over a six-month period from January through June 2012. Patients were included if they had a singleton gestation, were not diagnosed with overt DM (A1C less than 6.5%), and they had completed both A1C testing and testing for GDM (2h oral glucose tolerance test, OGTT).

The following data was also abstracted: maternal age, ethnicity, gravidity, parity, neonatal birth weight, and mode of delivery. Once a diagnosis of DM was made, the patient underwent dietary counseling, home glucose monitoring and close supervision for the duration of pregnancy. RESULTS: The mean A1C of patients who developed GDM was significantly higher than patients who didn't develop DM (5.5 ± .25% vs 5.2 ± .40% SD, P=0.0036). A value of 5.9% was predictive GDM in 100% of cases, while a value of 5.7-5.8% predicted a 50% chance. Thus 30% of patients were exposed to higher glucose levels early in pregnancy. There was no difference in birthweight or route of delivery between the groups; not unexpected since there was no early intervention. CONCLUSION: A borderline A1C at the first prenatal visit (<20 weeks) predicts patients who will go on to develop gestational diabetes. These patients may benefit from earlier intervention and treatment to reduce maternal and fetal morbidity.〈〈

The METHODS: In this cohort study, CRP was measured by ELISA in age-matched plasma samples throughout pregnancy from obese (BMI>30) and lean women. Maternal and fetal clinical variables were extracted from the medical record. The data was analyzed by ANOVA and clinical correlates were analyzed by Pearson correlations, multivariable regression and ROC curves. alpha=0.05. RESULTS: The groups (lean n=121, obese n=131) were similar in terms of median age, gravida, and racial distribution. Delivery characteristics between groups were similar in median gestational age at delivery, birthweight, and 1-minute APGAR scores. Maternal-fetal clinical conditions were higher in the obese group such as chronic hypertension (27. The difference in CRP between trimesters 1 and 2 (CRP 1-2 ) was moderately predictive of fetal macrosomia (AUC=0.77, p=0.013) and gestational diabetes (AUC=0.76, p=0.050). In this cohort, CRP 1-2 was very predictive of the development of preterm delivery (AUC=0.95, p=0.0095). DISCUSSION: Our results suggest that CRP is elevated in obese pregnant women throughout pregnancy. In contrast to previous studies, however, we did not find that CRP is an adequate predictor of preeclampsia, diabetes, or hypertensive disorders, which we found to be significantly more common in this obese cohort. Most significantly, CRP 1-2 was very predictive of the development of a preterm delivery. This finding strengthens the association between inflammation and preterm labor. CRP 1-2 may prove to be a significant, noninvasive tool in the prediction of preterm labor supporting the role of inflammation in its pathogenesis.

Maternal Obesity Influences Utero-Placental Development. Background: Lifelong maternal obesity is an epidemic associated with increased pregnancy complications. In a subset of such pregnancies, an increase in fetal growth restriction (FGR) has been observed. This is thought to be related to placental dysfunction although the exact underlying mechanisms involved are unclear. Objective: To investigate whether lifelong maternal obesity is associated with altered utero-placental development.

Methods: A life-long high fat diet (HFD) induced Sprague Dawley obesity rat model that mimics outcomes seen in the human population was used for these studies and compared with control diet (CD) at two crucial gestational ages 15 (GD15) and 18 (GD18). To characterize utero-placental malformations, systematic morphometric analysis was performed, by measuring the surface area of the specific layers of the utero-placental unit such as the deciduas basalis, the junctional zone and the labyrinth. Trophoblast uterine invasion and uterine vascular remodeling was assessed by profiling endovascular trophoblast and uterine vascular associated structural cells such as smooth muscle and endothelial cells.

The obese rat model revealed decreased neonatal birth weights. Morphometric analysis revealed an increase in labyrinth size at both GD15 and GD18 and increased decidua basalis thickness at GD18 in HFD rats when compared CD rats. Extravillous trophoblast invasion at GD15 was significantly increased in the HFD rats compared to CD rats. In addition, decreased vascular smooth muscle degradation was observed at GD18. Conclusion: Maternal obesity alters utero-placental growth patterns in such that premature trophoblast invasion and decreased uterine vascular remodeling are observed. These results coincide with gestational specific increases in placental size and failure of physiological thinning of the decidua basalis.

Maternal obesity's influence on utero-placental development is evident but further research is required to elucidate the mechanisms involved in obese pregnancy related complications.

CONCLUSIONS: Different windows occur for MO-induced, age-related dysfunctional changes in lipid metabolism in OFF. These windows constitute an opportunity to evaluate antecedent mechanisms underlying premature aging which may result from exposure to MO in pregnancy.

(1) J Physiol, 2010; 588(10):1791-9; (2) Br J Nutr, 2012; 107(11):1562-5. Objective: Borderline epithelial ovarian tumors are staged the same as invasive ovarian tumors. Some borderline tumors may not require complete surgical staging. We wanted to better understand and characterize those borderline tumors that are associated with a recurrence of disease. Methods: A retrospective cohort analysis was conducted looking at all cases of borderline ovarian tumors at a single institution between 1976 and 2005. Cases were confirmed by pathology report. Over fifteen variables were examined critically and analyzed using Fisher's exact test and other statistical tests.

In total, 146 cases of borderline ovarian tumor were identified. They were divided into serous, mucinous, and seromucinous. The risk of recurrence was 12%. Mean follow-up was 73.5 +/-72.1 months. A significant difference in the risk of recurrence between the three subtypes exists (p=.036). The relative risk of recurrence of serous vs. mucinous tumors was 3.87 (p=.049). 4% of mucinous tumors recurred and were initially unstaged and Stage III; one had a positive appendix and both were associated with pseudomyxoma peritonei. 2% of mucinous tumors had a positive appendix. No mucinous tumor had nodal disease. 15.5% of serous tumors recurred and were Stage I-III. 28.6% of seromucinous tumors recurred and they were Stage III. 15% of recurrent serous tumors exhibited micropapillary features. One patient with a recurrence of Stage IC serous borderline tumor died; this patient had a recurrence in the small bowel, large bowel, and omentum 12 years after diagnosis at the time of complete surgical staging. Discussion: Conventionally, ovarian cancer is staged by completing a hysterectomy, bilateral salpingo-oophorectomy, pelvic and para-aortic lymph node dissection, and omentectomy. In cases of mucinous tumors, an appendectomy is also performed. Based on our data, a low risk of appendiceal or lymph node involvement was observed in the mucinous tumors and a low risk of recurrence for mucinous tumors compared to serous or seromucinous tumors. Therefore, less aggressive staging may be considered if a mucinous tumor is identified with a normal appearing appendix in the absence of pseudomyxoma peritonei. In patients presenting with Stage III mucinous tumor, a serous or a seromucinous tumor, complete surgical staging is recommended.

Potential Targets for Immunotherapy in Epithelial Ovarian Cancer. Sayeema Daudi, Tony Miliotto, Adrienne Groman, Shashikant Lele, Kunle Odunsi. Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA.

The clinical response in epithelial ovarian cancer (EOC) from cytotoxic chemotherapy has reached an apparent plateau, which illustrates a need for alternative therapeutic options. In a tenacious search to identify novel targets for antigen specific immunotherapy, CT104.1 (POTE-D), TSK-1 and GPR-6 genes were identified through EST and SEREX database analysis. To study the potential for immunotherapy in EOC, we examined the expression and the prognostic significance of these antigens.

One-step reverse transcriptase PCR was performed with RNA from a panel of 23 normal and 83 EOC tissues obtained between 1992 and 2007. Specific primers were used to amplify a 235 bp product for POTE-D, a 225 bp product for TSK-1 and a 481 bp product for GPR-6. Clinico-pathologic correlation to antigen expression was assessed utilizing the Wilcox Rank Sum and Pearson Chi-square tests. The survival outcomes of antigen expression in EOC were analyzed by a multivariate Cox proportional hazard model, with a 95% confidence limit for the HR.

The expression of POTE-D, TSK-1 and GPR6 transcripts were demonstrated in 65% (54/83), 64% (53/83) and 55% (46/83) of the EOC specimens, respectively. Restricted expression in normal tissues was observed for all three genes. The median follow up was 109 months (range: 42-174). TSK-1 mRNA expression was associated with POTE-D and GPR-6 expression (p < 0.01). POTE-D expression was associated with platinum sensitive disease (p= 0.036), but did not translate into a significant improvement in survival. TSK-1 expression was associated with an improved survival [HR 0.55, 95% (CI 0.33-0.92), p= 0.023]. Conversely, GPR-6 expression was associated with a poor survival and disease recurrence [HR 2.26, , p= 0.015 and HR 2.42, 95% (CI 1.27-4.62), p= 0.007].

Restricted expression of these antigens in normal tissues and expression in a significant proportion of EOC patients makes these antigens an attractive target for ovarian cancer immunotherapy. Moreover, expression of TSK-1 and GPR-6 were significantly associated with survival. Studies evaluating the specific immunogenicity of each target are ongoing, with preliminary data suggesting strong therapeutic potential. Our demonstration of concomitant expression in these antigens suggests the induction of a gametogenic program in human ovarian cancer.

Primary A retrospective review of patient records was used to determine patient characteristics and outcome.

Results: a total of 7 patients with histologically confirmed primary carcinoid tumor of tha ovary were detected with a mean age of 52. Histological analysis of these carcinoid tumors revealed 2 (28%) were insular, 2 (28%) were trabecular, 2 (28%) were strumal carcinoid, and one was mixed insular and trabecular. 100% of the tumors were found to be unilateral. Tumors associated with teratomas were on average smaller in greatest diameter than those that were not. Patients presented with pelvic pain or pelvic masses prior to surgery. Recurrence, including hepatic metastasis, was noted in 1 of 7 cases. Positive cytology during primary surgery was noted in this patient with recurrence. Conclusion: Primary carcinoid tumors of the ovary should be included in the differential diagnosis of pelvic masses. Surgical approach should include cytology, removal of mass, and appendectomy. Further studies need to be performed to determine the usefulness of pelvic washings at time of surgery in predicting recurrence and the need for closer follow up afterwards. Ovarian cancer is the second most common gynecologic cancer in women and causes 5 % of cancer deaths in females. Epithelial ovarian cancers are the most common malignant ovarian tumors. Pregnancy is an important factor in epithelial ovarian cancer for risk reduction and improved survival. Persistence of fetal cells in the maternal circulation and organs for decades following pregnancy is known as fetal microchimerism. It has been implicated in some female malignancies such as cervical and breast with different hypothesized roles, based on the type of functional cells that fetal microchimeric cells have differentiated into. In this retrospective study, we aimed to determine if fetal microchimeric cells were involved in the progression of ovarian cancer. We investigated a well described archive of epithelial ovarian tumur formalinfixed and paraffin-embedded tissue sections from nulliparous and parous women. Fluorescence in situ hybridisation (FISH) was performed to detect male presumed-fetal cells, labelled by DNA probes for X and Y chromosomes. The outcome of blinded FISH analysis was correlated with the known reproductive history and the clinic-pathological features of the studied group. Fetal microchimeric cells were not detected in archived ovarian tumor tissue sections from the parous women included in this study. Interestingly, tumor cells were found to have multiple copies of X chromosome by FISH analysis, with more than two X signals detected in some nuclei. We also found some of the tumor cells had loss of X chromosome relative to the ploidy level. These findings imply that fetal microchimerism does not have a role in the progression of ovarian cancer. Instead, fetal microchimerism could have a role in preventing the development of ovarian cancer by sensitizing the maternal immune system to develop adaptive immunity against tumor cells that express onco-fetal antigens. A follow-up study with a larger sample size is necessary to verify our findings as this is the first study of fetal cell microchimerism on ovarian tumor tissues. Ovarian cancer is one of the most aggressive female reproductive tract tumors. Paclitaxel (PTX) is widely used for the treatment of ovarian cancer. However, ovarian cancers often acquire chemotherapeutic-resistance to this agent. We investigated the mechanism of chemoresistance by analysis of microRNAs using the ovarian cancer cell line KFr13 and its PTX-resistant derivative (KFr13Tx). We found that miR-31 was downregulated in KFr13Tx cells, and that re-introduction of miR31 re-sensitized them to PTX both in vitro and in vivo. miR-31 was found to bind to the 3?-UTR of mRNA of MET, and the decrease in MET correlated to higher sensitivity to PTX. Furthermore, cotreatment of KFr13Tx cells with MET inhibitors sensitized the tumor cells to PTX both in vitro and in vivo. In addition, lower levels of miR31 and higher expression of MET in human ovarian cancer specimens were significantly correlated with PTX chemo-resistance and poor prognosis. This study demonstrated miR31-dependent regulation of MET for chemoresistance of ovarian cancer, raising the possibility that combination therapy with a MET inhibitor and PTX will increase PTX efficacy. Epithelial-Mesenchymal Transition (EMT) is a critical step in ovarian tumor invasion and metastasis and correlates positively with a poor patient prognosis. Prior reports reveal that miRNAs have the potential to function as either tumor suppressors or oncogenes, and contribute to metastasis. We hypothesized that miRNA family genes are potent regulators of EMT and the resulting tumor cell adhesion, migration, invasion, metastasis. Ovarian cancer patient and controls were recruited, and total RNA isolated from their ovaries; yield, purity and integrity of RNA were determined using a nanospectrometer and the Agilent Bioanalyzer. The miRNA/mRNA transcriptom was determined using the Affymetrix miRNA 3.0 and Human whole-Transcript Expression Arrays, respectively, and then validated by appropriate assay. miRNA-mRNA interactions were sought by the MetaCore Enrichment tool using the overlapping miRNA/mRNA microarray data sets. We found that miR-205, mesenchymal markers (vimentin, Collagen I, N-cadherin) were significantly increased; TCF21, E-cadherin, and epithelial markers (Laminin 1, Collagen IV) were decreased in ovarian cancer. Then using miRanda and mirSVR bioinformatics tools, plus a reporter assay, we found that miR-205 targets TCF21 which suppresses E-cadherin, a hallmark of EMT progression. In summary, we found for the first time that miR-205 is significantly up-regulated in ovarian cancer, and that miR-205 targets and suppresses TCF21 resulting EMT progression mediated by E-cadherin. Our study suggests the miR-205/ TCF21/E-cadherin signaling cascade pathway may play an important role in ovarian cancer progression. Apoptosis-inducing factor (AIF) is a ubiquitously expressed flavoprotein that plays a critical role in caspase-independent apoptosis. AIF is normally localized to the mitochondrial intermembrane space and is released upon loss of mitochondrial membrane potential and rapidly translocates to the nucleus, preceding cytochrome c release from mitochondria, resulting in the morphological hallmarks of apoptosis. Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as nuclear translocation of AIF in the cerebral cortex of guinea pig fetus at term. The present study tests the hypothesis that hypoxia-induced translocation of AIF from mitochondria to neuronal nuclei in the cerebral cortex of guinea pig fetus at term is nitric oxide-mediated. Guinea pig fetuses were studied at term (60 days) gestation and grouped in normoxic (Nx, n=7), hypoxic (Hx, n=7) and hypoxic pretreated with a NOS inhibitor, N-Nitro-L-Arginine Methyl Ester (Hx+L-NAME, 30mg/kg i.p., n=6). Cerebral tissue hypoxia was documented biochemically by determining the levels of ATP and phosphocreatine (PCr). Mitochondria and neuronal nuclei were isolated and proteins separated on 10% SDS-PAGE and probed with specific anti-AIF antibody. ATP levels were 4.6±0.3 (Nx), 1.6±0.4 (Hx, p<0.001) and 1.6±0.2 (Hx+L-NAME). PCr values were 3.4±0.3 (Nx), 1.2±0.4 (Hx) and 1.1±0.2 (Hx+L-NAME). Expression of AIF in the mitochodria was 31.70±2.7 in Nx, 22.9±3.7 in Hx, and 21.81±3.01 in Hx+L-NAME. In the neuronal nuclei the expression of AIF was 37.10±4.0 in Nx, 123.13±12.9 in Hx and 45.09±4.6 in Hx+L-NAME, (p<0.05 vs Hx). The data show that hypoxia results in significant translocation of AIF from the mitochondria to the neuronal nuclei and that the administration of nitric oxide synthase inhibitor prior to hypoxia prevents the hypoxia induced translocation of AIF to the nucleus. We conclude that the hypoxia-induced translocation of AIF from mitochondria to the nuclei is NO-mediated. We speculate that the nitric oxide mediated, hypoxia-induced increased AIF in neuronal nuclei leads to increased DNA fragmentation and results in caspase-independent programmed neuronal cell death in the guinea pig brain at term.

Infusion of IL-17 Blocking Protein Blunts Hypertension and Oxidative Stress in Response to Placental Ischemia. James P Hogg, Denise Cornelius, Kedra Wallace, Janae Moseley, Babbette LaMarca. Obstetrics and Gynecology, University of Mississippi Medical Center, Jackson, MS, USA. Preeclampsia, new onset hypertension with proteinuria during pregnancy, is associated with chronic inflammation including autoimmune cells, TH17 subtype, secreting interleukin-17, and placental oxidative stress. We have previously shown that chronic IL-17 infusion increases blood pressure by stimulating placental oxidative stress (ROS) during pregnancy. The objective of our current study was to determine if chronic IL-17RC (recombinant receptor C) decreases hypertension and placental ROS in response to placental ischemia during pregnancy. To answer this question three groups of rats were examined: Normal Pregnant NP (n=18), Reduced Uterine Perfusion Pressure RUPP (n=21), and RUPP+ IL-17RC (n=14). On day 14, mini-osmotic pumps infusing 100 pg/day of IL-17RC were surgically implanted intraperitoneally into pregnant rats undergoing RUPP procedure. The RUPP procedure reduces the uterine perfusion pressure with the application of a constrictive silver clip (0.203 mm) to the aorta superior to the iliac bifurcation and clips (0.100 mm) to the bilateral uterine arcades at the ovarian end on ovarian collateral circulation to the uterus on day 14 of gestation under isoflourane anesthesia. On day 18 carotid catheters were placed and on day 19 blood pressure (MAP) was recorded, plasma, urine and tissue were collected for isolation of ROS detected by chemilluminescent technique and expressed as relative light units. Graphpad Prism 5 was used to analyze statistical differences, all data was analyzed via one-way ANOVA. MAP increased from 101 +/-3 mmHg in NP to 118+/-4 mmHg in RUPP, and decreased to 107+/-5mmHg in IL-17RC infused RUPP rats. Placental ROS increased from 410 RLU in NP to 609 RLU in RUPP, but decreased to 511 RLU in IL-17RC infused RUPP rats. Administration of IL-17RC blunted the hypertension and placental oxidative stress in the RUPP rat model of preeclampsia. Therefore, from these data we conclude that IL-17RC, possibly by suppressing circulating IL-17, decreases placental oxidative stress which may play an important role in mediating hypertension in response to placental ischemia during pregnancy.

Glucose Metabolic Pathway and ROS Production in Endothelial Dysfunction Induced by TNF-alpha: Clinical Implications. Rashmi Rao, Suvajit Sen, Gautam Chaudhuri. Obstetrics and Gynecology, UCLA, Los Angeles, CA, USA. SIGNIFICANCE: Intra-uterine infection and preeclampsia, which are associated with endothelial cell dysfunction (EC), are an important cause of perinatal morbidity and mortality. Increased levels of TNF α have been linked to EC dysfunction in these conditions. EC generates ATP mainly by glycolytic mechanisms unlike other normal cells which use oxidative phosphorylation. We focused on mechanisms that affect the function and viability of EC in the fetal circulation using HUVEC as the model system. This work focuses on the role of reactive oxygen species (ROS) and their association with either glycolytic or oxidative phosphorylation modes of ATP generation in HUVEC following their exposure to TNF α. Objective: We elucidated the metabolic flux between oxidative phosphorylation and glycolysis under conditions that induce EC dysfunction. Study Design: HUVEC cells were isolated and cultured from freshly collected umbilical cords. EC dysfunction was induced following chronic treatment with TNF α (10ng/mL) for seven days. Hydrogen Peroxide(H 2 O 2 )released in culture media was measured utilizing the oxidation of Amplex Red. Metabolomics of HUVEC cells were performed utilizing HPLC/MS techniques in collaboration with Metabolon. Extracellular flux analysis was determined by the extracellular flux analyzer, XF24, from SeaHorseBiosciences. Results: A decrease in the growth rate of HUVEC was associated with increased H 2 O 2 production following treatment with TNF α (.8 vs. 1.5 pm/hr). Metabolomic studies indicated that there was an increase in pentose phosphate pathway (PPP) intermediates such as: glucose-6-phosphate and 1-3 di-hydroxyacetone. This was associated with attenuated levels of the TCA cycle intermediates such as: succinate and citrate. This indicates that TNF α treatment compromised metabolic flux through the TCA cycle and subsequently favored increased flux through the PPP. This was further corroborated by observing enhanced spare respiratory capacity upon treatment with TNF α (11.5% vs. 19.8%). Conclusions: 1) Conditions leading to EC dysfunction, as mimicked by the chronic treatment of HUVEC with TNF α, shifted the metabolic flux from the TCA cycle towards the PPP. 2) Elevated levels of intermediates of the PPP indicate increased production of NADPH which apart from generating reducing equivalents may also serve as a substrate for NADPH oxidase (specifically NOX 4), which explains the increased rate of H 2 O 2 production observed S-148 Introduction: The importance of the thyroid hormone (TH) transporter, MCT8, to human neurodevelopment is highlighted by findings of severe global neurological impairment in subjects with MCT8 mutations. IUGR may be associated with milder neurodevelopmental deficits, which has been partly attributable to dysregulated TH action in utero secondary to reduced circulating fetal TH concentrations and decreased cerebral TH receptor expression. We postulate that changes in MCT8 expression are also implicated in this pathophysiology.

Methods: MCT8 was localized by immunohistochemistry in human fetal occipital and parietal cerebral cortices from mid-trimester. We objectively compared MCT8 immunostaining in the occipital cortex of IUGR (n=7) and appropriately grown for gestational age (AGA; n=5) stillborn human fetuses between 24-28 weeks gestation by quantifying the percentage area of cortical plate (corrected for cell number) and the proportion of subplate microvessel cross sections immunostained.

Results: There was widespread MCT8 immunostaining in the cortical plate, subplate, ventricular and subventricular zones, choroid plexus, ependyma and microvessels. MCT8 expression was significantly reduced by 5-fold in the cortical plate in IUGR (p<0.05) but not significantly changed in the microvessels compared with AGA. However, there was a significant positive correlation between the area of cortical plate immunostained and the proportion of microvessels stained (correlation coefficient=0.71, r²=0.27; p<0.01). Cortical MCT8 expression was negatively correlated with the severity of IUGR as indicated by brain to liver weight ratios (correlation coefficient=-0.64, r²=0.28; p<0.05). Conclusions: Our results support the hypothesis that reductions in MCT8 expression in the IUGR fetal brain could further compromise TH-dependent brain development.

Synthesis of Estrogen by the Ovine Fetal Adrenal. In the mammalian fetus, estrogen influences embryogenesis, cell proliferation and differentiation, fetal imprinting and, in late gestation, neuroendocrine mechanisms controlling fetal stress responses and initiation of parturition. Estradiol augments fetal ACTH secretion, and we have hypothesized that it provides one limb of a negative feedback mechanism by which the fetal hypothalamo-pituitary unit and the placenta communicate. Estrogen is synthesized in the ovine placenta, both de novo from cholesterol and possibly from precursors secreted by the adrenal cortex. The present study was performed to test the hypothesis that the ovine fetal adrenal has the biosynthetic capacity to synthesize estrogens and release them into the fetal bloodstream without involvement of the placenta. In experiment 1, we extracted steroids from tissue (120-130 days gestation fetal sheep) and found that both adrenal (782±470 pg/g, n=6) and placenta (392±12 pg/g, n=3) contained high concentrations of estradiol compared to concentrations in plasma (<50 pg/mL). In experiment 2, we extracted mRNA from fetal sheep adrenal glands, (80, 100, 120, and 145 days during gestation and 7 days postnatal (n=4-7/group), using q-RT-PCR to quantify expression normalized to the expression of β-actin. Aromatase (CYP19) was expressed throughout the latter half of gestation and was significantly increased in late gestation and in neonatal life (p=0.001 by ANOVA). For comparison, 17 α-hydroxylase (CYP17A) showed a highly significant increase in expression (p<0.001), while the apparent increased expression of aldosterone synthase (CYP11B2) was not statistically significant (p=0.076). Interestingly, expression of the ACTH receptor (MC2R) was not significantly changed with developmental age, but the Prolactin receptor and the LH receptor (PRLR and LHR, both expressed throughout) were significantly changed (p=0.05 and p=0.01, respectively, by ANOVA). LHR was highest at 80 days, while PRLR was highest at 120 days. Our lab attempted to examine FSH receptor expression in the adrenal as well; however, detection was unsuccessful using q-RT-PCR. Based upon our findings, we conclude that the adrenal has the capacity to synthesize estrogen. The temporal pattern and the mechanisms controlling estrogen biosynthesis by the adrenal is unknown, but could involve signaling by Prolactin or Luteinizing Hormone. Objective: Our goal was to investigate interburst interval (IBI) and burst duration (BD) as quantitative indices for fetal neurological maturation and compare them between normal pregnancies and ones at risk for adverse neurological development. Methods: Using non-invasive magnetoencephalography (MEG) the brain activity was examined. There were 23 low-risk along with 148 high-risk and 72 IUGR recordings with gestational age (GA) from 27-38 weeks. The discontinuous patterns in the brain activity were characterized by BD and IBI and plotted against GA. Further we determined the fetal states of quiet (1F) and active sleep (2F) and studied their variation with burst parameters.

Results: IBI showed a decrease with gestational age (-0.19 sec/wk, P=0.02) in low-risk fetuses; no significant change was observed in either the IUGR or the high-risk fetuses. The changes over GA were then compared between the groups. The trend in the low-risk group was significantly different from the IUGR (P=0.03) but there was no trend in the high-risk group (P=0.10). With respect to BD, no significant trend difference was detected. When fetal states were included the low-risk fetuses showed decreases in IBI with GA in the 1F state (-0.23 sec/wk, P=0.02) but not in 2F state. The high-risk and IUGR fetuses did not show any statistically significant changes. Comparing changes over GA revealed a difference between low-risk and IUGR (P=0.04) and marginal between low-risk and high-risk (P=0.06) for 1F state. No significant differences among IBI trends were noted in 2F state. No trends were observed in BD for either 1F or 2F between the groups.

The mean IBI and the length of discontinuity decreased in the lowrisk fetuses over GA (5.3s ;38 wk GA). In the case of IUGR the IBI is already as low as 5.6s at GA 27 weeks, which may point towards an early maturation/ dysmaturation. Further the high-risk group has a lower mean IBI at 27 weeks compared to low-risk but are higher than the IUGR.

The Ovine Fetal Adrenal as an Estrogen Target Tissue. Ashley Grapes, 1 Teresa Collins, 2 Charles E Wood. 2 1 Animal Sciences, University of Florida; 2 Physiology and Functional Genomics, University of Florida.

In the sheep, the adrenal cortex undergoes rapid growth in late gestation. The increased size and secretory capacity of the adrenal is an important factor in the rise in plasma cortisol concentrations that accelerates visceral development and culminates in the initiation of parturition. An important action of the cortisol that is secreted by the adrenal cortex is the induction of 17α-hydroxylase (CYP17) in the placenta, which in turn increases the secretion of both conjugated and unconjugated estrogens. Estrogens increase myometrial activity, but also increase concentrations of both ACTH and cortisol. Exogenous sulfoconjugated estrogens increase fetal plasma cortisol while decreasing POMC mRNA in the fetal pituitary as well as CRH mRNA in the fetal hypothalamus. The present study was designed to test the hypothesis that sulfoconjugated estrogens can act Background: Congenital renal disease is the leading cause of end-stage renal disease in Pediatrics. Often, this can be diagnosed prenatally and can worsen with gestational age. In-utero gene therapy may improve or prevent worsening of these conditions. However, the kidney has been difficult to target. The guiding hypothesis for this work is the rAAV9 vector can be used to target fetal kidneys by systemic administration in pregnant mice.

The rAAV9-CMV-dsGFP and rAAV9-NPHS1-eGFP vectors were created containing the cytomegalovirus (CMV) or the Nephrin (NPHS1) promoters driving the green fluorescent protein (GFP) gene. Recombinant AAV vectors were administered to 17 week-old pregnant mice at embryonic day 12.5 by tail vein injections (8.4 x 10 12 viral genomes for CMV-or 1.0 x 10 12 viral genomes for NPHS1-containing vectors). Dams were sacrificed at 8 weeks post-injection and pups at 2, 4, 6, 8, 10 and 12 weeks. Kidneys from both dams and pups were analyzed for transduction by quantitative PCR (qPCR and qRT-PCR) and expression by immunohistochemistry (IH) and immunofluorescence (IF). Results: Mice injected with the rAAV9-CMV-dsGFP vector showed high levels of GFP in the kidneys as well as the liver, brain, heart, and lungs. Kidneys from dams treated with rAAV9-CMV-dsGFP showed stable transduction of viral DNA at 8 weeks. Expression was verified by IH and IF. Similar results were obtained when the kidneys of treated pups were examined. Stable transduction of viral DNA was identified by qPCR at all time points. Staining of the kidney appeared to localize to the glomeruli and renal tubules. Kidneys from Dams and pups treated with rAAV9-NPHS1-eGFP vector showed an 18-fold and 6-fold increase in GFP mRNA, respectively, over saline-injected controls at up to 12 weeks, as well as robust IH and IF staining. Expression was limited to the kidneys.

The AAV9 vector appears to allow selective targeting of the kidney when containing the modified NPHS1 promoter. Further, this vector provides stable transfection of both maternal and fetal kidneys for up to twelve weeks post-delivery. These experiments demonstrate the first selective targeting of fetal kidneys with a gene therapy vector and provide a new method for the investigation and potential treatment of congenital renal diseases in mice. Placental insufficiency leading to fetal growth restriction (FGR) with chronic hypoxemia may result in cellular apoptosis and aberrant neuronal development in the fetal brain underlying risk for encephalopathy. We have determined the impact of fetal hypoxemia from ∼0.80 to 0.90 gestation in the ovine fetus, analogous to late onsetting FGR and previously shown to effect cardiovascular development, on measures of apoptosis, synaptogenesis and myelination in the fetal brain. Fetal sheep were chronically instrumented and either embolized (EMB group, n=12) daily between 117-134 days gestation (term 145 days) to maintain arterial O 2 sat <40%, or given saline (Control group, n=11). Animals underwent necropsy with brains perfusion-fixed, sectioned for histology through the hippocampus at the level of the entorhinal cortex, and subsequently analyzed for apoptosis indices: cleaved caspase-3 as an early indicator of cellular apoptosis and ApopTag TUNEL for DNA fragmentation. Sections were also analyzed for neuronal connectivity: synaptophysin (SYN) immunoreactivity, a synaptic vesicle protein involved in synapse formation, and luxol fast blue (LFB) staining, marking proteolipids within myelin. Mean O 2 sat measured 52±1% (SEM) for the Control group and 35±2% for the EMB group and for individual animals showed an inverse relationship to the brain:body weight ratio. TUNEL positive cells for the brain areas including cortical grey matter, periventricular white matter, germinal eminence, and hippocampal sub-areas were all extremely low averaging 0.1±0.1 cells per high power field (HPF) and with no significant differences between the EMB and Control group animals. Interestingly, cleaved caspase-3 positive cell numbers were considerably higher at 14.4±1.7 cells per HPF, but again with no significant differences between groups. SYN immunoreactivity and LFB staining per HPF for all brain areas studied were likewise unchanged between the EMB and Control groups. Chronic hypoxemia of a moderate degree, and known to disrupt growth and cardiovascular development in the ovine fetus in the latter part of pregnancy, does not appear to induce brain apoptosis or alter synaptogenesis or myelination, presumably due to the adequacy of neuroprotective mechanisms including the well known redistribution of brain blood flow at this time.

Agonist Estradiol (E2) plays a critical role in the ovine fetal hypothalamus to stimulate the hypothalamus-pituitary-adrenal axis at the end of gestation. E2 action is mediated through nuclear and membrane receptors that induce changes in gene expression. ICI 182 780 (ICI) is an estrogen receptor alpha and beta antagonist. The objective of this study was to evaluate the genomic effect of intravenous E2 or ICI in the ovine fetal hypothalamus. Chronically catheterized ovine fetuses (120-133 days gestation)were infused for 48 h with: saline (Control group), E2 500 ug/kg/day (E2 group), ICI 5ug/kg/day (ICI low) and ICI 5mg/kg/day (ICI high), then euthanized and hypothalami collected from Control (n=6) and each of the treatments groups (n=4). mRNA extracted from these samples was hybridized with a previously validated sheep 8 X 15 K array slide (Agilent 019921). Intensity results were imported and analyzed with the limma package for R. Significant differences in gene expression between each treatment group and the Control group were determined with t-statistics using the limma package. Significantly up-and down-regulated genes for each treatment group were subjected to network inference using the GeneMania(Cytoscape). The number of nodes for the resulting up-regulated/ down regulated networks was, respectively: E2: 344/242, ICI high: 388/343 and ICI low: 438/367. The resulting networks were merged to determine overlapped nodes (genes) between them. Unexpectedly, there was a strong overlap between the up-regulated genes for all treatments (153 nodes) and down-regulated genes for all treatments (85 nodes). The overlap was even more evident between the networks resulting from the E2 group and ICI high group. Very few genes overlapped between the up-regulated network from the E2 group and down-regulated network for the ICI high and low dose groups, and vice versa. Enriched biological processes (BP) for the overlapped genes for the up-regulated networks were cell development, myeloid cell differentiation, cell migration and angiogenesis. For the down-regulated overlapped genes, enriched BP were oxidative phosphorylation and mitochondrial respiratory chain. We conclude that intravenous administration of ICI in ovine fetuses for 48 hours has agonist effects in the ovine fetal hypothalamus similar to E2 administration for the same period of time.

The Exposure to dietary restriction during the periconceptional period in either normal or obese ewes results in increased adrenal growth and a greater cortisol response to stress in the offspring, but the mechanisms which program these changes are not fully understood. Activation of the IGF1R and STAT signalling pathways have been shown to stimulate adrenal growth and/or steroidogenesis.

was significantly higher in the GnRH agonist group (2.32 vs. 1, P<0.01) and epiregulin was higher in the GnRH agonist group albeit without statistical significance (1.92 vs. 1, P=0.16) . CONCLUSION Gene expression of enzymes which take part in steroidogenesis of estrogen and progesterone is already impaired at the time of oocyte retrieval in patients triggered with GnRH agonist. The decreased expression of VEGF and inhibin beta in the GnRH agonist group can explain the mechanism of early OHSS prevention in these women. The increase in amphiregulin expression in the GnRH agonist group may reflect egg quality and thus may be in correlation to the higher fertilization rate observed in the GnRH group. Background: γ-hexachlorocyclohexane (Lindane), a potential endocrinedisrupting chemical is used as an agricultural insecticide and as a pharmaceutical treatment for lice and scabies. It has been reported to interact with the pregnane X receptor (PXR). Human CYP3A4 enzyme has an essential role in hydroxylation of steroid hormones and is regulated at the molecular level by the PXR. Objective:To determine the effect of γ-hexachlorocyclohexane (γ-HCH) and other hexachlorocyclohexane (HCH) isomers on CYP3A4 transcriptional expression in the engineered human hepatoma cell line, DPX2.

Design: Laboratory-based study Materials and Methods: DPX2 cells, human hepatoma cells that are stably integrated with human PXR and a luciferase construct containing the CYP3A4 promoter, were exposed to HCH isomers at relevant concentrations in a 96 well plate. Activation of human PXR regulated gene expression was determined by luciferase activity (normalized to cell viability). All assays were measured with a BioTek Synergy2 plate reader. DMSO vehicle served as control Results: γ-hexachlorocyclohexane (Lindane) and other HCH isomers(1-100 µM) caused a significant activation of the CYP3A4 promoter, via the PXR compared to control (p<0.05)

Concentration α-HCH β--HCH γ-HCH Technical-HCH (mix of α,β,γ andδHCH isomers) 0µM Conclusions: γ-hexachlorocyclohexane (Lindane) and other HCH isomers, in relevant exposure ranges, induce the CYP3A4 expression in DPX2 hepatoma cells in vitro and may affect endocrine function by altering steroid hormone metabolism/action through a PXR mediated mechanism, in vivo

Artery Endothelial Cells. Thomas Hydrogen sulfide (H 2 S) is regarded as the third "gasotransmitter" that potently stimulate vasodilatation. Endogenous H 2 S is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We have reported that estrogen stimulates H 2 S biosynthesis in uterine artery endothelial cells (UAEC) via estrogen receptor (ER) dependent up-regulation of CBS and CSE expression. However, the specific role of ERα vs. ERβ remains unknown. Hypothesis: ERα and ERβ play different roles in regulating CBS and CSE expressions in UAEC. Methods: Primary UAEC from late pregnant (120-130d) ewes were cultured in MCDB131. UAECs were treated with 10 nM of estradiol-17β (E2) or agonists for ERα (PPT) and ERβ (DPN), or E2 with 1 µM of ICI 182, 780, or the specific antagonists for ERα (MPP) and ERβ (PHTPP) in phenol-red free M-199 with 2% charcoal-stripped serum for 48 hr. CBS and CSE mRNA and protein were assessed by real-time qPCR and immunoblotting, respectively. A novel fluorescent H 2 S probe and the methylene blue assay were used to determine H 2 S production. Luciferase reporter assays were used to determine if estrogen stimulation of CBS and CSE expressions occurs at the level of transcription. Results: Treatment with E2 stimulated CBS and CSE mRNA and protein expressions and H2S production (p<0.05), which was blocked by ICI 182, 780 and mimicked by treatment with PPT and DPN, and abrogated (p<0.05) by pretreatment with ERa or ERb antagonist, indicating both ER sub-type involvement. Using a novel fluorescent probe, H 2 S production was detected within 30 minutes post-E2 treatment and gradually increased (p<0.05) in a time-dependent fashion up to 120 min. Treatment with E2 for 24 hr stimulated a 3-fold increase in CBS and CSE luciferase reporter gene expressions, which was completely abbrogated by ICI 182, 780. Background : During human menstruation endometrial breakdown and repair occur simultaneously in adjacent areas. Breakdown is likely to be regulated during the late-secretory phase, whereas repair is instigated during menstruation. Delayed endometrial repair will lead to prolonged and heavy menstrual bleeding (HMB), a common disorder with a significant impact on quality of life. Hypotheses:(1) women with HMB have increased menstrual duration compared to women with normal loss (NMB) (2) women with HMB have significantly different menstrual phase gene expression compared to those with NMB. Methods: Women with regular cycles and no overt pathology completed a menstrual pictogram and objective alkaline-haematin measurement of their blood loss (n=44). A sub-set provided a menstrual endometrial sample; confirmed by histology and serum hormone levels (n=8). Ethical approval and informed consent was obtained from all participants. Illumina gene expression profiling was performed by the Finnish DNA Microarray Centre, Turku. R package LIMMA was used to perform rigorous statistical testing of differentially expressed transcripts. Filtering thresholds were set at P<0.01 and fold change of 1.2. Results were validated using Q-RT-PCR and statistically analysed using Mann Whitney tests. GeneGo in silico analysis was performed using MetaCore TM . Results: Women with HMB bled for 6 days on average, compared to 4 days for women with NMB (P<0.01). Microarray analysis of menstrual endometrium from women with NMB/HMB revealed 259 differentially expressed transcripts (P<0.01); 171 up-regulated in HMB and 88 down-regulated. Cluster analysis demonstrated segregation of the two groups. Many bioprocesses identified by in silico analysis were associated with repair; positive regulation of biological processes, leukocyte differentiation, regulation of apoptosis and response to stress/hypoxia. Q-RT-PCR validated the differential expression of SMAD-3, ACTG2, CXCR4, IDH1 and ESM1 between the two groups (P<0.05). Discussion: Women with objectively measured HMB have prolonged bleeding, consistent with delayed endometrial repair. To our knowledge, this is the first unbiased analysis of differently expressed transcripts in menstrual endometrium from women with NMB and HMB. We have revealed novel processes that may be involved in the pathology of heavy, prolonged menstrual bleeding. Recognition of the fetus by mother's immune system activates complement. Excess activation or reduced inhibition of complement puts pregnancy in danger. Cell surface complement regulatory proteins (CRP) DAF, CD46 and CD59 protect the self-cells by inhibiting the complement cascade. Trophoblast cells which encounter mother's immune system during early development express CRPs.

innate immune response is generated, which is regulated by the a2V with the concurrent polarization of M1 (inflammatory) and M2 (anti-inflammatory) macrophage. The objective of the present study to identify the role of a2V and its relationship in the regulation of apoptosis and the differential polarization of the innate immune response during preterm labor. Methods: CD-1 mice on day 14.5 of pregnancy underwent intrauterine injection of either saline or peptidoglycan (PGN, 0.3mg/mouse) plus poly (I:C) (1.0 mg/ mouse) to induce preterm labor. Animals were euthanized 8 hours after surgery and gestational tissues were harvested. RT-PCR and immunohistochemistry was performed for a2V, proapoptotic markers and M1/M2 macrophages markers. Results: RT-PCR and immunohistochemistry for a2V showed that its expression was significantly decreased in the placenta, uterus, and fetal membranes recovered from mice undergoing preterm labor compared to the controls. This decrease of a2V expression during preterm labor was associated with significant (p≤0.01) upregulation of the ratio of bax (proapoptotic)/bcl2 (anti-apoptotic) mRNA in both placenta and uterus. However, the expression of trophoblast development markers (Lif, Hoxa 10, Hbegf) remained unchanged in both groups. During preterm labor, macrophage polarization was skewed toward M2 (anti-inflammatory) in the uterus compared to the controls. In contrast, no specific change of M1/M2 polarization was observed in the placenta. Discussion: The results show upregulation of the bax/bcl2 mRNA ratio (i.e., a pro-apoptosis milieu) in both placenta and uterus is associated with the decreased in expression of a2V. However, the M1/M2 polarization data suggests that the M2 skewed polarization in the uterus is consistent with M2 macrophage role providing host defense against infection during the induction of preterm labor. Immune adaptation to accommodate pregnancy requires sufficient T regulatory (Treg) cells in the endometrium to prevent maternal immune rejection during the critical peri-implantation period. In women, reduced Treg cells are linked with preeclampsia and other immune-mediated pathologies of pregnancy. We have previously demonstrated that exposure to seminal fluid at coitus activates antigen presentation pathways to stimulate Treg cell proliferation and Treg recruitment into the uterus. In this study, we aimed to determine whether repeated exposure to seminal fluid can further expand the Treg cell pool. Female C57Bl/6 (B6) female mice were mated either once or four times to B6 males (syngeneic mating) or Balb/c males (allogeneic mating) over several weeks, and RU486 was administered on each day 3.5 pc to prevent progression to pregnancy. As a mating control, females were mated to seminal vesicle deficient and vasectomised (SVX/VAS) males. Uterine tissue and lymph nodes were harvested at day 3.5 pc after the final mating. Using immunohistochemistry and flow cytometry for the unique marker Treg cell transcription factor Foxp3, we found that Treg cell numbers were increased in both the uterus and the para-aortic lymph nodes after four allogeneic matings (2.2-fold and 4.1-fold higher than one mating respectively, P<0.05). Seminal fluid, as opposed to neuroendocrine or physical responses to mating were necessary for Treg cell pool expansion, since mating to SVX/VAS males failed to elicit the increase of Treg cells. However, no increase in the Treg cell population was observed after repeated mating to intact syngeneic males, indicating a necessity for male transplantation antigens in expanding the Treg cell pool. Collectively, these data provide evidence that repeated exposure to seminal fluid acts to expand the Treg cell pool in the uterus, and that active factors in seminal fluid are necessary to elicit the Treg cell response. This finding provides a mechanistic explanation linking clinical observations that longer duration of sexual cohabitation can protect from preeclampsia in women.

Pregnancy Is Associated with Greater PIBF Expression on Peripheral Blood CD8+ T-Cells In-Vitro. Nishel Shah, 1 Nesrina Imami, 2 Mark Johnson. 1 1 Surgery & Cancer, Imperial College London, London, United Kingdom; 2 Medicine, Imperial College London, London, United Kingdom. Background Progesterone induced blocking factor (PIBF) is a lymphocyte-derived immunemodulator that has been described as a mediator of the immune response in pregnancy. However its detection on lymphocytes has proven to be difficult.

This study aims to compare the expression of PIBF on γδ + T-cells from normal healthy pregnant and non-pregnant control subjects, and determine whether in-vitro stimulation with progesterone has any impact on the expression. Methods Peripheral blood mononuclear cells (PBMC) were obtained from healthy pregnant (Pr; n=9) and healthy non-pregnant controls (HC; n=4). These cells were cultured in the presence of progesterone (P4) at concentrations of 20 mcg/ml, 2 mcg/ml and 0.2 mcg/ml, as well as a control well with no added P4, for 24 hours in tissue culture medium. These cells were subsequently analysed by flow cytometry using surface markers CD3, CD8, TCR-γδ and PIBF. Appropriate isotype controls were used in each experiment. Statistical analysis was undertaken using Mann-Whitney U test for independent samples (Graphpad Prism 5.0). Statistical significance was defined as p<0.05.

The median maternal age and gestation of pregnancy was 33yrs (IQR 28. 5-36.5) and 29 weeks (IQR 24.5-36.1) respectively. The median age of HC was 29.5yrs ). In the absence of progesterone stimulation the expression of PIBF measured by MFI (mean fluorescence intensity) on CD8 + TCR-γδ + T-cells was significantly greater in pregnancy compared to controls (median 335, IQR 272-401 vs HC median 232, IQR 201-267; p=0.016). However this difference disappeared when comparing both HC and Pr following progesterone stimulation of cultured PBMC with P4 at the same concentrations. Overall the authors found culturing with progesterone had no significant impact on the expression of TCR-γδ + or TCR-γδ + PIBF + on CD4 and CD8 T-cells measured by percentage and MFI in pregnancy.

In summary the results show that pregnancy is associated with an increased expression of PIBF on CD8 + TCR-γδ + T-cells. The failure of in-vitro stimulation with P4 to increase PIBF expression on TCR-γδ + T-cells from pregnant donors may be due to the absence of paternal/fetal antigens in culture. Objective: Leukocyte infiltration in the decidua (maternal-fetal interface) before, during and after term (TL) and preterm (PTL) labor was studied in mouse. We also investigated the mechanism of peripheral leukocyte recruitment into decidua by analyzing the tissue cytokine profiles. Methods: (1) Uterine tissues were collected during mouse gestation, TL and post-partum (PP); (2) Inflammation-induced PTL was initiated on gestational day 15 (d15) by intrauterine injection of LPS (125µg); (3) Non-inflammationinduced PTL was initiated by the subcutaneous injection of RU486. Animals were euthanized during PTL or 24 hours PP. One uterine horn was used for mRNA analysis (by Real-Time PCR) and/or for protein analysis (by Luminex assay). The second horn was enzymatically dispersed for FACS analysis or used for immunohistochemistry (IHC). Markers of myeloid cell differentiation (Gr1, Ly6G, Neu 7/4, F4/80) were assessed to define tissue monocytes (M), neutrophils (N), and macrophages (Mac). Analysis of Mac and N/M infiltration (positive/total cells, %) was conducted using NewCast stereology software with systematic randomized sampling of 5% of the total decidual area. Results: Flow cytometry revealed a significant (p<0.05) increase in decidual Macs prior to TL; M and N numbers increased during TL and further increased during PP, which correlated with IHC data. Different mechanisms underlie LPS-PTL and RU486-PTL: a massive influx of N was detected in the decidua during LPS-PTL (5.1 vs 17.5%) but not during RU486-PTL (1.1 vs 2.0%); M/Macs numbers did not change during PTL. IHC analysis confirmed these findings.

The PP period appears similar in the two PTL models, with highest infiltration of M and N into decidua. Changes in decidual leukocytes were accompanied by an increase in multiple pro-inflammatory cytokines/chemokines (Il1b, Il6 and Ccl2) during TL and RU486-PTL and a robust pro-inflammatory response during LPS-PTL. PP period following TL and PTL was associated with further up-regulation of multiple cytokines/chemokines (p<0.05). Conclusions: Our data suggest a program of myeloid cells involvement in parturition with the pre-partum influx of Macs into the decidua contributing to the progression of labour, while the later influx of M and N contribute to PP decidual involution. Analysis 86 clinically relevant variables were selected. Variable reduction was undertaken in the development set by discarding any potential predictor not significantly (p<0.05) related to normal pregnancy then fitting backward stepwise logistic regression models (p<0.05). Subsequent significant key predictor variables were fitted to both validation cohorts using log-probability regression. Data analysis was conducted in Stata version 11.2.

Of the 5624 women, 3538 (63%) had an uncomplicated pregnancy. Significant potentially modifiable factors that altered the likelihood of an uncomplicated pregnancy are shown below. Significant non-modifiable factors included family history of hypertension in pregnancy, hypertension on oral contraceptive pill, any vaginal bleeding in pregnancy, increasing mean uterine artery resistance. Conclusions This study provides a novel evidence base to inform women, health care professionals and policy makers on modifiable factors that increase the likelihood of a healthy pregnancy outcome. Funding Tommy's Charity; Guy's and St Thomas' Charity

Development of a Dosing Regimen for Recombinant Human Antithrombin (rhAT) in Pregnant Patients. Joost DeJongh, 1 Johan Frieling, 2 Elizabeth Clark, 2 Simon Lowry, 2 Henk-Jan Drenth. 1 1 Consultant, LAP&P, Leiden, Netherlands; 2 Scientific Affairs, GTC Biotherapeutics, Framingham, MA, USA. Objective The thrombophilic disorder hereditary antithrombin deficiency (HD) substantially increases the risk of venous thromboembolism (VTE) during high-risk situations (ie, delivery, surgery). Replacement of antithrombin (AT) to adequate levels may prevent VTE. Population pharmacokinetic (PK) modeling was used to create rhAT dosing regimens that restore AT levels in HD patients.

From population PK modeling using data from 15 HD patients, an individualized dose regimen was derived to achieve target AT activity (80%-120% of normal) and validated in a prospective trial of 14 HD patients (9 delivery). Delivery was identified as an additional covariate, a second regimen was developed, and simulation-based therapeutic drug monitoring (TDM) with dose adjustment was derived to achieve target AT. Both algorithms were validated in an independent prospective study (18 HD patients, 12 delivery).

The rhAT regimen used in the first study involved a loading dose and subsequent continuous maintenance infusion based on weight (kg) and baseline (BL) AT activity.

Maintenance (IU/h): [(100-BL AT activity)×kg]/10. 2 The model was valid for surgery patients, but a covariate analysis indicated greater rhAT clearance and distribution in delivery patients. The model was updated to optimize rhAT dosing for delivery patients.

Simulations yielded a TDM method in which >99% of both groups achieved target AT activity within 12 hours after the initial infusion. The first sample is taken at 2 hours, followed by infusion adjustment +/-30% if AT activity is <80% or >120% of normal, respectively. Samples are then taken every 2 hours, followed by infusion adjustment until target AT activity is achieved. Thereafter, samples are drawn every 6 hours. A second external validation confirmed suitability of the model and algorithms for both groups. Population PK analysis of AT activity revealed that clearance and volume of distribution are also increased in pregnant HD patients treated with plasma-derived AT.

Population PK modeling and external validation revealed PK differences between delivery and surgery HD patients that are important for AT replacement. Distinct dosing algorithms with TDM were developed that restored target AT activity in both groups.

Mapping Adverse Pregnancy Outcomes: The Obesity Belt as a Predictor of Perinatal Indicators in the United States. Kacey Y Eichelberger, 1 Robert A Strauss, 1 Veronica Escamlla. 2 1 Department of Obstetrics & Gynecology, University of North Carolina, Chapel Hill, Chapel Hill, NC, USA; 2 Carolina Population Center, University of North Carolina, Chapel Hill, Chapel Hill, NC, USA. Objective: To determine the relationship between mapped obesity prevalence in the United States (the "obesity belt") and the prevalence of seven adverse perinatal indicators. Study Design: Using ArcGIS, we mapped the most recently reported state-level prevalence data from the National Vital Statistics System for obesity (BMI > 30), as well as the following: cesarean delivery, perinatal mortality, fetal mortality, teen pregnancy, low birth weight, very low birth weight, and preterm birth. We used geographically weighted regression to measure the spatial variation of the relationship between obesity and each perinatal indicator. A local regression model was measured for each state accounting for the effect of neighboring states using an inverse distance squared weighting mechanism. Results: The mapped prevalence of two of the seven perinatal indicators, as well as the geographically weighted regression modeling, is presented in Figure 1 .

Coefficients are interpreted as the average expected increase in adverse outcome for one unit increase in obesity. The red states in Figure A .1 have coefficients ranging from 0.36 to 0.47. This suggests that for each additional obese person per 100, there is an increase between 0.36 and 0.47 of teen pregnancies per 100. A significant relationship between obesity rates and adverse outcomes for individual states is demonstrated using asterisks of varying sizes to represent p < 0.05, p < 0.01, and p < 0.001. The obesity belt was most significantly associated with teen pregnancy and preterm birth, though demonstrated significant predictive value for all perinatal outcomes. Conclusion: Mapped prevalence of obesity in the United States is significantly associated with mapped prevalence of adverse perinatal outcomes. B (10/12) 47.8 days (p=0.25). In Gp B 5/12 [42%] cases were transferred to other facilities for further care compared to 1/11 [9%] case in Gp A (p = 0.15). The mortality was 6 /11 in Gp A vs. 2/12 in Gp B (OR: 6, 95% CI, Conclusions: In the later decade NIH cases were diagnosed and delivered earlier. Although mortality was lower, it was not significantly different from earlier decade.This might suggest that advances in perinatal care has not significantly impacted NIH outcome.

A Comparative Study of Early Multifetal Pregnancy Reduction from Triplets to Twins Versus Triplets to Singeltons Performed at 7-8 Weeks of Gestation. Jigal Haas, 1 Jehoshua Dor, 1 Ariel Hourvitz, 1 Yoav Yinon, 1 Adrian Shulman. 2 1 Department of Obstetrics and Gynecology, Chaim Sheba Medical Centre, Ramat Gan, Israel; 2 Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba, Israel. Objective: To compare the perinatal outcome of early transvaginal multifetal pregnancy reduction (MPR) from triplets to twins vs triplets to singletons.

A prospective cohort study of 60 trichorionic triamniotic triplet pregnancies who underwent early transvaginal MPR at 7-8 weeks of gestation. The choice to reduce to twins or singleton was according to patient decision. Group 1 included 44 women who underwent MPR to twins and group 2 included 16 women, who underwent MPR to singletons. The rates of early miscarriage, pregnancy loss before 24 weeks, preterm delivery prior to 32 weeks and 37 weeks of gestation, gestational diabetes, hypertension and IUGR were compared between the 2 groups.

Altogether 2 pregnancies were lost before viability. In general, the outcome of triplet pregnancies reduced to twins was comparable to the outcome of triplet pregnancies reduced to singletons. The rates of pregnancy loss prior to 24 weeks of gestation, preterm delivery prior to 32 weeks and preterm delivery prior to 37 weeks were similar among both groups (4.55 vs 0, 2% vs 0 and 47% vs 37.5% respectively). Similarly, there was no significant difference in the incidence of gestational diabetes (13% vs 6%), hypertension (18% vs 18%) and IUGR (2% vs 0) between the 2 groups.

The outcome of triplet pregnancies reduced to twins did not differ from that of triplets reduced to singeltons. This information should be taken into account when counseling couples with triplet pregnancies, who consider early transvaginal MPR.

A Retrospective Study on the Timing of Delivery and Outcome in Women with Placenta Previa. Hirotaka Hamada, Masatoshi Saitoh, Hidekazu Nishigori, Junichi Sugawara, Nobuo Yaegashi. Department of OBGY, Tohoku University Hospital, Sendai, Miyagi, Japan. Objective: In women with placenta previa, it has been recommended that delivery should occur at 37 weeks' gestation in order to reduce the risk of perinatal mortality. There are few studies, however, that have assessed optimal gestational age at delivery from a wider clinical outcome perspective. The aim of this study was to predict the optimal gestational age at delivery by characterizing maternal clinical episodes and clinical outcomes. Methods: Referring to clinical records, we retrospectively studied the clinical outcomes of 116 patients with placenta previa who delivered from 2006 to 2011 in our hospital. Mean Maternal age 32.9±4.9, mean gestational age 34.6±4.3weaks,and mean birthweight 2224±735g. There were 57 patients with total placenta previa(Group A), and 59 with partial or marginal placenta previa(Group B). Results: In both group A and B, the rate of emergent c-section and patients who had hemorrhage before operation was significantly higher in the group who had warning bleeding (Group A 87% VS 52%, 59% VS 40% p<0.05, Group B 64% VS 19%, 50% VS 11% p<0.05). 80% of Group A and 70% of Group B required an emergent c-section within 2 months from the first bleeding. In group B, the cervical length was significantly shorter in the warning bleeding group (28.7mm VS 37.4mm p<0.05), while Group A had no significant difference (30.0mm VS 30.8mm). Conclusion: We conclude that i)in patients with total plcenta previa, warning bleeding is predictive of a heightened risk of hemorrhage, suggesting a need for preterm cesarean section within 2 months from the first warning bleeding; and ii) Identification of a reduced cervical length may be a predictor of preterm labor in placenta previa patients.

Placental Pathology Analysis in Severe IUGR Pregnancies Fails To Reveal a Distinct Phenotype When Uterine Artery Doppler Studies Are Normal: Implications for Clinical Practice. Mary Higgins, 1 Melissa Walker, 1 Catherine Windrim, 1 Sarah Keating, 2 John Kingdom. 1 1 Maternal Fetal Medicine, Mount Sinai Hospital, Toronto, ON, Canada; 2 Pathology, Mount Sinai Hospital, Toronto, ON, Canada. Screening programs to identify pregnancies at risk of severe early onset growth restriction (sIUGR) generally combine uterine artery Doppler with maternal blood tests and clinical characteristics. Given the diverse range of placental pathology associated with sIUGR (Walker M, Placenta 2012) we tested the hypothesis that a significant proportion of sIUGR pregnancies have normal uterine artery Doppler and a discrete placental pathology phenotype that is distinct from utero-placental vascular insufficiency. We retrospectively identified 122 singleton pregnancies with sIUGR (estimated fetal weight <10th centile for gestation and gender and absent/reversed end diastolic flow in the umbilical arteries prior to delivery) that had uterine artery Doppler (UtAD) studies, delivery at Mount Sinai Hospital and placental pathology performed in the period 2004-2011. Outcomes were as follows; IUFD (41%), severe preeclampsia (45%), gestational age at delivery (median 28 weeks [range 19-36 weeks]) CS delivery (50%). 34/122 (28%) cases had normal UtAD (defined as mean pulsatility index <1.45). Placenta <10th centile for gestation was significantly more common in the abnormal UtAD group (62/88 vs 18/34, p=0.04) as was placental infarction (54/88 vs 13/34, p=0.01) and accelerated villous maturation (44/88 vs 5/34; p<0.01). Primary defective formation of placental villi (distal villous hypoplasia) was equally common in both groups. Our findings illustrate a major limitation of designing a sIUGR screening program around proximal uterine artery Doppler. The lack of a specific placental phenotype for sIUGR with normal uterine artery Doppler that could block maternal-fetal transfer suggests either that maternal blood flow dies not limit growth, or that important functional defects in syncytiotrophoblast function, mediated by processes such as hypoxia-reoxygenation injury, are not readily identifiable with conventional light microscopy.

Allostatic Load in Women Reporting Low Birth Weight in the National Health and Nutrition Examination Survey (NHANES). Vanessa J Hux, 1,2 Janet M Catov, 2,3 James M Roberts. 2, 3, 4 1 School of Medicine, Vanderbilt University, Nashville, TN, USA; Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA; 3 Epidemiology, University of Pittsburgh; 4 Clinical and Translational Research Institute, University of Pittsburgh. Background: Allostatic load (AL), a measure of cumulative biologic risk and chronic stress, is associated with adverse health outcomes. Few studies have examined AL and reproductive outcomes. We hypothesized that women with a history of low birth weight (LBW) deliveries with or without preterm birth have higher AL as a marker of chronic stress that likely preceded pregnancy. Methods: We calculated AL in non-pregnant, women ages 17-35 who responded to the reproductive health questionnaire (RHQ) in NHANES, 1999 NHANES, -2006 . Cutpoints for individual components of AL were calculated as above or below the 25th or 75th percentile. AL was calculated as a composite score of 0-9 with one point assigned for each of 9 components: SBP, DBP, BMI, HbA1c, cholesterol, HDL, albumin, creatinine clearance, and CRP. Women reported any LBW deliveries and indicated either term or preterm. We classified preterm-LBW and term-LBW as surrogates for PTB and SGA. A weighted analysis was performed to account for the complex survey design of NHANES. We utilized linear and logistic regression, calculating both unadjusted and adjusted values (age, race) for AL and odds ratios. Results: We identified 878 women with weighted percentages of SGA, PTB, and normal groups that were 2.1%, 10.4%, and 87.5%, respectively. Women in the SGA group had higher AL than those with normal weight outcomes (Adjusted means: 2.98 +/ -0.38 vs. 1.86+/-0.23; p = 0.004). There was no difference in AL in the PTB group compared to those with normal weight infants (Adjusted mean: 2.14 +/-0.23, p = 0.228). Women in the top quartile for AL had increased odds of SGA compared to those in the lower 3 quartiles (Adjusted OR: 3.48, 95% CI: 1.36-8.92) . Conclusion: In a representative United States population, allostatic load determined in non-pregnant women is associated with higher odds of SGA in prior a pregnancy that persists after controlling for age and race. Using AL as a proxy for pre-pregnancy health, this suggests a role for chronic stress and subclinical multisystem dysregulation in adverse birth outcomes.

Systematic Follow-Up of Pregnancies after Bariatric Surgery: A Prospective Controlled Study. Goele Jans, 1 Annelies Mattheus, 2 Sander Galjaard, 3 Annick Bogaerts, 4 Greet Vansant, 5 Roland Devlieger. 3 

An increasing number of women with a history of bariatric surgery are getting pregnant. The aim of this study is to evaluate the effects of intensive nutritional monitoring on maternal and neonatal outcomes in women with a history of bariatric surgery. METHODS A prospective controlled study was conducted. Pregnant women (N=49) with a history of bariatric surgery were prospectively enrolled during the first trimester and received intensive monitoring of dietary habits including dosage and correction of major nutrients (Fe, Folate, Mg, Vit A, D, E, K). A contemporary control group (N=50) was identified from the electronic data records from the U.Z. Leuven. Maternal and neonatal outcomes, micronutrient levels, and nutritional status (7 day records) were compared.

Baseline characteristics were comparable for both groups with the exception of mode of conception (IVF 10% in intervention group, 0% in controls (p=0.028). We found significant less hypertension (p=0.027) and induction of labour (p<0.001) in the intervention group compared to the controls. Low birth weight (< 2500 g) was significantly less frequent in the intervention group (4% vs 19%; p=0.023). Mean (SD) head circumference were larger in this group [34.5 (1.2) vs 33.6 (2.0) cm, p=0.018]. Less neonates showed meconial amniotic fluid (0% vs 11%; p = 0.025) resulting in less admissions to the neonatal intensive care unit (2% vs 21%; p=0.003). The intake of folic acid was higher in the control group during the first (30 % vs 8 %, p = 0.006), second (34 % vs 2 %, p < 0.0001) and third trimester (34 % vs 2 %, p < 0.001). In the 2 nd and 3 th trimester, more Fe-supplements were taken in the intervention group (TII: 52% vs 30%, p=0.026, T3: 52% vs 30%, p=0.026). No significant differences in nutritional deficiencies were observed. CONCLUSION A systematic and intensive clinical follow-up with nutritional monitoring results in improved pregnancy outcomes after maternal bariatric surgery. 

Recently several neonatal cases with severe intracranial bleeding have been reported, all possibly related to maternal vitamin K1 deficiency during pregnancy following bariatric surgery. The aim of this study is to investigate the effect of intensive follow-up and vitamin K1 supplementation on vitamin K1 blood serum levels in pregnant women after bariatric surgery. METHODS A multicenter prospective cohort study including 49 pregnant women with a history of bariatric surgery (malabsorptive or restrictive procedure) was conducted. Nutritional deficiencies were prospectively screened. In case of an observed vitamin K1 deficiency, a prescription for vitamin K1 supplement was given. Data on serum blood levels of vitamin K1, blood coagulation indicators and supplement intake were collected during all trimesters.

The New Management of Pregnancy of Women with Congenital Afibrinogenemia -The Fibrinogen Replacement Therapy with Evaluating Fibrinogen Clearance. Kotomi Nagahashi, Keiko Muramatsu, Kaori Yamazaki, Toshiyuki Uchida, Kazunao Suzuki, Kazuhiro Sugihara, Hiroaki Ito, Naohiro Kanayama. Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu City, Shizuoka-ken, Japan. Fibrinogen is a factor associated with the final stage of coagulation cascade and essential to hemostat. It is also an adhesion molecule important in maintenance of pregnancy. Without fibrinogen replacement therapy, pregnancy with afibrinogenemia always results in spontaneous abortion after 5 weeks. Congenital afibrinogenemia was first reported by Rabe & Solomen (1920) . It is an autosomal recessive disorder affecting boys and girls in equal proportions. We reported the first case of congenital afibrinogenaemia with elective caesarean delivery (Inamoto & Terao, 1985) and since then we experienced 5 cases and 7deliveries of the disease. The fibrinogen level which should be maintained during pregnancy was reported based on the experience (Kobayashi et al, 2001) . Furthermore, here we report another delivery of congenital afibrinogenemia with the new management of pregnancy. A woman experienced the third delivery, and we evaluate the fibrinogen clearance to determine the replacement amount of fibrinogen about recent two deliveries. Successfully, the woman delivered fine infants without abortion, abruption of placenta or genital bleeding. We suggest the fibrinogen replacement therapy of congenital afibrinogenemia with evaluating fibrinogen clearance is effective in maintenance of pregnancy. Objective: The maternal mortality ratio remains low in the United States. However, maternal morbidities remain constant. Recently, the study of near misses has become a more useful tool in efforts to reduce maternal morbidities. This study sought to determine the role of multidisciplinary obstetrical rapid response teams in the management of near miss conditions. Study Design: Two obstetrical rapid response teams were developed. One team is dedicated to manage obstetrical emergencies such as shoulder dystocia, eclampsia, placental abruption, and the second team was developed to manage obstetrical hemorrhage. Data was collected prospectively from 2009 -2011 on the location of call initiation, type of obstetrical emergency that prompted the call and need for transfer of the patient to an higher level institution. Results: From 2009 obstetrical rapid response calls were called. In 2009, 5/8 calls originated in Labor and Delivery (LD) and 3/8 calls originated in the Emergency room (ED). 5/8 calls were for obstetrical emergencies(OE) and 3 were for hemorrhage (H). In 2010, 3/10 calls originated in LD, 6/10 originated in the ED and 1/10 originated on the post partum ward (PP). 7/10 were for OE and 3/10 were for H. In 2011, 0/4 calls originated in LD, 3/4 calls originated in ED and 1/4 calls originated on PP. Of these 4 calls, 3 were for OE and 1 was for H 1 patient was transferred to a higher level institution in 2009. There were no transfers in 2010 and 2011. Conclusions: Our study suggests that the development of a multidisciplinary obstetrical rapid response team helps to reduce maternal morbidity in near miss situations by enhancing the skills of OB 1st responders. In a time when the overall delivery number increased in our institution by 10% (3000 to 3300), the number of obstetrical rapid response calls hospital wide decreased by 50%, the number of calls originating in LD dropped significantly (5 to 0, p < 0.5) and no patients required transfer to a higher acuity institution. We hypothesize that increased awareness of the entire hospital staff, particularly LD staff, regarding recognition and management of obstetrical emergencies through simulation drills and didactic sessions along with practiced ongoing dialogue with sub specialists (such as MFM, surgery, Intensivist) were responsible for our findings.

Does an Intense Prenatal Care Management Program Improve Perinatal Outcomes in a High Risk Population? Lin Wang, 1 Objective: To investigate the effect of an intense prenatal case management program, Partner's with Moms (PWM), on perinatal outcomes in a high risk obstetrical population. This program links each patient to a case manager who is present at each clinic visit. Medication, transportation and social work assistance along with nutrition and smoking cessation counseling are integral parts of the program. Study Design: This is a retrospective, case control study, from July 1, 2008 -June 30, 2010. All women were enrolled in a Medicaid MCO, called Priority Partners (PP), at least 56 days prior to delivery. The intervention group included women who also enrolled in an intense case management program, PWM, at least 56 days prior to delivery, and continued PWM enrollment until 30 days prior to the delivery. The control group included those women who were referred to PWM but did not enrolled in PWM or had enrolled in but dropped out of PWM > 30 days prior to delivery. Perinatal outcomes assessed were gestational age at delivery, birth weight and admission to the NICU. Data were abstracted from administrative claims and case management records. Results: There were 672 patients in the intervention group and 427 patients in the control group. There were no differences in maternal age or perinatal deaths between intervention group vs control group (26.4 yrs (6.4) vs 25.4 yrs (6.0); 3 vs 0, respectively). There were no differences in term (>37 weeks) birth rate, birthweight > 2500 g or NICU admissions between the intervention vs control groups (79% vs 83%; 82% vs 86%; and 17% vs 15%; respectively). However there was a lower rate of early preterm (< 34 weeks) birth, very low birthweight (<1500 g), and NICU stay >15 days, than those in the comparison group (23% vs 34%, p =.06;17% vs 24%, p = 0.05; 33% vs 41%, p = .2 respectively). Conclusions: Our study suggests that an intense prenatal case management program tends to improve the rates of early prematurity, very low birthweight and prolonged NICU stays. These findings highlight that such an intense program may reduce the burden of prematurity and fetal growth restriction that is often associated with high risk maternal conditions. Moreover, the data suggests that such a program may reduce the amount of NICU care that is needed in this population. LGA cust was defined as birth weight (BW) greater the 90 th customized centile. Analysis: Predictors were identified using stepwise multinomial logistic regression and area under the ROC curve (AUC) was calculated from the reduced model. Results: The incidence of LGA cust was 8.8%. In multivariate analysis clinical risk and protective factors at 14-16 and 20 weeks' gestation are listed in table1. Addition of fetal ultrasound variables improved prediction. Screening positive for GDM increased risk but did not improve prediction. 1.0 (0.9 -1.0) Head circumference (HC) (+1cm) 1.1 (1.0 -1.2) Waist hip ratio (+ 0.1)* 0.6 (0.4 -0.9) Random glucose (+1 mmol/L) 1.2 (1.1 -1.4) 0.63 20 weeks fetal scan measurements HC < 10th centile 0.3 (0.1 -0.7) Abdominal circumference (AC) ≥ 75th centile 1.6 (1.1 -2.2) AC ≥ 90th centile 2.7 (1.9 -3.8) 0.72 GDM screened +ve 1.6 (0.9 -3.1) 0.72 *There is an interaction between waist & hip, greater effect of hip measurement Conclusion: We have identified early and mid pregnancy factors which are associated with modest prediction of infants LGA by customised centiles who are at risk of adverse birth outcomes. This increased growth is also evident from 20 weeks. Funding: Tommy's Charity, National Institute of Health Research with shoulder dystocia were induced, compared to 1 out 7 in the non-diabetic group. Risk factor analysis for shoulder dystocia suggests caution with induction of labor in diabetic pregnant patients.

Labor Induction in Women with an Unfavorable Cervix: Randomized Controlled Trial of Double Balloon Catheter Versus Dinoprostone. Katarzyna Suffecool, Barak Rosenn, Janelle Forutan, Kimberly Herrera. OB/ GYN, Roosevelt Hospital, New York, NY, USA. Objective: There is little consensus on the best available method for cervical ripening and induction of labor.Studies of various balloon catheters have shown more effective dilation of the cervix with shorter induction-to-delivery time while studies of vaginal prostaglandins have shown that they increase the likelihood of delivery within 24 hours, but do not reduce the rate of cesarean delivery. The purpose of this study was to test the hypothesis that using a double balloon cervical catheter (DB) for induction of labor in nulliparous women will shorten the time to delivery compared to 10mg Dinoprostone vaginal insert, Cervidil. (C) Methods: Nulliparous patients admitted for induction of labor at ≥37 weeks with singleton gestations in vertex presentation and intact membranes and a Bishop score <6 were randomized to receive C or DB for cervical ripening. C was left in place for 12 hours (unless removal was indicated) followed by oxytocin infusion. Patients in the DB group had the catheter inserted and left in place for 12 hours (unless spontaneously expelled) and oxytocin infusion was started 6 hours after balloon insertion. Primary outcome was time from insertion to delivery. Secondary outcomes included vaginal delivery rate within 24 hours, cesarean delivery rate, time to active labor, and occurrence of adverse events. A sample size of 26 women per group was required to detect a 25% difference between the groups in mean time from induction to delivery, with β=0.8 and α=0.05. Statistical analyses were performed by intention to treat using Chi-square, Fisher's exact, and Student's t-test, as appropriate. Results: The study included 62 women (31 in each group). Patients' characteristics, indications for induction, gestational age, birth weights, and rates of macrosomia did not differ significantly between the two groups. Primary and secondary outcomes are presented in the Although postnatal glucocorticoid (GC) therapy has well established beneficial effects on pulmonary function, it may also result in growth restriction during treatment. The course of early childhood growth is believed to predict cardiovascular and metabolic diseases in adulthood. Therefore, we determined the effects of postnatal dexamethasone (DEX) or hydrocortisone (HC) treatment on patterns of postnatal growth until approximately 5 years of age.

In an observational cohort study of children born prematurely (<32 weeks of gestation) between 1993-1996, we compared growth patterns for body weight, height and head circumference of children who received DEX (boys n=26, girls n=13), HC (boys n=27, girls n=26) to a reference group that had not received postnatal GCs (boys n=50, girls n=47) using the five parameter Reed-2 model. Results DEX-treated boys showed a significantly different growth pattern for body weight (Fig. 1) , height, and head circumference (all p<0.01) compared to the reference group. DEX-treated girls also showed a significantly different growth pattern for body weight and height (both p<0.0001), but not for head circumference. HC-treatment significantly changed the growth pattern for height of boys and girls (both p<0.01), and also the body weight growth pattern for boys (p<0.05; Fig. 1 ). After HC-treatment, no changes were found in the growth pattern of head circumference for boys and girls.

Conclusion Postnatal DEX-and HC-treatment, with a dosing commonly used in the nineties, significantly changed body weight and height growth patterns of prematurely born boys between birth and approximately 5 years of age. The same effects were seen in girls, although their weight growth pattern was not different compared to the reference group after HC treatment. The observations may have impact on health in later life for those individuals treated with GCs in the neonatal period.

Neonatal Mortality after Early Elective Repeat Cesarean Delivery. Gustavo Vilchez, 1 Anushka Chelliah, 1 Pedro Argoti, 2 Ray Bahado-Singh. 1 

These data indicate that we need to reassess the implications of pyuria ≤10 wbc µl -1 and its occurrence may be an important marker of significant disease which should not be dismissed. Background: The use of synthetic polypropylene meshes to improve anatomical outcomes in the surgical repair of pelvic organ prolapsed has been limited by significant mesh complications. It is unclear how mesh characteristics affect the acute phase immune response and tissue turnover. The aim of this study is to compare matrix -2 and -9 activity between a medium weight nonabsorbable (Gynemesh, Ethicon), a partially absorbable lightweight (Gynecare Prolift+M, Ehticon) and an ultralight-weight (Smartmesh, Coloplast) synthetic mesh in vivo. MMP-2 and -9 are type IV collagenases which play a role in extracellular matrix remodeling and the inflammatory response. Gynemesh is a polypropylene mesh with a weight of 44g/m 2 and pore size of 2440µm. In comparison Gynecare Prolift+M is a composite of polypropylene plus poliglecaprone with a post-absorption weight of 31g/m 2 and pore size of 4000µm and Smartmesh has a weight of 19g/m 2 and a pore size of 2370µm. Methods: C57/Black6J mice underwent sham surgery, or placement of 1of 3 synthetic meshes onto the abdominal wall after full thickness injury and repair, with and without ovariectomy. The animals were sacrificed 26-weeks postoperatively and the abdominal wall complexes (ASC) harvested. ASC were mechanically disrupted in high-salt buffer. Protein extracts were run on 10% gelatin zymography, and stained with coumassie blue. Bands were quantified by UN-SCAN-IT (Silk Scientific) and compared to internal loading controls.

Statistical analysis was performed with by Mann-Whitney test.

Results: There were no differences in expression of any form of MMP-2 or -9 between sham and control animals, or with oophorectomy status within groups. No differences were seen in Pro-MMP-2 in any group, while Pro-MMP-9 was different from sham in both Gynemesh and Smartmesh, but not Gynecare Prolift + M. Both active MMP-9 and MMP-2 were significantly different from sham across all three mesh types. Activities were lowest with Smartmesh and increased with mesh weight. Conclusion: The decrease in mesh weight of Smartmesh resulted in a lower level of expression of active MMP-2 and -9 which may ultimately lead to differences in tissue response to synthetic mesh implantation. Further studies are needed to characterize the specific cellular response to these materials and how they impact resolution or prolongation of the acute phase response over time. Objective: Abnormalities of connective tissue structure may predispose women to Pelvic Organ Prolapse (POP). We reported earlier that the expression of proteins involved in elastin and collagen metabolism (i.e. LOX, and MMP1, 2, 7, 9, 12, 14 , ADAMTS2/PNP and PCP/BMP1) are altered in vaginal tissue of premenopausal women with severe POP vs asymptomatic controls. We hypothesized that vaginal fibroblasts (VFs) derived from patients with POP display altered functional characteristics as compared to VFs derived from asymptomatic women. We aim to characterize primary VFs and study the effect of continuous static mechanical stretch on VFs expression of enzymes regulating extracellular matrix (ECM) proteins assembly, synthesis and degradation. Methods: Premenopausal and postmenopausal Caucasian women undergoing total hysterectomy for benign conditions were recruited as controls while women with advanced POP (POP-Q stage ≥3) were recruited as patients. VFs were isolated by enzymatic digestion of vaginal biopsies, seeded at a density of 250,000 cells/well on 6-well collagen I (COL-1)-coated or pronectin (PN)coated bioflex® plates and subjected to static mechanical loading at 25% elongation for up to 48 hours. Cell attachment and proliferation rate were studied; total mRNA was analyzed by real-time PCR and stretch-conditioned culture media were collected for zymography. Results: Our preliminary results indicate (1) similar attachment of VFs from POP and controls to different ECM substrates.

(2) Control VFs show a higher proliferation rate than POP VFs when plated on both COL1 and PN. (3) LOX, LOXL1,2 mRNA were expressed by non-stretched VFs; LOXL1 gene expression was significantly decreased in POP VFs vs asymptomatic controls seeded on PN (p<0.05). (4) MMP2 and MMP14 mRNA expression was similar in non-stretched VFs and display a trend of induction after stretch for 24 and 48 hours in both patients and control groups on COL-1 and PN substrates. Conclusion: POP VFs display differential responses to ECM depending on substrate and mechanical loading. These data may provide a potential mechanism by which risk factors that induce vaginal stretch contribute to altered pelvic ECM composition in POP patients.

Retraction Technique for Urinary Catheterization of Women with Female Genital Mutilation. Abdulrahim Rouzi, Nora Sahly, Nedaa Bahkali, Hassan Abduljabbar. Obstetrics and Gynecology, King Abdulaziz University. Objectives: To evaluate the retraction technique for urinary cauterization of women with Type III female genital mutilation (FGM).

The hospital records of all women from Sudan, Somalia, Ethiopia, Egypt, Eritrea, and Chad who were admitted King Abdulaziz University Hospital, Jeddah, Saudi Arabia were reviewed. Women with Type III FGM who had urinary catheterization were identified and their records were examined. Results: During the study period, one hundred and sixty two women with Type III FGM had urinary catheterization by residents in our hospital. One hundred and twelve (69.1%) women had urinary catheterization by the standard procedure and 50 (30.9%) women had urinary catheterization by the retraction technique because of failure of the standard procedure. No attempts to use the technique were unsuccessful; that is, no procedures were converted to emergency defibulation. No complications occurred during insertion or while the catheter was in place (37.5 ± 5.6 hours).

The retraction technique provides a safe and effective option for urinary catheterization of women with Type III FGM.

Ertapenem in the Treatment of Overactive Bladder. Sheela Swamy, Kiren Gill, Anthony Kupelian, James Malone-Lee. Division of Medicine, UCL, London, United Kingdom.

The recent description of undetected urinary inflammatory signals in women with overactive bladder (OAB) opens a new avenue for research. The original 1957 Kass criteria for diagnosing urinary tract infections (UTI) using MSU culture, were based on patients with acute pyelonephritis (1) . The urinary leucocyte esterase and nitrite tests were calibrated to the Kass criteria and have been found to be extremely inaccurate (2) .However, intracellular bacterial colonisation has been demonstrated to lie at the core of chronic UTI implying that conventional treatments may not be adequate and single daily prophylactic treatment might be counterproductive. There is a growing interest in missed UTI in the aetiology of OAB. Methods:

We studied 27 women with recalcitrant OAB symptoms and pyuria (≥10 wbc µl-1(sup)). They were MSU culture negative. They failed to respond to antimuscarinics and oral urinary antibiotic regimes. Despite negative cultures, they were treated with intravenous Ertapenum 1g daily for 5 days. Ertapenem achieves good urothelial cell penetration, the probability of resistance was low and Extended-Spectrum Beta-Lactamases (ESBL) microbes were unlikely to survive. Following treatment, patients were placed on full dose oral urinary antibiotic therapy and reviewed two weekly. Results: 27 women (mean age =57 years sd=16) were studied through treatment. This small sample is not suitable for a statistical inference, but the observations are notable. Voiding symptoms were noted to fall in the immediate aftermath of treatment compared to OAB & pain. Urine microscopy showed a reduction first in uroepithelial cell shedding, followed by a fall in pyuria.

bought about an effective retrieval method for two antibodies requiring heatinduced epitope retrieval, cytokeratin and Protein gene product 9.5 without altering morphology.

This protocol allows for the continuation of previous three-dimensional reconstructive work illustrating the endometrial microvasculature [2] [3] [4] [5] . Previous studies have indicated that blood vessels and nerve fibres course throughout the body in an orderly pattern, often alongside one another 6 . The presence of these nerve fibres in women with pain symptoms strongly suggests that in women with endometriosis, the eutopic endometrium is involved in the generation of pain symptoms.

for the CD126 and GP130 receptors. These results provide additional surface antigen markers to both improve endometrial CSC isolation and to allow their specific targeting towards the development of novel therapeutic strategies. Introduction: Malignant transformation of endometriosis is a rare event. We report an extremely rare case of colonic wall mullerian carcinoma arising from foci of endometriosis where gene profiling assisted with diagnosis. Pubmed search of the English medical literature from the late 1940's through 2012 revealed that this is the first case with this clinical presentation. Case Presentation: 63yo G0 post menopausal female with a history of breast and stage Ia endometrial endometrioid cancer FIGO 1, Nuclear grade 2 presented for evaluation of a pelvic mass detected on a CT of abdomen and pelvis. At the time of exploratory laparotomy, a large mass was incorporated to the recto sigmoid wall Recto sigmoid resection with endcolostomy was performed. Frozen section revealed poorly differentiated carcinoma. Final pathology confirms a few nests of poorly differentiated malignant epithelial tumor within necrotic tissue. Final pathology favored its mullerian origin. This reported to be unrelated to the patient's previous endometrial cancer. Gene profiling analysis revealed the presence of TOP2A, TOPO1, ERCC1, MGMT and Her2/Neu. These results concluded a mullerian origin from foci of endometriosis in the colonic wall. Patient did not respond to Adriamycin and presented with tumor progression. Conclusion: Endometriosis of colonic wall can progress to malignancy. These factors have been proposed as the nidus for malignant transformation of endometriosis as well as TP53 mutations, and mutation of ARID1A. A greater understanding of the molecular profiling may lead to better diagnosis and treatment of this rare clinical presentation.

Poster Session: Inflammation, Infection (Saturday, 3/23/2013, 8:00 AM -10:00 AM) Scientific Abstracts Reproductive Sciences

299A maternal BMI, women with excessive GWG carrying female infants were significantly more likely to develop diabetes (aOR 1.70, 95% confidence interval [CI] 1.46, 1.99) and have fetal macrosomia

Pregnancy Alters Urinary Excretion of Myo-Inositol and D-Chiro-Inositol

Insulin resistance in non-pregnant subjects involves a dysfunction of insulin receptor transduction. Insulin resistance also entails changes in a putative alternative insulin-signaling pathway involving inositol phosphoglycan second messengers

This study was designed to test the hypotheses 1) that pregnancy alters MI and DCI excretion, and 2) that these alterations are enhanced in gestational diabetes (GDM)

These were matched with 61 normal gravidas (NG) by age, ethnicity, and pre-pregnancy body mass index. Urine MI and DCI levels were measured by gas chromatography / mass spectrometry

During pregnancy mean log urinary MI/Cr and DCI/Cr ratios in NG were significantly greater than PP (Fig. 1A, B). Additionally, inositol ratios in 2T were significantly greater than in 1T. Compared to NG, mean log MI/Cr ratios were significantly higher in GDM in 1T. Conclusion. Pregnancy increases excretion of MI and DCI, suggesting substantial alterations in the alternative insulin-signaling pathway

AM) Scientific Abstracts Reproductive Sciences

Poster Session: Renal, Adrenal, Brain (Saturday, 3/23/2013

MDRP 1 distribution: There is heavy staining in proximal tubules and light staining in distal tubules with almost no positive staining in collecting tubules, glomerulus or medulla. Immunoreactivity expressed as fraction (area immunostained ÷ area of the field x 100%) or density (arbitrary units) in kidney of fetal baboons from control mothers fed ad lib (CTR; n = 7,A) or IUGR(fed 70% CTR diet

Down-Regulated Leptin Receptor (ObRb) Peptide Expression in the Fetal Hypothalamic Paraventricular Nucleus (PVN) and Adrenal Cortex Explains the Observed Hypothalamo-Pituitary-Adrenal Axis (HPAA) Up-Regulation in Fetal Baboon Intrauterine Growth Restriction (IUGR)

250) showed leptin inhibition of the HPAA in a fasting mouse model. In the present study we hypothesized that IUGR down-regulation of ObRb peptide expression in fetal PVN and adrenal gland in comparison to ad lib fed controls (CTR) at 0.9 gestation (G) could explain our observed fetal HPAA up-regulation in IUGR baboons

= 10) or fed 70% CTR global diet (IUGR, n = 6) from 0.16 to 0.9 G with fetuses taken at c-section. ACTH and cortisol were measured by immunoassay and ObRb peptide expression determined by immunohistochemistry (IHC) with image analysis for % area immunostained (Fraction). Statistical analyses was via Student's t-test with α level set at 0.05 and data presented as mean ± SEM

05) in the fetal circulation, while ObRb peptide expression by IHC was decreased (p< 0.05) in PVN and adrenal cortex of IUGR fetuses vs. CTR (Fig. 1). Conclusion: The observed significant decreases with IUGR in ObRb peptide expression at the top and bottom of the final common pathway for cortisol secretion provide a mechanism, i.e., via decreased leptin signaling in comparison to CTR

Poster Session: Risk Factors Outcomes, Mortality (Saturday, 3/23/2013

Poster Session: Risk Factors Outcomes, Mortality (Saturday, 3/23/2013

Figure 1: Rate of adverse outcome per state (A -teen pregnancy, B -preterm birth). Local regression coefficients and significance for each adverse outcome by state

Model To Predict Spina Bifida Risk

This is a common debilitating birth defect with a prevalence of 0.5-10 per 1000 births worldwide. Pre-conception supplementation with folic acid clearly decreases the population risk of having a child with an NTD, although not entirely. One possible explanation for the remaining risk is that some women carry variants in folate/one-carbon pathway genes that impact their ability to metabolize folate. Objective: To describe a novel spina bifida risk model that was developed via examination of the full spectrum of variation within genes of the folate pathway. Methods: We first undertook a case-control study of 949 women reporting a spina bifida live birth and 1,166 controls. A total of 824 single nucleotide polymorphisms (SNPs) in 37 genes in the folate-homocysteine pathway were evaluated. Using the Lasso and cross-validation

Results: Within these "discovery" data, the model can significantly identify women at 6-fold risk increased for spina bifida affected pregnancy (95% CI: 240 SB case moms, and 3,900 controls and will also present these findings. Discussion: The described model would provide an important advance in

Prenatal Diagnosis and Neonatal Outcomes in Non Immune Hydrops: A Comparison of Two Decades at an Academic Center

1 AnnMarie Prabulos, 1 Naveed Hussain

3 patients received digoxin antenatally in Gp A vs none in Gp B. The GA at delivery, BW, MD, ApS and CpH were similar. The etiology was cardiac (30%), idiopathic (30%), genetic (20%), infectious (10%), hematological (10%) in Gp A vs. idiopathic (50%), genetic (25%), cardiac (16.7%), hematological (8.7%) in Gp B. The average NICU stay for cases discharged home in Gp A (5/11) was 26.2 days and in Gp Poster Session: Risk Factors Outcomes

Poster Session: Urogynecology/ Pelvic Floor (Saturday, 3/23/2013

Objective: Hydroxy-beta methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors, statins, have been proposed to decrease the risk of developing endometrial, prostate, and breast cancers. The role of statins in survival from endometrial cancer is unknown. The goal of our study was to evaluate the association between hyperlipidemia/statin use and overall survival in women with endometrial cancer. Method: A retrospective cohort study was performed at a single institution of all patients with a diagnosis of uterine cancer from

Kaplan Meier survival analysis and Cox proportional hazards models were used. Results: Of 987 patients with endometrial cancer

Reactive Oxygen Species (ROS) Play a Key Role in the Induction of Labor-Related Features. T Hadi, 1 M Bardou, 1,2 M Barrichon, 1 P Mourtialon, 3 M Wendremaire, 1,2 M Dumas, 1 P Sagot, 3 F Lirussi. 1,2 1 U866, INSERM; INSERM; 3 Obstetrics & Gynecology, CHU, Dijon. INTRODUCTION: Preterm labor physiopathology remains to be elucidated, but the process is known to involve uterine remodeling, with apoptosis induction (Caspase-3), Metalloproteinases induction (MMPs), angiogenesis (via VEGF) and prostaglandins synthesis (via COX-2). Inflammation, driven by a massive monocyte influx, has been identified as a key feature of labor onset, and evidences have highlighted a potential NFκB-driven effect. It is well established that inflammation is associated with ROS production, and cross talks between NFκB and NADPH oxidase (NOX, Producing ROS) pathways have been described. Thus, the aim of this study was to assess a potential role for ROS production in labor onset. METHODS: Myometrial biopsies obtained from women undergoing C-section, before labor onset, were cut into small strips and stimulated 48h with proinflammatory LPS (10µg/ml), pro-oxidant hydrogen peroxide (H2O2, 100µM), in presence or absence of NFκB or NOX respective inhibitors, Curcumin and DPI, or anti-oxidants Gluthatione and γ-tocopherol (100µM each). Primary myometrial cell (MC) lines and Th-P1-differentiated macrophages (Th-P1) were also stimulated. RESULTS: LPS induced a 3.5fold increase in ROS production assessed by DHE staining, an induction restricted to macrophages (CD115 positive cells). A 3fold increase in NFκB phosphorylation, an increase in MMP2, MMP9, VEGF and Cox-2 expression and Caspase-3 cleavage (x3, x10, x8.6, x4.5 and x3 vs. CTL respectively) were also observed by western blotting. Interestingly, while Background: MicroRNAs (miRNAs) are small non-coding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. Our objective was to identify differentially expressed miRNAs associated with acute fetal lung injury in utero following choriodecidual infection. Methods: Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term=172 days) received either choriodecidual inoculation of: 1) Group B Streptococcus 1 x 10 6 colony forming units (n=5) or 2) saline (n=5). Cesarean section and fetal necropsy was performed 4-7 days after infection to collect tissues. RNA was extracted from fetal lungs and profiled by mRNA and miRNA microarrays. Results were analyzed by multiple software platforms including Ingenuity Pathway Analysis (IPA) and validated by mRNA array and qRT-PCR. Results: Choriodecidual infection was limited with labor and chorioamnionitis occurring in only 2 of 5 GBS cases. Amniotic fluid (AF) culture and PCR were negative in all cases, but AF cytokines were significantly elevated in the GBS group and correlated with fetal lung injury. We identified 9 differentially expressed miRNAs (p<0.05, >1.5 fold change) in the fetal lung after choriodecidual infection; in GBS animals, 3 were significantly upregulated (miR-155, let-7i*, miR-769-3p) and 6 downregulated (miR-29c, miR-371-5p, miR-489, miR-514b-5p, miR-326, miR-4324), with 56 significant mRNA targets having inverse correlations with these miRNAs. Inflammatory, apoptosis, and angiogenesis miRNA-gene signaling networks predicted by IPA were matched to actual mRNA gene array expression (p<0.05, >1.5 Acknowledgements: We are very grateful to Barbara Sanborn for the kind gift of the PHM1 cell line.

Metformin Versus Insulin in Gestational Diabetes: A Meta-Analysis. Aneesha Varrey, 1 Joanne E Brady, 2 Heather Hume, 1 Shari E Gelber. 1 Introduction: Gestational diabetes (GDM) is an important complication of pregnancy, affecting 2-10 % of pregnant women. Insulin has been the gold standard for the treatment of GDM. However, the use of oral hypoglycemics has been increasing due to their low cost and ease of administration. Metformin (MET) increases insulin sensitivity. Due to this effect, the use of MET has also been evaluated in GDM. Objective: To determine maternal and neonatal outcomes of pregnancies treated with metformin versus insulin for GDM. The outcomes were cesarean section (CS), large for gestational age (LGA), neonatal hypoglycemia, gestational hypertension, pre-eclampsia and hyperbilirubinemia. Methods: We systematically searched PubMed, Medline, Embase, Cochrane, International trials registry, pharmaceutical industry registries using the search terms "metformin" and "gestational diabetes". We included RCT and casecontrolled studies on patients with GDM being treated with metformin or insulin that measured more than one of the outcomes. The data was abstracted by two authors and was evaluated for the presence of bias, study confounders and number of patients. We examined heterogeneity among studies and calculated a summary effect estimate for each outcome. Results: 206 relevant references were identified. From these, 8 eligible studies were selected -2 randomized trials and 6 case-control studies. Among women who received metformin, we found a decreased incidence of hyperbilirubinemia, (odds ratio (OR) 0.68, 95% confidence interval (CI) 0.48-0.96), LGA (OR 0.76, 95% CI 0.61-0.95) and neonatal hypoglycemia (OR = 0.48, 95%CI 0.35 -0.67). There was no statistically significant difference in gestational hypertension (OR:1.12, 95% CI 0.79-1.58) pre-eclampsia (OR:0.82 95% CI 0.57-1.17), or CS (OR = 0.96 95% CI 0.78-1.55). No significant heterogeneity existed between studies for the above outcomes. Conclusions: This meta-analysis evaluating the use of metformin compared to insulin for GDM demonstrated a decreased incidence of hyperbilirubinemia,LGA and neonatal hypoglycemia. Further evaluation of metformin for treatment of gestational diabetes is warranted.

Zambrano, 1 Carlos Ibanez, 1 Luis A Reyes-Castro, 1 Claudia Vega-Garcia, 1 Peter W Nathanielsz. 2 1 Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, DF, Mexico; 2 Center for Pregnancy and Newborn Research, University of Texas Health Sciences Center, San Antonio, TX, USA. We have shown increased adiposity index, leptin, and triglycerides (TG) in male OFF of MO mothers at postnatal day (PND) 150 (1). Since maternal low protein diets accelerate aging in rat OFF (2), we hypothesized that MO results in a more rapid aging of some or all these lipid indices. We determined lipid metabolism at PND 110 (early adults), 450 (mature adults) and 600 (early aging). METHODS: We fed female Wistar rats from weaning through pregnancy and lactation on chow (C) or high energy obesogenic diet (MO). Mothers were bred at PND 120 and diet maintained until OFF weaning on to C. All OFF ate chow from weaning. Male OFF (different litters) were euthanized at PND 110 (n=8), 450 (n=6) and 600 (n=6) by decapitation, and fat depots weighed, body weight (BW), serum leptin, TG and cholesterol measured. Statistics: two-way anova (Maternal diet and OFF age); p< 0.05. RESULTS: Body weight was higher in MO than C at 450 and 600 days and at all ages for fat, leptin and TG cholesterol was not different at 110 and 450 PND. By 110d there was already aging-accelerated change in fat, leptin and TG in MO OFF. 

Iwa Antonow-Schlorke, 1 Michael Brodhun, 2 Alexandra Helgert, 1 Peter W Nathanielsz, 3 Matthias Schwab. 1 

Co-stimulation of various TLRs and NOD2 results in enhancement of the inflammatory response in this in vitro model. These results suggest synergistic activity between different types of pattern-recognition receptors. Future work in this area will delineate the mechanism of synergy, and translational studies can help define the role of this interaction in parturition.

Myometrium. Natasha Singh, Kaiyu Lei, Suren R Sooranna, Mark R Johnson.Obstetrics & Gynaecology Department, ICL, Chelsea & Westminster Hospital, London, United Kingdom. Introduction: Myometrial contractions of labour signal the normal physiological end-point of pregnancy but the biochemical onset of labour may occur at or before term via a series of changes in expression of labourrelated genes. Expression of prostaglandin H synthase (PGHS-2), CXCL-8 and oxytocin receptor (OTR) have previously been used as markers of labour. Our aim was to determine how these genes change when a person enters labour. Methods: Samples of lower segment myometrium were taken from pregnant women undergoing LSCS either before or after the onset of labour. Term labour was divided into 2 groups with up to 2cm of cervical dilation being early labour (EAL), and 3cm or more classified as established labour (ESL). Samples were rapidly frozen at -70 o C for extraction of RNA (n=19 for TNL; n=19 for EAL and n=24 for ESL) and converted to cDNA. Copy numbers of GAPDH, PGHS-2, CXCL-8 and OTR were measured by quantitative realtime PCR using a Rotor-Gene TM (Corbett Research, Australia) . The results are presented as mean ± SEM and as a ratio of GAPDH. Protein was extracted from samples for western blotting for PGHS-2 and OTR. Results: Significant increases were seen in PGHS-2 (0.77 ± 0.14) and OTR (15.19 ± 2.38 ) expression when compared with TNL samples (0.26 ± 0.06 and 8.6 ± 1.61 respectively; p<0.05 in each case). These genes were not significantly increased in ESL samples (0.87 ± 0.54 and 14.61 ± 2.78 respectively). However, CXCL-8 was significantly increased only in ESL (TNL, 0.38 ± 0.09; ESL, 56.16 ± 46.01, p<0.05). Conclusions: These data show that PGHS-2 and OTR are increased in early labour whereas CXCL-8 is increased only in established labour. This study highlights the differences in gene expression at different stages of term labour.

Development of a Clinically Relevant Model of Perinatal Hypoxic Ischaemic Encephalopathy in Mice. Xia Zheng, 1, 2 Hailin Zhao, 2 Suren R Sooranna, 1 Daqing Ma, 2 Mark R Johnson. 1 1 Academic Obstetrics & Gynaecology, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom; 2 Anesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom. Objective: Hypoxic ischaemic encephalopathy (HIE) is one of the most common causes of neonatal death and long-term neurological handicap. Current management is suboptimal and novel therapeutic approaches are urgently needed. We have established a clinically relevant model of perinatal HIE in mice. Methods: On gestational day 18, pregnant mice were sacrificed by cervical dislocation. The pups from one uterine horn were delivered immediately (caesarean section [CS] ). The vessels of the second uterine horn were clamped and the uterine horn placed in a 0.9% sodium chloride water bath at 37 °C for either 10 or 15 min, after which, pups were rapidly delivered. All pups with or without hypoxia were transferred into an incubator at 37 °C for 1 hour whilst they received manual stimulation to to maintain spontaneous respiration. The pups were transferred to surrogate mothers who had given birth naturally. Some pups from the surrogate mothers were used as naïve controls. Pups were sacrificed on postnatal day (PND) 1, 3 and 7 and their brains analysed by immunohistochemistry and western blotting (n = 6 /group). Results: The survival rates at 24 hours after delivery for the naive, CS, 10 min HIE and 15 min HIE group were 100% (75 of 75), 100% (86 of 86), 96.9% (31 of 32) and 84.2% (32 of 38) respectively. Immunohistochemical and western blotting analysis revealed an increased activation of the inflammatory transcription factor, NFκB and of a marker of apoptosis, cleaved caspase-3, in both of the HIE groups up to PND 7 when compared to both control groups. Conclusion: We have established a clinically relevant model of perinatal HIE in mice. This model could be used to test novel therapeutic strategies for their ability to prevent and/or improve the outcome after HIE in humans.

Hydroxychloroquine Prevents Antiphospholipid Antibody-Induced Inhibition of Trophoblast Migration. Caroline R Albert, 1 Objective: Women with antiphospholipid syndrome (APS) are at high risk of recurrent pregnancy loss and late pregnancy complications. Antiphospholipid antibodies (aPL) target the trophoblast by binding beta2-Glycoprotein I (β2GPI) and alter human first trimester trophoblast function by: triggering a pro-inflammatory cytokine response; modulating angiogenic factor secretion; and inhibiting cell migration. Current management of obstetric APS is treatment with heparin, alone or with aspirin. While this may decrease the risk of miscarriage in APS patients, their risk of preeclampsia remains high. APS patients, especially those with lupus, are often treated with the anti-malarial drug, hydroxychloroquine (HCQ). While it is safe to use during pregnancy, little is know about its effects on aPL-associated adverse pregnancy outcomes. Therefore, the objective of this study was to determine the effects of HCQ on aPL-modulation of human trophoblast function. Methods: The human first trimester trophoblast cell line, HTR8, was treated with or without aPL (mouse IgG1 anti-human β2GPI mAb, 20µg/ml) in the presence and absence of HCQ (1µg/ml) for 48-72 hrs. Cell supernatants were analyzed for: pro-inflammatory cytokines IL-8 and IL-1β; pro-angiogenic factors VEGF and PlGF; and anti-angiogenic factors sFlt-1 and sEndoglin by ELISA. Cell migration was measured using a colorimetric two-chamber assay. Results: HCQ had no effect on basal trophoblast cytokine or angiogenic factor (VEGF, PlGF, sEndoglin, sFlt-1) secretion, nor did it have any effect basal trophoblast migration. The aPL-induced upregulation of trophoblast IL-8, IL-1β, PlGF, sEndoglin and VEGF (p<0.05) were not significantly altered by the presence of HCQ. However, the aPL-inhibition of trophoblast migration (p<0.001) was significantly reversed by the presence of HCQ (p<0.001). Conclusion: These findings demonstrate that HCQ is able to reverse the adverse effects of aPL on human first trimester trophoblast migration. Our data indicate the possibility that addition of HCQ during the period of trophoblast implantation and invasion may be beneficial in reversing the adverse effects of aPL in pregnancy. This work was funded in part by the Lupus Foundation of America and the March of Dimes. 3 Program in Reproductive and Adult Endocrinology, NICHD, NIH, DHHS, Bethesda, MD, USA. Objective: Trophoblast cells from the placenta appear in the cervical canal early in the first trimester and can be collected through noninvasive transcervical sampling (TCS). It is unclear whether these fetal cells originate from the villous syncytiotrophoblast, invasive extravillous trophoblasts or a population of Center, San Antonio, TX, USA. Brain development follows a species specific trajectory. The sheep is a major model to study functional and structural aspects of brain development. Objective; To translate to human development, we compared myelination in humans and sheep. Methods: Brain tissue specimens were taken from regular autopsies from five human fetuses at 26, 27 (n = 2), 34, 38 weeks gestation (wG) corresponding to 0.67, 0.7, 0.87 and 0.97 gestation, and from two infants at 4, 18 postnatal months (pM) that did not show any sign of clinical or neurological pathology. Sheep brains were taken from 47 fetuses at 0. 27, 0.40, 0.53, 0.63, 0.75, 0.87, 0.93 gestation (G, term 150 days, n = 4-12) . After formalin fixation, paraffin slices were stained against myelin basic protein (MBP), a marker for mature oligodendrocytes which produce myelin and cerebral myelin itself. Results: Humans: At 26 and 27 wG, scattered oligodendrocytes appeared in the intermediate zone (immature white matter) and cortical plate (immature cerebral cortex). At 34 and 38 wG, myelinated fibers were found in the deep white matter, and start to appear in the subcortical white matter. Myelination was advanced at 18 pM. Sheep: From 0.63 G, oligodendrocytes firstly appeared in the deep and subcortical white matter. At 0.75 G myelinated fibers firstly were found. From 0.87 G, dense myelinated fiber bundles occurred in all white matter tracts. Conclusions: Myelination is accelerated in sheep compared to humans. In terms of myelination of the subcortical and deep white matter, 34-38 wG, i.e. 0.87-0.97 G in humans compare to 0.75 G in sheep. Supported by the European Community FP7 HEALTH, Project 279281 (BRAINAGE).

Middle Cerebral Artery Doppler Ultrasonography in Fetuses with Congenital CNS Abnormalities and Prediction of Perinatal Death and VP Shunt Placement. Brian Brocato, Mauro Schenone, Giancarlo Mari. Obstetrics and Gynecology, University of TN Health Sciences Center, Memphis, TN, USA. OBJECTIVE: The objective of this study was to assess fetal middle cerebral artery (MCA) Doppler in fetuses with congenital central nervous system (CNS) abnormalities to determine whether it can predict perinatal demise or placement of a ventriculoperitoneal (VP) shunt. STUDY DESIGN: Pregnant women seen at our Fetal Center whose fetuses received the diagnosis of a congenital CNS anomaly from 2009 to 2012 were included in this study. The middle cerebral artery peak systolic velocity (PSV) was determined during the fetal ultrasound; data were expressed as normal or abnormal based on our reference ranges for these indices. We compared the PSV to determine whether this parameter could predict fetal or neonatal demise, and/or placement of ventriculoperitoneal shunt. The Fisher-exact test or chi-square when indicated was used for statistical analysis. A p < .05 indicated statistical significance. RESULTS:Twenty-seven fetuses were identified with CNS anomalies and MCA Doppler studies during the study period. Gestational age (GA) at the time of study ranged between 21 and 38 weeks (median: 31 weeks), GA at delivery ranged between 23 and 40 weeks (median: 36). Thirteen fetuses had either VP shunt placement (n=3) or they died in the perinatal period (n =10). In this group, 4/13 fetuses had an abnormal PSV, whereas none of the fetuses that survived or did not have a VP shunt had an abnormal PSV (p=.04). CONCLUSION: Our data indicate that middle cerebral artery peak systolic velocity may be a useful parameter in the prediction of perinatal death and/or VP shunt placement in fetuses with CNS abnormalities. This information can be used to counsel patients of expected outcomes, prepare neonatologist of the severity of the disease and ultimately delivery better care to the mother and affected neonate.

Fetal Inflammation-Induced IL-1β Is Not Essential for Fetal Brain Injury. Wance Firdaus, 1 Talaibek Borbiev, 1 Shorouq AlRebh, 1 Tahani Dada, 1 Michael Johnston, 2 Roger Reeves, 3 Irina Burd. 1 1 Gyn/OB and Neurology, Johns Hopkins School of Medicine; 2 Neuroscience, Kennedy Krieger Institute; 3 Transgenic Core, Johns Hopkins School of Medicine. Introduction: Exposure to intrauterine inflammation is known to lead to long-term neurodevelopmental disabilities in offspring. We have previously shown, in a mouse model of intrauterine inflammation, that there is a significant IL-1β upregulation in fetal brain which is accompanied by neuronal cortical injury. Maternally administered IL-1β antagonist appears to prevent fetal and perinatal cortical brain injury. The aim of this study was to investigate whether LPS-induced elevated level of IL-1β in fetal compartment is essential for fetal brain injury. Methods: IL-1β-deficient mice (B6.129P2-P2rx7 tm1Gab /J, Jackson laboratories) were mated and embryos were transferred to CD-1 (IL-1β-competent) pseudopregnant mice. A mouse model of intrauterine inflammation was utilized. Dams were randomized to either intrauterine LPS (50 ug) or normal saline (NS) infusions (n= 5 dams/treatment group). Fetal brains, maternal serum, placentas and amniotic fluid were harvested 6 h after infusions. Primary cortical neuronal cultures were created. Enzyme-linked immunosorbent assay (ELISA) for IL-1β was performed on all harvested maternal and fetal tissues. In primary neuronal culture, neurotoxicity was evaluated with immunocytochemistry for MAP2 and NF200. Results: Fetal neurons from LPS-exposed group exhibited statistically less dendritic processes than the ones from NS-exposed group at DIV 3 (p<0.05). Neuronal cell morphology appeared to be aberrant in LPS-treated group. IL-1β ELISA showed a statistically significant increase in IL-1β concentration in maternal serum, placentas and amniotic fluid of LPS-exposed group as compared to NS-exposed group (p<0.05 for all) but not in fetal brains. Conclusion: Fetal IL-1β production in response to inflammatory stimulus does not appear to be essential for fetal brain injury. Maternal IL-1β appears to cross placenta, as it is increased in amniotic fluid of IL-1β-deficient fetuses exposed to intrauterine inflammation. Furthermore, maternal inflammationinduced IL-1β upregulation appears to be associated with fetal neuronal injury in IL-1β-deficient fetuses. While further research is needed, this line of research could provide better understanding of therapeutic strategy needed for acute fetal brain injury in the setting of intrauterine inflammation. (Supported by NIH K08HD073315(IB)) at the fetal adrenal. Pregnant sheep were sacrificed on gestational age 80 (n=4), 100 (n=3), 120 (n=4), 130 (n=4), 145 (n=8), and 7 days postnatal (n=5). mRNA was extracted from whole adrenal glands and used for synthesis of cDNA. Expression of the following genes were measured using qRT-PCR: estrogen receptor alpha and beta (ERα, ERβ) and the deconjugating and conjugating enzymes steroid sulfatase (STS) and estrogen sulfotransferase (STF). The fetal adrenal glands expressed all of these genes throughout the latter half of gestation. ERα increased at 120 days, then decreased in late gestation and in newborn lambs (p=0.005). ERβ was expressed, but at a lower level than ERα and not significantly changing as a function of developmental age (p=0.26). STF expression was increased dramatically in late gestation (p<0.0001). STS was expressed, but was not significantly changed (p=0.198). We conclude that the fetal adrenal gland is a target tissue for estrogen in the late gestation fetal sheep. We speculate that estrogen might accelerate the development of the adrenal gland, and therefore play a supportive role in the development that promotes readiness for birth. (Nijland M J Physiol 588.8 (2010) . Cortisol plays a central role in maturation of many tissues in late fetal life in preparation for independent postnatal existence. Cortisol's impact on the developing kidney differs from other fetal tissues since high cortisol impairs MR function. Cortisol's effects are due to both circulating and local cortisol generation by 11-β HSD1 and 2. Renal MR function requires that local cortisol are lower than those required by the glucocorticoid receptor (GR). Local cortisol is kept low by conversion to cortisone by 11-βHSD-2. Cortisol increases11-βHSD-1 so we hypothesized 1) 11-βHSD1 and 2 present in the fetal baboon kidney at 0.9G and 2) the 11-βHSD-1:2 ratio would change in favor of 11-βHSD-1 in IUGR accompanied by high fetal cortisol. METHODS: Adult female baboons were fed ad lib (CTR) or 70% CTR global diet (IUGR) from 0.16 -0.9 G with fetuses obtained at CS under general anesthesia. Renal cortical 11-β-HSD1/2 protein was determined by immunohistochemistry and quantified by image analysis (ImageJ, NIH) for fraction (area immunostained ÷ area of field x 100%). Analysis was by Student's t-test; data M ± SEM, *p< 0.05.

Fig 1 shows distribution of 11-βHSD1/2.(CTR:A,D and IUGR:B,E) and that 11-βHSD 1(A,B,C) increases and 11 βHSD2(D,E,F) decreases in the IUGR fetal kidney. CONCLUSIONS: 11-βHSD1 is increased and 11-βHSD2 is decreased in the renal cortex in IUGR fetal baboons at 0.9G. Combined with higher circulating fetal cortisol, these changes would expose the fetal kidney to higher levels of cortisol then normal for the current stage of maturation potentially triggering a cycle of feed-forward induction of 11-βHSD1 with increased risk of adult renal disease and later life hypertension (HD 21350). Introduction: MDRP 1 is present in tissues that perform specialized barrier functions e.g. blood-brain, blood-testes and blood-placental barriers (Marchi, BMC Medicine 2004) . One role of placental MDRP 1 in rodents is to protect the developing fetus from maternally derived glucocorticoid. We have shown increased serum cortisol in IUGR baboon fetuses (Nijland M J Physiol 588.8 .2010) . Because of the need to maintain low renal cortisol concentrations to regulate mineralocorticoid receptor (MR) function, we hypothesized that IUGR fetal renal MDRP1 expression increases to protect against high fetal plasma cortisol.. Methods: From 0.16 to 0.9 G pregnant baboons were fed as ad lib controls (CTR; n = 7) or fed 70% of CTR diet to produce IUGR (n = 6) with tissue collection at CS at 0.9 G. Fetal kidney cortical MDRP1 protein was quantified immunohistochemically for fraction and density [arbitrary density units (DU)]. Statistical comparisons with rank sum test with alpha l set at 0.05. Results: Fetal renal cortical MDR1 expression increased in IUGR (Fig 1) .

In this study we aimed to investigate the effects of periconceptional dietary restriction on the IGF1R and its downstream signalling molecules and STAT isoforms in the adrenal cortex of offspring. Donor ewes were assigned to one of 4 nutritional treatment groups preconception: CC, 100% MER for 5 months; CR, 100%MER for 4 months followed by 70% MER for 1 month; HH, ad libitum for 5 months; HR, ad libitum for 4 months followed by 70%MER for 1 month. At 6-7 d postconception, single embryos were transferred into normal weight recipient ewes. Post mortem was conducted and adrenal glands collected from lambs at 4 months. There was a decrease in adrenal IGF1R and Akt protein in the CR, HH and HR groups when compared with the CC group. The adrenal p-Akt protein was also lower in the CR and HR groups. The adrenal mTOR protein was lower in the CR, HH and HR groups and this effect occurred in female lambs only. There was no change of the protein abundance of adrenal p-mTOR, RPS6, p-RPS6, 4EBP1 or p-4EBP1 in all of the treatment groups. Interestingly, adrenal STAT1 protein was higher in the CR female lambs, while adrenal p-STAT1 was higher in both CR female and male lambs. Adrenal p-STAT3 was also higher in the CR and HR groups and this occurred in female but not male lambs. These results demonstrate that maternal dietary restriction during the periconceptional period in normal or obese ewes resulted in an upregulation of adrenal STAT signaling pathway, which may contribute to increased adrenal growth and stress responsiveness in postnatal life. The decreased protein abundance of adrenal IGF1R and Akt might be the changes in consequences following a response to the increased adrenal growth in the dietary restricted groups.

Microchimerism from an Older Sibling? Hilary S Gammill, 1 Christine Luu, 2 Kimberly K Ma, 1 Vijayakrishna Gadi, 3 Anne M Stevens, 4 J Lee Nelson. 5 1 Obstetrics and Gynecology, University of Washington; 2 Clinical Research Division, Fred Hutchinson Cancer Research Center; 3 Division of Medical Oncology, University of Washington; 4 Division of Pediatric Rheumatology, Seattle Childrens; 5 Division of Rheumatology, University of Washington. Background: Microchimerism (Mc) refers to harboring a small amount of foreign cells or DNA by an individual and naturally occurs primarily from maternal-fetal exchange during pregnancy. Hypothetical alternative sources for Mc include a vanished twin, maternal transfer of cells acquired from a previous pregnancy (including miscarriage, abortion, or delivery of an older sibling), or unrecognized genetic mosaicism. A recent study found evidence supporting the possibility of older-sibling-derived Mc. We sought to further explore and quantify sibling Mc by measuring male Mc in the mononuclear cell compartment of umbilical cord blood from female neonates with and without an older brother. Methods: From a population of healthy pregnant women, umbilical cord blood was collected after delivery by sterile venipuncture from a double-clamped cord segment. DNA was extracted from Ficoll-purified mononuclear cells. Male Mc was quantified in females with a Q-PCR assay targeting a Y-chromosome sequence (DYS14) and compared based on the presence or absence of an older brother. Prevalence and concentration of Mc was measured using logistic regression (with adjustment for total number of genome equivalents tested) and the Wilcoxon rank-sum test, respectively. Results: Cord blood samples from 40 female neonates were tested for male Mc. Overall, 9/40, 22 .5% subjects had detectable male Mc. Detection of male Mc occurred at a statistically similar rate among subjects with and without an older brother (1/12, 8.3% vs. 8/20, 40% respectively, p=0.1) . The concentration of male Mc also did not differ between groups. Conclusions: Our data demonstrate occasional detection of male Mc in cord blood. Overall detection and concentration of male Mc was similar in female neonates with an older brother than those without an older brother. While these findings do not refute the possibility of acquisition of Mc from an older sibling, other pathways for acquisition of male Mc, including prior miscarriage, abortion, vanished twin, or unrecognized mosaicism may be of similar or greater relevance.

Notch Pathway Promotes and Controls Uterine Decidual Angiogenesis during Early Pregnancy in Mouse. Carmen M Garcia-Pascual, 1 Ralph C Zimmermann, 2 Hortensia Ferrero, 1 Carrie J Shawber, 2 Carlos Simon, 1 Antonio Pellicer, 1 Raul Gomez. 1 1 Pediatrics, Obstetrics and Gynecology, Fundación IVI/INCLIVA, Paterna, Valencia, Spain; 2 Obstetrics and Gynecology, Columbia University, New York, NY, USA. Decidua sustains the early phases of pregnancy by creating an extensive vascular network. VEGF/VEGFR2 is known to orchestrate decidual angiogenesis with several other factors being required to finely tune such a complex process. We recently confirmed the presence of Notch components in the decidua. Objective: Because Dll4/N1 pathway has been described to prevent vessel oversprouting by interacting with the VEGF/VEGFR2 system in ovary and retina, we wondered whether it might play a role in regulating decidual angiogenesis as well.Mat & Methods:Functional studies were performed by administering a single i.p 10µg/g dose of Dll4 blocking antibody(Genentech) (treatment group N=20) or unspecific Ig (control group N=8) to 6-8 week old female CD1 pregnant mice on ED 3.5. Mice were sacrificed on ED5. 5, 7.5, 9.5, 11.5, 13.5 . Number of implantation sites and embryos, average embryo weight and pregnancy loss rate were determined at all time points. Uterine decidual vascularization, Dll4, N1 and J1 expression, cellular proliferation and apoptosis were evaluated on ED7.5 and ED9.5. Results: Decidual blood vessels expressed N1 and its ligands Dll4, J1, mostly overlapping with vasculature (PECAM+). Pregnancy loss was observed in the treatment group with 30% of females showing no embryos at different ED assayed. We observed reduced embryo growth in the Dll4 ab (0.15+0.04g) treated vs control (0.30+0.07g average weight per embryo, p<0.01) when assessed on ED11.5 in mice in whom pregnancy had not been terminated. Paradoxically treated deciduas assessed on ED7.5 presented increased vascular density (11.14+3.1%) and Dll4 expression (8.65+3.5%) when respectively compared (2.53+1.2%,p<0.01) and (3.25+1.5%,p<0.01) to controls. Treated deciduas at this time point also showed higher percentage of proliferating (71.8+0.9% vs control 50.14+1.2%,p<0.01) and apoptotic cells (200+21 vs control 36 +21cells/mm2,p<0.01). Pattern especially visible surrounding the trophoblast. Conclusions: Blocking Dll4 in peri-implantation period causes Dll4 overexpression, which leads to increased, but likely non-functional vascularization and associated apoptosis. Our data suggest that Dll4/N1 pathway plays a role by preventing VEGF/VEGFR2 mediated oversprouting and chaotic vessel growth during decidual angiogenesis.

GnRH Agonist vs. hCG for Triggering of Ovulation -Differential Effects on Gene Expression in Human Granulosa Cells. Jigal Haas, Libby Ophir, Eran Barzilay, Gil M Yerushalmi, Yuval Yung, Ettie Maman, Ariel Hourvitz. Human Reproduction Lab and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Centre, Tel-Hashomer, Ramat Gan, Israel. Introduction GnRH analog triggering for final oocytes maturation and ovulation is an emerging more physiological approach, preventing completely the risk of OHSS but inducing luteolysis. It remains unclear how and when GnRH agonist triggering affects the steroidogenesis. Therefore, the aim of this study was to investigate the mRNA expression of genes related to steroidogenesis and OHSS in granulosa cells of patients triggered with GnRH agonist compared to patients triggered with hCG. METHODS Twelve women were treated with GnRH agonist for triggering of ovulation and twelve women were treated with hCG. Mural granulosa cells (GCs) were obtained at the time of oocyte retrieval for IVF procedures and different gene expression was analyzed using quantitative real time PCR.

The number of oocytes retrieved was higher in the GnRH agonist group (13.75 vs. 11.72) but with no statistical significance. The fertilization rate was higher in the GnRH agonist group (70% vs. 54%) with borderline statistical significance (P=0.07). The mRNA expression of aromatase (0.50 vs. 1, p<0.01), CYP 11 (0.6 vs. 1, P=0.02) and 3-beta-hydroxysteroidehydrogenase (0.39 vs. 1, P=0.03) was significantly lower in the GnRH group compared to the hCG group. The expression of STAR was similar in the two groups. The expression of VEGF (0.74 vs. 1, P=0.05) and inhibin beta (0.38 vs. 1 p=0.01) was significantly lower in the GnRH analog triggered group. FSH receptor expression was also significantly lower in the GnRH agonist group (0.4 vs. 1, P<0.01). LH receptor mRNA expression was similar in the two groups. Amphiregulin expression Hypothesis: Excess nitric oxide (NO) causes trophoblast injury in placenta by down-regulating CRPs, leading to inadequate placentation. Methods: Human decidual tissues were obtained from elective and spontaneous abortions of gestational age 7-12 weeks with patients consent. Exclusion criteria were fever, maternal infections and known chromosomal abnormalities. Expression of CRPs was assessed by RT-qPCR and immunofluorescence. First trimester Trophoblast cell line, HTR-8/sv.neo were exposed to exogenous or endogenous NO using NO donor DTNO or NO synthase (NOS) substrate L-arginine respectively and the expression of CRPs was assessed by RT-qPCR. Effect of NO on HTR cell lysis by complement was evaluated by estimating cell death using a calcein release assay. Anti HLA-G antibody and normal human serum (NHS) were used to trigger classical pathway and cell lysis respectively. Results: Expression of DAF and CD46 mRNA was 3 and 3.5-flod lower in decidua from spontaneous (n=06) compared to elective abortions (n=11). Immunofluorescence revealed predominant expression of DAF on anchoring extravillous trophoblast cells attached to decidua. DTNO dose dependently reduced the expression of CRP's mRNA in HTR cells. L-arginine also reduced the expression of CRP's and L-NAME, an inhibitor of NOS, reversed the effect of L-arginine. We further assessed if NO mediated decreases in CRPs in HTR cells leads to increased NHS induced lysis in presence of anti HLA-G antibody. DTNO or L-arginine increased HTR cell lysis by 2-fold compared to that of control (expressed as % lysis induced by Triton X-100) and L-NAME reversed the effect of L-arginine indicating an increased lysis of cells by complement upon NO tratement. Conclusion: Expression of CRPs in decidua from spontaneous abortion was significantly less and down-regulation of CRPs by NO in HTR cells made them more prone to complement lysis. Therefore, we conclude that spontaneous abortion may be associated with reduced expression of CRPs caused by excess NO. Objective: Embryo-derived PIF is essential for pregnancy. (AJOG2010,2011; RB&E2011; RBMO2011). Synthetic analog (sPIF) translates PIF's inflammation regulatory features (Endocr2011; JNS2011). sPIF targets naïve monocytes/leucocytes in activated T&B cells blocks MLR proliferation and creates Th2cytokine bias (AJOG2012). Herein examined mechanisms involved in PIF's orchestration of systemic inflammatory response on both innate (naïve) and adaptive (activated) immunity. Methods: PBMC isolated from non-pregnant and male donors (n=12) were incubated with sPIF or irrelevant peptide. We examined sPIF's effect on: PHA, anti-CD3-activated PBMC-induced proliferation & cytokine secretion, global-naïve & anti-CD3/CD28Mab, co-activated PBMC gene expression (oligoµarray), Ca++ ion mobilization using FlexStation. Statistical analysis: t-test, ANOVA. Results: sPIF blocked mitogen & antibody-activated PBMC proliferation and promoted pro-tolerance IL10 cytokine secretion. In both naïve and activated PBMC, sPIF preserved embryo tolerance by promoting HLA B,C and critical HLA-G expression. sPIF also increased in naïve PBMC FKBP1A and activated PBMC IDO, Cyclophylin-B. In naïve PBMC, inflammation was controlled by reduced macrophage-activation MIG,CD85,MCP3 and increased antipathogen genes expression. In activated PBMC, sPIF reduced oxidative stress ALOX5, TRX and promoted PDRX3, prevented protein misfolding uncreased HSPs and reduced platelet activation PECAM1,PF-4 genes expression. sPIF promoted T-cell activation (Fyn, LCK,) while markedly reducing PPFE2 a Ca++/calmodulin dependent gene expression. sPIF effect on both antibody and mitogen-activated PBMC is independent of calcium mobilization. Conclusion: sPIF adapts/tailors its response to the immune-activating agent. PIF action on PBMC is independent of calcium mobilization-a prime mechanism of immune suppressive agents. sPIF regulates differently systemic innate or adaptive immunity: sPIF preserves embryo tolerance and acceptance while enabling vigorous maternal response to fight pathogens and disease.

Roles of Uterine Natural Killer Cells in the Pregnant Uterus before Placental Formation. Barbara A Croy, 1 Alexander P Hofmann, 1 Edith M Lord, 2 Scott A Gerber. 2 Objectives: Constantly changing relationships between maternal immune cells, conceptus-derived trophoblasts and blood vessels characterize the early gestational uterus. Although uterine Natural Killer (uNK) cells become abundant during early decidualization, little is known regarding their early pregnancy functions. To characterize these role(s) fluorescent immunohistochemistry of live intact early implant sites was undertaken. Methods: Gd6.5, 8.5 and 9.5 implant sites of BALB/c-Rag2-/-Il2rg-/-(alymphoid) and BALB/c+/+ controls (BALB/c), allogeneically-mated by C57BL/6 males with ubiquitous, transgenic expression GFP, were compared by whole mount in situ immunohistochemistry. Adoptive transfer of BALB/c-Rag2-/-(NK+ B-T-) marrow to alymphoid mice was used to examine the effects of uNK cell reconstitution on the anomalies observed in alymphoid implant sites. Viable implant sites were halved sagittally and incubated (1h) with fluorescently-tagged CD31 and CD45 in PBA (PBS, 1% BSA, 0.1% sodium azide). Samples were mounted onto glass slides, examined and photographed under epifluorescence microscopy. Results: In comparison to gd-matched BALB/c, implant sites from alymphoid mice showed ∼24h delay in uterine lumen closure and in trophoblast invasion. Mesometrial angiogenesis was limited, pruning of vascular networks in decidua basalis was deficient, leukocytes failed to acquire expression of CD31 and myeloid cells localized to anomalous regions. Putative spiral arterial precursor vessels were present by gd8.5 in control lateral decidua but were absent from alymphoid decidua. All of these anomalies were reversed by adoptive transfer of NK+B-T-marrow. Conclusions: These data suggest that uNK cells set the pace for normal development of the decidua basalis. By optimizing gd-appropriate changes and supporting all phases of angiogenesis, uNK cells function not only in spiral arterial remodeling mid-pregnancy, but also in angiogenic signaling, decidual development and in new vessel pruning shortly after implantation. Similar roles for human decidual NK cells could be vital to promotion of healthy pregnancies. Background: First trimester human decidua, is comprised of decidual cells (DCs) (∼50%), interferon gamma (IFN-γ)-expressing CD56bright CD16(-) uterine natural killer (uNK) cells (∼30%), and tumor necrosis factor alpha (TNF-α)-expressing macrophages (∼10%). IP-10 promotes NK cell migration by acting as a ligand for CXCR3 expressed at high levels by peripheral CD56brightCD16(-) NK cells. We previously found that incubation of first trimester DC monolayers with TNF-α + IFN-γ synergistically increases IP-10 mRNA and protein expression. Objectives: Elucidate signaling pathway(s) that enhance IP-10 expression in response to IFN-γ and TNF-α added separately and together in cultured human first trimester DCs. Methods: First trimester DCs were primed with estradiol (E 2 ) + medroxyprogestrone acetate (MPA) for seven days to mimic decidualization, then switched to a defined medium (DM) with E 2 + MPA or IFN-γ, or TNF-α, or IFN-γ +TNF-α for 24 h. In cell lysates, immunoblotting assessed NF-κB, phospho (p)-NF-κB, STAT1, and p-STAT1 expression. In separate experiments, DCs were incubated with control or IFN-γ or TNF-α, or IFN-γ +TNF-α for 30 minutes. In select incubations with IFN-γ +TNF-α, DCs were pre-treated with 10 µM of a JAK1 inhibitor (JI) or JAK2 inhibitor (AG490) for 1h. Cell lysates were used for immunoblotting. Results: IFN-γ enhanced p-STAT1 but not p-NF-κB expression, TNF-α enhanced p-NF-κB but not p-STAT1 expression and IFN-γ + TNF-α enhanced p-STAT1 and p-NF-κB expression. Added separately, TNF-α increased IKB-α degradation, although IFN -γ had no such effect alone. It significantly enhanced TNF-α induced degradation of IKB-α. Moreover, inhibition of either JAK 1 and JAK 2 blocked IKB-α degradation. Conclusions: Taken together, these observations indicate that cross-talk between the JAK/STAT signaling pathway and IKB-α /IKK/NF-κB signaling pathway mediates the synergistic up-regulation by TNF-α plus IFN-γ of IP-10 expression in human first trimester DCs. Objective: Labor resembles an inflammatory response with infiltration of leukocytes into the decidual tissue at the maternal fetal interface. Previously we demonstrated that IL6 depletion delays delivery by 24 hrs, which can be restored by administration of IL6. Because IL6 has key roles in the inflammatory response and in regulating T cell differentiation, we hypothesized that IL6 deficiency is associated with altered numbers and phenotypes of decidual T cells and that IL6 administration during late gestation reverses these alterations. Methods: Decidual tissues were isolated from wild type C57Bl/6 (WT; n≥8) and IL-6 deficient (IL6 KO; n≥8) mice at gestational day (d) 18.5 post coitus (pc) and d14.5 pc. Decidual leukocytes were isolated from viable implantation sites after tissue dispersion. Several leukocyte subsets and cytokines were analyzed by flow cytometry using mAbs. Leukocyte subsets were quantified as a percentage and total number of CD45+, CD3+, CD4+ or CD8+ cells after excluding non-viable (DAPI+) cells. Phenotype differences observed between WT and IL6 KO mice on d18.5 pc were considered specific for late gestation because they were not present on d14.5 pc. Specific phenotype differences in WT and IL6 KO decidua were also evaluated on d18.5 pc following subcutaneous implantation of a microosmotic pump containing either 1 µg of hIL-6 (0.5 µl per hour, 7 days) or PBS on d11.5 pc. Statistical analysis was by Mann-Whitney U Test with significance defined as p≤0.05. Results: During late gestation IL6 KO mice had lower total number of macrophages (F4/80 + cells) and T cells (CD3+ cells) compared with WT mice. In IL6 KO mice, more decidual T cells expressed the activation marker CD69 than in WT mice. IL6 KO T cells contained a higher proportion of Th9 cells (CD4+IL9+ cells) while CD8+ Tregs (CD8+CD25+FOXP3+ cells) were diminished. IL6 administration did not restore the total number of macrophages and T cells; however, it decreased the proportion of Th9 cells and increased the proportion of CD8+ Tregs.

Conclusion: Interleukin-6 ablation impairs the progression of labor and this is linked with high proportions of Th9 cells and scarce CD8+ Tregs in the decidua. Exogenous IL6 does not alter the total number of macrophages and T cells, but it restores the proportion of Th9 cells and increases the proportion of CD8+ Tregs. These T cells may participate in regulating the timing of parturition.

Maternal Monocyte-Derived Cell Depletion Promotes Preterm Delivery in Mice. Nardhy Gomez-Lopez, [1] [2] [3] Objective: Pregnancy resembles an inflammatory response with infiltration of leukocytes into the maternal fetal interface. Monocyte-derived cells (MDCs), macrophages (MΦ) and dendritic cells (DCs), are major specialized antigenpresenting cells that contribute to fetal tolerance and other processes required for successful pregnancy. We hypothesized that depletion of MDCs during late gestation would alter the timing of delivery. Methods: CD11b-diphtheria toxin receptor (CD11b-dtr) mice were injected with either diphtheria toxin (DT, 25 ng/g) or PBS on gestational day (d) 16 post-coitus (n=8-9). Gestational length and duration of labor were determined by camera monitoring until delivery. Number, weight and viability of pups were also determined. In a second set of experiments, peripheral blood, myometrium, decidua, placenta and fetal liver were collected 24h after injection. Tissue leukocytes were isolated by tissue dispersion and phenotype was analyzed by FACS using mAbs: CD45, F4/80, Ly-6G, CD11c, Siglec-F, CD3, CD4 and CD8. In addition, serum progesterone was quantified and RT 2 Profiler PCR arrays were performed on decidual RNA from DT-and PBS-treated mice. Statistical analysis was by Mann-Whitney U Test with significance at p≤0.05. Results: DT-treated mice exhibited preterm delivery, delivering ∼20h earlier than PBS-treated control mice (d17.9±0.5 vs. d18.7±0.1). In DT-treated mice, duration of labor was longer than in control mice (3.9±1.6h vs. 1.3±0.1h). Pups of DT-treated mice had lower weight and poor viability compared with control pups. In DT-treated mice, MDCs were depleted by 95% in peripheral blood, myometrial and decidual tissues, and were also reduced by 40% in placenta. However, MDCs from fetal liver were unchanged. Interestingly, in peripheral blood and myometrial tissues granulocytes were higher in DTtreated mice than in control mice (p<0.036). In DT-treated mice, decidual tissues down-regulated Cd14, Myd88, TNFα, Il1β, Il6, Il9, Cxcl3, -9 and -10, and up-regulated Tlr1, Mapk8 and Ccr2 expression. Serum progesterone was not altered by DT treatment. Conclusion: Late gestation depletion of maternal monocyte-derived MΦ and DCs induces preterm delivery in mice. Preterm labor is not linked with proinflammatory gene induction in the decidua or progesterone decline. These data suggest that MDCs may exert suppressive effects constraining labor progression.

CD4-Lymphocytes and Anti-Müllerian Hormone Levels in a Cohort of HIV-Infected and -Uninfected Women. R Scherzer, 1 P Bacchetti, 2 G Messerlian, 3 D Seifer, 4 RM Greenblatt. 5 Background: Anti-Müllerian hormone (AMH) is produced by small ovarian follicle granulosa cells, and measured in blood, is an indicator of functional ovarian follicle mass. Serum AMH levels have been proposed as a predictor of menopause that is potentially applicable to women who have chronic illness or pharmacologically induced amenorrhea. We assessed patterns of AMH decline and associated factors in a cohort of HIV-infected and uninfected women. Methods: Clinical characteristics, lymphocyte subsets and AMH levels (via ELISA) were determined for 2621 HIV-infected and 941 uninfected women enrolled in the Women's Interagency HIV Study, utilizing specimens and data collected between 1994 and 2010. Statistical analyses used mixed models for left-censored repeated measures. Results: In age-adjusted models, HIV infection was associated with 29% lower levels of AMH (95%CI: -36 to -20, p<.0001). After controlling for current and nadir CD4+ counts, HIV infected women had ∼15% higher levels of AMH relative to uninfected women (p=0.030), regardless of HIV RNA detectability. The effect was strongest and statistically significant only in Caucasian women (AMH level 34.0% 95%CI: 4.1, 72.6; p=0.023). Additionally, AMH levels were negatively associated with current but not past use of hormonal contraceptives, and negatively associated with history of weight loss. Gravidity, amenorrhea, higher levels of CD4, and higher nadir total lymphocyte counts were associated with higher levels of AMH. Conclusions: Numbers of CD4+ lymphocytes are independently associated with higher AMH levels in both HIV-infected and uninfected women, a finding which appears to explain most of the difference in AMH levels between HIV infected and uninfected women. HIV infection, regardless of the presence of viremia, was associated with increased AMH levels in Caucasians after adjustment for CD4 cell counts and age. These findings support the concept that lymphocyte populations and products influence ovarian granulosa cell function. Background: The role of placental fetal macrophages (Hofbauer cells, HBCs) in the regulation of innate immune response at the placental/fetal interface remains unelucidated. HBCs are adjacent to fetal vessels in placenta and were reported to show a M2 (anti-inflammatory/pro-angiogenic) phenotype. We documented that HBCs express CD163 and folate receptor (FR)-β, key cellsurface proteins involved in the scavenging of free hemoglobin (Hb) and the transport of folate, respectively. The goal of the current study was to examine HBC function and inflammatory cytokine expression following treatment with poly (I:C), a TLR-3 agonist and viral dsRNA mimetic. Methods: HBCs were purified from human term placentas using Percoll gradients and negative immunoselection (n=4). Cells were maintained in serumfree medium with and without poly (I:C) and uptake of labeled hemoglobin (Hb):unlabeled haptoglobin (Hp) complexes, as well as CD163 localization, was examined by flow cytometry. Levels of CD163 and FR-β protein in cell lysates was determined by Western blotting following normalization to HSP90 levels. Levels of IL-6, IL-8, and CCL5 (RANTES) in conditioned media was measured by ELISA and normalized to cell protein.

Results: Flow cytometry revealed that uptake of labeled Hb:Hp complexes in HBCs was inhibited 80% by treatment with 1 µg/ml poly (I:C) for 48 h (P<0.001), and the expression of cell surface-associated and total cellular CD163 levels were similarly inhibited 61% and 65%, respectively. Western blotting revealed treatment of HBCs with poly (I:C) suppressed levels of CD163 and FR-β 85% and 71%, respectively (both P<0.001) and this effect was timedependent between 8 and 48 h, and dose-dependent between 10 ng/ml and 1 µg/ ml poly (I:C). In contrast, levels of IL-6, IL-8, and CCL5 in HBC conditioned media were significantly enhanced 220-, 3-, and 47-fold, respectively (all P<0.01) by treatment with 1 µg/ml poly (I:C) for 24 h. Conclusions: This study demonstrated that levels of CD163 and FR-β, as well as CD163 function, were markedly suppressed in HBCs in response to dsRNA. Surprisingly, dsRNA promoted a marked pro-inflammatory response in HBCs, a cell type reported to express a M2 phenotype. This suggests that viral infection of the placenta may shift HBC phenotype and function by curtailing scavenging/transport activity and enhancing the release of pro-inflammatory compounds to the umbilical circulation.

Role Objective: Infection-associated inflammation is a major cause of preterm premature rupture of membranes (PPROM). Cytokines such as IL-1β weaken the membranes by upregulating MMPs and inducing apoptosis, causing rupture. IL-1β secretion occurs after pro-IL-1β is processed into its active form. This is mediated by the inflammasome, a protein complex that activates non-apoptotic caspases, like caspase-1, leading to IL-1β processing. The objective of this study was to determine the mechanism by which fetal membranes produce IL-1β in response to various bacterial Toll-like receptor (TLR) and Nod protein agonists. Methods: Term chorioamniotic membranes collected from normal caesarean deliveries without labor (n=3) were treated with or without the following agonists: TLR2: peptidoglycan (PDG; 10µg/ml); TLR4: lipopolysaccharide (LPS; 100ng/ml); TLR5: flagellin (1µg/ml); Nod1: iE-DAP (100µg/ml); Nod2: MDP (10µg/ml) in the presence and absence of a casapse-1 inhibitor (1µM). After 24hrs, supernatants were measured for IL-1β by ELISA and tissues were analyzed for active IL-1β by western blot. Results: Treatment of fetal membranes with PDG, LPS, flagellin, iE-DAP and MDP all significantly induced IL-1β secretion when compared to the control (p<0.05), although the responses for LPS and PDG were 4-fold greater than MDP, 10-fold greater than flagellin and 50-fold greater than iE-DAP. Western blotting of the tissue showed expression of active IL-1β (17kDa) after treatment with PDG, LPS, flagellin, iE-DAP and MDP, while no expression was seen in the control. The caspase-1 inhibitor significantly reduced membrane IL-1β secretion induced by PDG by 76.1±10.8%, LPS by 54.4±14.6% and flagellin by 38.3±9.7% (p<0.05). However, the caspase-1 inhibitor had no effect on iE-DAP or MDP-induced IL-1β secretion. Conclusion: These findings show that bacterial TLR and Nod protein agonists trigger fetal membranes to generate an IL-1β response through its intracellular processing and secretion. While TLR2, TLR4 and TLR5 agonists induce IL-1β secretion via caspase-1, Nod1 and Nod2 agonists induced IL-1β secretion independent of caspase-1. Thus, fetal membrane IL-1β production in response to bacterial components is mediated through distinct mechanisms that are dependent upon the type of innate immune receptor involved.

First Trimester Decidual Cells Mediate Pro-Inflammatory-Induced M1 Macrophage Polarization-Implication in Preeclampsia. Min Li, 1 Chie-Pein Chen, 2 Chang-Ching Yeh, 3 Salley Pels, 4 S Joseph Huang. 5 . Previously, our laboratory found: i) excess Møs and Mø differentiation factors, granulocyte-Mø-colony-stimulating factor (GM-CSF) and M-CSF in the preeclamptic decidua; ii) that the PE-associated pro-inflammatory cytokines, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), enhanced GM-CSF and M-CSF expression in leukocyte-free first trimester decidual cells, the main cell type encountering Møs. However, the abundance of GM-CSF is greater than M-CSF. Thus, this study hypothesizes that the pathogenesis of PE involves Mø polarization regulated by elevated CSFs produced by IL-1β-or TNF-α-stimulated first trimester decidual cells. Methods: Immunofluorescent staining localized CSF expression in preeclamptic and gestation age-matched decidua. ELISAs assessed signaling pathways that regulate CSF production in IL-1β-or TNF-α-treated first trimester decidual cells. Expression of M1 and M2 markers (CD80, CD86 and CD163) and corresponding cytokines (TNF-α, MCP-1, TGF-β and IL-10) were assessed by flow cytometric analysis of peripheral monocyte-derived Møs incubated with decidual cell conditioned medium (CM). Results: Preeclamptic decidua displayed elevated CSF-positive decidual cell number. Inhibitors of MAPK and NF-κB signaling pathways blocked IL-1β-or TNF-α-induced GM-CSF and M-CSF production by first trimester decidual cells. An increase of cells expressing M1 surface marker (CD80 and CD86) and M1 cytokines (TNF-α and MCP-1) was observed in the Møs co-cultured with CM from IL-1β-or TNF-α-stimulated first trimester decidual cells. These effects were inhibited by either an anti-GM-CSF or -MCSF antibody. Conclusion: Enhanced GM-CSF and M-CSF secretion by pro-inflammatory cytokine-stimulated first trimester decidual cells involves MAPK and NF-κB pathway mediation and may regulate Mø polarization in PE.

Characterization of Ovarian Antibody Reactivity with Cellular and Exosomal Antigens in Patients with Endometriosis. Divya C Sridharan, 1 Douglas D Taylor, 1 Bruce A Lessey, 2 Cicek Gercel-Taylor. 1 1 Obstetrics and Gynecology, University of Louisville; 2 Obstetrics and Gynecology, Greenville Hospital System. Introduction Patients with endometriosis, defined as endometrial glands and stroma outside of the uterus, have an increased risk of endometrioid and clear cell subtypes of ovarian cancer. The exact molecular mechanisms transforming endometriosis to ovarian cancers are not understood. One hypothesis involves inflammation. Endometriosis fulfills most of the criteria for autoimmune disease, including polyclonal B cell activation, abnormalities in T and B cell function, and multiorgan involvement. Patients with endometriosis have an increased incidence of autoantibodies, including anti-endometrial and anti-ovarian antibodies, of which three and zero, respectively, have been identified.

Our purpose was to characterize anti-ovarian antibodies seen in patients with endometriosis. These may play a role in malignancies and thus be utilized to identify women with endometriosis who have an increased risk of ovarian cancer. Methods Using sera from patients with different stages of endometriosis, ovarian cancer patients, and controls, we performed Western immunoblots with ovarian tumor samples and exosomes released from those tumor cells.

Selective reactivity of benign and malignant ovarian tumor antigens were demonstrated in endometriosis patients as compared to controls. When endometriosis patient sera were reacted with ovarian and endometrial tumor cell lysates, high reactivity in all stages of endometriosis was seen with both tumor cell lysates. Unlike control sera, endometriosis patient sera did not recognize molecular weights above 70kD -only normal sera reacted with bands 80, 95, 140, 270kd. Normal sera reactivity with exosomes of benign ovarian tumors and a benign tubal tumor demonstrated the major band at 40kD, in addition to 25 kD and 75-78KDs. When different stage endometriosis sera were reacted with exosomes derived from sera of ovarian cancer, benign ovarian, breast, and fallopian cancer, similar reactivity to exosomal antigens was seen in the 45 to 67 kDa range. The 25 kDa was only seen in benign ovarian tumor cells.

The selective reactivity demonstrated in endometriosis patients to ovarian tumor cell antigens may define the role of immunologic factors involved in ovarian cancer development. Identification of these moieties may result in biomarkers and targets that can be used for risk identification and treatment. INTRODUCTION Human decidual (d)NK cells represent the most abundant leukocytes at fetal maternal interface. The CD56+CD16-NK subset is distinct from peripheral NK cells expressing unique marker with immunomodulatory potential. Meanwhile, dNK cells play a critical role in mediating spiral artery remodeling during pregnancy. However, their phenotypic and functional changes remain unclear in the 2nd trimester pregnancy. OBJECTIVE Quantitative flow cytometry was used to determine the number, activation status and function of uNK cells throughout the 1st and 2nd trimester. MATERIALS & METHODS Obtained decidual samples were identified, rinsed and disassociated to enrich mononuclear cells. Live/dead staining was applied to eliminate staining artifacts. Multi-color flow cytometry was performed to investigate dNK population and receptors. Intracellular cytokine staining was conducted to examine IFNG, VEGF and IL-8 of dNK cell. CD107a mobilization assay was applied to study dNK degranulation. RESULTS In the early pregnancy, 68% of CD45+ decidual leukocytes were CD56+CD16-dNK cells and this proportion remained constant through the second trimester. More than 95% of uNK cells were strongly positive for CD335, CD244 and only 15% was CD336+. With increasing gestational age, CD337, CD314 and NKp80 expression of dNK was significantly upregulated. The degranulation assay revealed that about 6-8% of 1st trimester dNK cells was CD107a+ and its expression was significantly increased after PMA stimulation. Compared to 1st trimester, 2nd trimester dNK cell showed similar VEGF secretion but significantly weaker reaction to PMA stimulated CD107a expression. Detailed Boolean gating found a functional shift of dNK cells between 1st and 2nd trimester pregnancy. Upon stimulation, the frequency of VEGF+CD107-IL-8-IFNG-subset was decreased while VEGF+CD107+/-IL-8-/+IFNG-/+ and VEGF-CD107+IL-8-IFNG-populations were greatly increased in 2nd trimester pregnancy. To conduct a retrospective cohort analysis among a Hispanic population determining ICP incidence and factors associated with recurrence risk. METHODS: Retrospective cohort analysis from January 1, 2004 to July 31, 2012 at MHC. Mild ICP defined as total serum bile acids (TBA) 10 and 39 micromol/L; severe ICP greater than 40 micromol/L. TBA were only drawn in symptomatic patients. RESULTS: 369 ICP cases identified, with 326 women affected. All women were Hispanic. Mild ICP complicated 234 (63%) pregnancies and severe ICP seen in 135 (37%). 60 women (16%) had subsequent pregnancies in the study period. ICP recurred in 37 of these pregnancies.

Eight women had two of more subsequent pregnancies complicated with ICP. No significant differences in severity of disease were seen among recurrent cases of ICP. Gestational age at diagnosis was similar in the index ICP pregnancy compared with the following ICP pregnancy (p=0.14). No differences were seen between neonatal intensive care admission rates between ICP pregnancies. More than 40% of the total study population subsequently underwent gallbladder surgery during the study period. However, the risk of recurrence of ICP was not associated with subsequent gallbladder surgery (p=0.886).CONCLUSION: To date, no study has described ICP in a large tertiary population and its rate of recurrence. ICP recurrence risk was 10%. No differences were seen in disease severity with subsequently affected cholestasis pregnancies. Additional studies are needed to identify risk factors associated with ICP recurrence.

Risk Factors for Unscheduled Delivery in Patients with Placenta Accreta. Zachary S Bowman, Tracy A Manuck, Alexandra G Eller, Marilee Simons, Robert M Silver. Obstetrics and Gynecology, University of Utah Health Sciences Center and Intermountain Healthcare, Salt Lake City, UT, USA. Objective: Emergent cesarean delivery is associated with increased maternal morbidity. Scheduled delivery for patients with suspected placenta accreta has been shown to improve outcomes. Our objective was to identify risk factors for unscheduled delivery in patients with placenta accreta. Study Design: Cohort study of women with antenatally suspected or pathologically confirmed placenta accreta who underwent a cesarean delivery (CD) at a tertiary care center from January 2000 through July 2012. Those women who underwent CD prior to a planned delivery date were compared to women who had a scheduled delivery (at term or at a predecided preterm gestational age). Data were analyzed using t-test, chi-square, and logistic regression. Variables included in regression analyses were PPROM, number of episodes of antenatal vaginal bleeding (VB), number of prior cesarean deliveries, interpregnancy interval, race/ethnicity, and parity. A p-value <0.05 was considered significant. Results: 76 accreta patients were identified, the majority of whom were antentally suspected (86.8 %). 41 (54%) had an unscheduled delivery. Demographics were similar between groups. Unscheduled patients delivered earlier (mean 32.0 vs. 35.7 weeks, p<0.0001) and were significantly more likely to have had VB (75.6% vs. 34.3%, p<0.001) and PPROM (26.8% vs. 2.9%, p<0.005; Figure) . In the final regression model, each episode of antenatal VB (OR 2.58, 95% CI 1.47-4.55) and PPROM (OR 16.7, 95% CI 1.86-150) were associated with increased risk of unscheduled delivery. Conclusion: Among women with placenta accreta, those with PPROM were significantly more likely to require unscheduled delivery. Additionally, for every episode of VB there was a 2.58-fold increased risk of unscheduled delivery. These data suggest that development of PPROM and multiple episodes of vaginal bleeding should be considered when determining the optimal delivery gestational age for women with placental accreta.

Risk Factors for the Development of Placenta Accreta. Zachary S Bowman, 1 Alexandra G Eller, 1 Tyler Bardsley, 2 Tom Green, 2 Michael W Varner, 1 Robert M Silver. 1 1 

Center and Intermountain Healthcare, Salt Lake City, UT, USA; 2 Epidemiology, University of Utah, Salt Lake City, UT, USA. Objective: Placenta previa and prior cesarean delivery are known risk factors for placenta accreta. However, other risk factors have not been identified. Thus, our objective was to examine risk factors for the development of placenta accreta in a large multicenter cohort. Study Design: Secondary analysis of a de-identified data set from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal Fetal Medicine Units Network Cesarean Registry. Patient characteristics in pregnancies with placenta accreta were compared to those without placenta accreta using univariate and multivariate analyses. Results: 196/73,257 (0.27%) had placenta accreta. As expected, women with increasing numbers of prior cesarean deliveries were more likely to have an accreta (p<0.0001) as were women with a placenta previa (OR 34.9, 95% CI 22. . Controlling for previa, patients with 1, 2 or 3 or more prior cesarean deliveries had an OR for accreta of 2.86 (95% CI 1.73-4.72), 4.61 (95% CI 2.62-8.11), and 12.57 (95% CI 6.86-23.0), respectively. In order to determine whether additional risk factors contributed to the development of accreta, we considered the subset of patients who had a placenta previa and examined the following variables: maternal demographic factors, prior cesarean deliveries, time interval between delivery, parity, BMI, tobacco use, and coexisting hypertension or diabetes. In this model, patients who had a previa and 2 prior cesarean deliveries had an OR for accreta of 4.93 (95% CI 1.71-14.3), and patients with a previa and 3 prior cesarean deliveries had an OR for accreta of 7.65 (95% CI 2.35-24.9). However, no other variables achieved significance in this model. Conclusion: Patients with placenta previa have an increased risk for placenta accreta that increases with the number of prior cesarean deliveries. Objective: The impact of breast cancer diagnosis on pregnancy outcomes is poorly understood. We sought to describe the maternal and neonatal outcomes of breast cancer diagnosed prior to and during pregnancy. Study Design: We conducted a retrospective chart review of women with a diagnosis of breast cancer prior to delivery and a singleton live birth at Brigham and Women's Hospital or Massachusetts General Hospital from January 1, 2000 to January 1, 2010. Chi-squared, students t, and wilcoxon rank sum tests were used to compare outcomes between women diagnosed prior to and during pregnancy. Results: 90 women with breast cancer diagnosis prior to pregnancy were identified: 39 with pregnancy associated breast cancer and 51 diagnosed prior to pregnancy. Women diagnosed in pregnancy were more likely to have invasive carcinoma (94% vs 60%, p < 0.05) and were significantly more likely to deliver preterm, (18% vs 67% for preterm birth<37 wks, 0% vs 8% for preterm birth<34 wks, p<0.05 for both). Most preterm births were due to indicated deliveries to facilitate cancer treatment. The incidence of spontaneous preterm birth prior to 37 weeks was similar in the two groups (10% vs 13%, p=0.6); although all spontaneous preterm births less than 34 weeks occurred in the pregnancy associated breast cancer group, this did not reach statistical significance (0% vs 5%, p=0.1) Fetal outcomes and pregnancy complication rates were similar. (Table) Conclusion: Women with breast cancer diagnosed during pregnancy were significantly more likely to deliver preterm than were those diagnosed prior to pregnancy, primarily due to late preterm birth to facilitate cancer treatment. Fetal outcomes were good, with low rates of growth restriction. There was no significant difference in pregnancy complications. Further investigation is needed regarding the association of breast cancer diagnosis during pregnancy and spontaneous preterm birth.

Maternal Cardiac Output as a Marker for Subclinical Aortocaval Compression in the Second and Third Trimesters. Kristen Buono, 1 Jerasimos Ballas, 1 Kristin Mantell, 2 Thomas Archer. 2 1 Reproductive Medicine, University of California, San Diego, CA, USA; 2 Anesthesia, University of California, San Diego, CA, USA. Objective: To assess cardiac output in different maternal positions as a marker for aortocaval compression in high risk pregnancies. Study Design: Patients underwent continuous cardiac output (CO) monitoring with the Cardiotronic Aesulon Electric Cardiometry system in three different positions: Full-left lateral (L90), full right lateral (R90) and supine. Maternal history, characteristics and diagnoses were recorded. Mean CO and ranges were calculated for each position. ANOVA and Student's t-test were performed where appropriate. Results: Forty patients were enrolled with a median GA of 27w6d. CO varied significantly with position in singleton pregnancies across all gestational ages. CO did not change significantly in severely and morbidly obese, normotensive women in the third trimester. With hypertension, L90 produces significantly greater CO in all classes of obesity. In non-obese women with multiple gestations, L90 and supine produced similar CO >28wks. Maximal CO was observed in R90 in obese women with multiple gestations across all gestational ages. Multiple Gestations (N=11) Non-obese (n=6) 9.8 (6.9-15.6) 8.6 (5.9-12.5) 9.1 (6.3-17.2) <0.01 GA <28 weeks 8.5 (6.9-11.1) 8.9 (6.7-11.0) 8.3 (6.3-10.2) <0.01 GA >28 weeks 11.4 (8.6-15.6) 8.0 (5.9-12.5) 10.8 (7.0-17.2) <0.01 Obese (n=5) 10.9 (7.5-15.6) 12.2 (6.6-18.7) 9.9 (7.0-12.9) <0.01 GA <28 weeks 8.9 (7.5-10.2) 8.3 (6.6-9.9) 8.4 (7.0-9.6) <0.01 GA >28 weeks 12.2 (9.9-15.6) 13.2 (6.6-18.7) 10.9 (9.4-12.9) <0.01 L90: Full left lateral; R90: Full right lateral Conclusion: Maternal characteristics should be considered when counseling a patient about optimal positioning to prevent aortocaval compression. Noninvasive CO monitoring may be useful in guiding maternal positioning.

Umbilical Cord Blood Gases: Predicting Fetal Acidemia Using Venous Cord Gas Parameters. Jessica Cantu, 1 Jeff Szychowski, 2 Xuelin Li, 2 Adi Abramovici, 1 Joseph Biggio, 1 Rodney Edwards, 1 William Andrews, 1 Alan Tita. 1 1 Obstetrics and Gynecology, University of Alabama at Birmingham; 2 Biostatistics, University of Alabama at Birmingham. Objective: Umbilical cord arterial blood gas (ABG) is a critical tool to objectively assess fetal status at birth. However, arterial samples are often not available especially in high risk settings. We used cord venous blood gas (VBG) parameters to identify VBG pH cut-off values for low likelihood of fetal acidemia. Methods: Retrospective cohort study of all singletons delivered at UAB with a paired arterial and venous cord gas from 2006 to March 2012. Universal collection of cord blood gas at every delivery is performed at our institution. Cord gas values that did not meet published criteria for valid collection were excluded: single blood gas value, absolute difference in ABG and VBG pH of <0.02 or pC02 <4 mmHg-indicating the same vessel was sampled. Fetal acidemia was primarily defined as cord ABG pH <7.0. Alternative definitions also examined: ABG pH <7.05 and <7.10. Logistic regression analyses were performed to predict probability of fetal acidemia by VBG pH values. ROC curves relating fetal acidemia to VBG were derived to determine predictive ability. Results: Of 23,506 births during the study period, 18,335 (78%) cord blood samples were reported by the lab. 4,185 (22.8%) did not meet criteria for valid collection and were excluded, leaving 14,150 for analysis. The ROC curve for the prediction of ABG (pH <7.0) from VBG is shown in Fig 1. For all three definitions of acidosis, VBG pH is a significant predictor for acidosis (AUC>95%; p<0.0001). The frequency of acidemia was 147 (1%) for ABG pH <7.0, 269 (1.9%) for pH <7.05, and 582 (4%) for pH <7.10. The VBG pH cutoffs associated with selected low probabilities of fetal acidemia are presented in Table 1 . Conclusions: Despite universal collection of cord gases approximately 1 in 4 births have incomplete results -typically representing vein-only collection. We demonstrate that VBG pH is a powerful predictor of ABG pH and likelihood of fetal acidemia. The data will be used to develop an ABG pH calculator (given VBG parameters) for validation.

During the first trimester 87.8% of women had a vitamin K1 deficiency (<0.8 nmol/l), during the second trimester 59.1% and during the third trimester 50.0%. The mean serum level of vitamin K1 during the third trimester (4.70 ± 10.31 nmol/l) was significantly higher than during the first (0.42 ± 0.26 nmol/l) (p<0.05). Activity of prothrombin time was significantly higher during the second or third trimester than during the first trimester (p<0.05). Activity of Factor X and Factor VIII was significantly higher during the second trimester than during the first trimester (p<0.05). Activity of factor VII was significantly higher during the second trimester (p<0.001) and third trimester (p<0.05) than during the first trimester. There were no differences in mean vitamin K1 serum levels or blood coagulation indicators between procedure group. CONCLUSION Intensive follow-up and nutritional screening for vitamin K1 deficiencies and vitamin K1 supplementation in pregnancies after bariatric surgery is effective for improving vitamin K1 serum levels and activity of vitamin K dependent coagulation factors throughout pregnancy.

Cesarean in the Second Stage of Labor: Pushing the Head or Pulling the Feet through a Low Segment Incision. Clark T Johnson, Andrew J Satin. Gynecology and Obstetrics, Johns Hopkins School of Medicine, Baltimore, MD, USA. Objective: Cesarean in the second stage of labor is associated with increased morbidity. The purpose of this investigation is to systematically review methods of cesarean with an engaged fetal head in an effort to elicit best practices. Study Design: Meta-analysis of all identified prospective randomized controlled trials of cesarean delivery in advanced labor using the search terms "reverse breech extraction," "push method cesarean," and "pull method cesarean" in the MEDLINE database. Two reviewers independently reviewed each identified articles, extracting data on rate of uterine extension, endomyometritis, maternal blood loss, and operative time. Results: Four studies met inclusion criteria and were included for meta-analysis. Studies compared using a "push" method of using a vaginal hand to disengage the fetal vertex for delivery, to the "pull" method where the vertex presenting fetus was extracted from the breech through a low segment hysterotomy. All studies included all four outcomes in their findings, with the exception of one that did not report estimated blood loss. All studies involved obstructed labor, with some obstructions lasting longer than others prior to cesarean. Two neonatal long bone fractures and 8 active site extensions were reported with the "pull" method. Meta-analysis of the four outcomes demonstrate a statistically significant (p<0.05) benefit in all using the "pull" method.Conclusion: Using the "pull" method, or "reverse breech extraction" for the well engaged fetal head is an alternative to using a vaginal hand to disengage a fetal head to enable vertex delivery. Although there may be a risk of long bone fracture or active site extension, this method may be associated with less maternal morbidity. Further studies evaluating the use of the technique are warranted.

Section. Jasmine Lai, Emily S Lukacz, Stephen A Hebert, Douglas A Woelkers. Department of Reproductive Medicine, University of California, San Diego, CA, USA. Objective: Visually estimated blood loss (EBL) at cesarean section is notoriously inaccurate and subject to physician bias. The objectives of this study were 1) to compare EBL to two alternative methods of estimation: quantitative blood loss (QBL) and calculated blood loss (CBL), and 2) to study the relationship of these estimates to clinical outcomes. Study Design: This was a prospective observational study of all C-sections at UCSD from 7/2011 to 3/2012, in which a QBL and EBL were recorded independently. EBL was assigned by the attending surgeon. QBL was determined by the circulating nurse, derived by adding the suction canister volume with the blood weight of laparotomy sponges. CBL was derived from the product of the maternal blood volume and the % of blood lost. Statistical analysis was performed by z-value comparison of Spearman's correlation coefficient. Results: 234 patients qualified for inclusion; median gestational age was 39+2 weeks. Primary indications for surgery were elective repeat (24%) and active phase arrest (23%). Median blood loss by EBL, QBL, and CBL were 900, 853, and 1150mL, respectively. The percent of women with hemorrhage (defined as BL ≥1000mL) by EBL, QBL, and CBL was 40%, 34%, and 59%. EBL and QBL were modestly correlated with each other (R=0.59, p<0.05), but less correlated with CBL (R=0.40 and 0.48, both p<0.05), and were not statistically different from one another (z-value 0.66). The sensitivity and specificity of a diagnosis of hemorrhage (BL ≥1000 mL) for transfusion, use of uterotonics, or length of stay (LOS) >4 days was not significantly different for QBL than EBL: LGA baby compared to AGA. Platelet count was reduced in PE+SGA but platelet volume significantly increased in all outcomes with PE. Conclusion: Biomarkers of trophoblast invasion measured close to delivery were not associated with adverse outcomes. Increased expression of markers for vascular condition and inflammation, alterations in pro and anti angiogenic factors and increased platelet volume were associated with the development of PE. Angiogenic factors were not different with SGA or LGA. Doppler findings were abnormal in preterm but not term PE. The association of Doppler RI and PI at 16 weeks with fetal size at delivery suggest they measure uteroplacental adaptation that determines fetal growth.

Womb Pregnancy loss is difficult to predict. Current models of risk prevention focus on adult lifestyle and anthropometric factors, but leave the majority of all stillbirths (loss after 28 weeks) unexplained. Fetal programming theory suggests that the intrauterine environment can have a lasting impact on adult reproductive health. Currently, the impact of a female's fetal development on her later reproductive success is largely unknown. Female common marmoset monkeys (Callithrix jacchus) are much more closely related to humans than typical animal models. They produce a variable number of fetuses per pregnancy. Marmoset triplets are a useful clinical translational model of a growth-restricted human fetus, because they experience a nutritionally-restricted prenatal environment compared to twins. To assess whether a marmoset female's birth weight and litter size at birth contributes to her adult fertility as measured by fetal loss, the effect of litter size on fetal loss was examined in 56 adult female marmosets (Southwest National Primate Research Center). Differences in loss rates between twins (n=29) and triplets (n=27) were measured using 2-sample Z-tests of proportion. Linear regression was used to determine the extent to which litter size predicted fetal loss even after accounting for the lower birth weight of triplets. Triplet females lost three times more fetuses than twins (38% vs. 13%, p=0.02) across all categories of birth weight. Litter size explained 18% of the variance in total loss rate, whereas birth weight explained only 7%. The high rate of fetal loss in triplet marmoset females may result from disruption of the growth and development of the reproductive system, a process that is seemingly decoupled from overall prenatal growth as reflected by birth weight. Using this primate model, we find support for the view that fetal loss in women stems from their own adverse prenatal environments. Hospital were identified. Cases were reviewed using our computerized database of medical records, as well as hand written Labor and Delivery logs. Potential risk factors for shoulder dystocia were obtained. All statistical analyses were performed using SAS Version 9.2 (SAS Institute Inc., Cary, NC). A Chisquare test was used to compare proportions of categorical variables between different patient groups. A two sample t-test was used to compare means of continuous variables between different patient groups. All statistical analyses were performed at the 0.05 level of significance.

Results: 154 patients with shoulder dystocia were non-diabetic, 20 patients had diabetes (3 patients with Type 1 diabetes; 6 patients with Type 2 diabetes; 5 patients with diet controlled gestational diabetes; 6 patients with gestational diabetes taking glyburide or insulin). 57% of our patients were augmented, 20% were induced, and 23% had spontaneous labor. Estimated fetal weight (EFW) was significantly lower in the diabetic group compared to the non-diabetic group (3304g versus 3600g respectively, p< 0.01). Moreover, 14% of the non-diabetic patients were induced compared to 55% of the diabetic patients (p< 0.0001). There was no significant difference in the non-diabetic group versus the diabetic group in: age, Body Mass Index (BMI), labor augmentation, macrosomia (weight> 4000g), large for gestational age (EFW> 90%), operative delivery or low fetal pH (pH< 7.00). Conclusions: Our data shows that diabetic patients had shoulder dystocia with significantly lower EFW than the non-diabetic patients. Most diabetic patients OBJECTIVE: Maternal smoking is a well-established risk factor for a variety of adverse pregnancy outcomes. Macedonia is one of the 5 countries with the highest globally reported smoking rates. Over 10% of the country's population consists of the underprivileged Roma minority, which suffers from both frequent maternal smoking as well as high rates of adverse pregnancy outcome. We aimed to determine whether Roma ethnicity is an independent risk factor for adverse pregnancy outcome or merely a mediator of maternal smoking. METHODS: Maternal demographics and pregnancy outcome were retrieved from the perinatal computerized database for all deliveries occurring during 2007-2011 at the only Clinical Hospital in Bitola, Macedonia. Multivariable logistic regression models were constructed to control for confounders. RESULTS: Of 6968 deliveries, 8.65% were of maternal Roma ethnicity and 14.6% were to smoking mothers. Forty percent of the Romani women admitted to regularly smoke during their current pregnancy and Romani ethnicity consisted almost a quarter of the smoking population. Both Roma ethnicity and maternal smoking were significantly associated with absence of any formal maternal education, history of abortions, lower mean birthweight and head circumference and higher rates of IUGR. Both maternal Roma ethnicity (OR, 2.46, 95%CI 1.79-3.38), as well as maternal smoking (OR, 1.37, 95%CI 1.02-1.85), were found to be independent predictors of IUGR in a multivariable regression model. Lower birthweight and smaller head circumference were both independently associated with the same predictors: Roma ethnicity, smoking, illiteracy and primiparity. CONCLUSION: Underprivileged ethnic background is a significant risk factor for IUGR, independent of maternal smoking status or education. Romani women exhibit several independent risk factors for IUGR and other adverse pregnancy outcome. Efforts to improve pregnancy outcome in this population should address not only maternal smoking but also other socio-demographic characteristics. To the best of our knowledge, this is the first publication focusing on pregnancy outcome in Romani Macedonian parturients.

Maternal 

Low maternal vitamin D levels in pregnancy were associated with an increased risk of preeclampsia, gestational diabetes mellitus, preterm birth and small-for-gestational age. Objective To compare changes in pelvic floor support from pregnancy to 1-year postpartum after unlabored cesarean delivery(UCD)and trial of labor (TOL). Design Prospective observational cohort study.

Setting Wenzhou Third People's Hospital, in Wenzhou, Zhejiang, China. Population Primiparous women undergoing UCD or TOL. Methods Pelvic organ prolapse (POP) was assessed at 36-38 weeks of gestation (G36-38w), postpartum 6-weeks (PP6w), 6-months (PP6m) and 1-year (PP1y), using the Pelvic Organ Prolapse Quantification (POPQ) system. Main outcome measure Comparisons of incremental changes in POP were made between groups at each time point and within groups at adjacent time points.Results Stage II POP was present in 37 and 35% of women in TOL and UCD at G36-38w. After delivery, the rate of stage II POP was significantly higher in TOL compared to UCD at PP6w, PP6m and PP1y, (48, 29 and 27% vs. 35, 10, and 7%, respectively, p<0.05). Points Aa (Ba) decided the final stage assignment in most cases. With the resolution of the pregnancy, 90% of the UCD women and over half of the TOL women with POP at 36-38w did not have POP at PP1y. Conclusion Factors unique to labor and delivery prevent normal pelvic floor remodeling and recovery that protect against sustained pelvic floor relaxation.Alterations in pelvic support occur prior to labor, however the presence of POP during pregnancy does not appear to predict long-term POP. Further investigation into the mechanisms leading to persistent or progressive POP after TOL are warranted.

Gender 

Previous studies indicated sex differences in the incidence and progression of kidney disease with a higher risk observed in males compared to age-matched premenopausal women It was also noted that male gender was associated with decreased renal function after kidney donation, including lower estimated glomerular filtration rate and increase in creatinine. The mechanism is not clear. Intrarenal rennin angiotensin system plays an important role in renal function. Previous studies demonstrated alterations in angiotensin converting enzyme (ACE) and ACE2 pathways might affect renal function. The currently study was designed to determine the effect of nephrectomy on renal ACE and ACE2 activity and if this effect shows gender differences.

Age matched male(N=6) and female(N=5) adult sheep were anesthetized and underwent unilateral nephrectomy at 1.8 years of age followed by necropsy and removal of the remaining kidney 3 weeks later. Renal cortex from first and remaining kidney was dissected on ice and frozen at -80 ºC for further study. ACE and ACE2 activities (expressed as fmol/min/mg protein) were assayed by radioactivity assay. All data were expressed as the means±SEMs. t test was used for data analysis. P<0.05 was used as statistical significance.

In 27% vs.19%) . This is the first time, when high rate of pre-pregnancy PFD was demonstrated in nulliparous women followed by high postnatal persistence. Among all postnatally symptomatics, PP predominated and their PFD scores were higher than in DNO group. Prepregnancy PFD is the major risk factor associated with postnatal PFD, while Caesarean Section had a protective role. Conclusion: This study demonstrated a high prevalence of different types of asymptomatic POP at one year post partum. There is a link between presence of uterine prolapse and collagen concentration. Serum ELISA test can be a simple and acceptable test for collagen quantification.

The Problem of Pyuria 1-9 wbc ul -1 ; Are We Missing Significant Disease? Kiren Gill, Anthony Kupelian, Sheela Swamy, James Malone-Lee. Division of Medicine, University College London, London, United Kingdom. Introduction There is growing interest in missed infections in the aetiology of overactive bladder syndrome (OAB). MSU cultures, urinary leucocyte esterase and nitrite screening tests are validated on Kass' 1957 criteria based on patients with acute pyelonephritis with no justification for application to other symptomatic groups 1 . Microscopic pyuria of fresh urine remains the best marker of UTI. However controversy exisits about the accepted threshold ≥10 wbc µl -1 for judging significance. Inflammation in the urinary tract can manifest through the expression of antimicrobials and cytokines. Lactoferrin chelates iron, essential to microbes and has been found in abundance in patients with acute UTI 2 . Urothelial cells constitutively express IL-6 and studies show rapid increases in IL-6 after the onset of infection. Urothelial cells when infected with microbes, signal their distress by expressing ATP 3 . We capitalised on the immune properties of these three proteins to scrutnise the significance of pyuria1-9 wbc µl -1 and ≥10 wbc µl -1 . Method A prospective observational study obtaining clean catch MSU samples from 182 women with OAB presenting to a specialist outpatient service from Feb 2011-July 2012, and 30 controls. Microscopy for pyuria was performed on fresh urine. Urinary IL6 and Lactoferrin was analysed by high sensitivity ELISA and ATP using a luciferin-luciferase assay. Results 182 patients (F=168; M=14; Age mean+sd 60+17.7y); 61 patients had OAB no pyuria, 64 had OAB pyuria 1-9 wbc µl -1 and 57 OAB ≥ 10 wbc µl -1 . Urinary IL-6 in those with pyuria 1-9 wbc µl -1 was well above controls (F=15.02, df=3, sig =.000).Very similar patterns manifest with Lactoferrin and but urinary ATP was only significantly elevated above controls when pyuria was ≥ 10 wbc µl -1 .

These patients, who exhibited microscopic pyuria (≥10 wbc µl-1) had negative routine culture had proved recalcitrant to antimuscarinics and antibiotics. Despite absent microbiological data, they were exposed to aggressive antibiotic treatments and showed a response. These data are only indicators but express a need to reappraise our whole approach to UTI in patients with OAB. References: 1. Arch. Intern. Med., 100 (1957) Objective: Thermal destruction of the endometrium may theoretically inhibit the development of endometrial cancer (EC). New Zealand White (NZW) rabbits have favorable uterine anatomy, are prone to spontaneous EC, and have been previously used as animal models in gynecology. We sought to develop and validate an animal model for radiofrequency endometrial ablation (EA), and to evaluate the histopathologic outcomes of EA in a cohort of New Zealand White (NZW) rabbits. Methods: A feasibility study was initially conducted in 4 euthanized NZW rabbits using a prototype radiofrequency EA probe. Uterine specimens were reviewed (H&E staining) to determine EA settings that correlated with optimal thermal coverage and depth. Subsequently, 4 NZW rabbits were treated with a modified radiofrequency EA probe using power of 5W/cm 2 for 7-24 seconds. An algorithm was developed to determine appropriate EA parameters. Bilateral EA was performed via laparotomy in 5 rabbits (E218-E222) using 5.2mm, 6.1mm and 7.1mm x 100mm EA probes. All rabbits were screened with urinalysis, abdominal palpation, suction biopsies and hysteroscopy for EC prior to EA. Rabbits were euthanized 3 weeks following EA. Histopathologic analysis of post-ablation hysterectomy specimens was performed. Results: Using an EA algorithm, bilateral EA was successfully performed in 4 out of 5 rabbits. Endometrial sampling and hysteroscopy were feasible in all rabbits. No ECs were identified. Uterine anatomy was variable among rabbits. Uterine perforation occurred in E218 limiting interpretation of the results. Excessive thermal ablation and transmural necrosis occurred in E219 (4.5W/cm 2 for 26seconds (s) right horn (R) and 28s left horn (L). In E218, EA was aborted in E2210 due to irregular anatomy. EA was performed at 4.5W/ cm 2 for 20s (R), 24s (L) and 27s (R), 30s (L) in E221 and E222, respectively. Partial ablation was noted in E221 and E222. All rabbits tolerated the procedure without major complications. Conclusions: Radiofrequency EA can be successfully accomplished via laparotomy in NZW rabbits. However, EA probe size and energy settings must be tailored to each rabbit's anatomy. A balance between overtreatment and under treatment is needed. Future application of EA in rabbits receiving high dose estrogen may address the relationship between EA and EC. Methods: This is a case report of a 59-year-old woman diagnosed with UPSC by endometrial biopsy. She was noted to have cellulitis of the left lower extremity that did not improve with antibiotics. A skin biopsy demonstrated metastatic UPSC. After a review of the literature, we described the first case of UPSC presenting as cutaneous disease complicated by tumor lysis syndrome (TLS). Results: The patient received two cycles of chemotherapy with carboplatin and docetaxel. After the first cycle she experienced extensive skin breakdown, infection and pulmonary embolism. We performed aggressive management with broad spectrum antibiotics, wound care, and physical therapy allowing the patient to receive her second cycle. Prior to the planned third cycle, the patient was admitted with creatinine of 1.74mmol/L, potassium 5mmol/L, phosphorus 6 mmol/L, uric acid 12.9 mg/dl. Based on these findings she was diagnosed with TLS. TLS was treated initially in the ICU with hydration, recombinant urate oxidase, and IV antibiotics. With this aggressive management the patient recovered. She was then maintained on allopurinol. However, her performance status deteriorated rapidly. Conclusions: Cutaneous metastasis of UPSC is rare with only 3 cases reported. This is the first case reported of UPSC with skin metastasis complicated by TLS, after an attempt to administer chemotherapy. The aggressive behavior of this tumor, the rapid cell turnover, the size of the metastasis and the response to chemotherapy were the factors involved in the development of TLS. Cytological sampling was inadequate in 7 cases (5%). The K value between cytology and histology was 98.4% for benign and 85.7% for malignant lesions. Notch-1 revealed a changing expression pattern: absent in benign lesions, focal and marked in atypical hyperplasia and widespread and marked in cancers. Moreover Notch-1 expression was mild and focal in originally cyto-hystologycal benign lesions which turned into atypical hyperplasia during follow up. In cancer cases, ER-α and PR-β were widespread and markedly expressed either in the glandular or stromal layer. Conclusions: Cytological analysis could be used as a screening test, at least for women at high surgical risk. Notch-1+ER-α+PR-β expression could be predictive for the risk of endometrial malignancy even at an earlier stadium than hyperplasia and could be used to identify the glandular or stromal origin of cancer thus helping in identifying women at increased risk of malignancy. Objectives Uterine serous carcinoma (USC) is a biologically aggressive variant of endometrial cancer. In GOG 177, over-expression of the human epidermalgrowth-factor receptor-2 (HER2) was reported in up to 61% of USC by IHC, suggesting trastuzumab (T) (anti-HER2 receptor MAb) as a potential treatment option for advanced/recurrent USC over-expressing HER2. However in GOG-181B there was a lack of clinical response with T alone. Trastuzumab emtansine (T-DM1, Genentech/Roche) is a novel antibody-drug conjugate that has demonstrated clinical activity in patients with refractory HER2 positive breast cancer. We evaluated for the first time T-DM1 activity against HER2 positive USC cells in vitro.

Methods 15 primary USC cell lines were assessed by IHC and flow cytometry for HER2 protein expression. C-erbB2 gene amplification was evaluated using fluorescent-in-situ-hybridization (FISH). Sensitivity to T-DM1 and T-induced antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated in 5-hrchromium-release assays. T-DM1 and T cytostatic and apoptotic activity were evaluated using flow-cytometry.

High levels of HER2 protein overexpression by flow cytometry and IHC and c-erbB2 gene amplification by FISH were detected in 30% of the USC cell lines. T-DM1 and T were similarly effective in inducing strong ADCC against all primary USC showing HER2 overexpression (mean cytotoxicity ± SEM, 61.6 ± 5.3% versus 58.4 ± 5.74%, T-DM1 vs T, P= 0.686), while negligible ADCC was detected against USC cell lines with low/negative HER2 expression. In contrast, T-DM1 was dramatically more effective than T in inhibiting cell proliferation and in causing apoptosis of USC showing HER2 over-expression (mean number of viable cells ± SEM, 26.9 ± 12.2% versus 92.4 ± 8.8 %, T-DM1 vs T, P=0.004). Importantly, T-DM1 showed significant activity against USC with de novo or acquired resistance to T.Conclusion T-DM1 shows promising anti-tumor effect in HER2 positive USC cell lines and its activity is significantly higher when compared to T. T-DM1 therapy may warrant clinical trials for HER2 positive advanced/recurrent and/or refractory USC. There were no significant differences in ileus rates (2 v 2%), DVT (0 v 2%), PE (0 v 1%), lymphocyst creation (1 v 0%), wound dehiscence (16 v 10%), MI (0 v 0%), pneumonia (1 v 0%), or other significant post-operative morbidity (13 v 9%). LH was related to a slight improvement in survival, although this may be explained by the effect of other covariates. The results of a regression analysis and QOL will be presented. Conclusions: LH for endometrial cancer appears to be a safe, has similar morbidity, requires longer operating time but is associated with shorter hospital stay. The small survival benefit may be related to the effect of other covariates. 

The detection of nerve fibres in the endometrium of women with endometriosis 1 has lead to an increased interest in studying their relationship with infertility and pain. All studies so far involved in the description of neuronal structures have relied on conventional histological tissue sections. However, neuronal networks have rarely been studied in three-dimensions. Biological systems exist and operate in three-dimensional surroundings; therefore it makes good sense to gain access to the three-dimensional quantitative information, based upon observations made on three-dimensional projections.

Utilising thick (≥50µm) formalin fixed endometrial sections, low temperature heat induced epitope retrieval, immunofluorescence and Laser Scanning Confocal Microscopy in order to visualize and quantatively assess the three-dimensional relationships of endometrial neuronal structures. Digital images were acquired using a Nikon Eclipse E800 microscope equipped laser attachment.

Computationally reassembled image stacks provided three-dimensional reconstruction of neuronal architecture and glandular epithelium of uterine endometrial section. Additionally, demonstrated that the utilization of a lower temperature 60°C combined with a longer time period (18-24 hours) hasFor patients with endometrioid histology there was a trend toward survival advantage (stratified log rank test p=0.08), and there was a survival advantage for women with non-endometrioid histology (stratified log rank test p<0.001) who were identified to be hyperlipidemic/statin users. The hazard of death in those with hyperlipidemia/stain use is estimated to be 59% (OR: 0.41, 95% CI: 0.29, 0.58) of that of women without hyperlipidemia/statin after adjusting for age, histologic type, clinical stage, grade, and metformin use. Increasing age, stage, grade, and type 2 histologic type were associated with increased hazard of death. Conclusion: These data suggest a survival advantage for women with endometrial cancer who were hyperlipidemic/statin users. Research is needed to clarify the protective effect of statin use verses hyperlipidemia on survival from endometrial cancer.

Fenretinide: A Novel Treatment for Endometrial Cancer. Mary Ellen Pavone, Navdha Mittal, Saurabh Malpani, Julian Shink, J Julie Kim, Serdar E Bulun. Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA. Objective: To Investigate the therapeutic effects of fenretinide (4-HPR) in endometrial cancer. Background: The primary treatment for endometrial cancer is surgery, with chemotherapy and hormonal therapies being the treatment options for advanced or recurrent disease. New therapeutic options either as alternatives to surgery or for recurrent cancers are needed. Previous studies have indicated that retinoic acid (RA) may be therapeutic for endometrial cancer. Fenretinide, a synthetic retinoid, has emerged as a promising anticancer agent based on numerous in vitro and animal studies, as well as chemoprevention clinical trials. However, it is unknown whether fenretinide has anticancer effects on endometrial cancer. In addition, the molecular mechanisms explaining how fenretinde works is unknown. Methods: Ishikawa cells were treated with fenretinide and assays for viability, proliferation, and apoptosis were done. Genes involved in retinoid metabolism and trafficking were measured using real-time RT-PCR. Ishikawa xenografts were established in nude mice and antitumor activity of fenretinide was measured by monitoring tumor volume and tumor weights. Results: Fenretinide led to a sustained dose and time dependent reduction in cell viability and increase in markers of apoptosis. This compound significantly interfered with the growth in tumor volume and tumor weight in xenografts.Real time RT-PCR revealed an up-regulation of STRA6, indicating that this compound may work by increasing retinol uptake into cells. Taken together, fenretinide inhibits cell proliferation and induces apoptosis through increased retinol uptake. Conclusions: Fenretinide causes an increase in cellular apoptosis and a decrease in cellular viability in-vitro as well as a decrease in tumor volume in-vivo. This suggests that fenretinide may have an anticancer potential against endometrial cancer.

Aldehyde Dehydrogenase Activity Earmarks Cancer Stem Cells in Endometrial Cancer. Marten van der Zee, 1 Andrea Sacchetti, 2 Medine Cansoy, 3 Leen Blok, 4 Riccardo Fodde. 5 Cancer stem cells (CSCs) are considered to play key roles in the maintenance and malignant behavior of a broad spectrum of cancers. Moreover, they are thought to be resistant to conventional cytotoxic treatment and to underlie dissemination from the primary mass and the formation of distant metastases. Recently, it was found that patients with endometrial tumors that display high levels of aldehyde dehydrogenase (ALDH), a detoxifying enzyme found characteristic of many progenitor and stem cells, have a reduced survival when compared to patients with endometrial tumors with low ALDH levels.Based on this observation we hypothesized that ALDH activity could be used to enrich for CSCs in endometrial cancer. Here, we found that ALDH can be employed as a marker to enrich for CSCs in endometrial cancer cell lines and primary malignancies, as indicated by the increased tumor-initiating capacity of ALDH hi cells upon transplantation into immune deficient mice. Also, ALDH hi cells revealed increased clonogenicity and organoid-forming capacity when compared with the parental cell line and ALDH low cells. Notably, the number of ALDH hi cells in both human endometrial cancer cell lines and primary human endometrial tumors inversely correlates with their differentiation grade. Gene expression analysis of ALDH hi vs. ALDH low tumor cells revealed that the IL-6 receptor subunits CD126 and GP130 are differentially expressed (upregulated in ALDH hi ). On the other hand, ALDH low tumor cells express IL-6, i.e. the ligand