key: cord-0007069-snmf8cvt
authors: nan
title: Scientific Abstracts
date: 2015-12-30
journal: Reprod Sci
DOI: 10.1177/1933719115579631
sha: a9719670ba12812aa1c4df175570c6bad4a9d336
doc_id: 7069
cord_uid: snmf8cvt

nan

Obese P-value 

Levels of cortisol and hormones that regulate its bioavailability are lower in obese pregnancy suggesting decreased activity of the HPA axis, though studies of cortisol clearance are required. The findings offer a novel mechanism underlying prolonged pregnancy in obesity.

compared with women £20mmHg, this threshold was determined based on previous small published studies. A second analysis was performed for resting tone >25mmHg, the 75th percentile. Outcomes included 1 and 5 minute APGAR <7, level II or III nursery admission, cesarean delivery, and neonatal acidemia. Poisson regression was used to adjust for relevant confounders. RESULTS: Of 4890 eligible laboring women, 2565 (52%) had a baseline resting tone >20mmHg, and 1347 (28%) had a baseline resting tone >25mmHg. Women with a resting tone >20 were more likely to have a 5 minute APGAR <7 (aRR 1.66, CI 1.11-2.49); but, the overall rate was low occurring in 75 (3%) infants. There was no difference in the 1 minute APGAR <7, level II or III nursery admission, acidemia, or rate of cesarean. The resting tone group >25mmHg had higher rates of 1 and 5 Minute APGARs <7 (aRR 1.23, CI 1.01-1.49; aRR 1.92, CI 1.32-2.79), but otherwise resting tone >25mmHg did not impact outcomes. Sensitivity analyses limited to women with an IUPC revealed identical results. methyltransferase (DNMT) inhibitor 5-Aza-dC and HDAC inhibitor Trichostatin A for 24 h. The expression levels of genes were measured by Real-time PCR in both tissues and primary cells. RESULTS: Quantitative PrimePCR array indicated that more than twenty genes were significantly down regulatedincluding RAD51, BRCA1, and BRCA2. In contrast, CHEK1 and RAD9A were significantlyup-regulated in F compared to Myo. Interestingly, the expression levels of RAD51, BRCA1, and BRCA2 were also downregulated in F primary cells compared to Myo primary cells. In contrast to the effect of Trichostatin A treatment, treatment with DNMT inhibitor 5-Aza-dC markedly increased the expression levels of RAD51 and BRCA1 in F primary cells, suggesting that DNMT activity plays an important role in regulating the expression pattern of DNA repair genes in uterine fibroids. CONCLUSIONS: Impaired expression patterns of DNA repair signaling occur in uterine fibroids, which may lead to failure to repair DNA damage and therefore result in DNA instability. DNA hypermethylation is likely involved in the regulation of DNA damage repair related genes. Further investigation is warranted in determining the role epigenetic regulation plays in DNA damage signaling pathways with respect to uterine fibroid formation.

was utilized to compare the growth capacities of distal fallopian tube epithelial cells isolated from mice that underwent either tubal ligation or sham operation.

The human tubal ligated distal fallopian tube epithelia had a significantly reduced population of proliferating epithelial progenitors *Figure(s) will be available online. In the murine model, distal fallopian tube epithelial cells isolated after tubal ligation had a significantly reduced growth capacity in an in vitro organoid assay. CONCLUSIONS: We show that tubal ligation is associated with a lower number and reduced proliferation of distal fallopian tube epithelial progenitors. We believe that tubal ligation induced quiescence of fimbriated epithelium demonstrated by our data may have protective effects against accumulation of genetic changes involved in genesis of serous carcinomas. oxytocin (1.51, 1.04-1.82, P=0.01). However, there was no statistically significant effect of stretch when explants were incubated with retosiban (P=0.3 & 0.2, respectively). In low stretch, incubation with retosiban had no statistically significant effect on the response to either KCl or oxytocin (P=0.8 & >0.9, respectively). In high stretch, incubation in retosiban resulted in a statistically significant reduction in the response to both KCl (0.74, 0.60-1.03, P=0.046) and oxytocin (0.71, 0.53-0.91, P=0.008). The greater the effect of stretch on explants from a given patient, the greater was the reduction in response induced by retosiban in high stretch (r= -0.65 P=0.02 for KCl and r= -0.73, P=0.007 for oxytocin). CONCLUSIONS: Retosiban inhibits the pro-contractile effect of prolonged mechanical stretch on isolated human myometrium explants in vitro. Retosiban is a potential approach to preventing preterm birth in multiple pregnancy.

weakening we have demonstrated that the pro-inflammatory cytokine, tumor necrosis factor (TNF) (modeling inflammation), and separately, thrombin (modeling abruption) weaken FM in a concentration dependent manner. Recently we identified granulocyte-macrophage colonystimulating factor (GM-CSF) as a critical intermediate compound in both the TNF and thrombin induced FM weakening pathways: 1. TNF and thrombin both induce GM-CSF in the choriodecidua; 2. When GM-CSF action is blocked by neutralizing antibodies neither TNF nor thrombin elicit FM weakening; and, 3. GM-CSF itself induces FM weakening in a concentration dependent manner. GM-CSF is thus part of an overlap in the pathways of inflammation and of abruption induced FM weakening.

Here we examined inhibitory effects of progesterone (P), medroxyprogesterone acetate (MPA) and 17α-hydroxyprogesterone caproate (HP) on GM-CSF induced FM weakening. METHODS: FM (amnion (A)+choriodecidua (CD)) from uncomplicated term, repeat Cesarian patients, mounted in Transwell inserts, were preincubated without, or with 10-7M P, MPA or HP added to the CD side of FM. GM-CSF (250ng/ml) was then added to the CD side and incubated for 48h. Medium was sampled and explant tissues strength tested using our established protocol. Medium samples were first screened semiquantitatively using Protease arrays. Proteases exhibiting 2-fold or greater differences between treatment groups were confirmed by Western Blot. Hierarchical cluster analysis of measured proteins in the maternal serum identified a profile of biomarkers associated with poor VFI. The angiotensin 1 / angiotensin 2 (ang1/ang2) ratio and VEGF/VEGF-R ratio was elevated (ang1/ang2: 2-fold increase; p<0.05, and VEGF/VEGF-R: 2.5-fold increase; p<0.05). Serum leptin was also increased by 2.8 fold in women with poor VFI (p<0.05). Additional analysis in the group of women with a poor VFI showed increased ang1/ang2 ratio only in women developing a placental syndrome.

The results of this pilot study show that women with a poor placental vascularisation at 12 weeks have an abnormal profile of circulating biomarkers and are predisposed to a placental syndrome. We speculate that poor VFI combined with increased ang1/ang2 ratio at 12 weeks reliably identifies women at risk for a placental syndrome.

The Role of Aryl Hydrocarbon Receptor Activation in Fetoplacental Endothelial Cell Function and Fetal Growth Restriction. Anna Palatnik, Hong Xin, Emily J Su. Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA. INTRODUCTION: Maternal cigarette smoking is a well-established risk factor for fetal growth restriction (FGR) and is also associated with elevated fetoplacental vascular resistance. Nicotine, often considered a central culprit in adverse effects of smoking, has been shown to have beneficial effects on endothelial cell (EC) function. In contrast, the role of polycyclic aromatic hydrocarbons (PAHs), abundant toxins in cigarette smoke that activate the aryl hydrocarbon receptor (AhR) pathway, has not been investigated in human fetoplacental ECs. METHODS: Human placental villous ECs were primarily isolated (N=3), cultured, and treated with vehicle or benzo[a]pyrene (BaP), a key PAH in cigarette smoke. ECs were subjected to qRT-PCR, western blotting, enzyme immunoassays (EIAs), tube formation assays, cyclooxygenase-2 (COX-2) inhibition, and RNA interference against AhR. Statistical analyses were performed with one-way ANOVA followed by multiple comparisons testing (p<0.05 considered significant). RESULTS: Fetoplacental ECs exposed to BaP demonstrated a dosedependent increase in COX-2 mRNA (p<0.001) and protein (p=0.004). In contrast, BaP had no effect on expression of COX-1 or downstream prostanoid biosynthetic genes including prostacyclin synthase or thromboxane synthase. BaP also had no effect on expression of several other key vascular genes such as endothelial nitric oxide synthase and endothelin. Thromboxane levels were undetectable by EIA, whereas BaP surprisingly led to a dose-dependent increase in 6-keto-prostaglandin F1alpha (6-keto-PGF1α), the stable metabolite for prostacyclin. This effect was ablated by pre-treatment with NS-398, a COX-2 inhibitor (p<0.001).

Similarly, RNA interference of AhR both inhibited BaP-mediated induction of COX-2 and production of 6-keto-PGF1α (p<0.001 for both). From a structural perspective, BaP exposure inhibited fetoplacental EC tube formation, which was rescued by AhR knock-down (p<0.05). CONCLUSIONS: AhR activation via PAH exposure impaired EC tube formation in fetoplacental ECs. Furthermore, it resulted in COX-2 induction and surprisingly led to increased 6-keto-PGF1α production with undetectable thromboxane levels. This suggests that AhR activation may contribute to elevated fetoplacental vascular resistance via its inhibition of proper angiogenesis and not via production of vasoconstricting factors. Future studies are needed to further delineate the mechanisms by which fetoplacental angiogenesis is impaired. A series of studies has demonstrated high-grade serous ovarian adenocarcinoma can arise from fallopian tubes. The p53 signature in the tubal epithelium is focused as a primary change of carcinogenesis and DNA-double strand breaks (DSBs) are frequently observed in the area of the p53 signature. However, the mechanism of introducing DSBs in fallopian tubal epithelium has not been fully understood. Hydroxyl radical produced from Fenton reaction catalyzed by iron ion is a potent molecule capable of inducing DSBs. Because Transferrin, a carrier protein of iron ion, exists in follicular fluid and retrograde menstrual blood, we investigated the potential of transferrin as a DSBs introducing molecule in fallopian tubal epithelium. METHODS: Transferrin receptor 1 (TfR1) and 2 (TfR2) expressions in human fallopian tubal epithelium were assessed by immunohistochemistry.

Dr.Drapkin (Dana-Farber Cancer Institute) and A2780 ovarian cancer cells were treated with holo-transferrin and γH2AX expression levels were determined as a marker of DSBs. To uncover specific involvement of TfR1 and/or TfR2 in forming DSBs, small interfering RNA was employed. RESULTS: Immunohistochemistry revealed both TfR1 and TfR2 were expressed in the tubal epithelium. Transferrin treatment increased the level of γH2AX in both cells. Transferrin also potentiated an extent of γH2AX expression in the presence of hydrogen peroxide served as a substrate in Fenton reaction. Those results suggest transferrin is a key molecule for forming DSBs through Fenton reaction. Knockdown of TfR1, but not TfR2, selectively reduced the uptake of transferrin and inhibited an increase of γH2AX by transferrin compared with siRNA group. TfR1 knockdown also suppressed synergistic generation of γH2AX by transferrin and hydrogen peroxide. CONCLUSIONS: Transferrin is suggested to potentiate DSBs via TfR1 in cell lines including FTSEC. Considering follicular fluid or retrograde menstrual blood contains transferrin abundantly, we propose transferrin -TfR1 axis is one of the pathway for the generation of DSBs in human fallopian tubal epithelium.

protein expression of secreted frizzled related protein (sFRP1) that may be responsible for FGR. Given the importance of Wnt signaling in placental and embryonic development, we hypothesized that extracellular inhibition of Wnt signaling by sFRP1 could to lead to FGR. METHODS: To evaluate sFRP1 gene and protein expression in human placentas, we used RT-PCR and immunoblotting. Soluble sFRP1 overexpression was achieved by tail vein injection of an adenovirus expressing a truncated form of sFRP1 to CD1 pregnant mice on E15. Fetuses were weighed on E19, and proliferation in the murine placentas was assessed by Ki67 staining. To examine the effects of sFRP1 on trophoblast function, a Matrigel invasion assay was performed. Statistical analysis was performed using GraphPad Prism5 software, and a p value < 0.05 was considered significant. RESULTS: We found that smoking mothers had elevated gene expression (13 fold increase, p<0.024) and protein (3 fold increase, p< 0.002) levels of sFRP1 in their placentas. Adenoviral overexpression of sFRP1 in pregnant mice resulted in fetal growth restriction (virus-treated animals: 1267 mg ±14 mg compared to controls: 1416 mg ± 12 mg, p< 0.0001), decreased placental β-catenin expression, and histological evidence of increased karyorrhexis and decreased proliferation in the murine placentas. In vitro, recombinant sFRP1 inhibited Matrigel invasion by trophoblastic cells by more than fifty percent. CONCLUSIONS: Increased placental levels of sFRP1 in smoking mothers may alter trophoblast invasion and proliferation and contribute to fetal growth restriction. This work was supported by funding from the Flight Attendants Medical Research Institute (AW), and Howard Hughes Medical Institute (SAK).

responses to phenylephrine (PE) and Acetyl Choline (ACh). Statistics performed were Mann-Whitney or Two-way ANOVA. RESULTS: Ageing mice had fewer pups at E17.5 (p=0.03), more resorptions (p=0.007) and more late fetal deaths (p=0.044) per litter (con n=8, AMA n=10). The pups of ageing mice had lower fetal weights (p=0.0002) and reduced crown-rump length (p=0.0051), abdominal (p=0.0136), and head circumferences (p=0.0195). Ageing mice had increased placental weight (p=0.05) and lower fetal:placental weight ratio (p=0.0002). Uni-directional maternofetal clearance of 14 C-MeAIB was reduced in older mice (µl/min/g placenta; p=0.015; n=8/group) compared to controls. UA from ageing dams showed no difference with constriction to PE, but pre-constricted arteries exhibited increased relaxation to ACh when compared to controls (p=0.0091; con n=7, AMA n=5). CONCLUSIONS: Ageing affected pregnancy outcome in C57 mice resulting in more early and late fetal losses, smaller litter sizes and growth restricted offspring, similar to findings in human studies. Reduced fetal:placental weight ratio and placental amino acid transport suggests reduced placental efficiency. Altered placental transport and increased UA relaxation provide evidence of placental dysfunction, which may affect delivery of nutrients to pups. These data support the use of the ageing mouse as a model for investigating the underpinning mechanisms linking AMA and poor outcome seen in humans. (p<0.01). T 3 alone stimulated, and IGF-1 alone suppressed, p21. NH3 powerfully inhibited the stimulation of p21 in the 135dGA, but not in 100dGA CMs. Reduced TR expression by siRNA recapitulated the actions of NH3. NH3 treatment alone did not alter TR gene expression. CONCLUSIONS: NH3 blocks T 3 's ability to suppress fetal CM proliferation, providing strong evidence that T 3 binds TRs rather than using non-genomic mechanisms. Stimulated phosphorylation of ERK/AKT by T 3 were not suppressed by NH3, suggesting the activation of a nongenomic pathway. NH3 blocked the dramatic increase in phospho-ERK/ AKT by T 3 +IGF-1 suggesting this activation pattern requires a functional T 3 -TR ligand receptor complex. Increased p21 in the presence of T 3 is not inhibited by NH3 in 100dGA CM but is powerfully inhibited the older CMs. Conclusion: T 3 signals in fetal CM via genomic and non-genomic pathways according to maturation level. , is thought to mediate maternal stress (MS) effects on the fetus because catechol do not cross the placenta. However, MS in early pregnancy induces growth restriction and is associated with pronounced later-life programming effects despite limited fetal glucocorticoid receptor (GR) expression at this stage of development. We hypothesized that, in addition to CORT, catechol transfer MS indirectly to the fetus by a decrease of uterine blood flow (UBF). METHODS: Pregnant sheep chronically instrumented with uterine ultrasound flow probes and maternal and fetal catheters at 0.75 gestation underwent 2h of isolation stress. We used adrenergic blockade with labetalol to examine if the decreased UBF is CATECHOL mediated. RESULTS: Ewes responded to isolation with an increase in blood pressure (BP) from 84±3 to 95±3mmHg for 14min and heart rate from 90±4 to 121±7bpm for 36min (p<0.05). UBF decreased from 346±6 to 272±13ml. min -1 (p<0.05) reflecting uterine vasoconstriction. Maternal CORT and noradrenaline (NA) increased for 15min from 45±14 to 115±9nmol.L -1 and 10.2±2.0 to 27.9±7.0nmol.L -1 , respectively (p<0.05). Time course and relative magnitude of fetal CORT increase were similar to the maternal CORT increase. Maximum fetal CORT concentrations were 8.1±2.1% of those in the mother. Fetal NA increased from 0.6±0.1 to 1.4±0.4 nmol.L -1 and BP from 41.5±1.6 to 48.5±3.5mmHg (p<0.05). Although UBF only decreased for 36min (p<0.05), fetal NA and BP but not CORT remained elevated over 2h (p<0.05). Fetuses developed a delayed and prolonged shift towards an anaerobic metabolism in the absence of fetal P O2 and P CO2 changes. Adrenergic blockade prevented the stress-induced UBF decrease and, consequently, the fetal NA and BP increase as well as the shift towards anaerobic metabolism. Fetal CORT was transiently increased from 11±2 to 19±3mmol.L -1 immediately after begin of MS reflecting a labetalol rather than stress effect. CONCLUSIONS: A CATECHOL induced decrease of UBF transfers MS to the fetus indirectly which may explain programming effects of MS in early pregnancy in the absence of fetal GR expression. Fetuses respond to the decreased UBF with an endogenous CATECHOL increase and a shift towards anaerobic metabolism independently of oxygen supply. Both may contribute to the fetal growth restriction and programming effects seen after MS.

Fetal NK Cells Contribute To Late-Gestation Fetal Fitness. Beverly Strong, Amanda Lee, Lucas Turner, Helen Jones, Aimen Shaaban. Center for Fetal Cellular and Molecular Therapy, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. INTRODUCTION: While maternal natural killer (NK) cells have a well-described role in promoting normal placentation, little is known about the role of fetal NK cells in pregnancy outcomes. Previous studies suggest that a deficiency in fetal NK cells was associated with reduced mid-gestation placental size. Therefore, we examined the contribution of fetal NK cells to late-gestation placental development in a murine model of selective fetal NK cell depletion.

intrahepatic injection of NK cell depleting (PK136) or isotype control mAb into fetuses (B6Ly5.1/Ly5.2) at E14 via a maternal (B6Ly5.2) laparotomy. Recipients were harvested at E18 and E19 for histologic and flow cytometric analysis of the fetus and placenta. Offspring were tracked for survival and weight. Statistical comparisons were performed using a two-tailed student t test assuming unequal variances. RESULTS: Fetal NK cells were identified in the placenta at E18, a novel finding. In vivo depletion effectively eliminated NK cells from the fetal liver and placenta at E18 ( Figure A) . Maternal decidual NK cells were minimally affected (FigB) . Fetal NK depletion resulted in a dramatic reduction in fetal survival when compared to controls (19% vs. 61%, p<0.05, Figure C ). However, fetal NK depletion did not lead to in utero growth restriction as there was no significant change in pup or placental weight at E19. Placental architecture was abnormal as highlighted by CD31 staining ( Figure D ). *Figure(s) will be available online. CONCLUSIONS: Fetal NK cells play a critical role in late-gestation fetal fitness. Abnormal placental architecture following NK cell depletion suggests that fetal NK cells may be involved in late-gestation placental remodeling or fetomaternal tolerance. Further study in this novel system will facilitate a better understanding of the role for fetal NK cells in fetal development. The insulin-like growth factors 1 and 2 (IGF1 and IGF2) are known to play an important role in placental and fetal growth. We sought to investigate whether the placental expression and DNA methylation of IGF and their binding proteins (IGFBPs) is altered in women delivering small for gestational age (SGA; birthweight <10 th percentile for gestation) and large for gestational age (LGA; birthweight >90 th percentile for gestation) neonates compared to those delivering appropriate for gestational age (AGA) neonates.

We included 73 pregnant women with singleton pregnancies; 37 with AGA, 16 with SGA and 20 with LGA neonates. Placental samples were obtained at delivery and stored at -80 0 C. Laboratory techniques involved qPCR and DNA methylation in CpGs upstream of transcription start site (TSS) of IGF1, IGFBP1, IGFBP2 and IGFBP3. RESULTS: Compared to AGA group, placental expression of IGF1 gene was found to be significantly lower in SGA neonates but no different in the LGA group. There was no difference in placental IGF2 expression between the groups. Placental IGFBPs 1, 2 and 3 expression was significantly higher in SGA neonates and significantly lower in the LGA group. IGF1, IGFBP1, IGFBP2 and IGFBP3 genes were found to be hypermethylated in the SGA group as compared to controls. Whether these alterations cause growth restriction or are an associated finding requires further investigation *Figure(s) will be available online. CONCLUSIONS: The placental expression of IGFs and their binding proteins differ in fetal growth disorders. Placental gene expression of IGFBPs 1, 2 and 3 was inversely correlated with birth weight. Placental gene expression of IGFBP1, 2 and 3 is inversely correlated with DNA methylation at CPGs upstream of the TSS. Whether these alterations cause growth restriction or are an associated finding requires further investigation.

GCV effectively reduced the expression of TGFb3, VEGF and IGF, compared to Ad-LacZ control. There was no detectable adenovirus DNA from any tested tissues other than inoculated leiomyomas. CONCLUSIONS: Ad-RGD/GCV, a fibroid-targeted adenovirus, effectively reduces angiogenesis and inhibit ECM formation in a leiomyoma mouse model without any detectable viral leakage beyond the inoculated leiomyomas. Adenoviral-based gene therapy may be a viable localized non-surgical option for effective and safe treatment of uterine leiomyoma.

Progesterone Dependent Paracrine Activation of RANKL/RANK Signaling in Human Uterine Leiomyoma Stem/Progenitor Cells Drives Tumor Growth. Ping Yin, Molly B Moravek, John S Coon, Ecem Esencan, Matthew T Dyson, Serdar E Bulun. Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA. INTRODUCTION: Uterine leiomyomas (fibroids) occur in the majority of women in the US and cause severe morbidity and recurrent pregnant loss. Progesterone (P), via its receptor PR, is essential for leiomyoma growth. However, the cellular mechanisms for P action remain unclear.

Here, we seek to define the distinct roles of tumor stem/progenitor cells and other populations in leiomyoma tissue with respect to P-dependent cell proliferation and tumor growth. METHODS: Uterine leiomyoma tissues, obtained from 60 premenopausal women (ages 32-47), were used for flow cytometry analysis, antibodybased cell sorting, xenografting under mouse kidney capsules for tumor formation, mRNA (real-time PCR), and protein (western blot) expression. RESULTS: Using a real-time PCR based cell surface marker array, we found significantly elevated CD34 and CD49b gene expression in a leiomyoma cell population with high expression of the stem cell transcription factors NANOG, OCT4, KLF4, and SOX2. After removing dead and hematopoietic cells, the leiomyoma cells were sorted into 3 biologically distinct populations: CD34 + /CD49b + stem/progenitor cells (+/+, ~5%), CD34 + /CD49bintermediately differentiated cells (+/-, ~7%), and CD34 -/CD49bterminally differentiated cells (-/-, ~88%). Although +/+ cells are deficient in estrogen receptor alpha (ERα) and PR, they were essential for estradiol (E)+P-dependent tumor formation in the mouse xenograft model. Interestingly, RANK (receptor activated NFκB) was mainly expressed in +/+ cells, whereas its ligand, RANKL, was primarily expressed in +/-cells. In vivo, RANKL expression was dramatically elevated in leiomyoma vs adjacent myometrial tissues. E+R5020 (P agonist) increased RANKL expression by over 100-fold in leiomyoma but not in myometrial tissue explants. Additionally, RANKL treatment robustly activated both NFκB and ERK signaling, increased Cyclin D1 and BCL2 expression, and significantly expanded the leiomyoma stem cell population. CONCLUSIONS: CD34 + /CD49b + cells are functional leiomyoma stem/ progenitor cells. RANKL/RANK signaling, activated in response to E+P, mediates stem cell function in a paracrine fashion. Our study may reveal potential drug targets that can be translated to novel therapeutics to prevent or shrink leiomyomas. Patients were on no hormonal treatments for at least 3 months prior to surgery. Levels of tissue miR-29c and its predicted target protein were determined by QRT-PCR and western blot analysis. The mRNA and protein levels of predicted targets of miR-29c were assessed in primary leiomyoma smooth muscle cells (LSMC) following adenovirus transduction for overexpression or knockdown of miR-29c. The direct interaction of miR-29c and 3' untranslated region of predicted target genes was confirmed by luciferase activity assay. The effects of inhibitors of NF-kb, SP1 and DNA methyltransferase on miR-29c and its target genes expression were measured by QRT-PCR, western blot and siRNA strategy.

Our results indicate that the expression of miR-29c is suppressed in leiomyoma (100%) as compared to matched myometrium, and expectedly the expression of Col3A1 and DNMT3A which are predicted targets of miR-29c are significantly increased in LYO (72.2%, 72.2%, respectively). Using luciferase 3' untranslated region reporter assay, we confirmed Col3A1 and DNMT3A are direct targets of miR-29c in isolated LSMC. Gain-of function of miR-29c through adenovirus transduction in primary LSMC decreased mRNA and protein expression of Col3A1 and DNMT3A, while knock-down of miR-29c increased the expression of Col3A1 and DNMT3A. Treatment of LSMC with inhibitors of NF-kB (Bay 11-7082), SP1 (mithramycin A) and methyltransferase blocker (Zebularine) induced miR-29c expression, while decreasing mRNA and protein expression of Col3A1 and DNMT3A. Knock-down of NF-kB, SP1 and DNMT3A by siRNA, increased miR-29c levels and decreased the expression of Col3A1 in LSMC.

These results indicate that suppression of miR-29c in LYO plays a significant role in the fibrosis and aberrant DNA methylation that characterize this tissue. Targeting miR-29c expression through inhibitors of NF-kB, SP1 and methyltransferase could have therapeutic potential for treatment of LYO. In a prospective, randomized clinical trial, we previously demonstrated that UPA significantly reduced fibroid volume. Leiomyoma and myometrial tissue samples were collected from patients who underwent hysterectomy at conclusion of the trial. In this current study, we determined the impact of UPA treatment on extracellular matrix (ECM) expression in leiomyomas compared to placebo treated patients. METHODS: Surgical specimens from a total of 10 patients (5 placebo, 5 treated with 10mg UPA) were analyzed. Four common proteins related to ECM production were evaluated: fibronectin (FN1), versican, collagen 1A (COL1A), and TGF-b3. Gene transcription was assessed by normalized RT-PCR analysis. Immunohistochemistry (IHC) was performed with paraffin-embedded tissue. Western Blot was used for confirmatory testing with cryopreserved tissue. RESULTS: RT-PCR results showed up regulation of gene transcripts on average in COL1A of 1.6 fold and versican of 1.7 fold in treated vs. placebo, but with slight down regulation in FN1. Preliminary IHC data demonstrated down regulation of versican, FN1, and TGF-b3 proteins in leiomyoma tissue while not consistently altering COL1A concentrations.

Myometrial tissue did not demonstrate any significant differences in ECM expression with UPA treatment. Western blot analysis demonstrated 3-fold reduction of COL1A and versican proteins with no significant change in FN1 protein in treated compared with placebo-treated patient surgical samples. CONCLUSIONS: Our results suggest that ECM gene expression and protein production are decreased in leiomyoma tissue with UPA treatment, which may contribute to volume reduction seen clinically. Our findings characterize a molecular mechanism for UPA treatment, and provide insight into progesterone-mediated leiomyoma growth. Dissection of UPA mechanism of action could provide improved targeting of therapy. 1002670) is a highly potent and selective progesterone receptor modulator that markedly reduced the growth of human leiomyoma tissue in a preclinical model of uterine fibroids. This double-blind, multi-center trial in healthy women assessed the pharmacodynamics (PD) and safety of vilaprisan. METHODS: Healthy women aged 18-45 years, sterilized by tubal ligation, were randomized equally to daily vilaprisan (0.1mg, 0.5mg, 1mg, 2mg, 5mg) or placebo tablets for 12 weeks starting at day 1 or 2 of the menstrual period. The primary study variable was the proportion of women with amenorrhea or spotting only (non-bleeding rate) in a daily diary. A dose-response curve (median point estimates) and 90% credible intervals were estimated using a Bayesian model. Secondary variables included return of bleeding during follow-up, size of follicles on transvaginal ultrasound and hormone levels. Safety assessments included adverse events (AEs), laboratory parameters and vital signs. RESULTS: All randomized women received at least 1 dose of vilaprisan (0.1mg, n=12; 0.5mg, n=12; 1mg, n=13; 2mg, n=12; 5mg, n=12) or placebo (n=12) and were included in safety analyses; 69/73 women were included in PD analyses. Observed non-bleeding rates increased with vilaprisan dose: 0.1mg (0%), 0.5mg (27%), 1mg (80%), 2mg (100%), 5mg (91%); 0% in the placebo group. The Bayesian dose-response analysis estimated a non-bleeding rate >60% at doses of 1mg or higher with a probability of 95%. At 2mg the maximum rate was reached with a point estimate >90%. Return of menstrual bleeding was observed in all women ≤52 days after study drug discontinuation. Follicular growth was not suppressed during treatment, minimum average estradiol levels remained >40pg/mL. Low progesterone levels (£1.57µg/L) indicating ovulation inhibition were observed in >80% of women in the 2mg and 5mg groups. No serious treatment-emergent AEs or study discontinuations due to AEs were reported. The most frequent AEs assessed as drug-related were headache, ovarian cyst (follicle ³30mm), fatigue and abdominal pain. Clinically relevant changes in laboratory parameters or vital signs were not evident. CONCLUSIONS: These results supported initiation of an ongoing phase 2 trial assessing vilaprisan efficacy and safety in patients with uterine fibroids. Fibroids are a common problem, affecting 70-80% of women by the age of 50. Controversy exists over the effect of therapeutic options for fibroids on ovarian reserve. However, no studies have been done to determine the effect of fibroids on ovarian reserve prior to treatment. Furthermore, an association of fibroids with reduced ovarian reserve might suggest shared risk factors. Our goal is to determine if the presence of fibroids is associated with serum AMH levels. METHODS: This is a cross sectional study of 1654 AAW initially recruited to the Study of Environment, Lifestyle and Fibroids (SELF). Inclusion criteria for participation in SELF were AA race, age 23-35 years and no known diagnosis of fibroids. Serum AMH levels were obtained using an ultra-sensitive ELISA assay. Each participant underwent a pelvic ultrasound. The true AMH was used for mean and frequency calculations and log-transformed AMH was used as the outcome in simple and multiple linear regression models. RESULTS: 364 women had fibroids identified on ultrasound. The mean age of women with fibroids was 30 ± 3 years (mean ± SD) vs. 28 ± 4 years in women without fibroids. The mean AMH in women with fibroids was 3.46 ± 2.73 ng/ml vs. 4.14 ± 3.66 ng/ml in women without fibroids.

In the simple linear regression model, the presence of fibroids was not significantly associated with AMH (β= -.102; SE 0.066; P=.123). There was no association between AMH levels and the number of fibroids (β= -.159; SE .091; P=.083) or uterine volume (β= -.0002; SE .0002; P=.375).

These observations were unchanged after controlling for age, current hormonal contraception use and Body Mass Index (BMI). CONCLUSIONS: Our findings indicate that for this population of young AAW, the presence of fibroids is not significantly associated with lower serum AMH. These findings do not support a common pathway for fibroid development and decreased ovarian reserve. The absence of any significant effect of fibroids on ovarian reserve suggests that any effect on ovarian reserve following treatment is likely to be solely secondary to the intervention.

The Immunoproteasome: A Novel Therapeutic Target for Spontaneous Preterm Birth. Stella Liong, 1,2 Helena C Parkington, 3 Martha Lappas. Preterm birth remains one of the most important issues facing perinatal medicine today. The aetiology of a significant proportion of preterm births involves bacterial and viral infections. Tolllike receptors (TLRs) play an important role in mediating the early innate immune response to invading pathogens. TLR activation by bacterial and viral products induces pro-inflammatory cytokines in human gestational tissues. In non-gestational tissues, inflammation is mediated by a multisubunit complex known as the immunoproteasome.

The effect of human term and preterm labour on the expression of immunoproteasome subunits LMP2 and LMP7 in fetal membranes and myometrium was determined by Western blot. The effect of the LMP7 inhibitor ONX-0914 on TLR-induced pro-labour mediators in fetal membranes and myometrium was measured by qRT-PCR and ELISA. The effect of LMP7 siRNA knockdown in primary myometrial cells on cytokine-and TLR-induced pro-inflammatory effectors was measured by qRT-PCR and ELISA. RESULTS: Pro-LMP7 protein expression was increased with term labour in myometrium but not in fetal membranes. There was no change in LMP2 protein expression in fetal membranes or myometrium with labour. In myometrium, ONX-0914 significantly attenuated LPS (TLR4 ligand) and flagellin (TLR5 ligand)-induced mRNA expression and release of pro-inflammatory cytokines (IL-6, IL-8), COX-2 and prostaglandins. Furthermore, siRNA knockdown of LMP7 in myometrial cells significantly attenuated IL-1β, TNF-α, flagellin and poly(I:C) (TLR3 ligand)-induced expression and release of pro-labour mediators. NF-κB activity was also diminished in LMP7-deficient myometrial cells. Excitingly, preliminary data has shown in vitro administration of ONX-0914 suppresses contractility in labouring human myometrial tissue. CONCLUSIONS: Pro-LMP7 protein expression is increased in labouring myometrium. LMP7 enhances TLR and cytokine-induced pro-inflammatory and pro-labour mediators in human myometrium via NF-κB. Myometrial contractility was suppressed with LMP7 inhibitor ONX-0914. Given that inflammation is associated with spontaneous preterm birth, our findings indicate a therapeutic potential for agents that target LMP7 in preventing spontaneous preterm birth.

Innate Immune Response To Vaginal Commensals. Amy M Valent, 1 Margaret K Hostestter. 2 1 Ob/Gyn, University of Cincinnati, Cincinnati, OH, USA; 2 Pediatrics, CCHMC, Cincinnati, OH, USA. INTRODUCTION: Candida albicans colonization of vaginal epithelium elicits cytokines like IL-1beta that promote Th17 cells and mediate preterm labor as well as inhibits factors (CCL20) that induce regulatory T cells that are essential to sustain pregnancy. We characterized the cytokine milieu induced by lactobacilli in the presence/absence of C. albicans and showed that lactobacilli restore the balance between pro-and antiinflammatory states. METHODS: VK2/E6E7 vaginal epithelial monolayers were colonized with 10 7 cfu/mL of Lactobacillus crispatus or L. reuteri at 37°C for 24hr. C. albicans strain BWP17-WT,10 6 cfu/mL, was added for an additional 24hr. In separate experiments, epithelial monolayers were exposed to the supernatant of cultured lactobacilli followed by C. albicans colonization for 24hr. IL-1beta and CCL20 released into epithelial supernatants were measured by flow cytometry multiplex bead technology or ELISA and analyzed using ANOVA with Bonferroni correction. RESULTS: IL-1beta expression was not induced by L. crispatus or reuteri colonization of vaginal epithelium but was increased significantly by C. albicans colonization. *Figure(s) will be available online. L. reuteri supernatant inhibited IL-1beta expression in the presence of C. albicans by 50%. Both L. crispatus and L. reuteri reversed the inhibitory effect of C. albicans and significantly increased CCL20 expression. Supernatants from cultures of L. reuteri, but not L. crispatus, were as effective as live bacteria in increasing CCL20 expression. CONCLUSIONS: In vitro, lactobacilli reversed the effects of C. albicans by blunting the IL-1beta response and restoring CCL20 release. The ability (CD45 + CD11b + CD11c + MHCII + ), indicating a maternal pro-inflammatory response even at 90µg LPS/kg. Apoptotic inflammatory genes were also upregulated in placentas. CONCLUSIONS: A subclinical maternal inflammation in late pregnancy causes placental pathology, fetal loss, and alterations in maternal inflammatory immune cells in the absence of overt signs of systemic infection. This model will serve as a base for our future studies investigating if resultant fetal loss or perinatal brain damage is immunemediated.

Maternal Malnutrition Impacts the Holobiome and Gut Transporters. K L Connor, 1 E Bloise, 2 A Altrichter, 3 C Chehoud, 3 T DeSantis, 3 S G Matthews, 1,2 S J Lye. INTRODUCTION: ABC transporters P-glycoprotein (Abcb1a/b) and breast cancer resistance protein (Abcg2) protect against potentially harmful exogenous compounds, regulating transport across the gut into maternal and fetal blood. Gut microbes have co-evolved with the host, respond to environmental cues and interact with host genetics to impact immune and metabolic status. Disruption of host-microbiota (holobiont) homeostasis can alter these interactions. Previously, we showed maternal diet to restructure the gut microbiome. We hypothesised that the dynamic nature of the holobiont would be reflected in altered relationships between gut transporters and microbiota after changes in maternal diet. METHODS: Mice (n=7/gp) were fed a control diet during pregnancy (CON) or a 30% calorie reduced diet from d5.5-18.5 of pregnancy (CR) or a 60% high fat diet from 8 weeks before, and during, pregnancy (HF). At d18.5 maternal small intestine (SI) structure (H&E), proliferation (Ki67) and gut expression of Abcb1a/b and Abcg2 mRNA were measured (p<0.05). Microbial abundance in maternal caecal contents was determined at d18.5 by G3 PhyloChip TM . Associations between the microbiome and transporters (Adonis; p<0.05, Spearman; q<0.05) were determined. RESULTS: Maternal SI crypt depth was longer in HF and trended shorter in CR. In maternal villi % of Ki67-positive cells was greater in CR. Maternal SI Abcb1a expression was >3.7-fold higher in CR, but lower in HF, an effect replicated in HF fetal gut. Abcb1a expression and % Ki67-postive cells in maternal villi were positively associated. CR decreased maternal SI Abcbg2 expression. The maternal gut microbiome was associated with ABC expression. Abundance levels of 17 families were associated with Abcb1a and Abcg2 expression in opposing directions. T. erythraeum abundance and Abcg2 expression was positively correlated in HF mothers. CONCLUSIONS: This is the first report of a relationship between ABC transporter expression and microbial abundance levels and shows altered gut structure by maternal diet. These data have implications for drug/ nutrient transport and immune defence, which may be of concern in human pregnancies complicated by disease or malnutrition. Understanding the pathway to optimal adaptation to pregnancy and its outcomes requires a systematic approach that includes the role of the microbiome and its interactions with the host.

Oral INTRODUCTION: A decrease in lactobacilli and an overgrowth of facultative anaerobic bacteria represent a disturbed vaginal microbiota termed bacterial vaginosis (BV). Probiotic L. rhamnosus GR-1 and L. reuteri RC-14 (GR-1/RC-14) reduces BV recurrence and restores the indigenous lactobacilli in non-pregnant women. We hypothesized that oral GR-1/RC-14 would modulate the vaginal microbiota and cervicovaginal cytokines in pregnant women with an abnormal Nugent score. METHODS: Pregnant women (n=86) with Nugent score≥ 4 at 13 weeks gestation were randomized to receive orally either GR-1/RC-14 (5x10 9 CFU /strain) or placebo for 12 weeks. Vaginal swabs were collected at 13, 28 and 35 weeks gestation. Vaginal microbiota were analyzed by sequencing the V6 region of 16S rRNA, and 27 cytokines were measured with a multiplex assay. Significance was assessed with t test or Two-Way Repeated Measure ANOVA with Holm Sidak test. RESULTS: Among the 93 distinct bacterial species detected, L. iners, L. crispatus, Gardnerella (G.) vaginalis and Atopobium (A.) vaginae were the most abundant vaginal bacterial species. The Nugent score returned to normal in 30% of the women in both groups by 28 weeks. Compared to 13 weeks gestation, the relative abundance of 26 species including several Lactobacillus spp and BV-associated bacteria, G. vaginalis and A. vaginae decreased in both groups at 28 and 35 weeks. In contrast, 21 species including Finegoldia magna and Prevotella micans increased variably between groups. There was no difference in the vaginal microbiota and cervicovaginal cytokines between the two groups. However, the inflammatory cytokines IL-6, CCL3 and CCL 4 decreased and the antiinflammatory cytokines IL-4 and IL-10 increased by 28 and 35 weeks in the probiotic group but not in the placebo group. CONCLUSIONS: Oral GR-1/RC-14 at 5x10 9 CFU /strain twice daily for 12 weeks does not change the vaginal microbiota or cervico-vaginal cytokines significantly in pregnant women with an elevated Nugent score, and these profiles are dynamic across gestation. (FM) in this process is unknown. Inflammation can be negatively regulated through activation of the TAM receptors (Tyro3, Axl, Mer) by Gas6. The objective of this study was to determine the effect of viral dsRNA on FM responses to LPS and the involvement of the TAM receptors. METHODS: Human FM explants (n=8) from normal term deliveries without labor were treated with or without viral dsRNA (Poly(I:C); 20µg/mL) for 24h. LPS (1ng/mL) was added or not and FMs incubated for another 24h. Supernatants were measured for cytokines by multiplex.

Tissue RNA was measured for Tyro3, Axl, Mer and Gas6 by qRT-PCR. FM explants were pretreated with media, blocking antibodies to Tyro3, These cells were subjected to normoxia and hypoxia (O2=1.5%), treatment with vehicle or VEGFA, adenoviral ARNT overexpression (or adenoviral control vector), real-time PCR, western blotting, tube formation, and wound healing assays. Student's t-test and one-way ANOVA with post-hoc testing were utilized, and p<0.05 was considered significant. RESULTS: In contrast to ECs from appropriately grown controls, FGRadv ECs continued to express lower ARNT levels (p<0.05). Treatment of FGRadv ECs with VEGFA resulted in more tube formation branch points (p<0.01) and total length (p<0.05). Similarly, in comparison to adenoviral control vector, ARNT overexpression in FGRadv ECs rescued VEGFA expression (p<0.01) in the setting of both normoxia and hypoxia while demonstrating trends toward improved EC migration (p=0.07) and improved tube formation branch points (p=0.05) and total length (p=0.10). CONCLUSIONS: Impaired EC-mediated angiogenesis in placentas from FGRadv pregnancies is ameliorated by direct VEGFA treatment or by rescuing of ARNT expression via adenoviral infection. Future studies to determine whether our findings persist when incorporated into a threedimensional model are needed. Early human placental and embryonic development occur in a physiologically low oxygen environment supported by histiotrophic secretions from endometrial glands. In this study we compared the placental metabolomic profile of the first, second and third trimesters to determine whether the energy demands are adequately met in the first trimester. We investigated whether HIF-1α might regulate transcription during the first trimester. METHODS: First and second trimester tissue was collected using a chorionic villus sampling-like (CVS) technique. Part of each villus sample was frozen immediately and the remainder cultured under 2% or 21% O 2 ± 1 mM H 2 O 2, and ± the p38 MAPK pathway inhibitor PD169316. Levels of HIF-1α were assessed by Western blotting and VEGFA, PlGF and GLUT3 transcripts quantified by RT-PCR. Term samples were collected from normal elective caesarean deliveries. Metabolomic profiles of first, second trimester and term placental samples were compared. RESULTS: There were no significant differences in concentrations of ADP, NAD + , lactate, and glucose, and in the ATP/ADP ratio, across gestational age. HIF-1α or HIF-2α could not be detected in time-zero CVS samples (Fig. 1) . Culture under any condition (2% or 21% O 2 ± 1 mM H 2 O 2 ) induced increases in HIF-1α and HIF-2α ( Fig. 1 ). HIF-1α and HIF-2α was additionally detected in stressed specimens retrieved after curettage (Fig.1) . HIF-1α stabilisation was accompanied by significant increases in VEGFA and GLUT3 and decrease of PlGF mRNAs. These effects were suppressed by the p38 pathway inhibitor PD169316. *Figure(s) will be available online. CONCLUSIONS: Our data suggest that first trimester placental tissues are not energetically compromised, and that HIF-1α is unlikely to play an appreciable role in regulating transcriptional activity under steadystate conditions in vivo. However, the pathway may be activated by stress conditions. INTRODUCTION: Uterine Natural Killer (uNK) cells are key regulators at the human maternal-fetal interface. Aberrations in uNK cell function are implicated in placental syndromes such as fetal growth restriction and preeclampsia. Functionally, uNK cells change markedly between the 1 st and 2 nd trimester of pregnancy to accommodate pregnancy-demanding adaptations. The precise mechanisms that regulate changes in uNK cell function are still poorly understood. METHODS: We isolated uNK cells (CD56 bright CD16 -CD3 -) from 1 st (7-8 w, n=6) and 2 nd trimester (13-14 w, n=6) decidual tissue by flow sorting. Conditioned uNK cell medium was used for functional analysis, including trophoblast (HTR-8/SVneo) invasion and endothelial (HMEC-1) network formation assays. Transcript profiling of uNK cells was performed by whole genome microarray (Agilent). RESULTS: 2 nd trimester uNK cells potentiated trophoblast invasion that was 2.4-fold stronger than trophoblast invasion induced by 1 st trimester uNK cells (p<0.05). In contrast, endothelial network formation (total tube length) was stimulated to a similar extent by 1 st and 2 nd trimester uNK cells. Microarray analysis identified 140 differentially expressed genes in 2 nd trimester uNK cells, of which the majority clustered into a group of interferon (IFN) stimulated genes. To investigate if this cluster is up-regulated by type I (IFNα/β) or type II (IFNγ) IFNs, uNK cells were stimulated in vitro for 24h with the various IFNs. Stimulation with type I but not type II IFNs dose-dependently increased IFN marker genes (p<0.001). Functionally, type I IFNs changed uNK cells from 1 st to 2 nd trimester uNK cells. Type I IFNs enhanced 1 st trimester uNK cell mediated trophoblast invasion (p<0.001). CONCLUSIONS: Type I IFNs mediate the functional change between 1 st and 2 nd trimester uNK cells to promote trophoblast invasion. The capacity of 1 st and 2 nd trimester uNK cells to stimulate endothelial tube formation is similar. We hypothesize that type I IFNs play a key role in determining uNK cell functions at the maternal-fetal interface to regulate processes such as spiral artery remodeling and trophoblast invasion. The prevalence of obesity (BMI ³30) in American women of childbearing age is still increasing. Epidemiological studies have found positive correlations between obesity and placental dysfunction. Brain derived neurotrophic factor (BDNF) is necessary for placental development and fetal growth. The action of BDNF is facilitated through tropomyosin related kinase B (TRKB), a receptor kinase that induces several signaling cascades. BDNF levels are lower in plasma of obese adults however the effect of adiposity on placental BDNF is not known. We hypothesized that placentas from obese women (OB; pre-pregnancy BMI>30) will have decreased BDNF expression and attenuation of TRKB autophosphorylation when compared to placentas from lean women (LN; BMI 18.5-24.9). METHODS: Placental villous tissues were collected, in a randomized manner, following delivery by C-section at term with no labor, from LN or OB women with either male or female fetuses (n= 10 each group). BDNF mRNA expression, mature BDNF protein expression (inactive monomer and active homodimer), total TRKB protein expression, and TRKB phosphorylated at tyrosine 515 and 817 were quantified in total RNA and placental homogenates by RT-qPCR and Western blotting. Estrogen and its metabolites are important mediators of angiogenesis during pregnancy and are substantially reduced in preeclampsia. We have reported (Jobe et al Hypertension 2011) that 4-Metoxyestradiol (4-ME 2 ) induces proliferation in ovine uterine artery endothelial cells (P-UAECs). However, unlike Estradiol-17β (E 2 ) and 4-OHE 2 , 4-ME 2 does not induce proliferation through the estrogen receptors or the adrenergic receptors. We also demonstrated (SGI 2013 ) that the signaling pathways modulating E 2 , 4-OHE 2 and 4-ME 2 induced proliferation of P-UAECs specifically converged at the Mitogen Activated Protein Kinases (MAPKs). Furthermore, our observation that 4-ME 2 rapidly (< 15 min of stimulation) induced monophasic activation/ phosphorylation of p42/44 MAPK led to the hypothesis that 4-ME 2 induced proliferation is mediated by a G-protein Coupled Receptor (GPCR) or another membrane bound receptor. METHODS: P-UAECs from late pregnant ewes were pretreated with or without 10 mmol/L of G-protein Coupled Estrogen Receptor-1 antagonist (G36), Aryl Hydrocarbon Receptor antagonist (CH-223191) or the EGFR Kinase inhibitor (AG 4178) followed by either 4-ME 2 or E 2 treatment (0.1 nmol/L) as these receptors are known to interact with estrogen and seem the most likely to mediate 4-ME 2 responses. In addition, we pretreated with Pertussis Toxin (PTX) (100 ng/ml) followed by either 4-ME 2 or E2 (0.1 nmol/L). Cell proliferation was evaluated using Click-iT EdU (5-ethynyl-2'-deoxyuridine) microplate assay.

We observed that pre-treatment with G36, CH-223191, or AG 4178 did not block the elevated proliferation induced by either 4-ME 2 or E 2 . However, pretreatment with PTX completely abolished (P<0.05) proliferation induced by both 4-ME 2 and E 2 . CONCLUSIONS: Thus 4-ME 2 induced proliferation is mediated via a Gi-dependent mechanism and both E 2 and 4-ME 2 utilize similar signaling pathways regardless of their independent receptors. These novel data further support the hypothesis that the signaling pathways of estrogen and its metabolites converge regardless of their receptor. Future experiments are needed to elucidate the upstream points at which the receptors and signaling mechanism of estrogen and its metabolites are divergent. HL49210, HD38843, HL87144, HL117341, R25GM083252, T32HD041921-07 biopsies were collected from healthy pregnant (n=15) and (early onset) preeclamptic women (n=24) and AT-1R expression was examined in the placenta using immunohistochemistry and PCR. RESULTS: Plasma Hx activity increased (p<0.05), while monocyte AT-1R expression was lower (p<0.01) and plasma AT-1R levels were higher (p<0.01) in healthy pregnant women compared with nonpregnant women.

In women with preeclampsia as compared with healthy pregnant women, plasma Hx activity decreased (p<0.05), monocyte AT-1R expression increased (p<0.01) and plasma AT-1R levels decreased (p<0.05). In addition, placental AT-1R expression was also increased (p<0.01) in preeclamptic women versus healthy pregnant women. CONCLUSIONS: Our data suggest that also in vivo active Hx sheds the AT-1R from tissues, such as monocytes and placenta. Therefore active Hx may play a role in the development of the decreased responsiveness to angiotensin II in healthy pregnancy, while the decreased Hx activity in preeclampsia may contribute to increased angiotensin II sensitivity in this disease. MBL is a component of the innate immune system active against bacterial and viral infections. A single nucleotide polymorphism (SNP) at codon 54 is associated with decreased MBL production. IFNg is produced in the female genital tract by activated T cells and by neutrophils and is a marker of immune system activation. A SNP at position +874 is associated with elevated IFNg production. We evaluated whether these genetic variations were associated with occluded fallopian tubes. METHODS: Women with laparoscopy-confirmed tubal occlusions were assayed by gene amplification and analysis for polymorphisms in the MBL (47 women) and IFNg gene (36 women). Controls were women with patent tubes (82 for MBL and 49 for IFNg). Clinical data were obtained after completion of all laboratory studies. Differences in allele and gene frequencies between groups were analyzed by Fisher's exact test. RESULTS: Women with occluded tubes had a higher frequency of the variant MBL B allele (21.3 % vs. 9.1%, p = 0.0081) and the variant IFNg T allele (27.8% vs. 14.3%, p = 0.0341) than did controls. Homozygosity for carriage of the wild type AA genotypes in both the MBL and IFNg genes was identified in 67.3% of women with patent tubes as opposed to only 36.0% of those with blocked tubes (p = 0.0136). Antibody analysis to determine probable causes of blocked tubes in individual women is ongoing. CONCLUSIONS: Genetic variations in the genes coding for MBL and IFNg increase susceptibility to develop fallopian tube occlusion. Lowered circulating MBL concentrations reduce anti-microbial cell-mediated immunity allowing for an increased likelihood of infection. Elevated IFNg levels signal an elevated pro-inflammatory immune response that may contribute to enhanced epithelial cell damage and scar formation in the fallopian tubes. The findings illustrate that the combined effect of two independent functional SNPs increases the likelihood of an adverse event and suggest that women with these variations may benefit from enhanced treatment of genital tract infections. Exposures detrimentally impacting ovarian reserve are not fully characterized, and this knowledge gap prevents the development of effective interventions toward prevention of premature ovarian aging. We evaluated for an association between hydrosalpinx and reduced ovarian reserve in a clinical cohort study. To evaluate for causality, we assessed the impact of induced hydrosalpinx on ovarian reserve in a murine model of Chlamydia infection.

The records of 657 consecutive women presenting for infertility and undergoing ovarian reserve testing between 2008 and 2012 were reviewed. Twenty women with surgically confirmed hydrosalpinx and 60 healthy women from couples with male factor infertility met inclusion criteria. In a preclinical protocol, the ovaries of 8 wk old C57/ BL6 mice with Chlamydia-induced hydrosalpinx at 42 days post-infection and those of unexposed age-matched controls were fixed and sectioned for microscopic ovarian follicle counts performed by three independent investigators blinded to cohort.

The mean age, gravidity, parity and BMI were similar between clinical cohorts. Significant reductions in AMH and total AFC were observed in women with chronic hydrosalpinx as compared to healthy women. Consistent with the clinical observation, the mean antral follicle count of murine ovaries exposed to chronic, hydrosalpinx-associated inflammation was significantly decreased compared to that of age matched control ovaries (p = 0.03). *Figure(s) will be available online. CONCLUSIONS: This study provides clinical and preclinical evidence for a significant association between chronic hydrosalpinx and reduced ovarian reserve. In addition to counseling implications, these findings support the investigation of anti-inflammatory approaches toward the protection of the ovarian follicle pool in at-risk populations. Similarly, after E2, obese but not NW women had a 29% increase in LH pulse amplitude (p=0.04), and 55% increase in FSH max response to GnRH (p <0.01). Most cytokines were significantly higher at baseline in obese women and reduced after E2 (-6% for IL-1B, p=0.02; -5% for IL-12, p=0.02), but not changed in NW. *Figure(s) will be available online. CONCLUSIONS: After transdermal E2, obese women exhibited improved gonadotrope sensitivity and reduced pro-inflammatory markers. Among obese women who were ovulatory after E2, there was a significant improvement in corpus luteum function.

Analysis of Nanosorted Oocyte Mitochondrial Subpopulations Reveals Active But Reversible Repression of Non-Respiring Mitochondria. Kshama Chandrasekhar, Julie A MacDonald, Deanna M Navaroli, Jonathan L Tilly, Dori C Woods. Department of Biology, Northeastern University, Boston, MA, USA. INTRODUCTION: ATP production by mitochondria in oocytes is essential for fertilization and embryogenesis. Mitochondrial activity changes throughout oocyte development, with subsets of mitochondria actively respiring while others reside in a resting state. The regulatory mechanisms that determine which and how many mitochondria in an oocyte are active or resting are unknown. To address this, we developed a novel nanoscale-sorting platform, termed fluorescence-activated mitochondrial sorting (FAMS), to identify and purify mitochondria for characterization. METHODS: cumulus-oocyte complexes were collected from 3-monthold C57Bl/6 mice after superovulation and denuded of cumulus cells. Mitochondria were isolated from oocytes using a custom-engineered BD FACSAriaIII containing a hybrid dual forward scatter mechanism powered by a photomultiplier tube and diode that distinguishes very small particles from debris and noise. Extensive validation using a 200-3000 nm calibrated bead strategy was initially performed. After purification, mitochondria were labeled with the mitochondrial membrane dye, JC-1, which remains monomeric in resting mitochondria (and fluoresces green) but aggregates in respiring mitochondria (and fluoresces red), and then segregated by FAMS into 'respiring' and 'non-respiring' mitochondrial subpopulations based on JC-1 emission spectra. RESULTS: mitochondria with high (respiring) and low (resting) membrane potentials were purified as non-overlapping entities from oocytes. Post-sort validation confirmed that active mitochondria generated ATP upon provision of ADP substrate. To our surprise, equivalent numbers of resting mitochondria were just as capable of producing ATP in the presence of ADP as their active counterparts. CONCLUSIONS: Our development of a platform that enables purification of mitochondria, and mitochondrial subpopulations, from oocytes has revealed that mitochondria normally found in a resting state are fully competent to generate ATP in the presence of substrate, but are actively repressed inside oocytes by an as-yet unidentified factor(s) that specifically targets these, and not respiring, mitochondria. The identity of this factor(s), as well as how mitochondrial subpopulations change during oocyte maturation and embryogenesis, are currently under investigation.

Antiphospholipid Women with antiphospholipid antibodies (aPL) are at risk for adverse pregnancy outcomes associated with poor placentation and placental inflammation. aPL bind to trophoblast expressing beta 2 glycoprotein I (b 2 GPI); consequently aPL alter trophoblast function.

Our previous studies demonstrated that aPL trigger human first trimester trophoblast to express elevated levels of miR-146a via activation of Toll-like receptor 4 (TLR4) and consequently secrete elevated levels of IL-8. TLRs can be negatively regulated through activation of the TAM receptors by the endogenous ligand, Gas6. The objective of this study was to determine the role of the TAM receptor pathway in aPL-induced trophoblast miR-146a expression and subsequent IL-8 secretion.

Human first trimester trophoblast cell lines (Sw.71 and HTR8) were treated with or without aPL (mouse anti-human b 2 GPI mAb) or an IgG isotype control (20mg/ml) for 48-72h in the presence and absence of recombinant (r)Gas6 (100ng/ml). miR-146a expression was measured by qRT-PCR. Expression of the TAM receptors: Tyro3, Axl, and Mer, was evaluated by qRT-PCR and Western blot. Cellular and secreted Gas6 and secreted IL-8 were measured by ELISA RESULTS: aPL treatment significantly downregulated trophoblast cellular expression and secretion of Gas6 by 88.5±2.6% and 74.4±6.6%, respectively, when compared to untreated controls (p<0.05). The isotype control had no effect on trophoblast cellular or secreted Gas6 levels. aPL had no effect on Tyro3, Axl, or Mer mRNA or protein expression. aPL treatment of trophoblast cells significantly increased miR-146a expression

Xanthine Oxidase (XO) Inhibition as Therapy for the Interaction Between Developmental Hypoxia and Ageing in Promoting Cardiovascular Disease Risk. B J Allison, 1 A D Kane, 1 E J Camm, 1 C Lusby, 1 R Nevin-Dolan, 1 J L Tarry-Adkins, 2 S E Ozanne, 2 D A Giussani. . However, therapeutic targets for their interaction have been little explored. We hypothesised that in offspring of hypoxic pregnancy: 1) maternal treatment with the XO inhibitor allopurinol would protect against accelerated vascular ageing; and 2) this antioxidant protection would involve the maintenance of vascular telomere length. METHODS: On day 6 of pregnancy, Wistar rats (n=8 per group) underwent normoxia or hypoxia (14% O 2 ) ± allopurinol (30 mg.kg -1 .d -1 i.o. via jelly) treatment until day 20 of gestation. Pups were born naturally, culled to 8 to standardise litter size and males reared until 4 (early adulthood) or 15 (aged) months. At both ages, femoral artery endothelial function (response to acetylcholine; wire myography) and aortic telomere length (Southern blot) were determined in 1 male per litter. RESULTS: In controls, ageing impaired endothelial-dependent relaxation and increased telomere shortening (Fig. 1A) . Offspring of hypoxic pregnancy showed accelerated endothelial dysfunction already at 4 months ( Fig. 1A) . Allopurinol in hypoxic pregnancy rescued endothelial function in aged but not in young adults (Fig. 1B) . This protection by allopurinol on programmed endothelial dysfunction was matched by vascular telomere length maintenance in aged but not in young adults (Fig. 1B) . CONCLUSIONS: Maternal allopurinol protects against endothelial dysfunction in aged but not in young adult offspring of hypoxic pregnancy or in aged offspring of control pregnancy. The data show that the contribution of XO-derived oxidative stress in mediating programmed endothelial dysfunction only becomes significant when two inductors of oxidative stress synergise, namely developmental hypoxia and ageing. British Heart Foundation *Figure(s) will be available online. We hypothesized that poor nutrition during prenatal development (70% calorie diet) accelerates cardiac aging, leading to cardiac dysfunction characterized by impaired left ventricular (LV) diastolic function, similar to heart failure with preserved ejection fraction (EF) seen in elderly. Clinical measures of diastolic dysfunction include left ventricular peak filling rate (LVPFR) and the three-dimensional sphericity of the left ventricle (3DSI). METHODS: Cardiac MRI measures of EF, LV volumes, 3DSI and LVPFR in IUGR were obtained in baboons. Three groups of baboons were studied: adult control (CTR): N=21 (11M, 10 F), age = 5.9+1.3 yr., IUGR: N=9(4M, 5F), age = 4.6 + 0.5 yr., and normal elderly adults (ELD): N=11(5M, 6 F), age = 15.3 + 2.4 yr. All data were analyzed using CMR 42 cardiac image analysis package (Circle Cardiovascular, Calgary, ED). LV end-diastolic volume (EDV) and end-systolic volume (ESV) were referenced to body-surface are (BSA). Data are presented as Mean+SD.

One-way ANOVA with multiple comparison correction was used on systolic and diastolic measures, with p<.05 deemed significant. Linear regression and correlation were performed between 3DSI and LVPFR data for each of the three groups. RESULTS: Systolic left-ventricular (LV) function of subjects in all three groups was normal. There were significant differences between the three groups for 3DSI and LVPFR by ANOVA. ( Linear regression and correlation were performed between 3DSI and LVFR data for each of the three groups. CTR: r=0.01(p=NS); IUGR: r=0.881 (p<.02); ELD: r=0.724 (p<.05). CONCLUSIONS: All subjects had normal systolic function. 3DSI was associated with LVPFR in the IUGR and ELD groups, but not in the CTR group. These data are consistent with data showing increased fibrosis and changes in the distribution of titin isoforms in IUGR fetal baboon heart tissues (personal communication). These results support the hypothesis that developmental programming accelerates the processes leading to diastolic dysfunction, which are associated with cardiac aging. A maternal high fat diet predisposes offspring to later life obesity and impaired insulin sensitivity. Low-grade inflammatory processes are associated with high fat diet-induced obesity, however the impact of this "meta-inflammation" on offspring metabolic health is poorly understood. Our objective was to examine the effects of high fat diet-induced obesity on offspring growth trajectory and metabolic profiles, and whether maternal supplementation with the anti-inflammatory lipid, conjugated linoleic acid (CLA), is beneficial for mothers and offspring. METHODS: Female Sprague-Dawley rats were assigned to control (CD; 10% kcal from fat), control with CLA (CLA; 10% kcal from fat, 1% CLA), high fat (HF; 45% kcal from fat) or high fat with CLA (HFCLA; 45% kcal from fat, 1% CLA) diets 10 days prior to mating and throughout gestation and lactation. Subcohorts of dams/offspring were culled at gestational day 20 or postnatal day 24 (n=6/group) for plasma and tissue collection. Plasma concentrations of insulin and proinflammatory cytokines IL-1β and TNFα were determined by ELISA. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Remaining offspring (n=10 litters/group) consumed a chow diet ad libitum from weaning until adulthood (postnatal day 150) and DXA scans and oral glucose tolerance tests were undertaken. RESULTS: CLA, HF and HFCLA dams were heavier than CD dams throughout gestation (P<0.05). Plasma concentrations of IL-1β and TNFα were elevated in HF dams, but resolved in HFCLA dams (P<0.05). Male offspring from HF dams were smaller as fetuses, but displayed accelerated growth and impaired insulin sensitivity at weaning; this was ameliorated in HFCLA offspring (P<0.05). Adult male offspring from HF dams had greater fat mass compared to all other groups (P<0.05). In response to glucose challenge, adult male HF offspring displayed hyperinsulinemia compared to all other groups (P<0.05). CONCLUSIONS: A maternal high fat diet is associated with low-grade inflammation and programmed impaired insulin sensitivity in male offspring, evident as early as the weanling stage and persisting into adulthood. Maternal CLA supplementation normalised these effects, demonstrating that there are critical windows of developmental plasticity in which the effects of an adverse early life environment can be reversed.

Gestational Protein Restriction Causes Hyperglycemia By Affecting IRS-1 Tyrosine Phosphorylation and the Dysregulation of Akt-GSK3 Signaling in Adult Female Offspring. Chellakkan S Blesson, 1 Vijayakumar Chinnathambi, 2 Kunju Sathishkumar, 2 Chandra Yallampalli. 1 1 OB/GYN, Baylor College of Medicine, Houston, TX, USA; 2 OB/GYN, UTMB, Galveston, TX, USA. INTRODUCTION: Gestational low protein (LP) diet causes hyperglycemia and insulin resistance in adult offspring but the mechanism is not clearly understood. In this study we explored the role of insulin signaling in gastrocnemius muscles of gestational LP exposed female offspring to understand their role in LP induced hyperglycemia. METHODS: Pregnant rats were fed control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery. Normal diet was given to mothers after delivery and to pups after weaning until sacrifice. Glucose tolerance test (GTT) was done at 3months. Offspring were sacrificed at 4 months and muscles were treated with insulin ex vivo for 30 min. mRNA and protein levels of insulin signaling molecules were assessed at 4 months in gastrocnemius muscles.

: LP offspring were smaller at birth (5.3±0.1 g in LP vs. 6.2±0.1 g in controls) but showed rapid catchup growth by 4 weeks. GTT in LP offspring at 3 months showed elevated serum glucose levels (p<0.01; glycemia ∆AUC 342±28 in LP vs.155±23 in controls, mmol/L*120min) without any change in insulin but fasting glucose and insulin levels were similar. Pancreas of LP rats showed smaller islets with less number of β-cells compared to controls suggesting the reason for the absence of increased insulin production in response to hyperglycemia. In skeletal muscles, LP rats showed reduced tyrosine phosphorylation (Tyr608 and 895) of IRS1upon insulin stimulation due to the overexpression of tyrosine phosphatase SHP2. However, serine phosphorylation of IRS1 was unaffected at Ser 318 and 612 positions but was blunted at Ser307. Further, insulin induced phosphorylation of Akt (Ser473 and Thr308), GSK3α (Ser21) and GSK3β (Ser9) were severely diminished in LP rats. Interestingly, LP rats displayed an increased basal phosphorylation (inactive form) of glycogen synthase (Ser641). Unlike males, insulin induced Glut4 translocation was unaffected, even though basal levels of AS160 were upregulated. CONCLUSIONS: Our study shows that gestational protein restriction causes impaired insulin production and a series of phosphorylation defects in skeletal muscle in a mechanism involving IRS1, Akt and GSK. Thus, LP rats have an overactive GSK3 signaling indicating insulin resistance causing altered glycogen synthase activity leading to distorted glucose homeostasis. (GC) in the perinatal period is associated with alterations in glucose tolerance in animals and in people. A common feature in the fetal programming literature is the existence of sex differences in the response to the triggering effect. The aim of the present study was to further characterize the effects of antenatal GC on glucose handling by studying the effects of antenatal steroids and opbesity in males and females. METHODS: Pregnant sheep were treated with two IM doses of betamethasone (Beta, 0.17 mg/kg) or vehicle (CTR) 24-hs apart at 80 days gestational age and allowed to deliver at term. At 9 mo of age, female sheep were randomly allocated to be fed at either 100% of recommended nutritional allowance or ad libitum for three months. Sheep were chronically instrumented under general anesthesia to place intravascular catheters. Insulin sensitivity was evaluated by iv glucose tolerance test (IVGTT). Data are presented as Mean±SEM and were analyzed by ANOVA and/or two sample t test. RESULTS: Ad lib fed sheep gain > 50% of the original weight. In the lean state we found a significant sex difference on the effects of Beta on insulin sensitivity expressed as the Insulin:Glucose ratio ( I/G). A Beta effect was significant in female (Panel A) but not in male (C) sheep. Superimposed obesity had a significant effect on the I/G in of all 4 groups (B and D). Obesity significantly impaired I/G in CTR and Beta both in females and males (P<0.05 Two way ANOVA). Antenatal BM was associated with an elevation in plasma insulin values thus an increase in I/G. *Figure(s) will be available online. CONCLUSIONS: Our data show that in our animal model prenatal exposure to a single course of GC at 0.55 gestation has long-term effects in glucose metabolism regulation in a sex dependent manner. Female Beta sheep exhibit alterations in insulin levels and the insulin to glucose ratio. Surprisingly, males were not affected. However, using superimposed obesity as a "second hit" we were able to unmask the metabolic alterations associated with Beta in males and to induce a worsening of the effect in females. HL 68728 and HD 04784.

Obstetrical Mode of Delivery and Childhood Mental Illness and Behavior in a British Cohort. Eileen A Curran, 1 John F Cryan, 2 Louise C Kenny, 1 Timothy G Dinan, 3 Patricia M Kearney, 4 Ali S Khashan. 1,4 1 Obstetrics and Gynaecology, University College Cork, Cork City, Cork, Ireland; 2 Anatomy and Neuroscience, University College Cork, Cork City, Cork, Ireland; 3 Psychiatry, University College Cork, Cork City, Cork, Ireland; 4 Epidemiology and Public Health, University College Cork, Cork City, Cork, Ireland. INTRODUCTION: Given the high rates of birth by Caesarean section (CS), it is important to understand any possible long-term effects on child development. Our objective was to assess the association between planned CS and autism spectrum disorders (ASD), attention-deficit/hyperactivity disorder (ADHD) and behavioral difficulties among 7 year old children.

We used data from the Millennium cohort study, a representative UK cohort. ASD and ADHD were parent-reported based on a diagnosis by a doctor or health care professional. Behavioral difficulties were measured using the Strengths and Difficulties Questionnaire (SDQ) scores. The impact of mode of delivery and induction of labor on ASD, ADHD and abnormal SDQ scores were modelled using logistic regression, and adjusted for variables relating to pregnancy and delivery, maternal health and mental health and demographic/socioeconomic factors. 

We found no association between mode of delivery and ASD or ADHD in this cohort. Induction of labor was associated with behavioral difficulties in unadjusted analysis, but this relationship was not significant in the adjusted model. pregnancies while on oral contraceptives (OCs) and even more will purposefully conceive within a few months of stopping OC use. In both of these instances, there may be inadvertent exposure of the fetus to exogenous sex hormones circulating in the mother's body from continued or recent OC use. Yet, little is known about the health effects of OC use just before or during pregnancy on offspring. METHODS: Using multiple Danish registries, we conducted a cohort study of livebirths between 1996-2011 to investigate whether OC use up to three months before or during pregnancy was associated with an increased risk of major birth defects. We collected OC use from the National Prescription Register and conservatively assumed that a woman was exposed up to the date of her most recently filled prescription. We defined never users as those who never filled an OC prescription since the study period began in 1996. Given the high prevalence of OC use, never users are likely a highly selected group of individuals and therefore may not be the best reference group. Based on the previous literature, particularly on the proposed mechanism, we then modeled OC exposure using the following categories: >3 months before pregnancy onset (reference), 0 to £3 months before, and after pregnancy onset. RESULTS: Our study cohort was made up of 883,112 livebirths with 23,409 major birth defects. Compared to women with latest use of OC more than 3 months before pregnancy onset there was no increased risk of birth defects in women with use in the last 3 months before pregnancy onset (prevalence odds ratio, 0.97; 95% confidence interval, 0.92, 1.02) or after pregnancy onset [0.97 (0.87, 1.09). These results were consistent, even after adding pregnancies ending as stillbirths and induced abortions to the cohort [1.04 (0.88, 1.21)] and in a sensitivity analysis using propensitymatching [0.93 (0.82, 1.06)]. CONCLUSIONS: OC exposure just before or during a pregnancy is unlikely to cause a fetus to develop a major birth defect; this should reassure both patients and physicians.

The INTRODUCTION: National studies using county-level measures of hazardous air pollutants (HAPs) have shown associations with adverse pregnancy outcomes. The aims of our study were to 1) analyze whether such county-level data could provide enough geospatial discrimination to discern differences in exposures amongst a population of pregnant women receiving care within large urban area, and 2) analyze whether adverse pregnancy outcomes were associated with HAP exposures. METHODS: A population-based nested cohort study was conducted at a large tertiary care center in the third most populous city in the U.S. between August 2011 and July 2014. Patients with validated 5-digit zip codes that were within the city and bordering counties were included. HAP concentrations were obtained from the EPA's National-Scale Air Toxics Assessment (NATA) Ambient and ASPEN model. Non-parametric means testing and Spearman's correlations were performed using the 95th percentile exposure concentrations for each HAP in relation to a subject's county of residence and each individual's zip code, respectively.

Utilization Data analysis was based on the total weighted sample of women with either a primary or secondary ICD-9 code categorized as a menstrual disorder. Statistical analysis included weighted frequencies to account for complex sampling design and to allow for national estimates, as well as reporting of total cost of encounter. To examine factors associated with admission to hospital, we used multivariable logistic regression to generate odds ratios and associated 95% confidence intervals. RESULTS: There were 1,388,806 encounters for menstrual disorders from 2009-2011. Inclusion of primary and secondary diagnoses accounted for 81.9% of all encounters that documented menstrual disorders. The three most common diagnoses were dysfunctional uterine bleeding (38.8%), dysmenorrhea (17.8%) and excessive menstruation (12.6%). 94.9% percent of encounters resulted in treatment and release from the ED. The mean age of women presenting was 29.4 years (SE 0.02), with 38% percent of encounters from zip-codes with median household income <$39,000. The most common primary payer was Medicaid (32.9%) followed by private insurance (30%) and self pay (28.6%). The average total charges to ED services were $2,061 (SE $4.55). Menstrual disorder encounters cost a total of approximately $2.5 billion over the three years. Encounters for excessive menstrual bleeding were significantly more likely to result in admission compared to other menstrual disorders (Adjusted OR 7.1, 95% CI: 6.9-7.2). CONCLUSIONS: The majority of visits for menstrual disorders resulted in discharge from the ED and did not require inpatient admission. While the ED is often perceived as an easier and more visible access point for health care, helping women seek care in the appropriate settings could shift the burden of care and cost out of the acute setting to the ambulatory.

Charles E Wood, 1 Belen Rabaglino, 1 Eileen I Chang, 1 Maureen Keller-Wood. 2 1 Physiology & Functional Genomics, Univ. Florida Coll. Med., Gainesville, FL, USA; 2 Pharmacodynamics, Univ. Florida Coll. Pharmacy, Gainesville, FL, USA. INTRODUCTION: Estradiol circulates in the blood of fetal sheep as both unconjugated steroid (E) and estradiol-3-sulfate (ES). We have reported that the plasma concentrations of ES are 40-100 times higher than concentrations of E. Fetal brain regions express steroid sulfatase (STS: the enzyme that converts ES to E), leading us to propose that ES acts within the fetal brain after deconjugation and binding to the estrogen receptor alpha (ERα) . Both E and ES stimulate increases in fetal cortisol concentration. We have recently analyzed the transcriptomics response of the fetal hypothalamus to ES and concluded that the conjugated steroid does not likely act via the classical ERs. The objective of the present investigation was to determine if the cortisol response to ES is independent of the classical ER.

We performed two studies on chronically catheterized late gestation fetal sheep (125-133 days gestation). Plasma cortisol concentrations were measured by ELISA (Oxford Biomedical). ES and STX64 were purchased from Sigma Chemical Co.

In the first study, we infused ES or vehicle with or without a blocker of STS (STX64) for 2 days, measuring the fetal plasma ACTH and cortisol concentrations before and during infusion. STX64 (1 mg/ day) alone significantly increased fetal cortisol to 11.7±4.5, compared to 3.0±3.2, 6.8±3.5, and 9.0±3.5 ng/mL in vehicle, ES, and ES+STX64treated fetuses, respectively (p<0.05). In the second study, we chronically infused vehicle or STX64 into the lateral cerebral ventricle (0.02 mg/day) of the fetal sheep. STX64 significantly increased fetal plasma cortisol to 14.9±3.6 ng/mL compared to 5.9±1.6 ng/mL in vehicle-infused fetuses (p<0.05). CONCLUSIONS: We conclude that: 1) blockade of STS does not block the cortisol response to ES; 2) the cortisol response to ES is consistent with an action at a membrane receptor in the fetal brain; and 3) the cortisol response to STX64 is consistent with increased ES availability at the receptor. We have shown that estrogen (E 2 ) suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces C 19steroid precursors, e.g. dehydroepiandrosterone sulfate (DHAS), that are aromatized to E 2 within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators provide a mechanism by which E 2 regulates fetal adrenocortical development.

Fetal adrenal glands were obtained on day 165 of gestation (term = day 184) from baboons (Papio anubis) untreated (n=6) or treated on days 100-164 with the aromatase inhibitor letrozole (0.115 mg/kg BW/ day, n=6) which decreased serum E 2 by >95%, or letrozole and E 2 (each at 0.115 mg/kg BW/day, n=6). RESULTS: Weight of the fetal adrenal glands was 35% greater (P<0.01, ANOVA) in letrozole-treated (501±25 mg) than in untreated (366±12) mg) animals and restored by letrozole and E 2 administration. Levels of cyclin D1 mRNA, quantified by RT-PCR, in the whole fetal adrenal were increased 50% (P<0.05), number of fetal adrenal definitive zone cells immuno expressing cyclin D1 protein was increased 2.5-fold (P<0.05), while total number of Ki67 immunopositive fetal zone cells and fetal serum DHAS levels were elevated 2-fold (P<0.05) in letrozole-treated baboons and values restored to that in untreated animals by letrozole plus E 2 . However, fetal adrenocortical mRNA levels and immunoexpression of cyclin D2, cyclin-dependent kinases (Cdk) 2, 4 and 6, and Cdk regulatory proteins p27 Kip1 and p57 Kip2 were not changed by letrozole or letrozole ± E 2 administration. CONCLUSIONS: We suggest that E 2 controls growth of the fetal zone of the fetal adrenal by downregulating cyclin D1 expression by and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone ( Fig. 1) . We propose that E 2 restrains growth and function of the fetal zone via cyclin D1 to maintain E 2 levels in a physiological range during primate pregnancy. Supported by NIH R01 DK93950 and R01 HD13294. J DOHAD 4:328, 2013). However, vitamin C is a comparatively weak antioxidant and only effective in doses incompatible with human treatment. Therefore, there is an urgent clinical need in identifying alternative antioxidant therapy translatable to the human condition. We targeted mitochondria-derived ROS as a candidate therapy against fetal origins of cardiovascular disease during hypoxic development. We used the chick embryo as it the only species which permits isolation of the direct effects of therapy on the fetus, independent of effects on the maternal and placental physiology. METHODS: Fertile chicken eggs were incubated from day 0 under normoxia (N) or hypoxia (H; 14% O 2; term is 21 days). From days 13-18 eggs were treated daily with vehicle (water) or the mitochondrial targeted antioxidant MitoQ (0.2mg/kg/day). At day 19 embryos were euthanised by spinal transection. Cardiac and femoral vascular function was investigated using the Langendorff preparation and in vitro wire myography, respectively. A separate cohort of embryos was perfusion fixed (4% paraformaldehyde at 2.66kPa) and the cardiac morphology established by unbiased stereology. RESULTS: Chronic fetal hypoxia promoted brain sparing, impaired cardiac systolic function, endothelial dysfunction and led to dilated cardiomyopathy . Treatment of hypoxic embryos with MitoQ maintained brain sparing, while restoring endothelial and cardiac function and improving the LV lumen to wall volume ratio. CONCLUSIONS: Mitochondrial derived oxidative stress contributes to fetal cardiovascular dysfunction during hypoxic development. Targeting mitochondria-derived ROS is an attractive candidate therapy against fetal origins of cardiovascular disease in human high risk pregnancy. INTRODUCTION: Endometriosis affects 10% of reproductive aged women. Heterogeneity in pathogenesis and presentation presage poor response to conventional therapy. We found that transcription factor KLF11, implicated in uterine diseases, epigenetically regulates endometriotic fibrosis via deacetylation of Collagen1 promoter histones. KLF10, a KLF11 paralog, also recruits epigenetic mechanisms. Although frequently co-expressed, KLF10 and 11 drive divergent biological mechanisms as evidenced by inflammatory or fibrotic disease progression with surgical induction of endometriosis in Klf10 or 11-/-mouse models respectively. We investigate here the role of inflammation and immunology in endometriosis in vitro and in novel animal disease models. METHODS: Endometriosis was induced by suturing autologous 0.5 mm everted uterine segments on to the parietal peritoneum of 8 week Klf 10-/-,11-/-or wildtype mice. Lesion phenotype was assessed after 3 weeks by morphometry and mRNA expression. Immune cell infiltrate was assesed by peritoneal fluid flow-cytometry and IHC for FOXP3, interleukins 10, 17, and lineage representative CD antigens. This was supported by in vitro evaluation of Jurkat lymphocyte cells transfected with KLF10, EV, KLF10siRNA or control by PCR and western blot for FOXP3 expression.

In contrast to lesion regression in wildtype and prolific fibrosis in Klf11-/-mice, Klf10-/-developed cystic/vesicular lesions as seen in humans. Lesions had abundant neutrophil and scarce lymphocytic infiltrate. KLF10 epigenetically activates transcription factor FOXP3, also implicated in endometriosis, via promoter histone methylation. FOXP3 directs development of T-regulatory cells and adaptive immunity. Loss of Klf10 regulation in the disease model results in a compensatory, exaggerated innate immune response and vesicular lesions. Congruently, KLF10 upregulated and siKLF10 repressed FOXP3 expression in vitro. CONCLUSIONS: KLF11 is associated with human diabetes, leiomyoma and endometriosis. This is the first study to describe a role for its coexpressed and closely related paralog KLF10. Strikingly, rather than an expected compensatory role, KLF10 mediates distinct inflammatory pathways that are regulated epigenetically and hence potentially reversible with pharmacotherapy. Molecular characterization of distinct disease phenotypes offers a critical new dimension for individualized treatment of endometriosis, a complicated and heterogeneous disease. In order to investigate the function of specific genes in human models of endometriosis and ovarian cancer, a model is needed that will allow in vivo modification. We describe a method of genetic manipulation ex vivo of human tissue which will facilitate such studies.

For proof of concept, we made reporter Green Fluorescent Protein (GFP) and luciferase expressing lentiviruses. We collected fresh endometrial tissue from consented patients undergoing elective gynaecological surgery, removed excess blood and cut the tissue into 1mm 3 fragments. To allow adhesion of the lentiviral particles to the cells we maximised the concentration of virus by using 10 6 or 10 5 infectious units of GFP or luciferase lentivirus, respectively, in a total of 50µl of media supplemented with hexadimethrine bromide. To ensure survival of cells in 50µl we incubated the tissue overnight at 4°C. To allow transduction we added 2ml of serum-supplemented medium and incubated the tissue at 37°C for 2hr. We used confocal microscopy to serially image the tissue for GFP. *Figure(s) will be available online.

Once we validated the transduction protocol, we modified it by using luciferase lentivirus and injected the tissue subcutaneously into Balb/c nude mice. We monitored tissue growth using in vivo bioluminescent imaging for up to 6 weeks. RESULTS: GFP was undetectable by confocal microscopy 24 hours after lentiviral transduction but was detected to a depth of 150µm 72 and 96 hours post transduction. Similarly, we did not detect luciferase in the nude mouse 24 hours after tissue implantation, but we detected a bioluminescent signal after 96 hours. This could still be detected up to 6 weeks later.

We have shown that human endometrium can be genetically modified ex vivo and this modification persists for at least 6 weeks after tissue transplant. Thus luciferase can be introduced ex vivo and used to monitor endometriotic lesion or tumour growth. This can be adapted to allow investigation of the role of specific genes in models of endometriosis and ovarian cancer with human tissue in mouse models.

Platelets Are Responsible for Impaired Natural Killer Cell Cytotoxicity in Endometriosis. Sun-Wei Guo, Yanbo Du, Xishi Liu. Gynecology, Shanghai OB/GYN Hospital, Fudan University, Shanghai, China. INTRODUCTION: Natural killer (NK) cells play an important role in immune surveillance with the ability to neutralize target cells by recognizing major histocompatibility complex (MHC) class I-antigen. In endometriosis, decreased NK cell cytotoxicity has been well-recognized and is thought to be involved in its pathogenesis. The aberrant expression of both killer inhibitory receptors (KIRs) and killer activating receptors (KARs) in NK cells in women with endometriosis has been reported. However, the cause(s) for these aberrations remain unknown. Following our finding that platelets are an unindicted culprit in endometriosis development, we hypothesized that platelets may play role in NK cell dysfunction in endometriosis. METHODS: A mouse experiment using platelet and/or NK cell depletion, in vitro cytotoxicity assay of NK cells against endometrial stromal and epithelial cells, quantitative RT-PCR and fluorescence-activated cell sorting (FACS) analysis of KIR2DL1, NKp46 and NKp44 expression in NK cells, immunofluorescent staining of platelets and endometriotic stromal cells and MHC-I, measurement of TGF-b1 concentration by ELISA and the percentage of P-selectin-positive platelets in peritoneal fluid from 33 women with endometriosis and 36 controls by FACS, and in vitro cytotoxicity assay of NK cells co-cultured with peritoneal fluid taken from women with endometriosis. RESULTS: Thrombocytopenia reduced lesion size, along with immunoreactivity to TGF-b1, but this was reversed by NK cell depletion in mice with induced endometriosis. The NK cytotoxicity against endometrial cells treated with activated platelets was significantly reduced, and the expression of KIR2DL1 and NKp46, but not NKp44, was significantly altered in NK cells co-cultured with activated platelets. Endometrial cells were found to be coated with platelets, causing enhanced MHC-I expression. In peritoneal fluid, the percentage of activated platelets correlated with TGF-b1 concentration. Neutralization of TGF-b1 in NK cells co-cultured in the peritoneal fluid of women with and without endometriosis resulted in increased cytotoxicity. CONCLUSIONS: Activated platelets, through the release of TGF-b1 and also providing physical shield, inhibits KIRs while boosting KARs in NK cells, resulting in decreased NK cell cytotoxicity in endometriosis. Thus, our data provide a novel explanation for well-documented NK cell dysfunction in endometriosis, and suggest that anti-coagulant therapy may hold promise in treating endometriosis. INTRODUCTION: Endometriosis is defined by the presence of endometrial tissue outside the uterus. Non-human primates are the best animal models for endometriosis research because they menstruate and can develop endometriosis spontaneously. We aimed to discover altered protein expression after induction of endometriosis in baboons, to gain insight in the pathogenesis and find potential biomarkers. METHODS: Endometriosis was induced by intrapelvic injection of menstrual endometrium in 9 female adult olive baboons at the Institute of Primate Research (Kenya). Plasma samples were collected before and after induction in the luteal and menstrual phase, and were pooled accordingly. Pools were enriched for low-abundant proteins using ProteoMiner (Bio-Rad), applied onto the Thermo Scientific Orbitrap Q Exactive MS and analyzed with Progenesis LC-MS (Nonlinear Dynamics). For identification of pepetides, Mascot was used to search against the SwissProt database (April 2014).

In the luteal phase, 35 proteins were identified with at least 1.5-fold change in expression after induction of endometriosis, including 18 up-and 17 downregulated biomarkers. 2 proteins (Hepatocyte growth factor activator precursor, Fibulin-5 precursor) were only expressed before induction (not after induction), whereas 1 protein (Serotransferrin precursor) was only expressed after induction (not before induction).

In the menstrual phase, 52 proteins were at least 1.5-fold differentially expressed after induction, including 40 up-and 12 downregulated biomarkers. 2 proteins (Ig kappa chain V-I region BAN, Procollagen C-endopeptidase enhancer 1 precursor) were only expressed before induction (not after induction), and 8 proteins only after induction (not before): delta-aminolevulinic acid dehydratase, Keratin type I cytoskeletal 9, Glyceraldehyde-3-phosphate dehydrogenase, Tropomyosin beta chain, Peroxiredoxin-2, Alpha-actinin-1, Hemoglobin subunit theta-1 and Tropomyosin-1 alpha chain. CONCLUSIONS: Experimental induction of endometriosis in baboons resulted in altered protein expression levels. Some proteins were only expressed either before or after induction, especially during the menstrual phase, and can be strongly considered as potential endometriosis biomarkers. stimulates cell survival and is elevated in peritoneal fluid and serum from women with endometriosis. Inhibition of MIF activity reduces endometriotic implant survival in experimental animal models for the disease. MicroRNA-451 (miR451) is a putative regulator of MIF which is also mis-expressed in endometriosis. The objective of the current study was to examine the relationship among miR451 and MIF in eutopic and matched ectopic endometriotic tissue and to determine if miR451 regulates MIF expression and modulation of cell survival. METHODS: Endometrial biopsies and corresponding endometriotic implants were obtained from 30 women with (rAFS) stage III/IV endometriosis. Stage of menstrual cycle was taken into account but did not affect data allowing data to be collapsed across stage of menstrual cycle. miR451 and MIF transcript expression were examined by qRT-PCR while MIF protein expression was evaluated by Western blot. Cyclin E1 (CCNE1) and PTEN expression were assessed by qRT-PCR as an index of cell proliferation/survival. To demonstrate that miR451 regulates MIF expression and in turn cell survival, a human endometrial epithelial cell line transfected with miR451 (or non-targeting control) mimics was subjected to 3'UTR luciferase reporter assays as well as cell proliferation/ survival assays. RESULTS: miR451 expression was significantly increased in endometriotic implant tissue compared to corresponding eutopic endometrium (P<0.05; 14.3-fold increase) and this increase was associated with significantly (P<0.05) reduced levels of MIF mRNA and protein expression. Associated with this pattern of expression was a significant reduction (P<0.05) in CCNE1 expression and significant (P<0.05) elevation in PTEN expression in endometriotic implant tissue, consistent with a reduction in cell proliferation. 3' UTR reporter assays confirmed that miR451 targets the MIF 3'UTR and that miR451 mimics significantly reduced endometrial epithelial cell survival and this was also associated with reduced MIF protein expression. CONCLUSIONS: We conclude that elevated miR451 expression in endometriotic implant tissue may be part of an endogenous mechanism to limit endometriosis survival by reducing MIF expression and function. Endometriosis is an estrogen-dependent disease affecting reproductive women. Metformin could have a therapeutic effect on endometriosis through regulation of local estrogen production. The aim of this study was to investigate the molecular and cellular mechanism by which metformin regulated cytochrome p450 aromatase (CYP19A1) and steroidogenic acute regulatory protein (StAR) expression in human endometriotic stromal cells (ESCs). METHODS: ESCs and endometrial stromal cells (EMs) were derived from the cyst walls of ovarian endometriomas (n=6) and normal endometrial tissues (n=6) from the same individuals. ESCs were cultured with metformin and prostaglandin E2 (PGE2). Real-time RT-PCR was used to quantify CYP19A1 and StAR mRNA. Pregnenolone, progesterone, and estrogen production were measured by ELISA kits. Aromatase activity was determined by using aromatase activity assay. Western blot assay was done to detect StAR, AMP-activated protein kinase (AMPK), cAMP response element binding protein (CREB) and CREB regulated transcription coactivator 2 (CRTC2) protein expression. Coimmunoprecipitation and chromatin immunoprecipitation were used to assess the effect of the CREB-CRTC transcription complex. RESULTS: 1) CYP19A1 mRNA and StARm RNA were strikingly higher (approximately 512-fold and 264-fold, n= 6, p<0.01) in ESCs, whereas they were much lower or nearly absent in Ems. 2) Metformin downregulated the CYP19A1 and StAR mRNA expression (by 32% and 31.7%, n=3, p<0.05) stimulated by PGE2 (4.5-fold and 2.4-fold, n=3, p<0.05) in ESCs. 3) PGE2 induced CRTC2 translocation and enhanced its association with CREB to form a transcription complex that binded to the cAMP response element (CRE) region of CYP19A1 and StAR promoter. 4) Through increasing AMPK phosphorylation, metformin prevented the nuclear translocation of CRTC2 and then decreased the transcription complex formation between CRTC2 and CREB. This disruption attenuated the CREB-CRTC2 complex binding to the CYP19A1 and StAR promoter. CONCLUSIONS: We have demonstrated a detailed mechanistic analysis of CYP19A1 and StAR expression regulated by metformin in ESCs. Our data highlighted a role for CRTC2 in the mechanism by which metformin inhibited CYP19A1 and StAR expression.

Notch Signaling Is Activated in Inflammation-Induced Preterm Labor and Its Inhibition Reduces the Pro-inflammatory Response. Varkha Agrawal, 1 Mukesh K Jaiswal, 2 Sahithi Parmarthy, 2 Kenneth D Beaman, 2 Emmet Hirsch. 1 1 Obstetrics and Gynecology, NorthShore University HealthSystem, Evanston, IL, USA; 2 Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA. INTRODUCTION: Notch signaling is evolutionarily conserved and critical for cell-fate determination, differentiation and many other biological processes. Notch signaling pathways exert effects throughout pregnancy, playing an important role in placental development and trophoblast function. Notch signaling is activated in response to tolllike receptor (TLR) ligands, which amplify the inflammatory response by enhancing NF-κB signaling. To identify the role of Notch signaling in preterm labor (PTL), placental Notch receptor (Notch1), its ligand (Delta-like (Dll)1) and the Notch-induced transcriptional repressor Hey1 were assessed. METHODS: PTL was initiated on gestation day 14.5 by one of two methods: 1) inflammation-induced (IPTL): intrauterine (IU) injection of lipopolysaccharide (LPS, a TLR4 agonist); and 2) non-inflammation (hormonally) induced (NIPTL): subcutaneous (SC) injection of the progesterone receptor antagonist mifepristone. Placentas were collected from treated and control animals 10 hrs after IU or SC injection. Singlecell suspensions of placentas from gestation day 14.5 mice were also prepared and treated with either LPS or PBS with and without gamma secretase inhibitor (GSI) (an inhibitor of Notch receptor processing) ex vivo for 10 hrs. RESULTS: Notch1, Dll1 and Hey1 were significantly elevated in placentas of IPTL mice but remained unchanged in NIPTL compared to respective controls. In placental cells cultured ex vivo, GSI significantly diminished the LPS-induced expression of TNF-α and IL-6. CONCLUSIONS: Taken together, these results show that in IPTL (but not NIPTL), placental Notch signaling is activated. Inhibition of Notch signaling in placental cells ex vivo suppresses the pro-inflammatory response induced by LPS. Thus, suppression of Notch signaling in IPTL might be a useful method of suppressing inflammation-induced preterm labor.

RU486, maximal cervical dilation was increased modestly with ER agonist (from 7 ± 1.1 to 10.8 ± 1.0 DPN; 11.8 ± 1.3 mm, PPT) and dramatically with combination treatment (13.1 ± 0.5 mm, p < 0.01). Similarly, cervical stiffness decreased from 0.08 ± 0.01 to 0.053 ± 0.028 (PPT), 0.055 ± 0.013 (DPN), and 0.026 ± 0.006 N/mm with combination. These dramatic changes in cervical biomechanics, however, were not accompanied by increased length of the pubic ligament resulting in dysfunctional and difficult labor. Importantly, IL-8 and COX-2 gene expression were induced 7-and 5-fold (p < 0.05) in cervical stroma with combination treatment but not with RU486 or ER agonist alone. 15-PGDH was also decreased 6.5-fold with combination treatment. Interestingly, ERα and ERβ mRNA levels were unchanged. CONCLUSIONS: Collectively, these data indicate that functional progesterone receptors and local estrogen inactivation are crucial for cervical competency during pregnancy. We suggest that progressive loss of cervical PR and increased estrogen responses culminate in cervical ripening and dilation both at term and preterm. Our primary objective was to investigate changes in amniotic fluid (AF) telomere fragments as an indicator of fetal cell senescence in term labor. We also investigated the mechanistic role of short telomere oligomers (T-oligos) in inducing senescence and inflammation. METHODS: In this cross sectional nested cohort study, telomere fragment concentrations were quantitated by qPCR in amniotic fluid samples from subjects at term in labor (L) (n=50) and term not in labor (NIL) (n=51). In vitro models, using primary amnion cells and myocytes were treated with 40uM telomere mimetic sequence (T-oligo, [TTAGGG] 2 ), or control (C-oligo, [AATCCC] 2 ). p38MAPK, senescence activation phenotype, and inflammatory cytokines were examined using western blots, senescence-associated β-Galactosidase [SA-β-Gal] staining, and ELISAs for IL-6 and IL-8, respectively. COX2 expression in myocytes was determined by qPCR. Generalized linear regression and ANOVA were used for statistical analysis. RESULTS: Telomere fragment levels were higher in L [Median 2.3 (IQR2.1)] compared to NIL [1.7 (1.5 ]. There was a significant association between telomere fragments and L compared to NIL after adjustments (p=0.045). In amniocytes, T-oligos induced p38MAPK activation, increased SA-β-Gal positive cells, and higher IL-6 and IL-8 compared to C-oligos. This effect was minimized by coincubation with a p38 inhibitor (SB203580). p38MAPK but not SA-β-Gal was activated by T-oligos in myocytes; however, they increased cytokine and COX-2 expression. CONCLUSIONS: Telomere fragment concentrations are significantly higher in L than NIL AF. T-oligos cause p38MAPK induced amnion cell senescence. We postulate that at term, physiologic senescence of fetal amnion cells generates telomere fragments that serve as a signal to initiate parturition by enhancing inflammation or directly activating myocytes.

Restriction in Mice. F Rosario, 1,2 I Aye, 2 TL Powell, 3 T Jansson. (FD) during pregnancy is associated with neural tube defects and IUGR; however the underlying mechanism remains to be fully established. The mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 signaling pathways regulate cell growth, proliferation and metabolism. We recently discovered that mTOR functions as a novel folate sensor in cultured primary human trophoblast, MCF-7 and HEK293 cells (Reprod Sci 19 Suppl: 380A) We hypothesized that maternal FD inhibits placental mTOR signaling and amino acid transporter activity and causes fetal growth restriction in vivo. METHODS: ICR female mice were fed a control diet (folic acid (FA), 6 mg/kg) or a folate-deficient diet (FA 0 mg/kg) for 6 wks prior to mating and throughout pregnancy. At E18.5, dams were killed and placenta, maternal and fetal organs were collected. Trophoblast plasma membrane (TPM) vesicles were isolated from placentas. The activity of mTOR signaling was assessed by the determining phosphorylation of downstream targets for mTORC1: T389-S6K, S235/236-S6 ribosomal protein and T37/46/70-4EBP-1 and mTORC2: S473Akt and S422-SGK1) using Western blot. System A and L expression and activity were measured in TPM. RESULTS: FD mice had lower serum folate (-60 %) and packed cell volume (PCV, -12 %). However, FD did not affect maternal fasting blood glucose, food intake or weight gain in pregnancy. At E18.5, fetal weight in the FD group was decreased (-17 %, p<0.05), whereas placental weight, litter size and crown rump length were unaltered. Maternal FD inhibited placental mTORC1 and mTORC2 signaling and decreased TPM System A and L amino acid transporter activities and transporter isoform expression. FD decreased the weight of maternal liver, heart, spleen and kidney and inhibited mTORC1/C2 signaling in maternal heart, liver and muscle. Furthermore, maternal FD decreased fetal PCV (20%), heart and liver weight and inhibited fetal heart mTORC1/C2 signaling. CONCLUSIONS: We report for the first time that maternal FD inhibits placental mTOR signaling and amino acid transporter expression and activity resulting in fetal growth restriction. Because we have previously shown that trophoblast mTOR inhibition is mechanistically linked to down-regulation of amino acid transport, our data provide a novel link between maternal folate availability and fetal growth.

accumulation in lipid droplets, detected by Oil red O staining. Mean placental triglyceride content of the CGI-58 KO (24.33 ± 6.55 nmol/ mg) was significantly (p<0.0001) higher than the ATGL KO (6.62 ± 1.76 nmol/mg) or wild type (0.99 ± 1.00 nmol/mg). Importantly, accumulating triglycerides in the CGI-58 KO placenta were enriched for LCPUFAs, when compared to wild type placenta. This finding is attributed to CGI-58 deficiency, because placenta-selective overexpression of CGI-58 in the CGI-58 KO background abolished triglyceride accumulation and the selective enrichment of LCPUFAs. CONCLUSIONS: CGI-58 plays a central role in triglyceride storage and the selective trafficking of LCPUFA in the placenta. Supported by Kobe University, Japan (MM) and NIH P01HD069316 (YS). Maternal obesity during pregnancy results in an adverse intrauterine environment and affects placental function. We have shown that maternal obesity induces oxidative stress in the human placenta and decreases syncytiotrophoblast mitochondrial respiration. Sodium selenite (NaSe) binds to the active site of the selenoprotein, antioxidant enzymes glutathione peroxidase and thioredoxin reductase and increases their activity. We tested the hypothesis that NaSe could improve mitochondrial respiration in isolated trophoblasts under conditions of maternal obesity and oxidative stress. METHODS: Placental tissues were collected at term (~40 weeks) via cesarean section prior to labor to avoid the confounding effects of labor on induction of placental oxidative stress s from two groups of patients based on pre-pregnancy body mass index (BMI): lean -BMI 22.3+0.2 (n=5) and obese -BMI 36.0+0.3 (n=4) and with matched gestational ages. Cytotrophoblasts were isolated, plated and allowed to syncitialize. At 48 hours, cells were treated with 50nM, 100nM, 200nM, or 400nM NaSe for 24 hours with subsequent addition of 400uM H 2 O 2 for 2 hours to induce oxidative stress. Mitochondrial respiration was measured with the XF24 Extracellular Flux Analyzer from SeaHorse Bioscience. RESULTS: Maternal pre-pregnancy obesity significantly decreased (P<0.05) mitochondrial respiration parameters as previously reported.

In syncytiotrophoblasts from placentas of lean mothers, H 2 O 2 treatment significantly decreased (P<0.05) basal respiration, ATP coupled respiration, maximal respiration, and spare capacity compared to control cells, an effect that could not be prevented by pretreatment with NaSe. However in syncytiotrophoblasts from placentas of obese mothers, basal and maximal respiration were significantly decreased (P<0.05) with H 2 O 2 alone while 50 and 100nM NaSe prior to H 2 O 2 had no effect, but 200nM NaSe prior to H 2 O 2 prevented the decrease. There were no differences in ATP coupled respiration and spare capacity in the presence of H 2 O 2 alone or with NaSe+H 2 O 2 in trophoblast from an obese pregnancy. CONCLUSIONS: NaSe protects trophoblasts from oxidative stress only under conditions of maternal obesity and potentially could serve as an intervention to restore mitochondrial respiration. Antioxidants such as NaSe may improve both mitochondrial and placental dysfunction in complicated pregnancies. INTRODUCTION: Human amnion fibroblasts produce abundant prostaglandins (PG) toward the end of gestation, which is one of the major events leading to parturition. Cyclooxygenase-2 (COX-2) is an inducible key enzyme catalyzing PG production. Glucocorticoid receptor (GR) and cAMP response element binding protein (CREB) have been shown involved in the induction of COX-2 expression by glucocorticoids in human amnion fibroblasts. However, whether other transcriptional factors also participate in the induction of COX-2 expression by glucocorticoids is not known. Signal transducer and activator of transcription 3 (STAT3) is reported to interact with GR in the regulation of a number of genes. Whether STAT3 could interact with GR to induce COX-2 expression in human amnion fibroblasts is unknown. In this study, we investigated the role of STAT3 in the induction of COX-2 expression by cortisol in human amnion fibroblasts. METHODS: Primary human amnion fibroblasts were isolated from amnion collected at c section without labor at term. The phosphorylation level of STAT3 was examined with Western blotting after treatment with cortisol in the presence or absence of protein kinase A inhibitor H89 and SQ22536. The role of STAT3 in the induction of COX-2 expression by cortisol was studied with siRNA-mediated knock-down and STAT3 inhibitor S3I-201. Chromatin immunoprecipitation (ChIP) was conducted to study the binding of STAT3 to COX-2 promoter. The interaction of STAT3 and GR were examined with co-immunoprecipitation (co-IP). RESULTS: Cortisol increased the phosphorylation level of STAT3 in a time-dependent manner with the maximal phosphorylation at 6 h. The phosphorylation of STAT3 by cortisol was attenuated by protein kinase A inhibitor H89 and SQ22536. Furthermore, the induced expression of COX2 by cortisol could be blocked by either STAT3 inhibitor S3I-201 or knockdown of STAT3 expression. ChIP showed that cortisol increased the binding of STAT3 and RNA polymerase II to COX-2 gene promoter.

Co-IP revealed GR and p-STAT3 in the same nuclear protein complex upon cortisol stimulation. CONCLUSIONS: Cortisol increases phosphorylated STAT3 level via activation of protein kinase A, and cortisol enhances the binding of STAT3 to the COX-2 promoter thereby stimulating COX-2 expression in human amnion fibroblasts. STAT3 is one of the transcriptional factors involved in the induction of COX-2 expression by cortisol in human amnion fibroblasts. INTRODUCTION: Viral infection of the placenta is associated with fetal inflammation and adverse pregnancy outcomes. There have been limited studies on how placental macrophages in the villus and adjacent fetal umbilical endothelial cells respond to a viral insult. This study aims to evaluate the communication between Hofbauer cells (HBCs) and HUVECs, and the activation of the later, during viral infection. METHODS: Human HBCs (n=4), isolated from normal term placentas, were uninfected or infected for 1 h with murine gammaherpes virus (MHV)-68 (10 5 PFU/ml), fresh media was added, and after 24 h conditioned media (CM) was collected and IL-1β measured by ELISA. HUVECs were then exposed to HBC CM for 24 h and mRNA levels of IL-8, VCAM-1, ICAM-1, and E-selectin (i.e. neutrophilic response markers) were measured by qPCR (n=3-5). Secreted IL-8 was evaluated by ELISA.

The effects of HBC CM on HUVEC activation were further examined by neutralizing IL-1β using 10 mg/ml blocking and isotype-matched control antibody (n=5). Statistical significance was defined as P <0.05. Heavy menstrual bleeding (HMB) affects up to 30% of reproductive age women and may cause extensive financial, social and psychological disruption. Traditional methods of measuring blood loss have been replaced by assessing "patient experience" using patientreported outcome measures (PROM). PROMs provide insight into how patients perceive their health and the impact that treatments or lifestyle adjustments have on quality of life (QOL). This study aimed to compare bleeding-related QOL between women with/without HMB using a newly developed and validated PROM for HMB. METHODS: Premenopausal women with a subjective complaint of HMB (n=13) and a control group (n=12) with no menstrual complaints were recruited following informed consent. Those with known pelvic pathology (eg fibroids) were excluded. Participants reported bleeding experiences in the prior month using the short form of a validated PROM for HMB, the Menstrual Bleeding Questionnaire. This instrument has 22 items in 4 domains: heaviness (8 items), regularity (3 items), pain (1 item) and bleeding-related QOL (10 items). Differences were examined using Mann Whitney U tests. Heavy menstrual bleeding (HMB) is commonly defined as >80 mL blood loss per menstrual cycle, but the diagnosis is not straightforward. The 'gold standard' technique for measuring menstrual blood loss (MBL) is the alkaline hematin (AH) method but it is not in routine clinical use. In recent years other diagnostic methods have been developed, including pictorial assessment methods (e.g. the pictorial blood loss assessment chart [PBAC] ) that can be used by patients to measure MBL. However, not all of these have been fully validated. Many studies have used modified PBACs and/or different feminine items and cut-off scores for diagnosis of HMB that have not been certified against the AH technique. We review pictorial methods for MBL assessment, with particular focus on the validation requirements.

We performed systematic searches of Medline and Embase, using predefined search criteria to identify publications describing pictorial methods for assessment of MBL. These searches were supplemented with material from our libraries. RESULTS: Seven validated PBACs for assessing MBL were identified. The three most commonly used were originally developed for nowobsolete feminine hygiene products. Another method used unspecified menstrual towels; two others were essentially derivative PBACs. A new menstrual pictogram (SAP-c version) has recently been validated for a widely used, contemporary ultra-thin towel that contains superabsorbent polymers. Based on our systematic search data, we present the crucial requirements for PBAC validation that will ensure their suitability for use in both clinical studies and in primary care. CONCLUSIONS: Several semi-quantitative pictorial methods exist for assessing MBL, but many are outdated because of advances in sanitary product technology; others have not been scientifically validated. In clinical practice, it is essential that verified PBACs are used correctly to diagnose women presenting with HMB; this guides decision-making for appropriate therapy and provides a standard tool for research. INTRODUCTION: Studies of ovarian torsion (OT) have shown that even in necrotic-appearing ovaries, function is preserved in 88-100% of cases if de-torsion is performed, suggesting that oophorectomy may be avoided in most cases. We hypothesized that certain clinical factors may be associated with an increased likelihood of having an oophorectomy at the time of surgery in patients with suspected OT. METHODS: Patients who presented to the Boston Medical Center ED between 1/1/09 and 7/31/14 with a diagnosis of OT who had surgery within 1 week of presentation were retrospectively identified by ICD-9 code. Patient data (age, gravidity, parity, BMI, history of prior tubal ligation and/or oophorectomy, and affected ovary size by imaging) as well as surgical data (time from presentation to surgery, work shift when surgery occurred, intra-operative findings, pathology findings, estimated blood loss, and subspecialty of surgeon) were collected. Fisher exact test, t-test, and Mann-Whitney U tests were used to identify factors that were significantly associated with having an oophorectomy at the time of surgery. RESULTS: Sixty cases were identified. 48/60 (80%) of the suspected torsion cases had OT diagnosed at surgery. Oophorectomy was not performed in any of the non-OT cases. In patients diagnosed with OT, 31/48 (64.6%) had an oophorectomy and 15/48 (31.3%) underwent ovarian cystectomy. Patient factors associated with a higher rate of oophorectomy among the OT cases were older age (p = .001), higher parity (p = .001), and larger size of the affected ovary on imaging (p = .002). Surgeon subspecialty was significantly associated with performance of oophorectomy in OT cases: gynecologic oncologists completed 10/48 (20.8%) of the cases and were more likely than generalists and other subspecialists to remove the affected ovary (p = .003). One OT patient who underwent oophorectomy had a mucinous borderline ovarian malignancy. In other OT cases where oophorectomy was performed, the pathologic findings were teratoma (29%), necrotic ovary (25.8%), cystadenoma (19.4%), follicular/simple/hemorrhagic cyst (12.9%), fibroma (6.5%), and a benign Brenner tumor (3.2%). CONCLUSIONS: Older age, higher parity, larger size of the affected ovary on imaging, and having a gynecologic oncologist perform the surgery were significantly associated with a higher likelihood of oophorectomy in OT cases. Awareness of these clinical factors may ultimately reduce unneeded oophorectomy and promote ovarianconserving surgery.

Ovarian Cancer Exosomes Are Carriers of Autoantibody. Diogo Torres, 1 Douglas D Taylor, 1 Lynn P Parker, 2 Daniel Metzinger, 2 Cicek Gercel-Taylor. 1 1 Obstetrics, Gynecology, and Women's Health, University of Louisville, Louisville, KY, USA; 2 Gynecologic Oncology, University of Louisville, Louisville, KY, USA. INTRODUCTION: Early diagnosis and outcome failures are associated with current lack of non-invasive, reliable markers. Recent evidence indicates that exosomes released by tumors play roles in the pathogenesis of ovarian cancer. In addition, the protein and RNA cargoes of exosomes are specific for the disease and form the basis of markers that can be utilized for management of disease. Our aim was to study the antibody response against tumor exosomes in ovarian cancer patients. METHODS: Exosomes were isolated from patient plasma by chromatography and centrifugation. Appropriate fractions (void volume) were characterized by nanoparticle track analysis (NTA) . For analysis of the presence of autoantibody, exosomes were incubated with anti-human IgG conjugated with quantum dots prior to analysis with NTA. Western blotting was performed to study exosome-associated proteins. RESULTS: Exosomes were isolated from three benign cases: a borderline tumor, a cyst adenofibroma, and a cystadenoma. The total numbers of particles were 20.41+/-6.27 X 10e8 per ml. Of these, 10.60+/-7.30% was labeled with anti-human IgG. We also studied six malignant cases that included a serous, endometrioid, primary, and recurrent ovarian malignancy. Total particles in these samples were 11.59+/-3.27 X 10e8 per ml. Further analysis of these exosomes demonstrated that 44.37+/-22.81% of them were positive for human IgG. Exosome populations studied were CD63 positive by western blotting. CONCLUSIONS: Our findings demonstrate that tumor exosomes carry autoantibody recognizing aberrant antigens. The presence of circulating exosomes with IgG bound appears to be a marker of malignant disease. Characterization of these antigens of exosomes may lead to markers of interest while the effectiveness of the antibody response would be of importance in designing novel therapies.

Relationship Although previous studies have demonstrated a link between bacterial vaginosis and IUD use these may be confounded by age and behavior. As use of LARC among younger patients is increasing and the demographics of IUD users is changing,we sought to determine whether there is an association between IUD use and bacterial vaginosis in an ethnically diverse urban population.

This was an analysis of the BV-IDEAS study: a cross sectional study in an urban GYN clinic. Patients filled out a questionnaire about sexual behaviors, medical records were reviewed and a vaginal swab was collected for gram stain. BV was determined by Nugent score greater than 7. Chi square analysis was performed to test the association of BV with progesterone IUD, copper IUD, no hormonal contraception, age, race, partner circumcision, postpartum state, condom use and number of sexual partners in the past year. Multivariate binary logistic regression was performed to control for confounding factors. RESULTS: 446 patients were enrolled and we had Nugent score data for 433. The median age was 31 (18yrs-64yrs), 15.7% were Caucasian 31.6% were African American, 45.3% were Hispanic and 7.4% were other. 211 (48.7%) had a diagnosis of BV. 32 women (7.4%) used progesterone IUD(PIUD) 17 women (3.9%) used copper IUD and 231 women (53.3%) used neither IUD or hormonal contraception (NHC). On univariate analysis, partner circumcision (PC) and race were found to be statistically significantly associated with BV. PC (LR 0.51 (95%CI: 0.32-0.81) p= 0.004); African American (LR: 2.6 (95% CI:1.33-5.29) p= 0.005) Hispanic (LR 2.8 (95%CI:1.47-5.45) p = 0.002). However, IUD use was not associated with BV. After stratification by these confounders there was no statistically significant difference between BV and PIUD ( p= 0.77); CIUD(p= 0.19) or NHC (p=0.60). CONCLUSIONS: In our population, IUD use was not associated with an increased risk of BV. As the demographics of IUD use changes, older epidemiologic data may be less applicable. Future studies investigating the effects of IUD on acquisition of abnormal flora are warranted. Chronic pelvic pain (CPP) is a common symptom in women that is frequently difficult to treat. It is well known that CPP is associated with a reduction in quality of life (QoL). We aimed to identify whether QoL relates directly to pain intensity in women with CPP and whether other factors, modifiable or not, also impact QoL. METHODS: A detailed questionnaire was sent to all new referrals to a tertiary CPP clinic over a 27 month period. Data collected included pain scores; medical, social and abuse history; validated psychological measures; and SF-36 (a QoL measure). Only responses received prior to attending clinic were analysed. Multivariable regression models were obtained to identify significant predictors of QoL. RESULTS: 90 questionnaires were returned; 66 contained complete data sets for regression analysis. Women with CPP had reduced QoL compared to age and gender-matched population norms. The effect was greater for the physical component scores (PCS) than the mental component scores (MCS) (34 vs 43, p<0.001). Regression models demonstrated that reduced PCS scores (i.e. worse physical QoL) were predicted by an increased affective component of pain (McGill Pain Questionnaire), increased depression, reduced anxiety and a history of symptoms being disbelieved. Reduced MCS scores were predicted by increased depression and a lack of co-morbid medical conditions. Only 40% of the variance of PCS and 57% of MCS scores was predicted by these models. The strongest predictor of both subscales was depression (PCS: β=-0.613, MCS: β=-0.485). CONCLUSIONS: This study demonstrates that a number of factors contribute to QoL in women with CPP. Given that conventional treatment frequently fails to provide pain relief for these women it is reassuring to note that their QoL is not solely determined by pain intensity. Moreover, our results highlight the importance of believing patients from their first contact with medical personnel and the need for targeted psychological therapies focusing particularly on depression as a component of multidisciplinary treatment packages. The amount of variance in QoL not explained by our models suggests further work be undertaken to identify other predictors of QoL in women with CPP in order to optimize treatment strategies and design meaningful patient report outcome measures (PROs) for future clinical trials. Chronic Pelvic Pain (CPP) is common, but its management is difficult and little is known about the underlying pain mechanisms. Gabapentin is increasingly being prescribed for CPP despite no supporting evidence and no understanding of how it produces analgesia. Functional Magnetic Resonance Imaging (fMRI) of the brain is a sensitive tool that has been successfully used to understand central mechanisms in other chronic pain conditions and assess efficacy in early stage drug trials. METHODS: An fMRI study of the central effects of gabapentin in women with CPP was nested within a pilot RCT (GaPP). Primarily we aimed to assess whether gabapentin alters brain activity in women with CPP. 11 women (placebo n=5; gabapentin n=6) underwent a 3T fMRI brain scan after ³12 weeks treatment. Brain activity at rest, and in response to noxious thermal (arm/abdomen) and punctate (abdomen) stimulation were recorded. Data were analysed with FSL and SPM8. Analyses assessed resting state networks (RSNs:known to be altered in chronic pain and by centrally acting drugs) and stimulus-related activity in the insula (thought to encode pain intensity) and the PAG (frequently upregulated in chronic pain and potentially modulated by gabapentin).

The posterior insula was activated by abdominal punctate stimuli in both groups, but to a greater extent in the placebo group (p=0.04). All stimuli produced PAG activation in the placebo group but deactivation in the gabapentin group (t-test: non-significant trend). Differences in connectivity within two RSNs between groups approached significance (p=0.087; p=0.089). CONCLUSIONS: Despite the small sample size, our results support an effect of gabapentin on brain activity in women with CPP. Furthermore, they suggest a central component to CPP that can be suppressed with gabapentin. The observed disruption of RSNs is similar to that seen in fibromyalgia where it is predictive of a response to pregabalin. We believe these results justify both a definitive clinical trial and a larger mechanistic fMRI study to investigate the role of gabapentin in CPP further. Benign gynecological diseases are characterized by growth dependence on sex steroids in case of fibroids or a modulation of the normal hormonal activity observed by relative progesterone resistance in endometriosis. Selective progesterone receptor modulators (SPRMs) may have beneficial effects in these disorders as already reflected by clinical data. Objective of the outlined studies was to compare the progesterone receptor (PR) antagonistic activity of different SPRMs, including testing of the novel SPRM, vilaprisan, in an endometriosis model. METHODS: Different SPRMs have been analyzed on their PR activity in the endometrial transformation assay in rabbits and implantation efficacy test in rats. Ulipristal and vilaprisan have also been evaluated in an estrogen and progesterone stimulated myometrial growth assay in mice (as surrogate marker for fibroid growth). A disease model for hormonedependent endometriosis in the rat has been performed and lesion size analyzed after vilaprisan treatment. RESULTS: Oral administration of different SPRMs to rats showed full inhibition of implantation starting from 0.3 mg/kg*d (lonaprisan/ ZK230211) up to 10 mg/kg*d (ulipristal) . Antagonistic effects on endometrial transformation could be observed at doses from 1 mg/kg*d (lonaprisan, vilaprisan) up to 10 mg/kg*d (mifepristone, ulipristal) .

Higher PR antagonistic activity of vilaprisan in comparison to ulipristal was reflected by a stronger dose-dependent decline in the myometrial height of mice (p<0.005). Hormonally stimulated autologous endometrial tissue sutured onto the peritoneal wall and treated with different doses of vilaprisan for 4 weeks showed a statistical significant reduction of lesion area of ca. 75% at 1 mg/kg*d (p<0.01). CONCLUSIONS: All compounds are efficacious in the tested in vivo models, with vilaprisan and lonaprisan being the most potent SPRM representatives. Owing to the reduction of endometrial lesion size and formerly reported shrinkage of fibroid growth by vilaprisan further advancement of this compound in clinical development will eventually demonstrate whether its preclinical profile translates into a safe and efficacious long-term treatment of gynecological diseases like uterine fibroids and endometriosis.

in Cycle-13 (> 251 days). Thirty-six subjects had a history of combined hormonal contraceptive (CHC) use in the cycle preceding CVR use and 70 subjects had no history (naïve users).

The values increased for all subjects at 13 cycles of treatment for Factor VIII, Fibrinogen, and SHBG, and decreased for Protein S. The baseline values for Factor VIII, fibrinogen, and SHBG were higher for previous users compared to naïve subjects. The observed changes for Factor VIII, Fibrinogen, and Protein S at cycle13, as compared with baseline, were all small and within the normal range. Mean SHBG levels increased by nearly 100%. For each hepatic factor, the analysis of variance over the time of CVR use (Global F-Test for the fixed effect of time) was statistically significant, and the p value, based on Bonferroni Correction was; Factor VIII: p=0.0005; Fibrinogen: p=0.0052; Protein S =0.0043; SHBG : p< 0.0001 In naïve users, at cycle6, Protein S decreased (p=0.002) and Fibrinogen (p=0.002) and SHBG (p<0.001) increased, and were similar at cycle13 in naive users. Baseline changes were not significant in previous users, except for SHBG. Previous use of EE-based contraceptives modified proteins at baseline. CONCLUSIONS: Prior exposure to CHC impacts the changes induced by a new contraceptive. A wash-out of 6-8 weeks after previous CHC use is recommended before testing the effects of a new hormonal method on estrogen-dependent proteins.

Joseph C Chen, Sahar Houshdaran, Shaina Balayan, Juan C Irwin, Linda C Giudice. OBGYN / Reproductive Sciences, University of California, San Francisco, CA, USA. INTRODUCTION: Studies in women have suggested higher risk of HIV acquisition in women using progestin-containing contraceptives, including the levonorgestrel intra-uterine system (LNG, Mirena TM ), medroxyprogesterone acetate (MPA, Depo Provera TM ), and northindrone acetate (NEA, oral tablet). Mechanisms to account for these observations are unclear, but may involve progestin-mediated cellular pathways in the upper female reproductive tract (FRT) . The objectives of the current study were to determine the effects of trans-activating doses of the progestins LNG, MPA, and NEA on expression of pro-inflammatory factors by endometrial stromal fibroblasts (eSF) in vitro. METHODS: Endometrial biopsies were obtained from premenopausal women undergoing benign gynecological procedures and oocyte retrieval. Samples were digested and eSF were isolated using size fractionation and selective attachment. eSF were passaged twice and plated into 10 treatment groups/sample: 1µM progesterone (P 4 ); 10nM estradiol (E 2 ), E 2 +P 4 (E 2 P 4 ), 54.7nM LNG; 6nM MPA; 0.294µM NEA; E 2 +LNG (E 2 LNG); E 2 +MPA (E 2 MPA); E 2 +NEA (E 2 NEA); and ethanol vehicle (veh). Cells were treated for 14d (with 48 hr medium exchanges), after which RNA and conditioned media collected for transcript and protein analysis. RESULTS: E 2 P 4 , E 2 LNG, E 2 MPA, and E 2 NEA all stimulated mRNA and protein production of IGFBP1, a marker of eSF decidualization. P 4 , LNG, MPA, and NEA (without E 2 ) resulted in reduced IGFBP1 mRNA expression and protein production (p<0.05) compared with each compound + E 2 . Up-regulation of PR expression confirmed the bioactivity of E 2 treatment compared to veh (p<0.05). MPA increased (p<0.05) IL6 protein secretion compared to other progestins and E2P4. Combined treatment with E 2 attenuated this effect, resulting in comparable levels to E 2 P 4 . Similar patterns were observed for VEGFA. All progestins, with or without E 2 , increased (p<0.05) CCL2 protein compared to E 2 P 4 . CONCLUSIONS: These data suggest that there are specific effects of each progestin on the production of factors associated with inflammation, immune cell recruitment, and immune cell activation. These effects may contribute to mechanisms of immune modulation in the upper FRT, creating a niche conducive to HIV infection. JCC and SH equal contribution. Supported by NIH AI083050 (WCG/LCG) and F32HD074423 (JCC). INTRODUCTION: Unscheduled breakthrough-bleeding ('spotting') is a common problem in progestin only contraceptive (POC) users. A major feature of POC-endometria is the presence of 'fragile' luminal epithelium (LE), susceptible to shedding/bleeding. We propose that disturbances in Wnt signalling, mediated by signals from the decidualized stroma in POC-endometria, causing LE alterations, underlying fragility and bleeding events. METHODS: Wnt5a and 7a, β-catenin, E-cadherin and DKK1 examined by immunohistochemistry in POC-and normal endometrium. Primary human endometrial stromal cells (HESC) treated with estrogen (10 -8 M) and progesterone (MPA, 10 -7 M), prolactin (PRL) and DKK1 were assessed by ELISA in decidualized (DEC, day 12) versus non-DEC (day 2) media. Phospho(P)-GSK3 β, Crumbs and Wnt7a localization was examined by immunocytochemistry in recombinant human (rh)-DKK1 treated endometrial epithelial cells (EEC). P-GSK3β abundance was also determined by western immunoblot (WB). The impact of DECversus non-DEC media and rhDKK1 on real-time EEC proliferation and migration was determined using the xCelligence system. The impact of a decidualizing stroma on epithelial tight junctions was determined by taking transepithelial resistance measurements RESULTS: Wnt-5a, Wnt7a, β-catenin and E-cadherin localized to luminal/glandular epithelial cells in cycling endometrium. DKK1 localized to luminal epithelium in proliferative-phase, and stroma in secretory-phase endometria. In the POC-endometrium, Wnt7a, β-catenin and E-cadherin had disappeared/displayed abnormal localization while DKK-1 strongly localized to DEC-stroma. DEC-HESC secreted elevated levels of PRL and DKK1 versus non-DEC. Treatment of endometrial epithelial cells (EEC) with DEC-media or rhDKK1 reduced EEC proliferation and increased migration. rhDKK1 treatment of EEC resulted in a down-regulation of Wnt7a, crumbs and P-GSK3β WB also demonstrated a reduction in P-GSK3β-catenin upon rhDKK1 treatment. Co-culture of epithelial cells with decidualizing stromal cell decreased tight junction integrity. CONCLUSIONS: Wnt signalling molecules are altered in the POCendometrium and rhDKK1 treated EEC. Alterations in Wnt signalling pathways impact cell function. This data suggests that stromal signals (DKK1) impact on signalling and function in the epithelium, contributing to epithelial fragility in POC-endometria. The efficacy of long-acting reversible methods of contraception (LARC), especially intrauterine devices/system (IUD/IUS) in reducing unplanned pregnancy is widely recognized. In cohort studies post-abortal contraception by means of IUD/IUS has been associated with greatly reduced risk (approx. 70%) of repeat abortion. In the present randomized trial we study the long-term effect(s) of routine provision of either the levonorgestrel-releasing intrauterine system or copper-releasing intrauterine device in comparison to initiation of oral contraceptives at the time of abortion (either medical or surgical). METHODS: Adult women requesting termination of an unwanted pregnancy of up to 12+0 weeks of gestation between October 2010 and February 2013 (n=2305) were informed about the study. Half of them (49%) were interested in intrauterine contraception and 756 (66%) were enrolled. They were randomized into two groups. In the intervention group (n=377) women received an IUD/IUS at the time of surgical abortion or at follow-up visit 2-3 weeks after medical abortion. In the control group (n=376) oral contraceptives were started with a follow-up in the primary health care. Repeat abortions were monitored by means of the abortion registry of the Finnish National Institute of Health and Welfare. The follow-up was continued until first repeat abortion or the end of follow-up (end of 2013). The median follow-up time was 28 months. RESULTS: There were fewer subsequent abortions in the intervention group when analyzed by either intention-to-treat (ITT) or per-protocol (PP) basis. The existence of a close and complex relationship between chronic pelvic pain and endometriosis is widely recognized. Peritoneal lesions of endometriosis are characterized by an increased density of nerve fibers, compared to normal peritoneum. These nerve fibers are proposed to play a role in generation and transmission of pelvic pain in endometriosis; however, neurogenesis and pain generation mechanisms of the disease remain unclear. Therefore, this study aims to quantify the expression of neuronal guidance and nerve growth molecules in peritoneal endometriotic lesions.

In this ongoing study, peritoneal endometriotic lesions (n=35) and uninvolved peritoneum (n=16) were immunohistochemically stained with PGP9.5 (nerve fibers); neuronal guidance molecules Semaphorin 3E and slit-2, their receptors Plexin-D1 and Robo-4 (respectively); and neurotrophins Persephin, GDNF, Neurotrophin-3 (NT-3) and NT-4. Staining is being analyzed using MetaMorph software. RESULTS: Nerve fibers were detected in and around lesions. Preliminary results show variable expression of neuronal guidance molecules and neurotrophins in lesions and uninvolved peritoneum, as summarized in Table 1 .

Semaphorin 3E ++-+++ 0-+ 0-+ We ascertained information on first hospitalization for surgery for endometriosis, restricting to cases where records were linked to data in HES. Women for whom a new recorded entry of surgery following initial surgery was recorded in HES were identified as having potential recurrence of endometriosis. To calculate the recurrence rate, follow-up started the day after first surgery and ended at the time of new surgery or death or in March 2011. The recurrence rate was calculated using the person-time contribution among the subset of all women with a first surgery. RESULTS: Among the subset of 902 women with incident endometriosis in THIN who were also recorded in HES, we ascertained first hospitalizations for surgery for endometriosis by manual review of hospital records and retained 436 women. In total, 100 women had potential recurrence (new recorded entry of surgery). After manual review, 86 (20%) were considered to have had a second surgical procedure for endometriosis -i.e., recurrence. Of these, 51 women (59%) had ≥ 2 surgical procedures. Most (71%) underwent recurrent surgery within 3 years of the initial procedure. The most common type of surgery was hysterectomy (44%). The recurrence rate was highest in women aged 12-20 years at the time of first surgery (7.6 per 100 women-years). CONCLUSIONS: Data on endometriosis recurrence are scarce. In this study, the first systematic analysis using a primary care database, one in five women undergoing surgery for endometriosis underwent surgery for disease recurrence, with most of these procedures taking place within 3 years of initial surgery. Women aged 12-54 years between January 2000 and December 2010 who had an incident diagnosis of endometriosis, had been enrolled with their PCP for ≥5 years and had a computerized prescription history of ³3 years were identified by automatic review of Read codes. To examine treatment patterns following diagnosis, we searched for surgical procedures and medical therapies used to treat endometriosis.

+-+++ 0-+ +-++ Slit-2 +-+++ 0-+ 0-+ Robo-4 ++-+++ +-+++ +-+++ Persephin ++-+++ 0-+ +-++ GDNF 0-++ 0-+ +-+++ NT-3 0-+++ +-++ 0-+ NT-4 +-++ 0-+ +-++ (0 =

In total, 4833 women with endometriosis were identified in THIN; 23.8% underwent surgery or an invasive procedure within the first year and the most frequent procedure was hysterectomy (11.8%). Within 1 year of diagnosis, 15.7% of those who had undergone surgery or an invasive procedure had one or more further procedure. Among all women with endometriosis, 2522 women (52.2%) received hormonal treatment within the first year after diagnosis. The most commonly prescribed hormonal therapies were combined oral contraceptives (21.4%; 8.7% started treatment after first recording of endometriosis), gonadotropinreleasing hormone analogues (16.8%; 82.2% started treatment after first recording of endometriosis) and the levonorgestrel-releasing intrauterine system (9.7%; 43.2% started treatment after first recording of endometriosis). Within the first year after diagnosis, the proportion of women receiving analgesics was 44.9%, with non-steroidal antiinflammatory drugs (34.3%), codeine (22.2%) and tramadol (8.7%) the most frequently prescribed. The proportion of women receiving analgesics decreased with increasing time following diagnosis of endometriosis.

The burden of endometriosis is high. Data from THIN suggest that, within the first year of diagnosis, nearly a quarter of women with endometriosis were treated with surgery or an invasive procedure and more than half were prescribed hormonal treatment. In addition to altered estrogen production, aromatase inhibitor treatment has shown to result in marked microRNA alterations in various cancers, but this molecular mechanism of action has not yet been characterized in endometriosis. The purpose of this study was to evaluate the association between aromatase inhibitor treatment and let7 microRNAs expression in endometriosis. METHODS: Human endometrial stromal cells (HESC) were obtained from women with a histological diagnosis of endometriosis, and Ishikawa cells were cultured in conditioned media and each were treated with letrozole at concentrations of 0.0, 0.1, 1.0, 10.0, and 20.0µmol/L. After 48 hours, RNA was extracted and expression of microRNAs let7a-f were evaluated using qPCR. Further, after transfection with mimics of let7 subtypes of interest for 48 hours, aromatase expression was determined using qRT-PCR and western blot and the effects on cell migration were evaluated using Millicell cell culture insert system.

In Ishikawa cells letrozole treatment led to increased expression in a dose dependent manner of let-7b and let-7f (P=0.034 and P=0.001, respectively). Similarly, let-7 expression was higher in cultured HESCs from patients with endometriosis after treatment with 1.0 µmol/L letrozol, and let-7f showed the most significant difference between the control after 10 µmol/L treatment (P=0.026). After transfection of let-7f mimics for 48 hours, aromatase expression was decreased in both Ishikawa cells and HESCs using qRT-PCR and western blot. Let-7f transfection also led to a significant decrease in number of migrating cells in both HESCs and Ishikawa cells (7.5 vs 4.3, P=0.032, and 13.7 vs 6.1, P=0.009, respectively). CONCLUSIONS: Aromatase inhibitor treatment significantly increased expression of let-7f in Ishikawa cells and HESCs from patient with endometriosis. Enhanced lef-7f expression effectively decreased endogenous aromatase expression and reduced the migration of endometrial cells. One mechanism of action of aromatase inhibitors is through altered lef-7f expression. Direct modulation of miRNAs involved in the pathogenesis of endometriosis may have therapeutic potential. Endometriosis is defined as the growth of endometrial tissue outside the uterine cavity. It is well-known that ectopic endometrium is characterized by estrogen dependence and progesterone resistance. Epigenetic mechanisms may play an important role in the etiology of endometriosis. The modification of histones by methylation of lysine residues has been shown to regulate gene expression by changing chromatin structure. We have previously shown that endometriotic lesions had higher levels of histone acetylation and methylation than control tissues. We aimed to determine the levels of histone marks such as H3K27me3, a well-known repressive mark, in endometriosis by Immunohistochemistry (IHC) of an endometriosis-focused Tissue Microarray (TMA). We also determined the expression profile of ER and PR receptors and correlate them to histone mark expression.

TMA was used to determine the expression profile of ovarian steroid receptors and epigenetic markers. RESULTS: Based on the percent of positive nuclei, we detected differences in the expression of ER, PR, and H3K27me3 by lesion type. In addition, we were able to document differences in cell specificity (i.e., glands vs. stroma) and confirmed the nuclear localization of these markers. We observed that lesions (ovarian, fallopian, and peritoneal) are characterized by higher % of H3K27me3 positive nuclei compared to eutopic endometrium from patients. Positive and negative correlations between H3K27me3 levels and those of selected hormone receptors (i.e ERα, ERβ, and PR) were observed in lesions and eutopic endometrium from patients and from controls. CONCLUSIONS: These results will help to better understand the molecular mechanisms at play in the observed dysregulations in hormonal responses that characterize endometriosis Our aim was to test the hypothesis that FM is present in eutopic and ectopic endometrium (EM) from women with endometriosis but not in eutopic EM from women without endometriosis. METHODS: 31 women with endometriosis (paired eutopic and ectopic endometrium; n=19 including 17 women on hormonal treatment) and women without endometriosis (eutopic endometrium; n=12 including 6 women on hormonal treatment) had delivered a son before laparoscopic surgery for infertility and/or pain and were selected for this study. They had given informed consent and this study was approved by the institutional ethical review board at the University Hospital Gasthuisberg.

Paraffin-embedded specimens of eutopic and ectopic endometrium were cut into 3µm sections. Tri-color interphase FISH analysis was then performed using CEP Y (DYZ1, satellite III) SpectrumAqua (SA) and CEP X (DXZ1) SpectrumGreen (SG)/CEP Y (DYZ3) (alpha satellite) SpectrumOrange (SO) probes. 500 cells were counted for each specimen on Zeiss Axioplan 2 imaging microscope (100x magnification) using CytoVysion (version 4.5.3.9) . Metasystems Isis software (version 5.4.9 V3.10) was used for imaging. For a positive control, a male bowel tissue was analysed, and for a negative control the probes were left out throughout the experiment. RESULTS: The male bowel control had 92% XY (SG + SO + SA signals), 2% XY (SG + SO, lack of SA signal), 7% X (single SG) and 1% XX (double SG) chromosomes. Ectopic endometrium from women with endometriosis had 75% XX chromosomes (double SG signals) and 25% X chromosomes (single SG signal). Y chromosomes were not observed in any of the EM tissues from cases or controls. CONCLUSIONS: We were unable to confirm our hypothesis that fetal microchimerism (FM) is present in eutopic and ectopic endometrium (EM) obtained from women with endometriosis. The obesity epidemic has led to an increase in the incidence of endometrial adenocarcinoma. Although endometrial cancer is usually treated with surgery and has an excellent prognosis, metastatic disease has a poor prognosis. There is hope that new drug therapies may target oncogenes identified by next generation DNA sequencing. However, these targeted therapies rely on metastatic tumors having the targeted mutations; therefore, sequencing the readily available primary tumor, or only one metastasis may not yield the necessary genetic information.

Our objective was to compare the mutational profiles of paired primary and metastatic tumors. METHODS: For this pilot study, we identified 10 paired cases. Tumor rich foci were identified within H&E slides and the corresponding areas within each archived paraffin-embedded block from the primary tumor (grade 1-3, excluding UPSC and clear cell carcinoma) and most tumor rich metastasis were cored for DNA. Only one metastasis was tested for each case. Samples were sequenced using Ion Torrent Technology to generate the mutational profiles of 37 oncogenes commonly involved in solid tumors. Data were analyzed by X 2 as the presence or absence of a mutation in the metastasis versus the primary. Grade and stage were potential covariates, but the available sample size was insufficient for regression modeling. RESULTS: As expected, PIK3CA and PTEN mutations were common in both the primary and metastatic tumors. We observed a 50% discordance (21/42 loci) in paired samples with a relative loss of mutations ( 

The Metastatic endometrioid ovarian cancer model was generated through intrabursal AdCre-infected LSL-Kras G12D/+ Pten fl/fl mouse model. LSL-K-ras G12D/+ Pten fl/fl Axl +/+ and LSL-K-ras G12D/+ Pten fl/fl Axl -/were used to evaluate the role of AXL.

Of the 80 patient-derived primary ovarian tumor specimens, 76% (61) of advanced stage, high-grade epithelial ovarian cancer specimens had 3+transmembrane AXL staining whereas 24% (19) of advanced stage, high-grade epithelial ovarian cancer specimens had no AXL staining. When correlated with overall survival, patients with tumors that had no AXL expression had a median survival of 52.9 months vs. 26.7 months for those with 3+AXL expression (p=0.03). The majority of intrabursal AdCre-infected LSL-K-ras G12D/+ Pten fl/fl Axl +/+ mice had primary (78%) and metastatic tumor formation (86%) whereas the majority of LSL-K-ras G12D/+ Pten fl/fl Axl -/had primary tumor formation (64%) but few had metastatic tumors (28%) (p<0.05). There was no difference in primary tumor weight between the two groups: 120mg for Axl +/+ vs. 110mg for Axl -/-. Metastatic tumors were microscopic and therefore unable to be resected and weighed prior to identification by H&E staining. Western blotting of primary and metastatic tumors from LSL-Kras G12D/+ Pten fl/fl Axl +/+ showed high AXL expression compared to no AXL expression in the ovaries of normal mice. CONCLUSIONS: AXL expression in advanced stage epithelial ovarian cancer correlated with lower overall survival. Primary ovarian and metastatic tumors in this intrabursal AdCre-infected LSL-K-ras G12D/+ Pten fl/fl mouse model express AXL. Deletion of the receptor tyrosine kinase, AXL, in this conditional genetically engineered mouse model of endometrioid ovarian cancer resulted in inhibition of metastatic tumor formation. AXL is a critical factor driving metastasis in this genetically-engineered murine model of metastatic ovarian cancer. Targeting cell motility, which is required for dissemination and metastasis, has a therapeutic potential for treating ovarian cancer, and molecular regulation of cell motility needs to be uncovered for developing novel therapeutics. Ceramide, one of sphingolipids, has been emerging as a bioactive lipid, and we discovered novel role for ceramide in cell motility. In the present studies, we investigated the molecular mechanism by which ceramide suppresses cell motility and potential of ceramide-based therapy for ovarian cancer. METHODS: Invasive ovarian cancer SKOV3 cells spontaneously form protrusions, such as lamellipodia, required for generating locomotive force in cell motility. Cell motility was determined by lamellipodia formation. Ceramide interacting proteins were determined by pull down assay using ceramide analogue probes. An ovarian cancer SKOV3 cell xenograft mouse model was established for testing therapeutic efficacy of ceramide liposomes. RESULTS: Small interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation. Treatment with cell permeable C 6 -ceramide significantly decreased the number of cells with PI3KC2ß-driven lamellipodia, the levels of PI3KC2ß products, and Akt phosphorylation. Interestingly, ceramide was revealed to interact with PI3KC2ß and affect its compartmentalization, thereby suppressing cell motility. To determine the potential of ceramide as a therapeutic agent, the effects of C 6 -ceramide liposomes on cell motility and in vivo ovarian cancer metastasis were tested. Ceramide liposomes exhibited not only an inhibitory effect on cell motility, but also therapeutic efficacy against peritoneal dissemination and metastasis in an ovarian cancer cell xenograft mouse model. CONCLUSIONS: Taken together, our studies identified ceramide as an inhibitory lipid for PI3KC2ß-controlled cell motility, and they raise therapeutic potentials of ceramide for treating ovarian cancer dissemination and metastasis. Hypoxia is the key regulator of cancer progression, inducing epithelial to mesenchymal transition (EMT) which is an initiating process of ovarian cancer metastasis. The exosome bioactivity (i.e. release and content) is regulated by oxygen tension; however, the effect of exosomes on EMT has not been established. The aims of this study were to establish the effect of ovarian cancer cell-derived exosomes released under hypoxic condition on EMT of target cells; and to determine their exosomal content (protein and miRNA). METHODS: Exosomes were isolated and purified by differential and buoyant density centrifugation from SKOV-3 cell conditioned media cultured under oxygen tensions of 0.1% (exo-SKOV3-0.1%) and 8% (exo-SKOV3-8%) for 48 h (BioSpherix). Exosomes were characterised by their density, presence of exosome specific markers (CD63 and TSG101) using Western Blot, size distribution (Nanosight™) and shape with electron microscopy. BeWo cells were used to establish the effect of exo-SKOV-3 on EMT. EMT was validated by the ratio of E-cadherin (epithelial marker) to N-cadherin (mesenchymal marker). The effects of exo-SKOV-3 on target cell migration were determined using a live-cell imaging system (Incucyte). The exosomal protein and miRNA profiles were established by mass spectrometry (MS/MS) and RT-PCR, respectively. RESULTS: Exosomes were identified as spherical vesicles with a typical cup-shape, diameters ranging from 50 to 100 nm with the expression of endocytic markers. Hypoxia (i.e. 0.1% O 2 ) increased the exosome release from SKOV-3 cells by ~3-folds and induced a mesenchymal phenotype (increased N-cadherin/E-cadherin ratio) of BeWo cells compared to exosomes released under 8% O 2 . BeWo cells displayed an epithelial phenotype; however, exo-SKOV-3 induced the expression of mesenchymal marker and cell migration. Finally, we identified hypoxicspecific exosomal proteins and miRNA profile in exosome isolated from SKOV-3 exposed to 0. 

In this study, we showed that the combination of EGCG and curcumin significantly reduced SKN cell proliferation more than either drug alone. The combination induced apoptosis at a much lower curcumin concentration than previously reported. EGCG enhanced the incorporation of curcumin. Curcumin induced autophagy while EGCG decreased autophagy. Curcumin increased extracellular signal-regulated kinase 1/2 (ERK1/2) activity while EGCG decreased ERK1/2 activity in SKN cells. CONCLUSIONS: These experimental findings suggest that combination treatment reduces uterine LMS cells viability and induces apoptosis in uterine LMS cells by increasing the cellular incorporation of curcumin in the presence of EGCG. Moreover, EGCG may play an important role in the cross-regulation between autophagy and apoptosis of cucumin in SKN cells. In our previous study, we reported that cytokeratin (CK19) and annexin A8 (ANXA8) mRNAs are optimal markers for onestep nucleic acid amplification (OSNA). In this study, we further explored which of these two targets provided superior detection of sentinel lymph node metastases in cervical cancer. METHODS: CK19 and ANXA8 mRNAs were evaluated with OSNA assays using 22 histopathologically positive (squamous cell carcinoma: 11, adenocarcinoma: 11) and 217 histopathologically negative lymph nodes (LNs). The cutoff values for CK19 and ANXA8 mRNAs were established by the sensitivity and specificity of serial cutoff values (25-5000 copies/ µL) in receiver operating characteristic curves (ROC). By comparison of the accuracy to histopathological examinations, the optimal marker was determined. RESULTS: To establish a cutoff value for the OSNA assay using histopathologically positive and negative LNs, we evaluated the sensitivity and specificity of serial cutoff values (25-5000 copies/µL) in ROC curves.

In this evaluation, cutoff values of both 100 and 250 copies/µL showed superior sensitivities and specificities for CK19 mRNA than other tested cutoff values. When considering histological procedures as the gold standard, the specificity, positive predictive rate, and concordance rate were superior when the cutoff value was 250 copies/µL compared with those when the cutoff value was 100 copies/µL. Based on this analysis, we set the cutoff value for CK19 mRNA in the OSNA assay to 250 copies/µL. For ANXA8 mRNA, cutoff values of both 75 and 100 copies/µL showed superior sensitivities and specificities compared with the other tested cutoff values. When considering histological procedures as the gold standard, no significant differences in sensitivity, specificity, positive predictive value, negative predictive value, or concordance rates were observed between cutoff values of 75 and 100 copies/µL. Therefore, the cutoff value for ANXA8 mRNA was 75-100 copies/µL. To determine which of these was the optimal marker, we compared the accuracy of CK19 mRNA versus ANXA8 mRNA; CK19 mRNA showed significantly improved specificity, positive predictive value, and concordance rate. CONCLUSIONS: Our results indicated that CK19 mRNA was an optimal marker for use in the OSNA assay in cervical cancer.

The High stromal content of tumours has been found to predict adverse clinical outcome in a range of epithelial tumours. This study aimed to assess the prognostic significance of tumour/stroma ratio (TSR) in endometrial adenocarcinomas and to investigate its relationship with other clinico-pathological parameters. METHODS: TSR was measured in a series of 400 endometrial adenocarcinoma cases using systematic spot-counting software on digitised hysterectomy specimens. TSR prognostic significance (overall survival; OS) and disease free survival (DFS) were determined using Cox Proportional Hazards regression analysis and Kaplan-Meier curves generated. Associations of TSR with other clinico-pathological parameters were determined using non parametric tests and corrected for multiple comparisons using the Holm-Bonferroni method. RESULTS: TSR as a continuous variable related to worse OS (P = 0.034) in univariable Cox regression analysis. Using an optimal cut-off TSR value of 1.3, TSR-high (stroma-poor) tumours were associated with significantly worse OS (HR 2.507; 95%CI 1.22-5.14; P = 0.012) and DFS (HR 2.18; 95%CI 1.15-4.160;P = 0.017) in univariable analysis. However, TSR did not have independent prognostic significance in multivariable analysis. A highly significant positive association was found between TSR and tumour grade (P<0.001) as well as with lymphovascular space invasion (P<0.001), both of which were confirmed to be of independent prognostic value.

This study suggests that TSR has limitedindependent prognostic value in endometrial adenocarcinoma. However, the significant association of high TSR with independent adverse prognostic parameters in endometrial cancer indicates that the negative prognostic influence of high tumour stromal content is not universal amongst epithelial tumours. 

These pilot data indicate these compounds binds to CB1,CB2 receptors(AEA,OEA,PEA) and TRPV1 receptor(AEA,CAP) reduce the number of Ishikawa cells in a time and pseudo dosedependent manner. The effect is probably through inhibition of cell proliferation rather than cell death, because cells recover in longterm culture. These suggest that increased plasma and tissue AEA/PEA concentrations observed in EC patients may be a 'counter mechanism' against further cancer growth and suggests that modified endocannabinoids may provide new therapeutics against EC.

UHPLC-MS/MS in these tissues. Results were statistically analysed using Prism 7. Statistical analysis was performed using Student's t-test ; p<0.05 was considered statistically significant. RESULTS: All components of the ECS were present in leiomyoma. The expression (mean±SEM) of all transcripts (except CB2 and TRPV1) were decreased in leiomyomal tissues when compared to healthy myometrium but those decreases failed to achieve statistical significance (p>0.05): CB1(0.59±0.18vs2.01±1.10); FAAH(0.46±0.14vs1.34±0.86); NAPE-PLD(12.53±3.84vs14.08±2.0); GPR55(1.12±0.32vs5.61±3.28). For CB2 and TRPV1, transcript levels were increased in the leiomyomal when compared to healthy myometrium: CB1 (2.18±0.70vs0.87±0.47), TRPV1(4.58±2.0vs3.72±0.77) but again differences were not statistically significant (p>0.05). Although UHPLC-MS/MS analysis of leiomyomal extracts showed AEA, OEA and PEA concentrations to be higher than that in normal myometrial extracts, these increases failed to reach statistical significance; there was a clear order of increment among the three endocannabinoids, PEA>OEA>AEA. CONCLUSIONS: These pilot data suggest that ECS (ligands, receptors, regulatory enzymes) are expressed in leiomyoma and some components may be differentially expressed. We conclude that the presence of this system in leiomyoma (with some differential expression) indicates that it may be involved in the pathogenesis of fibroids. Further studies are required; firstly to confirm our findings and secondly to investigate a possible role for the ECS in leiomyoma.

The However, the effect is restrictive and new treatment for endometrial cancer is strongly desirable. A strong rationale exists for the use of retinoic acid (RA) in cancer therapy and chemoprevention. There are two types of receptor, retinoic acid receptor (RAR) and retinoid X receptor (RXR), and these receptors have three subtypes α,β,γ respectively. It has been reported that the expression of RARα was considered as good prognostic factor in breast cancer. We previously reported that immunohistochemical staining of RAR subtypes in endometrial cancer didn't have significant correlation to pathological findings. However, the details of the status of these retinoid receptors and the correlation between retinoid receptors have not been studied.In this study, we initially examined the effects of RA in endometrial cancer cell lines. And then, we checked the expression of RAR subtypes and analysed functional difference for each RAR subtypes.

In this study, cell proliferation assay using endometrial cancer cell lines (RL95-2, ishikawa, Hec1A, and Sawano) were performed using cell counting kit, and we examined apoptosis assay using caspase 3/7 Glo assay and annexinV detection assay treated with all-trans retinoic acid (ATRA). And then, real-time PCR was performed to analysis the expression of RAR subtypes. In addition, we performed knock down of RAR subtypes in RL95-2, which expressed all kinds of RAR subtypes. RESULTS: Each cell lines had different kinds of the expression for RARs.

Cell proliferation assay showed that the proliferation of RL95-2 was significantly suppressed by ATRA. Apoptosis assay showed that apoptosis in RL95-2, which expressed both RARα and RARβ, was induced by ATRA. And the knock down for not only RARα but also RARβ induced cell suppression using ATRA. CONCLUSIONS: We found RA suppressed the cell proliferation and induced apoptosis in RL95-2.This study showed that RARα might have important roles for the treatment of endometrial cancer, which was same as breast cancer. Moreover, we thought RARβ might be responsible for the treatment of endometrial cancer. Pregnancy is the most successful form of graft tolerance, involving multiple mechanisms that protect the semi-allogeneic fetus from attack by the maternal immune system. We have previously shown that maternal T cells are activated in pregnancy complications in a mouse model and hypothesized that a similar immune response leading to "graft rejection" may be involved in patients with preterm labor (PTL).

We enrolled patients with PTL and healthy term controls and analyzed peripheral blood mononuclear cells (PBMCs) from maternal and cord blood. We performed a phenotypic analysis of T cells to define naïve (N: CCR7 + CD45RA + ), effector (EM: CCR7 -CD45RA -) and central memory (CM: CCR7 + CD45RA -) subsets. We also performed Mixed Lymphocyte Reactions (MLR) with maternal and fetal PBMCs and determined the cytokine production of individual T cells using flow cytometry.

the cord blood of women with PTL (n=27) compared to term controls (n=24), indicating early maturation of the fetal immune system during PTL. 2) Increased percentage of CM Th1 cells (CXCR3 + CCR6 -) that produce IFN-γ, an inflammatory effector cytokine, in PTL. 3) Increased percentage of fetal T cells that react against maternal antigens in MLR assay in women with PTL compared to controls. 4) Significant increased production of IFN-γ among proliferating T cells, only in the context of PTL. 5) Increased production of IFN-γ in maternal CD8 + T cells of patients with PTL.

The presence of IFN-γ producing T cells in the cord blood of patients with PTL suggests an immune response from the fetus to exit a hostile uterine environment. On the maternal side, the IFN-γ secretion would represent a similar breakdown of maternal-fetal tolerance leading to an immunogenic response against the fetus. Determining whether pregnancy complications involve activation of graft rejection has important clinical implications for the treatment of preterm labor. Adiponectin is a hormone specifically expressed in adipose tissue; levels decrease as obesity increases, and low levels of adiponectin are associated with background inflammation and propensity to develop metabolic syndrome. We hypothesize that elevated inflammatory biomarkers and low levels of adiponectin may be associated with unexplained RPL. METHODS: Women with RPL (cases; n=136) were compared to healthy, parous controls (n=137). RPL was defined as two or more pregnancy losses and no more than one prior live birth. Women with positive antiphospholipid antibodies, uterine malformation, or parental balanced translocation were excluded. Controls were defined as at least one live birth and no more than one prior pregnancy loss. Clinical and obstetric history and BMI were obtained. Inflammatory markers including ferritin, IL-6, and adiponectin were assessed and levels were analyzed using linear regression including case status and relevant clinical data in a regression model. Uterine vascular adaptations, including angiogenesis, are essential during early pregnancy. Impaired adaptations contribute to increased risk of pregnancy complications such as preeclampsia and intrauterine growth restriction. Circulating levels of relaxin peak in early pregnancy in humans and are thought to contribute to uterine angiogenesis. This study investigated the role of relaxin in uterine vascular adaptations during early pregnancy using relaxin deficient mice. METHODS: Uterine tissues were analyzed before implantation (days 1-4) in wild type (Rln +/+ ) and relaxin-deficient mice (Rln -/-). Genes included angiogenic factors (VegfA, and its receptor Vegfr2; hypoxiainducible factor-1α, Hif-1α; egl nine homolog-1, Egln1), genes involved in extracellular matrix remodeling (matrix metalloproteinase-14, Mmp14; tissue inhibitor of metalloproteinase-3, Timp3), and receptors for relaxin (Rxfp1) and estrogen (Esr1, Esr2). Immunohistochemistry with antibodies to BrDU (labels proliferating cells) and the endothelial cell marker CD31 was used to visualize the proportion of vessels containing proliferating endothelial cells. Circulating progesterone concentrations were measured by mass spectrometry. RESULTS: Expression of Egln1, Esr1, Hif-1α, Mmp14, Rxfp1, VegfA and Vegfr2 mRNA were significantly increased on day 1 of pregnancy in Rln -/compared to Rln +/+ mice. By day 4 of pregnancy, differences in mRNA expression were no longer detectable between the genotypes for most genes. Proliferating endometrial endothelial cells were observed on day 4, but not day 1, of pregnancy; there was no significant difference in the percentage of vessel profiles containing proliferating endothelial cells between Rln +/+ and Rln -/mice. Plasma progesterone concentrations were significantly elevated at day 4 of pregnancy in Rln -/mice compared to Rln +/+ mice (p=0.0134). CONCLUSIONS: These studies demonstrate that relaxin-deficiency impacts on uterine gene expression in early pregnant mice, but does not cause abnormal endometrial endothelial cell proliferation prior to implantation. The functional consequences of the increased mRNA expression and circulating progesterone levels in Rln -/mice are unknown, but may reflect compensating mechanisms that prevent disruptions to uterine angiogenesis in early pregnancy. A better understanding of processes related to blastocyst formation could lead to novel identifiers of viable embryo development and eventually influence assisted reproductive techniques. We compared messenger RNA levels in cleavage and blastocyst stage embryos to identify differential gene expression during blastocyst development of human embryos. METHODS: Embryos frozen at the pronuclear stage and donated for research were used for this study under approval of our institutional review board. Three good-quality, non-fragmented day 3 cleavage-stage embryos and 3 good-quality day 5 blastocyst-stage embryos were studied individually. RNA was extracted, and after linear amplification, genes were detected using Affymetrix Human U133 Plus 2.0 arrays. We compared messenger RNA levels between these day 3 and day 5 embryos (n = 3 for each group), with statistical significance assigned using a standard t-test and p-values <0.001. The data were validated with independent embryonic gene datasets available in the Gene Expression Omnibus (GEO). The Kyoto Encyclopedia of Genes and Genomes was used to perform pathway analyses for validated genes (p <0.01 considered statistically significant). RESULTS: Fifty genes were found to be differentially expressed between day 3 and day 5 embryos, with fold changes ranging from -5.75 to +18.04. Of 47 genes available for validation in the GEO Datasets, 23 had significant differential expression, with 21 in the same direction as our discovery set (kappa 0.8). Pathway analysis revealed that day 3 and day 5 differentially expressed genes largely represented amoebiasis and cancer pathways (FDR < 0.1). CONCLUSIONS: Our findings suggest that expression of genes involved in embryonic migration, adhesion, and invasion are important in the development of human preimplantation embryos and are critical for the processes necessary to facilitate implantation. Future validation studies and functional analyses will assist in uncovering gene markers that could be used to identify viable blastocyst-stage embryos for in vitro fertilization. Several studies report differences in oocyte and embryo quality in obese women, and alterations in the follicular environment due to obesity may lead to suboptimal oocyte development. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, have been identified in human follicular fluid but they have not been described in the context of obesity. To determine whether obesity alters miRNA expression patterns in follicular fluid, we examined miRNA expression in follicular fluid from lean and obese women. METHODS: Lean (BMI<25; n=4) and obese (BMI>30; n=4) women undergoing in vitro fertilization for male-factor infertility were selected for study with approval of the institutional review board. Total RNA was purified from the follicular fluid and 100 ng samples from each patient were reverse transcribed and preamplified prior to assessment of expression on A-set miRNA TaqMan Low Density Arrays (Life Technologies). miRNA expression was normalized against the mammalian U6 endogenous control and differential expression determined via the conventional ∆∆Ct method. Statistical significance was assigned using a standard t-test with unequal variances, and significance was set at p < 0.05. Validated miRNA targets (through reporter assay, Western blot, or qPCR) for differentially expressed miRNAs were taken from miRTarBase and TarBase. RESULTS: We detected expression of 138 miRNAs in human follicular fluid, of which 24 miRNAs (17%) had statistically significant differential expression. Among these, 23 were significantly downregulated in the obese group. These differentially expressed miRNAs had over 150 validated targets, many relating to relevant functions such as oocyte metabolism, DNA methyltransferase activity, COX2 expression, metalloprotease expression, and granulosa cell apoptosis. CONCLUSIONS: Differential expression in miRNAs occurs in follicular fluid of lean and obese women, indicating an altered follicular environment in obesity. Moreover, there appears to be a nearly universal decrease in miRNA expression in obese women, which may negatively affect the ability of the follicular environment to optimize oocyte development at a post-transcriptional level. Future studies of follicular fluid from obese infertile and fertile women may further refine the identities of miRNAs and targets associated with obesity and infertility. (Hirota, et al., J Clin Invest, 2010) . p53 deficiency is also associated with decidual senescence and provokes growth restriction and terminal differentiation of dedidual cells with increased levels of p21, and senescence-associated β-galactosidase activity (SA-β-gal). These results suggested that premature decidual senescence is a cause for the pathogenesis of preterm labor/birth. Our aim was to determine whether the chorioamniotic membranes (including the decidua parietalis) from women who had undergone preterm or term labor show upregulation of senescence-associated genes and increased SA-β-gal staining. METHODS: Chorioamniotic membranes were collected from different groups of women: 1) who underwent term spontaneous labor (TSL), 2) who delivered term without labor (TNL), 3) who underwent preterm spontaneous labor (PTL), and 4) who delivered preterm without labor (PTNL) (n=7-9 each). Total RNA was extracted and mRNA expression of 84 senescence-associated genes was quantified by qRT-PCR. Staining of SA-β-gal was performed in the chorioamniotic membranes, and its intensity was semi-quantified using a slide scanner. RESULTS: 1) Senescence-associated genes were differentially expressed among groups; 2) seven senescence-associated genes (IRF5, PCNA, IFNG, TWIST1, AKT1, IGF1, and CCND1) were downregulated, and GADD45A was upregulated in the TIL group when compared to the TNL group; 3) twenty-eight senescence-associated genes (E2F3 , IRF5,  CDKN1A, MAP2K1, MAP2K6, CHEK2, MDM2, GSK3B, IFNG, CCNE1,  IRF3, TP53BP1, IRF7, MORC3, TP53,PTEN, TERF2, RBL1, CALR, RB1,  AKT1, PIK3CA, RBL2, IGF1R, SIRT1 , ATM, MAPK14, and CDK2) were downregulated, and SERPINB2 was upregulated in the PTL group when compared to the PTNL group; 4) nine senescence-associated genes (IFNG, CCNE1, COL1A1, CCNA2, COL3A1, CCNB1, FN1, GADD45A, and CCND1) were upregulated in the PTL group when compared to the TIL group; and 5) staining of SA-β-gal was observed only in the choriodecidua, and it was higher in the PTL than in the TIL group. CONCLUSIONS: Upregulation of several senescence-associated genes and SA-β-gal activity in the chorioamniotic membrane is associated with preterm labor when compared to term labor. However, a drastic downregulation of senescence-associated genes is observed in chorioamniotic membranes from women at term and in preterm labor when compared to women without labor. Our preliminary data using a murine model demonstrate that advanced maternal age causes dystocia and increases the duration of active labor, rate of stillbirth, and rate of neonatal mortality (Sanchez-Rodriguez et al, unpublished data). The immunological mechanisms whereby older mothers undergo adverse pregnancy outcomes are unknown. The aim of this work was to investigate the effects of advanced maternal age on the T-cell repertoire in the mother and offspring. METHODS: Older (20-24 weeks old, n=10) and young (8 weeks old, n=10) pregnant B6 mice were euthanized prior to delivery. Spleen and thymus were collected from mothers and fetuses. Decidual tissues were also harvested from mothers. Additionally, neonates (1-week old) from older and young mothers (n=19 each) were euthanized and splenocytes were harvested for immunophenotyping. The T-cell repertoire: Th1 (CD4+IFNγ+), Th2 (CD4+IL4+), Th9 (CD4+IL9+), and Th17 (CD4+IL9+) cells, CTLs (cytotoxic T cells, CD8+IFNγ+), Tc17 cells (CD8+IL17+) and Tregs (regulatory T cells, CD4+Foxp3+) were determined by flow cytometry. RESULTS: 1) Prior to delivery, decidual tissues from older mothers contained lower proportions of Th1 and Th9 cells, when compared to young mothers (p=0.05 and p=0.003); 2) decidual tissues from older mothers contained lower proportions of CTLs and Tc17 cells, when compared to young mothers (p=0.023 and p=0.015); 3) older mothers had lower proportions of thymic Th9 cells than young mothers (p=0.035); 4) fetuses from older mothers had increased proportions of activated CD25+ (p=0.001), IL4+ (p=0.007) and IL9+ (p=0.007) CD8+ T cells, Tc17 cells (p=0.05), and CTLs (p=0.007) in the thymus than fetuses from young mothers; and 5) neonates from older mothers had lower proportions of Tregs (p= 0.04) and higher proportions of Th1 (p=0.001) and Th17 cells (p=0.001), and CD8+IL9+ T cells (p=0.002) than neonates from young mothers. CONCLUSIONS: Advanced maternal age has adverse effects on the T-cell repertoire of the mother and offspring. Advanced maternal age impairs the pro-inflammatory response at the maternal-fetal interface required for on-time parturition, and drives a systemic pro-inflammatory state in the offspring. These novel findings may explain why older mothers undergo dystocia and their offspring die shortly after birth. (EPAB) is an RNA-binding protein expressed exclusively in germ cells and early embryos prior to zygotic genome activation (ZGA). EPAB is required for translational activation of maternally-stored mRNAs in the oocyte and Epab -/female mice are infertile due to impaired oocyte maturation. In addition, folliculogenesis is disrupted in Epab -/females and cumulus expansion and ovulation rates are significantly impaired. The aim of this study was to characterize the molecular mechanisms of follicular somatic cell dysfunction in Epab -/mice. METHODS: First, we determined whether the defect in cumulus expansion and ovulation in Epab -/mice is due to factors inherent to the oocyte. To do this, we utilized a co-culture system of oocytectomized cumulus oophorus complexes (OOX) with denuded oocytes. Cumulus expansion was evaluated following 16 h of culture with or without 10ng/ ml epidermal growth factor (EGF), which mediates luteinizing hormone (LH) action during cumulus expansion. qRT-PCR was performed to assess the expression of downstream mediators of LH/EGF [prostaglandinendoperoxide synthase 2 (Ptgs2 or Cox2), betacellulin (Btc), and tumor necrosis factor alpha induced protein 6 (Tnfaip6)] in cumulus cells. Western analysis was used to assess the activation of ERK cascade in WT and Epab -/granulosa cells in culture after stimulation with LH (1mg/ml), EGF (10ng/ml) or amphiregullin (AREG, 100ng/ml) for 5 min or 10 min. Expression of oocyte derived growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) was similarly tested. RESULTS: EGF treatment of WT OOX/WT oocyte co-culture resulted in normal cumulus cells expansion with increased expression of Ptgs2, Btc and Tnfaip6 (p<0.05 for each transcript). In contrast, when WT OOX was co-cultured with Epab -/oocytes, cumulus expansion did not occur and the expression of Ptgs2, Btc, and Tnfaip6 did not change. Compared to WT, Epab -/oocytes did not show a difference in GDF9 or BMP15 expression. Granulosa cells of Epab -/mice showed impaired response to LH, EGF and AREG treatment, with decreased expression of downstream mediators p-MEK1/2, p-ERK1/2, and p90RSK (p<0.05).

In conclusion, our findings demonstrate that EPAB is required for the oocyte to send appropriate signals to surrounding somatic cells for cumulus cell expansion to occur. Importantly, EPAB acts through a pathway that is independent of the GDF9/BMP15 system but is required for EGF signaling in cumulus and granulosa cells.

Characterizing INTRODUCTION: Success in fertility treatment often depends upon the ability of gametes and early embryos to adapt appropriately to the stress imparted on them from exposure to a culture environment. Early embryos express the constituents of several well-known stress response pathways in an effort to mitigate adverse responses to culture. Our research focuses on p66Shc, a cellular stress sensor protein that regulates mitochondrial reactive oxygen species production. We hypothesize that p66Shc is integral to the early embryo's culture adaptive stress response mechanisms. As a prelude to defining p66Shc function in preimplantation development and in response to culture, we have investigated p66Shc expression and localization and the influence of culture on these parameters. METHODS: p66Shc transcript abundance and protein localization in mouse preimplantation embryos collected in vivo and after in vitro culture was characterized by RT-qPCR and immunofluorescent confocal microscopy. Mature oocytes and zygotes were collected from the oviducts of female mice, while 2-cell, 4-cell, 8-cell, morula and blastocyst-stage embryos were either collected in vivo from oviducts or after in vitro culture at 37ºC/5% O 2 /5% CO 2 humidified microenvironment. RESULTS: In vivo, p66Shc transcript abundance significantly decreased from the mature oocyte to 2-cell stage (p<0.05) and remained low to the blastocyst stage. In vitro, p66Shc transcript abundance also decreased from the 2-cell stage to the blastocyst stage (p<0.05). Interestingly, p66Shc expression significantly decreased in in vitro cultured 2-cell embryos compared to in vivo stage matched controls (p<0.05). Similarly, in vivo 2-cell embryos displayed enhanced immunofluorescence intensities of total p66Shc compared to in vitro cultured 2-cell embryos. Serine 36-phosphorylated (pSer36) p66Shc localized to the nuclei from the 2-cell stage onwards in both in vivo and in vitro cultured embryos. CONCLUSIONS: These results suggest that p66Shc transcript is accumulated and stored in the oocyte and may be degraded prematurely and inappropriately during in vitro culture. Unexpectedly, pSer36-p66Shc was localized to the nucleus in early embryos. Future work will determine the role of p66Shc during preimplantation embryo development and during the in vitro adaptive stress response. Human IVF embryos of different developmental stages and competencies were analyzed using immunohistochemical, microscopic, and cytogenetic techniques to quantify and characterize such nuclear abnormalities and determine their relevance to developmental potential. METHODS: Three types of IVF embryos with different developmental potential were fixed, stained, and imaged using confocal microscopy: 89 cryopreserved "good" quality cleavage stage IVF embryos, 44 cryopreserved "good" quality IVF blastocysts, and 13 "poor" quality fresh embryos that had arrested. DNA staining (Hoechst) was used to identify extra nuclear DNA and centromere antibody (CENP-A) staining was used to better understand the mechanism in which it was formed (DNA fragment, whole chromosome). 19%) blastomeres contained centromere negative abnormalities, and 21/103 (20%) blastomeres contained both centromere positive and centromere negative abnormalities. Six additional "good" quality cryopreserved cleavage stage embryos were thawed, imaged, and blastomeres dissociated and karyotyped. Of the three embryos containing microscopic nuclear abnormalities, 9/12 (75%) blastomeres were aneuploid and 3/12 (25%) blastomeres were euploid. Of the three microscopically normal cleavage stage embryos, 2/18 blastomeres (11%) were aneuploid and 16/18 (89%) blastomeres were euploid.

The presence of abnormal nuclear formation in IVF is common, negatively associated with developmental potential, and may be associated with aneuploidy. Meiosis is central to germ cell development and requires a single round of DNA replication followed by two successive steps of chromosome segregation. Anomalies in chromosome segregation lead to aneuploidy, an abnormal chromosome number. Aneuploidy is a major cause of pregnancy loss, infertility, and birth defects, yet the molecular basis of the defects is poorly understood. Sister chromatid cohesion, a process mediated by proteins called cohesins, is crucial to ensure accurate chromosome segregation. Here, we present novel interactions between cohesin proteins and CHTF18 during meiosis. CHTF18 is a member of the Replication Factor C -like (RLC) complexes that play a role in DNA replication and sister chromatid cohesion in yeast and in human cell lines. We recently demonstrated essential roles for CHTF18 in mammalian meiosis. Mutant mice lacking CHTF18 (called Chtf18-null) are subfertile, meiotic recombination is defective, and homologous chromosomes separately prematurely during prophase I. Our objective in the current study was to investigate the mechanism underlying premature homologue disjunction in Chtf18-null mice. METHODS: Testis nuclear and whole cell protein extracts were prepared from wild-type and Chtf18-null male mice at postnatal day 16. Coimmunoprecipitation experiments were performed and protein expression was determined by western blot analysis using antibodies to CHTF18 and cohesin proteins. Triton-X-100 and centrifugation were used to fractionate cells into soluble proteins and insoluble chromatin-bound proteins. Protein levels were quantitated with densitometry and statistical analyses were performed with the unpaired t-test. RESULTS: CHTF18 co-immunoprecipitated with all of the cohesin complex subunits. Although absence of CHTF18 did not affect cohesin protein expression, meiosis-specific cohesins bound to chromatin were significantly decreased during prophase I. Furthermore, the decrease in meiosis-specific cohesins bound to chromatin was sensitive to increasing salt concentration. CONCLUSIONS: CHTF18 stabilizes or maintains meiosis-specific cohesins on chromatin during prophase I, likely ensuring accurate chromosome segregation during mammalian meiosis. These findings have important implications for the molecular basis of aneuploidy in humans.

Attenuation of Fibroblast Growth Factor Signaling By Poly-N-Acetyllactosamine Type Glycans. While we attempted to remove glycans from living human spermatozoa using glycosidases, we found human sperm cells treated with endobeta-galactosidase (EBG), which specifically hydrolyzes poly-Nacetyllactosamine type glycans (polyLacs), moved more rapidly than untreated sperm.We investigated the mechanism underlying EBGtriggered sperm motility and found that seminal fluid polyLacs down regulate sperm motility by impairing fibroblast growth factor-dependent signaling. METHODS: Sperm and seminal fluid samples were obtained from healthy 20 study volunteers. RESULTS: Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor (FGF) signalling.

Plate assay and glycan array demonstrated positive binding between FGFs and polyLacs. Furthermore, heparin did not inhibited polyLac binding to FGFs, suggesting that FGF has distinct binding sites each for heparin and polylacs. Since both FGFs and polyLacs are expressed widely in mammalian tissues and in many cell types, we tested effect of polyLacs on FGF signaling using human kidney epithelial HEK293T cells, which showed down-regulation of tyrosine phosphorylation of FGF receptor. CONCLUSIONS: These results suggest that polyLacs may regulate FGF signalling in a variety of cell types and that sperm cells employ a conserved mechanism requiring highly concentrated polyLacs to regulate motility 2 Resolution of inflammation at menses is critical to limiting tissue damage and efficient endometrial repair. Apoptosis and clearance of apoptotic cells are necessary for this process and involve neutrophils.

Our aim was to study the processes of neutrophil recruitment, apoptosis and resolution of inflammation in a mouse model of induced menses. We hypothesise that, as in its human counterpart (unpublished data), mouse endometrium will show significant apoptosis preceding resolution of inflammation and tissue repair. METHODS: C57Bl/6 mice underwent induced menstruation via sequential oestrogen and progesterone (P 4 ) exposure and decidualisation via intrauterine oil (Cousins et al., PLOS ONE 2014) . P 4 withdrawal induced bleeding and menstrual-like endometrial changes.

Mouse uterine tissue sections (n=39) were immunostained for cleaved caspase-3 (an apoptosis marker) and Ly6G (a mouse neutrophil marker) at several time points after P 4 withdrawal (0h, 4h, 8h and 24h). Caspase-3 expression was assessed by semi-quantitative histoscoring; neutrophil abundance was determined by semi-and fully-automated cell counting strategies. RNA was extracted from matched uterine samples (n=20), and TNFα and CXCL1 transcription was assessed by qRT-PCR and normalised to β-actin. RESULTS: Cleaved caspase-3 was observed to be increased significantly at 8 and 24 hours post-P 4 withdrawal, particularly in the decidualised endometrial stroma.

Neutrophil abundance increased at 8 and 24 hours post-P 4 withdrawal, before and during tissue shedding and regeneration. CXCL1, a neutrophil chemokine, was significantly up-regulated at 24 hours post-P 4 withdrawal, as was TNFα, an inducer of inflammation and apoptosis. CONCLUSIONS: Decidual stromal apoptosis became widespread before shedding of endometrial tissue and persisted into repair of the endometrial surface. Apoptosis preceded all other features of menstrual-like changes, including neutrophil recruitment and CXCL1 and TNFα up-regulation. The observation that neutrophils were recruited prior to chemokine CXCL1 induction is of interest and suggests this chemoattractant may be important for the resolution of menstruation. 

HESCs from proliferative phase were treated with vehicle or thrombin at baseline or during decidualization using cAMP and MPA. Thereafter, mRNA and secretory protein were analyzed by qPCR and MAGPIX ® Beads ELISA assays. RESULTS: As expected, two marker of decidualization (IGF-1 and prolactin (PRL)) were induced 15-and 9-fold 72 h after initiation of cAMP/MPA (p<0.01). Likewise, secreted PRL increased from 166 ± 65 to 12389 ± 429 pg/mL (p<0.01) and IGFBP-1 from 30 ± 6 to 247 ± 37 pg/mL (p<0.01). Treatment with plasma thrombin (0.5-6 U/ml) decreased transcripts encoding IGF-1, IGFBP-1, and PRL at baseline (71-73%, P<0.04) in a dose-and time-dependent manner with maximal effects at 72h. Interestingly, thrombin (2 U/ml) also inhibited decidualizationinduced expression of IGF-1 as well as decreased PRL and IGFBP-1 mRNA from 9.7±1.5 to 5.5±0.6 R.U./GAPDH (p=0.057) and 18.9±3.6 to 12.5±1.3 R.U./GAPDH (p<0.01) respectively. Similarly thrombin decreased secreted prolactin and IGFBP-1 at baseline (54-50%) and 72 h after decidualization (13-14%). Thrombin induced MMP1 at baseline (7-fold, p<0.01) and during decidualization (11.4-fold, P<0.01). Basal COX-2 mRNA was also induced 8-fold and amplified in cells undergoing decidualization (from 4-to 7.7-fold). In contrast, thrombin decreased expression of collagen 1a1 50-65% and lysyl oxidase, but not fibronectin. Recombinant thrombin mimicked the effects of plasma thrombin suggesting that these effects were not due to contamination. Like thrombin, PAR-1 agonist inhibited IGF-1, PRL, LOX, and Col1a1. CONCLUSIONS: Taken together, our data indicate that thrombin adversely affects decidualization in vitro primarily through PAR-1. Furthermore, thrombin not only activates genes involved in matrix degradation (MMPs) and inflammation (COX-2), but also suppresses genes important for collagen formation (Col1a1, LOX). The results suggest that intrauterine bleeding and generation of thrombin disrupts matrix homeostasis of the endometrium, impairs decidualization, and endometrial support of early pregnancy.

Roles The age at which women deliver their first child has increased steadily in recent years, particularly in Western societies. Aging is associated with poor pregnancy outcomes, and studies have shown that changes in the uterine microstructure affect implantation success. Little is known about age-related changes occurring between the peri-implantation period and term pregnancy. Here, we investigated pregnancy loss at several periods throughout gestation in young and aged rats to enable detailed mechanistic investigations into causes of age-related pregnancy loss. METHODS: Aged female Sprague Dawley rats (9.5 months; approximately equivalent to a 35 year old woman) and young controls (4 months) were mated with young males. Implantation was assessed on gestational day (GD)5, GD8, GD15, and GD20. RESULTS: On GD5 pregnancy success was lower in aged rats (young: 100%; aged: 66%), although preliminary data suggest that of the rats that were pregnant, the number of implantation sites between young and aged dams was not different. On GD8, 9/9 young dams were pregnant, compared to 7/11 aged (P<0.05 by Chi-square test). Aged pregnant dams had 39% fewer implantation sites (young=15.2±0.8, vs. aged=9.3±1.7 implantation sites, P<0.01), and of those implantation sites, more of them were undergoing various stages of resorption (young=0 vs. aged=5.6±2.0 resorptions, P<0.01); similar results were observed on GD15. Overall, by GD20, aged dams had a reduced capacity to carry a viable pregnancy to term (young=90% vs. aged=50%), and had reduced litter sizes (young=15.0±0.57 pups vs. aged=8.5±1.6 pups, P<0.01). CONCLUSIONS: Pregnancy in aged dams is impaired early, which is attributed to both implantation failure and miscarriage. These results are dramatic given the aged rats used in this study are otherwise healthy, without existing comorbidities. This model of advanced maternal age will enable the assessment of mechanisms underpinning the pregnancy loss, including those pertaining to oocyte quality, embryo developmental competence, spiral remodelling and depth of trophoblast invasion. family. TRP channels are cation conduction ion channels that serve an important role in cellular sensing. However, the distribution of TRP channels in the human endometrium is unknown.

Here, we investigated the functional expression pattern of TRP channels in primary human stromal cells by quantitative realtime PCR (qRT-PCR), immunocytochemistry, calcium microfluorimetry, and whole-cell patch-clamp experiments. RESULTS: Starting from frozen endometrial biopsies obtained from the window of implantation, qRT-PCR revealed the expression of a large subset of the TRP channels. Thereafter, their expression was assessed in purified primary stromal cell cultures set up from freshly isolated endometrial biopsies obtained during the luteal phase, and mRNA expression from TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4, and TRPM7 was identified. The lack of specific pharmacology confined our further analysis to TRPV2, TRPV4, TRPC6, and TRPM7. Immunohistochemistry showed clear expression of TRPV2 and TRPM7 in the plasma membrane as well as in the cytoplasm. TRPV4 could be detected in the plasma membrane and showed cytoplasmic vesicle, whereas TRPC6 protein was more abundantly expressed in the perinuclear area. Further functional characterization by Ca 2+ -microfluorimetry and whole-cell patch-clamp techniques using specific pharmacology, provided evidence for functional expression of TRPV2, TRPV4, TRPC6, and TRPM7 in human stromal cells. CONCLUSIONS: For the first time, we have demonstrated via different experimental approaches the functional expression of TRPV2, TRPV4, TRPC6, and TRPM7 in human endometrial stromal cells. Further research is required to investigate the possible role of these ion channels in the epithelial-stromal crosstalk during the process of embryo implantation.

The Vaginal secretions were dissolved in PBS and scanned with a 9.4T NMR scanner with 5mm BBO probe using a Watergate water suppression pulse sequence. Identified metabolites in the 1 H NMR spectra were integrated and normalized to the total spectrum integral (omitting the water signal).

Vaginal microbial DNA was extracted and amplified with QIAamp DNA mini kit and genus-specific primers to examine for a range of commensal and potential pathogenic species. PCR amplicons were verified by gel electrophoresis. RESULTS: Five metabolites (lactate, glucose, acetate, alanine, glutamine/ glutamate) were identified. There was a significantly higher acetate integral in preterm SYM women compared to the other groups (2-way ANOVA, p<0.05). Although not significant, term ALR women showed the highest lactate integral with a trend to lower lactate in term AHR (p=0.29) and the lowest for the preterm SYM groups (p=0.06). No other differences were found in relation to delivery outcomes. PCR revealed several commensal Lactobacillus sp. and PTB associated organisms. The later were found in a higher proportion of SYM group. CONCLUSIONS: Higher amounts of vaginal lactate may be associated with reduced risk of PTB, while high acetate levels in vaginal secretions appear to be associated with an increased risk of PTB. Progesterone is essential for establishment and maintenance of pregnancy and its withdrawal converts the myometrium to the laboring state. Human parturition is triggered by a functional progesterone withdrawal through changes in the progesterone receptor (PR) signaling in myometrial cells. The human PR exists as two main isoforms, PR-A and PR-B. We hypothesize that functional progesterone withdrawal is at least in part mediated by post-translational modifications of the PRs; especially serine phosphorylation (pSer). We tested this hypothesis by determining the changes in the pattern of serine phosphorylation on PRs in association with labor status in term human myometrium.

The abundance of pSer-PR isoforms in term myometrium was determined by immunoblotting using commercially available pSer-PR antibodies on lysate from myometrial tissue obtained from women (with informed consent) undergoing c-section delivery at term before (n=7) and after (n=7) the onset of labor. The regulation of PR pSer by progesterone was examined in the hTERT-HM A/B immortalized human myometrial cell line. RESULTS: Immunoblotting using commercially available pSer PR antibodies showed that pSer at serine 345 was the most abundant form of pSer-PR and was only detected on PR-A. pSer345-PR immunoreactivity was localized to the nucleus of myometrial cells. The relative abundance of pSer345-PRA decreased significantly (P<0.05) in association with the onset of labor. In hTERT-HM A/B cells, pSer345 was induced by progesterone within 15 minutes and in a dose dependent manner and was predominantly on PR-A. CONCLUSIONS: In term human myometrium serine 345 is phosphorylated on PR-A. In other studies pSer345 causes PRs to tether with other transcription factors such as AP-1 and SP-1. This modification may be involved in the process of parturition through its potential effects on the transcriptional activity of PR-A in myometrial cells. treatment. An initial screen of the splice factors hnRNPA1 and SR proteins (SRSF1, SRSF2 and SRSF5) were found to be gestationally regulated in the pregnant mouse myometrium. P4 treatment had profound effects on the gestational profile of these splice factors. CONCLUSIONS: This study will define the splicing mechanisms and factors that ultimately change the functionality of ERα to produce proteins of very different and sometimes contrary functions as they relate to the pregnant uterine myocyte. , is a polycarbonate compound used in plastic products and is a major environmental pollutant. It has documented pro-estrogenic properties but its impact on risk for spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) is unclear. Therefore, we evaluated the association between maternal plasma (MP) and amniotic fluid (AF) BPA levels and risk of PTB and pPROM.

In this nested case-control study, AF and MP samples from women in term labor (n=45), preterm labor that ended with preterm delivery (n=36) or who had pPROM (n=36) were assayed for BPA using an enzyme immunoassay. BPA concentrations were evaluated by univariate analysis and correlations with pregnancy outcomes analyzed with multinomial logistic regression with R. Results are presented as odds ratio with 95% CI before (cOR) and after (aOR) adjustment for maternal confounders. The CSE increase at term is P 4 -independent, while CDO is P 4 responsive in term and preterm labor. We propose that coordinated regulation of L-Cys availability by P 4 and other unidentified signals are part of the gestational clock. This is the first report of gestation-regulated changes in L-Cys metabolism, and as H 2 S production may be increased at term, our data suggest a role other than tocolysis for uterine H 2 S expression. At parturition increased PR-A represses the anti-inflammatory actions of PR-B leading to tissue-level inflammation, which induces labor. We examined the functional interaction of these processes using mathematical modeling on existing transcriptome datasets from laboring and quiescent term myometrium. METHODS: Differential equations were used to express PR-B transcriptional activity as a function of transcription rate and capacity to inhibit inflammation, and inflammation as a function of its generation rate and its inhibition by PR-B. The equations were applied to two published transcriptome datasets of laboring and quiescent term myometrium.

Surrogates were used to reflect PR-B activity (FOXO1A or FKBP5) and inflammation (IL-1ßor IL-8). Phase space analysis was used to determine a probability of labor for each sample. Predictive power was assessed by cross-validation where one dataset served to build a classifier to predict labor and the other was tested on that classifier. Precision and recall statistics were compared to single-gene predictors.

The qualitative dynamics of our model mimic those observed in human pregnancy and parturition with respect to P4 responsiveness and tissue level inflammation. PLSDA score plots showed much greater separation of cases and controls at 20 weeks than 15 weeks with the 2 cases that delivered earliest furthest from controls. Random forest classification showed much improved classification at 20 weeks versus 15 weeks with error rates as low as 0.221. Quantifying dysregulation by outlier methods also shows that much greater dysregulation of metabolomic profiles is evident at 20 weeks than 15 weeks. Furthermore the two cases that are closest to delivery at 20 weeks having gestational age at 22 and 23.7 weeks show markedly increased dysregulation compare to other cases. CONCLUSIONS: Our preliminary findings show that the closer to delivery the more markedly perturbed the metabolomic profile. We consistently find through a vareity of analysis methods that the two cases in our cohort that deliverd at 22 and 23.7 weeks show highly dysregulated profiles at the 20 week time point and are the most discriminatory cases as seen on PLSDA score plots. This points towards the possibility of developing a highly specific and sensitive test to predict SPTB that will occur within 4 weeks. We await validation data to confirm our findings and to further investigate this possibility. INTRODUCTION: Preterm birth is defined by birth of a baby at less than 37 weeks of gestational age and is the most common catastrophic and costly complication of pregnancy. Numerous studies have provided compelling evidence that IL-6 produced by the decidua in response to inflammation plays a role in the pathophysiology of preterm labor. However, there are no appropriate therapeutic interventions available to circumvent inflammation-mediated preterm labor. Curcumin, a naturally occurring polyphenolic compound, has been widely used as an alternative therapeutic agent to treat inflammatory and infectious diseases. Yet, very little is known about the anti-inflammatory effect of curcumin in the uterine environment. Therefore, the goal of this study was to identify whether curcumin can be used as a safe and effective pharmacological compound in the prevention of inflammation-mediated preterm birth.

To examine the effect of curcumin on preterm labor, we utilized a novel injectable sustained release curcumin microparticle.

Female mice were challenged with LPS on day 14.5 of pregnancy in the presence or the absence of curcumin microparticles and observed for signs of labor and the number of pups born. To further understand the molecular mechanisms of curcumin action, the effect of curcumin on the expression and regulation of IL-6 was evaluated in human primary decidual cells and a rodent decidual cell line. Involvement of NF-kB and p53 transcription factors was evaluated.

Our in vivo studies clearly demonstrated that supplementation of curcumin microparticles effectively prevented LPS-induced preterm labor and fetal loss. Cell culture studies demonstrated that curcumin completely inhibited the expression of IL-1β-induced IL-6 as well as gp130, whereas expression of IL-6R was not affected. Furthermore, curcumin treatment inhibited phosphorylation of STAT3, which is a downstream effector of IL-6 signaling. We also found that curcumin completely attenuated IL-1β-induced IL-6 promoter activity in decidual cells. Our results further demonstrated that curcumin inhibited IKK phosphorylation, NF-kB expression and nuclear localization, while upregulating p53 in decidual cells, which was correlated with inhibition of IL-6. CONCLUSIONS: Taken together, our results strongly suggest that curcumin effectively prevented LPS-induced preterm labor and this may be mediated through the inhibition of IL-6 at the level transcription by a mechanism involving opposing effects of NF-kB and p53. We defined a decidual hemorrhage SPTB (PTB/DH) phenotype based on clinical data, including the presence of vaginal bleeding at anytime during pregnancy, placental pathology and other factors. Cases with PTB/ DH were compared first to a subset of the term controls without DH (TB/ No DH). We also performed an interaction test including all cases and controls to identify SNPs that interact with hemorrhage to result in PTB. Significance of each analysis was determined by logistic regression, adjusted for three principal components to account for population stratification. The results of each test were analyzed for genelevel association using VEGAS, which calculates significance levels for 17,744 known genes. All genes with p<0.05 in the VEGAS results were analyzed for pathway enrichment using InnateDB. RESULTS: 698 mothers with a history of PTB (cases) and 715 mothers with no history of PTB (controls) were included. Among them, 30% of cases and 3.4% of controls had DH. Although no SNPs achieved genome-wide significance (p<5e-8) in either analysis, the best result from each analysis falls near SNPs previously associate with age at menarche. VEGAS identified one gene (ZNF681, p<1e-6) as significantly associated in the test of cases with hemorrhage versus controls without. 900 genes with a p< 0.05 were included in the pathway analysis. The "co-stimulatory signal during t-cell activation" pathway was significantly over-represented among PTB/DH cases (adj. p=0.006). 92 pathways had p<0.05 pathways were not significantly over-represented in women with PTB/DH. CONCLUSIONS: Although no single SNP reached genome-wide significance, the ZNF681 gene and "co-stimulatory signal during t-cell activation" pathway were significantly associated with PTB/DH. that have yet to be fully characterised. The myometrial primary cell culture model is well established for studying the mechanisms of progesterone action in vitro. However, it is limited by the significant reduction of progesterone receptor levels when compared to tissues and the need for the use of supra-physiological progesterone doses (10µM) to elicit a response to cytokines such as IL-1β. This study sought to establish an alternative ex vivo model to study the mechanism of progesterone action. METHODS: Myometrium samples obtained from consenting women undergoing elective caesarean section at term were dissected into explants measuring 3x3x3mm 3 and placed in DMEM supplemented with L-glutamine and penicillin-streptomycin. The explants were treated for 6 hours with progesterone (1 or 10µM) or ethanol vehicle prior to addition of IL-1β (10ng/mL) or water vehicle for a further 24 hours, at which point all tissues were snap-frozen. Whole tissue protein extracts were prepared via bead homogenisation and western blotting was used for COX-2, 11βHSD1 and GAPDH estimation. RNA was extracted and following cDNA synthesis, qPCR was conducted for COX-2 and GAPDH.

The addition of IL-1β led to a significant increase in COX-2 protein versus vehicle control (p<0.05). The addition of progesterone, at a concentration of both 1 (p<0.05) and 10µM (p<0.05), led to a significant reduction in IL-1β-driven COX-2 protein expression levels. Preliminary RNA data also corroborates these findings. The addition of progesterone (1 (p<0.05) and 10µM (p<0.05)) led to a significant increase in 11βHSD1 protein.

CONCLUSIONS: Myometrium in explant culture has been shown to respond both to progesterone as well as to IL-1β. The use of shortterm myometrial explant cultures offers a novel method of assessing the molecular mechanisms of progesterone action offering many advantages over primary cell culture. Using this model, further studies will be undertaken to establish the mechanism of action of progesterone via selective pharmacological inhibition of the progesterone and/or glucocorticoid receptors. Our work showed that stiffness varies along the length of the cervix and there are aligned and organized scattering sources in the cervix. These observations suggest that acoustic attenuation likely varies along the length of the cervix and that attenuation is likely dependent on the angle of interrogation relative to the scattering sources. However, these sources of variance were not accounted for in previous studies. In this work, we test the hypothesis that spatial location along the length of the cervix and angle of interrogation relative to the canal contribute significantly to attenuation coefficient estimates, and by controlling for these sources of variance attenuation estimates are more sensitive to cervical ripening status. METHODS: Cervix specimens were obtained from premenopausal women undergoing hysterectomy for benign reasons. There were three groups of women: those unripened (n=5), those experiencing uterine bleeding (n=5), and those given 400 mg of misoprostol 10-12 hours prior to surgery (n=4). The specimens were positioned so that the endocervical canal was parallel to the transducer face and scanned with a Siemens Acuson S2000 and an 18L6 linear array transducer. Radiofrequency echo signals were collected with the acoustic beams steered from -32 o to +32 o in steps of 4 o and attenuation was calculated using the Reference Phantom Method. The internal os and external os locations were used to group the attenuation measurements among specimens based on fractional distance from the external os. Attenuation anisotropy (angle of interrogation dependence of attenuation estimates) was estimated by subtracting the attenuation estimate for 0 o data and comparing results as a function of beam steering angle. RESULTS: There was a significant difference (p=0.007) in attenuation estimates among spatial locations along the cervix. The attenuation also varied with angle of interrogation. Attenuation anisotropy increased linearly with steering angles (0.6 dB cm -1 MHz -1 range at the mid-proximal location). Accounting for spatial location and angle of interrogation, there was significantly different attenuation estimates among ripening states. (p < 0.001). CONCLUSIONS: These results are consistent with the presence of spatially-dependent aligned collagen in the cervix and suggest great promise for monitoring cervical change.

influence the susceptibility. In genetic linkage analysis, we identified a linkage peak close to the C-X-C chemokine receptor type 3 (CXCR3) gene. CXCR3 is a transmembrane protein present on T cells, natural killer cells, macrophages and airway epithelial cells. In here, the objectives were to elucidate the role of CXCR3 in singleton SPTB and to analyze the levels of CXCR3 and its ligands in the onset of delivery.

The only common single nucleotide polymorphism (SNP) within CXCR3 (rs2280964) was genotyped. The frequency of this SNP in SPTB cases and controls of the discovery and replication populations was compared. Family-based association test was performed. Samples from umbilical cord blood, placental tissue and fetal membranes were collected from preterm and term deliveries. The concentrations of CXCR3 ligands were measured from the cord blood and the expression of CXCR3 in preterm placentas was visualized by immunohistochemistry. RNA and proteins were extracted from the preterm and term placentas for quantitative PCR and Western analyses, respectively. The Mann-Whitney U or Kruskal-Wallis tests were used to study the association between the SNP and expression of CXCR3. RESULTS: CXCR3 SNP rs2280964 associated with SPTB in infants from families with multiple preterm births (p=0.009); family-based association test was also significant (p=0.007). The minor allele (A) had a protective role. By immunohistochemistry, CXCR3 was detected mostly in cytotrophoblasts, syncytiotrophoblasts and decidual and chorionic trophoblasts. Staining intensity of trophoblasts depended on the fetal rs2280964 allele, with A allele associating with weaker staining. Both at the mRNA and protein level, statistically significant increase in the expression levels of CXCR3 were observed in SPTB placentas. The concentration of CXCR3 ligand (CXCL9) increased in cord blood of SPTB infants, as well.

The genetic, immunohistochemical and quantitative analyses suggest the involvement of CXCR3 mediated signaling in the early onset of labor and preterm birth. The gene encoding hBD2, DEFB4, is part of a cluster on chromosome 8 that is variable in copy number (CN). Increased serum hBD2 is associated with increased DEFB CN. We hypothesized that variation in DEFB CN will be associated with preterm birth.

In a retrospective, case-control study, genomic DNA (n=378) and serum (n=140) were extracted from blood collected from White European women at 11-13 weeks' gestation attending King's College Hospital between March, 2006, and September, 2010 . DEFB copy number was determined by paralogue ratio test. Serum hBD2 concentration was measured by ELISA. Data were analysed using Pearson correlation (Excel) and binary logistic regression (SPSS). RESULTS: DNA was available for 102 women who delivered preterm in the index pregnancy or had a history of preterm delivery; these women were considered cases. 152 women had had at least one previous term delivery and delivered at term in the index pregnancy; these 152 women had no prior history of preterm birth and were considered controls. Modal CN was 4 (range 2-7). Serum was available for 140 women. Median hBD2 concentration was 761.5 pg/mL (IQR 449.6-1232.0). There was no association between DEFB copy number and preterm birth. There was no correlation between copy number and serum hBD2 concentration. CONCLUSIONS: Although variation in hBD2 protein expression in the first trimester may be of use to evaluate preterm birth risk, there is no association between DEFB copy number and preterm birth. Correlation between DEFB copy number and serum hBD2 expression, observed in non pregnant adults, is not present in the first trimester of pregnancy; this may be due to variation in regulatory sequences -some of which are progesterone and oestrogen sensitive -between individual copies.

to characterise the intrauterine response to E.coli lipopolysaccharide (LPS) exposure in a chronically catheterised ovine model of second trimester pregnancy. Human parturition is regarded as an inflammatory process, in which the pro-inflammatory cytokines, such as IL-1β, are involved. A number of transcription factors, including NF-κB and AP-1 are activated, which lead to the expression of labour-associated genes such as COX-2. Progesterone (P4) probably exerts an anti-inflammatory role to maintain uterine quiescence during pregnancy and P4 withdrawal leads to the onset of labour. Our previous data showed that P4 acts via GR to repress IL-1β-driven COX-2 expression. In this study, we have investigated other IL-1β responsive genes and aim to explore the mechanisms by which P4 represses the effect of IL-1β in human myometrial cells. METHODS: Cultured of human myometrial cells were transfected with siRNA against PR or GR and exposed to different stimuli at day 4 post-transfection, including 5ng/mL IL-1β and10uM P4, either alone or in combination for 6 h. Total RNA was extracted and 3 representative samples, which expressed varying PR levels, but similar levels of GR, was subjected to Affymetrix Human Genome U133 plus 2.0 Array analysis. To confirm microarray data, qPCR was performed on candidate genes.

The list of IL-1β-driven genes from the array was compared to the list of genes that was previously found to be increased by p65 overexpression and 20 were increased by both p65 overexpression and IL-1β (greater than two fold) and none were repressed by the addition of P4. These findings were confirmed by qPCR validation of 4 randomly chosen genes. Conversely, of the IL-1β-driven genes that were repressed by P4, none were increased by p65 overexpression. Cells were treated with PMA, an AP-1 agonist, but which also activates p65 and then studied a selection of IL-1β-driven genes. Generally, genes that were driven by p65, IL-1β and PMA were not repressed by P4 but those genes that were driven by IL-1β and PMA but not p65 were repressed. The nuclear receptor through which P4 repressed IL-1β-driven genes was investigated and P4 appeared to act exclusively via GR. In the gene array, for some IL-1β-driven genes (eg.IRAK3), the addition of P4 further enhanced their expression. This was also mediated via GR.

These data show that although IL-1β activates both AP-1 and NF-κB and that P4 appears to repress most of the AP-1 related genes, but has no effect on NF-κB related genes and this repression acts mainly via GR. significantly decreases expression of mPRα (Paqr7) in uterus harvested eight hours after injection in CD-1 mice. We hypothesize that decreased expression of mPRα contributes to the diminished P4 responsiveness before labor. Therefore, the aim of this study is to examine the expression of mPRα and other reproduction-related genes in uterus, placenta and fetal membrane at different gestational ages (GAs) in mice. METHODS: RNA samples were extracted from uterus, placenta, and fetal membrane (n=2-6) collected at E12.5, E14.5, E15.5, E16.5, E17.5, E18.5, or postpartum (E20.5) according to timed pregnancy. The levels of transcripts were detected by RT-PCR and normalized to the levels of Gapdh. Statistical testing was by nonlinear or linear regression followed by stepdown bootstrap analysis. RESULTS: Mouse uterus demonstrates polynominal expression patterns for Paqr7, steroid receptor coactivator 2 (Ncoa2) and cyclic AMPresponsive element-binding protein 3 (Creb3), with expression increasing with advancing GA, peaking by E16.5 and declining before the initiation of labor (at E18.5). In addition, COX-1 (Ptgs1) expression in uterus increases with advancing GA to E18.5 and declines postpartum to a level similar to that of E12.5. Uterine progesterone receptor membrane component 1 (Pgrmc1) and Il-1β levels rise significantly after labor. In placenta and fetal membrane, the levels of Paqr7, Ncoa2 and Creb3 rise with GA and do not decline before labor. CONCLUSIONS: Uterine mPRα steadily increases in early pregnancy and is down-regulated preceding both term and induced preterm labor in mice, whereas mPRα in placenta and fetal membrane rises through pregnancy and does not decline before labor. We speculate that regulated expression of Paqr7 and nPR coactivators Ncoa2 and Creb3 might contribute to altered P4 sensitivity in uterus, placenta and fetal membrane throughout pregnancy and as labor approaches. Parturition is an inflammatory process mediated to a significant extent by macrophages. Progesterone maintains quiescence in pregnancy, and a proposed functional withdrawal of progesterone classically regulated by nuclear progesterone receptors (nPRs) leads to labor. Progesterone can impact the functions of macrophages despite the absence of expression of nPRs in these immune cells. Therefore, in this study we investigated the effects of activation of the putative membraneassociated progesterone receptors (mPRs) on macrophage functions and discuss the implications of these findings for pregnancy and parturition. METHODS: Murine macrophage cells (RAW264.7) were pretreated without or with 20 µM PD98059, a specific inhibitor of MEK1/2, or 30 µM H89, a known PKA inhibitor, for one hour. This was followed by incubation with control or P4 modified to be active only extracellularly by conjugation to BSA (P4BSA, 100nM) for two, 15 and 30 minutes for signal transduction experiments or for four hours for mRNA and protein isolation. RT-PCR was used to examine mRNA expression levels and western blotting was used to evaluate phosphorylation levels of signal transduction components and protein expression. RESULTS: Activation of mPRs by P4BSA (100nM) caused a proinflammatory shift in mRNA expression profile, with significant upregulation of the expression of cyclooxygenase 2 (Ptgs2), Il-1B, and Tnf and down-regulation of mPR-alpha (Paqr7) and oxytocin receptor (Oxtr). Pretreatment with PD98059, a MEK 1/2 inhibitor significantly reduced P4BSA-induced Ptgs2, Il-1B, and Tnf mRNA. Inhibition of protein kinase A (PKA) by H89 blocked P4BSA-induced Il-1B and Tnf mRNA levels. Pretreatment with either PD98059 or H89 also significantly diminished P4BSA-induced IL-1β protein expression. In addition, P4BSA induced rapid phosphorylation (within 2 mins of treatment) of MEK1/2 at Ser217/221 and CREB at Ser133 as determined by immunoblotting, and PD98059 and H89 pretreatment reduced P4BSA-induced MEK1/2 and CREB phosphorylation, respectively, confirming the involvement of MEK1/2 and PKA in this pathway. CONCLUSIONS: Activation of mPRs leads to decreased Paqr7 expression and increased MEK1/2-and PKA-dependent inflammatory responses. We speculate that changes in mPR expression or activation by progesterone may represent a novel pathway that contributes to the regulation of inflammatory responses in macrophages. Identifying robust combinations of informative biomarkers could benefit from advanced metabolomic analytical tools. In order to address this, an unbiased and untargeted metabolomic study of both term and preterm amniotic fluid samples was performed using several analytical and data processing strategies. METHODS: State-of-the-art gas-phase separations using ion mobility and mass spectrometry were utilized in the analysis of amniotic fluid samples of both term (n = 5; >37 weeks) and preterm (n = 5; 22-32 wks) pregnancies. Reversed-phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC) coupled to ion mobilitymass spectrometry (IM-MS) (nanoAcquity, UPC 2 and Synapt G2-S, Waters, MA) provide a great advantage of higher metabolome coverage since separations are based upon different physicochemical properties. This increases both selectivity and sensitivity in assessing significant differences between sample groups. A self-organizing map (SOM) approach (MEDI, Molecular Expression Dynamics Investigator) was used to filter the high dimensional data into analytes that distinguish the sample groups. Specifically, the MEDI approach compares and groups over-and under-abundant metabolites to prioritize differences between term and preterm metabolome. RESULTS: MEDI images show significant differences between the metabolome of term and preterm amniotic fluid samples, where several putative metabolites were detected. These images cluster metabolites with similar expression profiles and converts complex multidimensional data into two-dimensional heat maps. Out of 2049 molecular features detected by both RPLC and SFC, 298 metabolites were found to significantly differ with a p-value of <0.01 between term and preterm samples.

The workflow presented here demonstrates a rapid assessment of the phenotype of term and preterm amniotic fluid samples.

Differences can be observed with data analysis and imaging software, which helps prioritize relevant features in both groups. The use of threepronged approach to biomolecular inventory generation expanded the metabolome coverage detected. (control, n=10) 13 to 17 days post-coitum. Prior to delivery, mice were euthanized, uterine and decidual tissues were obtained, and leukocytes were isolated for immunophenotyping. Within the macrophage (CD11b+F4/80+) or neutrophil (CD11b+Ly6G+) gates, the expression of MHCII, iNOS, Arg1, or IL4 was determined by flow cytometry as proportions. MMP-9 activity in cervical leukocytes was determined by in situ zymography. Plasma progesterone, estradiol, and cytokine concentrations were quantified using ELISA or a multiplex assay. Additionally, pregnant mice pretreated with VP4 or Replens were injected with an endotoxin (10mg, n=10 each). Mice were monitored via infrared camera until delivery. A p-value of £0.05 was considered significant. RESULTS: 1) Administration of VP4 caused a reduction of MHCII+ and IFNγ+ neutrophils in the uterine tissues (p=0.03 and 0.021, respectively); 2)administration of VP4 caused a reduction of M1-like (iNOS+IFNγ+) macrophages in the uterine tissues (p=0.06); 3) cervical leukocytes that express active MMP9 were fewer in mice treated with VP4 than in the controls (p=0.032); 4) plasma IL1β concentrations were lower in mice treated with VP4 than in the controls (p=0.05); 5) no differences in plasma progesterone or estradiol concentrations were seen between mice treated with VP4 and Replens; and 6) administration of VP4 caused a reduction in PTB in mice injected with an endotoxin (40% vs. 90%). CONCLUSIONS: Administration of VP4 results in an anti-inflammatory micro-environment at the maternal-fetal interface by reducing proinflammatory macrophages and neutrophils. In addition, administration of VP4 reduces the infiltration of active MMP-9+ leukocytes in the cervix and decreases the rate of endotoxin-induced PTB. These findings provide further evidence about the mechanisms of action of VP4 in preventing PTB. 

Both T-oligo and CSE treatments resulted in intense 3-NT staining, p38MAPK activation, SA-β-Gal stained amniotic cells and in higher AF concentrations of all cytokines compared to saline and C-oligo groups. Minimal p38MAPK activation and SA-β-Gal were seen in placenta. Co-treatment with SB203580 decreased p38MAPK activation and cytokine release CONCLUSIONS: OS-induced telomere fragments from senescing cells creates a positive feedback loop and enhances fetal cell senescence through p38MAPK resulting in sterile inflammation (uterotonic cytokines increase). These data corroborated with our in vitro findings in human amnion cells. Telomere fragments may act as physiologic fetal signals of parturition at term due to natural aging and fetal maturity whereas they indicate premature senescence triggering preterm labor. 

To study gestational changes and interrelationships between circulating and urinary RAS components, we measured plasma and urinary prorenin, active renin, angiotensinogen (AGT), and angiotensin converting enzyme (ACE) levels in Indigenous pregnant women. Nine, 31 and 60% of the samples were collected in the first, second and third trimesters, respectively and samples were taken from 55, 33 and 11% of women on 1, 2 or 3 occasions. Further data relating to blood pressure was collected from antenatal records.

There was a positive correlation between gestational age and plasma angiotensinogen (r=0.32,P <0.01). Gestational age was also related to plasma cystatin C (r=0.55,P <0.01) and diastolic pressure (r=0.26, P = 0.01). There was a negative correlation between plasma Na + /K + and gestational age (r=-0.18,P =0.01). Cystatin C was directly related to plasma AGT (r=0.27,P < 0.01) and urinary protein:creatinine (r=0.21,P =0.01). Plasma angiotensinogen and urinary protein:creatinine were also correlated (r=0.18,P =0.01) as was plasma and urinary Na + /K + (r=0.19,P = 0.01). Urinary Na + /K + was inversely correlated to both systolic and diastolic pressure (r=-0.23, 0.02 and r=-0.27, P =0.005 respectively). There were no associations between components of the circulating RAS and their urinary counterparts. Urinary ACE and AGT were correlated (rho =0.53,P <0.01), as were urinary ACE and prorenin (r=0.26,P =0.02). Urinary protein correlated with urinary albumin, ACE, AGT, prorenin and active renin (all P £0.01). Urinary AGT, prorenin and ACE were related to albumin levels (r=0.36,P <0.01, r=0.24,P =0.05, r=0.21,P =0.04).

The intrarenal RAS is activated in pregnancy and influences the excretion of sodium with secondary effects on blood pressure. Associations between urinary protein levels and the urinary RAS might relate to the poor renal health of these Indigenous Australian women.

Factors In women with a urinary protein/creatinine ³30 mg/mmol, BPs, circulating renin-angiotensin system (RAS) components and inflammatory markers were similar to those measured in women without proteinuria. However, their albumin/creatinine levels were higher (P=0.01) as were their urinary angiotensinogen, ACE, prorenin and active renin/creatinine ratios (P<0.01, 0.01, 0.02 and 0.01, respectively). CONCLUSIONS: In this population of Indigenous Australian women, the incidence of high BP is low, but there is an increased prevalence of proteinuria. Systolic BP appears to be affected by the presence of inflammation as evidenced by the relationships between systolic BP, CRP and wbc count. The higher urinary angiotensinogen/creatinine in pregnant women with proteinuria is in accordance with observations made in non-pregnant populations. As well, the activity of the intrarenal RAS is increased, indicative of previously undetected severe renal dysfunction. Our objective was to assess whether maternal serum levels of heavy metals were associated with spontaneous PTB. METHODS: As a secondary analysis of a multi-center trial of vitamins C/E to prevent preeclampsia in low risk women, we randomly selected 75 women with spontaneous PTB <37 weeks gestation (cases) and 75 women with term delivery (controls). Logistic regression was used to estimate associations between 1st trimester serum levels of arsenic, cadmium, lead, manganese, mercury and uranium and spontaneous PTB.

In cases and controls, median lead and manganese levels were significantly higher among Hispanics than among Africans Americans.

Demographics were similar between case and control groups. Median levels of each metal were not significantly different between cases and controls. Odds ratios indicate no associations between increasing measured unit values of any metal and risk for PTB subcategories (Table) . CONCLUSIONS: In this exploratory study, higher serum levels of heavy metals were not associated with spontaneous PTB. Our study is strengthened by its reliance on prospective 1st trimester measurements rather than using proxies such as occupational/residential history or postpartum serum measurements. . 10 of these also increased in chPTL samples compared to PNL, and a further 3 (IL6, CXCL2 and CCL1; p£0.05) increased in both chPTL and iPTL. Interestingly eotaxin-3, TNFα and CCL25 only increased in chPTL (p£0.05) but not TL, and MIG increased in iPTL only (p£0.05). Furthermore increased expression of chemokines/cytokines was not observed in twins PTL or abruption PTL. CONCLUSIONS: Cytokine/chemokine-mediated inflammation appears to be a consequence rather than the cause of TL, where it is most likely involved in the reparative & remodelling of the uterus. Both chPTL & iPTL are associated with acute inflammatory response with chPTL displaying a more intense inflammatory response than iPTL. PTL secondary to twins or abruption does not appear to be mediated by an inflammatory response but by other pathways which are still yet to be identified. There is no current evidence from randomised trials to inform best practice for delivery when malposition complicates the second stage. The American College of Obstetrics and Gynaecology has recommended training in instrumental deliveries to control and reduce rates of emergency CS. Manual rotation (MROT) is widely used to correct malposition in the second stage, but no modern data is available on how best to carry out the procedure, or on efficacy or safety. METHODS: Anonymous questionnaire was administered to obstetricians focusing on preferred method for rotational delivery and the use of, indication and documentation of MROT. 1862 deliveries requiring assistance for malposition at full dilatation were analysed during the 6 year retrospective/prospective study, this included 365 MROT deliveries. We compared fetal and maternal outcomes for MROT with all other forms of operative assisted deliveries for malposition, rotational ventouse (RV), Kielland forceps (KF) and 2 nd stage CS without a trial of instrumental delivery (pEMCS).

A total of 150 obstetricians responded, 55 documenting RV as their preferred method of rotational delivery. 124/150 use MROT, with 10 participants not consistently documenting the use of MROT at any time and 12 documenting only successful MROT. The prospective groups had similar failure rates despite method of rotation used (MROT 17.5%; RV 17.2%; KF 13.8%). The incidence of massive obstetric haemorrhage was highest following CS in both the prospective and retrospective groups. The overall incidence of anal sphincter injury was much lower than nationally expected; KF carried the highest risk at just 2.6%. The rates of cord pH <7.1 were seen at the highest level post MROT (9.1%) in the prospective cohort. The rates of low Apgars (<5 at 5 minutes) were extremely low in all groups studied. CONCLUSIONS: MROT is widely used and preferred by many obstetricians, as highlighted by our questionnaire study. The studies in our tertiary centre confirm MROT's use and comparative outcomes with other methods of delivery when malposition complicates the second stage. Therefore these data compels us to take urgent steps in setting guidelines for performing and training MROT, auditing outcomes to regulate safety and efficacy. Formal training programmes urgently need to be introduced to unify the skill of MROT allowing best practise to be undertaken.

Interleukin ( We recently found an increase in mRNA expression of IL-1R1, IL-1R2, IL-1RAcP and IL-1RAcPb in rat uterus in late gestation. Previously AcPb was found only in brain. IL-6 and its signaling component glycoprotein (gp)130 also play a key role in the onset of parturition. We showed that PGF 2α stimulation induces output of IL-6 by HMSMC. In this study, we hypothesize that HMSMC express the four IL-1R genes and that they are regulated by PGF 2α and IL-1β. We also investigate whether IL-1β, with or without PGF 2α treatment, stimulates IL-6R and gp130. METHODS: Myometrial biopsies were retrieved from women not in labor at term, following caesarean section and HMSMC prepared (n=5-9). Cells were pre-treated with 5 ng/ml IL-1β for 24h, followed by PGF 2α (10 -7 to 10 -5 M) for 6h, or treated only with PGF 2α . mRNA abundance of IL-1R1, IL-1R2, IL-1RAcP, IL-1RAcPb, IL-6R, gp130 and housekeeping gene GAPDH was evaluated by RT-PCR. ANOVA with Tukey HSD post-hoc tests or t-tests were performed for statistical evaluation after log 10 transformation. RESULTS: Both IL-1R1 and IL-1R2 expression increased in a doseresponse manner to PGF 2α (p<0.001 and p<0.05). IL-1AcP displayed a similar trend (NS), while IL-1RAcPb demonstrated an inverse relationship, with PGF 2α stimulating expression at lower doses (p<0.05). IL-1β stimulated a significant increase in expression of these 4 genes (p<0.001). Addition of PGF 2α after IL-1β pre-treatment did not induce a further rise in expression. IL-6R expression was increased by IL-1β, with an additive effect of PGF 2α (p<0.001). There was no effect on gp130 expression.

The expression of IL-1R1, IL-1R2, IL-1RAcP, IL-1RAcPb is regulated by PGF 2α and IL-1β in HMSMC. PGF 2α has an additive effect on IL-6R expression after IL-1β treatment. IL-1β stimulates its own receptors and accessory proteins, as well as IL-6R. Furthermore, these are the first data showing presence of IL-1RAcPb outside the human brain. Together, these observations add a level of complexity to the feedforward aspect of uterine transformation for labor. Acknowledgements: FWO, CIHR, MOD, GAPPS We previously found significantly higher decidual prorenin expression and protein levels in women carrying a female compared to a male fetus. We postulate that this decidual prorenin interacts with the amnion (pro)renin receptor and influences the amniotic expression of transforming growth factor-b1 (TGFB1) and prostaglandinendoperoxide synthase 2 (PTGS2), which are involved in maintaining membrane integrity and initiation of labour. We propose, since decidual prorenin is highest in pregnancies with a female fetus, that amniotic TGFB1 expression is also higher, which may explain the lower incidence of premature rupture of membranes and preterm birth in pregnancies with a female fetus. METHODS: All tissues were collected from term uncomplicated singleton pregnancies delivered by elective caesarean section. Amnion explants were treated with 0, 5 or 50 ng/ml of rhProrenin in DMEM-F12 media for 0, 0.5, 4, 16, or 24 h at 37 o C. Gene expression data were obtained by qRT-PCR using previously verified primers.

In vivo, decidual prorenin expression and protein is lower in women with a male fetus (P<0.05). In vitro, only amnion of women with a male fetus responded to rhProrenin treatment, showing decreased ATP6AP2 ((pro)renin receptor) and TGFB1 expression (both P<0.05). Additionally, amnion PTGS2 expression was inhibited by amniotic fluid and dexamethasone, but unchanged with rhProrenin treatment. Furthermore, a correlation exists between decidual prorenin and amnion PTGS2 expression.

We postulate that irrespective of fetal sex, amnion PTGS2 expression is inhibited, perhaps by secretion of steroids into the amniotic fluid. The female fetus however has an additional pathway that helps prevent premature rupture of membranes and preterm labour. Specifically, higher decidual prorenin acting on the amnion (pro)renin receptor in females stimulates the sustained production of amniotic TGF-B1, and its profibrotic products, which aids in maintaining membrane integrity. This may account for the lower incidence of female compared with male preterm births and suggests that the observed fall in decidual prorenin expression seen in women with a female fetus, may in fact be crucial for labour to progress.

An INTRODUCTION: Each delivery is preceded by an invasion of circulating leukocytes into the uterine myometrium and decidua. They are attracted by a chemotactic factor that we identified in the fetal membranes (amnion, chorion) and decidua. However, the regulation of expression for this factor is unknown. Therefore we developed an in vitro human tissue culture model to study the regulation of its expression. Our aim is to describe regulation of the chemotactic factor. METHODS: Tissue cultures: Fetal membranes with attached decidua vera from term, not in labour placentas delivered by elective cesarean section were used. The protocol was approved by the AHS/UA REB and consent granted in each case. Intact tissues were cut into 12mm(dia.) pieces (100mg) by a punch. After washing, the pieces were placed into 6 well culture plates (6.9 cm 2 ) and incubated for 24h at 37°C in DMEM/F12 maintained at pH 7.4. Following 48h the media were discarded and new DMEM/F12 containing either agonists or vehicle was added for 24-96h. The media were collected and stored at -80°C for subsequent leukocyte migration activity (LMA).

LMA: Peripheral blood (5mL) was obtained from women immediately following term spontaneous vaginal delivery. The leukocytes were separated by Hetasep TM and centrifugation. Unseparated leukocytes (10 5 ) were inserted into the upper chamber of a modified Boyden chamber and approximately 25µL(125µg protein) of medium containing chemoattractant was placed into the lower chamber. The compartments were separated by a filter containing 3 µm pores. The chambers were incubated for 90 min at 37°C. Afterwards, the number of cells that migrated to the lower chamber were quantified by FACS analysis. RESULTS: Tissue DNA and RNA remained steady over 96h suggesting the minced tissues remained viable. There was a time-dependent increase in the release of chemoattractant activity into the media from 24h to 96h when it reached a plateau. Upwards of 45,000 cells migrated when chemoattractant was present. Blanks were negligible.

We have developed a new model for studying the regulation of the uterine cheomoattractant. Future studies will assess the responsiveness of human fetal membranes in this model system to a number of inflammatory and non-inflammatory agonists that mediate the processes of parturition and preterm birth. Acknowledgement:CIHR During pregnancy, the myometrium remains quiescent but at term, switches to a state capable of producing a series of coordinated contractions for the delivery of the fetus. There is evidence that components of the cAMP-signalling pathway are up-regulated in the human myometrium during pregnancy to promote myometrial relaxation.

Our aim was to determine which cAMP-associated genes are important during gestation by exposing myometrial cells to forskolin and performing a gene array, and then to validate these results by investigating the cAMPassociated genes in human primary myometrial cells with different agents that are capable of raising intracellular cAMP levels. METHODS: Primary cultures of human myometrial cells were grown from biopsies obtained during planned caesarean section at term. Cells were treated with forskolin (100uM), and total RNAs were extracted. The concentration and purity of RNA were determined by value of OD260:280. Three samples were chosen as representatives for Affymetrix Human Genome U133 plus 2.0 Array analysis. Genes were further filtered and only those with p< 0.05 and fold change >1.5 were analysed by qPCR. 8 genes were up-regulated (11βHSD1, MKP-1, PKAR2α, GPR125, GaS, PDE4B, CCL8 and PTGES), and 5 genes were down-regulated (OTR, CREB3L1, GUCY1A3, GPR124 and PRKG1). Subsequently primary myometrial cells were treated with forskolin, 8 bromo cAMP, rolipram and dibutyryl-cAMP. Total RNA were extracted, and levels of cAMPassociated genes were measured. RESULTS: Among the genes that are up-regulated by cAMP as determined by the gene array, treatment with 8 bromo cAMP, rolipram and dibutyryl-cAMP was able to increase the mRNA levels of 11βHSD1, MKP-1, PKAR2α, GPR125, GaS, PDE4B, CCL8 and PTGES compared to control. Among the genes that are down-regulated by cAMP, treatment with 8 bromo cAMP, rolipram and dibutyryl-cAMP was associated with the reduction of OTR, CREB3L1, GUCY1A3, GPR124, PRKG1 mRNA levels compared to control. CONCLUSIONS: These data show that all 4 agents that increase the intracellular levels of cAMP have similar effects on cAMP-associated genes, suggesting that these agents are useful tools for studying the effects of raise intracellular levels of cAMP in human myometrium during pregnancy and parturition.

The (OEA) , and N-palmitoylethanolamide (PEA)] concentrations are controlled by the activity of a hydrolysing enzyme fatty acid amide hydrolase(FAAH), which is down-regulated progesterone (P4) and oestradiol (E2) action. An inverse relationship is thus expected between eCBs and FAAH, and between FAAH and both steroids. As eCBs increase during labour, we tested the hypothesis of an inverse relationship in pregnant women at high-risk for preterm birth(PTB) METHODS: FAAH activity (radioactive substrate conversion rates) was determined in blood obtained from high-risk pregnancies(n=18) and emergency admissions with threatened PTB(n=25). Plasma E2, P4(n=36) and eCB concentrations were measured by ELISA and UHPLC-MS/MS, respectively. Pregnancy outcome (PTB & term delivery; <37 &≥ 37 weeks respectively) was recorded. Correlations of FAAH activity with AEA, OEA, PEA, E2, P4 and E2/P4 were examined. FAAH expression (RT-qPCR and immunohistochemistry(IHC)) in placentae obtained from spontaneous labourers at preterm and term, and non-labourers (elective CS) at term and preterm (n=6/group) RESULTS: Plasma E2, FAAH, P4 and the E2/P4 ratio were all higher in the PTB group compared to the term group, but the differences did not reach significance (P>0.05). There was a weak correlation between FAAH and E2(r=0.04; P=0.78), P4 (r=0.05; P=0.74) and E2/P4 ratio (r=-0.009; P=0.9), and none between FAAH activity and eCBs levels. FAAH transcript levels were respectively 70% and 68% lower in placenta from term and PTB compared to non-labouring women. Prematurity was associated with reduced FAAH expression (33%) in placentae of PTB labourers and 1.38-fold higher in those who had preterm CS; observations also confirmed by IHC CONCLUSIONS: The data suggest E2 and P4 may not be involved in the regulation of peripheral FAAH in, but is explained by the lower FAAH placental expression. Placental FAAH expression could therefore be important in PTB, but not related to circulating E2 or P4 .29) had a significant positive correlation with age. One metabolite differed significantly in GDM cases vs, controls: adipic acid (P=0.008; FDR=0.14).

This study was the first to investigate the GDMrelated hair metabolome. The identification of hair metabolites that correlate with age and BMI justifies the continued matching of cases and controls using these two factors in future studies. Adipic acid was found at significantly higher levels in the hair of GDM cases when compared with controls. Elevated levels may indicate omega oxidation resulting from lipid peroxidation consequent of the oxidative stress environment observed in GDM. This pathway should be explored in future studies for use in screening or prevention strategies.

The 

The findings of this study suggest that CAM and IHC are independently associated with adverse neonatal outcomes and that CAM with IHC may enhance the risk of these outcomes. The knowledge gained can help us understand disease etiology and implement effective intervention strategies. INTRODUCTION: There has been concern for increased neonatal morbidity and mortality associated with antenatal magnesium sulfate (MgSO4) exposure, particularly in very preterm infants. We hypothesize that there is no clinically significant increased risk of short-term neonatal morbidity or mortality with the use of antenatal MgSO4 prior to very preterm birth.

We analyzed a large, retrospective cohort of infants born 23 0/7 to 31 6/7 weeks gestation within a multicenter healthcare system in Utah, January 2003 to December 2013. We excluded multiple gestations, fetal anomalies, and aneuploidy. Infants who received antepartum MgSO4 for any indication were compared to those who were not exposed to MgS04. The primary outcome was composite neonatal morbidity (intraventricular hemorrhage, bronchopulmonary dysplasia, necrotizing enterocolitis, and/or death prior to hospital discharge). RESULTS: 1,246 infants were included in the analysis. 457 (36.7%) were exposed to MgSO4 and 789(63.3%) were not. Maternal demographics were similar between groups, except for BMI, which was lower in the group exposed to MgSO4 ( (Obstet Gynecol 1988; 72:746 ) established a correlation between worsening of patterns (form type 0to4) and risk of neanatal acidemia. The aim of our study was to assess its reliability in a much wider population and to evaluate the relationship between different CTG patterns and neonatal acidemia at birth. METHODS: 2-year retrospective cohort study of 2297 singleton pregnancies who delivered vaginally at our Institution. Inclusion criteria were: at least the final 60 minutes of continuous and technically interpretable trace in the first stage, a technically interpretable trace in the second stage lasting at least 10 minutes and umbilical artery gas analisys at birth. CTGs were extracted prospectively and analyzed retrospectively blind to the clinical information. Neonatal acidemia was defined by the presence of umbilical arterial cord pH of 7.10 or less. Data were analysed by multiple logistic regression analysis. RESULTS: Only 50% of our tracings in the II stage could be classified according to the original 6 types proposed by Piquard. 46.8% of tracings fit within a single class (1, 0, 2A), 53.3% were a combination of types (most frequently: 0-1). Type 1 and the combination of type 1 and 4 were associated with a fourfold and ninefold increased risk of acidemia respectively. In type 1 tracings the presence of malignant criteria, such as frequent, repetitive, prolonged or deep decelerations, were strongly associated with academia. A length of II stage >120 minutes and induction of labor were risk factors for acidemia (OR:4.3 and 1.76 respectively). The only independent risk factor for acidemia was maternal BMI (<18.5 and >25 kg/m 2 ); while maternal and pregnancy-induced disease, parity, male sex and neonatal weight at birth were not associated with acidemia. Gestational age was a protective factor. CONCLUSIONS: Our study shows that: only 50% of our tracings could be classified according to the classification of Piquard whereas the other represented a combination of more than 1 type; acidemia at birth could be predicted by the presence of the so called malignant criteria. These results suggest the need of a novel classifcation for the fetal heart rate in the second stage of labor The mean gestational age at evaluation was 20.6 ± 1.7 weeks. The mean CL was 3.7 ± 0.8 cm without contrast and 3.7 ± 0.8 cm with contrast (p = 0.341). All quality criteria were seen in 46 (61%) of patients without contrast versus 52 (69%) with contrast, (p = 0.23). In 7 patients (9%), £2 of these metrics were clearly seen without contrast, but ³3 were identified after application. All landmarks were clearly visualized in 34/50 patients (68%) with 10mls applied versus 18/25 (72%) with 20 ml of methylcellulose, (p = 0.8). CONCLUSIONS: CL measurement was not altered by imaging contrast in this low risk cohort examined by experienced sonographers. Improved visualization of cervical anatomy was noted for a minority of patients, but was not statistically significant. This methodology may benefit inexperienced sonographers or more challenging cases but requires further study to define benefit.

Who Among women with a nonrecurrent indication for the first CD, 35% underwent TOLAC and 77% had a VBAC. Among women who had their first cesarean for arrest disorders, 27% underwent TOLAC and 68% had a VBAC. Of women with a prior vaginal birth, 45% underwent TOLAC and 91% had a VBAC. CONCLUSIONS: Women whose characteristics predict a higher likelihood of VBAC are more likely to choose TOLAC; however, the majority of these women still choose ERCD. While patient preference should remain paramount, improving counseling to ensure that optimal candidates understand their likelihood of VBAC may improve the decision making process, which may ultimately decrease the CD rate and help avoid the morbidity associated with TOLAC ending in CD. Two measurements greater than the 95th percentile were required to establish the diagnosis of a large fetal stomach. Ultrasound, antepartum, delivery and neonatal records were reviewed. Fetuses with associated major structural anomalies were excluded. RESULTS: A total of 470 cases of large fetal stomach were identified. 272 cases met inclusion criteria (singleton pregnancy, no GI or other major anomalies diagnosed on ultrasound i.e. gastroschisis, omphalocele, duodenal atresia, major cardiac or neurologic anomalies). Median gestational age of large stomach diagnosis was 21 weeks. Large fetal stomach was seen on only one ultrasound and subsequently resolved in 159 cases (59%). It persisted over two or more ultrasounds and subsequently resolved in 27 cases (10%). It persisted over two or more ultrasounds and remained enlarged on the last ultrasound prior to delivery in 36 cases (13%). Follow-up ultrasounds were not available in 49 cases (18%). Neonatal outcome data was available in 151 cases (56%). Two neonates had GI problems diagnosed postnatally; in one case, malrotation and GERD were diagnosed during infancy; in the other case, distal esophageal stricture with tracheobronchial remnants on pathology was diagnosed during childhood. In both of these cases, workup was initiated because of postnatal symptoms. In one case a postnatal GI workup was initiated solely on the basis of ultrasound findings and revealed no abnormalities. There were no fetal deaths in utero or neonatal deaths. Twelve of the neonates had prolonged hospital stays after birth, but never for GI problems. Twenty six had known normal karyotype, 3 had abnormal karyotype, and the remainder did not have any testing for aneuploidy. CONCLUSIONS: Large fetal stomach is often associated with other anomalies and in those cases should prompt a detailed fetal evaluation. This retrospective study suggests that there are no postnatal consequences when enlarged fetal stomach is seen as an isolated finding on ultrasound. To evaluate the compliance to and tolerability of daily cranberry capsule ingestion for asymptomatic bacteriuria (ASB) prevention in pregnancy.

A total of 49 pregnant women from two sites were randomized to two doses daily of cranberry or matching placebo at gestational ages less than 16 weeks and followed monthly for urinary tract infection (UTI) until delivery. Up to seven monthly visits were scheduled for each subject. Delivery data were evaluated. RESULTS: Of 38 evaluable subjects, the mean compliance rate over the study period was 82% (range 20 to 100%). This compliance rate and the 74% of subjects achieving good (i.e., ³ 75%) compliance were comparable between those who received cranberry capsules and placebo. Compliance evaluation revealed that most subjects stopped capsule consumption after 34-38 weeks of participation. Multivariate logistic regression and longitudinal analysis showed a significant interaction time effect with cranberry treatment. However, cranberry consumption was not a significant predictor ofgastrointestinal intolerance or study withdrawal. While 30% of subjects withdrew for variety of reasons, only one subject withdrew due to intolerance to the cranberry capsules. Loss to follow-up was mostly due to provider change (9/49, 18%) and therapy disinterest (4/49, 8%). Seven cases of ASB occurred in five subjects, with 2/24 (8%) in cranberry-and 3/25 (12%) in placebo-treated subjects. No cases of cystitis or pyelonephritis were observed. *Figure(s) will be available online. CONCLUSIONS: One-third of pregnant women were unable to complete the study protocol for various reasons. Compliance and tolerability to cranberry capsule ingestion appears good, providing a potentially effective means to prevent ASB in pregnancy. Further studies with large samples are necessary to confirm findings.

Relationship were more likely to score in the lowest quartile of health literacy. These same factors were associated with scoring in the lowest quartile for each cognitive domain. Conversely, there were no differences in any cognitive domain based on parity or gestational age (among pregnant participants). Postpartum status was associated with limitations in processing speed (aOR 3.79, 95% CI 1.32-10.93) and inductive reasoning (aOR 4.07, 95% CI 1.21-13.7). CONCLUSIONS: While differences in reasoning and processing speed were associated with postpartum status, there were no differences in cognitive function across pregnancy itself. Rather, differences in cognitive functioning and health literacy were highly associated with demographic characteristics. These disparities suggest that it is these differences that need to be addressed and acknowledged in perinatal education in order to optimally improve understanding throughout pregnancy.

Is The mechanism by which preterm premature rupture of membranes (PPROM) occurs is unclear but usually results in preterm birth (PTB). Plasma anandamide (AEA) levels increase in women at highrisk for PTB, suggesting that the cannabinoid receptors (CB1 and CB2) or regulatory enzymes, NAPE-PLD and FAAH (the endocannabinoid system: ECS) may be involved. Thus, the expression of these proteins in human fetal membranes (FM) at term and preterm was examined METHODS: FM acquired from four groups of women; spontaneous labourers having vaginal delivery at term and preterm and non-labouring women having elective Caesarean section at term or prematurely (n=6 per group) were subjected to immunohistochemistry (IHC) for the presence of CB1, CB2, FAAH and NAPE-PLD and RT-qPCR for transcript levels (presented as means) RESULTS: Labour had similar effects on CB1 and CB2 expression with CB1 being increased 5% and 35% in term and preterm FM, respectively whilst CB2 transcript levels decreased by 59% and 36%, respectively. The expression of NAPE-PLD also increased (473% and 39% in term and preterm FM, respectively). By contrast, FAAH expression increased by 15% in term FM, BUT decreased by 49% in preterm FM. These observations were confirmed by IHC. The increase in NAPE-PLD expression correlates with the observed increase in AEA at term. The massive decrease in FAAH expression in PTB explains the plasma AEA increase in women who deliver preterm The effect of prematurity (in the absence of labour) on ECS gene expression was more striking. CB1 was decreased by 44% (0.56 vs1.0), CB2 increased by 76% (1.76 vs1.0), NAPE-PLD increased by 42% (1.42 vs1.0) and FAAH expression decreased by 33% (0.66 vs1.0).

The FM at term appears to be fully controlled by the ECS with decreased FAAH and increased NAPE-PLD expression consistent with the known increased plasma AEA that occurs at this time.

Although prematurity increases NAPE-PLD expression, FAAH expression starts at a lower level that is exacerbated by labour. Additionally, the expression of CB1 is reduced, whilst CB2 is increased, supporting data from the CB1 KO mouse that delivers prematurely. These data suggest that the ECS is dramatically dysregulated in the FM of women at risk of PTB. and its synthesizing enzyme NAPE-PLD are expressed in the placenta, the effect of labour on that expression remains unknown. That the CB1 knockout mouse undergoes PTB, suggests CB1 expression is altered in women at risk of PTB. Placental CB1, CB2 and NAPE-PLD expression in women delivering prematurely or at term (<37 & ³37 weeks respectively) was therefore determined to see if this was affected as predicted. METHODS: Samples (6 per group) collected from 4 groups of patients: spontaneous vaginal deliveries (TVD) term and preterm (PTVD), nonlabouring elective Caesarean sections (CS) at term and preterm were subjected to RT-qPCR and immunohistochemistry (IHC) for CB1, CB2 and NAPE-PLD expression. RESULTS: CB1 transcript levels decreased by 86% in TVD and 91% in PTVD compared to CS samples, suggesting that CB1 expression is affected by labour. When PTVD was compared to TVD, CB1 expression was respectively 44% and 18% lower in non-labouring and labouring states. Similarly, IHC showed placental CB1 expression in the TVD and PTVD samples to be 5-10 times lower than the in non-labouring states. In concert with the CB1 data, CB2 was decreased 76% in the TVD samples, but interestingly increased 500-fold in the PTVD samples. However, CB2 transcript levels were increased in non-labouring (1.4-fold) and laboring (3000-fold) preterm samples; observations confirmed by IHC. NAPE-PLD levels decreased 38% in the TVD group but increased 1.6-fold in the PTVD group. Prematurity caused a 33% decrease in NAPE-PLD expression in labouring placentae and 75% in non-labourers. Observations of reduced NAPE-PLD at term and increase at preterm were confirmed by IHC. CONCLUSIONS: Increased plasma AEA levels in women in labour or who deliver prematurely are explained by alterations in NAPE-PLD expression and reduced CB1 expression. The massive rise in placental CB2 expression in preterm samples suggests CB2 activation could be important in the development of PTB. Placental endocannabinoid system changes seem to be linked to the development or progression of PTB.

Are METHODS: This is a retrospective cohort study of all elective HICs placed from January 2011 to December 2013 at a single institution. We included all patients that had an elective HIC placed prior to evidence of cervical shortening, received serial transvaginal cervical length evaluation during pregnancy with images available for reviewing, and whose outcomes were available. Multifetal gestations were excluded. All images were reviewed by two independent physicians. Sonographic evidence of CI was defined as the appearance of asymptomatic cervical shortening and/or funneling to the level of the cerclage on transvaginal ultrasound prior to 32 weeks gestation. Traditional criteria for the placement of a HIC were defined as three or more second trimester losses (STL) or early preterm births (EPTB). RESULTS: Eighty-three HICs were identified with fifty-five patients meeting inclusion criteria. Of these, twenty-six were found to have sonographic evidence of CI. Baseline characteristics of the two groups were similar except for the number of previous STL/EPTB.

Age (mean ± SD) 32 ± 5 33 ± 5

Gravidity (median, range) 5 (2-9) 4 (2-14)

BMI (mean ± SD) 29.2 ± 6.3 29.1 ± 6.3 The incidence of cervical insufficiency is reported to be up to 1%. Insertion of transvaginal cervical cerclage (TVC) has been used to prevent preterm birth in these women. Although placement of a transabdominal cerclage (TAC) following failure of a TVC remains debatable, regulatory bodies recommend their placement in this population. We sought to examine the outcomes of pregnancy after TAC compared with TVC in women with a failed TVC in a preceding pregnancy. METHODS: MEDLINE, EMBASE and the Cochrane Library (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) were searched without restrictions. Included studies reported maternal and perinatal outcomes in women with TAC/TVC placed as an interval procedure or <20 weeks, after a failed TVC leading to a second trimester miscarriage or early preterm birth. We excluded small studies (n<10) and those precluding TVC use. Meta-analyses of proportions was performed (Stata v10) using the metaprop command (random effects).

Eighty-five studies were identified and 10 met the inclusion criteria; nine were retrospective case series and one was a retrospective cohort study. Five studies reported TAC via laparotomy with an outcome of delivery <24 weeks, of which 4 were included in the meta-analysis (n=161). The overall proportion of birth before 24 weeks was 0.043 (95% CI 0.012-0.074). The excluded study did not report any preterm births <24 weeks (n=10). Two studies reported delivery <24 weeks in women with TVC insertion (n=64); the overall proportion of birth <24 weeks was 0.063 (95% CI 0.025-0.15). Five studies reported TAC insertion via laparotomy or laparoscopy with delivery <34 weeks (n=117). The overall proportion of preterm birth <34 weeks was 0.16 (95% CI 0.05 to 0.27). There were more serious operative complications reported after TAC insertion. CONCLUSIONS: Despite a twelve-year interval since the last published systematic review, there remains a paucity of data to guide clinical practice. Whilst the numbers remain small, regulatory bodies are recommending the use of TAC in this population. Although TAC does appear to be associated with a lower risk of delivery <24 weeks, it has a higher risk of operative complications. A multicenter randomized controlled study in this population is warranted.

The N (%) 7 (14) 7 (14) 12(23.5) 6 (12) 9 (18) 5 (10) 5 (10)

Bleeding 4 (8) 3 (6) 8 (16) 3 (6) 6 (12) 2 (4) 0

Persistent GTD 3 (6) 1 (2) 2 (4) 0 2 (4) 0 2 (4) Hysterectomy 2 (4) 0 4 (6) 1 (2) 0 0 0

The only association of significance was a decrease in vaginal bleeding in women delivering after 39 weeks (OR 0.177 [CI 0.033-0.940] p = 0.038).

Logistic regression revealed no relationship between gestational age of delivery and the rate of any comorbidity considered. CONCLUSIONS: In gestations complicated by CMCF analysis failed to show that delivery prior to term decreased perinatal morbidities and post-partum complications. In the absence of evidence of improved outcome, it may be reasonable to await spontaneous labor at term to avoid complications of iatrogenic preterm delivery. Non-cephalic fetal presentation occurs in 4% of term pregnancies, and these neonates are generally delivered via cesarean delivery (CD). Our institution offers planned vaginal breech deliveries to a select group of women following extensive counseling and evaluation.

Our objective was to compare the demographic characteristics of women who achieved a vaginal breech delivery (VBD) to those who opted for scheduled cesarean.

This was a retrospective cohort study of women with breech-presenting singleton pregnancies who delivered at our institution between 2002-2014. We excluded cephalic presentations, deliveries at < 37 weeks gestation, women with a prior CD, fetal congenital anomalies, and pregnancies complicated by placenta previa or other contraindications to vaginal delivery. Data on demographic characteristics, as well as details of the pregnancy and delivery, were extracted from our perinatal database. Chi square analyses were used to compare proportions among women who had a planned VBD versus those who chose scheduled CD. Of these, 30 (61.2%) were exposed to 17OHPC and 19 (38.8%) were unexposed. There was no difference in stillbirth or neonatal death by exposure groups. The groups were similar by chorionicity, maternal age, BMI, and race. However, the 17OHPC-exposed group had more baseline infections and Hispanic women, and less women with baseline hypertension. The mean gestational age at delivery was greater in those exposed to 17OHPC compared to unexposed (33 6/7 weeks versus 33 2/7 weeks respectively, p=0.083). 66.7% of the unexposed group compared to 52.6% of the exposed group delivered at <35 wks (RR 0.56, 95% CI 0.48, 1.03). The relative risk of PTB<32 weeks was 0.83 (95% CI 0.55, 1.25) and at <28 wks was 0.47 (95% CI 0.19,1.14). After adjusting for group differences and confounders, the clinically significant 24% and 38% reductions in PTB at <35 and <32 weeks were not statistically significant. 

We did not find a benefit to 17OHPC, perhaps due to a small, fixed sample size. We required 96 patients in each group for adequate power. However, these data suggest that further research is warranted to assess 17OHPC in this cohort.

An INTRODUCTION: Immediately following a preterm birth (PTB), the inpatient setting offers a unique opportunity to provide critical counseling regarding risk of recurrent PTB and prevention strategies. We sought to analyze the adequacy of PTB counseling following PTB prior to hospital discharge in a tertiary care center with maternal-fetal medicine services and level III newborn intensive care unit. METHODS: This is a secondary analysis of an RCT assessing uptake of highly effective reversible contraception following focused family planning counseling or usual care after PTB. Women were recruited following a PTB at 24 0/7 to 36 6/7 weeks gestation, during their inpatient postpartum stay. Adequate counseling was defined as participant report of a provider discussing 1) PTB recurrence risk, 2) recommended inter- Pregnancies of women with an uterine anomaly or a history of cervical surgery (including conisation, trachelectomy, first trimester cerclage, or prepregnancy abdominal cerclage) were excluded, as well as pregnancies that ended preterm by iatrogenic means. Differences between groups were analyzed by independent t-tests.

Of 422 pregnancies with a preceding spontaneous preterm delivery, 72 were excluded. In 350 pregnancies, 2221 cervical lengths were measured (mean ± SD, 6.4 ± 2.9). *P <0.05 compared to Term delivery **P <0.05 compared to Spontaneous delivery < 37 weeks CONCLUSIONS: Women with subsequent spontaneous preterm deliveries have significantly shorter mean cervical lengths between 12 and 28 weeks of pregnancy as compared to those who deliver at term. Women who spontaneously deliver < 28 weeks have a significantly lower mean cervical length in early pregnancy compared to those who deliver at a later gestational age. Longitudinal cervical length measurements may predict the risk of recurrent spontaneous preterm birth and are possibly a useful tool in a prospective risk model.

The 

We investigated the role of epithelial to mesenchymal transition (EMT) as mechanism of amniotic membrane rupture, and used an in vitro pressure chamber to measure the applied pressure at which the amnion undergoes rupture. Etodolac, a pharmacological inhibitor of EMT, was used to study EMT during the rupture of amniotic membranes. Briefly, freshly isolated amniotic membranes were subjected to a flow derived pressure to cause rupture, in the presence and absence of etodolac, with and without TNFα. RT-PCR, western blotting and histochemical analysis was performed on membranes taken from freshly isolated amniotic membranes before and after these experiments.

We observed that amniotic membranes collected from women with medically uncomplicated pregnancy at the time of SVD contained a markedly increased mesenchymal to epithelial cell ratio when compared to those obtained from patients undergoing C/S. Increased vimentin expression in SVD was further confirmed by western blotting of full thickness amnion. The number of epithelial cells (ECadherin expressing) from amniotic membranes obtained from C/S was significantly less than the number of mesenchymal cells (vimentin expressing). Pre-incubation of the amnion with TNFα led to increased rupture with less pressure applied. Incubation with etodolac (a known EMT inhibitor) with and without TNFα, led to protection, increasing the pressure required for membrane rupture. CONCLUSIONS: These results suggests EMT during the process of normal labor and amniotic rupture. We hope to evaluate this in future studies to determine the mechanistic pathways that convey increased susceptibility to EMT and disruption of the amniotic membranes. If our research demonstrates that increased EMT accounts for PPROM, it would lay the foundation for a trial with EMT inhibitors to assess whether these agents also prevent PPROM. The onset of parturition involves changes in the morphology of the cervix. Currently 2 nd trimester cervical length (CL) is used as a predictive tool for preterm birth (PTB). This may be improved through the addition of further parameters assessing change in cervical morphology, such as cervical volume (CV). This prospective longitudinal study aimed to establish a reference range of CV in healthy pregnancy. Women with pre-pregnancy excisional cervical treatment (CT) are at increased risk of PTB (RR 2.19). We therefore aimed to investigate the effect of CT on cervical volume, compared to low risk controls. METHODS: Women were recruited from antenatal booking (8+0 to 15+0 weeks) and followed up at 4 timepoints in pregnancy; 16+0-19+6, 20+0-24+0, 27+0-30+0 and 32+0-36+0 weeks. At each visit CL and CV measurements were taken by transvaginal ultrasound (TVUS), with an empty bladder, using 2D and 3D/4D modalities on a Voluson E ultrasound. Data analysis for CV was performed using the Virtual Organ Computeraided AnaLysis (VOCAL TM ) software. RESULTS: 77 women had serial TVUS at 5 timepoints (n=57 controls, n=20 CT). Age, BMI and parity were comparable among the two groups. All delivered at term. CL decreased with advancing gestation while CV increased; mean CL decreased from 34 mm at timepoint A (8-15 weeks) to 22mm at timepoint E (32-36 weeks; p<00.1) and CV increased from 25cm 3 to 45cm 3 respectively *Figure(s) will be available online.

Women with previous CT have a lower CV (23cm 3 ) than controls at the end of the first trimester (timepoint A). This difference continues through to the third trimester (timepoint E, 32+0-36+0weeks) where the mean CV at in women with previous CT is 42cm 3 CONCLUSIONS: This study establishes a reference range of cervical volume in normal pregnancy. We reveal a reduction in cervical volume in women with previous CT compared to low risk controls. Cervical volume may be used as an additional screening parameter in the prediction of preterm birth. Further investigation into cervical volume may be useful in women at risk of preterm birth Few studies have examined the potential benefits of activity restriction (AR) during pregnancy for women at low risk for preterm delivery. Recent reports have condemned AR outside of a research trial because there is no proven pregnancy benefit and adverse maternal effects have been documented. We hypothesized that AR during pregnancy may be associated with decreased risk of preterm delivery (PTD) in lowrisk women, and tested this hypothesis in a large prospective cohort study. METHODS: This is a secondary analysis of the MFMU's Preterm Prediction Study, a multicenter prospective cohort study designed to study risk factors of preterm birth. We included women with a non-anomalous singleton gestation. We excluded women who at their initial visit (23-25 weeks) met the following criteria: contractions, severe back pain, US cervical length <15 mm, spotting, protruding membranes, or positive fetal fibronectin (fFN). Women were assessed for whether or not they had been on AR at 27-28 weeks. Associations between activity restriction and PTD (<37 weeks) were examined through logistic regression models after adjustment for confounders. RESULTS: There were 1,639 women met inclusion criteria for the lowrisk cohort, of whom 10.8% (n=177) delivered preterm. Within this low risk cohort, 4.9% (n=80) women were recommended activity restriction, with 25.0% (n=20) of those women having a preterm delivery and 16.3% (n=13) having a spontaneous preterm delivery. The associations between activity restriction and preterm delivery are shown below. *Figure(s) will be available online. CONCLUSIONS: Activity restriction in a low risk cohort was associated with an increased risk of PTD. These findings corroborate previous reports statements that the recommendation of AR during pregnancy should be discouraged.

Predictors 

It is difficult to characterize women most likely to respond to indomethacin. However, co-administration of nifedipine, and use among women with BV are associated with increased likelihood of pregnancy prolongation by at least 2 days. These data should be prospectively studied in larger cohorts and combined with genetic data to identify the ideal tocolytic for individuals with threatened SPTB.

The 

Total time spent pushing during the second stage was prospectively collected in a cohort of 300 nulliparous women. Enrollment was restricted to live singleton pregnancies delivering vaginally at term (³37 weeks gestation) without maternal indications for an assisted second stage. The primary comparison was between women of advanced maternal age (³35 years) versus younger controls. A secondary comparison between women in the three age groups of 18-29, 30-34, and 35+ years was also performed. To determine sample size, a clinically significant difference was considered to be 20 minutes. RESULTS: Time spent in the active second stage was not normally distributed and ranged from 3 to 297 minutes, with 95% of women having delivered by 212 minutes. The median time spent in the active second stage was 68 minutes (95% CI 62-76) for the entire cohort. There was no significant difference in median time spent in the active second stage between women of advanced maternal age and younger controls (70 and 67 minutes respectively, p = 0.43 using Mann-Whitney U test), nor was significance reached among the secondary comparison groups (p = 0.06 using Kruskal-Wallis test). The frequencies of potential confounders (regional anesthesia, operative delivery, starting station, fetal head position at delivery, infant birthweight >3999g, BMI >29, gestational age, induction, augmentation, intrapartum use of magnesium sulfate, and chorioamnionitis) were similar for both the primary and secondary comparison groups. Regional anesthesia was used by 80% of participants, and the rate of operative vaginal delivery was 8%.

The median time spent actively pushing in the second stage of labor is 68 minutes and not significantly affected by advanced maternal age. Previous studies found a relationship between perinatal outcomes and umbilical cord indices. Hypocoiling is associated for example with meconium staining, low Apgar scores and NICU admissions of babies. We sought to assess the differences in cord parameters related to fetal gender.

In this prospective study we collected full-length umbilical cords from liveborn singleton infants, born after uncomplicated pregnancies in our institution. The umbilical cord was dissected at its insertion from the placenta and approximately 5 cm from the abdominal wall of the infant, and clamped on both sides. Cord length and number of complete (360°) helices were measured. The UCI (umbilical cord index) was calculated as the number of these coils divided by the cord length in centimeters. Cord parameters were compared between the two genders. Data regarding obstetric history and pregnancy outcome were also collected. The study was approved by the local Institutional Review Board. RESULTS: A total of 154 umbilical cords were collected, 84 from male and 70 from female deliveries. Obstetric history and pregnancy outcome were similar in the two groups. We found that the number of coils was significantly lower in males compared to females (10.17±3.96and 12.25±5.31, respectively, p=0.007). Umbilical cord index was also lower for males (0.157±0.05 and 0.183±0.06, respectively p=0.006). No differences in cord lengths were found. (Table 1) . 

Interestingly, we found a significant difference in umbilical cord index between male and female fetuses. The difference was due to higher number of coils, whereas cord length was similar in both genders. This finding may imply that umbilical cord index is a gender specific property. Measurements were from 210 nulliparous women at <13 to >37 weeks gestation, 40 postpartum women (40-60 days after delivery) and 25 non-pregnant women of reproductive age at indeterminate stages of menses. Also included were 10 nulliparous pregnant women followed longitudinally (3 to 4 CA measures at early, mid-and late gestation). These studies were approved by the Hospital Ethics Committee. RESULTS: CA correlated with gestational age (r = 0.1755, P <0.01). CA in early gestation (n = 34, <13 weeks = 6.00 ± 0.10 cm 2 ) was smaller (P < 0.001) than the CA in late gestation (n = 54, 28-36 weeks = 6.28 cm 2 ± 0.16 SEM and n = 19, >37 weeks = 6.85 cm 2 ± 0.39). Similarly the CA of postpartum (4.30 cm 2 ± 0.17) and non-pregnant groups (4.59 cm 2 ± 0.24) was smaller than the CA at all gestational times (p<0.001). There was no relationship (P>0.05) in CA and BMI. In patients studied longitudinally CA progressively increased in gestation in 6, CA increased from early to mid-gestation and then declined in 3 and in 1 CA decreased from early to late gestation. CONCLUSIONS: 1) CA slowly and progressively increases to about 30% during pregnancy and then reverts back to lower CA postpartum.

2) The change in CA probably reflects changes in cervical volume and remodeling. 3) Longitudinal studies support the concept that CA increases during gestation but are preliminary. 4) CA might be used to estimate abnormal function of the cervix in pregnant and non-pregnant women or be predictive of successful vaginal delivery, induction or cesarean section. *Figure(s) will be available online. INTRODUCTION: Cervical shortening is a major risk factor for preterm delivery. Despite identifying this sign, many women with short cervices go on to have term deliveries. Changes in the cervical stroma and extracellular matrix occur prior to labor and may delineate which women will deliver at term versus preterm. Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to increase with advancing gestation and in labor. We hypothesized that women with short cervices would have increased levels of endocervical HA when compared to women with a normal cervical length (CL) during the midtrimester.

A prospective cohort of women between 17 and 23 weeks without risk factors for preterm delivery were enrolled. An endocervical swab was obtained followed by transvaginal sonographic cervical length (CL) measurement. The short cervix group (n=12) included subjects whose cervix was <25 mm. Controls (n=12), with a CL ³25 mm, were matched for parity and gestational age (GA). The concentration of HA in the endocervical fluid, our primary outcome, was determined using an ELISA specific for high molecular weight (>35 kDa) HA. The two groups were compared using unpaired Student's t-tests (two-tailed).

The mean (± SD) CL in the short cervix group was 20.3 ± 5.0 mm vs. 39.5 ± 6.4 mm in controls (p<0.0001). The mean (± SD) GA for both groups was 19.7 ± 1.5 weeks. The mean (± SD) HA concentration in women with a short cervix was 893.2 ± 597.9 ng/ml, whereas the parity and GA matched controls had an HA of 905.0 ± 481.7 ng/ml (p= 0.96).

Linear regression analyses showed no correlation between CL and GA (r 2 = 0.02) nor between endocervical HA concentration and CL (r 2 < 0.01).

Similarly, when we regressed endocervical HA levels by GA we observed no correlation (r 2 = 0.05). Post hoc analysis indicated that our pilot study with 12 subjects per group was powered to detect a 50% difference in HA levels, based on standard α = 0.05 and 1-β = 0.80 parameters. CONCLUSIONS: In this small cohort of pregnant women, a difference in endocervical HA concentrations was not observed when comparing participants with a short cervix to those with a normal cervical length. These findings lead us to conclude that between 17 and 23 gestational weeks endocervical HA levels are independent of cervix length in women without other risk factors for preterm delivery. The risk of operative delivery (cesarean and assisted vaginal) increases with advancing maternal age. We previously hypothesized that prolonged, cyclical, pre-pregnancy exposure to estrogen and progesterone contributes to uterine ageing (Smith et al, PLoS Med 2008;5:e144). This hypothesis makes 3 predictions: (1) later age at menarche would be associated with a reduced risk of operative delivery, (2) the association would disappear following adjustment for the menarche to birth interval, (3) the risk of operative delivery would increase with longer menarche to birth interval.

We used the Norwegian Mothers and Baby Cohort study, and analyzed the relationship between age at menarche and the risk of operative delivery in 38,069 first labors with a singleton infant in a cephalic presentation at term. The relationship between age at menarche and menarche to birth interval (as continuous variables, scaled for a 5y increase) and the risk of operative delivery was assessed using multivariate logistic regression analysis. Confounding by maternal characteristics was assessed by adjustment for maternal height, BMI, smoking, marital status, onset of labour, week of GA, BW percentile, infertility treatment, diabetes, educational status and household income.

A five year increase in age at menarche was associated with a reduced risk of operative delivery (unadjusted odds ratio [OR, (95% CI), P]: 0.79, (0.72-0.86), P<0.001). This was due to a reduced risk of both emergency cesarean delivery and operative vaginal delivery. Adjustment for the age at first birth slightly strengthened the association ( CONCLUSIONS: These observations support the hypothesis that age related deterioration in the outcome of first labour is related to prolonged pre-pregnancy exposure to the female sex hormones. This hypothesis suggests that the method of contraception used to delay first pregnancy may affect the process of first parturition.

Isolating the Direct Adverse Effects of Antenatal Glucocorticoid Therapy on the Fetal Heart and Circulation. N E Teulings, 2 Y Niu, 1 T Garrud, 1 K L Skeffington, 1 C Beck, 1 N Itani, 1 J B Derks, 2 D A Giussani. Here, we isolated the direct effects of AGT in the chick embryo, the only model in experimental medicine that permits investigation of the effects of therapy on the fetus independent of effects on the maternal and/or placental physiology. METHODS: Fertilized eggs were randomly divided into two groups. Dexamethasone was injected into the egg air cell using a human clinically relevant dose (0.1 mg/kg) on day 14 of the 21 day incubation period. At day 19, the embryos were euthanized. Cardiac function was assessed in isolated hearts under a Langendorff preparation. Second order femoral artery reactivity was studied via in vitro wire myography. A separate cohort of embryos was perfusion fixed (4% paraformaldehyde at 2.66kPa) and cardiac morphology established by unbiased stereology. RESULTS: Dexamethasone treatment restricted relative fetal growth (C: 38.3±0.6, D: 31.9±0.8 %, P<0.05), increased LV end diastolic pressure (LVEDP, Fig.1A ), impaired LV inotropic responses to carbachol (muscarinic agonist) and to isoprenaline (b 1 adrenergic agonist, Fig not yet fully understood, with many clinical and biochemical risk factors associated with idiopathic PTB. It therefore remains difficult to identify the women and babies at risk. Metabolomics, the study of low molecular weight molecules, provides a broad spectrum analysis of biochemical pathways potentially involved in disease manifestation and an unbiased hypothesis generating approach to identify pathophysiological processes. We applied this technology to identify early gestation biomarkers of PTB in maternal serum samples. Prediction of preterm delivery at an early stage of pregnancy, combined with appropriate treatment, could significantly reduce the disease burden on both mother and child. METHODS: A nested case (PTB<37 weeks; n= 56) control (> 37 weeks of gestation; n=112) study was performed on maternal serum samples, from the Irish Cohort of the Screening for Pregnancy Endpoints (SCOPE) biobank, obtained at 15 and 20 weeks' of gestation. PTB samples were matched against two sets of controls; one set was matched for maternal age only and the other for both maternal age and BMI. Samples were analysed using gas chromatography coupled with mass spectrometry. RESULTS: Preliminary analysis showed a significant change in maternal serum metabolite profile at 20 weeks of gestation, but no alterations could be found at 15 weeks. 15 metabolites were significantly increased in PTB cases when compared with controls matched only by age, and 9 when matched by age and BMI (p<0.05; organic acids, amino acids and antioxidants). 7 discriminatory metabolites were common for both sets. False discovery rate ranged from 0.18 to 0.25. CONCLUSIONS: An untargeted metabolomic approach identified an altered metabolite signature in early pregnancy maternal serum related to PTB development. Validation studies will be performed on a separate cohort (New Zealand cohort of the SCOPE biobank). Developing a highthroughput diagnostic tool has the potential to significantly impact both the diagnosis and subsequent treatment of PTB. The aim of this study was to determine the relationship between EMG of the rectus abdominis muscles in pregnant women during pushing and the duration of the second stage of labor. METHODS: EMG of the abdominal muscle was recorded during the 2nd stage of labor in 41 untreated (no anesthetic) nulliparous pregnant women.

Surface electrodes were placed on the abdominal surface and EMG of electrical bursts (5 to 150 Hz range) recorded for 30 minutes and analyzed (AD Instruments, New South Wales, Australia). Examination of muscle electrical bursts was performed using power density spectrum (PDS) and root mean square (RMS) analysis. The duration of the second stage of labor and other maternal and fetal characteristics were also recorded. RESULTS: Pushing events are characterized as high frequency and amplitude signals occurring during pushing ( Figure 1 ). There is a significant correlation between the duration of the second stage of labor and the EMG PDS (r =-0.919, P<0.001) and RMS (r = -0.879, P<0.001) ( INTRODUCTION: Action potential propagation recruits and coordinates the contraction of cells for participation in tissue-level contractions. However, the distance a single action potential can travel is uncertain.

In vitro data in rodent suggest action potential propagation distances are restricted to several centimeters. If true in human, this limit would divide the pregnant uterus into many "regions", which we suggest are functional units organized by electrical coupling. Here we search for regions using newly designed electrodes, called "area electrodes", which are modifications of Laplace electrodes. Area electrodes probe perpendicular to the skin surface, allowing precise location of the EMG signal within the abdomen. METHODS: Multichannel monopolar EMG recordings were obtained on 10 women in term labor. Three to five electrodes with areas varying between 12 and 300 cm 2 , were placed on the subject's abdomen and monopolar recordings were obtained over 30 minutes. Uterine bioelectrical signals were isolated by filtering with a 0.3 to 2 Hz window. Monopolar channels were subtracted in pairs to yield multiple pseudobipolar tracings. Signal "fingerprints" were identified. Data were analyzed by identifying the fingerprints in the pseudo-bipolar tracings, and assigning each bioelectrical burst to an area electrode. RESULTS: Electrodes acted independently when the areas were less than 100 cm 2 . Fingerprints were not be repetitive from contraction to contraction, but the expression of signals arising from each electrode was consistent over several contractions. Bursts occurred both independently from, and simultaneously with, bursts from other electrodes. *Figure(s) will be available online. CONCLUSIONS: Regional contractions of the term uterus in labor can be measured using area electrodes. Regions demonstrate physical stability over 30 minutes. Multiple regions can contract synchronously or asynchronously.

Weeks of Gestation(W/G). Eyal Zohav, Isca Landesberg, Larissa Feinmesser, Michael Kupferminc. Department of Obstetrics and Gynecology,, Sourasky Medical Center, Lis Maternity Hospital, Tel Aviv, Israel. INTRODUCTION: Data in the literature indicate that 50% of women with PPROM < 34 W/G deliver within one week. The aim of this study was to validate our policy and confirm the length of the latency period with our protocol.

In our study all PPROM cases of singleton pregnancies that were admitted to our "High Risk pregnancy" unit before 34 weeks gestation were included. The study group was further divided to 3 subgroups according to gestational age at PPROM occurrence. Group A prior to 28 W/G. Group B between 28-32 W/G and Group C > 32 W/G. Maternal and fetal data were abstracted from the computerized institutional database. Statistical analysis of fetal and maternal outcome was performed according to study subgroups by using, T test and Chi square tests and survival analysis. RESULTS: 87 women with singleton pregnancies complicated by PPROM delivered between years 2011-2014 and were included. Overall the median latency time from admission with rupture membranes to delivery, was 7 days. Nevertheless, in group A the median latency was surprisingly 24 days, while in group B it was 10 days and 3 days in group C. *Figure(s) will be available online.

The latency period of women with PPROM before 34 W/G that remains undelivered.

The latency period of women with PPROM stratified by W/G. CONCLUSIONS: Overall, the median latency period in women with PPROM before 34 was 7 days. However, our findings of a median latency period of 24 days in a subgroup of women with PPROM before 28 W/G is crucial when consulting women with PPROM regarding time of delivery and maternal and fetal outcome. To determine concentrations of acyl-carnitines and amino acids in serum during pregnancy in normal or obese women with or without GD. METHODS: Women included in the Mexico City's Perinatal Cohort were selected according to pregestational BMI and three groups were analyzed: 1. Women with normal BMI (≤ 24.9) (N), 2. Obese women (BMI ³30) (O) and 3. Obese women who developed gestational diabetes (OD).

Concentration of the acyl-carnitines, free carnitine and amino acids were determined by mass spectrometry in monthly samples of serum obtained from week 14 of gestation. A metabolomic approach using principal components analysis was used to evaluate changes. RESULTS: Glucose, insulin concentration and HOMA index were significantly higher in second trimester in OD group.OD group had higher acyl-carnitines concentrations during second trimester. Amino acid concentrations were significantly higher in the second trimester and there was a significant increase in the gluconeogenic and ketogenic amino acid concentrations in OD group. CONCLUSIONS: A broad group of metabolic changes along gestation reflecting the metabolic plasticity in pregnancy were found. Metabolic changes associated to disruption of plasticity resulting in gestational diabetes appeared as a cluster of modifications in a specific window during weeks 22 to 26. This results may be used to identify changes in the metabolic adaptation in pregnancy that may be useful for early diagnosis of gestational diabetes.

Ana 

To compare acyl-carnitine species in serum between women with normal pregestational BMI <= 24.9 (NWP) and obese pregestacional BMI >= 30 (OP) along pregnancy. METHODS: Serial samples of serum were obtained from women in the Mexico's City Perinatal Cohort. Serum concentration of the acylcarnitines and free carnitine was determined by flow injection targeted tandem mass spectrometry, during pregnancy in NWP group (n=19) and OP group (n=19). A principal component analysis and correlations within principal component analysis was performed in order to identify potential biomarkers. RESULTS: Principal component analysis demonstrates a significant variability in C3 and C8:1 between OP and NWP groups across pregnancy, particularly between 15-20 gestational weeks. In addition, a significant negative correlation was observed between glucose and total acyl-carnitines, free carnitine, C2 and C3 during the third trimester of pregnancy. CONCLUSIONS: Increase in seric acyl-carnitines reflects a reduction in glucose utilization, increased β-oxidation, increased use of branchedchain amino acid oxidation and mitochondrial dysfunction. C3 and C8:1 could be good candidates as biomarkers of metabolic disruption during pregnancy associated to obesity.

Marian C Aldhous, 1 Max Bylesjo, 2 Jane E Norman. Gestational diabetes (GDM) has a long-term effect on the offspring, possibly via epigenetic mechanisms. We aimed to determine DNA-methylation patterns in cord-blood samples of babies exposed in utero to well-controlled GDM (n=9) and controls, born at term. Each sample was matched with two controls (n=18) by sex of baby (18M:9F), gestational age (within a week), parity (parous vs. non-parous), maternal age and whether the mother laboured (n=6 labour, EmCS, n=21 no labour, ELCS).

Cord-blood DNA was extracted, bisulphite treated and run on an Illumina® Infinium Human Methylation450 BeadChip. This measures methylation of 485,000 CpG sites across the genome.

We found no significant differences in cord-blood DNAmethylation by whether the mothers had had GDM or not. Genes implicated by KEGG pathways comparing differential DNA-methylation of GDM-exposed samples vs. controls showed no differences in expression by qPCR according to GDM-exposure but did by offspring gender (P<0.05). Differential methylation was also found in samples from offspring born by EmCS (labour) compared with those born by ELCS (no labour): 5400 CpG sites were significantly (FDR-adjusted P<0.05) differentially methylated in labour vs. no labour; 3611 of these on known genes. Mothers undergoing ELCS were older than those having EmCS (mean±SD age 33.0±4.5 vs. 30.3±4.9 years, P<0.0001). The average difference in methylation of the top 50 most significant CpG sites was 6.6% (range 2.6-14%). KEGG-pathway analysis of differentially methylated genes (by labour) indicated a variety of affected genes and pathways that might be relevant to later development of asthma and/ or allergies. Other babies delivered by EmCS (n=10) or ELCS (n=26), showed higher white cell counts in cord blood from EmCS than ELCS, especially neutrophils (mean± sd, 8.38±3.2 vs. 4.74±1.5 x10 9 /l, P=0.0002). CONCLUSIONS: These results suggest that well-controlled GDM may not affect offspring cord-blood DNA methylation patterns. However, offspring gender and labour had a significant effect on cord-blood DNAmethylation patterns and therefore both need to be taken into account for DNA-methylation studies. Further research into these effects is required, as it has been suggested that mode of delivery of a baby might affect its immunity during childhood.

Evaluating . GWG was defined as follows: maternal pre-pregnancy body mass index (BMI) category (underweight, normal weight, overweight, and obese) was determined and then the BMI-specific recommended weekly GWG range was utilized to categorize GWG as above, within, or below IOM guidelines. This approach generated gestational-age adjusted GWG ranges. PTB was defined as any delivery before 37 weeks of gestation. Stata 12.1 was used to compute the standard errors for final weighted analyses and statistical tests to account for the complex survey design of PRAMS. Weighted multivariable logistic regression was used to calculate an adjusted odds ratio (aOR) and 95% confidence interval (CI) for each GWG variable and PTB, after controlling for confounding variables. RESULTS: Overall, 60.4% of women gained above the IOM guidelines, whereas 16.2% of women were below the IOM guidelines. Gaining above the IOM guidelines was significantly associated with PTB across all maternal BMI categories: underweight (aOR=1.30, 95% CI: 1.07-1.14); normal weight (aOR=1.21, 95% CI: 1.14-1.29); overweight (aOR=1.25, 95% CI: 1.14-1.37); and obese (aOR=1.30, 95% CI: 1.19-1.41). Gaining below IOM guidelines was also associated with preterm birth for overweight and obese women. Results were similar when restricting PTB to deliveries before 34 weeks of gestation. *Figure(s) will be available online. CONCLUSIONS: In the PRAMS population, excess GWG is prevalent and associated with PTB across all maternal pre-pregnancy BMI categories. Based on our estimates, 16% of PTBs among obese women can be attributed to GWG. Furthermore, there was a U-shaped association between GWG and PTB among overweight and obese mothers, whereby gaining below guidelines was also associated with risk of PTB.

Environmentally In this study we investigate the effect of maternal BPA exposure on the expression of key epigenetic regulators for both methylation and hydroxymethylation in the rat ovary. DNA methyltransferases (Dnmts) and other methylation binding proteins establish and maintain methylation patterns. Ten-eleven translocation (TET) enzymes initiate DNA demethylation by oxidation of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc). METHODS: Rats housed in a BPA free environment were fed with either an AIN control or BPA 250µg/kg diet for a minimum of 20 days, then sacrificed. The ovaries were snap-frozen and stored at -80°C. Primers were designed for the following genes: Dnmt1, Dnmt3a, Dnmt3b, TET1, TET2, and TET3. Total RNA was isolated using a mirVana kit (Life Technologies). Reverse transcription was performed using the Smartscribe RT kit (Clontech Laboratories) to generate cDNA template for real-time (RT) polymerase chain reaction (PCR). RNA quality was verified with denaturing RNA agarose gel electrophoresis. Quantitative RT PCR was performed using the ViiA™ 7 RT PCR System with the Power SYBR Green PCR Master Mix (Life Technologies). Quantification of epigenetic genes was calculated relative to the transcription of housekeeping gene RPL-19 using Microsoft Excel. Gene expression fold differences with p<0.05 were considered statistically significant. RESULTS: A significant increase in Dnmt1 expression in the BPA exposed group (fold change 14.04, p=0.02) was noted. A trend toward down regulation of all three TET enzymes was also noted, although significance was not achieved. CONCLUSIONS: Increased ovarian expression of Dnmt1 in BPA exposed rats could result in increased methylation, methylation maintenance dysregulation, and down-regulation of key genes necessary for folliculogenesis. A trend toward down regulation of TET enzymes in the exposed group may correlate to the overall inability to de-methylate the maternal genome. Further studies will investigate the BPA effect on global hydroxymethylation as a result of down regulation of TET enzymes. In 10 pregnancies, animals were put down at 138 days. Following biometry, plasma and tissues underwent biochemical analysis. The other 10 pregnancies delivered naturally and offspring maintained until 9 months. These adult offspring were chronically instrumented with catheters and a femoral flow probe and experiments were performed in vivo and ex vivo (wire myography) to determine cardiometabolic function.

Offspring of hypoxic pregnancy showed IUGR (a) with postnatal accelerated growth (b). At adulthood, they were hypertensive (c) with femoral endothelial dysfunction (d), enhanced constrictor reactivity (e) and with glucose intolerance (f). Vitamin C in hypoxic pregnancy prevented many of these effects by promoting a greater fetal antioxidant defence (g) and a right shift in the maternal oxygen-haemoglobin dissociation curve (h). CONCLUSIONS: Vitamin C is not the antioxidant of choice for human therapy, however this work provides proof of principle offering translational targets for human clinical intervention against the programming of disease in offspring of high risk pregnancy. Support: British Heart Foundation *Figure(s) will be available online. Epidemiological and animal studies demonstrate that prepregnancy obesity leads to increased anxiety, decreased spatial learning and lower IQ in offspring. This study aims to determine the role prepregnancy obesity has on fetal brain programming using a wellestablished mouse model.

For three months before breeding the CD-1 female mice were fed either high fat diet (34.9% fat, HF group) or standard chow (5.8% fat, SF group). Offspring from both groups of mothers was weaned onto standard chow. Male offspring were randomly selected from each litter to represent as n of 1. They were sacrificed at 6 month of age and brains were collected. Half of the brain was fixed in paraformaldehyde (4%) and processed for Nissl staining for evaluation of cortical morphology and neuronal count. RNA was extracted from the primary somatosensory cortex using the other half of the brain. Quantitative RT-PCR was used to measure mRNA expression of genes involved in glial and neuronal status: glial fibrillary acidic protein (GFAP), reelin (RELN), and myelin basic protein (MBP). Student t-test or Mann-Whitney test were used as appropriate for statistical analysis (significance P<0.05). RESULTS: GFAP, RELN and MBP expression was upregulated in HF group, though not statistically significant. Neuronal distribution and counts were significantly lower in cortex from offspring males born to mothers in HF group (P=0.002). CONCLUSIONS: Maternal prepregnancy obesity alters fetal brain programing in somatosensory cortex. Our study is first to demonstrate an abnormal count and distribution of neurons in adult offspring of mothers exposed to prepregnancy high fat diet. As prepregnancy obesity rates continue to rise further, investigations into mechanisms of the abnormal fetal brain programming with this condition are urgently warranted. *Figure(s) will be available online.

In In utero tobacco exposure has been shown to increase offspring risk for pediatric and adult obesity. However, critical biomarkers predicting such adverse outcomes and their potential mechanistic pathways remain poorly understood. The goal of this pilot study was to evaluate the effects of maternal smoking on the fetal programming of glucose metabolism and inflammatory gene expression in neonates. METHODS: Foreskin samples were collected from a total of 55 non-anomalous neonates. Maternal smoking status was determined by self-report. Foreskins were grossly dissected into epidermal/dermal and hypodermal layers. Samples were flash frozen, and RNA was isolated from the epidermal/dermal layer. Nine epidermal/dermal samples were excluded (n=4 from twins; 2 from preterm infants; 3 due to RNA degradation). RNA expression levels were analyzed using the NanoString Technologies nCounter system. Expression of genes involved in glucose/lipid metabolism and inflammatory response was quantified and normalized to housekeeping genes. The average gene expression levels among infants with (n = 15) and without (n = 31) maternal history of smoking during pregnancy were compared, using Student's t test. RESULTS: Expression levels of cytochrome P450 1B1 (p < 0.01) and Cu-Zn superoxide dismutase (p = 0.058), inflammatory stress response genes, were increased in babies born to smokers compared to controls. The foreskin samples of infants born to smokers had significantly increased protein-tyrosine phosphatase expression (p < 0.05), a negative regulator of insulin signaling, suggesting an increase in insulin resistance. Altered glucose utilization was also suggested by a significantly increased expression of chemerin (p < 0.01), apolipoprotein D (p < 0.05), cAMP responsive element binding protein 1 (p < 0.05) and significantly decreased expression of the inhibitory protein, fatty acid transporter protein 4 (p < 0.05), suggestive of possible altered fatty acid metabolism. CONCLUSIONS: Genes involved in the inflammatory response as well as energy metabolism were deferentially expressed in the foreskins from infants born to smokers compared to nonsmokers. Further studies are warranted to evaluate if these changes will be sustained in the long term through epigenetic interactions. Fetal perirenal adipose tissue (PAT) has characteristics of both white (WAT) and brown (BAT) adipose tissue thus resembling a "beige" fat phenotype. We previously reported a significant impact of LTH on PAT gene expression in the late gestation ovine fetus, with increased expression of both BAT and WAT-associated genes. However, by post-natal (PN) day 14, LTH lambs exhibit a decrease in the BAT/beige molecular program while retaining or amplifying genes of the WAT phenotype compared to controls. This suggests a decreased beiging and/or potential loss of beige adipocytes. This study was designed to determine the effect of LTH on miRs that target genes regulating the beige phenotype. We quantified miR155 (repression of PGC1α) and miR133a and miR346 (repression and activation, respectively of RIP140). PGC1α is a key co-activator while RIP140 is a key co-repressor of BAT/beige genes. METHODS: Pregnant ewes were maintained at high altitude (3,820 m, LTH) from ~ 40 days' gestation (dG). Fetuses were delivered on 137-140 dG; additional animals were allowed to deliver and tissue collected from lambs on 12-14 days PN. PAT was analyzed for PGC1α, RIP140, miR155, miR133a and miR346 by qRT-PCR. Data are expressed as mean ± SEM, in fg mRNA/50 ng RNA for adipose genes and fold change from the housekeeping gene SNORD61 for miRs. RESULTS: Table 1 .

PGC1α ( We reported that lambs exhibit a leptin surge on day 6-9 of postnatal life. In rodents, this neonatal leptin surge plays a central role in development of the hypothalamic appetite control centers. Maternal obesity (MO) in the ewe obliterates this leptin peak and also elevates lamb blood CORT. Offspring of MO ewes exhibit increased appetite, weight gain and adiposity to ad lib feeding. Hypothesis: Since CORT alters perinatal adipose tissue development we hypothesized that exogenous CORT administration to normal newborn lambs would raise their plasma CORT to values seen in lambs born to MO ewes, eliminating their leptin peak. METHODS: Newborn male lambs were assigned randomly to the following treatments: One ml i.m. injection of 3 µg CORT/ kg body weight (CORT3, n=3), 10 µg CORT/ kg body weight (CORT10, n=3), or vehicle (CON, n=3). Half the original CORT dose or vehicle was administered 24 h later. Two ml jugular blood samples were collected at birth, at 2 h intervals for 12 h, and daily until postnatal day 11 and plasma stored at -80°C until analyzed for CORT and leptin via validated RIA. Data were analyzed as repeated measures using the Proc Mixed procedure of SAS (SAS Inst. Inc., Cary, NC). RESULTS: Plasma CORT levels of CORT10 animals were elevated (P<0.05) from 2 to 4 h post treatment on day 1 compared to CON animals, while CORT levels of CORT3 lambs were intermediate (Fig.  1A) . Thereafter, CORT in the three groups was similar. Plasma leptin levels were similar across groups through postnatal day 5. Thereafter, plasma leptin in CON animals increased (~2 fold) to peak on days 7-9 before declining on day 11 (Fig. 1B) . CON leptin levels were markedly higher (P<0.01) than CORT10 animals from day 6 through day 11. CORT3 leptin levels were intermediate between CON and CORT10 levels from day 7 through day 11. CONCLUSIONS: These data are consistent with the concept that the markedly elevated blood CORT seen at birth in MO lambs is responsible for the elimination of the early postnatal leptin spike in this species. *Figure(s) will be available online.

Effect Maternal malnutrition is associated with metabolic programming of the offspring. We have shown that perinatal maternal high-fat diet (HFD) programs obesity, hyperleptinemia and central leptin resistance in male rat offspring. Obesity is associated with over activation of the endocannabinoid system (ECS). ECS components are anandamide and 2-arachidonoylglycerol (ligands), cannabinoid receptors (CB1 and CB2) and monoacylglycerol lipase (MAGL). Over activation of ECS is associated with leptin resistance. However, it is unknown whether ECS participates in the developmental origins of obesity during gestation and lactation. We hypothesize that perinatal maternal HFD will alter the ECS components in the adipose tissue of male and female rat offspring at weaning.

We fed female Wistar rats a standard diet (C; 9% of calories as fat) or high-fat diet (HF; 28% of calories as fat) during 8 weeks before mating and throughout gestation and lactation. At weaning (21 days), male and female offspring were killed. Serum was used for leptin serum concentration measurement by radioimmunoassay. Retroperitoneal white adipose tissue (RWAT) was used for western blot of the ECS components.

Results are reported as % of change compared to sex-matched controls (*p<0.05). Male and female HF offspring had increased body weight (+31%* and +27%*, respectively), RWAT mass (4 fold*) and serum leptin (+53%* and +84%*, respectively). Maternal HDF increased CB1 (+52%*) and decreased CB2 (-18%*) in RWAT of only female offspring. Maternal HFD increased both FAAH and MAGL abundance (+90%* and +40%*, respectively) only in male offspring. CONCLUSIONS: Maternal HFD induces early obesity and hyperleptinemia in both male and female rat offspring, with sex-specific changes in the ECS of RWAT. CB1 receptor is involved lipogenesis while CB2 receptor is involved with inflammatory pathways. FAAH and MAGL metabolize endocannabinoids. We speculate that the endocannabinoid system is over activated in both HF male and female offspring through down regulation of CB2 and up regulation of CB1 receptors in females and increased of both MAGL and FAAH in males as adaptive mechanisms. and Type 2 Diabetes. It is well established that estrogenic chemicals interact with the ligand-binding site of estrogen receptors to disrupt transcriptional control of estrogen responsive targets, such as the liver. However, the mechanism by which BPA in utero exerts biological effects on the developing offspring liver has yet to be studied. We hypothesize that short-term prenatal BPA exposure creates lasting effects via developmental programming that alters hepatic gene expression and regulation. METHODS: Twelve pregnant CD-1 mice were continuously treated with BPA (5 mg/kg/day) or vehicle control via osmotic mini pump on days 9-18 of gestation. Six weeks after birth, female offspring were ovariectomized. At eight weeks, female pups were treated with a single IP injection of 300 ng estradiol (E2) or vehicle, and the livers were removed for RNA isolation and analysis. Affymetrix GeneChip Mouse Gene 2.0 ST Array was used to survey the genome-wide expression profile. MatLab was used for bioinformatics and statistical analysis. RESULTS: Of the 24,990 genes on the microarray, 113 genes were significantly altered by a 1.75 fold change in BPA-exposed livers compared to controls (43 genes were significantly upregulated, 70 were significantly downregulated, p<0.05). Many of the genes were found to be involved in hepatic metabolism, including upregulated glucose-6-phosphatase (G6pc) and downregulated sodium-dependent citrate transporter (Slc13a5). Changes in gene expression in BPA-exposed livers versus controls were validated using quantitative real-time PCR (qRT-PCR). CONCLUSIONS: Significant changes in mRNA expression of genes were observed in the livers of female mice exposed to BPA in utero. Geneenvironment interactions driven by BPA alter the normal developmental programming of the estrogen-responsive liver. Endocrine disruptors can potentially cause lasting changes to hepatic development and subsequent metabolic function, long after chemical exposure has ceased. Since developmental processes required for efficient pulmonary gas transfer are incomplete at birth, the lung and pulmonary circulation are particularly vulnerable. We extend the concept of developmental programming to chronic mountain sickness (CMS), a debilitating syndrome marked by severe polycythemia, hypoventilation, pulmonary hypertension (PHTN) and possible right-heart failure. We hypothesized that perinatal hypoxia impaired ventilatory or pulmonary vascular function and led to CMS later in life.

Our approach was to recruit 123, 18-24 yr old male highlanders (La Paz, Bolivia, 3600-4100m) with a preclinical form of CMS (excessive erythrocytosis, EE, Hb>18.3 g/dL, n=67) or without (controls, CON, n=66) to avoid the confounding effects of advancing age. Medical records and maternal interviews provided data on perinatal complications. We measured Hb by the cyanmethemoglobin method, arterial O 2 saturation (SaO 2 ) by pulse oximetry, pulmonary artery pressure (P PA ), and right heart function by transthoracic echocardiography, and respiratory function by spirometry and whole body plethysmography. ANOVA or chi-squared tests were used to identify differences between EE vs. controls. Logistic regression was used to determine the relative contribution of pregnancy, perinatal or other characteristics to EE. RESULTS: EE vs. CON were 3-times more often born to mothers with preeclampsia (30.8% vs. 10.3%, p<0.05), and had a 4-fold greater incidence of hypoxia-related perinatal complications (32.7% vs. 7.7%, p<0.05) but birth weights and the frequency of prematurity did not differ. Compared to CON, EE had lower ventilation (V A ), SaO 2 , reduced pulmonary diffusion capacity (DL CO ), and a higher incidence of PHTN (p<0.05) and RV enlargement (p<0.05). Hypertensive pregnancy and perinatal hypoxia remained significant predictors of EE after accounting for DL CO, V A and P PA .

Our findings indicate that adverse perinatal oxygenation increases susceptibility to polycythemia in adulthood that may be due, in part, to disrupted alveolarization or microvascular development leading to impaired gas exchange. After weaning pups in both groups were placed SF chow. Starting at 1 month of age pups underwent monthly scanning using micro-computed tomography scanner. The muscle radiodensity (MR) of left and right three-dimensional sections of quadriceps was analyzed. At 6 months of age animals were sacrificed and quadriceps were collected. Quantitative RT-PCR was used to measure mRNA expression of genes involved in fatty acid transport: fatty acid translocase/CD36 (CD36), calveolin-3 (CV3) and fatty acid transporter-1 (FATP1). Statistical analysis was performed using repeated 2-way ANOVA and Mann Whitney test (significance: P£0.05).

In males, in both groups, longitudinal micro-CT results demonstrated a decrease in MR as animals aged, indicating increasing fat infiltration. mRNA expression of CD36, CV3, FATP1 was not different between SF and HF males at 6 months of age. In females, MR was decreasing only in HF group, but did not reach statistical significance. mRNA expression of CD36 (P=0.04), CV3 (P=0.02), FATP1 (P=0.05) were significantly higher in HF females ( Figure) , indicating activated fatty acid transport into muscle. CONCLUSIONS: We demonstrate the developmental trajectory of fat infiltration into skeletal muscle in an animal model of developmental metabolic syndrome. In females, this was confirmed by increased mRNA expression of fatty acid transport regulating genes. Our study provides novel evidence of systemic alterations in offspring after exposure to maternal pregnancy obesity. *Figure(s) will be available online.

Baboon Kidney miRNA Changes at 0.9 Gestation (G). while only 43 miRNAs were differentially expressed (P<0.05; 9 up and 34 down) in males. Further, miRNA target filtering identified 221 inversely related gene/miRNA pairs in males, considerably more than females (99 pairs). Network analysis results show that gene networks involved in cell death and survival, cellular development and function, as well as cellular assembly and organization are affected in males, while no specific network was identified in females. CONCLUSIONS: Effects of MNR in pregnancy on signaling pathways related to nutrient sensing and cell proliferation in the late gestation are greater in male than female kidneys. Inverse expression of miRNAs known to target genes in these pathways suggests that miRNA mechanisms may play a mechanistic role. These results need further validation at the protein level. *Figure(s) will be available online. The nutritional environment of the oocyte and embryo during the periconceptional period has been shown to determine cardiovascular health in fetal and postnatal life. In this study we aim to investigate whether in vitro embryo culture and transfer, which are manipulations of the nutritional environment during the periconceptional period, result in dysregulation of blood pressure regulation. METHODS: Embryos were either transferred to an intermediate ewe (ET) or cultured in vitro in the absence (IVC) or presence of human serum (IVCHS) and a methyl donor (IVCHS+M) for 6 days. Controls were nnaturally mated (NM) ewes. Mean arterial pressure (MAP) and heart rate (HR) were measured before and after phenylephrine (PE) bolus doses of 4, 8, 16, 20 µg/kg. The relationship between MAP and HR was analysed using the logistic function and the maximal gain was calculated. Two way ANOVA was used to analyse the slope from each curve and delta MAP and HR (at maximal response to PE). RESULTS: Basal MAP and HR did not differ between the treatment groups. There was no significant difference between the maximal gain coefficient (P= 0.5), upper plateau (P=0.059), and MAP50 (P=0.11). With the low doses of PE (4 and 8 µg/kg), there was no difference in change in MAP but the largest drop in HR was observed in the IVCHS group (P=0.015).There was no difference in delta MAP with high doses of PE (16 and 20 µg/kg). The maximum drop in HR was, however, attenuated in the IVC and IVCHS+M group compared to the other groups (P=0.010) *Figure(s) will be available online.

The results suggest that embryo transfer and in vitro embryo culture do not alter basal MAP, HR or baroreflex sensitivity. The diminished change in HR in IVC and IVCHS+M groups at maximal response from baseline after PE may suggest abnormal regulation.

Joining the Dots: Genetics, the Environment, Developmental Programming, and Obesity. The 'U-shape' relationship between birth weight & adult obesity is well established; however, not all fetuses exposed to adverse environments develop obesity. We have shown that the 'U-shape' relationship only occurs in individuals with poor HPA-axis responsiveness to stress as young adults; those with normal HPA function demonstrate no relationship between birthweight and obesity. Aim: to identify a genetic risk profile that increases the likelihood of developing poor HPA-axis function and subsequent obesity as a result of developmental programming. METHODS: An extensively phenotyped cohort, the Western Australian Pregnancy (Raine) Study, conducted Trier Social Stress Tests (TSST) in 1137 participants at 18yrs. A GWAS was performed on change in blood cortisol concentration during the TSST (GWAS+TSST data n=758).

Regions of interest were identified from Manhattan plots and SNPs p<10 -5 that were not in LD were used to generate an individualised risk score which was modelled against the odds of having a non-responsive vs. responsive HPA axis at 18yrs.

We identified 4 regions in chromosomes 7, 8 & 14 with multiple variants p<10 -5 (n=97) with promising regional plots. When SNPs in high LD were removed and the remaining SNPs were modelled for independence, 4 SNPs remained; these are in the CHN2, PRKCH, BAALC & NUMB genes. A risk score was developed based on 0 to 8 adverse alleles [median 5.96 IQR 1.33]. For each increase in risk-score of 2, the odds of havng a non-responsive HPA-axis increased by 1.44. These non-responders were at increased risk of adult obesity with OR up to 4 at the ends of the birthweight spectrum.

We have identified genes that influence HPA responsiveness in adults. A risk-score based on these genes identifies those at high risk of poor HPA function and subsequent adult obesity. At birth, the ability to identify both small and large babies at high risk of developmental programing offers new unique opportunites to target and personalise interventions to reduce adult obesity. 

Time-dated pregnant rats were divided into four groups: 1) nicotine, 2) saline, 3) nicotine plus a ROS inhibitor, N-acetylcysteine (NAC, 500 mg/kg/day), and 4) saline plus NAC. Nicotine was administered to pregnant rats starting at d4 of gestation till 10 days after birth via a subcutaneous osmotic minipump. NAC was added in the drinking water throughout gestation. Male offspring were studied at 3 months old. RESULTS: Angiotensin II (Ang II)-induced blood pressure responses were significantly higher in antenatal nicotine-treated group than in saline control group. However, with the NAC treatment there were no significant differences of Ang II-induced blood pressure responses between the nicotine and saline control groups. Antenatal nicotine exposure significantly enhanced both Ang II-and PDBu (PKC activator)induced contractions of aortic rings, as compared with those in the saline control, and this difference was blocked by the NAC treatment. In addition, acetylcholine-induced relaxations were significantly decreased in aortas isolated from the nicotine-treated offspring, as compared with those in the control animals, which was also inhibited by the NAC treatment.

The results suggest that antenatal nicotine exposureinduced programming of hypertensive response in adult offspring may be mediated by heightened ROS signaling and oxidative stress in the vasculature. (Supported in part by the California Tobacco-Related Disease Research Program Award 22XT-0022 and NIH grants HL118861, DA032510). 

Late gestation rat uteroplacental insufficiency was induced (Restricted) or sham (Control) surgery in F0 females. F1 males born of normal and low birth weight were mated with normal females. F2 body weights were measured, metabolic function characterised (fasting glucose tolerance test and insulin challenge) and pancreatic β-cell and islet mass quantified. In 16mo males arterial stiffness of small renal and mesenteric arteries was assessed (pressure myography) and blood pressure measured (telemetry). RESULTS: F2 body weights were not different at birth but Restricted males were heavier than Controls at 4-6mo with no differences in organ or adipose weights. Males from Restricted fathers had higher area under glucose curve and reduced first phase insulin secretion at 6mo, which was not sustained to 12mo. There were no changes in pancreatic β-cell or islet mass. Restricted F2 males had elevated systolic blood pressure at 16mo, which were not driven by changes in left ventricle COL1A2, COL3A1, Tgfβ-1, Tgfβ-2, Smad2, Mmp1a, Mmp2, FGF2 expression or renal and mesenteric arterial stiffening. There were no differences in any parameters studied between F2 females born to Control and growth restricted fathers. CONCLUSIONS: We demonstrated that in the absence of low birth weight, F2 males born to F1 growth restricted fathers had accelerated growth during early adulthood without obesity. They had altered glucose control at 6mo. Hypertension emerged with exacerbation of age to 16mo although this was not sufficient to drive changes in gene expression that promote cardiac hypertrophy or fibrosis. Paternal growth restriction programs male cardiovascular and metabolic dysfunction, which may worsen with further adverse diet and lifestyle factors. Females from fathers born small appear to be protected from dysfunction, although these phenotypes may emerge under the stress of a future pregnancy. Advanced maternal age is associated with increased maternal and perinatal morbidity/mortality, and an increased risk of complications such as preeclampsia and intrauterine growth restriction.

Evidence from both humans and animal models demonstrate that offspring from adverse pregnancies are at an increased risk of cardiovascular disease as adults. However, little is known about the effects of advanced maternal age on the long-term cardiovascular health of adult offspring. We hypothesize that adult rat offspring born from aged dams will have compromised cardiac function and impaired vascular reactivity compared to offspring from young dams. METHODS: Aged female Sprague Dawley rats (9.5 months; ~ 35 year old woman) and young controls (4 months) were mated with young males. Maternal blood pressure was measured by CODA tail cuff at gestational day (GD) 19. In offspring, biometrics were measured and males and females aged to 4 months; the isolated working heart model and wire myography were used to assess cardiac function and vascular reactivity. 

Bisphenol A (BPA) Exposed Offspring. have been attributed, in part, to programming effects resulting from developmental exposures. Bisphenol A (BPA), which is widely used in plastics (e.g. water bottles) is an endocrine disruptor that is detectable in plasma of pregnant women, fetal plasma and amniotic fluid. Liver plays a central role in the control of lipid metabolism, and monounsaturated fatty acids (MUFA) are the preferred substrates for triglyceride synthesis. We hypothesized that maternal BPA exposure causes upregulation of hepatic stearoyl-CoA desaturase enzyme-1 (SCD-1), which converts saturated fatty acids to MUFA for triglyceride synthesis. METHODS: Female rats had access to filtered drinking water (control group) or drinking water containing BPA (5mg/L; BPA group) two weeks prior to mating and throughout pregnancy and lactation. Standardized litters were nursed by the same dam. At end of nursing period (3 weeks of age), liver tissue was collected from male and female offspring for fatty acid extraction and analysis by GC/MS. GC/MS peaks were used to calculate the desaturation index (DI: MUFA/saturated fatty acid), the elongation index (EI: C18 stearate/C16 palmitate) and the de novo fatty acid synthesis index (palmitate/linoleate). Protein expression of hepatic SCD-1 was determined by Western Blot and plasma triglyceride concentrations were determined by ELISA. Data were compared by ANOVA. RESULTS: BPA females had increased plasma triglyceride levels (33±3 vs 14±3 mg/dL; P<0.05) and an increased EI (1.37±0.07 vs 1.14±0.05; P<0.01). BPA females showed no differences in the DI. However, there was a trend towards increased SCD-1 expression (1.9±0.3 vs 1.0±0.3; P=0.07), and a trend towards decreased de novo fatty acid synthesis (5.30±0.65 vs 6.98±0.73; P<0.07) in BPA females compared with controls.

In contrast, BPA males had normal triglyceride levels with normal fatty acid indices and SCD1 expression. CONCLUSIONS: Together, these data suggest that maternal BPA has sex-specific effects on offspring liver fatty acid metabolism. Increased fatty acid elongation and SCD-1 expression in the presence of unchanged desaturation indices suggest increased utilization of MUFA products to promote triglyceride synthesis in female offspring exposed to BPA perinatally. Among the two primary fat depots, white adipose tissue (WAT) stores excess energy, whereas brown adipose tissue (BAT) expends energy via thermogenesis. Maternal obesity increases the risk of newborn and adult offspring obesity, though it is unclear if this is a physiologic or pathologic mechanism. We hypothesized that maternal obesity programs offspring adipogenesis via specific effects on both newborn WAT and BAT. We determined the proliferation rate and protein expression of distinguishing genes in WAT and BAT in newborn males of control and obese dams. METHODS: Female mice were fed either a control (10% k/cal) or high fat (45% k/cal) diet to create maternal obesity (MO) prior to mating, and diets continued throughout pregnancy and lactation. Newborns were delivered spontaneously, males sacrificed at day one of life, and inguinal WAT and interscapular BAT were collected. Primary WAT and BAT preadipocytes were cultured for proliferation (MTT assay). WAT and BAT protein expression (Western blot) of thermogenic gene (UCP1), adipogenic transcription factors (PPARγ, C/EBPα) and mitochondrial marker (ATP5A) were determined. RESULTS: At 1 day of age, MO males were heavier (1.55±0.09 vs 1.31±0.01 g; P<0.05) than Controls. In WAT, MO males exhibited decreased preadipocyte proliferation (0.8-fold; P<0.05) though with increased expression of PPARγ (1.3-fold; P<0.05) and C/EBPα (1.8fold; P<0.05). Conversely, in BAT, MO preadipocyte proliferation was increased (1.3-fold; P<0.05) as was the expression of UCP-1 (1.5-fold; P<0.05) and ATP5A (1.7-fold; P<0.05).

In MO newborn males, increased WAT PPARγ and C/EBPα expression is consistent with enhanced adipogenesis and lipid storage while increased BAT UCP-1 and ATP5A indicate enhanced newborn thermogenesis potential. These findings suggest a predictiveadaptive response in MO newborns facilitating both acute (BAT) and chronic (WAT) benefits of adiposity. Adipogenic programming effects of MO may have evolved for survival benefit. Perinatal over-or under-nutrition results in obese adult offspring with programmed enhanced adipogenesis. We have shown that increased adiposity in both offspring groups is mediated via early upregulation of adipogenic transcription factor, PPARΥ, which promotes adipocyte differentiation. Induction of PPARΥ and adipogenesis is regulated by factors including Wnt10b signal transduction. Activation of Wnt10b signaling inhibits adipogenesis through both canonical β-catenin dependent and non-canonical β-catenin independent pathways. We hypothesized that inhibition of Wnt10b contributes to increased PPARΥ and enhanced adipogenesis in offspring exposed to maternal obesity and under-nutrition. We further determined whether these effects were mediated via β-catenin pathway. METHODS: Female rats were fed either a high fat (HF; 60% k/cal) or control (10% k/cal) diet prior to mating, and throughout pregnancy and

lactation. An additional group of dams were 50% food-restricted (FR) from pregnancy day 10 to term to produce growth-restricted newborns. After birth, HF pups were nursed by the same dam whereas FR pups were crossfostered to Control dams. All pups were weaned to normal diet. Adipose tissue was obtained at 1 day (inguinal) and 9 months (retroperitoneal) of age from male offspring. Protein expression of Wnt10b and β-catenin were analyzed. RESULTS: As compared to controls, both HF and FR male newborn PPARΥ levels were upregulated (2-fold; P<0.05) and Wnt10b was downregulated (0.5, 0.6-fold, respectively; P<0.05). β-catenin expression was decreased in HF (0.7-fold; P<0.05), though increased in FR newborns (1.4-fold; P<0.05). These changes persisted in adults with HF males exhibiting decreased Wnt10b (0.2-fold; P<0.05) and β-catenin (0.7fold; P<0.05) whereas FR males showed continued increased β-catenin (2-fold; P<0.05). CONCLUSIONS: Both perinatal over-and under-nutrition promote adipose PPARΥ expression via suppression of Wnt10b signaling. Whereas enhanced adipogenesis likely occurs via the canonical Wnt10b/β-catenin pathway in response to overnutrition (ie, HF), the adipogenic stimulation is mediated via a non-canonical β-catenin independent pathway in response to undernutrition (ie, FR). The aim of the present study was 1) to study the effect of antenatal GC on arterial stiffening as a factor for the development of hypertension and 2) to determine if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to GC. METHODS: Pregnant sheep were treated with two IM doses of betamethasone (Beta, 0.17 mg/kg) or vehicle (CTR) 24-hs apart at 80 days gestational age and allowed to deliver at term. At 9 mo of age, male (M) and female (F) sheep were randomly allocated to be fed at either 100% of recommended nutritional allowance or ad libitum for three months. Sheep were chronically instrumented under general anesthesia to place intravascular catheters. BP is expressed as the average over 48 hours. ASI was calculated using the bisector regression method (Sym_Slope) on two individual data sets of 1400 points/day/sheep. Data are presented as Mean±SEM and were analyzed by ANOVA and two sample t test.

We found a significant Beta and Obesity effect of MAP and Sym_Slope. As shown on Figure 1 , we found no sex differences on the Beta effect in lean (A) or obese (B) sheep on MAP. In contrast, a sex difference in the magnitude of the Beta effect was observed on Sym_Slope in lean animals (A), but not on obese (B) sheep ( Figure 2 ). *Figure(s) will be available online. CONCLUSIONS: Our data show that superimposed obesity significantly worsens the effects of antenatal steroids in both female and male adult sheep. Interestingly, Syn_Slope an index of arterial stiffening is significantly more affected by Beta in male sheep, but the sex difference is obliterated by the effect of superimposed obesity. HL 68728 and HD 04784. INTRODUCTION: Our laboratory and others have shown that maternal protein restriction causes reduced placental and fetal weights and induces hypertension in adulthood offspring. However, the underlying mechanisms, especially those present in maternal and placental compartments remain unclear. In normal pregnancy, insulin resistance in the mother facilitates nutrient supply to the growing fetus and is counterbalanced by expansion of beta cells driven by placental lactogen.

Serotonin and associated signaling pathway play an important role in beta-cell expansion in response to pregnancy and placental lactogen. Our previous study showed that in mid-and late pregnancy plasma levels of tryptophan, precursor of serotonin were decreased in pregnant rats fed a low protein (LP) diet, coincident with reduced plasma levels of serotonin.

In this study we hypothesized that expansion of pancreas is impaired in pregnant rats fed LP diet due to disrupted serotonin signaling in pancreas.

In Study One, time-scheduled pregnant SD rats were fed a normal (control; CT) or LP from Day 3 of pregnancy until sacrificed at Days 10, 14, 18,19, 21 and 22 (n=6-10 rats/diet/day). Plasma was collected for ELISA on insulin and glucose measurement by Accu-Check glucose meter. In Study Two, pancreas and plasma were collected at 8-10 am in the morning at Day 21 (n=10 rats/diet). The abundance of 5-hydroxytryptamine receptor 2A and 2B (HTR2A, HTR2B), tryptophan hydroxylase 1 (TPH1), insulin and beta-actin (loading control) in pancreatic protein was analyzed by Western blotting. The effect of LP diet on these parameters was analyzed by one-or two-way ANONA. RESULTS: Results include: 1) Plasma glucose levels were unchanged in the LP group compared to the CT group at all days; 2) Plasma insulin levels were unchanged in the LP group at Day 10, but reduced (P < 0.05) by 1.6-, 4.5-, 2.0-, 2.3-, 6.9-fold, respectively, at Day 14, 18, 19, 21, and 22 compared to the CT group; 3) At Day 21, pancreas weight and the ratio of pancreas to body weight in LP rats were reduced (P < 0.05) by 1.26-and 1.32-fold, respectively, compared to the CT group; 4) The abundance of HTR2A and HTR2B in pancreas in the LP group was decreased by 2.7-(P < 0.001) and 1.5-(P < 0.05) fold, respectively; 5) The abundance of TPH1 was unchanged in LP rats.

These results indicate that expansion of pancreas in LP pregnant rats was impaired due to reduced serotonin receptors and plasma serotonin levels.

Maternal Our goal is to examine the impact of maternal diet on DNA methylation and expression in offspring liver to identify regulatory candidates linking adverse exposures and adult metabolic phenotype. METHODS: SD dams were fed: standard chow (Con), calorie restricted (pair-fed 60% kcal/d) from gestational day 11 through lactation (CR) or western diet from 3 wks old through gestation/lactation (WD). Litters culled to 8 were fed standard chow post weaning. MPS-based HELPtagging assay was used examine cytosine methylation levels at >1.6 million loci in liver of 9 wk old female offspring and 20 mo Con (OLD) (n=6/grp). Differentially methylated regions identified at 9 wks between Con vs CR + WD were mapped to genes and uploaded to GSEA (Broad Institute) to determine ontology/pathway enrichment. Relevant targets were evaluated for expression changes in 6mo Con, CR, WD and OLD using qPCR.

Comparisons of methylation profiles of 500 differentially methylated loci (p<0.01, for Con vs CR and Con vs WD at 9wks) show that CR and WD share a more similar profile with Old than Con *Figure(s) will be available online.

Limited impact on expression is observed at 9 wks, but the same genes show more significant changes in expression at 6 months compare to Con and are more homologous to those in Old CONCLUSIONS: Using an unbiased approach, we identified methylation changes at candidate loci in liver of young females that may be functionally important later in life. Maternal nutrition may lead to changes in young offspring that are predictive of an aging phenotype as suggested by the expression changes in exposed adult and unexposed old animals. Epidemiological data show that urinary levels of the endocrine disrupter chemical bisphenol A (BPA) are associated with hypertension, a major risk factor for cardiovascular disease. Components of the renin-angiotensin system (RAS), a major regulator of systemic blood pressure, are expressed in liver (angiotensinogen; AGT) and kidney (renin, angiotensin-converting enzyme; ACE), whereas the major vasopressor effects of angiotensin II are mediated through its receptor type 1 (AT1R) present in kidneys and vasculature. Using a rat model of maternal BPA we have demonstrated hypertension in male, but not female offspring as early as 6 weeks of age. We hypothesized that maternal BPA increases offspring arterial blood pressure via increased expression of RAS components. We determined protein expression of RAS components in liver, kidney and aorta from BPA and control offspring. METHODS: Female rats had access to drinking water (control group) or drinking water containing BPA (5mg/L; BPA group) from two weeks prior to mating and throughout pregnancy and lactation. Standardized litters were nursed by the same dam. At the end of the nursing period (3 weeks of age), liver, kidney and aorta were collected from male offspring. Protein expression of hepatic AGT, renal ACE and aortic AT1R was determined by Western Blot. Protein expression of renin and AT1R in the renal cortex was assessed by immunohistochemistry. Image-Pro software was used to quantify positive area/total area in 4 consecutive sections. RESULTS: At 6 weeks of age, systolic blood pressure was higher in BPA males (148±2 vs 131±3 mmHg; P<0.05) but not females (127±5 vs 128±3 mmHg) compared with controls. At 3 weeks of age, BPA males had lower expression of liver AGT (0.5-fold; P<0.05) and higher expression of renal renin (2-fold; P< 0.01), but not ACE. Both renal cortex and aorta demonstrated greater expression of AT1R (2-fold; 3.5-fold, respectively; P<0.05) in BPA males as compared to controls. CONCLUSIONS: Taken together, the data suggest altered expression of components of the classical renin-angiotensin system may contribute to the increased systolic blood pressure in male offspring exposed to BPA during pregnancy and lactation.

Perinatal The use of plastics (e.g., water bottles) containing the endocrine disrupter BPA has accelerated in parallel to obesity rates. BPA is detectable in plasma of pregnant women, and more importantly in fetal plasma and amniotic fluid, in which the levels are almost five-fold greater.

We have demonstrated that perinatal exposure to maternal BPA induces sex-specific effects on weanling (end of nursing) cardiometabolic function. We hypothesized that perinatal BPA programs long-term cardiovascular and metabolic dysfunction that persists in adult offspring, despite no further exposure to BPA. METHODS: Two weeks prior to mating, and throughout pregnancy and lactation, rat dams consumed filtered (control) or BPA-containing (5 mg/L) drinking water. After birth, litter size was standardized and pups nursed by the same dam until weaning, after which no further BPA exposure occurred. At 9 months of age, body composition (DEXA), fasting plasma glucose, glucose tolerance (D-glucose 1mg/kg body weight, i.p.) and arterial blood pressure were determined. Data were analyzed by ANOVA. RESULTS: Despite no differences in adult body weights (male: 614±38 vs 579±14 g; female: 323±10 vs 316±6 g) or % fat mass (male: 21±3 vs 19±2 %; female: 20±3 vs 17±1 %) between BPA and control offspring, BPA programmed sex-specific changes in offspring cardiovascular and metabolic function. Fasting plasma glucose levels were higher in BPA than control male (102±2 vs 87±2 mg/dL; P<0.001), but not female offspring (92±4 vs 100±3 mg/dL, respectively). However, there were no differences in the area under the glucose clearance curve between BPA and control offspring in either sex (male: 3308±849 vs 2536±319; female: 1359 ±281 vs 808±270 mg.dL -1 min -1 ). Both BPA males and females had increased systolic (146±1 vs 128±3 mmHg; P<0.001; 137±3 vs 125±2 mmHg; P<0.001, respectively) and diastolic (120±1 vs 102±3 mmHg; P<0.001; 110±3 vs 103±1 mmHg; P = 0.056, respectively) blood pressure. CONCLUSIONS: Despite similar adult body weights, maternal BPA programmed sex-specific changes in cardiovascular and metabolic function -all offspring were hypertensive, whereas fasting glucose levels were increased in males only. Together with human studies, these findings suggest that maternal BPA exposure has long-term deleterious effects on cardiovascular and metabolic development of the offspring and should be limited during pregnancy and lactation.

Gender We investigated the gender difference in the influence of birthweight on cardiovascular risks in Japan.

The relationship between birthweight and the following parameters was investigated in 1241 subjects aged 40-69 years (males 521, females 720): waist ≥90 cm (males) and ≥80 cm (females), blood pressure (SBP/DBP ³130/85 mm Hg and/or current use of antihypertensives), fasting blood glucose ³110 mg/dl and/or current use of insulin or oral diabetes medication, triglyceride ³150 mg/dl and/or current use of cholesterol-lowering medication, HDL cholesterol <40 mg/dl. Subjects were classified based on birthweight in the maternal notebook (<2500, 2500-3500, >3500 g) and on the examinee's memory ("light", "medium" and "heavy" body weight).

The maternal notebook correlated well with the examinee memory (r=0.73; p<0.025). "Light" body weight was a risk for hypertension (OR 2.0; 95%CI 1.02-4.09), hypertriglyceridemia (OR 3.0; 95%CI 1.06-9.77), low HDL cholesterol (OR 3.80; 95%CI 1.08-4.09) in females when controlling for age, BMI, smoking, alcohol, menopause. The percentage of "light", "medium" and "heavy" females fulfilling the glucose criteria was 14.1%, 11.6% and 4.8% (p<0.05), respectively. CONCLUSIONS: Females with a light birthweight had greater risk for metabolic syndrome than heavy ones for blood pressure, HDL-cholesterol, and glucose level. These phenomena were not observed in males. Although maternal diabetes is a risk factor for congenital heart defects, most fetuses exposed to hyperglycemia have structurally normal hearts. Our goal is to assess parameters of cardiac function in adult offspring of diabetic dams. METHODS: Hyperglycemia was induced in WT 8 wk old CD1 female mice with a single injection of 150 mg/kg IP of streptozotocin (STZ) prior to mating. Offspring consisted of exposed males (DM-m, n=9) and females (DM-f, n=9) and unexposed males (Con-m, n=9) and females(Con-f, n=8). Left ventricular echocardiograms were performed and included: interventricular septum diameter (IVS), left ventricular end diameter (LVED), area, volume, stroke volume, ejection fraction (EF), cardiac output (CO) and heart rate. Student t-test was used for statistical analysis. P<0.05 was considered significant.

Only DM-f exhibited differences in heart function. As shown in the table below, the findings of LVEDD, LVESD, area, volume and compensatory increase in EF of DM-f vs. Con-fare consistent with restrictive cardiomyopathy. 

Impaired cardiac function is evident in the adult female progeny of diabetic dams. More exploration needs to be done to elucidate the mechanism for these observed sex-specific differences in cardiac function.

High We reported that mice born to hypertensive mother, lacking endothelial nitric oxide synthase (NOS3), are hypertensive. We hypothesized that offspring born to hypertensive mother and placed on high fat diet (HFD) will undergo altered metabolic programming that is time dependent. METHODS: NOS3 knockout (KO) and wild type (WT) mice were crossbred to produce heterozygous offspring: maternal-derived (KOM) born to KO mother (hypertensive) and paternal-derived (KOP) born to WT mother (normotensive). KOM and KOP offspring were placed on HFD or control diet (CD) for 4 or 8 wks duration after weaning. KOM-HFD, KOM-CD, KOP-HFD and KOP-CD offspring were obtained. Offspring weight, OGTT and BP were obtained at 12-13 wks of age. 1-way-ANOVA/t-tests were used for statistical analysis. CONCLUSIONS: Offspring on HFD developing in an adverse uterine environment due to maternal HTN are more prone to develop HTN and glucose intolerance compared to their genetically identical counterpart. Maternal HTN is a risk factor for abnormal fetal metabolic programming. High fat intake and timing of its introduction in offspring nutrition play a role in worsening adult hypertension. This mouse model maybe suited to evaluate metabolic syndrome and timing of intervention to prevent adult cardiovascular disease. INTRODUCTION: The present study tested the hypothesis that estrogen (E 2 ) programs mechanisms in the nonhuman primate fetus that promote insulin sensitivity in offspring after birth. METHODS: An iv glucose tolerance test was performed at 6-12 month intervals (data averaged) between postnatal ages 1 and 3½ years in prepubertal offspring delivered from baboons untreated (n=6) or treated on days 100-165 of gestation (term is 184 days) with the aromatase inhibitor letrozole (0.115 mg/kg BW/day, sc, n=5) which decreased fetal E 2 levels by 95% or letrozole plus E 2 (each 0.115 mg/kg BW/day, n=4). Basal (i.e. non-insulin challenged) levels of insulin receptor (IR) signaling components were quantified by Western immunoblot in skeletal muscle obtained on day 165 of gestation from fetuses of baboons untreated (n=5) or treated with letrozole on days 100-164 (n=5). RESULTS: Peak 1 min blood glucose (210±3 mg/dl) and insulin (113.8±18.7 µIu/ml) levels after iv bolus of dextrose were greater (P<0.05) in offspring deprived of E 2 in utero than untreated animals and restored by letrozole + E 2 . Homeostasis model assessment of insulin resistance was 2.5-fold greater (P<0.02) in offspring from letrozole-treated (2.20±0.70) than untreated (0.65±0.09) animals and partially returned by letrozole + E 2 (1.14 ±0.27). Skeletal muscle basal levels (vs GAPDH) of phospho-Akt (0.30±0.06), -IRS-1 (0.39±0.15) and -GLUT-4 (3.26±1.33) in fetuses from letrozole-treated baboons were similar to values in untreated animals. Whether responsivity of the IR pathway to insulin challenge is altered in E 2 -deprived offspring remains to be determined.

The exaggerated rise in glucose and insulin levels after glucose challenge in baboon offspring deprived of E 2 in utero is indicative of insulin resistance. We propose that E 2 programs the mechanisms in the primate fetus that promote insulin sensitivity after birth ( Fig. 1) . Supported by NIH R01DK93950. *Figure(s) will be available online. We have shown that prenatal testosterone exposure leads to sex-specific cardiovascular dysfunction in adult offspring with more pronounced hypertensive effect in males than females. However, the impact of elevated maternal androgens on adult life metabolic disorders is not known. We examined if there are sex differences in the development of glucose intolerance in the adult rat offspring of dams exposed to elevated testosterone levels.

Dawley rats were injected with vehicle or testosterone propionate (TP) (0.5 mg/kg/day from gestation day 15-19) to increase plasma testosterone levels twofold, similar to that observed in preeclampsia. In the 6 months old offspring, oral glucose tolerance test was performed to measure plasma glucose and insulin responses. Rat pancreatic islets were isolated and in vitro cultured for 24 hour to assess insulin secretion.Plasma glucose and insulin responses were calculated using trapezoidal rule. Plasma glucose and insulin responses were expressed as glycemia area under the curve (AUC) (mg/dL*240 minx10 -3 ) and insulin AUC (ng/ml*240 min), respectively. RESULTS: Fasting blood glucose levels were similar; however the fasting insulin levels were significantly increased in both TP males ( Insulin secretion from islets in TP females was not affected. CONCLUSIONS: Prenatal testosterone exposure induced glucose intolerance in adult males but not in females. Glucose-induced insulin response was unaffected in males but exaggerated in females. Isolated islets revealed a clear constraint in insulin secretion in prenatal testosterone exposed males. Obesity is strongly linked to low testosterone (T) levels in men. However, how T replacement affects insulin secretion and action in obese men remains unclear. We examined the high fat fed obese rats to determine if castration-induced T deficiency and T replacement affect metabolic functions including glucose and insulin tolerance, pancreatic insulin secretion and adiposity. METHODS: Three month old male SD rats were divided into 3 groups: control, castrated (for T deficiency) and castrated with T replacement (subcutaneous 100 mg T pellets, 90 day release). All rats were fed with 45 kcal % high fat diet. Ten weeks later, all rats were assessed for glucose and insulin tolerance, feed intake, body weight and fat distribution. Pancreatic islets were isolated and cultured for 24 hour to assess insulin secretion. Plasma glucose and insulin responses were expressed as glycemia area under the curve (AUC) (mg/dl*240 min x10 -3 ) and insulin AUC (ng/ ml*240 min), respectively. RESULTS: Fasting insulin and glucose levels were significantly lower in castrated rats (insulin: 0.42±0.05 ng/ml; glucose: 114±3.69 mg/ dl) and T replacement increased insulin (0.82±0.28 ng/ml) to controls levels (0.75±0.23 ng/ml) but did not reverse glucose levels (112±4.5 vs 129±6.05 mg/dl in controls). Glucose tolerance was similar in castrated rats (glycemia AUC: 0.49±0.05, Insulin AUC: 5.6±0.8) and controls (glycemia AUC: 0.44±0.05, Insulin AUC: 7.9±1.3) but T replacement induced significant glucose intolerance with elevated glucose (glycemia AUC: 0.68±0.07) and impaired insulin responses (Insulin AUC: 5.83±0.4). Whole body insulin sensitivity was not different between groups, however insulin secretion from isolated islets were significantly impaired in T replaced rats (0.71±0.1 ng/ml) compared to castrated (2.30±0.5 ng/ ml) and control rats (2.81±0.1 ng/ml). T replaced animals weighed significantly smaller (595±15.1 g) than controls (815±33.8 g) and castrated rats (647.4±23.9 g). T replacement significantly decreased the amount of peritoneal fat (14.27±0.83 g) and subcutaneous fat (12.69±1.24 g) compared to controls (peritoneal: 40.94±2.34 g; subcutaneous, 26.13±2.36 g) and castrated rats (peritoneal: 32.06±2.67 g; subcutaneous: 31.42±3.02 g). Feed intake was not different between groups. CONCLUSIONS: Replacement of T in high fat diet fed obese rats decreases adiposity but induces glucose intolerance and inhibits pancreatic insulin secretion.

The Two reviewers independently screened citations and undertook study quality assessment and data extraction. We obtained summary estimates of adverse fetal events that were classified as potentially fatal, clinically significant morbidity or minor adverse event. We identified 602 titles and reviewed 30 articles for inclusion and exclusion criteria. Meta-analysis was performed on seven studies (3 cohort, 3 case-series and 1 case-control).

Of the 922 cases of statin exposure in pregnancy, 27 exposures were associated with lethal or clinically significant fetal morbidity and 10 with minor adverse events. Statin exposure was limited to the first trimester in all but two cases. The pooled rate of lethal or clinically significant fetal abnormalities in pregnant women exposed to statins was 0.01 (95% CI 0.00-0.04), less than the European rate of 0.026 (95% CI 2.54-2.57) EUROCAT. The rate of fetal abnormality for simvastatin was 0.03 (95% CI 0.00-0.08), atorvostatin 0.11 (95% CI 0.00-0.52), pravastatin 0.01 (95% CI 0.00-0.2) and lovastatin use 0.04 (95% CI 0.00-0.28). Systems based anomalies were also calculated, congenital heart disease was 0.8 (95% CI 0.02-0.12) compared with the background rate of 0.79 (95% CI 0.78-0.80).

The published data suggests that statins may not be teratogenic when given inadvertently during pregnancy and prospective studies such as The StAmP Trial may provide more data. INTRODUCTION: MNR in guinea pigs results in placental structural abnormalities and dysfunction, and we have recently shown that fetal growth restricted (FGR) animals in these pregnancies have increased erythropoietin (EPO) and hemoglobin as indirect markers of chronic hypoxia. We now hypothesize that MNR in guinea pigs as a causative factor for FGR will increase placental/fetal hypoxyprobe-1 (HP-1), a pimonidazole hydrochloride that is reduced in hypoxic cells and widely used as a direct marker for tissue hypoxia, further implicating chronic hypoxia as an additional mechanism for growth restriction in these animals.

Guinea pig sows were fed ad libitum (Control) or 70% of the control diet pre-pregnant switching to 90% at mid-pregnancy (MNR). Near term, HP-1 was injected into pregnant sows and 1.5 hr later animals were necropsied for fetal-placental growth measures, blood metabolites, and HP-1 levels in the fetal kidney, liver and placenta using immunohistochemical techniques. Results are presented for grouped means with significance assumed for p< 0.05.

Eight control (28 fetuses) and 12 MNR (42 fetuses) animals were necropsied with MNR fetal and placental weights decreased 28 and 23%, respectively, placental to body weight ratios increased 10%, and ponderal index increased 11%. Select control (n=20) and MNR (n=25) fetuses underwent full necropsy, with brain to liver weight ratios increased 48%, blood glucose values decreased 28%, and Hb values increased 9% in MNR fetuses. In these fetuses, HP-1 levels were increased 158% and 117% in the kidney and liver, respectively, while there was no significant change in the placenta. CONCLUSIONS: MNR in guinea pigs results in FGR with placentas that are large relative to body weight and likely a reactive process to nutrient deficiency, and livers that are small relative to body weight and likely reflecting a degree of blood flow redistribution; changes which are also characteristic of the asymmetrical FGR observed in association with "placental insufficiency". These fetuses also have HP-1 levels which are substantially higher in the kidney and liver and along with the higher EPO and Hb values we have previously reported, further implicate chronic hypoxia in these animals as an additional mediator for growth restriction and altered development. INTRODUCTION: MNR prior to and during gestation in guinea pigs results in fetuses which display evidence of chronic hypoxia in association with fetal growth restriction (FGR). An impact of decreased oxygen availability during fetal development is increased oxidative stress which could act as a further mediator of altered growth and development. We postulated in association with FGR, MNR fetuses would display impaired anti-oxidant capacity and markers of oxidative stress near term. METHODS: Guinea pig sows were fed ad libitum (Control) or 70% of the control diet pre-pregnant switching to 90% at mid-pregnancy (MNR). Animals were necropsied near term for fetal-placental growth measures and blood metabolites. Oxidized and reduced glutathione (GSSG/GSH) and peroxiredoxin (PRDX) protein levels as measures of anti-oxidant capacity, and malondialdehyde (MDA) as a measure of lipid peroxidation and thereby oxidative stress were assessed in the fetal liver and placenta.

Results are presented for grouped means with significance assumed for p< 0.05. RESULTS: Eight control (28 fetuses) and 12 MNR (42 fetuses) animals were necropsied with MNR fetal and placental weights decreased 28 and 23%, respectively. Select control (n=20) and MNR (n=25) fetuses underwent full necropsy with MNR-FGR animals showing GSSG/GSH ratios that were decreased by 56 and 107% in the liver and placenta, respectively, indicating increased conversion of glutathione to its reduced form as seen with scavenging reactive oxygen species (ROS). PRDX protein levels increased 143% in the fetal liver indicating a reactive compensatory increase in anti-oxidant capacity, although there were no changes in the placenta. Surprisingly, MNR-FGR MDA levels decreased 42 and 32% in the liver and placenta, respectively. CONCLUSIONS: MNR prior to and continuing through pregnancy in guinea pigs results in FGR animals with altered anti-oxidant capacity likely in response to an increase in ROS. This provides evidence that MNR, resulting in chronic fetal hypoxia, triggers an oxidative stress response during gestation. While we found no evidence of ROS lipid peroxidation, this may reflect the impact of nutritional impairment on lipid levels and turnover. Thus, it is probable that other cellular processes are interfered with thereby contributing to longer term adverse development. Little is known about the impact of growth discordance on TTTS outcome. The aim of this study was to determine the association between growth discordance and adverse perinatal outcomes in TTTS pregnancies.

We conducted a retrospective cohort study of all TTTS patients evaluated and treated by laser from 2005-2013. Exclusion criteria include triplets, significant congenital malformation and chromosomal abnormalities. Growth discordance was defined as estimated fetal weights >20% different when compared to the larger twin. Outcomes considered were stillbirth, preterm delivery <34 weeks, <32 weeks and <28 weeks. Data were stratified into concordant group with <20% discordance and discordant group with >20% discordance. Data was analyzed using Chisquare or T-test as appropriate. RESULTS: 439 patients met our inclusion criteria. There was no significant difference in base line demographics among concordant and discordant TTTS (Table) . Donor twins in the discordant group when compared to the concordant group had elevated head circumference/ abdominal circumference (HC/AC), lower birth weight, higher rate of selective growth restriction (49% vs. 14%, p<0.001) and lower survival rate (73% vs. 83%, p=0.036). Both concordant and discordant TTTS pregnancies had increased risk of preterm delivery but there is no different between the two groups (Table) . Results were the same between the two groups when selective IUGR was excluded. *Figure(s) will be available online. CONCLUSIONS: In TTTS pregnancies treated with laser, growth discordance is a risk factor for donor adverse perinatal outcome. This is important in patient counseling. This pilot study assesses the feasibility of in utero MRgHIFU delivery in a late-gestation rabbit model and determines the safety, accuracy and tissue response to the treatment. METHODS: High resolution 3T MR images on a Phillips Sonaleve system of an ~30 days gestation rabbit litter (term=32 days) were acquired to identify placental (n=3) and fetal lung, brain and heart targets. *Figure(s) will be available online.

Clusters of HIFU energy were applied to these targets guided by MR and thermal imaging. All HIFU exposures were averaged as power (watts)±sd of the treatment. Tissues were processed for histological analysis.

The placentas were exposed to 35±15W for 20.5±0.03s reaching 64°C, 62.5±13.9W for 20.5±0.01s reaching 70°C, and 68.3±18.6W for 22.6±7.1s reaching 75°C, respectively. The histological changes in the placentas were acute with a circumscribed area of hemorrhage. The brain was exposed to 40±10W for 20.5±0.02s reaching 74°C in the thalamus resulting in no visible histological changes. The lungs were exposed to 70±0W for 20.5±0.04s reaching 75°C to cause hemorrhagic foci with areas showing damage to large vessels, bronchi and alveoli.

The heart was exposed to 70±0W for 20.5±0.06s reaching 74°C resulting in vascular engorgement, focal disruption and hemorrhage into the myocardium with wavy fibers and evidence of early necrosis.

The late gestation rabbit model suitably delineates targets and evaluates MRgHIFU application in utero as treatment of fetal lesions and abnormal placental circulation. Future studies will focus on further characterizing and quantifying HIFU induced tissue changes. Previous studies demonstrated that female offspring were resistant to fetal stress-induced programming of ischemic-sensitive phenotype in the heart. Yet, the mechanisms remain unknown. The present study tested the hypothesis that estrogen plays a role in protecting females in fetal programming of heart vulnerability. METHODS: Pregnant rats were divided into normoxic and hypoxic (10.5% O 2 from day 15 to 21 of gestation) groups. Ovariectomy (OVX) and estrogen (E2) replacement were performed in 8-week-old female offspring. Hearts of 4-month-old females were subjected to ischemia and reperfusion injury in a Langendorff preparation. RESULTS: OVX significantly decreased post-ischemic recovery of left ventricular function and increased myocardial infarction. No difference was observed between normoxic and hypoxic groups. The effect of OVX was rescued by E2 replacement. OVX decreased the binding of glucocorticoid receptor (GR) to glucocorticoid response elements (GREs) at angiotensin II type 1 (AT1R) and type 2 (AT2R) receptor promoters, resulting in a decrease in AT1R and an increase in AT2R in the heart. Additionally, OVX decreased estrogen receptor (ER) expression in the heart and inhibited ER/GR interaction in binding to GREs at ATR promoters. Consistent with the changes in angiotensin II receptors, OVX significantly decreased PKCε and phospho-PKCε abundance in the heart. These OVX-induced changes were abrogated by the E2 replacement.

The results indicate the lack of effect of postnatal ovarian hormones on the sex dimorphism in fetal programming of heart ischemic vulnerability, but suggest a novel mechanism of estrogen in regulating cardiac AT1R/AT2R expression patterns and protecting female hearts against ischemia and reperfusion injury. (Supported by NIH grants HL118861, DA032510).

Invasive We sought to determine how often invasive prenatal diagnosis is being offered. METHODS: Pregnant women enrolled in an RCT of an "informed free choice" approach to prenatal testing were asked at 24-30 weeks whether they had discussed invasive prenatal testing with their providers, whether it was clear that such testing was optional, and whether the provider had initiated the discussion. We used univariate and multivariable logistic regression to determine predictors of these outcomes. We tested interactions between predictors of interest and treatment group, recognizing that the likelihood of discussing testing with providers might vary by randomization group.

Of 710 women in the study, 415 (58%) reported discussing invasive testing with their providers. 289/558 (52%) of women under 35 and 125/152 (83%) of women older than 35 reported discussions. Of those who reported discussing testing, 363/415 (87%) reported that their provider initiated the discussion. 244 of the 710 women 34% stated that they did not know, or were unsure, whether CVS, amniocentesis or both were optional. In multivariable models, women 35 or older were more likely to report having discussed invasive testing (aOR 3.7, 95% CI 2.2-6.0); only among women in the usual care group, those who were publicly insured were less likely to report having discussed testing (aOR 0. INTRODUCTION: ACOG recommends that obstetricians' prenatal genetic screening discussions include mention of the false positive rates, advantages, disadvantages and limitations of screening, and the availability of diagnostic testing. We examined discussions of prenatal genetic screening to assess how obstetricians currently counsel and how this counseling impacts patients' screening acceptance. METHODS: Transcripts from audio recordings of 200 first prenatal visits were coded using an iterative process to determine compliance with ACOG recommendations and to categorize discussion elements. Chi-squared, t-tests and Wilcoxon rank sum tests were used to determine the impact of demographic factors and discussion codes on women's decisions to pursue genetic screening or diagnostic testing. RESULTS: Providers offered genetic screening at 91.5% of visits. 71% of patients underwent genetic screening. Patients offered screening were significantly more likely to undergo screening than patients who were not offered screening (p<0.001). Few providers (1.5%) mentioned all of the ACOG recommended topics during the screening discussion. Inclusion of more of the ACOG recommended topics increased patient acceptance of screening (p=0.04). While discussion of the advantages of genetic screening increased screening acceptance (p=0.003), discussion of the disadvantages and limitations did not. Conversations varied from brief mention of available tests to explanations of the a woman's baseline risk of Down Syndrome, the types of genetic abnormalities assessed by screening, the screening process, the type of result obtained and options for diagnostic testing. Only 38.8% of the conversations included discussions of what a positive result from genetic screening meant and how a patient might use this information. CONCLUSIONS: Although the majority of patients were offered and underwent screening, most providers did not sufficiently explain the implications of screening to the patients. While this did not appear to impact patient acceptance of screening, it is uncertain that patients have a clear understanding of what additional stress and burden they may face if they receive positive or indeterminate testing results.

Urban and placenta are particularly vulnerable to toxins due to immaturity of the blood-brain-barrier barrier and diminished biotransformation enzyme activity. Our hypothesis is that newborns from rural areas are prenatally exposed to different levels of essential and toxic elements than their urban counterparts. This study has considerable significance as there have been no studies conducted comparing urban and rural exposure to these metals in umbilical cord blood.

A comparative cross-sectional study was conducted on 172 patients, 79 rural and 93 urban. Rural and urban locations were based on Rural-Urban Commuting Area Codes, determined by the U.S. census tracts. Umbilical cord blood was collected at the time of delivery, between April 2013-February 2014, and analyzed for 20 inorganic elements.

Patients were asked during their hospital stay the source of their drinking water. Comparisons and p-values were by one-way ANOVA. RESULTS: Significant differences were found between urban and rural samples for four elements: Arsenic (p=.03) and magnesium (p=.04) were higher in rural samples, copper (p=.03) and molybdenum (p=.004) were higher in urban samples. No difference between groups occurred for: barium, cadmium, calcium, cobalt, lead, lithium, manganese, mercury, selenium, strontium, or zinc. Barium was higher than the recommended level in both rural and urban samples. All samples were devoid of platinum, silver, thallium or uranium. Nickel was detected in 3 of 172 samples, all reported city water use during pregnancy. There were no significant differences of elements found when comparing those who drank city water (n=155) versus well water (n=12). CONCLUSIONS: Health disparities exist between individuals living in predominantly urban compared to rural environments. Rural areas of Appalachia have a higher incidence of birth defects than the general population, with a 42% increase reported in mountaintop coal mining areas. Our study showed statistically significant differences in urban and rural prenatal exposure to copper, magnesium, and molybdenum, as well as the toxic metal arsenic. Further study is needed to determine if there is a causal link between specific birth defects and prenatal exposure to these elements.

The , also known as trisomy 21, with reduced self-renewal of haematopoietic stem cells and accelerated senescence, offering a possible explanation for accelerated aging in DS. Usp16, a deubiquitizing enzyme that plays a role in cell cycle progression and gene expression, was independently observed as significantly increased in our gene expression profiling of DS placentas. We sought to improve understanding, at the molecular level, of the role of Usp16 upregulation in DS placentas.

We collected second trimester samples (14-22 weeks) of the decidual-placental interface from DS cases (n=8) and age-matched, euploid, control pregnancies (n=4). FISH was used to confirm ploidy, and a microarray approach enabled global transcriptional profiling in trisomies vs. controls. Confirmation of Usp 16 upregulation in DS was done at the protein level. RESULTS: Microarray analyses revealed upregulation of Usp16 mRNA expression in trisomy 21 (T21) placentas as compared to euploid controls (> 2 fold). Immunolocalization of Usp 16 showed that T21 was associated with highly upregulated expression of this molecule in the stromal cores of chorionic villi and in trophoblast cell columns that anchor the fetalplacental unit to the uterine wall. These findings were confirmed by immunoblotting, which also showed upregulated expression. See *Figure(s) will be available online. CONCLUSIONS: Inadequate formation and function of the placenta are associated with adverse pregnancy outcomes. Our findings showed altered expression of Usp 16 in Down syndrome placentas at the level of the villous stroma and cell columns. The consequences could include dysregulated cell growth and inhibition of cytotrophoblast migration. Therefore, Usp16 might play an important role in placentation. The risk of chromosomal abnormalities or structural defects in live births are 3%-5%. Current testing modalities for noninvasive prenatal testing (NIPT) are limited to 10 weeks gestational age (GA) and do not provide an intact fetal genome. Up to 2000 extravillous trophoblast (EVT) cells can be isolated non-invasively by TRIC as early as 5 weeks GA. It has been suggested that EVT cells exhibit a high degree of mosaicism. EVT cells obtained by TRIC were evaluated for mosaicism to determine whether TRIC can be used for NIPT. METHODS: Endocervical specimens were obtained from pregnant patients between 5 and 20 weeks GA for isolation of EVT cells by TRIC. Briefly, HLA-G positive EVT cells were separated from maternal cervical cells using a column-free, immunomagnetic nanoparticle separation procedure. Approximately 50 isolated EVT or maternal cells were spun onto slides and evaluated by fluorescent in situ hybridization (FISH) with probes for chromosomes 13, 18, 21, X, and Y (Aneuvysion, Abbott). Medical records were reviewed to select pregnancies with male fetuses lacking evidence of aneuploidy. Purity of the trophoblast specimens were determined by βHCG staining and percentage of cells staining for Y chromosome. Trophoblast retrieval and isolation from the cervix (TRIC) could provide an alternative approach for early non-invasive prenatal testing (NIPT) with cell free fetal DNA. Trophoblast cells have been retrieved from the cervix of pregnant patients as early as 5 weeks gestation and have been used to identify fetal gender. NIPT is negatively impacted in patients with a high body mass index (BMI) and early gestational age (GA). Prior to employing TRIC for prenatal testing, it is important to assess whether it is impacted by BMI, maternal age, parity, or GA. METHODS: Cervical specimens were obtained using a ThinPrep kit and cytobrush on 63 pregnant patients from 5 weeks 2 days to 20 weeks 3 days GA. Trophoblast cells were isolated with anti-human leukocyte antigen (HLA)-G antibody coupled to magnetic nanoparticles. Purity of the isolated trophoblast cells was assessed by staining for β human chorionic gonadotropin. Trophoblast yield was recorded for each patient. Using Pearson's correlation, relationship between BMI, parity, maternal age, and gestational age to trophoblast yield was evaluated. RESULTS: Average GA of the specimens was 12.8 weeks ± 4.27, average trophoblast yield was 647 ± 485, average parity was 1.22 ± 1.37, average age was 27.3 ± 5.6, and average trophoblast purity was 94.6% ± 5.57%. Statistical analysis revealed non-significant Pearson's correlation coefficients for comparisons between trophoblast yield and BMI (r= 0.12, P=0.35), maternal age (r= 0.13, P=.31), parity(r= 0.06, 0.64), or GA (r= -0.09, P=.48). Subanalysis comparing trophoblast yield between nulliparous and multiparous patients revealed no significant differences by an independent t test (p= 0.14). CONCLUSIONS: Trophoblast cells can be isolated from the endocervical canal of ongoing pregnancies with a high degree of purity. Unlike NIPT, acquisition of fetal DNA by TRIC is not negatively affected by early GA or elevated BMI. Additionally, parity and maternal age do not effect recovery. TRIC could provide a highly reliable platform for early, NIPT that is unaffected by BMI and GA between 5 and 20 weeks GA. . Objective criteria of VD were met in 42/60 (70%). Correlation between ventricular ratios and neonatal findings was tested overall and for each cardiac anomaly group. In none of these combinations did the objective criteria improve prediction of neonatal problems or of normal NE. CONCLUSIONS: Subjective VD, regardless of the objective measurement, is associated with cardiac defects as well as other adverse outcomes. A policy of follow-up FE appears justified based on the frequency and required perinatal management of associated cardiac and other abnormalities.

Nomograms To determine if patients with a negative first trimester screen (1TS) but with an aneuploidy risk greater than the age related risk (ARR) are at risk for poor pregnancy outcomes. Also, to determine if physician and patient perceptions of the "gray zone" result warrant further testing.

This was a retrospective study using a laboratory database from 10/2012-12/2013 of negative 1TS results (risk of aneuploidy <1:50). A result was defined as falling in the "gray zone" if it was <1:50 but greater than the ARR. Patients with results falling in the gray zone (Group 1) were matched 2:1 by maternal age and date of sampling to patients not in the gray zone (Group 2). Invasive testing included CVS and amniocentesis. An abnormal ultrasound finding was defined as a fetal malformation, increased nuchal translucency (NT>2.5mm), and small for gestational age (SGA, <10 th % estimated fetal weight). Outcomes were compared using Student's t-test and chi-square. CONCLUSIONS: This data suggests that physician and patient concern may be warranted when 1TS results fall in the gray zone. Further testing may be indicated in these cases, including cffDNA and an early (11-14 week) anatomical scan of the fetus. 

(HIE) is the occurrence of reactive astrogliosis and inflammation. During this process the activity of GFAP, STAT3 and NFκB are increased. Light emitting transgenic mice, where luciferase expression is controlled by a surrogate promoter are used to provide an in vivo readout of disease processes. STAT3 and NFkB activated or GFAP promoter driven luciferase reporter constructs were delivered to brains of neonatal mice as a form of assessing the amount of astrogliosis and inflammation in HIE.

A rodent GFAP promoter, and STAT3 and NFκB response elements were each cloned into a lentivirus vector upstream of the genes encoding a codon-optimised luciferase. Lentivirus were pseudotyped with either gp64 or VSV-g. Lentiviruses were injected intra-cranially into neonatal (P0) mice, and luciferase expression was monitored by bioluminescence imaging. Lentivirus carrying the NFκB response element were also injected into P0 mice, which underwent HIE surgery. RESULTS: Pseudotypes; gp64 and VSV-g show different cellular tropisms; with VSV-g targeting neuronal cells and gp64 targeting astrocytes. There was a significant correlation between the luciferase expression 24hr after surgery and weight (P = 0.0007) in mice which received the gp64 NFκB biosensor; the most severely affected HIE mice had the lowest luciferase expression and weight. However, the HIE mice which had received the VSV-g pseudotyped NFκB biosensor failed to show this correlation (P=0.479). *Figure(s) will be available online.

The expression of luciferase under the control of NFκB in astrocytes is predictive of brain injury. 

A retrospective study of microcystic CPAM cases from two tertiary care academic medical centers over a 10-year period. Subject demographics, antenatal imaging studies, obstetric and neonatal outcomes were abstracted from the medical record. CPAM volumes were normalized to their initial size at diagnosis and the growth trajectory modeled using a non-linear fit to estimate population characteristics. Descriptive statistics and non-parametric analysis (Mann-Whitney test) was performed. RESULTS: Of 21 cases of microcystic CPAM identified during the study period, 20 cases had a CPAM volume-to-head ratio (CVR) <1.6. Mean gestational age at diagnosis was 22.3 weeks (range 17.9-30.2 weeks). Growth trajectory of this CPAM cohort was best fit by the second order equation Y=91.05 + 5.94X-0.05X 2 . Maximal CPAM size was reached about 60 days after diagnosis, followed by regression, often close to the initial volume. All cases were born at term, and exposure to antenatal corticosteroids did not significantly impact CPAM growth trajectory (p=0.9) or CVR at delivery (p=0.2). Five fetuses had associated extrapulmonary anomalies (primarily cardiac defects) that were the only predictive factors for NICU admission. CONCLUSIONS: Microcystic CPAMs with CVR<1.6 exhibit a generalizable growth trajectory with a peak in volume followed by regression. Perinatal outcomes are most influenced by the presence of associated congenital anomalies. Decreased frequency of sonographic surveillance may be acceptable for microcystic CPAMs and should exclude associated congenital anomalies. CMA was more often discussed with women who primarily spoke English (p<0.001), self-identified as white (p=0.004), and did not receive prenatal care in a health center (p<0.001). There were no differences in CMA uptake by race/ethnicity, language, insurance or provider type. Multiparous women were less likely to accept CMA than nulliparas (OR 0.41, 95% CI 0.1-0.9). CONCLUSIONS: Women who undergo invasive fetal testing are more likely to undergo CMA if the indication for invasive testing is a fetal structural anomaly. There may be important demographic differences in women who are offered CMA compared to those who are not offered this test. These disparities bear further exploration. We now offer surgical intervention fetal ONTD diagnosed in the mid-trimester, as is has been shown toimprove outcomes. Traditionally, 2nd trimester maternal serum alpha feto-protein (MSAFP) and sonographic surveyhas been used to screen for detection of ONTD. As we continue to make strides in early aneuploidy screening,MSAFP levels may not be obtained routinely. The aim of this study was to employ a large contemporary pediatricneurosurgical referral center database of confirmed cases to determine the antenatal rate and means of ONTDdetection. METHODS: Using a retrospective study design, we generated a database of all repaired ONTDs (n=107) from July2007-February 2013. Detailed antenatal data was abstracted from the maternal record and in direct patient interview,and rates of detection for each modality of screening were determined. ORs were calculated to assess the relativediagnostic reliability of each screening modality. RESULTS: Of the 107 total cases 10.3% (11/107) were not detected antenatally and alternately found at the time ofdelivery. Consistent with prior findings, the OR for ultrasound-based detection of ONTD was 5.9 (95% CI 2.2-15.5).However, this was largely due to ubiquitous use of sonogram as 90.9% (10 of 11) of antenatal missed diagnoses hadprenatal care, and 80% (8 of 10) had documented "optimal" ultrasounds. In contrast, only a single missed case ofONTD had a false-negative MSAFP. Missed detection by ultrasound was likely not secondary to limited visualization,as the mean BMI was not significantly different (27.84 kg/ m2 vs 27.76 kg/m2). CONCLUSIONS: Following the MOMS trial, any ONTD that goes undiagnosed prenatally is a missed opportunity forintervention. We have observed that 10.3% of ONTD in our regional referral center were not detected antenatally andhad a lower utilization of MSAFP screening. We speculate that optimal detection requires the historically advocatedtwofold approach of mid-trimester MSAFP with sonographic screening. Neonatal abstinence syndrome (NAS) is characterized by withdrawal symptoms in a neonate who was exposed to drugs in utero. Buprenorphine dose does not correlate with NAS severity. The purpose of this study was to determine if metabolic differences play a role in NAS following buprenorphine treatment during pregnancy using two CYP450 SNPs. METHODS: Cord blood from MARC patients was collected at birth. Buprenorphine and norburprenorphine levels were quantified using gas chromatography-mass spectrometry. Cord blood DNA was extracted and quantified. SNP analysis was performed using PCR. An allelic discrimination was performed to determine the genotype of the SNPs rs28451617 and rs2740574. These were analyzed for Hardy-Weinberg equilibrium (HWE) using Chi-squared with two degrees of freedom. Expected genotypic frequencies were compared with those observed using Chi-squared with one degree of freedom and a desired P-value of 0.05. NAS was evaluated using Finnegan scoring. RESULTS: Genotyping of SNP rs28451716 (CYP3A7*1E) were all homozygous for the major allele. Six samples were heterozygous for the minor allele CYP3A4*1B (6.8%). This SNP was in HWE within our study population with a Chi-squared value of 0.23 and a p-value of 0.888. There was a significant difference between the observed and expected genotypic frequencies with a p-value of 0.03. ANOVA showed no correlation between NAS score or length of NICU stay. CONCLUSIONS: The CYP3A4 SNP had an increased allele frequency in the study population (6.8% versus 2.5%) with a significant difference between the observed and expected genotypic frequencies, indicating a possible enrichment. This population may have increased levels of buprenorphine compared to the general population due to decreased CYP3A4 metabolism of buprenorphine to norbuprenorphine. Increased buprenorphine levels may predispose those with the CYP3A4 SNP to drug abuse or even addiction due to increased nociceptive binding compared to those with normal buprenorphine metabolism. A correlation between NAS score or length of NICU stay was not observed.

Antenatal The cumulative test discordant rate for non-trisomies with FORTE was 0.08% compared to 1.07% with the Z-statistic. CONCLUSIONS: Analysis using FORTE to incorporate fetal fraction in cfDNA testing yields a >10 fold reduction in discordant results compared to the Z-statistic. This will become increasingly relevant as cfDNA testing gets more broadly used in the general pregnancy population. were Aneuploidy, 2 were normal, 2 were unknown and 2 were pending for the confirmation. Among 941 negative screening results, 1 newborn was identified as Triploidy and another was presented as microcephaly without karyotyping. Inconclusive results were found in 39 (4.04%) of the patients and the conclusive rates after repeated NIPT were achieved 98.9%. 14 patients were known to be exposed to prescribed medications or substances during pregnancy and none of them had any interference with the NIPT results. CONCLUSIONS: The use of NIPT is rapidly expanded into clinical practice. The NIPT Registry will continue to provide an important and independent evaluation of the accuracy of NIPT results in clinical setting. of women and is associated with poor outcomes in pregnancy, birth and offspring neurodevelopment. Inflammation is associated with depression in non-pregnant populations, with normal pregnancy, and with pregnancy morbidities such as gestational diabetes, obesity, preeclampsia and preterm birth. Studies have suggested an association of inflammatory markers TNFα and IL-6 with peripartum depressive symptoms. In the present study, we hypothesized that women with self-reported depressive symptoms may exhibit elevated serum inflammatory markers, including TNFα, IL-6, IL-1β, and CRP, supporting a role for inflammation in peripartum depression. METHODS: Sixty-three healthy gravidas were recruited at an academic medical center in their third trimester. At baseline and follow-up at 3 and 6 months postpartum, participants completed a demographics survey, the Edinburgh Postnatal Depression Scale (EPDS), and provided serum samples. Collected demographics included pre-pregnancy BMI, ethnicity, education level, smoking status, employment, sources of income, and socioeconomic factors. Key serum inflammatory makers were quantified by Luminex multiplex array at baseline, and at follow-up 3 and 6 months postpartum. Multiple linear mixed effects were used to model longitudinal trends of biomarkers with the EPDS score, throughout the third trimester and the postpartum period. RESULTS: Elevated serum TNFα was associated with a lower EPDS total score (β=-0.90, p=0.046) after adjusting for demographic factors and medication use. In contrast, IL-6, CRP and IL-1β did not demonstrate statistically significant associations with depression or anxiety symptoms as quantified by the EPDS in either crude or adjusted models. CONCLUSIONS: Increased inflammatory markers have been associated with depressive symptoms in non-pregnant and postpartum populations. Contrary to our hypotheses, we report that elevated serum levels of TNFα are associated with lower self-reported depression during the peripartum period. Relevant literature evaluating a role for inflammation in depression in the unique context of pregnancy is both limited and inconsistent, therefore further exploration and understanding is merited. The early 2 nd trimester is a key stage of pregnancy that includes the 2 nd wave of trophoblast invasion. Insufficient invasion is involved in the pathogenesis of disorders such as pre-eclampsia. Perturbations of the normal metabolic trajectory may predict poor outcomes. Analysis of the metabolome at this stage of pregnancy is increasingly being used for biomarker identification. METHODS: Plasma samples and detailed clinical metadata were collected at 12, 16 and 20 weeks from pregnant women from a mixed ethnic background who experienced a normal term delivery (n=359). 1 H-NMR spectra were acquired for samples using standard 1D and 2D pulse sequences. Spectra were processed, calibrated to TSP and analysed using multivariate approaches. Metabolites of interest were identified using a Bruker NMR Metabolic Profiling Database and in-house and public databases.

Unsupervised modelling of the plasma metabolic profiles by principal components analysis (PCA) revealed a shift in the metabolome associated with advancing gestation. Differences between the plasma metabolome of the 1 st and mid-2 nd trimester were also examined by Partial Least-Squares Discriminant Analysis (PLS-DA) (R 2 X cum =0.205, Q 2 cum =0.463). *Figure(s) will be available online. Samples from the mid-2 nd trimester had higher concentrations of lipids, lipoproteins, unsaturated fatty acids and branched-chain amino-acids. Ethnic specific differences in the metabolic profile were observed in the patient cohort. Black women (n=43) had consistently higher creatine and lower choline levels than White (n=253) and Asian women (n=26). CONCLUSIONS: The maternal plasma metabolome undergoes characteristic alterations during the early 2 nd trimester to support the maternal and foetal energy requirements and placental development. Biomarker discovery studies on clinical outcomes need to take these physiological changes and ethnics differences into account. (MO) has a significant impact on the health of the mother and OFF. Exposure to MO at conception and during pregnancy leads to excessive OFF weight and adiposity. We hypothesized that maternal biochemical parameters at breeding correlate positively with OFF metabolism. METHODS: F0 female Wistar rats ate control or obesogenic diet from weaning through lactation. Half of each group wheel ran 30 min/day, 5 times/week from 90 d through pregnancy beginning (120 d) providing four groups (n=8/ group): control (C), obese (MO), exercised controls (CEx), and exercised obese (MOEx). At breeding serum insulin and leptin were measured. After weaning F1 OFF ate C diet. At day 110 d OFF (n= 8 of each sex from different litter) serum insulin and triglycerides (TG) were measured. Pearson correlations were done between maternal and male and female OFF parameters. RESULTS: Maternal serum insulin and leptin at breeding correlate positively (p<0.001) with male and female OFF TG (Fig 1B r=0 .9, D r=0.8, F r=0.7, H r=0.7) and with male OFF insulin ( Fig 1A r=0 .7, C r=0.6) but not female ( Fig 1E r=0. 2, G r=0.2). *Figure(s) will be available online. CONCLUSIONS: Maternal biochemical markers can predict OFF metabolic parameters. A strength of this study is the heterogeneity of the starting points in the four groups at breeding -normal or MO and with or without exercise. Despite this heterogeneity some markers correlated very well. This approach will provide insights into which markers are most useful. In addition it is clear that MO negatively programs offspring metabolic phenotype in a sex specific manner. CONACyT 155166. Our objective was to determine the regulation of vitamin D in long-term hypoxic sheep. METHODS: Normoxic and long-term (~140 days)-hypoxic Western sheep were studied near term. Control non-pregnant normoxic and longterm hypoxic sheep were also studied. 25-hydroxy and 1,25-dihydroxyvitamin D metabolites were analyzed in maternal and fetal plasma using ELISA. CYP24a1 (degrading enzyme), CYP27b1 (activating enzyme), and vitamin D receptor (VDR) mRNA and protein levels were studied by real-time PCR and immunoblotting. CYP24a1 promoter methylation was studied by bisulfite sequencing. RESULTS: Pregnancy was associated with a significant increase in both 25-hydroxy vitamin D and the active metabolite 1,25-hydroxyvitamin D, compared to non-pregnancy. Fetal levels of the bioactive metabolite were 3-4 fold higher than maternal levels. Hypoxic sheep had more than double the levels of the stable precursor 25-hydroxy-vitamin D and ~90% higher levels of the bioactive metabolite than normoxic sheep. The changes in vitamin D status were partly due to pregnancy induced upregulation of renal CYP27b1 and downregulation of CYP24a1. Placental (cotyledon) downregulation of CYP24a1 was associated with promoter methylation. Hypoxia was also associated with increased maternal liver levels of CYP2R1 that regulates 25-hydroxyvitamin D levels. CONCLUSIONS: Western sheep experience similar increases in vitamin D as reported for pregnant women. Long-term hypoxia further increased vitamin D levels by upregulating renal CYP27b1, liver CYP2R1 and methylation induced downregulation of placental CYP24a1. To report risk factors, clinical presentation, cardiac findings, and maternal outcomes in coronary angiography proven myocardial infarction (MI) during pregnancy and postpartum (PP). METHODS: Women who underwent angiography in pregnancy or the PP period at a large tertiary hospital between 1998 and 2014 were studied. Data collected included: maternal demographics, known risk factors for MI, laboratory tests, and cardiac evaluation (EKG, Echo, coronary angiography and cardiac catherization findings), and maternal outcomes. RESULTS: 14 patients underwent coronary angiography for evaluation of a suspected MI: 10 (71%) had angiography proven MI. Four (40%) of which occurred before delivery and 60% were in the postpartum period. Mean maternal age was 36.7 years (Range: 35-41). Table1 summarizes known risk factors, symptoms and cardiac findings in these 10 patients. All 10 women had advanced maternal age plus at least 2 known recognized risk factors for MI. Coronary angiography revealed dissection in 6 (60%), atherosclerosis in 3(30%), and one had thrombosis. The vessel most commonly involved was the left anterior descending (LAD) at 90% of the time and the remaining 10% in an aberrant right anterior descending. There was one maternal death at six years after the event.

Age>=35 10 (100) Chest Pain 10(100) 

The rate of SIP-SF in this study was 11%. To our knowledge this is the first study to report it. Surprisingly pregnancy outcomes were similar in those with and without SF. We suggest a prospective observational study to answer the optimal timing for delivery. We hypothesise that Indigenous Australian women have a genetic profile that promotes high circulating cytokine levels leading to activation of the renin angiotensin system (RAS) which might predispose to chronic kidney disease (CKD). We aimed to determine whether cytokine gene polymorphisms in Indigenous pregnant women were associated with circulating and/or urinary RAS levels, inflammatory markers (C reactive protein, CRP), blood pressure or urinary protein excretion. METHODS: Whole blood and urine were collected from Indigenous Australian women (n = 122). Genomic DNA was extracted from whole blood and SNPs genotyped using Taqman SNP allelic discrimination assays. The following SNPs were genotyped: IL-1B (rs16944), IL-4 (rs2243250), IL-6 (rs1800795), IL-1RN (rs419598) and TNF (rs1800629). We also measured RAS proteins in blood and urine, markers of inflammation, cardiovascular and renal disease in the third trimester of pregnancy.

Only IL-4 and IL-1RN SNPs showed any significant effect on RAS components, CRP, blood pressure or proteinuria. The frequency of the IL-4 SNP was: CC (67%), CT (25%) and TT (7.5%). The T allele in the IL-4 SNP was associated with decreased CRP levels (2.93 vs. 7.56 mg/L, P=0.014), decreased diastolic blood pressure (62.1 vs. 69.7 mmHg, P=0.004) and a trend towards decreased urinary protein/creatinine (8.35 vs. 13.6 mg/mmol, P=0.067). The frequency of the IL-1RN SNP was CC (4%), CT (42%) and TT (54%). The TT genotype in this SNP was associated with a decrease in urinary angiotensinogen/creatinine (P=0.029) and urinary ACE/creatinine (P=0.026) compared with CT heterozygotes. CONCLUSIONS: Two polymorphisms associated with increased production of anti-inflammatory cytokines were associated with decreased inflammation, decreased blood pressure and decreased activity of the intrarenal RAS. These polymorphisms may protect these women from CKD. Our data highlight the important interactions between inflammatory and RAS pathways in pregnancy which may influence pregnancy outcome.

Reflex. C J Shaw, 1 K J Botting, 1 Y Niu, 1 A Brew, 1 I Rivens, 2 G ter Haar, 2 C C Lees, 3 D A Giussani. and abdominal access requires direct uterine manipulation. There are no human or animal data on the physiological responses of the mother and fetus to uterine manipulation under anaesthesia. We determined changes in maternal and fetal blood gas status and continuous cardiovascular function during surgical uterine manipulation in late gestation ovine pregnancy for the first time.

Under general isofluorane anaesthesia, 9 ovine pregnancies were surgically instrumented with maternal and fetal vascular catheters, uterine, fetal carotid and femoral Transonic flow probes at 116±1d GA (term~145d). Maternal and fetal cardiovascular function and blood gas status were established during 0.5h baseline, 0.5h uterine manipulation (gentle displacement without rotation comparable to packing) and 0.5h recovery. RESULTS: Uterine manipulation reduced uterine blood flow and increased uterine artery vascular resistance (Fig.1A ,B) and triggered redistribution of the fetal cardiac output away from the femoral circulation while maintaining perfusion to the carotid vascular bed. This suggests induction of the fetal brain sparing reflex, expressed as an increase in the ratio of carotid to femoral blood flow ratio (CBF:FBF, Fig.1C ). Fetal brain oxygen and glucose delivery was maintained by this response (Fig.1D ,E). *Figure(s) will be available online. CONCLUSIONS: Uterine manipulation during surgery represents an insult which requires fetal haemodynamic compensation. These data should be considered when planning elective surgery in pregnant women.

Trust and the Genesis Research Trust. Nocturnal dipper status was also significantly different between pregnancies complicated with PE and those that were not: MAP 3/9 vs 9/10 (p=0.02) and DBP 5/9 vs 10/10 respectively (p=0.03). CONCLUSIONS: Significant differences in the diurnal BP variation were observed in women who developed PE compared to those who did not. The absence of a nocturnal dip and higher nocturnal MAP levels <20weeks gestation might be a useful adjuvant test to identify women at increased risk of PE. RESULTS: 1) CGRP, AM, IMD and BK produced dose-dependent relaxation of OA from both NP and P women; 2) Responses (Emax) to CGRP were significantly enhanced in pregnant (56.5 +/-1.8) compared to NP women (30.3 +/-5.1); 3) Vasodilatory responses to IMD were also significantly enhanced in P (48 +/-2.6) compared to NP women (30.1+/-4.8); 4) No significant differences were observed in responses to AM and BK between two groups; 5) CRLR and RAMP1, 2, and 3 are localized to smooth muscle cells, with increased density for all of them in P compared to NP women; and 6)mRNA for CRLR, RAMP2 and 3 are increased in OA from P compared to NP. CONCLUSIONS: CGRP family peptides relax human omental arteries and these vasodilatory responses are increased during pregnancy. The relaxation actions are mediated via enhanced expression of their receptors in the smooth muscle cells. These data suggest vascular adaptions during pregnancy could involve CGRP family peptides. (IUGR) is a condition of placental etiology and associated with increased risk for the long-term development of cardiovascular disease in the mother. However, it is still unclear if IUGR patients exhibit global diastolic dysfunction during pregnancy. The aim of this study is to assess the vasodilatory responses of omental arteries from normal and normotensive IUGR pregnancies to calcitonin gene related peptide (CGRP) and its family members adrenomedullin (AM) and intermedin (IMD), and assess expression of CGRP family peptide receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMP 1, 2 and 3) . METHODS: Omental arteries (OA), collected during caesarian section from women with normal pregnancy (n=9) and normotensive IUGR (n=3) were cleaned, cut into rings and mounted on a wire myograph for isometric tension recording. Rings were pre-contracted with U46619 (1µM), and cumulative dose-response relaxation curves were constructed for CGRP, AM, IMD (0.1nM-100nM), and bradykinin (BK, 0.1nM -1µM) as percent inhibition of U46619 induced contraction. QPCR and immunofluorescence localizations were used to measure mRNA and proteins for receptor components in OA. RESULTS: Both CGRP and BK induced OA relaxation responses are similar in women with normal and IUGR pregnancies. However, maximal responses (Emax) were significantly (P<0.05) lower in IUGR compared to normal pregnancies for both AM (18.2 +/-6.7 vs 38 +/-2.5) and IMD (26.9 +/-6.7 vs 48 +/-2.6). CRLR, RAMP1, 2 and 3 are localized to smooth muscle cells in OA. mRNA levels for RAMP1 and 3 are decreased, CRLR are increased and RAMP 2 are unchanged in IUGR, compared to controls. CONCLUSIONS: IUGR did not alter relaxation of OA to CGRP and BK, but led to decreased responses to AM and IMD, implying impaired specific receptor components in vascular smooth muscle in normotensive IUGR without affecting endothelial functions. Reduced OA vasodilatory responses to AM and IMD may implicate similar mechanisms in other vascular beds and thus reduce blood flow to fetus in IUGR. Further studies are needed to clarify the potential nature of long-term cardiovascular risk that may arise due to impaired AM and IMD responses in these women. Exercise increases transmembrane glucose transporter expression in liver and skeletal muscle leading to improved glucose homeostasis. While exercise is encouraged to promote a healthy lifestyle, the effect of exercise during pregnancy on modulating fetal programming remains poorly understood. This study was undertaken to evaluate the effects of exercise on glucose transporter (GLUT) 1, 2, and 4 gene expression during pregnancy in mice. METHODS: Eleven-week-old female ICR mice were split into exercise and sedentary cohorts. Exercise mice were housed in cages with voluntary access to a running wheel for 1 week prior to mating and throughout pregnancy. Mating was timed such that the effects of exercise during pregnancy could be evaluated at a specific gestational age. Body composition was measured on gestational day 16, and maternal tissue and serum samples were collected for in vitro analyses on gestational day 18. RNA was isolated from maternal liver, gastrocnemius muscle, retroperitoneal fat, placenta, and fetal liver (n = 8 per group for each tissue). RNA expression levels were quantified using the NanoString Technologies nCounter system and normalized to housekeeping genes. The average gene expression level was compared among sedentary and exercise groups, using Student's t test. RESULTS: Exercising mice showed significantly improved body composition with increased lean mass (p = 0.003) and decreased fat percentage (p < 0.001). They also had significantly reduced serum leptin (p = 0.002) levels. Maternal voluntary exercise had differential effects on regulation of transmembrane glucose transporters, based on tissue of origin. Maternal exercise significantly upregulated GLUT4 in the fetal liver (p = 0.008), while GLUT1 was significantly upregulated in the maternal liver (p = 0.028). Likewise, GLUT 2 was significantly upregulated in the placenta (p = 0.017), but downregulated in the maternal liver (p = 0.011). There were no significant differences in the GLUT expression in fat or skeletal muscle. CONCLUSIONS: Voluntary exercise during pregnancy can significantly alter GLUT gene expression; protein levels and translocation must be measured to further elucidate possible effects. Further studies are warranted to explore the effect of maternal exercise on epigenetic changes in the offspring.

Pharmacokinetics of Ranitidine and Its Metabolite During Pregnancy. METHODS: Ten pregnant and 9 non-pregnant women who were taking ranitidine for reflux or nausea and vomiting were recruited in this study.

The pregnant women were studied either in the 1 st or 2 nd , or 3 rd trimester, or both. Plasma samples (n=12) were collected over one dosing interval (12 h for bid or 24 h for qd dosing) and plasma concentrations of ranitidine and its major metabolite of desmethyl ranitidine were analyzed using a sensitive and validated HPLC-MS/MS method. Non-compartment pharmacokinetic analysis was performed using Phoenix Winnonlin. RESULTS: Pharmacokinetic parameters of ranitidine were not significantly different between early and late pregnancy and non-pregnant subjects. The ratio of the area under the plasma concentration-time curve (AUC) of desmethyl ranitidine, the primary metabolite to ranitidine was not significantly different among pregnant and non-pregnant subjects.

Pregnant mean±SD Non-pregnant mean±SD n=9 1 st and 2 nd trimesters n=5 3 rd trimester n=8 Calcitonin gene related peptide (CGRP) and its family members adrenomedullin (AM) and intermedin (IMD) play important roles in maintaining vascular adaptation during pregnancy, but its effect on human omental arteries (OA), an important component of the maternal systemic vasculature, and the mechanisms involved remain unknown. The aim of this study was to determine the vasodilatory responses of OA to CGRP family peptides in pregnant women and assess the mechanisms involved.

The OA were collected from pregnant women during caesarian section (n=9). The OA rings mounted on a wire myograph system for isometric tension recording, were pre-contracted with U46619 (1µM), and cumulative dose-response curves were constructed for CGRP, AM and IMD (0.1nM -100nM), and bradykinin (BK, 0.1nM -1µM) with or without specific blockers of relaxation pathways, added 30 minutes prior to peptide additions to muscle bath. The relaxation responses were calculated as percent inhibition of the U46619 induced contraction, and compared before and after incubations with blockers. RESULTS: 1) CGRP, AM, IMD and BK produced dose-dependent relaxation of human OA obtained from pregnant women; 2) Vasodilatory responses to CGRP are reduced by nitric oxide synthase inhibitor L-NAME (0.1mM), adenylyl cyclase inhibitor SQ22536 (10 nM), calcium-dependent potassium channel blocker tetraethylammonium (TEA, 10mM), and ATP sensitive potassium channel blocker glybenclamide (10 µM); 3) The cyclooxygenase inhibitor, indomethacin (10µM) partially inhibited the concentration-response curve to CGRP, and combination of L-NAME and indomethacin plus non-selective K+ channel blocker tetrabutylammonium (TBA, 1mM) completely abolished CGRP-induced relaxation; 4) Vasodilatory responses to AM were also reduced by L-NAME SQ22536, and TEA, but not by glybenclamide; 5) Vasodilatory responses to IMD were reduced by L-NAME and SQ22536 as well, but not by TEA and glybenclamide. CONCLUSIONS: CGRP family peptides relax human omental arteries from pregnant women. The relaxation actions of these different peptides, CGRP, AM and IMD, were differentially mediated via multiple mechanisms, involving nitric oxide, cyclic AMP, cyclo-oxygenase derived prostanoids, and endothelium-derived hyperpolarizing factors. In late pregnancy, catabolism is predominant in nutrient metabolism and the stored nutrients in the mother are mobilized to support rapid fetal growth. Protein and amino acids are critical for fetal growth and development. Our previous study showed that in mid-and late pregnancy, plasma levels of total amino acids remained stable in pregnant rats fed a low protein (LP) diet, coincident with reduced plasma levels of essential amino acids and elevated non-essential amino acids. In this study we hypothesized that protein degradation in skeletal muscles is enhanced to maintain the homostasis of amino acids in pregnant rats fed LP diet. METHODS: Time-scheduled pregnant SD rats were fed a normal (control; CT) or LP from Day 3 of pregnancy until sacrificed at Days 10, 19 and 22 (n=6-10 rats/diet/day). Gastrocnemius muscle (GM) was collected for RNA and protein extraction. Expression of Fbxo32, Trim63 (protein degradation marker), Ccnd1 (skeletal muscle cell proliferation marker), Myod1, Ckdn1 (differentiation marker) and R18s (housekeeping gene) were analyzed by qPCR. The abundance of total and phosphorylated mTOR and AKT (key enzymes in protein synthesis), NDUFB8, SDHB, UACRC2, MTCO1 and ATP5A (key enzymes in oxidative phosphorylation) and beta-actin (loading control) in GM was analyzed by Western blotting. The effect of LP diet on these parameters was analyzed by one-or two-way ANONA.

The results include: 1) mRNA levels of Fbxo32 were unchanged in the LP group at Day 10, but increased (P<0.001) by 3.1-and 4.3-fold, respectively, compared to those in the CT group; mRNA levels of Trim63 followed the same pattern of changes as Fbox32; 2) mRNA levels of Ccnd1 were reduced (P<0.05) by 1.4-, 2.4-and 1.5-fold in LP rats at Day 10, 19 and 22, respectively; 3) mRNA levels of Myod1 were unchanged in LP rat at Day 10, but reduced (P<0.05) by 2.2-and 1.9-fold at Day 19 and 22, respectively; in contrast, mRNA levels of Ckdn1a were reduced (P<0.001) by 2.2-and 1.9-fold at Day 19 and 22, respectively; 4) The abundance of mTOR and AKT, both total and phosphorylated, and the ratio of phosphorylated to total proteins were unchanged in LP rats at all 3 Days; 5) The abundance of NDUFB8, SDHB, UACRC2, MTCO1 and ATP5A was unchanged in LP rats at all 3 Days.

These results indicate that in late pregnancy, protein degradation is enhanced in skeletal muscles in pregnant rats fed LP diet while protein synthesis is unaffected. and activity of ACE protein in lungs in pregnant rats, which may cause fetal and placental growth restriction. In this study we investigated the effect of LP diet on angiotensin II production in non-pregnant rats. We hypothesized that LP diet induces elevated angiotensin II production in the lung of non-pregnant rats by increasing Ace expression and ACE activity. METHODS: Virgin female SD rats were fed a normal (control; CT) or LP diet for 8, 12 and 17 days, respectively (n=6 rats/diet/day), to mimic the time course of diet treatment for pregnant rats in our previous studies. Angiotensin II levels, ACE and ACE2 (angiotensin I converting enzyme 2) activities were measured in plasma. Total RNAs and proteins were extracted from lungs and kidneys and expressions of Ace and Ace2 were analyzed by quantitative real-time PCR and Western blotting. Activities of these proteins in lungs, kidneys and plasma, were measured by fluorimetric methods. The effect of LP diet on these quantitative parameters was analyzed by one-or two-way ANONA.

The results include: 1) Plasma angiotensin II levels were elevated (P < 0.05) by 1.24-fold in the LP group compared to the CT group at Day 8 and 12, but not Day 17; 2) In lungs, mRNA levels of both Ace and Ace2, the abundance and activity of both ACE and ACE2 proteins were unchanged in LP rats at all 3 Days, except that ACE activity was increased (P < 0.01) by 2.1-fold at Day 12; 3) In kidneys, mRNA levels of both Ace and Ace2, the abundance and activity of both ACE and ACE2 proteins were unchanged in LP rats at all 3 Days, except that mRNA levels of Ace were increased (P < 0.05) by 1.6-fold at Day 17 and that ACE2 activity was reduced (P < 0.05) by 1.7-fold at Day 12; 4) In plasma, the abundance and activity of both ACE and ACE2 proteins were unchanged in LP rats at all 3 days, except that ACE activity was reduced (P < 0.05) by 1.6-fold at Day 12. CONCLUSIONS: These results together with our findings in pregnant rats fed LP diet indicate that unlike pregnant rats, non-pregnant females fed LP diet had elevated angiotensin II level temporally due to the contribution of multiple organs including lung, kidney and plasma. INTRODUCTION: HELLP syndrome is regarded as a subset of preeclampsia characterized by hemolysis, elevated liver enzymes and low platelet count. We examined regional cerebral blood flow differences using MRI comparing women with a history of non-HELLP preeclampsia (PRE) with women with a history of HELLP syndrome prior to and during subsequent pregnancies to examine the brain hemodynamic adaptations. METHODS: Paired pre-pregnancy (PP) and third trimester (3P) MRI data from 6 PRE and 6 HELLP were acquired using a Philips Achieva 3T MRI. Regional cerebral blood flow was measured using pseudocontinuous arterial spin labeling in 5 brain regions. INTRODUCTION: This present study was performed to determine the involvement of the nitric oxide (NO) signalling pathway in the increased susceptibility to infection thought to occur in pregnancy. Previously we reported that NO levels were attenuated in pregnant animals in endotoxaemia compared to non-pregnant (NP) controls. When taken with the hypotensive response observed, these data suggested an increased sensitisation to the vasodilator action of NO. We aimed to further explore both vascular function in pregnancy and the roles of DDAH1, an endogenous inhibitor of ADMA, and NO in pregnancy using DDAH1 knockout (DDAH1 -/-) mice. METHODS: Vessel function and sensitivity to vasoactive substances were assessed by wire myography in aorta and uterine artery. Pregnant (E16) or NP CD1 mice received either vehicle (PBS) or LPS (10µg 0111:B4) i.p. 12 hours before culling. Female DDAH1 -/mice or wildtype littermates (WT) were implanted with PA-C10 radio-telemetry device into the aorta. NP baseline recordings were taken for 48h. Mice were then timed-mated and recordings were resumed at E0 until labour. Further cohorts of mice were used for tissue harvest at 6 and 12 hours post LPS or vehicle i.p.

in both aorta (P<0.001) and uterine artery (P<0.05) in pregnant mice, suggesting that vessels in pregnancy are not more sensitive to the vasodilatory action of NO. Conversely, there appeared to be a reduced response to the vasoconstrictor action of phenylephrine. In pregnant mice, cGMP levels were increased compared to NP controls. LPS did not cause any further increase. In female DDAH1 -/mice, NP baseline MAP was significantly increased compared to WT controls (116±2.8 mmHg and 108.6±0.9 mmHg respectively, P<0.05). This increase was not maintained throughout pregnancy; however MAP remained high in early pregnancy (E4-E7) and increased again dramatically after labour. ADMA was increased in pregnant DDAH1 -/compared to WT controls but circulating NO was not significantly altered. CONCLUSIONS: As the effects of NO are primarily mediated by cGMP, increased levels of cGMP may have conferred increased sensitivity; however we did not see any evidence to support this. Furthermore, it may be that the hypotensive response to LPS in pregnancy is mediated through decreased sensitivity to vasoconstrictors. Overriding factors drive the decrease in MAP during pregnancy in DDAH1 -/mice. 

Our data suggests obesity, in young nulliparous women, is associated with variations in cardiovascular function that have been linked to subsequent preeclampsia. This includes increases in blood pressure and pulse wave velocity (an index of vessel stiffness) outside of pregnancy. Obesity, indexed as BMI or body fat, also was associated with increased pulse and CO. These associations may underlie the increased risk for preeclampsia associated with obesity.

hormones that dilate various vascular beds with the greatest response in the uterine artery (UA). Circulating estrogens are significantly elevated to stimulate uterine vascular development and to prepare for embryo implantation during the menstrual cycle. The objective of this study is to test a hypothesis that H 2 S biosynthesis is increased during the proliferative phase of human menstrual cycle. METHODS: Intact UA was obtained from hysterectomies of premenopausal women in the proliferative (UApro) or secretory (UAsec) phase (n=5/group). CBS/CSE mRNA was assessed by qRT-PCR and protein was assessed by immunoblotting and immunofluorescence microscopy. H 2 S production was determined by the methylene blue assay w/wo specific inhibitors of CBS (CHH, 1 mM) and CSE (BCA, 1 mM). RESULTS: UApro CBS mRNA and protein were 2.12 ± 0.20 and 2.17 ± 0.19 -fold greater (p<0.05) than that of UAsec, respectively. CBS protein was localized in UA intima and media, with 2.46 ± 0.39 and 2.16 ± 0.14fold greater (p<0.05) expression in UApro than UAsec, respectively. High levels of CSE mRNA and protein were detected in UApro and UAsec, but with no difference. CBS protein in the proliferative myometrium was 2.68 ± 0.32-fold greater (p < 0.05) than that of the secretory myometrium and CSE protein was unchanged. CBS and CSE proteins were localized in myometrial microvessels with no changes between the phases. UApro H 2 S production was significantly greater (p<0.05) than that of UAsec, which was blocked by incubation with CHH or its combination with BCA, but not BCA alone. CONCLUSIONS: Significantly elevated H 2 S biosynthesis via selective CBS upregulation in human UApro implicates a role of a novel CBS/ H 2 S pathway in estrogen-induced uterine vasodilatation during the proliferative phase of the menstrual cycle (supported by NIH RO1 HL 70562 and R01 HD08783). worldwide are anemic and half of these cases are attributed to iron deficiency. Daily oral iron and folic acid supplementation (30-60mg of elemental iron, once daily) is recommended to reduce the risk of low birth weight, maternal anemia and iron deficiency. Double dose is recommended by many obstetricians in cases of iron deficiency anemia. We sought to assess the efficacy of doubling the daily supplement of iron in anemic pregnant women. METHODS: A prospective randomized controlled trial was conducted at a community woman's health center from January 2009 to May 2013. A total of 302 pregnant women with iron deficiency anemia were randomized to receive one or two capsules of iron supplements (Aktiferrin F: Serine 129 mg; Iron (Ferrous Sulfate) 34 mg; Folic Acid 0.5 mg) daily from 18 weeks to delivery. Iron deficiency anemia was defined as hemoglobin (hb) concentration lower than 105 g/L along with ferritin level of less than 15 ng/ml after exclusion of other causes such as vitamin B12 deficiency or thalassemia. the major outcome measure compared between the two groups was maternal hb concentration at 35 weeks. Minor outcome measures included ferritin levels at 35 weeks, birth-weights, preterm birth rate and gastrointestinal side effects. RESULTS: 150 women were randomized to receive one capsule (group A) and 152 received two capsules (group B). Both groups had similar mean hemoglobin concentration (101gr/l) and ferritin levels at allocation (9.4 ng/ml and 9.3 ng/ml, respectively). Hemoglobin concentrations were similar also at 35 weeks (108.1 gl/l and 107.9 gl/l) as were ferritin levels (13.8 ng/ml and 14.2 ng/ml). Constipation was more common among women from group B (12% and 8%). No significant differences in birthweight (3258gr and 3303 gr) and preterm birth rate (<37weeks) (7.8% vs 7.4%) were found. CONCLUSIONS: A single dose iron supplement was found to be equally efficient with less side effects compared with double dose. are reported in spontaneous abortion, preeclampsia gestational diabetes (GDM); with GDM associated with elevated levels of AM. However, it is not known if IMD expression is altered in GDM placentas and if placental expression of AM, IMD and their shared 7TM receptor, calcitonin receptor like receptor (CRLR) and its 3 receptor activity modifying proteins (RAMP1, 2 or 3) are fetal sex dependent. This study was designed to assess if fetal sex has a role in regulating placental expression of AM, IMD and their receptor components in normal pregnancy and in pregnancy complicated with gestational diabetes. METHODS: Eleven placentas from normoglycemic and 12 diabetic women were collected term delivery. GDM was defined as two or more of the four plasma glucose concentrations exceeding ADA criteria(Report of the Expert Committee 1997).Villous tissue was used for gene expression and immunolocalization studies using qPCR and immunofluorescence respectively. RESULTS: 1) AM and IMD mRNA levels in villous tissue are not fetal sex dependent in normal pregnancy; 2) AM and IMD mRNA levels are significantly higher in female compared to male placenta in GDM (p<0.005);3) AM immunoreactivity is localized to cytotrophoblast cells in villous tissue and its intensity is similar in normal and GDM placenta, whereas IMD immunoreactivity is localized to both cytotrophoblast cells and vascular endothelial cells of fetal vessels and its staining intensity is higher in GDM compared to normal placental villi; 4) CRLR and RAMP1 mRNA in villous tissue are higher in placenta of female fetus compared to male in normal pregnancy (P<0.05); whereas, RAMP2 and RAMP3 mRNA levels remain similar; and 5) RAMP1and RAMP3 mRNA are significantly higher in placenta of female fetus compared to male (P<0.005) with no change in CRLR and RAMP2 mRNA expression. CONCLUSIONS: Placental expression of AM and IMD receptor components and not AM or IMD peptides are fetal sex dependent in normal pregnancy. Elevated placental levels of AM, IMD and their receptor component RAMP1 and RAMP3 in pregnancy with female compared to male fetuses, suggests involvement of these peptides in the sexual dimorphism in pregnancies complicated with GDM.

The Fetal growth restriction (FGR) describes the process through which the fetus fails to reach its predetermined growth potential and is known to be associated with increased incidence of perinatal morbidity and mortality. Placental dysfunction is thought to play a critical role in the pathogenesis of FGR. Perturbed trophoblast apoptosis has been linked to the pathophysiologic pathways encountered in placental failure or dysfunction. Through using stereology and concurrent image analysis, we aimed to quantify and characterize trophoblast apoptosis in placentas from normal pregnancies, and those complicated by hypertensive and normotensive FGR. METHODS: Placentas were weighed, randomly sampled, formalin-fixed and paraffin-embedded. Multiple representative full-thickness sections were labelled with monoclonal antibody M30, an epithelial apoptotic marker specific for a neo-epitope on cytokeratin 18. They were then digitally scanned, virtual slides reviewed and analyzed using Aperio ImageScope software. Thirty two fields of vision, randomly selected throughout the maternal and fetal aspects of each placenta, were analyzed by Positive Pixel Count algorithm to quantify the amount and grades of positive stain (weak, moderate, strong). Inter-group differences were sought using appropriate non-parametric statistical tests. RESULTS: Both hypertensive and normotensive FGR placenta expressed significantly higher levels of trophoblast apoptosis compared to control (22.6% vs 20% vs 6.5%, p<0.0001). Hypertensive FGR placenta showed significantly increased proportion of strongly positive stain compared to normotensive FGR placenta (11.9% vs 8.8%, p=0.01). Quantitative fetal/maternal zone discrepancies were not evident in any of the placental pathologies. CONCLUSIONS: This study emphasises the magnitude of the apoptotic burden in FGR placenta, more so when hypertensive disease is present. Furthermore, combining stereology and image analysis, to examine immune-labelled slides, is a powerful and a reproducible tool to quantify and characterize expression patterns of placental dysfunction. Autophagy is a tightly regulated intracellular lysosomal-mediated recycling process leading to the removal of old and/ or dysfunctional cytoplasmic organelles. It is essential for normal cell growth, development, and homeostasis, serving to maintain a balance between synthesis and degradation. Its involvement in reproduction and placental development has not been extensively studied but is thought to be linked to placental dysfunction implicated in the pathogenesis of preeclampsia and fetal growth restriction. We aimed, through combining stereology and image analysis techniques, to characterise and quantify placental expression and distribution of LC3, an autophagy marker. METHODS: Placentas from pregnancies complicated by preterm preeclampsia (PPE) and term preeclampsia (TPE) were matched to those from normal pregnancies. They were weighed, randomly sampled, formalin-fixed and paraffin-embedded. Four representative full-thickness sections from each placenta were labelled with LC3, digitally scanned and analyzed using Aperio imageScope software. Thirty two fields of vision, randomly sampled throughout the maternal and fetal aspects of each placenta, were analyzed by Positive Pixel Count algorithm set to quantify the amount and the grades of positive staining (weak, moderate, strong). Appropriate statistical tests were used to seek inter-group differences. RESULTS: PPE placenta had a significantly higher proportion of strong positive stain compared to control (13% vs 8%, p=0.0003). On the other hand, TPE placenta showed a consistently higher levels of weak positive stain expression compared to control (p=0.0003). In normal placenta, LC3 distribution displayed maternal side abundance (p<0.0001), but this distribution was reversed in PPE placenta, with the fetal side showing higher expression (p=0.02). CONCLUSIONS: These results show that preeclamptic placenta had a higher autophagy burden compared to control, indicating an enhanced necessity for recycling unwanted intracellular waste before the apoptotic cascade is activated. Results also emphasize that not only placental dysfunction enhances autophagy expression, but also reverses the maternal/ fetal zonal distribution. The eNOS -/mouse provides a well characterized model of fetal growth restriction (FGR) with altered uterine artery reactivity and reduced utero-placental blood flow. Oxidative stress is implicated in vascular dysfunction in human FGR but it remains to be established whether oxidative stress is elevated in eNOS -/mice compared to their wild type counterparts. We hypothesize that eNOS -/mice demonstrate increased placental oxidative stress and that a dietary anti-oxidant therapy (pomegranate juice-PJ) will restore uterine artery function.

Oxidative stress markers (HSP70, SOD-1 and SOD-2) were investigated in the placental labyrinth and basal zone of C57Bl/6J (WT) and eNOS -/mice at embryonic day (E) 18.5 by quantitative immunohistochemistry (IHC) using HistoQuest software (Tissue Gnostics). Uterine arteries (UtA) were isolated from WT and eNOS -/mice treated from E12.5-18.5 with either PJ or isocaloric control (apple juice -AJ). Artery function was assessed by measuring contraction to phenylephrine [PE](0.1-10000nM) and endothelium-dependent (acetylcholine [Ach]; 0.1-10000nM) and -independent (sodium nitroprusside [SNP]; 0.1-10000nM) relaxation using wire myography. RESULTS: Both WT (n=5) and eNOS -/-(n=5) placentas showed higher expression of all 3 oxidative stress markers in the basal zone compared to the labyrinth zone; HSP70 p<0.0001, SOD-1 p=0.0199, SOD-2 p=0.0081. Furthermore, eNOS -/placentas had decreased levels of SOD-1 compared to WT in both the basal zone and labyrinth (p=0.043 and p=0.022 respectively). HSP70 and SOD-2 expression did not differ between genotypes (p=0.122 and p=0.337 respectively). PJ treatment (n=7) did not alter uterine artery reactivity compared to AJ (n=9) in WT mice. However, preliminary data (n=3) suggest that in eNOS -/mice, PJ treatment reduces UtA constriction in response to PE. CONCLUSIONS: The data suggest that eNOS -/mouse placentas have reduced anti-oxidant capacity compared to WT mice and highlight the potential importance of the basal zone in oxidative stress balance. Despite PJ not affecting uterine artery reactivity in WT mice, it shows promise as an anti-oxidant therapy in the vascular compromised eNOS -/mouse.

Localization Assessment of nanoparticle turnover and localization in vivo is vital for development of a less-invasive, lower risk delivery system. METHODS: Texas Red-conjugated polymer was used for nanoparticle formation with a plasmid containing hIGF-1 under the control of the PLAC1 trophoblast-specific promoter. The complex was tracked throughout the organs of pregnant mice and their fetuses via ex vivo and microscopic imaging techniques at multiple time points following intraplacental or intravenous injection. Mouse organs were harvested and hIGF-1 expression measured using RT-PCR. Data were analyzed by the Student's t-test and a P≤ 0.05 was considered significant. RESULTS: Intraplacental Treatment: Fluorescence intensity of maternal liver and kidneys and placenta was increased compared to sham 2 hours post-injection, however fluorescence levels had returned to background levels 96 hours post-injection. Human IGF-1 RNA was expressed in the treated mouse placenta compared to control (ddCT 2 -12.96 vs 0, p<0.05,n=3), and to much lesser extent in maternal lung (13 fold) and ovary (70 fold), with no expression in brain, kidney or untreated sibling placentae. Intravenous Treatment: Whole organ fluorescence intensities in treated animals remained at background 48 and 72hrs post-injection. However, following sectioning, treated maternal lungs showed greater intensity at 72 hours; no other tissues fluoresced at that time point. At 72 hours post-intravenous treatment no hIGF-1 expression was measurable in any organs of the treated mouse. CONCLUSIONS: Successful hIGF-1 expression in the treated placenta following intraplacental nanoparticle injection substantiates the hIGF-1 nanoparticle as a viable treatment for FGR. Dispersal patterns of the nanoparticle following the more general intravenous injection, as well as gene expression in the intraplacentally-treated lung and ovary, must be controlled however. Future development of a peptide-sequence specific to the placenta for targeted-delivery will ensure that hIGF-1 expression is concentrated in the placenta, thus reducing any off-target effects in both the mother and the fetus. control key cellular processes such as development, growth and inflammation. This study was performed to determine whether alterations in placental miRNA expression are associated with maternal obesity and fetal overgrowth, independent of glucose intolerance. METHODS: A cross-sectional study was performed to investigate miRNA expression in placentas obtained from: (1) normal weight women (BMI 20-24.9 kg/m 2 ) with infants of normal birth weight (BW) (2700-3500 g) (n= 20), (2) normal weight women with macrosomic infants (BW ≥ 4000 g) (n= 10), (3) obese women (BMI ≥ 35 kg/m 2 ) with infants of normal BW (n= 16), and (4) obese women with macrosomic infants (n=10). All women had a term delivery (37-41 weeks) and normal glucose tolerance (1 hour GCT < 130 mg/dl). Exclusion criteria were: diabetes, chronic hypertension, preeclampsia, and chronic steroid use. Placental samples were collected after delivery, stored in trizol at -80C, and RNA was extracted using Qiagen miRNeasy kits. The expression of 5639 human miRNAs was assessed using the Affymetrix GeneChip miRNA 3.0 Array. Differential miRNA expression was determined using two-way ANOVA and pairwise contrasts, with the Benjamini-Hocherg (BH) correction. MiRNAs with bias-corrected absolute Z scores ≥ 2 or BH < .05 were considered significant. RESULTS: One hour glucose challenge test values were overall similar among groups. Placental miRNA expression was not statistically different among the groups. For all 5639 miRNAs investigated, Z-scores were ≤ 2 and BH > .05. Principal components analysis demonstrated that the global miRNA expression profiles were similar in all groups [ figure 1 ]. *Figure(s) will be available online. CONCLUSIONS: Placental miRNA profiles were not altered by maternal obesity or macrosomia, independent of glucose intolerance. miRNA modulation of gene expression is an unlikely mechanism underlying fetal overgrowth related to maternal obesity, and other pathways should be explored. The progenitor cytotrophoblast of the human placenta can differentiates into one of two main pathways forming either the syncytiotrophoblast or the invasive extravillous trophoblast (EVT). The diversity of mRNA essential for cell differentiation and development is mediated in part by post transcriptional alternative splicing coding for protein isoforms. Rbfox2 plays a major role in controlling myoblast fusion. Interestingly, Rbfox2 also regulates invasive EMT (epithelial to mesenchymal transition). We suggest that Rbfox2 may act as a molecular switch directing cytotrophoblast differentiation to either syncytiotrophoblst formation or EVT. Thus the placenta may present a singular system of development where these two diverse pathways of Rbfox2 regulation may be studied.

We screened publicly available data for trophoblast cell lineage expression of RNA binding proteins, splicing and alternative splicing proteins. Immunohistochemistry for Rbfox2 was performed on three trimesters of normal placenta, preeclampsia, complete hydatidiform mole and placenta accreta sections. Rbfox2 immunofluorescence was performed on isolated primary trophoblasts differentiated to syncytiotrophoblast. RESULTS: Rbfox2 is expressed in a trophoblast lineage specific manner in both normal and pathological placentas. Rbfox2 is expressed in the progenitor cytotrophoblast, reduced in the terminally differentiated syncytiotrophoblast (formed by cell fusion) and conversely, highly expressed in the invasive trophoblasts in an increasing gradient of expression towards the more invasive cells. Recapitulation of these observations in primary trophoblast syncytium in cell culture displayed reduced Rbfox2 expression. CONCLUSIONS: Rbfox2, an alternative splicing factor, is differentially expressed in specific trophoblast cell lineages and may act as the molecular switch determining the trophoblast differentiation pathway.

Environmental (III)) exposure affects millions of people worldwide. In the United States, roughly 4 million individuals drink well water that exceeds the As(III) levels recommended by the EPA. As(III) exposure is associated with an increased risk of impaired fetal growth and stillbirth, suggesting that As(III) impairs placental function. We sought to determine if As(III) leads to trophoblast injury and interrogate if altered DNA methylation contributes to arsenic-induced trophoblast injury. METHODS: Primary human trophoblasts (PHT cells) were isolated from women delivering at term using an established protocol. PHT cells were exposed to varying concentrations of As(III) for 48 hours. DNA and RNA were extracted with TriReagant and methylation of ERVWE-1 (Syncytin 1) measured with bisulfite genomic sequencing (n=3). Gene expression was measured by qPCR (n=5). RESULTS: Environmentally relevant concentrations of As(III) altered transcription of select genes in a manner consistent with trophoblast injury. *Figure(s) will be available online.

To explore an epigenetic mechanism for trophoblast injury, we characterized the methylation of the regulatory region of ERVWE-1 (Syncytin 1), a retroviral gene regulated by DNA methylation. Arsenic, at both doses interrogated, led to increased methylation at the regulatory region of Syncytin 1. Expression of Syncytin 1 was decreased in a manner consistent with increased methylation of the regulatory region. CONCLUSIONS: As(III) exposure is associated with a number of important perinatal complications, yet the impact of As(III) on trophoblast biology remains unclear. We demonstrate that As(III) leads to trophblast injury. Additionally environmentally relevant dose of arsenic caused epigenetic dysregulation of a gene critical to fusion of cytotrophoblasts to form syntiotrophoblasts. Taken together these data suggest that epigenetic perturbations are mechanistically involved in As(III)-induced trophoblast injury. (Winn et al, 2007) identified TncRNA as increased in term placental basal plate (BP) relative to second trimester; whether this increase actually represents an increase in NEAT1 or MENß is unknown. We sought to clarify the relative expression of these lncRNAs in the placenta during gestation and hypothesize that changes in NEAT1 expression are responsible for our earlier results.

Under IRB-approved protocols, placentas from second trimester terminations (early, 14-18 wk, n=5; mid, 21-24 wk, n=5) were obtained. These groups were comprised of samples used in the prior microarray study. Placentas from healthy term pregnancies delivered by cesarean section before labor (37-40 wk, n=4) were collected immediately after delivery. TncRNA, NEAT1, and MENß expression in both BP and CV was examined by real-time (RT) PCR using a series of distinguishing primers. Term pregnancies were compared to early and mid-second trimester. Results were normalized to 18S rRNA. Student's t-test was used to compare experimental groups with statistical significance set at p<0.05. RESULTS: By relative quantification, NEAT1 increased 2.4-fold in BP at term versus early and mid-second trimester (p<0.05). A trend towards decreased NEAT1 expression in CV at term was observed, but this change was not significant. There were no significant differences in MENß expression. TncRNA levels increased in BP in a manner similar to NEAT1. CONCLUSIONS: NEAT1 expression increases in placental BP at term relative to second trimester, but not in CV. The similar increase in TncRNA (part of NEAT1)indicates that our prior results were in fact due to changes in NEAT1 expression. These findings suggest a role for NEAT1 in the regulation of placental gene expression during normal pregnancy. Given the potential role of NEAT1 in regulating RNA processing, effects of NEAT1 on placental gene expression are under investigation. Infection-associated pre-term labor (PTL) commonly leads to preterm birth, which is the leading cause of neonatal mortality and morbidity. PTL is characterized by increased levels of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6. Besides pro-inflammatory cytokines, PTL has also been associated with increased levels of the soluble fms-like tyrosine kinase-1 (sFLT-1) -an anti-angiogenic factor. Inflammatory responses and angiogenesis can be regulated upon activation of the PPAR-γ pathway. Therefore, we hypothesized that a PPAR-γ agonist (rosiglitazone) will attenuate the inflammatory response caused by lipopolysaccharide (LPS) in the placenta. METHODS: First trimester placental explants (n=4) were collected and exposed to 1ug/ml or 10ug/ml of LPS in presence or absence of 10µM of Rosiglitazone (PPAR-γ agonist). The expression of different PPAR-γ regulated and inflammation-associated genes was quantified by qPCR in placental explants. Levels of sFLT-1 and heme oxygenase-1 (HO-1) were determined in the supernatants by ELISA. RESULTS: Incubation with LPS up regulated the expression of TNF-α, IL-8 and COX2 in first trimester placental explants. Concentrations of sFLT-1 increased in the supernatant upon LPS incubation, while concentration of heme oxygenase-1 (HO-1) reduced. Treatment with Rosiglitazone significantly down-regulated the expression of IL-8 and TNF-α (≥ 3.0 folds, p£0.05). Expression of COX2 was also down regulated but not significantly. Rosiglitazone induced the transcription and secretion of HO-1 in the supernatants (mRNA levels ³2.0 folds, p value £0.05) and concentration of sFLT-1 was also reduced upon treatment with Rosiglitazone.

The activation of PPAR-γ by Rosiglitazone inhibits the expression and secretion of TNF-α, IL-8 and COX2 induced by LPS in first trimester placental explants. These findings suggest that the PPAR-γ pathway is a new molecular target for future preventative methods for infection-induced preterm birth. Preeclampsia is a common disease of human pregnancy and a leading cause of both maternal and neonatal morbidity and mortality. Inadequate trophoblast invasion and deficient remodeling of uterine spiral arteries are associated with preeclampsia. This syndrome is thought to be almost certainly the result of multiple factors. Therefore, we investigated candidate genes associated with possible mechanism of preeclampsia using transcriptome.

We isolated total 6 human amniotic epithelial (HAE) cells from placenta of normal pregnancy and preeclampsia and prepared total mRNA from each cells. 3 preeclampsia RNAs and one cocktailed RNAs sample from 3 normal patients were used for transcriptome. Illumina was utilized for a unique "bridged" amplification reaction that occurs on the surface of the flow cell. A flow cell containing millions of unique clusters is loaded into the HiSeq 2000 for automated cycles of extension and imaging. Solexa's Sequencing-by-Synthesis was utilized. Transcripts with a fold induction ≥ 2 and Benjamini-Hochbery adjusted p-value ≤ 0.05 were considered significant. Pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online resource. RESULTS: We obtained several differentially expressed genes(DEG) through a comparative analysis of HAE. Groupings were performed as increasing and/or decreasing genes in preeclampsia. Next, Grouped genes were subdivided according to the classification, biological function, cellular component, and molecular function. Also, since KEGG pathway mapping provide pathway maps representing on the molecular interaction and reaction networker of higher-level systemic functions, we independently investigated KEGG pathway mapping using transcriptome data. Pathway networking maps of preeclampsia according to the several classification were obtained. CONCLUSIONS: Our study demonstrates that candidate genes could be provided and useful as novel disease biomarkers. These findings could provide valuable information for understanding uncertain molecular mechanism of preeclampsia and developing targeted therapy. 

We collected placentas from women at 38 weeks of gestation (term) and placentas collected at 41 weeks of gestation (post-dated). We compared the two groups for: telomere length measured by real-time PCR, oxidative DNA damage assessed by immunohistochemistry and for mTOR and autophagic pathways using immunohistochemistry and western blots. RESULTS: Telomeres from placentas at 41 weeks were shorter (P=0.05) and immunohistochemistry revealed significantly (p=0.008) increased 8-hydroxy guanosine compared to those at 38 weeks of gestation. Phos-AKT (S473) was reduced in post-dated placentas (p=0.04) and correlated with phos-mTOR s2481 an mTORC2 marker (R 2 =0.4212, p=0.002); phos-p70S6 kinase (T389), a substrate for mTORC1 rose (p=0.0008), and phos-p70S6K and AKT were strongly negatively correlated (R 2 = -0.4018, p=0.003). Western analysis showed that LAMP2, a lysosomal marker, rose in post-dated placentas (p=0.005) and immunohistochemistry demonstrated a change in the distribution of LAMP2 to the basal side of the syncytiotrophoblast in post-dated placentas *Figure(s) will be available online. Finally IGF2R, a major component of the lysosomal pathway, increased (p=0.02) in post-dated placentas. CONCLUSIONS: Our data are consistent with the hypothesis that placental ageing is associated with the late gestation increase in fetal death. (n=24), posterior (n=13) and fundal (n=1). Women with a previa had a higher median number of prior cesarean sections compared to those without a previa. Patients with a previa were more likely to have a prenatal diagnosis of accreta than those without a previa (81% vs. 42%, p<.001). The rate of unscheduled hysterectomy in the previa group was 47% compared to 71% in the nonprevia group (p=.01). Median gestational age at delivery was earlier in the previa group compared to the non-previa group (34 vs. 35 weeks, p=.045). There were no differences between the previa and non-previa group in maternal morbidities. Neonates of women with a previa were more likely to receive antenatal corticosteroids and had longer length of stays (p=.02), however there was no difference in birth weights. Patients with a previa were more likely to be diagnosed with an increta or percreta on pathology than patients without a previa (44% vs. 18%; p=.005).

The presence of a placenta accreta without a previa is less likely to result in the prenatal diagnosis of accreta and more likely to result in unscheduled hysterectomy. The maternal morbidities associated with non-previa placenta accreta are similar to those of a placenta accreta with a previa. RESULTS: 95 prenatally diagnosed placenta accreta cases were identified over the study period. 51.6% of the women required hospitalization for an episode of antepartum vaginal bleeding. There were no differences in maternal age, number of prior cesarean sections, or the presence of placenta previa between the bleeding and no-bleeding groups. In women with antepartum bleeding, there was a trend toward an increased rate of unscheduled cesarean hysterectomy (40.8% vs. 23.9%; p=.08). Median gestational age at delivery was earlier in the antepartum bleeding group (33.9 weeks) than the no-bleeding group (35.0 weeks) (p<.001). Delivery at <32 weeks occurred more often in the bleeding group (22.4% vs. 8.7%; p=.09). Neonates of women with an antepartum bleeding were more likely to receive antenatal corticosteroids (p=.02). Neonatal birth weight was lower and length of stay longer in the bleeding group compared to the no-bleeding group. There were no differences between the bleeding and no-bleeding group in maternal morbidities including estimated blood loss, need for transfusion, intraoperative organ injury, ICU admission and postoperative length of stay.

Approximately one-half of women with a prenatally diagnosed placenta accreta will require hospitalization for antepartum vaginal bleeding. Women with antepartum bleeding are delivered at earlier gestational ages and therefore have increased neonatal morbidities despite an increase rate of administration of antenatal corticosteroids. Given the crucial regulatory role ofthe placenta in glucose uptake and modulation of metabolism, molecular characterization of gene expression couldlead to the identification of novel biomarkers to improve diagnosis and management of diabetes in pregnancy. Wepreviously applied whole transcriptome RNA sequencing (RNA-Seq) and robust computational analysis to a discoverycohort (n=27). In this current study we aimed to significantly expand this analysis using an expanded subject cohort inorder to identify robust molecular markers specific for GDMA1, GDMA2, and Type II DM. METHODS: 100 placental samples from subjects with comprehensive clinical data were rigorously collected at delivery and snap frozen until use (n=25/group from control non-diabetics, GDMA1, GDMA2, and Type II). mRNA was extracted and subjected to whole transcriptome analysis (RNA-Seq, Illumina HiSeq, n=27) or validation with RT qPCR (TaqMan, n=100 We have previously demonstrated reduced placental blood flow in our nonhuman primate (NHP) model of Western-style diet (WSD) consumption during pregnancy. Moreover, maternal WSD results in increased production of inflammatory mediators within the placenta. These mediators undoubtedly modulate the types of immune cells recruited to the placenta and influence their function. Yet the immunological changes that occur within the WSD exposed placenta are poorly understood.

Our objective was to examine the impact of WSD on the frequency and phenotype of the immune cells that infiltrate the placenta. METHODS: Pregnant Japanese Macaques maintained on control (14% fat, CON n=3) or WSD (36% fat, n=4) underwent cesarean section at gestational day 130 (term is 175 days). Maternal decidua and fetal villous were collected from placental tissue for digestion and immune cell separation using a percoll gradient. Flow cytometric analysis was performed to delineate innate and adaptive immune cells. Specifically, cells were stained with anti-CD3, CD4, CD8, CD20, CD14 and CD16 to delineate T cells (CD3+CD4+ or CD3+CD8+), B cells (CD3-CD20+) and macrophages (CD3-CD20-CD14+). Macrophages were further divided into classical (CD16-) and non-classical (activated CD16+). RESULTS: Maternal WSD consumption during pregnancy results in increased recruitment of leukocytes in the decidua and villous. Our data indicate that the majority of leukocytes in the villous are macrophages.

Relatively few T and B cells were found in the villous compared to decidua. More importantly, WSD resulted in a greater accumulation of activated macrophages in the villous and increased accumulation of macrophages, memory CD4 and CD8 T cells. In addition, we demonstrate increased natural killer and dendritic cells in the decidua of WSD compared to control animals. CONCLUSIONS: We propose that the increased prevalence of activated leukocytes at the maternal-fetal interface with WSD consumption will increase local production of inflammatory cytokines resulting in altered placental vasculature and increased risk of ischemic injury. In these studies we utilized an invasive cps V strain of GBS (GB37) to assess macrophage metabolism and polarization, as well as the ability of this strain to alter the production of reactive oxygen intermediates. METHODS: Following infection with GB37 (or control), a multianalyte microphysiometer (MAMP) was utilized to dynamically measure glucose and oxygen consumption, lactate production, and extracellular acidification in vitro with murine macrophage-like cell line, RAW 264.7. Ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS) was also carried out to assess changes in the metabolome using human, macrophage-like THP-1 cells. GB37 was used in vitro to stimulate human monocyte derived macrophages to assess alterations in macrophage polarization by qRT-PCR using the M1 target CCR7 and the M2 targets CD209 and MRC1. Human term placental macrophages were used to address reactive oxygen intermediate generation when stimulated with GB37. RESULTS: GB37 stimulation induced an M1 phenotype in monocyte derived macrophages by expression of CCR7, significantly suppressed ROS in human term placental macrophages (p < 0.05) at 2hr, and altered the metabolism of murine RAW macrophages in vitro. Lactate production and extracellular acidification both decreased over time when exposed to GB37. MEDI heat maps were generated from the UPLC-MS data, showing significant differences in THP-1 cells when comparing PMA treatment with GB37 stimulation. These differences covered a wide range in terms of chemistry, where putative metabolites include PC's, small peptides and other long aliphatic chains according to metabolite databases. CONCLUSIONS: These studies demonstrate significant shifts in macrophage metabolism and activation during confrontation with GBS.

The extent to which these changes contribute to disease pathogenesis remain to be determined. Maternal obesity is associated with systemic inflammation and oxidative stress, leading to placental morphological and physiological changes that can affect pregnancy outcomes. In this study, we investigated the effect of prepregnancy obesity in the placenta on three markers related to aging: telomere relative length, Klotho and p53 genes. Telomere shortening has been associated with increased aging and activation of p53, an apoptosis marker, while the Klotho gene exhibits anti-aging properties.

We utilized an established mouse model where, over 3 months, the CD-1 female mice were fed either high fat diet (34.9% fat, HF group) or standard chow (5.8% fat, SF group). Mice were then bred. Placenta was obtained (n = 5-7 per group) on day 18 of pregnancy.

Telomere relative length, Klotho gene and p53 mRNA levels were measured using genomic DNA analysis and quantitative RT-PCR. T/S ratio indicative of relative telomere length was calculated from the relative input amount of the telomere PCR divided by the relative input amount of the 36B4 gene PCR of the same sample. Statistical analysis was performed using Student t-or Mann-Whitney test as appropriate (significance P£0.05). RESULTS: T/S was significantly lower in placenta from HF group (SF 1.7 ± 0.3 vs. HF 0.8 ± 0.2, P=0.04). The p53 mRNA levels were significantly increased in HF placenta (SF 0.6 ± 0.1 vs HF 1.2 ± 0.2, P=0.03, Figure) . Klotho gene mRNA expression was lower in HF group, though not statistically significantly (SF 6.7 ± 1.6 vs sFlt1 3.6 ± 2.1, P=0.4). *Figure(s) will be available online. CONCLUSIONS: Our data demonstrates for the first time that prepregnancy obesity resulting from high fat diet contributes to modifications in aging pathways in the placenta, which in turn leads to placental dysfunction. Reduced anti-aging properties in the high fat group in combination with an increase in apoptosis markers demonstrate clear placental changes. Our study reveals novel insight to the physiological effects of obesity in pregnancy. and IUGR. Secondary to this maldevelopment, placental malperfusion may result in a repetitive ischaemia/hypoxia-reperfusion type of injury resulting in oxidative stress, which can compromise normal function of cellular components including mitochondria and endoplasmic reticulum (ER). We have shown that exposure of placentas to labour can provide a useful in vivo model for studying cellular changes induced by oxidative stress as seen in the preeclamptic placenta. In this study we further tested whether the labouring process in placentas from healthy pregnancies can also induce ER stress. METHODS: 16 placental samples were used for the study, including 8 caesarean section controls and 8 labour placentas. All placentas were delivered at term by standard vaginal delivery or by elective non-labour caesarean section (CS) from normotensive healthy singleton pregnancies. For each placenta, small pieces of tissue from separate lobules were randomly taken and snap-frozen in liquid nitrogen within 10 min of delivery; the samples were stored at -80°C. Protein levels of a number of ER stress markers including GRP78, GRP94, PDI, XBP-1 and p-eIF2α

were measured with western blotting. Immunohistochemistry was used to identify cellular localization of ER stress using GRP78. RESULTS: Western blotting showed significantly higher protein levels of GRP78, XBP-1 and p-eIF2α in placentas exposed to labour compared to CS controls. Immunohistochemistry showed strong reactivity for GRP78 in laboured placentas, but not in the CS controls. The majority of the positive staining was found in the syncytiotrophoblast, while no or only weak staining was observed in stromal and endothelial cells. CONCLUSIONS: Elucidating the role of ER stress in PE is confounded by the presence of the maternal pro-inflammatory milieu, making it difficult to distinguish between cause and effect. Exposure to labour may therefore present the opportunity to study placental cellular changes to oxidative and ER stresses in the absence of maternal factors. Our results further suggest that caution should be taken when interpreting gene expression or other data obtained from placentas exposed to labour, as they may be subjected to stress-induced protein synthesis inhibition and other downstream consequences of ER and oxidative stress.

striking resemblance between atherosclerosis and placenta specific acute atherosis might suggest a common pathway in an already fragile cardiovascular system. The extensive inflammation in both conditions could be the possible denominator in the pathophysiology.

We investigated vascular and inflammatory pathology of spiral arteries (SA) in the placental bed of 29 healthy and 29 preeclamptic patients. Per patient, four placental bed punch biopsies were sampled during caesarean sections, stained for the presence of T-cells, NK cells and macrophages, and systematically studied using a newly developed scoring system to evaluate SA remodelling and several other characteristic features of the placental bed. In addition we compared modifiable cardiovascular risk factors directly postpartum. RESULTS: 232 biopsies were collected. Women with PE showed lower presence of T-cells in both decidua and myometrium (p=0.006 and p=0.047 respectively). An increase of macrophages was also observed but statistically not significant. Complete SA remodeling was significantly less often present in the PE group (p=0.008 Intensive expression of PHB1 was also localized by immunostaining in the trophoblast layer in PE compared to normal placentas. TCs transfected with PHB1 siRNA exhibited increased CuZnSOD expression, but decreased MnSOD expression, p<0.05, and produced significantly less 8-isoprostane than the control cells, 0.50± 0.05 vs. 0.78±0.07pg/µg cellular protein, p<0.01. CONCLUSIONS: Expression of PHB1 and PHB2 was increased in PE placentas. PHB1 siRNA induced an increase in CuZnSOD, but a decrease in MnSOD, expression in primary isolated placental trophoblasts. The increased CuZnSOD expression was associated with reduced 8-isoprostane production in PHB1 siRNA transfected cells. These results provide evidence that PHB is involved in theregulation of anti-oxidative activity in placental TCs, and increased PHB expression may contribute to increased oxidative stress in PE placentas. (n=14) by Western blotting and real-time qPCR. ELISA was used to determine angiogenic factors levels in plasma and first-trimester (8-12 weeks gestation) human placental explants. Time pregnant mice were treated with CBS inhibitor, aminooxyacetic acid (AOA). Mean arterial blood pressure (MBP), histological assessments of placenta and embryos were performed. RESULTS: Placental CBS expressions were significantly reduced in women with FGR. Inhibition of CBS activity by AOA reduced PlGF production from first-trimester human placental explants, Administration of AOA to pregnant mice had no effects on blood pressure, but caused fetal growth restriction, which was associated with reduced placental PlGF production. Histological analysis revealed a reduction in the placental junction zone, within which trophoblast giant cells and glycogen cells were less prominent in CBS inhibitor-treated animals. Furthermore, H 2 S donor GYY4137 treatment restored fetal growth in pregnant mice exposed to high level of sFlt-1. CONCLUSIONS: These results imply that placental CBS is required for placental development and that dysregulation of CBS activity may contribute to the pathogenesis of FGR but not preeclampsia opening up the therapeutic potentials of H 2 S therapy in this condition. INTRODUCTION: Approximately 15% of women continue to smoke during pregnancy due to the highly addictive nature of nicotine. While nicotine replacement therapies are considered to be a safer alternative, nicotine alone has been found to cause hypoxia and compromised placental development. Although the underlying mechanisms remain elusive, recent studies identified that augmented endoplasmic reticulum (ER) stress preludes placental insufficiency. Moreover, ER function depends on proper disulfide bond formation -a partially oxygendependent process mediated by protein disulfide isomerase (PDI) and ER oxidoreductases. Given that: (1) nicotine induced hypoxia in the rat placenta, and (2) hypoxia and placental insufficiency were associated with poor disulfide bond formation and ER stress, we hypothesized that maternal nicotine exposure leads to both placental ER stress and impaired disulfide bond formation. METHODS: Female rats received daily subcutaneous injections of either saline (vehicle) or nicotine bitartrate (1mg/kg) for 14 days prior to mating and during pregnancy. Placentas were harvested on embryonic day (e) 15 for analysis. Protein and mRNA expression of hallmarks of ER stress (e.g., phosphorylated eIF2α, GRP78, ATF4, and CHOP), key players in the disulfide bond formation pathway (e.g., PDI, QSOX1, VKORC1), and hypoxia marker, HIF-1α, were quantified via Western blot and/or Real-time PCR.

Maternal nicotine exposure led to increased protein expression of phosphorylated eIF2α and GRP78 (p<0.05) in the rat placenta, demonstrating the presence of augmented ER stress. Nicotine also led to increased mRNA and protein expression of ATF4 and CHOP (p<0.05), suggesting activation of ER-stress apoptotic pathways. Finally, decreased mRNA and protein expression of PDI, VKORC1, and QSOX1 (p<0.05) reveal an impaired disulfide bond formation pathway, which may be due to nicotine-induced hypoxia as indicated through elevated protein expression of HIF-1α (p<0.05). CONCLUSIONS: This study is the first to connect maternal nicotine exposure with both placental ER stress and hypoxia-induced disulfide bond impairment, providing novel insight into the deleterious effects of nicotine exposure during pregnancy on placental health. investigation, emerging research points to the role of progesterone, which is synthesized in the placenta. Previous studies reported significantly lower serum progesterone levels in PE which was associated with increased endothelin and reduced uteroplacental blood flow. Gas chromatography-Mass Spectrometry (GC/MS) can be used to determine the levels of urinary Pregnanediol (PD) and Pregnanetriol (PT), the metabolites of Progesterone (P4) and 17-OH-Progesterone respectively. Our goal is to establish whether the levels of these metabolites differ in patients with preeclampsia compared to normal pregnant controls.

We conducted this study at SUNY Downstate Medical Center and Kings County Hospital Center. Urine samples were obtained from11 patients with preeclampsia and 17 controls in the immediate postpartum period. IRB-approved consent form was obtained from each patient prior to the specimen collection. We identified patients who met the diagnostic criteria for preeclampsia with elevated blood pressure and proteinuria. Steroid conjugates were extracted from urine, hydrolyzed, and subjected to further derivatization to form Methoxime-trimethyl silyl (MO-TMS) derivatives which were then injected and separated to obtain levels of PD and PT.

The PD and PT levels were significantly decreased in PE when compared to normal controls (p= 0.003, 0.02 respectively). The ratios of PD/Cholesterol and PT/Cholesterol were also significantly decreased in PE when compared to normal controls (p= 0.003, 0.004 respectively). There was no statistically significant difference between the subjects with preeclampsia compared to control subjects with respect to age, BMI and gestational age at time of delivery (p= 0.8, 0.44, 0.09 respectively). However, the neonatal birth weight was lower in preeclampsia (p=0.04).

The decreased levels of urinary progesterone metabolites in preeclampsia suggest impaired placental progesterone synthesis and/or metabolism. Further investigations with larger studies are needed to confirm these findings. (9), and preeclampsia (6), were cleaned, cut into rings and mounted to a wire myograph for isometric tension recording. Rings were pre-contracted with U46619 (1µM) and cumulative dose-response relaxation curves were constructed for CGRP, AM, and IMD (0.1nM -100nM) and Bradykinin (BK, 0.1nM-1 µM) as percent inhibition of U46619 induced contraction. mRNA levels for CRLR, RAMP 1, 2 and 3in OA were measured using QPCR and expressed relative to GAPDH. Preeclampsia is a serious multi-system disease occuring in 5% of pregnancies caused by the placental release of sFlt-1 into the maternal bloodstream. The only efficacious treatment is delivery, which leads to significant morbidity and mortality associated with prematurity. A therapeutic able to quench or stabilize the disease process would be a major advance.

Sulfasalazine is an anti-inflammatory and immune modulating drug safe in pregnancy that has recently been shown to activate the potent cytoprotective enzyme heme oxygenase 1 (HO-1).

To assess whether sulfasalazine can 1) reduce anti-angiogenic factors sFlt1 and sENG 2) reverse endothelial dysfunction in vitro.

Increasing doses of sulfasalazine were administered to primary human umbilical vein endothelial cells (HUVECs) and placental explants and sFlt1 and sENG release assessed.

Endothelial dysfunction was induced with TNFα to HUVECs and the effect of sulfasalazine on VCAM (vascular cellular adhesion molecule) was assessed. xCELLigence was used to assess HUVEC migration in the presence of sulfasalazine ± sFlt1.

We observed a significant dose dependent reduction in both sFlt1 and sENG release from primary HUVECs and placental explants. Importantly, mRNA expression of newly described human and placental specific variant sFlt1-e15a mRNA was significantly decreased, as was MMP14 mRNA expression (cleavage protease of endoglin). As expected, sulfasalazine also significantly increased mRNA expression of HO-1 (Figure 1:A,B) . Treatment of HUVECs with TNFα induced significant upregulation of VCAM which was potently reversed by sulfasalazine. XCELLigence demonstrated improved migration of VEGF-stimulated HUVECs and enhanced proliferation in the presence ± sFlt-1 (Figure 1 :C,D). *Figure(s) will be available online. CONCLUSIONS: Sulfasalazine is a novel agent, safe in pregnancy that significantly quenches sFlt1 and sENG release from primary human cells and tissues and improves endothelial dysfunction. This provides strong evidence to suggest that sulfasalazine may be able to quench preeclampsia and could be a novel effective treatment for the disease. reactivity was measured using a DMT myograph where endotheliumintact aortic rings were pre-contracted with phenylephrine and relaxed with acetylcholine or sodium nitroprusside. qRT-PCR reactions were performed using the human extravillous cytotrophoblast cell line (CTBs) SGHPL-4 and formalin-fixed human and mouse placentas. Placentas from all mouse groups were collected at gd 18 and H&E staining was performed. For determination of proteinuria, the total albumin and creatinine concentrations were measured. RESULTS: miR-155 expression was significantly increased in placentas from patients with PE, CTBs treated with poly I:C and pregnant WT mice treated with poly I:C (P-PIC WT). Poly I:C treatment induced hypertension in P-PIC WT (gd 17 SBP: 147±4.5 mmHg) compared to P WT mice (103±2.7 mmHg), but had no effect in P-PIC miR-155 KO mice (101±2 mmHg). P-PIC WT mice exhibited endothelial dysfunction, splenomegaly, proteinuria and placental necrosis compared to P WT mice and these were also attenuated in P-PIC miR-155 KO mice. Poly I:C treatment significantly induced fetal demise in P-PIC WT mice and this was also prevented in P-PIC miR-155 KO mice. CONCLUSIONS: These data suggest that TLR3-induced miR-155 expression contributes to inflammation leading to PE. INTRODUCTION: Hypertension during pregnancy is still a serious obstetrical problem. Hypertension and elevated levels of circulatory TNF-α are strongly associated with PE but the pathophysiology of preeclampsia is unclear. Our previous data suggested that an inadequate CGRP-mediated compensatory vasodilatory response may play a role in the pathophysiology of hypertensive disorders during pregnancy; however, the mechanism involved in the action of CGRP peptides remains unknown. The present study was designed to determine the effects of TNF-α on interactions of receptor components, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1, 2 and 3, on the cell membrane of rat mesenteric artery smooth muscle cells (RMASMCs), and assess if CGRP family peptides CGRP, adrenomedullin (AM), and intermedin (IMD) can rescue TNF-α-induced alterations. METHODS: RMASMCs were grown on Lab Tek chamber slides with or without TNF-α (1ng/ml) for 16 h, and then treated with CGRP/AM/ IMD at 10nM. Proximity Ligation assay (PLA) was performed using a DuolinkII Fluoresence kit. Fixed cells were incubated with primary antibodies to CRLR and RAMPs, PLA probes, and mixture of ligation and amplification. Images were observed under fluorescence microscope and red spots were counted using CellSense Digital Imaging software in 3 -5 randomly selected fields.

(1) TNF-α significantly inhibits proximity association of CRLR with RAMP1, 2, and 3 (p< 0.01); (2) CGRP reversed TNFα-inhibited CRLR/RAMP1 associations when compared with controls (p=0.007), without significant effects on associations of CRLR/RAMP2 and CRLR/PAMP3; (3) AM treatment rescues the complex formation of CRLR with RAMP3 when compared with controls (p=0.02), without significant effects on associations of CRLR/RAMP1 and CRLR/RAMP2; and (4) IMD significantly reverses the complex formation of CRLR with all three RAMP1, 2, and 3 (p= 0.01, 0.001, and 0.0001, respectively). CONCLUSIONS: TNF-α impairs associations between CGRP family peptide receptor components CRLR and RAMPs.CGRP family peptides differentially rescue TNF-α-induced reduction in proximity association of their receptor components. These findings may reveal novel opportunities to develop function-specific therapeutic drugs for preventing and treating pregnancy complications such as preeclampsia. There is growing evidence for the involvement of bioactive lipid, sphingosine-1-phosphate (S1P) in trophoblast differentiation and invasión, uterine angiogénesis; all the cellular and physiological processes necessary to establish healthy placenta. We hypothesize that dysregulation of SPHK1/ S1P pathway during early placentation process may contribute to the onset of the disease. METHODS: Hypoxia was induced in human JAR cells or in extravillous placental explants by oxygen deprivation (1% PO2) for 3, 6, 16 and 24 hrs. IUGR in pregnant rabbits was induced by uteroplacental vessels ligature. Five mg of human or rabbit placental tissues were used for protein isolation and RNA extraction. Protein and mRNA levels were determined by Western blot and real-time PCR. RESULTS: Our preliminary results show that SPHK1/S1P pathway is altered in human term placentas of PE subjects and as well in rabbit placentas of IUGR model. Sphingosine kinase-1 (SPHK1) mRNA and protein abundance are decreased in human PE and rabbit IUGR placentas as compared to controls, suggesting that synthesis of angiogenic S1P is impaired in ischemic placentas. In in vitro model of placental hypoxia (JAR trophoblastic cells or extravillous placental explants, exposed to low PO 2 ), SPHK1 levels are increased in 1% oxygen in a time-dependent manner as compared to normoxic conditions. While it is known that SPHK1 and S1P are upregulated in hypoxic conditions to promote angiogénesis in cáncer cells, our results may suggest that inherited or induced downregulation of SPHK1/S1P pathway during early gestation is a potential cause of impaired uterine angiogénesis and decidualization observed in PE patients.

Our results demonstrate that bioactive and angiogenic lipid, sphingosine-1-phosphate and its synthetic/metabolic pathway may contribute to pathogenesis of PE and may serve in the future as potential biomarkers of the disease. (PE) is a leading cause of maternal morbidity and mortality during pregnancy, yet there is no specific treatment for its prevention or cure. Excess placental production of soluble fms-like tyrosine kinase-1 (sFlt1), an antagonist of vascular endothelial growth factor (VEGF), is strongly implicated in the development of PE. However, neither the specific cellular source nor the mechanisms of sFlt1overproduction in the placenta have been clearly determined.

In this study, we collected placental bed biopsies and fullthickness placental samples from the central basal plate area from 15 normal and 12 preeclamptic pregnancies at the time of cesarean delivery and examined the expression and cellular localization of sFlt1 by in situ hybridization (ISH) with 35 S-labeled riboprobes. PE was defined as systolic blood pressure of >140 or diastolic blood pressure > 90 mm Hg more than once and significant proteinuria (> 300 mg/24 h, 1+ on dipstick, or urine protein/creatinine ratio of > 0.19). Immunohistochemistry (IHC) was performed for identification of trophoblasts (cytokeratin 7) and proliferating cells (Ki-67), and an in situ cell death detection kit (TUNEL) was used for detection of apoptotic cells.

We found thatsFlt1 was primarily expressed in the invading extravillous trophoblasts (EVTs) in the basal plate in both normal and preeclamptic women,but there was a dramatic increase in sFlt1 expression in PE. There were also significant (p<0.01) increases in cellularity, proliferation index, and thickness of the sFlt1-expressing EVT cell layer in the basal plate in PE. However, the number of apoptotic cells was the same in both normal and preeclamptic women. CONCLUSIONS: These results for the first time suggest that the expansion of the EVT layer in the basal plate may contribute to the observed increases in circulating levels of sFlt1 in PE. Only the percentage of male-gendered babies differed between complete and incomplete HELLP Syndrome (65.7% and 46.4%, respectively). CONCLUSIONS: There are few differences between complete and incomplete HELLP syndrome with respect to newborn physiology and maternal placental pathology. Similar results were observed with analysis of each individual trait (data not shown). CONCLUSIONS: Genetic predisposition to common diseases did not increase the likelihood of preeclampsia in our cohort. Independent genetic factors may contribute to risk. Future work will examine common and rare genome-wide variation in patients with prior preeclampsia to identify novel genetic loci. A medical therapy that stimulates growth of the pre-eclamptic placenta and quenches sFlt1 and soluble endoglin production could be an efficacious treatment. Granulocyte macrophage colony stimulating factor (GM-CSF) and Epidermal Growth Factor (EGF) may offer new therapeutic options given both are safe in pregnancy and contribute positively to pregnancy outcome; GM-CSF deficiency adversely impacts fetal and placental development, whilst addition to IVF culture media promotes embryo health and IVF success. EGF levels rise during pregnancy, and deficiency results in pregnancy loss in mice. This study aimed to assess whether GM-CSF or EGF could induce trophoblast proliferation and reduce sFlt1 secretion in primary human tissues. METHODS: mRNA expression of CD116 (GM-CSF receptor) and EGF receptor (EGFR) was assessed in severe early onset pre-eclamptic placentas (n=17) and gestationally matched controls (n=19). Proliferation kinetics of trophoblast cell lines, JEG3, BeWo and JAR cells were assessed with increasing concentrations of GM-CSF or EGF using the xCELLigence system. Primary trophoblast cells were purified, and the capacity of GM-CSF and EGF to improve cell viability assessed using an MTS assay. sFlt1 secretion was assessed by ELISA. RESULTS: mRNA expression of both CD116 and EGFR was significantly (p<0.05) upregulated in pre-eclamptic placentas. GM-CSF and EGF induced a significant increase in trophoblast cell line proliferation. Similarly, cell viability of primary trophoblasts was enhanced following treatment with GM-CSF. Excitingly, both GM-CSF and EGF induced a potent dose dependent reduction in sFlt1 release from primary human trophoblasts. The top dose of GM-CSF decreased sFLt1 levels by >90% * Figure( s) will be available online. CONCLUSIONS: This is the first data to demonstrate that GM-CSF and EGF promote trophoblast proliferation and potently reduce sFlt1 secretion from placental cells. We propose that GM-CSF and EGF may have potential as novel therapeutics to treat preeclampsia. (2013) reported increased NEAT1 expression in chorionic villi (CV) in growth restricted pregnancies. Given the underlying abnormal placentation common to many cases of growth restriction and preeclampsia, we hypothesize that placental NEAT1 expression is also altered in preeclampsia.

Under IRB-approved protocols, placentas from healthy term pregnancies (38-41 wk, n=5), spontaneous preterm deliveries (26-31 wk, n=5), and pregnancies complicated by preeclampsia (preterm 27-32 wk, n=5; term 37-39 wk, n=5) were collected immediately after delivery. All were delivered vaginally. NEAT1 and MENß expression in both BP and CV was examined by real-time (RT) PCR. Preeclamptics were compared to controls of similar gestational age. Results were normalized to 18S rRNA. Student's t-test was used to compare experimental groups with statistical significance set at p<0.05. RESULTS: NEAT1 was 60% lower in CV from term preeclamptics compared to controls (p<0.05), with no difference seen in the corresponding BP samples. NEAT1 levels were not significantly changed in either BP or CV from preterm preeclamptics compared to preterm labor controls. There were no significant differences in MENß expression in any comparisons. CONCLUSIONS: NEAT1 expression decreases in CV from term preeclamptics compared to controls, but not in BP. Placentas from preterm preeclamptics show no change in NEAT1 compared to gestational ageappropriate controls. These findings suggest a role for NEAT1 in the regulation of placental gene expression in preeclampsia seen at term. Given the potential role of NEAT1 in regulating RNA processing, effects of NEAT1 on placental gene expression in preeclampsia will be explored further. and September 2013, were adjudicated by two maternal fetal medicine specialists. Histopathologic findings are determined by a perinatal pathologist. We selected 7 cases with severe PE and histolopathologic findings of placental insufficiency and 13 controls. Subjects affected by chronic hypertension or pre-existing or gestational diabetes were excluded. RNAs extracted from placental tissues and analyzed by RNA-seq, mapped using STAR, and then DEseq was used for normalization and differential expression analysis.

Among the 17,606 genes detected by RNA-seq, 112 genes were differentially expressed in severe PE compared to controls (p<0.05). Among these genes, 33 were upregulated and 79 were downregulated. PE specific genes, such as HTRA4 (fold change: 4.2, p<0.01), LEP (fold change: 5.1, p<0.01), PAPPA (fold change: 1.7, p<0.05), and ENG (fold change: 1.5, p<0.05) were significantly upregulated. Functional enrichment analysis showed that 33 of the 112 differentially expressed genes were located in the plasma membrane, including 5 up-regulated genes (including ENG and VTCN1) and 28 down-regulated genes. Also, 21 of the differentially expressed genes were extracellular, with 4 (including LEP and PAPPA) upregulated and 17 downregulated. CONCLUSIONS: The first placental RNA-seq results identified several genes up-regulated in PE, the majority of the differentially expressed genes were down-regulated, with most coding for membrane-associated or secreted proteins. In our study, careful selection of cases based on both clinical and histopathologic criteria has resulted more detail to develop a better understanding of PE pathogenesis. Intracellular S1P is exported by ABC-type transporters and signals through S1P2/S1P3 receptors on vascular smooth muscle cells (VMSCs) to stimulate constriction. Thromboxane A2 (TXA2), a vasoconstrictor, releases S1P from platelets and is elevated in preeclampsia. Whether TXA2 utilizes S1P to increase constriction is unknown. We hypothesize that TXA2 will activate SK in VSMCs of intact arteries increasing S1P release. S1P will engage S1P2/S1P3 receptors to stimulate constriction. METHODS: Murine uterine and mesenteric arteries mounted on a pressure myograph, were treated in the bath with the TXA2 mimetic, U46619 (1-100nM), with or without inhibitors to SK (1µM SK-II), ABC transporters (1µM F9054, 10µM MK571 or combined), S1P1/S1P3 (1µM VPC23019) or S1P2 (10µM JTE013). Arteries from S1P3-/-or S1P3+/+ wildtype mice were also used. In some cases phenylephrine (PE; 0.01-10 µM) replaced U46619. Constriction was measured by diameter change from baseline. respectively. U46619 or PE-induced constriction was significantly inhibited in uterine arteries (to 25.2±5.1%; 12.6±7.4% respectively) from S1P3-/-mice but remained unaffected in mesenteric arteries. Thus, U46619 or PE signal through the S1P pathway to induce uterine artery constriction through S1P3 and in part via S1P2 in mesenteric arteries.

Notably, the inhibition of U46619 or PE-induced constriction mediated by JTE013 was partly relieved by the NOS inhibitor L-NAME, which had no effect in the absence of JTE013. Thus, JTE013 may activate NOS along with antagonizing S1P2 receptors.

We reveal a novel mechanism through which U46619 or PE induces constriction with vascular bed differences. Targeting the S1P pathway may be useful for the treatment of vascular-related disorders like preeclampsia and intrauterine growth restriction. Supported by CIHR. is an insulin-like growth factor binding protein-5 (IGFBP-5) protease known to be elevated in preeclampsia (PE). IGFBP-5 binds IGF-1 and -2. Upon cleavage, IGF is released. IGF-1 and -2 levels are reduced in PE but whether placental or circulating PAPP-A2 account for alterations in IGF is not known. Therefore, we sought to establish the relationship between placental and serum PAPP-A2 with IGF-1 and -2 in maternal and fetal serum. METHODS: Chorionic villi (CV) samples, maternal and cord sera were obtained from PE and gestational-age matched normotensive singleton pregnancies, 24-41 wks gestation. PAPP-A2 immunoblot was performed on CV protein lysates and sera. IGF-1 and-2 sera levels were assessed by ELISA. Data CONCLUSIONS: PAPP-A2 in placental lysates and maternal serum is elevated in PE, but not detected in fetal circulation. Maternal IGF-1 and -2 are decreased in PE, as is fetal IGF-1. The correlation of CV, and not maternal serum, PAPP-A2 with IGF levels supports that it is primarily placental PAPP-A2 that impacts systemic maternal and fetal IGF availability, likely by releasing IGF from the systemic circulation for local utilization. Alternatively, CV PAPP-A2 levels may increase in response to low circulating IGF levels as a compensatory mechanism to combat the placental dysfunction and growth restriction that can be seen in PE. Pre-eclampsia is associated with increased maternal urate serum levels. Hyperuricemia might lead to longterm maternal cardiovascular risk and to fetal programming. The regulation of the placental urate transport system is not yet understood, but is likely to be predominantly regulated by GLUT-9 (glucose-transporter 9), besides the mulittransporter hOAT4 (human organic anion transporter). The aim of this study was to investigate the placental urate transport system and to characterize its transporter GLUT9.

Electrophysiological techniques and radioactive ligand up-take assay were used to measure transport activity of GLUT9 overexpressed in Xenopus oocytes, and its activity under various conditions. We further analysed the placental GLUT9 expression using novel self-raised antibodies against human isoforms GLUT9a and -b.

In the Xenopus oocytes system chloride replacing chloride with iodine resulted in a complete loss of urate transport. In radioactive up-take experiments iodine had no effect on urate transport. Both GLUT9 isoforms are present in the microvillous membrane (MVM) of the syncytiotrophoblast cells, but not in the basal membrane (BM). CONCLUSIONS: Our results indicates that urate transport in the placenta is unidirectional in the feto-maternal direction, because GLUT9 isoforms are expressed exclusively in the microvillous (apical) membrane of the trophoblast and because the organic anion transporter isoform 4 (OAT4) is known be localized on the basal membrane and to transport urate unidirectionally into trophoblast cells. Furthermore, GLUT9 uric acid transport activity is iodine-regulated by changing the mode of up-take from an electrogenic to an electroneutral transport. This may allow regulating urate transport by the change of an active to a passive transport.

Functional Down-Regulation of Placental Glucose Transporter (GLUT)-1 in Pre-Eclampsia. Transplacental glucose supply is predominantly regulated by GLUT-1 transporter. Altered expression and function of GLUT-1 may affect the intrauterine environment and could compromise fetal development. We speculate that placental GLUT-1 expression is impaired in pre-eclampsia (PE), a disease known to be associated with GDM and IUGR, and could potentially lead to impaired glucose transport and altered fetal programming. METHODS: Placentae were obtained after elective caesarean sections following normal and PE pregnancies. Syncytial basal membrane (BM) and apical microvillus (MVM) fractions were prepared from syncytiotrophoblast (STB). Protein expression was assessed by western blot analysis and mRNA levels were quantified by rt-PCR. Radiolabeled glucose up-take assay and a transepithelial transport model using primary cytotrophoblasts (CTBs) were established to determine glucose transport activity. RESULTS: GLUT1 protein expression was significantly down-regulated in PE placentae compared to control placentae, while mRNA expression was unchanged. Glucose up-take into MVM was significantly reduced in PE compared to control. In a transepithelial transport model, phloretinmediated inhibition of GLUT1 at the apical side of primary cytotrophoblast cells showed a significant shift of the transepithelial glucose transport.

Using phloretin at IC 50 concentration, 50% of 3-OG transport is reached after 424.6±66.5 min; while without phloretin inhibition is reached after 343.9±34.5 min. CONCLUSIONS: Our study for the first time shows that in PE, GLUT1 is down-regulated on protein level and glucose transport activity is decreased. Altering glucose transport activity by inhibition of apical GLUT-1 indicates that transplacental glucose transport might be regulated on the apical side of the STBM. These results might help to elucidate the regulation of GLUT1 transporter and to develop preventive strategies to modulate glucose transport in PE, aiming to reduce the risk for metabolic and cardiovascular diseases for the child later in life. There is evidence that a history of increased physical activity is associated with a reduced risk of subsequent preeclampsia. We sought to determine if fitness, as estimated by VO2 max, is associated with a physiologic profile that has been associated with reduced risk for subsequent preeclampsia when identified prior to pregnancy.

We examined 88 women, including 30 with a history of prior early-onset preeclampsia (pPE) and 58 nulliparous women. All women were examined in the follicular phase (cycle day 9.5 ± 3.5).

Measures included pulse (P), blood pressure (BP), mean arterial pressure (MAP), brachial and popliteal pulse wave velocity (PWV, an index of vessel stiffness), and cardiac output (CO). Plasma volume was assessed by the Evans Blue Dye method and corrected for BMI. Maximal oxygen consumption (VO 2 max) assessment by bicycle ergometry was completed to estimate fitness. Associations between VO 2 max and cardiovascular measures were assessed using Spearman correlation coefficients. Results are presented as mean ± SD and p<.05 accepted for significance. RESULTS: Mean subject age was 31.5±4.3 years, and did not differ between subsets. BMI and MAP were significantly higher in pPE (nullips: 24.3 ± 5.4, pPE= 28.2 ± 6.3 kg/m 2 ; MAP, nullips= 86.6 ± 7.3, pPE= 94.9 ± 8.9 mmHg). For all subjects, increased VO 2 max is associated with increased age (R 2 = 0.21, p=.05), lower BMI (R 2 = -0.72, p<.001), lower resting P (R 2 = -0.48, p<.001) and lower supine MAP (R 2 = -0.32, p=.002).

Higher VO 2 max was also associated with higher PV/BMI (R 2 = 0.54, p<.001) in both groups. CONCLUSIONS: Higher VO 2 max indicates improved physical fitness. Low PV/BMI, a known risk factor for recurrent preeclampsia, is observed in both nulliparous and pPE women with lower fitness levels. Additionally, higher MAP was significantly associated with decreased fitness and is associated with subsequent preeclampsia. These physiologic findings are also linked to hypertension outside of pregnancy as well as contributing to the recognized physiologic profile of women at risk of developing preeclampsia.

Overall decreased fitness appears to be associated with these physiologic risk factors. Increased prepregnancy cardiovascular dysfunction may be the mechanism through which reduced fitness increases the risk for preeclampsia.

Exploring The heme oxygenase/carbon monoxide(CO) system has been suggested in both in vivo and in vitro studies to be involved in the pathophysiology of preeclampsia. Exogenous CO may be a potential non-invasive therapeutic treatment method. Data from our group has demonstrated that chronic exposure to 250ppm CO was effective in the prevention of hypertension and proteinuria in a sFlt1-induced mouse model of preeclampsia. This study aims to determine the effects of acute doses of CO on blood carboxyhemoglobin (%COHb) and end-tidal breath carbon monoxide (EtCO) levels in healthy female volunteers. METHODS: Healthy female volunteers (n=4) were exposed to 250ppm CO for 45 minutes; an initial dose of 15 minutes followed by six hourly doses of 5 minutes. %COHb and hemoglobin (Hb) were measured by head space gas chromatography and Hemocue respectively every hour following CO dosing. End-tidal breath CO was measured using a piCO Smokerlyzer non-invasive breath test both before and just following CO dosing. Data are presented as mean±SEM. Analysis was performed by repeated measures one-way ANOVA with post-hoc Dunn's test with significance of p<0.05. RESULTS: Blood %COHb increased from 0.73±0.06% at baseline to 3.05±0.27%. Increases in blood %COHb differed significantly from baseline at the 6 th and 7 th dose (p<0.05). End-tidal breath CO levels increased from 1.50±0.29 ppm at baseline to 11.63±0.90ppm, 12.00±0.79ppm and 12.50±0.84ppm after the 5 th , 6 th and 7 th doses respectively. Both blood %COHb and EtCO normalized to baseline values (p>0.05) 24 hours following initial CO dose. CONCLUSIONS: This preliminary data demonstrates that intermittent dosing of inhaled 250ppm CO is effective in raising blood %COHb and breath EtCO levels in healthy female volunteers. Translation of CO dosing protocols from animal models to healthy volunteers is the first step towards exploring the potential of CO as a gaseous therapeutic for conditions such as preeclampsia. This study is currently ongoing. Preeclampsia is a vascular disorder in pregnancy and is biochemical characterization by high soluble Flt-1 and low placenta growth factor as well as an imbalance in redox homeostasis. During conditions of high oxidative stress, cysteine residues on key proteins are reversibly altered by S-glutathionylation, modifying their function. Glutaredoxin-1 (Glrx) enzymatically catalyzes the removal of S-glutathione adducts, conferring reversible signaling dynamics to proteins with redox-sensitive cysteines. The role of Glrx in preeclampsia is unknown. METHODS: Immunohistochemistry and Western blot analysis for Glrx or glutathione were conducted on human placenta samples collected pre-term from early onset preeclamptic patients (n=10) or non-preeclamptic induced deliveries (n=9). Human endothelial cells were infected with adenovirus encoding Glrx or LacZ prior to the cells being exposed to hypoxia (0.1% O 2 , 24h) to measure changes in soluble Flt-1 (sFlt-1). Quantitative PCR and ELISA were used to measure sFlt-1 at mRNA and protein level. RESULTS: Immunohistochemical staining for GSH revealed lower S-glutathionylation adducts in preeclampsia placenta in comparison to controls. Glrx expression, which catalyses de-glutathionylation was enhanced in early onset preeclampsia compared to pre-term control samples. In contrast, no change was observed in preeclamptic and IUGR placentas at full term. In endothelial cells overexpressing Glrx, sFlt-1 expression was dramatically enhanced at mRNA (3-fold P<0.05) and protein level (5 fold P>0.01, n=4) after hypoxia andoverexpressing Glrx in mice enhanced levels of circulating sFlt-1 during in vivo ischemia. CONCLUSIONS: Enhanced Glrx expression in preeclamptic placenta in line with an apparent decrease in S-glutathionylation may leave key proteins susceptible to irreversible oxidation in conditions of high oxidative stress. The spectrum of visceral pain symptoms experienced by endometriosis patients suggests a dialogue between endometriosis lesions and the central nervous system (CNS). Sustained inflammation and production of prostaglandins is a mechanism for activation of nerve fibers innervating lesions. We hypothesize that prostaglandins play a role in establishing changes in the CNS that leads to enhanced pain perception in endometriosis. METHODS: Sensory behavior was analyzed in a validated mouse model of endometriosis compared to naïve and OVX+E2 control mice using a choice test (explorative behavior) and the Von Frey filaments test (mechanical allodynia). 5 mg/kg TG6-10-1 (EP2 antagonist) and 10 mg/kg L-161982 (EP4 antagonist) were injected i.p and the Von Frey test performed. Single immunohistochemistry, dual-label immunofluorescence, and QPCR were performed on peritoneum, lesions and L5-6 dorsal root ganglia (DRG). Western blot analysis was performed on lower lumbar spinal cord. RESULTS: Mice with endometriosis exhibited decreased explorative activity (p<0.01) and increased sensitivity when Von Frey filaments were applied to the abdomen and hindpaw (p<0.05 and p<0.01) compared to controls. EP2, EP4 and COX1 mRNAs were elevated in lesions compared to the peritoneum (p<0.001, p<0.01, p<0.001). COX2 mRNA was elevated in the peritoneum (p<0.05) and lesions (p<0.01) of endometriosis mice compared to peritoneum from naïve mice. Immunolocalisation studies confirmed these findings. EP2, COX1, SCN11A and TRPV1 mRNAs were increased in DRG from endometriosis mice (p<0.05) compared to controls. Dual immunofluorescence revealed increased TRPV1 expression in peripherin and neurofilament-200 positive DRG. Western blot analysis revealed elevated COX2 protein (p<0.01) in the spinal cord. CONCLUSIONS: Endometriosis mice exhibited significant changes in sensory behavior and an increase in the expression of nociceptive ion channels in DRG. This is linked to expression of COX2 and thus an increased ability for prostaglandin synthesis in the lesion and peritoneal environment (peripheral) and in the spinal cord (central). These data suggest prostaglandin receptor antagonists may alleviate endometriosisassociated pain.

The 

The rat "dyspareunia model" was adapted from the method of Berkley et al. In this model endometriosis is produced in cycling rats by an auto-transplantation of uterine tissue punches onto abdominal arteries of the mesentery and the colon where they develop into cysts. The surgery induces vaginal and visceral hyperalgesia, whose severity is greatest in proestrus and which can be measured by the vaginal distension method. Furthermore, the cysts as well as dorsal root ganglia (DRG) from animals undergoing surgery were analyzed by an immuno-histochemical approach in order to validate the innervation of the cysts and the activation of the respective DRGs. RESULTS: Our data indicates that we developed a robust model to measure hyperalgesia in order to test clinical candidates for the treatment of endometriosis. Immuno-histochemical analysis of the marker PGP9.5 (nerve fibers) and CGRP (C-fibers) showed a high innervation of the cysts. This finding might underline the measured hyperalgesia in this model. Immuno-histochemical analysis of DRGs has revealed important information about the expression levels of different pain markers relevant for disease progression. CONCLUSIONS: With the development of a rat "dyspareunia model" and the histological approach for target validation we attained useful tools to test clinical candidates for the treatment of endometriosis. are considered significant sources of stress, and we have recently demonstrated that stress can worsen an animal model of endometriosis. In many chronic conditions exercise can act as a stress buffer, both exercise and stress known to influence pain perception. Depending on the nature of stressors, different mechanisms may be activated leading to analgesia/ hyperalgesia. We tested the effect of forced swimming (a physical stressor) on pain perception and receptor expression in endometriosis. METHODS: Endometriosis (endo) was induced in female rats by suturing uterine horn tissue next to the intestinal mesentery. Sham rats had sutures only. Some rats were exposed to swim stress for 7 consecutive days; no stress were left in home cage. Fecal pellets were counted (FPC) during the stress protocol as a measure of anxiety, and corticosterone levels (CORT) measured. Changes in pain perception were assessed using the hot plate test for hyperalgesia and Von Frey test for allodynia. Mu opioid receptor (MOR) and NK-1R expression in the spinal cord was measured by immunofluorescence. At sacrifice tissues were collected and the vesicles measured. RESULTS: Stress groups gained less weight during the protocol. The endo-stress group had higher anxiety levels (FPC) than endo-no stress (p<0.01) and sham-no stress (p<0.05); the sham-stress group also had higher anxiety levels (FPC and CORT) than no stress groups (p<0.05). Endo-stress rats had higher colonic damage (p<0.05) and more cellular infiltration. Surprisingly, vesicles in the endo-stress animals were smaller than in endo-no stress. Forced swimming helped ameliorate hyperalgesia in sham but not endo whereas it improved allodynia in both endo and sham animals. MOR expression was significantly higher in endo-stress versus endo-no stress (p<0.01) similar to sham-no stress levels. In 40% of women, no underlying pathology is identified, and the pain is very difficult to manage. Gabapentin has been shown to be effective for other chronic pain conditions. It is being increasingly prescribed for CPP despite no evidence of efficacy.

We carried out a pilot RCT assessing the efficacy of gabapentin compared with placebo in the treatment of CPP to inform the planning of a future RCT. The primary objective was to determine recruitment and retention rates in two UK centres within defined inclusion/exclusion criteria. The secondary objectives were to determine the effectiveness and acceptability to patients of the proposed methods of recruitment, randomisation, drug treatments and assessment tools. A probabilistic decision analytical model was used to estimate the potential cost-effectiveness of gabapentin. RESULTS: 34% (47) of eligible women were randomised into the study (September 2012 to 2013), and 53% were followed up until six months with completed pain scores. The majority of participants described their overall trial experience favourably: positive responses were recorded for the recruitment approach used, paperwork burden for participants and the experience of randomisation. Analysis of one of the pain scores (BPI questionnaire) indicated that patients randomised to gabapentin perform better than placebo: 1.72 points, 95%CI: 0.07 to 3.36 at six months (p=0.041). We showed that, at the typical threshold for willingness to pay in the UK, there is a greater probability of gabapentin being cost-effective compared with no active treatment. CONCLUSIONS: Our results underline the need for a definitive multicentre trial evaluating the clinical and cost effectiveness of gabapentin in women with pelvic pain, and reinforces the need to adopt a conservative estimate for recruitment and randomisation, and use innovative ways of dealing with missing data.

The The lymphatic dissemination theory of endometriosis is proposed to explain the presence of endometriotic tissue in lymphatic vessels and lymph nodes, lesions in rare sites and high reoccurrence rates following treatment. Despite the likely importance of the lymphatic system in many aspects of endometriosis, detailed knowledge is lacking. Therefore, we aimed to examine endometrial-like cells and immune cell populations in uterine-draining lymph nodes (throughout the menstrual cycle) and nodes associated with deep infiltrating bowel endometriosis. METHODS: Immunohistochemical staining was performed on paraffinembedded uterine-draining iliac lymph nodes (endometriosis = 6, control = 9) and deep infiltrating endometriotic bowel lesion-associated nodes (n = 12). Cell numbers (percentage positive area) and antigen expression (optical density) of CD10+ endometrial-like cells; CD3+, CD4+, CD8+ and FoxP3+ T cells; DC-Lamp+ and DC-Sign+ dendritic cells; CD20+, CD79+ and plasma B cells; CD68+ macrophages and CD57+ natural killer cells were measured. RESULTS: In uterine-draining iliac nodes, DC-Sign+ numbers and CD4 expression peaked during menstruation (p=.027, p=.03; respectively), and CD20 expression significantly decreased between the proliferative and secretory phases (p=.04).In deep infiltrating bowel lesion associated nodes compared to iliac nodes, CD10+ endometrial-like cells were decreased (p=.002), as were DC-Lamp+ (p=.001), CD20+ (p<.001) and CD57+ (p=.005) numbers and DC-Lamp (p<.001), CD79 (p=.011) and CD57 (p=.012) expression; while CD3+ (p=.002) and CD4+ (p=.007) numbers, and FoxP3 (p=.018) expression were increased. CONCLUSIONS: This study provides new evidence for lymphatic and immune system involvement in the development and progression of endometriosis. Cyclical immune changes in uterine-draining iliac lymph nodes are linked to their essential roles in the regulation of the inflammatory process of menstruation. In bowel lesion-associated nodes, endometrial-like cells are present and immune responses are altered compared to uterine-draining nodes indicating decreased capacity to contain endometrial-like tissues. This may contribute to spread and/or reoccurrence of the disease. INTRODUCTION: Glycosylation is one of the most common posttranslational modifications of eukaryotic proteins and is known to undergo dynamic changes in a wide range of biological processes. To date, however, the glycan expression profiles in endometriosis are largely unknown. The objective of the study was to identify the panel of glycans that were aberrantly expressed in endometriosis, a hormone-dependent disease.

The glycan expression profilesin primary cultured human endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) were determined by lectin microarray analysis. Distribution of Wisteria floribunda agglutinin (WFA)-binding glycans in ovarian endometriotic cysts and eutopic proliferative phase endometrium were assessed by lectin histochemistry. The expressions of N-acetylgalactosaminyl transferases that synthesize WFA-binding glycans were evaluated in ECSCs and NESCs.

We found that the levels of WFA-binding glycans were decreased in ECSCs. Lectin histochemistry revealed that WFA-binding glycans were decreased only in the stromal components of the ovarian endometriotic cysts, but not in the epithelial components, compared to the eutopic proliferative phase endometrium. The expressions of N-acetylgalactosaminyl transferases that synthesize WFA-binding glycans were downregulated in ECSCs. CONCLUSIONS: Utilizing lectin microarray analysis and lectin histochemistry, we found that WFA-binding glycans were decreased in endometriosis. The synthetic enzymes of WFA-binding glycans were significantly downregulated in ECSCs. It is suggested that reduced expression of N-glycans with WFA-binding properties on ECSCs is a novel characteristics of endometriosis.

Revealing Even though it is widely accepted that certain cell types participate differently in its pathogenesis, gene expression studies are often performed on tissue homogenates, potentially leading to wrong estimates of expression changes. In this study we aim to contribute to a better disease understanding of endometriosis by analyzing mRNA expression separately in the pathological relevant cell populations.

For gene expression analysis eutopic and ectopic endometrial specimens from patients with endometriosis as well as normal endometrium from healthy control donors were received from the Department of Gynecology and Obstetrics of the University Hospital in Vienna. The separation of epithelial and stromal endometrial cells was performed by laser capture microdissection (LCM) on 10 µm thick fresh frozen tissue sections. After mRNA isolation, potential differences in its expression were subsequently examined by an Affymetrix Human Gene 2.1 ST array.

The mRNA expression analysis in separated cell populations of secretory phase eutopic and ectopic patients' endometrium versus normal control endometrium identified over 500 genes which were significantly differentially expressed among study groups. A subsequent pathway analysis identified candidates of the WNT pathway and nitric oxide signaling, as well as mast cell specific genes, growth factors and prostaglandins to significantly differentiate between eutopic and ectopic endometrial tissue in stromal and/or epithelial cells. CONCLUSIONS: Detailed data evaluation needs to reveal whether one or more candidates could serve as a biomarker or potential target structure for the treatment of endometriosis.

Alexis Laux-Biehlmann, Janina Boyken, Markus Koch, Henrik Dahllof, Thomas M Zollner, Jens Nagel, Nicole Schmidt. GDD-TRG Onc/GT Gynecological Therapies, Bayer Pharma AG, Berlin, Germany. INTRODUCTION: Retrograde menstruation describes the escape of menstrual debris including endometrial tissue through the fallopian tubes into the pelvis. The general accepted hypothesis is that such uterine tissue delivered to the peritoneum via retrograde menstruation causes inflammation and that additional mechanisms such as tissue repair, proliferation and scar formation potentially lead to the development of endometriosis (Sampson's Theory). However, the pathology and the underlying biologic processes of this phenomenon cannot be investigated so far due to the lack of an appropriate animal model. Here we show that retrograde menstruation mimicked by inoculation of uterine tissue in the peritoneal cavity of mice induces an inflammatory environment in the peritoneum as well as spontaneous pain. METHODS: Syngeneic uterine tissue has been injected from donor mice into recipient mice. Inflammation has been analyzed by detection of proinflammatory cytokines in the peritoneal lavage and in the injected tissue using a multiplex ELISA. Immune cells of the peritoneal lavage have been analyzed using flow cytometry. Non-evoked pain responses have been detected by the newly developed dynamic weight bearing system. RESULTS: Pelvic inflammation characterized by elevated levels of pro-inflammatory cytokines in the peritoneal cavity was present four days after inoculation. Furthermore, murine retrograde menstruation triggers a rapid elevation of neutrophils into the peritoneum followed by an influx of other innate immune cells underlying the inflammatory process induced by the presence of uterine tissue in the peritoneal cavity. Spontaneous pelvic pain behavior has been detected within the four days after inoculation of the uterine tissue. In addition, we show that the amount of uterine tissue present in the peritoneum impacts the intensity of both inflammation and pain. CONCLUSIONS: Our findings indicate that uterine tissue present in the peritoneal cavity induced by retrograde menstruation can induce processes associated with inflammatory pain in dependence of the amount of uterine tissue with possible implications for the mechanisms underlying the development of endometriosis. In this study, we tested the hypothesis of whether serum cytokines, lipids or their combination has better discrimination potential.

Detection and identification of serum lipids was done via mass spectrometry-based lipidomics. Internal standards were used for relative quantification of >150 phospholipids and sphingolipids. 27 immunomodulatory compounds were assayed using the Luminex technology. Data analysis of cytokine data was carried out using fiveparameter logistic regression modeling. Support Vector Machines, Orthogonal Partial Least Squares and Receiver Operating Curve were used for modelling and feature ranking.

The top ranked 5 lipids gave AUC of 0.712. The top ranked 3 cytokines performed the worst (AUC=0.571). IL-6 alone was better (AUC=0.611). Combining 5 lipids and 3 cytokines gave the best diagnostic sensitivity and specificity among severe endometriotic patients and controls (AUC=0.804). However, cytokines gave marginal increase in predictive power when combined with the 5 selected lipids. CONCLUSIONS: Here, we report an integrated approach to discovering non-invasive predictors to derive a focused set of molecular markers that correlated well with endometriotic patient outcomes. We found that serum molecular markers are not uniformly diagnostic. Contrary to other reports, cytokine levels have marginal diagnostic value. Conversely, serum lipids hold great predictive potential in diagnosing endometriosis. Further validation of these lipids are required.

Circulating Endometriosis is a common condition affecting an estimated 6-10% of reproductive-aged women, with an increased prevalence in women with infertility (25-50%) and pelvic pain (70%).

Unfortunately, diagnosis is often delayed by several years, and the gold standard for diagnosis remains laparoscopic identification of endometriotic lesions. There is a need for a non-surgical diagnostic test, but as of yet, no such test has been elucidated. We aimed to identify serum biomarkers of endometriosis that could serve as a minimally invasive means of diagnosis. METHODS: Serum was obtained from 12 women with stage III/IV endometriosis and 12 non-endometriosis controls. Sera was analyzed using a validated serum proteomics approach and analyzed via mass spectrophotometry. Peak heights were analyzed between cases and controls using student's t-test. Potential biomarker panels were identified using forward selection logistic regression. RESULTS: 39 potential biomarkers were found, and 6 of these were identified using MS/MS fragmentation. Endometriosis is an inflammatory disease that affects up to 10% of reproductive age women. Our objective was to dissect the pathophysiology of endometriosis contributed specifically by the epithelial and stromal cells of the eutopic endometrium. METHODS: Each proliferative, eutopic, endometrium sample was digested and sorted for epithelial and stromal cells by FACS and divided for paired RNA sequencing (RNA-Seq) and miRNA microarray. Cell type specific differentially expressed genes and miRNA were validated, with statistical significance, and analyzed by Ingenuity Pathway Analysis networks (IPA). Within each cell type, miRNAs and their targets were identified and validated with miRNA mimics and antagomirs. RESULTS: Over 23,900 genes and 1300 miRNAs were detected from the epithelial and stromal cells collected from normal and endometriosis subjects, with distinct clustering between endometrial epithelial versus stromal cells and normal versus disease. Compared to control, there were 220 and 154 differentially expressed genes from endometriotic epithelial and stromal cells, respectively, with only five common differentially expressed genes between these two cell types (CSF3, LTBP4, SERPINA1, ASH1L, PIK3R1). There were 16 and 9 differentially expressed miRNAs from endometriotic epithelial and stromal cells, respectively (Epithelial: miR-375, 1827, 504, 548n, 4450; Stromal: miR-200b, 204, 1297, 548o, 4445) . Specifically, endometriotic epithelial cells expressed genes and miRNAs that stimulate immune cell trafficking, inflammation, and vascularization through the IL1/AKT pathways. In contrast, endometriotic stromal cells expressed genes and miRNAs known to modulate immune cell trafficking, inflammation and phagocyte maturation through the NFkB/ERK pathways. Crucially, the differentially expressed miRNAs were shown to regulate the pathways of IL1/AKT and NFkB/ERK in endometriotic epithelial and stromal cells respectively. CONCLUSIONS: Eutopic endometrial epithelial cells and stromal cells have distinct differential gene and miRNA expressions and unique pathways in endometriosis patients. Distinguishing between epithelial and stromal cells has clarified the two distinct sets of genes and miRNAs that are differentially expressed in endometriosis yet have common functions of immune cell trafficking and inflammation. Although several studies have identified multiple contributory factors responsible for uterine fibroid growth and proliferation, it is not well documented as to the role vitamin D3 plays in modulating the expression of proteins in uterine fibroid cells.

The focus of this study is to evaluate protein factors that are modulated by vitamin D3 in uterine fibroid cells. METHODS: Immortalized human uterine fibroid (HuLM) cells were serum starved and treated with or without vitamin D3 (100 nM) for 48 hours. Protein lysates were prepared from these treated cells, digested in gel with a proteolytic enzyme to generate shorter peptides, and then analyzed for protein expression using shotgun proteomics. This quantitative method relies on separation after the digestion steps and takes advantage of tandem mass spectrometry to infer the amino acid sequence of individual peptides. The key SNP (rs13394619) on chromosome 2 is located within GREB1, which is a critical regulator of hormone-dependent breast cancer growth. Increased GREB1 expression has been reported previously in endometriosis. The aim of this study is to examine the mRNA transcripts and protein expression levels of GREB1 that are affected by the genetic variants associated with endometriosis risk.

We collected endometrial tissues (curette) from surgically confirmed cases and controls from 136 women. Blood samples were also collected from the same individuals for genotyping rs13394619. GREB1 mRNA expression was analysed using the Fluidigm qPCR platform. GREB1 protein expression was analysed by immunohistochemistry (IHC) and Western blotting. RESULTS: IHC localised GREB1 protein to endometrial glandular epithelium and stroma (both nuclear and cytosolic). GREB1 protein expression was observed to change across the menstrual cycle.For gene and protein expression studies, we did not observe any significant changes in GREB1 expression in endometriosis cases compared to controls. We then conducted eQTL analysis to examine if there is any effect of the key SNP (rs13394619) on GREB1 expression in the endometrial samples.

Our results showed a trend of small increase in GREB1 gene and protein expression in the individuals carrying the risk allele for endometriosis. However, this change was not significant after multiple testing corrections. Furthermore, menstrual cycle stage did not affect GREB1 expression for eQTL analyses. CONCLUSIONS: Further studies, including functional GREB1 investigations and larger sample sizes in each genotype group, are required to unequivocally decipher a role for GREB1 in endometriosis risk. As SNPs most strongly associated with disease risk are generally located in non-coding regions, key SNPs in regulatory regions of the genome will be prioritized for further analysis to examine the functional role of these SNPs on gene regulation. About 6 % of all endometriosis patients received a tissue/organ repair and/or a bowel resection during surgery. Patients with ICD-9 coding for endometriosis of the vagina/rectum or the intestine received most frequently an organ repair and/or bowel resection during surgery. The total cost per admission and the length of the hospital stay varied between the different ICD-9 617.X and combined CPT codes. CONCLUSIONS: Endometriosis is a heterogeneous disease. Severity and the associated total cost of hospitalization differ between the endometriosis sub-entities. To our knowledge this is the first study to investigate systematically the proportion and cost impact of two subentities of endometriosis.

Under The incidence of endometriosis is difficult to establish because diagnosis requires laparoscopy, ideally with histological confirmation, and women with mild or moderate symptoms may not undergo these procedures. The objective of this study was to estimate the incidence of endometriosis and to examine the potential for underrecording of endometriosis among patients with dysmenorrhea and menorrhagia (two symptoms of the disease), and patients treated with hysterectomy in a primary care database.

In The Health Improvement Network (THIN), symptoms, diagnoses and procedures are recorded using Read codes, and primary care physicians (PCPs) can provide additional information in free-text comments. We identified women in THIN database aged 12-54 years between Jan 2000 and Dec 2010 with a first recording of a Read code indicating endometriosis. Among those without a record indicating endometriosis, three separate follow-ups were performed to identify women with a Read code for hysterectomy, dysmenorrhea and menorrhagia. For a random sample of women in each of these groups, a validation process was undertaken to assess the potential for false negatives.This was achieved by the manual review of free-text comments from medical records. For women with a record of hysterectomy, the responses to questionnaires sent to their PCPs were analyzed. RESULTS: From a cohort of 866,295 women, 5087 women were identified with a record of endometriosis, an incidence of 1.02 (95% CI: 0.99-1.05) per 1000 person-years. The proportion of women with endometriosis among those with a code for endometriosis, dysmenorrhea, menorrhagia and hysterectomy was 99.4%, 3.5%, 0% and 9.5%, respectively. Extrapolating these percentages, we estimated that the number of women with endometriosis among those with a code for endometriosis, dysmenorrhea and hysterectomy was 5057, 950 and 1428, respectively. Based on this, the resulting incidence of endometriosis was estimated to be 1.49 (95% CI: 1.46-1.52) per 1000 person-years. CONCLUSIONS: Using Read codes as the only strategy to identify women with endometriosis in THIN results in underestimation of the incidence. This study shows the utility of complementing Read codes with additional data such as free text comments and information from PCPs. METHODS: Donor mRFP1 transgenic female mice were medically induced to development of menstrual endometrium, which was harvested for intraperitoneal injection into menstrually synchronized wild-type C57BL/6J recipient females. Fluorescent endometriotic lesions in recipients were measured using an in vivo imager. Recipients with and without endometriotic lesions were superovulated and mated to evaluate the impact of induced disease on number of oocytes retrieved. Immunohistochemistry was performed on lesional biopsies to validate endometrial and donor origin. RESULTS: Endometriosis was successfully induced in wild-type recipients after inoculation with mRFP1 menstrual phase endometrium.

Lesions observed at non-invasive fluorescent imaging and recovered from the peritoneal cavity of recipients were grossly consistent with endometriosis [ Figure 1 ]. Immunohistochemistry validated lesions to be endometriotic as evidenced by the presence of cytokeratinpositive epithelial cells and vimentin-positive stromal cells [ Figure 2 ]. Immunostaining for mRFP1 confirmed the donor origin of lesions. CONCLUSIONS: This model not only physiologically mimics disease pathogenesis in humans, but also provides an in vivo design for quantifying and longitudinally monitoring disease burden. The model is well suited for efficacy studies exploring novel treatments for endometriosis. * Figure(s) On day 29, prior to the second treatment period, geometric mean plasma concentrations of LNG and ATZ, respectively, were 0.228 µg/L and 12.5 µg/L for the low dose, 0.269 µg/L and 19.8 µg/L for the mid dose and 0.384 µg/L and 37.3 µg/L for the high dose; results were similar on day 57. Over the treatment period, mean FLS sizes were higher compared to pre-treatment; more women in the mid and high dose groups had FLS³30 mm during day 1-28 and day 29-57 (32%-45%) than those in the low dose group (14%-24%). A dose-dependent decrease in ovulation frequency was observed; during day 29-57, 57% of women ovulated in the low dose group compared with 32% in the mid dose and 25% in the high dose groups. No women had E2 serum level <20 pg/mL in the low dose group whereas one woman in each of the mid and high dose groups had low levels during treatment. All ATZ and LNG combinations showed good tolerability. CONCLUSIONS: Anticipated exposure levels were reached for both components of the IVR. The safety profile of the ATZ and LNG IVRs corresponded well with that known from other dosing regimens of the study drugs. These results facilitated the optimization of the effective dose and exposure of ATZ and LNG for further clinical studies.

The (1)To establish an appropriate time for urine collection. Samples are collected early morning and during laparoscopy.

(2)To establish differences between the urinary proteomes from women with and without endometriosis. (M) in that they produce excessive and disorganized extracellular matrix (ECM) proteins. Increased ECM production suggests increased transcription of the genome and a loss of the regulatory mechanisms that control ECM production. This increased transcriptional activity directly contributes to the bulk of these tumors and symptomatology. The Polycomb group proteins are a group of histone associated protein complexes that regulate the epigenetic control of transcription. One set of proteins, PRC2, function to suppress transcription by modifying histones. EZH2 is the histone methyltransferase (HMT) of PRC2, that adds methyl groups to histone H3 at lysine residue 27, creating H3K27me3. This modification results in condensed chromatin, limiting accessibility of the genome to transcription factors. Tri-methylation of histone H3 at lysine residue 4 yields a mark of increased transcription, H3K4me3. Careful regulation of H3K27 and H3K4 methylation may be essential for normal ECM production. Our objective is to study the role of tri-methylated H3, and the relationship between histone modification in human L cells, and the control of ECM production. Progesterone plays a key role in regulating the biologic behavior of uterine leiomyomas (fibroids). The objective of this study was to delineate the key mechanisms by which activation of the progesterone receptor (PR) promotes leiomyoma growth.

After obtaining IRB approval, total RNA was prepared from matched specimens of human myometrium and leiomyomas using the miRVana Kit (Ambion). Gene expression was profiled using the Illumina WG Bead Chip v2.6. After quantile normalization, patterns of gene expression were compared using two-way Anova with multiple hypothesis testing adjustment by Benjamini-Hochberg procedure. Levels of specific gene products were confirmed by Western blot (WB) and quantitative real-time PCR (qPCR). ChIP-Seq was used to identify PR binding sites. RESULTS: 175 distinct gene products were either up-(n=143) or down-regulated (n=35) >1.5-fold when leiomyomas were compared to myometrium (n=8 each, p<0.05). A novel bioinformatic algorithm integrating patterns of gene expression with GeneMania networks pinpointed SFRP4 as a key target a) overexpressed 2.6-fold in leiomyomas (P<0.01) and b) whose expression is >2-fold greater in specimens collected during the proliferative phase as compared to specimens from secretary phase and/or menopause(P<0.01). Using 26 additional matched specimens, we confirmed that overexpression of SFRP4 during the proliferative phase is a key feature of uterine leiomyomas that persists in primary culture. Levels of SFRP4 in primary cultures of leiomyoma were found to increase concomitantly with the loss of PR expression induced by serial passage. ChIP-Seq and ChIP-PCR confirmed direct interaction of PR with the SFRP4 promoter region (n=4).

The robust overexpression of SFRP4 found in uterine leiomyomas is dramatically suppressed by PR activation, potentially contributing to their enhanced growth during secretory phase of the menstrual cycle. Future work will focus on determining the mechanisms by which PR-induced suppression of SFRP4 impacts uterine leiomyomas and assessing whether these pathways can be used to target smooth muscle tumorigenesis.

Development 

Human uterine fibroids received from surgical interventions and with written informed consent from the patients were cut into small pieces and transplanted subcutaneously into E2 + P4 supplemented SCID mice. Grafts were grown up to 60 days and analyzed. RESULTS: Human uterine fibroid xenografts showed a clear dependency on optimal concentrations of E2 and P4 for their growth in SCID mice. When analyzed by immunohistochemistry, BrdU incorporation showed homogeneous growth of the grafts. They retained the characteristic deposition of ECM proteins and the whirl-like interwoven organization of the smooth muscle cells. Growth of the grafts is slow and heterogeneous over time, as has been shown for uterine fibroids in situ. Treatment of mice with the antigestagen mifepristone and the mTOR inhibitor rapamycin inhibited uterine fibroid growth in this model.

We developed an optimized animal model for the growth of human uterine fibroid tissue in SCID mice. The model retains all typical characteristics of uterine fibroids analyzed so far. Mifepristone showed the expected growth inhibiting activity seen for antigestagens in women in the clinics. Furthermore, for the first time we could show inhibition of growth of naïve human uterine fibroid tissue with the mTOR inhibitor rapamycin, proving the value of this model to validate new treatment paradigms for the human disease. This characteristic enables them to serve as potential serum biomarkers. The role of miRNA in ovulation has been researched, but the specific miRNAs involved and their mechanism of action are not known. We aimed to discover their expression profile in human ovulation. METHODS: Women diagnosed with polycystic ovarian syndrome (PCOS) and normally ovulating women undergoing IVF treatment were enrolled. Serum samples were collected at early follicular phase and at the ovum pick up day along with granulosa lutein cells (GLC) originated from follicular fluid aspirates. Total RNA was extracted from GLCs and serum using a specific protocol. A global analysis approach was employed to uncover miRNA expression profile using nCounter miRNA expression assay at NanoString Technologies. A total of 800 human miRNAs were simultaneously assayed. nSolver software was used for analysis of the results and quantile for data normalization.

A unique set of 17 miRNAs was differentially expressed in the serum of normal ovulating women between early follicular phase and immediate post ovulation. Five miRNAs expression were decreased and 12 were elevated during the cycle. Pathway analysis revealed that these miRNAs target key players in the PI3K/AKT and TGFβ signaling pathways. Comparing PCOS patients to normal ovulating women serum at the early follicular phase identified 29 differentially expressed miRNAs. Twenty seven circulating miRNAs were differentially expressed between the groups immediate post ovulation. Similar pattern of reduced expression was found for 6 miRNAs in the PCOS group and 10 in the normal ovulating, in GLC isolated from these women. CONCLUSIONS: There is a unique miRNAs expression profile during different phases of the human ovarian cycle. These miRNAs can be detected in the human serum. Major cellular signaling pathways might be regulated by these miRNAs. Anovulatory women exhibit altered miRNA expression profile. This unique profile might be a reflection of an aberrant ovarian function in anovulatory women. miRNAs expression profile might serve as a novel serum-based biomarker, potentially offering insight into the human ovulation. (SCD) is a hematologic condition that may result in endocrine dysfunction, such as in the thyroid or pancreas; however, little is known about its effect on ovarian function. In this prospective study, we aim to characterize ovarian function in reproductive age women with SCD. METHODS: Reproductive age women (<51) with SCD were solicited for measurement of endocrine parameters related to the hypothalamopituitary-ovarian axis (n=19). All patients were on progestin only contraception, precluding appropriate timing of blood draws to cycle day 3. Random assessment was made of FSH, LH, estradiol, AMH, and progesterone. Ferritin was used as an iron overload marker and hemoglobin electrophoresis to characterize percent HbF/HbA2/HbA/HbS. Patients were divided into two groups for analyses: group 1 with serum profiles suggestive of ongoing ovulation (low/normal FSH <20 U/L, high estradiol and elevated progesterone) and group 2 with elevated FSH and concurrent low serum estrogen and progesterone.

While FSH was elevated in 42% of patients (n=8), similar levels of iron overload were observed between groups (p=0.29). We observed differences in the mean age between the normal and elevated groups, 32.7 and 39.1 years, respectively. * Figure(s) While abnormal chromosome complement of the conceptus has been accepted as the most common cause for all miscarriages including recurrent miscarriages, immunologic causes have been proposed to be the cause of the majority of heretofore unexplained recurrent pregnancy losses. The most frequently studied risk factors to identify an immunologic cause have included elevated serum levels of antiphospholipid antibodies (APA) and natural killer (NK) cell killing. The purpose of this study is to determine the prevalence of elevated APA and NK cells among 420 women with histories recurrent pregnancy loss and to compare the frequencies of normal and abnormal karyotypes among the abortuses of women with a diagnosis of immune with non-immune causes of their recurrent pregnancy losses.

Clinical histories of 420 women with a history of recurrent pregnancy loss were reviewed and results of laboratory tests including antiphospholipid antibodies and circulating natural killer cells were recorded as well as karyotypes of their products of conception .

The prevalence of immune risk factors among recurrently aborting women was 29% (123/420) Of the 309 karyotypes of products of conception from women with a history of recurrent miscarriage, 231 (75%) had a normal chromosome complement and 78 (25%) were abnormal. Among the normal karyotypes, 121 (53%) were 46XX and 110 (47%) were 46XY. Similarly, when karyotypes of abortuses from women with immunologic risk factors were evaluated, 76% (80/105) were normal and 24% (26/105) were abnormal and 43% (34/80) of normal karyotypes were 46XX and 57% (46/80) were 46XY. The frequency of chromosomally normal pregnancy losses among women with a history of recurrent pregnancy loss is significantly elevated compared with sporadic abortion is significant (P<0.0001). CONCLUSIONS: Recurrent pregnancy loss is associated with immunologic risk factors in 29% and a 75% frequency of chromosomally normal pregnancy losses. years and now reside in all states of the US. Proximity to major roadways and outdoor levels of particulate matter less than 10 microns (PM 10 ), between 2.5 and 10 microns (PM 2.5-10 ), and less than 2.5 microns (PM 2.5 ) were determined for all residential addresses for 36,294 women from 1993 to 2003. Infertility was defined by report of attempted conception for ³12 months without success. Participants were able to report if evaluation was sought and if so, offer multiple clinical indications for infertility. Multivariable adjusted Cox proportional hazard models were used to estimate the relation between distance to road or PM exposures and infertility risk. Polytomous regression was used to assess potential differences in associations with primary and secondary infertility. RESULTS: Over 213,416 person-years, there were 2,508 incident reports of infertility. Results for overall infertility were mixed, however there were statistically significant elevations in risk in the group of primary infertility. The multivariable adjusted HR (95%CI) comparing those living closer to a major road vs living farther from a major road with primary infertility was 1. Median hormone levels (IQR: Q3-Q1) were significantly different between groups (Table) . Serum relaxin was undetectable in patients lacking a CL (<7.8 pg/ml). Those with one CL exhibited a median relaxin value of ~180 pg/ml. As the number of CL increased, so did the serum concentration of relaxin. Relaxin is significantly associated with egg number retrieved in autologous IVF cycles (Pearson Coefficient=0.42, p=0.008). CONCLUSIONS: Relaxin levels in early pregnancy varied depending on the number of corpora lutea. Further study is needed to examine the physiological impact of absent or supraphysiologic levels of relaxin associated with fertility treatments as currently employed. .01), a larger number of follicles >14mm at the time of IUI (p< 0.01) and higher pregnancy rates (6.6%) versus the ovulation induction group (6%) and natural cycle group (4.4%), (p= 0.03). Despite higher pregnancy rates, the live birth rate in the gonadotropin group did not differ significantly. When controlling for confounding factors, logistical regression revealed that the type of stimulation cycle still predicted pregnancy success rates (p= 0.03). CONCLUSIONS: Donor sperm IUI is a good option for women trying to conceive with an overall pregnancy rate of 17% per cycle. Gonadotropins produced higher pregnancy rates over ovulation induction and natural cycles. Live birth rates, however, were not statistically significant between the groups. This suggests natural cycle IUI may be as effective as gonadotropin injections in this population of women.

The INTRODUCTION: It has been well established that estrogen, hypothalamic progesterone synthesis, activation of the progesterone receptor, and the kisspeptin neuron are all necessary components of estrogen positive feedback which results in the LH surge; however, the specific molecular mechanisms involved within the kisspeptin neuron are unclear. The objective of this study was to characterize the progesterone receptor (classical membrane progesterone receptor (PR) or membrane progesterone receptors derived from the progestin and adipoQ receptor family (PAQR)) involved in the rapid cell signaling that occurs after progesterone (P 4 ) stimulation in the kisspeptin neuron. METHODS: Immortalized Kiss1 cells derived from adult, female mouse hypothalamus were thawed and plated on poly-D-lysine coated glass coverslips and maintained in DMEM/ 10% FBS at 37 o C with 5% CO 2 for 2-3 days. The cells were then loaded with a calcium (Ca 2+ ) indicator dye (4.5mM Fluo-4 AM) for 45 minutes and subsequently placed into a quick exchange platform for imaging on the LSM 710 confocal microscope. Gravity perfusion treatment with HBSS, 1nM P 4 , and 10nM RU486 (a P 4 antagonist acting at the classical PR) was performed with continuous vacuum suction. Fluo-4 AM imaging was performed using a waterimmersion objective with 488nm laser excitation and emission monitored through a low-pass filter with a cutoff at 505nm. The increase in Ca 2+ fluorescence (measured in relative fluorescence units or RFU) following P 4 stimulation with or without RU486 pretreatment was compared with 2-way ANOVA. In both groups, despite quality blastocysts, aneuploidy was detected by CGH in 27% of GS blasts and 74% of Infertile blasts. Moreover, 12 out of 82 Infertile women did not produce any chromosomally normal blasts; 85% of which were complex aneuploidy. Clearly, morphologic description alone, without PGS, has a poor predictive value for top quality euploid blastocysts. CONCLUSIONS: This study confirms a high rate of naturally occurring aneuploidy in normal fertile women. Only 25% of GS oocytes developed into healthy euploid blastocysts. However, in the female factor infertility group, only 8% of oocytes developed into healthy euploid blastocytes. Despite both groups producing good numbers of oocytes, we found a high occult rate of aneuploidy in both groups. These results suggest that embryo selection for transfer is difficult at best on Day 2 to 3, will be improved by morphologic progression to good quality blastocyst and optimized by the addition of PGS. We have previously characterized fibroblasts isolated from adhesion tissues (ADF) to manifest a persistent pro-oxidant state as compared to normal peritoneal fibroblasts (NPF) isolated from the same patient(s). Hypoxia and resultant oxidative stress causes NPF to irreversibly acquire the adhesion phenotype. Moreover, ADF manifest an overexpression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a key superoxide producing oxidant enzyme. Increased NADPH oxidase activity has also been associated with a tyrosine to histadine switch resulting from a specific C242T SNP (rs4673). The objective of this study is to determine the presence of this SNP in ADF and whether hypoxia induces this SNP in NPF. We will also evaluate expression of cytidine deaminase, an enzyme responsible for converting cytosine bases to uracil, in NPF and ADF cells under normal and hypoxic conditions. METHODS: Primary cultures of fibroblasts were previously established from normal peritoneal and adhesion tissues from the same patient. The NADPH oxidase SNP (rs4673) was analyzed using specific TaqMan probes that quantitate the abundance of this polymorphism in NPF and ADF before and after exposure to hypoxia (2% O 2 , 24 hrs). Assessment of cytidine deaminase mRNA levels utilizing real-time RT-PCR was also performed.A Student's t-test was used and values of p < 0.05 were considered statistically significant. RESULTS: ADF exhibited increased in the NADPH oxidase polymorphism (53.7%, p<0.05) as compared to NPF (25.2%, p<0.05). Exposure of NPF to hypoxia significantly increased acquisition of this polymorphism, from 25.2% to 51.8% (p<0.05). Levels of cytidine deaminase were significantly increased in both NPF exposed to hypoxia (28%, p=0.0452) and in ADF (37%, p=0.0187). CONCLUSIONS: Hypoxia resulting from tissue injury triggers a genotype switch in NADPH oxidase, and possibly other key oxidants and antioxidants, leading to the persistence of a pro-oxidant state, which contributes to the development of postoperative adhesions. The mechanism of this genotype switch involves the upregulation of cytidine deaminase. Scavenging oxidants as well as targeting cytidine deaminase may have beneficial preoperative effects. infertility; to evaluate risk of cancellation and ovarian hyperstimulation; and to compare pregnancy and live birth rates among women using ART with endometriosis-associated as compared to male factor infertility.

We assessed endometriosis trends among all ART cycles performed in the United States between 2000-2011 (n=1,589,079) using the National ART Surveillance System. Log binomial regression was used to compare complication rates (per cycle start) and success rates (per embryo transfer) among fresh, non-donor ART cycles performed for couples with endometriosis-associated, excluding concurrent male factor, infertility (n=112,475) as compared to male factor, excluding concurrent endometriosis-associated, infertility (n=375,557). Generalized estimating equations were used to account for clustering by clinic. 

The percentage of ART cycles with a diagnosis of endometriosis decreased over the 12-year study period. As compared to male factor, endometriosis is associated with an increased cancellation and decreased hyperstimulation risk. Among non-cancelled cycles, the likelihood of live birth is statistically different between cycles associated with either endometriosis or male factor infertility. Here, we analyzed mt content in granulosa cells of normal weight (NW) and obese (OB) infertile women. Moreover, we evaluated the possible effect of supplementation with the insulin-sensitizer myo-inositol (MI) together with alpha-lipoic acid (ALA), a natural antioxidant, co-factor in the mt respiratory chain, able to reduce OxS both in animal models and in humans.

were recruited at scheduling for IVF. In the 2 months preceding IVF, all women were supplemented with folic acid (FA). Four OB women were randomly assigned to FA +MI +ALA (Sinopol, Laborest Spa).mtDNA levels, accounting for the mt content, a marker of OxS, were measured in granulosa cells obtained by centrifugation of the follicular liquid, by Real Time PCR relative quantification.

Women age, as well as characteristics of infertility, was not different among groups. OB supplemented only with FA presented significantly higher mtDNA levels compared to NW-FA. OBsupplemented with FA+MI+ALA had significantly lower levels than OB-FA and they were not significantly different from NW-FA. *Figure(s) will be available online. CONCLUSIONS: Clarifying the mechanisms responsible for adverse effects of obesity on reproduction is needed for novel treatment approaches. These preliminary data show significant differences in mtDNA levels of granulosa cells between OB and NW assuming routine FA. This might represent the increased OxS occurring in the OB ovarian compartment. Supplementation with FA +MI +ALA in OB seems to reduce mtDNA levels, which might lead to an improvement of the altered panel occurring in obesity.

Further studies are ongoing to confirm these preliminary data.

Thresholds. Molly B Moravek, John Zhang, Randall B Barnes, Jared C Robins. Reproductive Endocrinology and Infertility, Northwestern University, Chicago, IL, USA. INTRODUCTION: It is widely thought that in vitro fertilization (IVF) pregnancy rates increase with increasing numbers of oocytes retrieved and, therefore, oocyte or embryo banking may increase overall pregnancy rates. We sought to develop an age-specific prediction model of pregnancy rate (PR) based on the number of oocytes retrieved. METHODS: Data were collected from IVF retrievals at a single academic institution over 2 years. Variables included patient age, AMH, total FSH used, number of oocytes obtained, number of lead follicles, number of embryos transferred and PR. Data were analyzed using Chi square, linear regression, and stepwise logistic regression with SPSS software. RESULTS: A total of 874 cycles were reviewed with an overall PR of 55.5%. The number of oocytes retrieved in a single cycle was highly predictive of PR (p<0.01), and remained statistically significant when controlling for age, AMH and total FSH used. However, the number of oocytes retrieved in a single cycle demonstrated an age-related threshold effect above which increased oocyte yield did not further improve PR: >15 oocytes at <36 years, >10 oocytes between 36-38 years, and >5 oocytes at >38 years. Cumulative PR by the sum of oocytes retrieved over two cycles was compared to the nomograms for single cycles; for a given number of oocytes, the pregnancy rate was higher if the oocytes were derived from a single cycle rather than over the course of two cycles. *Figure(s) will be available online. CONCLUSIONS: While PR improves with higher oocyte yield, there is an age-specific threshold beyond which no further benefit is noted. The establishment of clinic-specific nomograms can be used to counsel patients on their individual fecundability during an IVF cycle based on ovarian response. Furthermore, these data suggest that egg or embryo banking, particularly in older patients, may not significantly improve overall pregnancy rates.

The Anti-Mullerian hormone (AMH) and antral follicle count (AFC) are two promising marker for prediction of ovarian response in women treated with IVF. There are little and inconclusive data evaluating the prognostic value of AMH and antral follicles in predicting the ovarian response in intrauterine insemination (IUI) cycles. We conducted a retrospectively analysis in order to evaluate the role of AMH and of antral follicles' different cohorts (follicles of 2 to 4 mm, 2 to 6 mm, 2 to 9 mm) in predicting the ovarian response in 275 infertile women aged <40 years who underwent their first cycle of controlled ovarian hyperstimulation (COH) for IUI treatment.

Ovarian stimulation was achieved with gonadotropin. Follicular development was monitored by ultrasound and E2 plasmatic levels. Hyporesponse was defined as only one follicle >17 mm (and no other follicles ³14 mm); normal response was defined as two or three follicles ³14 mm (maximum two follicles >17 mm) and no need for dosereduction during stimulation; hyperresponse was defined as more than three follicles ³14 mm (and maximum two follicles >17 mm) on the day of HCG or dose reduction during stimulation. A single IUI per cycle was performed at 34-36 hours after hCG injection.

One-way analysis of variance (ANOVA) or Kruskal-Wallis test was used for analyses of a trend across response group. Separate logistic regression analyses were used to predict ovarian hypo-and hyperresponse. RESULTS: We found that 83/275 (30%) were hyporesponse, 148/275 (54%) normal response and 44/275 (16%) hyper response. Significant trends across groups were observed in terms of AMH, AFC of 2 to 4 mm, AFC of 2 to 6 mm, AFC of 2 to 9 mm. AFC of 2 to 9 mm (odds ratio (OR) 1.21, 95% confindence interval (CI) 1.03-1.43) was significantly predictive of hyperresponse, whereas AFC of 2 to 6 mm (OR 0.2, 95% CI 0.06-0.61) and AMH (OR 0.19, 95% CI 0.06-0.59) were significantly predictive of hyporesponse. CONCLUSIONS: In conclusion, AMH or AFC of 2 to 6 mm and AFC of 2 to 9 mm should be considered as predictors of ovarian response in COH for IUI cycle, therefore suggesting the best dose of gonadotropin to use in starting the ovarian stimulation. (2) to investigate the relationship between levels of antioxidant capacity in blood-free FF samples and ART prognosis. METHODS: After formal consent, 194 women undergoing fertility treatments were enrolled in the study. FF samples, from the largest and first follicle aspirated, were collected during oocyte retrieval. If present, this oocyte was placed in the insemination medium and fertilization procedure was done according to the clinic criteria. FF samples were carefully analyzed by spectrophotometric scan to measure hemoglobin levels and antioxidative status was determined by catalase activity (CAT). The quality of the ensuing oocytes and embryos was evaluated by fertilization rate and embryo score. T-test and Pearson Correlation were used to access the level of significance (p<0.05).

A total of 40% of the follicle aspirates yielded oocytes. CAT activity was significantly lower in FF that did not contain oocytes. By analyzing hemoglobin bands (412-418 hm) we identified 50 samples that presented more than 0.1% of hemoglobin contamination. Importantly, CAT activity was increased (20%) when contaminated samples were excluded (P<0.001). Therefore we repeated all analyses excluding all contaminated samples and no differences were found between the groups. No correlations were found between antioxidative status and the age of the patients, dose of FSH used, fertilization rate and embryo quality once all contaminated FF were excluded. CONCLUSIONS: In this work, CAT activity did not correlate with ART outcome. Without a standard methodology to eliminate contaminated samples, the results from ROS markers will be unreliable and difficult to compare among the groups. The human endometrium must undergo extensive remodelling during a woman's menstrual cycle to create a receptive environment for embryo implantation and development. Following menses, the endometrium regenerates and enters the proliferative phase when the glands initially develop. Glands and their secretions are essential for embryo implantation and development, which occurs in the subsequent secretory phase. Hence, the proliferative phase, during which the endometrium regenerates, is critical in the formation of a stable foundation for endometrial receptivity and embryo implantation. The proliferative phase and its significance in determining female fertility or infertility has been rarely examined. We hypothesised that alterations in the cytokine profile in uterine fluid of idiopathic infertile women would indicate a disturbance in endometrial regeneration during the proliferative phase, rendering the endometrium unable to achieve receptivity. Our aim was to determine the concentrations of cytokines, chemokines and growth factors in the uterine fluid of fertile and infertile women.

A bead-based multiplex immunoassay was used to examine the expression of 41 cytokines in uterine fluid collected by lavage during the proliferative phase from fertile (n=15) and infertile (n=15) women. The samples were further grouped according to age; fertile <35 years (n=5), fertile ³35 years (n=10), infertile <35 years (n=7) and infertile ³35 years (n=8). Immunohistochemistry was performed to localise transforming growth factor alpha (TGFα) within the endometrium of fertile (n=12) and infertile (n=11) women. RESULTS: Interleukin-1 alpha (IL-1α) was significantly increased (p=0.034) in infertile women compared to fertile women. Significant elevation of CCL11 (p=0.048), TGFα (p=0.049), interferon gamma (p=0.033) and IL-1α (p=0.047) was evident in infertile women compared to fertile women of <35 years. There were no significant differences in the ³35 years' group. TGFα was localised within the glandular epithelium of the fertile and infertile proliferative phase endometrium. CONCLUSIONS: This study has shown that an altered proliferative phase expression of cytokines, most notably among younger age women with idiopathic infertility, may underlie a subsequent inability to achieve a receptive endometrium.

Ovarian catalyses the interconversion of active glucocorticoids (GC) and their inactive metabolites. Glucocorticoids are implicated in several processes that underline successful embryo implantation and placentation. However, several evidences indicate that excess GC may also lead to abnormalities in placental development, highlighting a tight control of GC levels at the fetal-maternal interface. We have previously shown that first trimester trophoblast cells express high levels of the GC-metabolizing enzyme 11beta-HSD2. In this study, we investigated the potential contributions of decidual cells to 11beta-HSD activity in the early pregnancy by assessing the expression and steroidal regulation of 11beta-HSD isoforms in cultured first trimester decidua cell (FT-DC). METHODS: Isolated, purified FT-DCs cultures (n=5), obtained from elective termination between 6 and 12 weeks of gestation, were treated for 8 days in a defined medium with either control vehicle, 10-8 mol/L E2, 10-7 mol/L MPA and E2+MPA alone or together with the antiprogestin RU486. RNA was purified and relative mRNA levels of 11beta-HSD1 and 11beta-HSD2 were assessed by quantitative real-time RT-PCR.

The expression of 11beta-HSD1 and 11beta-HSD2 was demonstrated in all the cultures tested. In steady-state conditions, decidua 11beta-HSD1 mRNA levels were 12 fold higher than those of 11beta-HSD2. E2 did not stimulate 11beta-HSD activity while treatment with E2+MPA induced 11beta-HSD1 maximally (34 fold; p<0.001) but had no effect on 11beta-HSD2 mRNA levels. MPA alone stimulated suboptimally of not at all, suggesting the need of E2-enhancement of progesterone receptor levels among FT-DCs. Furthermore, RU486 counteracted almost all of the E2+MPA-augmented increase in 11beta-HSD1 indicating a progesterone receptor-mediated control of gene expression.

The present study demonstrated that purified FT-DCs preferentially express 11beta-HSD1 indicating the existence of a difference in tissue distribution of 11beta-HSD isoforms at the fetalmaternal interface in the first trimester. Furthermore, the observation that E2+MPA regulate 11beta-HSD1 expression in FT-DCs highlights the role of progestins in the control of the physiological functions of 11beta-HSD in the early pregnancy. is a large cell surface glycoprotein/adhesion molecule consisting of α and ß subunits derived from a single gene. While ß-DG is anchored within the plasma membrane, α-DG attaches to ß-DG extracellularly. The central region of α-DG is highly glycosylated and mediates adhesion; however, this region is masked by its large N-terminus (α-DG-N, 312 amino acid long). We hypothesized that α-DG-N on the plasma membrane is a barrier for embryo attachment and that endometrial receptivity requires α-DG-N removal by a protease, proprotein convertase 5/6 (PC6) which is specifically up-regulated in the receptive phase. In addition, α-DG-N that is removed from the membrane will be detected in uterine fluid and may serve as a potential biomarker for receptivity.

We engineered two distinct DG constructs: wild type (WT) and mutant (Mut, in which the PC6-cleavage site was mutated). Ishikawa cells stably expressing these DG constructs were established and studied for their adhesiveness to fibronectin and trophoblast spheroids (surrogates for human embryos). Uterine lavages from women were analysed by Western blot and a purpose-built ELISA to detect α-DG-N.

Mutating the single PC6-clevage site on the DG protein prevented the removal of α-DG-N from the plasma membrane, confirming the high-specificity of PC6 cleavage to shed α-DG-N from the human endometrium for receptivity. Ishikawa cells expressing the Wut-DG retained α-DG-N on the plasma membrane and showed significantly reduced receptivity to fibronectin and trophoblast spheroids attachment. In women in vivo, the α-DG-N cleaved from the endometrium was detected in the uterine fluids, and importantly Western blot and a purpose-built high throughput ELISA detected significantly higher levels of α-DG-N levels in uterine lavages in the receptive phase. CONCLUSIONS: DG plays an important role in endometrial receptivity and its adhesive function is regulated post-translationally by PC6-mediated proteolytic cleavage of α-DG-N. The α-DG-N removed from the plasma membrane is a potential biomarker for endometrial receptivity in women. Human endometrium is a complex tissue whose composition changes dynamically over the cycle, potentially confounding transcriptome analysis of whole tissue. The most commonly used transcriptome analysis method, DNA microarray, has dynamic range disadvantages in comparison to newer RNA sequencing methods. We sought to study cyclic changes in the endometrial transcriptome using cell separation and RNA sequencing. METHODS: Human endometrial biopsies in the mid-late proliferative (MLP-n=5) and mid-secretory phases (MSE-n=5) were obtained, timed by cycle day and urine LH testing. Samples were divided for whole tissue analysis and cell separation using enzymatic digestion and unigravity sedimentation. Total RNA was used for directional RNA-Sequencing. Libraries were prepared using the Illumina TruSeq Stranded mRNA protocol and sequenced (100 bp paired ends) using the Illumina HiSeq 2000 platform. Reads (~50-60 million/sample) were quality trimmed (error probability 0.001) and then mapped to the NCBI genome (Homo sapiens GRCh37.p13) using CLC Genomics Workbench. Reads per kilobase transcript per million values were evaluated using the EDGE method. Microarray data from normal endometrium used for comparison was obtained from GEOdatabase. RESULTS: Whole endometrial RNA-Seq analysis revealed 1489 differentially expressed genes between MLP and MSE phases, while similar comparisons using isolated stromal and epithelial cells yielded 927 and 851 genes respectively. From 1839 genes, 859 (47%) were unique to a specific cell type comparison, while 448 differentially expressed genes were seen in all comparisons. RNA sequencing yielded fewer genes than that published for microarray of whole endometrium (6837 vs 1489), but yielded unique differentially expressed transcripts. Among these unique genes identified by RNA-Seq was beta-1,4-N-acetyl-galactosaminyl transferase 2 (high in MSE), a protein suggested to be essential for murine embryo implantation. CONCLUSIONS: This is the first study comparing the genes expressed in human endometrium using RNA-sequencing to different compartments of human endometrium and to microarray data. Identification of novel genes involved in normal endometrial physiology may provide new insights into endometrial physiology.

Aryl Environmental toxicant exposure data as well as polymorphism and gene expression studies strongly implicate a role for the aryl hydrocarbon receptor (AhR) and its exogenous ligand, TCDD, in the pathophysiology of endometriosis. Recently, lipoxin A4 (LXA4) has been proposed as an endogenous AhR ligand and its biosynthetic enzymes (ALOX5 and ALOX15B) have been shown to markedly increased in the eutopic endometrium of women with endometriosis. We sought to examine the interrelationship between AhR and LXA4 synthesis and action.

The ishikawa cell line was subjected to AhR gene deletion using the CRISPR/CAS9 system and both the parent cell line and the AhR-/-cell line were treated with the AhR ligand TCDD or solvent control in triplicate. Gene expression was compared using microarray and real-time RT-PCR. Microarray results were analyzed using Affymetrix HuGene2-1st arrays. Patterns of significantly differentially expressed genes were determined using Significance Analysis of Microarrays(SAM). 2 class unpaired t-test with multiple corrections were run comparing the following conditions: 1)dmso-treated with and without AhR expression, 2) LXA4/TCCD/WKY-treated samples with and without AhR expression. Genes found to be significantly differentially expressed were based on a FDR of 0. RESULTS: Deletion of AhR resulted in at least 2-fold upregulation of 277 genes and at least 2-fold down-regulation of 124 genes after setting FDR to 0. Deletion of AhR was confirmed. Expression of ESR1 (an alternate LXA4 receptor) was significantly decreased while expression of ALOX5 was increased by AhR deletion. RT-PCR demonstrated a 5-fold increase in ALOX5 and a 47.5-fold increase in FPR2 in AhR deleted cells. Treatment of control cells with TCDD resulted in increased expression of Cyp1a1, Cyp1b1, ALDH3a1, and TIPARP, all known AhR targets, but no further effects on LXA4 pathway genes. Further analysis of these data are underway. CONCLUSIONS: Increased expression of AhR in the eutopic endometrium of women with endometriosis could underlie the profound changes in LXA4 synthesis and signaling we have previously described (SRI Meeting 2014) and contribute to the chronic inflammation seen in women with this disease.

The Stromal cell-derived factor-1 (SDF-1) is an important chemotactic factor for endothelial, inflammatory and hematopoetic progenitor cells. SDF-1 may be involved in inflammatory cell recruitment at the maternal-fetal interface.

We hypothesized that endometrial SDF-1 expression increases with decidualization, and that endometrial expression is mediated by increased intracellular cAMP.

A well-characterized line of telomerase-immortalized human endometrial stromal cells, which maintain differentiated function, was used to test these hypotheses. Replicate wells were incubated for 72 hours in phenol red-free culture media without or with the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate [8-br-cAMP (0.5 mM)] to increase intracellular concentrations of cAMP. Cells were also decidualized for 14 days, after which replicate wells of decidualized cells were incubated with 0.5 mM 8-br-cAMP for 72 hours. Cell numbers in each well at the end of the 72 hour incubation periods were determined.

Three independent experiments, each in triplicate, were performed. SDF-1α protein concentrations in conditioned medium from each well were measured using a human SDF-1α specific enzyme immunoassay. Data are expressed as pg SDF-1α per 100,000 cells. Statistical assessments of the data were performed using Student's t-tests since data were normally distributed. A p-value of less than 0.05 was considered significant. RESULTS: Decidualization significantly stimulated expression of SDF-1α to 352.2±46.9 pg/10 5 cells (Mean±SE), compared to 137±12.3 pg/10 5 in nondecidualized endometrial cells (p=0.0067). Increased intracellular cAMP stimulated expression of endometrial SDF-1α to 15.7±1.4 pg/10 5 cells, significantly greater than that of control endometrial cells (6.8±0.05 pg/10 5 cells; p=0.0001). CONCLUSIONS: SDF-1α may be an important factor in endometrial/ decidual recruitment of immune cell types and its expression appears to be regulated by increased intracellular cAMP. 

Levels of SIRT1 were examined in endometrium from patients with versus without endometriosis as well as in a non-human primate animal model using Western blot and immunohistochemical analysis. Endometrial SIRT1 was examined in women having surgery for endometriosis before and after resection. In the baboon model, endometrium was compared during progression of diseaase. RESULTS: Levels of SIRT1 were significantly higher in the eutopic endometrium from women with endometriosis as compared to women without disease in both the proliferative and secretory phases by western blot and immunohitochemistry. SIRT1 protein was also strongly detected in the ectopic lesions from women with endometriosis. Interestingly, the expression of SIRT1 observed in eutopic endometrium from women with endometriosis was remarkably reduced after surgical excision of endometriosis. By sequential analysis in a baboon animal model of induced endometriosis, the expression of SIRT1 was increased in the eutopic endometrium from the same animals during progression of disease CONCLUSIONS: Our results suggest that the aberrant overexpression of SIRT1 may play an important role in pathogenesis of endometriosis. Supported by NIH R01 HD067721 (SLY, BAL) and R01 HD057873 (JWJ). *Figure(s) will be available online. Hypoxia inducible factor (HIF-1) regulates the cellular response to hypoxia. HIF-1α is stable in hypoxia and is present in the human endometrium exclusively in the peri-menstrual phase. We hypothesize that HIF-1 drives efficient endometrial repair to limit menstrual blood loss (MBL).

Endometrial biopsies (n=43) were collected with consent and ethical approval from women with objectively measured MBL. Human endometrial HIF-1α protein was detected by Western blot and downstream genes by qRT-PCR. HIF-1 was pharmacologically manipulated during the late secretory/ menstrual phases in the murine model of simulated menstruation (Cousins et al 2014). Group 1: HIF-1 inhibition with 1mg/kg echinomycin (n=14). Group 2: HIF-1α stabilization with 8mg dimethyloxaloylglycine (DMOG, n=13). Group 3: vehicle (n=14). Repair was histologically graded 8 and 24h post progesterone withdrawal and vascular density (CD31) and proliferation (BrdU) quantified with Image J. RESULTS: Women with HMB (>80ml) had significantly less endometrial HIF-1α nuclear protein at menses than those with normal loss (p<0.05).

Menstrual expression of downstream targets (VEGF/CXCR4) was also decreased in women with HMB (p<0.001). Women with HMB bled for 2 days longer than normal counterparts (p<0.01), consistent with delayed repair.

To establish causality, we utilized a mouse model of menstruation. Murine endometrial VEGF mRNA was decreased with echinomycin administration (HIF-1 inhibition) and increased with DMOG (HIF-1α stabilization)(p<0.05). Echinomycin resulted in significantly delayed endometrial breakdown and luminal re-epithelialization (p<0.05). CD31 staining density was not altered by HIF inhibition or stabilization but vessels in echinomycin treated animals had an abnormal, tortuous appearance. BrdU analysis revealed decreased endometrial staining in echinomycin treated mice versus control and DMOG groups. CONCLUSIONS: These data provide evidence that HIF-1 is required for efficient endometrial repair at menstruation and suggest women with HMB have an aberrant hypoxic response. HIF-1 is an attractive non-hormonal therapeutic target for this common, debilitating condition. Endometrial scratching or injury has been reported to improve pregnancy rates in women with infertility. The objective of this study was to examine our own experience in unexplained infertility (UI) and compare the time to pregnancy after scratching to laparoscopic surgery (L/S). METHODS: Women with UI were included if they were ovulatory, had partners without significant male factor, and had at least one patent fallopian tube. Three groups of patients were compared: UI controls, subjects who underwent endometrial biopsy, and patients who underwent diagnostic L/S. Monitored cycles and time to pregnancy were compared between the groups. The primary outcome measure was time to successful pregnancy over 6 months of followup. RESULTS: A total of 62 subjects with and 103 control-subjects without UI endometrial biopsies were evaluated. Ninety-eight subjects had diagnostic L/S for evaluation and treatment of UI. Monitored cycles and pregnancy outcomes were compared between each group. Demographic characteristics for each group was similar for race, BMI, parity, and type of treatments. Controls had a lower overall gravidity (p = 0.0002). Compared to controls, subjects undergoing endometrial scratching were older (p < 0.001). The overall time to pregnancy was significantly shorter in the biopsy group compared to controls (p = 0.001). Significant difference (p < 0.05) between control and biopsy groups was only evident in the first 2 months. Interestingly, the short-term benefit, based on life table analysis, for endometrial biopsy and diagnostic L/S were surprisingly similar (p = 0.11) *Figure(s) will be available online. CONCLUSIONS: Endometrial biopsy improves short term cycle fecundity in women with unexplained infertility compared to controls to the same degree as diagnostic laparoscopy. Endometrial biopsy should be considered as a first line procedure for the evaluation and potential treatment of women with UI and suspected endometrial receptivity defects. 

The results from this pilot study provide novel information regarding the existence and functional relevance of different bacterial species in the human endometrium during the window of implantation. This opens new insights on the improvement of the endometrial factor in infertile patients. of women with recurrent pregnancy loss, infertility and amenorrhea following dilatation and curettage or other uterine procedures. We recently reported that while multiple hysteroscopic procedures were required to repair more severe cases of intrauterine adhesions, there was no difference in cumulative pregnancy, miscarriage or live birth rates following repair. We now compare these outcomes with a reproductive patient control group with a normal uterine cavity.

This retrospective case-control cohort study included all hysteroscopic lysis of adhesions performed by a single surgeon between January 2000 and June 2012. Charts with complete followup were available on 86 of 160 subjects. 82 controls with a normal sonohysterogram were randomly selected from the same surgeon's practice over the same time period. Data assessed included age, FSH, AMH, BMI, Chlamydia serology, reproductive and surgical history, and all subsequent reproductive outcomes and fertility treatments. Chi Square and ANOVA statistics were performed on Systat 13 software. RESULTS: Intrauterine adhesions were associated with increased gravity, recurrent pregnancy loss, prior curettage, myomectomy, hypothalamic amenorrhea, low BMI and prior exposure to chlamydia. There were no associations found with age, FSH, AMH or Parity. While there appeared to be trend toward higher cumulative miscarriage rates after treatment of severe intrauterine adhesions, there were no significant differences in reproductive outcomes between normal controls and patients treated for mild, moderate or severe intrauterine adhesions. Menstruation is a complex process involving tissue breakdown and repair. Our aim was to identify differentially expressed genes in human endometrium during menstruation and from these identify key pathways and novel regulatory genes controlling this process. METHODS: As part of a study examining heavy menstrual bleeding (HMB), women with or without HMB (n=24 total) were recruited (following informed consent) and endometrial samples collected by Pipelle biopsy on days 2 (n=9), 3 (n=9) or 4 (n=6) of menstruation. All samples were included, irrespective of comorbidities. RNA was extracted and analysed by genome wide expression Illumina array (HT-12 v4). Bioinformatics analysis included filtering the probes using RESULTS: 738 genes were differentially expressed between days 2 and 3 of menstruation, 1176 between 2 and 4, and 559 between 3 and 4. Using the functional clustering tool in DAVID (which clusters similar enriched pathways derived from sources such as KEGG pathways and GO terms), we identified key clusters related to pathways known to be important in menstruation including immune response, cytokine-chemokine signalling, and the complement/coagulation cascade. We have also identified many novel genes that have not previously been examined in the context of menstruation.

This study reports the first gene array analysis to examine gene expression during menstruation. In depth analysis of the genes and gene pathways identified will provide a basis for understanding normal versus abnormal menstruation, including menstrual bleeding disorders such as HMB. To validate estrogen regulation of DMBT1, immunohistochemistry was performed on endometrium from ovariectomized mice exposed to variable estrogen doses. RESULTS: DMBT1 gene expression was significantly down-regulated (fold change 6.2; p = 0.03) in mid-secretory relative to late proliferative phase endometrium with peak levels of DMBT1 transcript observed in the late proliferative phase immediately prior to the LH surge. Immunohistochemistry localized DMBT1 to the glandular compartment of human proliferative phase endometrium and DMBT1 gene expression showed an estrogen dose dependent increase in both RL95-2 cells and in ovariectomized mice. (Figure 1 ) CONCLUSIONS: DMBT1 localizes to the endometrial glandular compartment of human endometrium and is maximally expressed during the late proliferative phase. RL95-2 cells and murine endometrium exposed to increasing estradiol both demonstrated increased DMBT1 transcript levels supporting estrogen regulation of expression. There are several plausible roles for DMBT1 in the human endometrium, and this work represents the first characterization of endometrial DMBT1 expression across the menstrual cycle in human endometrium and provides a basis for comparison in endometrial neoplasia. *Figure(s) will be available online. INTRODUCTION: PGRMC-1 is a small membrane protein that mediates non-genomic action of Progesterone (P4). However, PGRMC1 has been implicated in a diversity of biological events other than P4 signaling, including apoptosis (1). In humans, PGRMC-1 protein abundance decreases in the receptive endometria (2), but its localization and function remains unknown. Our aim was to describe the protein localization in decidualized and non-decidualized endometrial stromal cells (hESC), to investigate how the overexpression could alter the decidualization process and to investigate the PGRMC1 anti-apoptotic role in endometrial epithelial cells (hEECs). METHODS: Primary hESCs obtained from endometrial biopsies were decidualizated using MPA and cAMP during 3 days. PRL was measured by ELISA to check decidualization and PGRMC1 protein localization was analyzed by immunofluorescence. PGRMC1 overexpression on hESCs primary culture (n=8) was carried by transfection using a pCMV6-AC-GFP plasmid and then hESCs were decidualized. We also analyzed the cytoskeleton reorganization using a F-actin staining with phaloidin, and hEEC apoptosis was measured after PGRMC1 overexpression with In Situ Cell Death Detection Kit. We have previously reported that women with a history of recurrent miscarriage (RM) have altered serum levels of TIMP-2, MMP-2 and RLX-2 in a subsequent pregnancy. In this study, we have developed a 3-dimensional in vitro tissue culture model of the endometrium to investigate the role of TIMP-2, RLX-2 and related molecules in implantation and the pathogenesis of RM and how these molecules influence trophoblast-endometrial interactions. Hypothesis: 3-D in vitro endometrial models that mimic the endometrium in vivo can be used to examine trophoblast-endometrial interactions during early implantation.

A 3-D endometrial model was developed using human uterine endometrial stromal cells embedded into a collagen connective tissue and topped with endometrial cell lines of varying receptivity.

The 3-D model was analysed alongside human endometrium using immunohistochemistry. JAR spheroids (human trophoblast cell line) were used to study the attachment and invasion stages of implantation using endometrial cells cultured as monolayers and as 3-D models. Levels of MMP-2 and -9 released by all cell lines were measured using zymography and ELISA.

The 3-D model comprising a monolayer of epithelium and a stromal layer containing collagen-embedded fibroblasts was successfully developed and the phenotypes confirmed by positive identification of specific cell surface markers such as galactin-9. JAR spheroids attached to all endometrial cell lines cultured as monolayers with the following affinity: Ishikawa> RL95-2 > endometrial stromal > HEC-1-A cells. JAR spheroids also attached to the 3-D models. Analysis of the culture supernatants from all cell lines by zymography showed marked levels of both pro-and active MMP-2 although these were most abundant for endometrial stromal cells. These data were confirmed by ELISA which showed that MMP-2 was more highly expressed by endometrial stromal cells compared to the other cell lines whereas MMP-9 was expressed at low levels.

A 3-D endometrial model provides a suitable platform for studies to investigate trophoblast attachment and invasiveness to the endometrium. Altered levels of MMP-2 may be important in implantation. A statistically significant relationship between AMH and AFC (p<0.0001) persisted when adjusting for age, BMI, and treatment history. CONCLUSIONS: There is a significant positive correlation between AMH level and random AFC in women with cancer. These findings support the use of these measures in this patient population, where urgency and time constraints often preclude testing on day 3.

Julian K Christians, Avery Y King, Monika D Rogowska. Biological Sciences, Simon Fraser University, Burnaby, BC, Canada. INTRODUCTION: Insulin-like growth factors (IGFs) contribute to the regulation of ovarian follicular development. The availability of IGFs is reduced by IGF binding proteins (IGFBP) and increased by proteolysis of IGFBPs. Pregnancy associated plasma protein A2 (PAPP-A2) is a protease of IGFBP-5, and a number of studies have found association between SNPs in the bovine PAPP-A2 gene and female fertility traits in cattle. In mice, deletion of PAPP-A (a paralog of PAPP-A2) reduces litter size and the number of oocytes ovulated. PAPP-A2 might also be expected to influence lactation since IGFBP-5 is known to play a role in mammary gland development.

We compared the fertility and fecundity of female mice homozygous for a PAPP-A2 knockout allele (N = 17) with that of controls (N = 15). We also examined the offspring of knockout males (N = 10) compared with controls (N = 6) to account for effects of offspring genotype. RESULTS: PAPP-A2 knock-out females were 12% lighter than controls at the time of mating (KO: 20.7 ± 0.4 g vs. controls: 23.5 ± 0.5 g; P < 0.0001), consistent with previous work. All females produced a first litter and PAPP-A2 deletion did not reduce the probability of having a second litter (15/17 KO females vs. 8/14 control females). PAPP-A2 deletion did not have a significant effect on the number of days between mating and the birth of the first litter (KO: 24 ± 2 vs. controls: 27 ± 2), or the number of days between the birth of the first litter and the birth of the second litter (27 ± 1 for both genotypes). Likewise, PAPP-A2 deletion did not affect the number of pups in the first litter (KO: 7.8 ± 0.6 vs. controls: 8.1 ± 0.6) or the second litter (KO: 8.1 ± 0.5 vs. controls: 7.6 ± 0.6). Offspring of female knockouts were significantly lighter than those of control females at birth (KO: 1.32 ± 0.01 g vs. controls: 1.39 ± 0.01 g; P = 0.002) and at 3 weeks of age (KO: 8.3 ± 0.1 g vs. controls: 9.1 ± 0.1 g; P < 0.0001). However, the mass of offspring of male knockouts was also reduced compared with controls at birth (KO: 1.28 ± 0.02 g vs. controls: 1.38 ± 0.02 g; P = 0.01) and at 3 weeks of age (KO: 8.4 ± 0.1 g vs. controls: 8.9 ± 0.1 g; P = 0.003), suggesting that these effects were due to offspring genotype. CONCLUSIONS: We found no evidence that deletion of PAPP-A2 affects the fertility or fecundity of mice, and are examining whether compensatory mechanisms maintain normal ovarian IGFBP-5 levels in PAPP-A2 deletion mice. There is evidence of their reproductive role in ovarian development, folliculogenesis, steroidogenesis and oocyte maturation. We previously reported the role in the mammary tissue of the miR-424(322)/503 cluster on multiple pathways (IGF1R, BCL-2, PI3K-AKT/PKB; Ras-MAPK, TGF-β). Since these pathways have a role in primordial follicle activation that may lead to premature ovarian failure, we hypothesize a possible interference of this cluster with the ovarian ageing process. The objective of our study was to determine if there is a difference in ovarian ageing morpho-histological markers between knockout mice (Ko) and the wild type (Wt) for the miRNA-424(322)/503 cluster.

To study this cluster in vivo we generated a miR-424(322)/503 knockout model in two mice strains (C57BL/6; FVB/N). The Ko and Wt groups were subdivided by age (< 1 (A) and >= 1 (B) year old). Ovarian specimens were stained for H&E and 10 microscopic fields per ovary were evaluated for markers of ovarian ageing in the cortex (Cx) (invaginations; thickness; medullar-cortex blurring; follicle number, corpora) and medulla (Md) (stromal fibrosis hyperplasia, and vasculature hyalinization). Statistical analysis used the Student's t-test, Chi square, and ANOVA or Fisher's exact tests applied when appropriate. A P<0.05 was considered statistically significant. RESULTS: A total of 57 mice were analyzed [Ko, n=37 (A=24; B=13); Wt, n=20 (A=17; B=3)]. There were no phenotypic differences between the Ko and Wt groups in appearance, size, except for an higher Ko weight noted past the 2nd month. There were no significant differences noted in the morpho-histological assessment for all Cx and Md markers of ageing (p=0.071), and unchanged post age-stratification (p=0.37), between Ko and Wt groups. CONCLUSIONS: There is growing evidence linking miRs to ovarian regulation and development. Even though we did not find a difference in the ovarian morpho-histological markers of ageing in our study, this miR cluster with preferred ovarian granulosa and oocyte expression may have an important role in its function that should be integrated in future research.

The little is known about the role of the long-noncoding RNA H19, which is known to be expressed in the mammalian ovary. We have shown that H19 negatively regulates the let-7 family of microRNAs, which themselves inhibit gene expression at the posttranscriptional level, and that let-7 can itself bind to and destabilize H19. Moreover, we have shown that estradiol (E2) increases expression of H19 via the AKT signaling pathway in a human granulosa cell line. E2 is known to stimulate PI3K/AKT signaling, and activation of the PI3K/AKT signaling alters phosphorylation of KSRP, a key protein involved in regulating let-7. Thus, the objective of this work is to characterize the mechanisms by which E2 regulates H19/let-7. We propose that E2 activates PI3K/AKT signaling, which alters KSRP protein level and leads to reduced let-7 processing, hence decreasing production of let-7 and increasing level of H19.

In order to determine whether let-7 expression is regulated by E2, KGN cells (a human GC-like tumor line) were cultured in αMEM and treated with E2 (10 -8 ) for 48 hours, after which time let-7 expression was measured via RNA extraction and RT-PCR. Western blot and immunofluorescence assay were performed to assess changes in KSRP protein levels after treatment with E2. Lastly, H19 RNA stability in the presence of E2 was determined by exposing KGN cells to actinomycin-D (at 5 ug/ml) for 1, 2, and 6 hours after E2 treatment. RESULTS: Extended culture of KGN cells with E2 led to a nearly fivefold downregulation of let-7 (coinciding with an increase in H19) compared to control cells (p<0.05). KSRP expression decreased 2-3-fold 48h following estradiol treatment; this was confirmed by Western blot and immunofluorescence analysis. CONCLUSIONS: We provide evidence that the E2-responsive lncRNA H19, which reduces the bioavailability of let-7 and is itself regulated by let-7, is regulated by E2 via the AKT; specifically via a decrease in KSRP protein level. The finding that E2 regulates the H19/let-7 axis, which is essential for cellular growth and development, opens the door to a more detailed exploration of novel, ncRNA pathways by which estrogen may affect the reproductive tract. This information allows, for the first time, for development of a new theory of follicular development based on E2 stimulation of lncRNA pathways.

Diffused We have recently shown that ROS deteriorate oocyte quality by altering the microtubule morphology (MT) and chromosomal alignment (CH). However, the exact mechanism by which these oxidants affect oocyte quality remains elusive.

Here, we examine the effect of increasing concentrations of peroxynitrite (ONOO -), the product of the diffusion-limited reaction between nitric oxide and superoxide, on the microtubule-organizing center (MTOC) of metaphase II mouse oocytes (N=200 cumulus, 200 noncumulus). The treated oocytes along with the control were fixed and subjected to indirect immunofluorescence, and deterioration in oocyte quality was assessed by the changes in the MTOC, MT and CH.

Our results showed significant alterations in the MT and CH from the control that displays a characteristic symmetrical barrel shape assembled around the equatorial chromosomes plate with the MTOCs condensed at both spindle poles. Indeed, oocytes treatment with low concentrations of ONOO -(<2.5 µM) showed collapse and elongation of the MT, disruption of the CH, and the MTOC scattered from a tight condensation to a dispersed arrangement of proteins loosely associated with each spindle pole. Whereas, in oocytes treated with higher concentrations of ONOO -(>2.5 µM) the central attachments between sister microtubules (kinetochores) are strained and unevenly break causing bending of the spindle, which creates an acute angle. The CH is scattered at this point, and the MTOC proteins are further dispersed and loosely attached to one other.

The damage to the MTOC mediated by ONOO-, which deranges the microtubular scaffold at the time of assembly and separation, causes the deterioration in oocyte quality which in turn lead to poor reproductive outcomes. Despite modest success with the use of progestational agents, there remains a significant population of women who experience repeated preterm deliveries. The objective of this study is to compare women with recurrent PTB less than 37 0/7 weeks gestation and those with recurrent term birth to delineate factors present in the first pregnancy, other than whether or not that pregnancy ends prematurely, that might be associated with recurrent preterm birth in subsequent pregnancies.

The study population was 7, 116 women who had their first delivery at the University of Vermont Medical Center from 1/01/04 to 12/31/13. Analysis was performed on the first pregnancy in a subset of women including 59 women with two or more preterm and no term deliveries in their total birth history and 326 women chosen at random from 2258 in the study population who had two or more term deliveries and no preterm deliveries. Modeling included univariate analysis and logistic regression to test the association between first pregnancy characteristics and membership in the recurrent term or recurrent preterm group. Hypothesis testing used p<0.05 as the level of significance.

The preterm birth rate in the study population was 10.97%. A preterm gestational age of delivery in the first pregnancy was significantly associated with recurrent PTB (p<0.001). Univariate analysis also revealed an association between recurrent PTB and maternal RH-ve status (p=0.033) and pre-pregnancy BMI (p=0.035). These factors were not independently associated with recurrent PTB. Logistic regression analysis suggested that the odds of experiencing recurrent PTB are increased 2.27 fold (CI 1.1-4.6) when the first baby delivered is a male when controlling for maternal BMI, birthplace and education and conditioning on the gestational age of this first pregnancy. CONCLUSIONS: While preterm birth in a first pregnancy is a powerful predictor of subsequent preterm birth, the sex of the first baby may also contribute to the risk of recurrent PTB. Fertility is impaired in women with rheumatoid arthritis (RA) and is related to disease activity and anti-rheumatic treatment. However, for the occurrence of miscarriages in RA, results have varied greatly, and the association of miscarriage with RA disease activity or anti-rheumatic medication is unclear. We aimed to study the association of miscarriage in RA with RA serology, disease activity and anti-rheumatic treatment, and to study disease activity and reproductive outcome after a miscarriage. METHODS: Within a nationwide prospective cohort study on pregnancy in RA (PARA study, 2002 (PARA study, -2010 , patients were followed from preconception until 6 months after delivery or miscarriage. We performed univariate analyses on preselected variables, and used logistic regression analysis with covariates included at p<0.20 and subsequently excluded at p>0.10 RESULTS: Amongst 162 pregnancies 28 (17%) miscarriages occurred.

Women who miscarried were older compared to women with an ongoing pregnancy (p=0.022), and more often were anti-citrullinated protein antibody (ACPA) positive (p=0.058), had higher disease activity scores (p=0.166) and more often had used methotrexate (MTX) in the past (p-0.174), though all not significantly. Logistic regression showed a tendency towards a higher OR to miscarry for increasing age (OR 1.12 per year (95%CI 0.99-1.25), p=0.065), and presence of ACPA (OR 2.47 (95%CI 0,86-7.08), p=0.092). After miscarriage 33% of women had a flare of RA. Within one year 68% of women became pregnant again. Fourteen percent of the subjects stopped trying to conceive and 11% were lost to follow up. Life birth rate of the subsequent pregnancy was 90%.

The miscarriage rate in the PARA cohort is comparable to that in the general population. However, this might be biased by a healthy cohort effect found earlier for this study. The associations between miscarriage in RA and the presence of ACPA, disease activity scores and MTX use did not reach statistical significance. After miscarriage, the majority of patients achieved a pregnancy resulting in a live birth.

Radek Bukowski, Lisbet Lundsberg, Jessica Illuzzi. Obstetrics and Gynecology, Yale University, New Haven, CT, USA. INTRODUCTION: Woman's risk of cardiovascular disease (CVD) has been shown to be associated with her own low birth weight (m-LBW) as well as with low birth weight of her children (c-LBW). Here we determine which of the two mechanisms of CVD dominates. METHODS: Risk of CVD in relation to m-LBW and c-LBW was evaluated in a prospective cohort of 404 women with prior live birth. CVD was a first diagnosis or death due to ischemic heart disease or cerebrovascular accident. Woman's own birth weight <2500g defined m-LBW; c-LBW was birth weight <2500g of at least 1 live born infant. Cox proportional hazards regression analysis was used to evaluate the impact of m-LBW and c-LBW on the risk of CVD. . We estimated risk ratios (RR) for teen pregnancy (<20 years old) using log-binomial models adjusted for age, race, and socioeconomic status, examining the following potential risk factors: gender expression/conformity, identity-related stress [e.g., worry that others might think they are LGB (lesbian, gay, bisexual)], bullying victimization and perpetration, and childhood abuse. Among sexual minorities, we also examined sexual orientation disclosure, LGB community involvement, and developmental milestones (e.g., age first identified as a sexual minority). RESULTS: Our sample included 140 teen pregnancies (2% of participants), of which 45 occurred among sexual minorities (4% of minorities). Bullying perpetration and each of the childhood abuse items were associated with approximately three-fold greater risk of teen pregnancy. Childhood abuse was a stronger risk factor in sexual minorities than in heterosexuals (among sexual minorities, physical abuse multivariable RR=3.13, 95%CI=1.62, 6.02; among heterosexuals, RR=1.90, 95%CI=1.10, 3.77, p-interaction=0.003). CONCLUSIONS: The higher prevalence of teen pregnancy among sexual minorities is driven by established risk factors, particularly childhood abuse, rather than unique risk factors, thus prevention efforts should focus on these factors. Similar trends are seen with hookah use, where as many as 18% of high school seniors use hookah. The goal of this study was to qualitatively assess baseline knowledge and perceptions of pregnant women of e-cigarette and hookah use during pregnancy. A secondary aim was to assess other terms used to refer to e-cigarettes. METHODS: Nine focus groups with pregnant women were conducted following regularly scheduled Centering Pregnancy meetings at 3 distinct clinics in Houston, TX. Ten semi-structured questions facilitated discussion of e-cigarettes and hookah knowledge. A minimum of two facilitators audio recorded and transcribed the sessions which were translated in Spanish as necessary.

Contradicting themes emerged from discussions on perceived risks of e-cigarette use in pregnancy. Some participants believed e-cigarettes were more dangerous than regular tobacco cigarettes, while others felt they were safer. One strong theme which emerged across groups in all clinics is the perception that e-cigarette use during pregnancy is a better alternative to smoking tobacco cigarettes. A common colloquialism for e-cigarettes is "Blu." Strong themes emerged concerning hookah use during pregnancy, notably, that it is not safe and likely more dangerous than traditional cigarettes. *Figure(s) will be available online. CONCLUSIONS: This study is the first to identify uncertainty about the benefits and risks of e-cigarette use among women receiving prenatal care.

No data exist concerning the safety of e-cigarettes in pregnancy, or their efficacy as smoking cessation devices. A strong public health message is necessary to inform women that no amount of nicotine is currently known to be safe in pregnancy. weeks' gestation or more occur each year. Despite protocols for fetal surveillance, there is a well-known increased risk of stillbirth in fetuses with various congenital anomalies. There are limited data to provide to patients once a congenital anomaly is diagnosed & even less about the trends in stillbirth rates as metrics of the success of our surveillance and interventions for these fetuses. Our purpose here was to evaluate trends in stillbirth rates in the US over several years to determine if the stillbirth rate has decreased in anomalous fetuses over time.

METHODS: This is a population based cohort study based on fetal death data files from the US national Vital Statistics System. Cases complicated by congenital anomalies were selected from 2003-2011. From this group, live birth and stillbirth cases were identified. Frequencies were compared using chi-square with p <0.05 with considered significant.

The stillbirth rate in the United States from 2003-2011 was 6.52 per 1000 live births. The rates of stillbirth among the 4 congenital anomalies studied are different (p<.05). There may be a slight downward trend for stillbirth in anencephaly, but no trend for the other anomalies, except for omphalocele with a sharp discontinuity, peaking in 2007 (41.6%). The frequency of stillbirth in omphalocele has remained elevated since then. *Figure(s) will be available online.

Our data suggest that while the stillbirth rates for most anomalies have been stable over this recent 9 year period, with perhaps a slight decrease for anencephaly, the stillbirth rate for fetuses with omphalocele acutely more than doubled, from ~15% to ~35%. Given that omphalocele is associated with some chromosomal abnormalies and other congenital anomalies, possible reasons for this increase in prenatal death are unclear. Continued surveillance and more detailed study in series of cases are warranted as we attempt to improve outcomes for children with this anomaly.

Cross In the past decades evidence accumulated that women with reproductive and pregnancy related disorders are at increased risk of developing cardiovascular disease (CVD) in the future. Up to now there is no standardized follow-up of these women since guidelines on cardiovascular risk management for this group are lacking. Therefore, the Dutch Society of Obstetrics and Gynaecology initiated a multidisciplinary working group (gynecologists, cardiologist, vascular internist, radiologist, general practitioner, and epidemiologists) to develop a guideline on cardiovascular risk management after reproductive and pregnancy related disorders.

The guideline was developed using the "Appraisal of Guidelines for Research and Evaluation" instrument. The guideline addresses the cardiovascular risk consequences of gestational hypertension, preeclampsia, preterm delivery, small-for-gestational-age infant, recurrent miscarriage, polycystic ovary syndrome or premature ovarian insufficiency. The best available evidence on these topics was gathered by systematic review. Recommendations for clinical practice were formulated based on the evidence and a consensus of expert opinion in de working group.

Based on the quality of the currently available evidence and a consistent relative risk >2 of developing CVD, a structured follow-up is only recommended for women with a history of preeclampsia. The recommendation is that a full cardiovascular risk profile is to be obtained at the age of 50 years by the general practitioner. Assessment of CVD risk and treatment of cardiovascular risk factors is according to the Dutch guideline for cardiovascular risk management. For the other reproductive and pregnancy related disorders optimization of modifiable cardiovascular risk factors to reduce the risk of future CVD, was recommended.

In this guideline we present the recommendations for cardiovascular risk management after reproductive and pregnancy related disorders. To the best of our knowledge we are the first to make such recommendations in a national guideline.

Overweight Many women with subfertility are overweight or obese and they are counseled to lose weight prior to initiating fertility treatments. Our objective was to assess preferences for weight loss interventions among overweight and obese women with subfertility. METHODS: This was a cross-sectional survey of overweight and obese women presenting for fertility consultation between 7/13 and 1/14. Women were asked to complete a 13-item survey on current weight loss efforts and preferences for weight loss interventions. RESULTS: 104 overweight and obese women responded to the survey.

(24/103) 79% reported trying to lose weight. Of those trying to lose weight, 73% reported exercise as part of their strategy. The majority reported walking (54%) or jogging (25%). 88% of women trying to lose weight reported dieting. Most women (62%) reported calorie counting on their own as opposed to following a commercially-available diet program. Only 2 women reported seeing a dietician. 84% reported successful weight loss in the past, but 61% felt their current efforts were hindered. Stress, discipline and time were the most commonly named factors hindering success. 67% reported they would be interested in a program if it were offered by their fertility clinic. Of those, most women (71%) would participate if the program was offered on-line with meal guidelines, plans and recipes, and most reported it would help if their partner participated (67%). Most women would prefer to exercise alone (80%) rather than in a group. Few women supported the idea of meal replacements (29%). CONCLUSIONS: Many overweight and obese women presenting for fertility care have tried losing weight, but feel their efforts are hindered. These women are enthusiastic about participating in weight loss interventions, and support the idea of programs offered by their fertility clinic. Informed, fertility-centered weight loss interventions may find great success among these women.

The Lead is a known environmental toxin that has several adverse maternal and fetal effects, such as gestational hypertension, low birth weight and impaired neurodevelopment. While the degree of maternal lead exposure can be identified through a serum sample, the amount of lead that the fetus is exposed to is not easily known. Our aim was to understand the relationship between lead content found in maternal and umbilical cord blood, and develop a model to determine how much lead a fetus, and therefore a newborn, is exposed.

This was a prospective cohort study of pregnant women receiving care at an urban, county hospital in 2010-2011. Our primary outcome was lead levels in maternal and umbilical cord blood. Maternal serum specimens were collected during pregnancy; umbilical cord samples were collected at delivery. Pearson's correlation coefficient and univariate linear regression were performed to develop a model of the relationship between maternal and umbilical cord blood lead content. RESULTS: Paired maternal and umbilical cord blood lead samples were obtained for 59 mother-infant pairs. Mean lead in maternal serum was 0.73 ug/dL (range: 0.23 -2.74 ug/dL) and in umbilical cord blood was 0.48 ug/dL (range: 0.1 -1.69 ug/dL). The Pearson's correlation coefficient was 0.93 (p=<0.001). After controlling for maternal birthplace, linear regression demonstrated that for every unit (ug/dL) increase in maternal lead levels, umbilical cord lead levels increased by 0.66 ug/dL (95% CI 0.59-0.72, p <0.001). A maternal lead level of 3.0 ug/dL could therefore predict a cord blood lead level of 2.0 ug/dL. This relationship is described in figure 1. *Figure(s) will be available online. CONCLUSIONS: There is a strong positive correlation between lead found in maternal and umbilical cord blood, even at low levels. This relationship suggests that maternal serum lead content can be used as a proxy for the amount that is delivered through the umbilical cord to the fetus. This information can be used to anticipate the amount of lead a fetus has been exposed to in-utero and identify which newborns need screening for excess lead exposure after birth. 

In a consecutive nested cohort design, 7508 pregnancies with over 4900 clinical variables from the 3rd most populous U.S. county were analyzed. Geospatial analysis was conducted utilizing 5-digit residence ZIP code data. ZIP-level vehicle miles travelled (VMT) data were aggregated based on regional commuter loop thoroughfares into inner ("urban") and outer ("suburban") areas. PTB was categorized as early (22-34 wks GA) or late preterm (34 1/7-36 6/7 wks GA).

The overall prevalence of PTB in our study population was 13.3% (9.6% late & 3.7% early PTB). Early PTB significantly varied with residential location, exhibiting the highest incidence in the outer loop (4.8% vs 2.5%,RR=1.9,adjusted p=0.005). *Figure(s) will be available online. Also, early PTB was associated with lower VMT/acre (165±85 vs 180±80, adjusted p=0.002). These differences persisted after adjusting for various confounders in mulitple logistic regression analysis. This could not be explained by differences in GA at presentation, number of prenatal visits, education, income, or school quality.

PTB is a multifactorial event. Our study demonstrates a possible link between a woman's urban/suburban location and her risk for early PTB. This provides a potential opportunity for community-based education and interventions such as improvement of patients' regional access to health care as well as providing a focus for exploring interactions with environmental exposures. 

We utilized mixed methods to evaluate the NIH funded DuPage Patient Navigation Collaborative (DPNC) intervention for uninsured women in Chicagoland's largest suburb who are in need of breast or cervical cancer screening and follow-up. In response to community concerns that Spanish-speaking women were vulnerable to follow-up delays following an abnormal cancer screening result, we used medical records review and patient surveys of 477 women to assess differences in delayed follow-up between Spanish and English-speaking patients, controlling for other risk factors for delay such as low patient activation, low health literacy, and distrust of the health care system. In addition, semi-structured interviews were conducted with 19 stakeholders across the county to explore key process measures assessing the DPNC's role in enhancing delivery of clinical services. RESULTS: Spanish-speaking women had lower health literacy, lower patient activation, and more health care system distrust than their Englishspeaking counterparts. Despite the prevalence of timeliness risk factors, we observed no differences in likelihood of delayed (>60 day) follow-up by language when Spanish-speaking women were assisted by patient navigators. Stakeholder interviews suggested that navigators enhanced clinical services by facilitating patient-provider communications and referrals, particularly by giving clinicians insight into patients' personal circumstances, identifying community resources to address patient concerns, and supplementing information that women received in the clinic. CONCLUSIONS: Patient navigators may play increasingly diverse and impactful roles across the continuum in women's health care delivery, especially for vulnerable populations. We report here the results of surveys on the emergency transport or evacuation status of obstetric patients conducted in Miyagi prefecture, one of the major disaster areas of the Great East Japan Earthquake and tsunami.

The surveys examined the damages to maternity institutions, evacuation status and transport of pregnant women, and prehospital childbirths, conducted in 50 maternity institutions and 12 fire departments in Miyagi.

Two coastal institutions were completely destroyed, and four institutions were partially destroyed by tsunami, forcing them to stop medical services. In the two-month period after the disaster, 217 pregnant women received hospital transport or gave birth after evacuation. Satisfactory perinatal outcomes were maintained. Emergency obstetric transport increased to approximately 1.4 fold the number before the disaster. 23 women had prehospital childbirths, indicating a marked increase to approximately 3 times the number of the previous year. CONCLUSIONS: For reconstruction of the local community, it is important to preserve the environment for women to have safe childbirths. Thus, it is important to treat pregnant women as people with special needs in disaster situations, to broaden training of healthcare professionals providing childbirth services, and to have systems for transport and childbirth services in emergencies or disasters.

Universal Umbilical Blood Consequently, this study set out to conduct the first cost-effectiveness analysis of introducing universal UCBGA.

Analysis was based on 42,100 consecutive deliveries >/= 23 weeks gestation at a single tertiary obstetric unit. Within four years of UCBGA's introduction there was a 45% reduction in term special care nursery (SCN) admission >2499 grams. Incurred costs included initial and ongoing costs associated with universal UCBGA. Averted costs were based on local diagnosis-related grouping costs for reduction in term SCN admissions. Incremental cost-effectiveness (ICER) and sensitivity analysis were also reported.

Under the base-case scenario, the adoption of universal UCBGa was less costly and more effective than selective UCBGA over four years and resulted in saving of AU$641,532 (US$576,089) while adverting 376 SCN admissions. Sensitivity analysis showed that UCBGA was cost-effective in 51.8%, 83.3%, 99.6% & 100% of simulations in years 1,2,3 and 4. These conclusions were not sensitive to wide clinically possible variations in parameter values for neonatal intensive care unit and SCN admissions, magnitude of averted SCN admissions, cumulative delivery numbers, and SCN admission costs. CONCLUSIONS: Universal UCBGA is associated with significant initial and ongoing costs; however, potential averted costs (due to reduced SCN admissions) exceed incurred costs in most scenarios. There was equal distribution of very early (gestational age of < 32 weeks) and early preterm birth (32-33 weeks). However, late preterm birth was significantly greater in the urban surroundings (1% vs 12 %, p<0.001). CONCLUSIONS: First 24 hrs seem to be the most crucial period of neonatal life in rural surroundings. Poor domiciliary care practices in rural areas could influence the postnatal care of mother as well as the new born. Furthermore, cultural beliefs like feeding sugar and water or honey to the new born, delaying breast feeding (considering colostrum as harmful) could also amplify the effect. Instigating community programs to train local midwives and other providers responsible for newborn care carries potential for positive outcome of neonatal survival.

The . Recent work in our laboratory has observed neurocognitive injury in PM-exposed offspring mediated via a C5a-C5a receptor pathway. We hypothesize that fetal cerebral vasculature may be modified in malariaexposed offspring through a C5a-C5a receptor dependent pathway.

We used a mouse model of PM that replicates the pregnancy outcomes and placental pathology of human malaria (Neres 2008). Pregnant mice, BALB/c wild type and C5a receptor knockouts (C5ar-/-), were infected on gestational day 13 with P. berghei ANKA. At gestational day 18 each fetus was perfused with radio-opaque silicone rubber contrast agent. Specimens were scanned using a Bruker SkyScan1172 high resolution Micro-CT scanner. Three-dimensional Micro-CT data were reconstructed using SkyScan NRecon software.

We identified all major cerebral vessels in fetuses (Figure 1) .

To assess the impact of malaria-exposure on small vessel development, we examined fetal cerebral vasculature with automated vessel tracking of the 3D images(Rennie, 2012). Automated vessel tracking did not reveal a significant impact of in utero malaria-exposure on vessel length or segments in wild-type mice. In malaria-exposed C5ar -/offspring at G18 vessel tracking revealed a significant increase in small vessel length and segments compared to unexposed C5ar -/controls (p < 0.05). CONCLUSIONS: To our knowledge, this is the first time micro-CT has been used to visualize fetal cerebral vasculature. These results are consistent with multiple lines of evidence that complement regulates angiogenesis and that blockade of C5a receptor may increase vascular development.

[ Figure 1 ] *Figure(s) will be available online.

To review the complications of female genital mutilation (FGM) in children and adolescent girls. METHODS: Data for this review were identified by literature search in the electronic databases MEDLINE and PubMed and the references from relevant articles through April 1, 2014 using the search terms "complications" "FGM", "FGM/Cutting", "female circumcision" "children", and "adolescent girls." Only articles published in English were included. RESULTS: Most of the literature focuses mostly on the need for defibulation to allow vaginal delivery and in some cases to allow sexual intercourse. Detailed guidance on the procedures being used has been published. The most frequent delayed complication is epidermal clitoral inclusion cysts. Unfortunately, due to many reasons including ignorance, unfamiliarity, and under-reporting, it is characterized in some reports as a rare or even a very rare complication. Urinary problems are common complication of FGM. Progressive scar shrinkage may result in urethral strictures, urine retention, slow urine streaming, and urinary calculi. Dysmenorrhea and menorrhagia are other typical complications. Finally, there are very few reports on the psychological complications in children and adolescent girls. CONCLUSIONS: Efforts and strategies are required to emphasize the importance of preventive medical management of pre-pubertal girls with FGM before long-term consequences befall them.

The Value of an External Assessment in the Training for Medical Specialist. Sophie I Velthuis, 1 Jacqueline Bustraan, 1 Arnout Jan . Therefore not only development, but also assessment of these generic competencies needs more attention during residency. We aimed to evaluate the added value of external assessment of generic competencies midterm residency training. METHODS: An assessment for learning at midterm residency was used, performed by an external assessment agency. The assessment focused on general competencies like communication, management and professionalism, and consisted of interviews and capacity and personality tests. We assessed twenty residents from different residency programs, among which five obstetrics and gynecology residents. We evaluated the assessment perceptions of residents and their supervisors using semistandardized interviews and questionnaires RESULTS: Preliminary analysis of the interviews shows that the external assessment, combined with competency observation by supervisors, provided-both resident and supervisor-insight into competencies. It gave direction to personal development planning of residents and provided a (new) perspective on the resident's career in which qualities, ambition and reality are in proper balance. Further evaluation of the current study results should focus on identifying factors that support or hamper the perceived added value. Issues like specialty, how the resident performs, what the resident expects and the exact timing of the assessment are possible influencing factors.

The external assessment has the potential to be a valuable tool: to give information about strengths and weaknesses ( more self-understanding), to give insight into the generic competencies, to encourage reflection and discussion and, in the end, to make motivated choices for the second half of residency. (4). 38 week old mice that had been treated with P4 implants from 8-36 weeks (n=10) or vehicle (n=5). Samples were obtained two weeks after OVX (or a sham operation in those having OVX in early life) and following a 1 week course of estradiol and P4. Transcript profiling was carried out using Affymetrix Gene ST 1.1 arrays. Data were normalised, and differentially regulated transcripts were identified using Cyber T and Rank Products, and analysed using Ingenuity Pathways Analysis (IPA).

We identified 60 differentially expressed transcripts in group 1. We validated these in group 2 (P for overlap = 1.0x10 -45 ). Early OVX prevented the age-related changes in myometrial transcript profile compared with sham operated controls [ Figure 1A ].

Similarly, long-term ovarian suppression using P4 implants prevented the age-related changes in myometrial transcript profile compared with vehicle controls [ Figure 1B ]. Interferon regulatory factor 7 (Irf7) mRNA was regulated by age and hormonal exposure, and was identified as a predicted regulator of the other differentially expressed transcripts by both promoter sequence and literature based (using IPA) canonical pathway activation analysis (P= 5.8x10 -5 and P<10 -15 , respectively). Immunohistochemistry demonstrated expression of IRF7 protein in both mouse and human myometrium. CONCLUSIONS: 1. Myometrial ageing in mice is associated with reproducible changes in transcript profile. 2. Age-related changes in transcript profile can be prevented by interventions which inhibit cyclical changes in the female sex hormones. 3. Irf7 may be an important regulator of myometrial function and ageing. Preterm birth is a disorder with multiple etiologies however, one of its causes is preterm premature rupture of the membranes (pPROM). It is known that pPROM is preceded by collagen degradation and amniotic cell apoptosis, thus we aimed to understand if a possible strategy to improve the strength of fetal membranes could be to increase the numbers of cells that control collagen turnover, by basic Fibroblastic Growth Factor (bFGF) treatment.

IRB approval. They were cultured in DMEM/F12 media with 10% FBS and once 50% confluent, treated with bromodeoxyuridine(BrdU) (30ng/mL), and bFGF (0, 0.01, 0.1, 1.0, 10, 100 ng/mL) for 24 hrs. Immunocytochemistry (ICC) was used to detect BrdU, and cell proliferation calculated as the percentage of cells with incorporation. Data were analyzed by 1-way ANOVA and Tukey's post-hoc test. MTT assay was used to confirm the cell proliferation data. Immunohistochemistry (IHC) (n=4), was used to show bFGF receptor (bFGFR) expression (dilution 1:300) and qRT-PCR was used to confirm both bFGF and receptor expression in cells.

Receptor and ligand expression were confirmed by qRT-PCR for both cell types in vitro. AEC expressed more of the bFGFR gene, while AMC expressed more of the ligand. bFGF increased the proliferating numbers of both AEC and AMC. Quantitation of BrdU incorporation showed AMC proliferation at all bFGF concentrations, reaching significance at 0.1, 1, 10, 100ng/mL (p=0.0006). AEC also proliferated at all of the bFGF doses, but only reached significance at 100ng/mL (p=0.0561). MTT assay confirmed the effect of bFGF treatment on proliferation. CONCLUSIONS: Amniotic cells express bFGFR and are capable of responding to bFGF in vitro. The importance of these data is that although bFGF and its receptor are endogenously expressed in vivo in the amnion, these cells do not proliferate in the third trimester. Thus it is thought that this lack in proliferative capacity may contribute to the weakening of the membranes towards term as the numbers of cells decrease and these cells can not contribute to collagen maintenance. INTRODUCTION: While a higher maternal BMI is associated with preterm birth, it is unclear whether excess gestational weight gain (GWG) or obesity drives increased risk. We and others have shown that the placenta harbors microbiota, which are significantly different among preterm births. Our aim in this study was to investigate whether the preterm placental microbiome varies by virtue of obesity or by excess GWG. METHODS: Placentas (n=320) were collected from term and preterm gravidae. Genomic DNA was extracted and subjected to metagenomic sequencing. Data was analyzed by clinical covariates, including the 2009 Institute of Medicine's GWG guideline and obesity. RESULTS: Analysis of 16S rRNA-based metagenomics revealed no clustering by virtue of obesity (p=0.161). Among gravidae who delivered preterm, there was no clustering by obesity (p=0.480), but there was clustering by excess GWG (p=0.022, Figure 1C ). Among preterm births, detailed analysis identified microbial genera and bacterial metabolic gene pathways that varied among gravidae with excess GWG (Figure 2A , 2B, and 2C). Notably, excess GWG was associated with decreased microbial folate biosynthesis pathways and decreased butanoate metabolism (LDA >3.0-fold, Figure 2D ). 

Although there were no alterations in the microbiome by virtue of obesity, excess GWG was associated with an altered microbiome and its metabolic profile among those who experienced a preterm birth. Cytokines are known to regulate inflammatory responses and nicotine has been shown to alter cytokine synthesis. The object of these studies was to evaluate the influence of LPS and nicotine on production of cytokines from rat placenta in vitro.

Rat placental explants (4/mid-uterine placenta) from rats (n=4) on gestational day 14 were incubated in Dulbeco's modified Eagle's medium (DMEM) alone (controls), or DMEM with LPS (1 ug/ml), with LPS +nicotine (10 uM) and with nicotine alone in a 5% CO2 incubator.

After 24 hours, culture media were collected and analyzed in duplicate for cytokines using a Luminex® assay. Wet weight was used to normalize differences between each explants. Statistical analysis was done using One-Way ANOVA and Student's T test. RESULTS: Media from control samples show low levels (ca. 1 to 2 pg/ ml) of IL-1, IL-2, IL-6, IL-17 and TNF-α with higher IFN-γ (ca. 15 pg/ ml) and IL-10 (ca. 4 pg/ml, Figure) . Media from explants treated with LPS alone show significant increases all cytokines compared to controls (p<0.05 to <0.001). Whereas treatment with nicotine + LPS significantly decreases cytokines compared to LPS alone (p<0.05 to <0.001), except IL-10 and IL-17 (p>0.05). Explants treated with LPS + nicotine were not significantly different compared to controls (p>0.05), except for IL-2 and TNF-α (p<0.05). Similarly, nicotine treatment alone did not change cytokines compared to controls (p>0.05), except TNF-α which was higher (p<0.01). INTRODUCTION: Exposure to prenatal inflammation is associated with both short and long term adverse neurobehavioral outcomes. We have previously demonstrated that low dose intrauterine inflammation (IUI) leads to a specific neuronal insult in the fetal brain that persists through gestation. However, the pathways responsible for mediating this injury have not been elucidated. Without understanding the mechanism of injury, therapeutic approaches cannot begin. Therefore, the objectives of this study were 1) to discover novel immune pathways acutely activated after exposure to IUI; 2) to determine if immune pathways remained activated 48 hours after exposure. METHODS: Timed-pregnant CD-1 mice received an intrauterine injection of lipopolysaccharide (50 µg/dam, LPS, n=10) or saline (n=10) on E15 of gestation. Six and 48 hours post-injection, placentas (PL) and fetal brains (FB) were harvested. RNA isolated from FB and PL (at 6 hours) was profiled using immune arrays on a microfluidic platform. The most differentially regulated targets in the PL discovered via the array were then investigated at 48 hours at the RNA level by QPCR. Changes in protein expression were assessed in the PL at 6 and 48 hours by ELISA. RESULTS: Immune profiling in the FB revealed altered expression in response to IUI: IL-12β was decreased 9-fold (P=0.007), IL-1β was increased 3.6 fold (P=0.003) and VEGF-α was increased 1.5 fold (P=0.02) compared to controls. In the PL, the expression of 6 inflammatory targets was significantly increased at 6 hours. At 48 hours, the expression of 4 of those targets was still significantly elevated. Protein levels of IL-1β, CCL3 and CCL5 mirrored the LPS-induced increase in mRNA expression at both 6 and 48 hours. *Figure(s) will be available online. CONCLUSIONS: Placental chemokine expression is markedly elevated, acutely after intrauterine inflammation and increased expression persists during the time period that fetal neuronal injury is observed. Understanding the role placental chemokines play in fetal brain injury from exposure to prenatal inflammation may lead to new therapeutic strategies.

Erythromycin 

Cells. Liping Feng, 1 Terrence K Allen, 2 Matthew K Nazzal, 1 Amy P Intraamniotic inoculation of UP in the rhesus monkey results in chorioamnionitis and preterm delivery. We demonstrated that UP adheres to primary fetal membrane cells and induces a strong pro-inflammatory response including interleukin 8 (IL-8), resulting in alteration of metalloproteinase expression and activity including metalloproteinase 9 (MMP9). We also demonstrated that P4 attenuate TNFα induced inflammation in fetal membranes. This P4 function is mediated through PGRMC1 which found to be down-regulated in the fetal membranes collected from PPROM patients (our published data). In this study, we investigate the role of P4 and PGRMC1 in inhibiting UP induced proinflammatory response in amnion and chorion cells. METHODS: Primary amnion and chorion cells (8 subjects) were transfected with scramble or validated PGRMC1 siRNA. After transfection (72h), cells were treated with UP at 10 6 cfu for 4h with or without pretreatment with P4 or Ethanol (vehicle) at a dose of 10 -7 M for 1h. Cell lysates were harvested for Western blotting to determine knockdown of PGRMC1 expression. Culture media was harvested for measuring MMP9 activity by zymography. IL-8, MMP9 and cyclooxygenase (COX-2) mRNA expression were quantified by real time PCR. RESULTS: IL-8, COX-2, MMP9 mRNA level and MMP9 activity are significantly higher in UP infected cells except MMP9 mRNA level in chorion cell was no difference compared to control group. IL-8 and COX-2 in response to UP stimulation were significantly accelerated in the PGRMC1 knockdown chorion cells compared with scramble siRNA group but not in amnion cells. The up-regulation of IL-8 and COX-2 by UP exposure was partially inhibited by P4 pretreatment and the inhibition was lost in PGRMC1 knock-down chorion cells with statistical significance but not in amnion cells. The effects of P4 and PGRMC1 on MMP9 are not significant. CONCLUSIONS: P4 inhibited UP induced IL-8 and COX-2 in chorion cells mediated by PGRMC1. However, the function of PGRMC1 in this process can be independent on P4. This indicates they function through separate, but overlapping signaling pathways. PGRMC1 is critical for balancing the inflammatory response of chorion cells to UP infection but not amnion cells. The regulation of MMP9 expression and activity might be deferent mechanism. Cytokines and chemokines are thought to be involved in the preparation of various tissues for onset of labor. The aim of this study was to determine the changes of pro-inflammatory, antiinflammatory and chemokines in placenta, decidua, myometrium and fetal membrane tissues of women in labor vs. not in labor.

Human placenta, decidua, myometrium and fetal membrane tissues were obtained from women (n = 10 each group) who delivered healthy, singleton infants at term (>37 weeks gestation) undergoing elective caesarean section in labor or not in labor. Luminex assay was used for cytokine and chemokine analysis to measure and characterize the profiles (totally nine cytokines) in each tissue. This study was approved by the Ethics Committee for Guangzhou Women and Children's Medical Center.

During labor the myometrium produces significantly higher amounts of interleukins IL -6 (p=0.031), IL-8 (p=0.031), and chemokine MCP-1β (known as C-C chemokine motif ligand 2 or CCL-2, p=0.030) compared to myometrium from non-laboring patients (Figure, A to D) . This increase coincides with the influx of microphage cells into the myometrium. In addition, IL-1β (p=0.037) and IL-6 (p=0.038) are also up regulated in fetal membranes during labor vs. non-labor. However, cytokines in placenta and decidua did not change significantly in labor vs non-labor patients (p>0.05). There was also no significant difference in the four tissues assayed for the anti-inflammatory factor IL-10 comparing labor to non-labor patients (p>0.05). . CONCLUSIONS: 1) Pro-inflammatory mediators in human fetal membranes and myometrium may play important roles in the onset of labor. 2) Up-regulation of cytokines in fetal membranes may be associated with rupture of membranes. 3) Increased expression of MCP-1β in myometrium may indicate that macrophages are important cells in labor onset. * Figure( s) will be available online. INTRODUCTION: Premature cervical remodeling is likely an obligatory step in the pathogenesis of spontaneous preterm birth. An inflammatory challenge to cervical epithelial cells has been shown to increase production of cytokines and disrupt the epithelial barrier leading to cervical remodeling. Statins have been shown to prevent cervical remodeling by inhibiting complement activation and increasing HO-1 gene expression leading to carbon monoxide production. We hypothesize treating cervical epithelial cells with statins or a carbon monoxide releasing molecule would mitigate inflammatory induced functional changes to the cervical epithelial barrier. METHODS: Immortalized ectocervical (ECTO) and endocervical (ENDO) cells were plated at a concentration of 9.6 x 10 4 per well for 24 hours. Cells were treated with 20ug/ml of Pravastatin (PRAV) or 30uM of the carbon monoxide releasing molecule tricarbonylchloro(glycinato) ruthenium III (CORM2) for 2 hours prior to treatment with 25ug/ml of lipopolysaccharide (LPS). Media was collected at 6 and 24 hours. The cytokine IL6 was assessed at both time points. The effect of LPS, PRAV, LPS + PRAV, CORM2, or LPS + CORM2 on the integrity of the epithelial cell barrier was assessed using an in vitro permeability assay. In this study, we tested the hypothesis that in utero inflammation causes long-term effects on hippocampal synaptic transmission and long term synaptic plasticity (LTP).

We prepared acute brain slices from saline and LPS (50 mcg) in utero exposed CD1 male mice (44-59 DPN), and made extracellular recordings of presynaptic fiber volleys (FV) and field EPSPs (fEPSPs) evoked by varying intensities. By plotting the initial slopes of fEPSPs against the FV amplitudes, we determined the input-output (I/O) ratio for CA3-CA1 synapses, a measure of synaptic strength. To determine the probability of presynaptic release, a paired pulse facilitation (PPF) stimulus protocol was used. Field LTP of CA3-CA1 neurotransmission was induced by stimulating CA3 axons with 3 sets of 100 stimuli delivered at 50 Hz. Statistical significance was determined by Mann-Whitney test or Student's T-test. RESULTS: Compared to controls, offspring mice that were exposed displayed increased synaptic strength, a higher I/O ratio (0.17±0.02, n=11 control; 0.55±0.13, n=11 LPS; p<0.05). The paired-pulse facilitation (PPF) ratio in LPS exposed mice compared to controls was reduced (1.6±0.2, n=2 control; 1.1±0.0, n=7 LPS, p<0.02). Decreased LTP was observed in LPS exposed mice compared to saline controls (159±8.8, n=4 control; 124.9±10.7, n=5 LPS; p<0.04). CONCLUSIONS: LPS exposed mice displayed increased synaptic strength that can be explained by an increase in the probability of glutamate release from pre-synaptic fibers, as evidenced by the reduced PPF ratio. LPS exposed mice also had lower levels of LTP compared to controls. This finding could be due to disrupted AMPA receptor trafficking in CA1 neurons, similar to ischemia-induced impairment of LTP. These novel findings of changes in hippocampal synaptic transmission may underlie the cognitive and behavioral deficits observed in survivors of preterm birth. 

The second trimester fetus is highly susceptible to UP, with intrauterine infection characterised by high AF titres, fetal bacteraemia, and significant fetal but not AF inflammation. These data underscore the importance of gestation-appropriate models of infection in pregnancy. We previously reported that thrombin in combination with bacterial LPS induces a synergistic effect on human endometrial endothelial cell (HEEC) secretion of the inflammatory cytokines/ chemokines: IL-8, IL-6, and G-CSF. The objective of this study was to further investigate the mechanisms by which thrombin augments LPSinduced HEEC inflammation by assessing the role of thrombin-specific protease activated receptors (PAR) receptors, and the negative inhibitors of Toll-like receptor function, suppressors of cytokine signaling (SOCS) 1 and 3. METHODS: HEECs were pretreated with or without either a specific PAR-1, PAR-2, or PAR-4 agonist (1mM) for 1hr followed by treatment with or without LPS (1µg/ml) for 48hrs (n=3). Supernatants were measured for IL-8, IL-6 and G-CSF by ELISA and multiplex analysis.

HEECs were also treated for 24hr with or without either thrombin (0.25U/ ml) or the PAR-1 agonist (1mM), after which RNA was collected and qRT-PCR performed for SOCS1 and SOCS3. RESULTS: Pretreatment of HEECs with the PAR-1 agonist significantly augmented LPS-induced secretion of IL-8 by 1.5±0.0 fold, IL-6 by 2.5±0.3 fold, and G-CSF by 2.5±0.2 fold when compared to LPS alone (p<0.05).

In contrast, neither the PAR-2 nor the PAR-4 agonist had a significant effect on HEEC LPS-induced IL-8 secretion. Treatment of HEECs with thrombin significantly decreased SOCS1 mRNA levels by 36.7±12.0% when compared to the untreated control. Similarly, treatment of HEECs with the PAR-1 agonist significantly decreased SOCS1 mRNA levels by 57.5±15.4% compared to the untreated control (p<0.05; n=3-4). In contrast, treatment with either thrombin or the PAR-1 agonist had no significant effect on HEEC SOCS3 expression. CONCLUSIONS: Thrombin augments LPS-induced HEEC inflammatory cytokine/chemokine secretion through activation of PAR-1 and diminished expression of SOCS1. These findings suggest a novel mechanism by which preterm birth in the context of abruption and bacterial infection may be associated with an aggravated inflammatory response. Supported by PO1HD054713. We evaluated IL-6 and receptors changes in fetal membranes (FM) in response to diverse microbial type and load. METHODS: FM collected from normal term (n=6), not in labor, were maintained in an organ explant system and treated with heat inactivated Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), Gardnerella vaginalis (GV) and combinations of CFUs: MH+UU(10 6 ), MH+UU+GV(10 6 ), MH+UU(10 6 )+GV(10 3 ), MH+UU(10 3 )+GV(10 6 ) for 24h to simulate PIAC. IL-6 and sIL-6R supernatants were measured by Multiplex assay. mIL-6R and gp130 expressions were evaluated by qPCR. Data were analyzed by linear mixed effects models. RESULTS: Stimulation by UU or GV alone and all polymicrobial combinations increased IL-6 (p<0.05, 0.01 and 0.05 respectively) compared to control. Highest dose of GV combination had the maximum IL-6 levels. MH alone had no effect on IL-6 and receptors. UU alone or combined with MH increased sIL-6R (p<0.01). GV alone or with Mycoplasmas didn't change sIL-6R. GV treatment increased mIL-6R and gp130 expressions (p<0.001); but, combined with Mycoplasmas didn't change either. CONCLUSIONS: Mono and polymicrobial treatment of FM produced distinct IL-6 signaling response. Increased IL-6 and sIL-6R by genital Mycoplasmas indicated inflammation; but, lack of mIL-6R or gp130 reflects limited IL-6 biofunction. GV produced proinflammatory IL-6 signaling that was curtailed in the presence of Mycoplasmas suggesting an anti-inflammatory milieu created to minimize IL-6 biofunction by other pathogens. Bacterial species, load and combinations determine IL-6 response. Predict pregnancy outcome and design interventions based on IL-6 levels without consider other signaling molecules may be ineffective. Preterm birth is believed to be a final common pathway of a multitude of factors, and it is clear inflammation plays a significant role in prematurity. However, the precise mechanisms of preterm parturition remain elusive. Normally, bacteria are not present in amniotic fluid. If bacteria gain access to the amniotic space, an inflammatory response may ensue in cells present in the amniotic fluid, specifically amniotic fluid mesenchymal stem cells. This study sought to assess if an inflammatory challenge with lipopolysaccharide (LPS) altered signal transduction pathways in these cells.

Human amniotic mesenchymal stem cells were plated at a concentration of 10 4 cells per cm 2 and treated with 2.5, 25, and 250ug/ ml of LPS. Media was collected at 24 hours and assessed for IL6 using an enzyme-linked immunosorbent assay. RNA was also collected from cells treated with 250ug/ml of LPS and an innate and immune response array was performed, profiling the expression of 84 genes involved in the response to pathogens. RESULTS: Amniotic fluid cells treated with all three doses of LPS resulted in an increase in IL6 levels (p<0.005). Additionally, treatment with 250 ug/ml of LPS altered expression of eight genes in the innate and immune response array. Notably, LPS treatment led to a 48.37 fold increase in the expression of CSF2 (p<0.001) which is associated with macrophage production, differentiation and function. Other genes associated with inflammation that were upregulated include IL1B (p<0.005), IL6 (p<0.05), IL8 (p<0.05), NFKB1 (p<0.05), and TLR3 (p<0.01). *Figure(s) will be available online.

ascending infection is the initiating event in preterm birth, the increase in expression of these pathways may play an essential role in the pathogenesis of preterm birth. HO-1 is produced by a number of tissues, including the placenta, and may play a key role in regulating inflammation at the maternal-fetal interface. Mice that are genetically deficient in HO-1 have increased fetal wastage and low birth weight that is reversed by the enzyme's product, carbon monoxide. HO-1 is controlled by the transcription factor nrf-2. Studies with other culture systems have shown that trigonelline, a component of coffee, inhibits nrf-2 activity but its effects on the placenta are unknown. We hypothesized that trigonelline inhibits HO-1 production and enhances production of proinflammatory cytokines by the placenta. METHODS: Placental explant cultures were prepared from tissues of women undergoing elective Cesarean section at term (n=12). Cultures were incubated with 0 to 10 -3 M trigonelline, incubated for 24 h, treated with 10 7 CFU/ml heat-killed E. coli or an equivalent volume of sterile medium, and then incubated for an additional 24 h. Concentrations of IL-1b, TNF-a, IL-10, and HO-1 in the conditioned medium were then quantified by ELISA. Viability of the explants was ascertained using the MTT assay. Data were analyzed using linear mixed effects models. RESULTS: E. coli significantly increased IL-1b, TNF-a, and IL-10 production (P < 0.001) but no effect of bacteria on HO-1 production was detected. Trigonelline significantly inhibited HO-1 production by both unstimulated (P = 0.047) and E. coli-stimulated cultures (P < 0.001, Figure  1 ) but had no significant effect on cytokine IL-1b or TNF-a production. IL-10 tended to be lowered by trigonelline for E. coli-stimulated cultures but results did not reach statistical significance. Trigonelline also tended to reduce viability of unstimulated (P = 0.044) and E. coli-stimulated cultures (P=0.088) but results were not dose-dependent. *Figure(s) will be available online. CONCLUSIONS: Trigonelline reduces production of HO-1 but has little or no effect on placental cytokine production in vitro. , an acute phase protein, may be used as a predictor of chorioamnionitis while other studies failed to support this conclusion. The aim of this study was to assess the prognostic value of the fold change of CRP level at delivery in prediction of chorioamnionitis rather than CRP cut-off level.

A prospective study which included 50 women with PPROM for whom we had initial CRP level at admission and CRP measurements before delivery. During the study period of time the diagnosis of chorioamnionitis was based on clinical data and leukocytosis while the data on CRP level was not available to the medical staff. Chorioamnionitis was defined as clinical presentation supported by histological evidence for chorioamnionitis. Women who delivered spontaneously or had induction of labor within 24 hours from admission were excluded. We calculated the fold change in CRP level at time of delivery compared to admission. RESULTS: Ten women 0ut of the 50 women with PPROM had chorioamnionitis (20%). The mean level of CRP among women with chorioamnionitis was significantly higher compared to women without chorioamnionitis (5.2±6.7 vs 14.6±12.8 dl/ml, respectively, p<0.05) but it was very difficult to determine the optimal cut off for diagnosis of chorioamnionitis. The fold change among women with chorioamnionitis was significantly higher compared to women without chorioamnionitis (3.3±4 compared to 11.8±15, respectively, p<0.05). If we define a cutoff that is not a CRP value but fold change >5 we obtained a sensitivity of 60%, a false negative of 40%, a specificity of 90% and false positive 10%. Importantly, if we add the 5 fold change rule to the leukocytosis, we increase the sensitivity of 90% but the false positive increases up to 25%.

The prognostic value of CRP in prediction chorioamninitis is unclear yet. We suggest using the fold change of CRP in prediction of chorioamnionitis rather than a cut-off value of CRP level. We found that a combination of the fold change of CRP and elevated WBC may improve our ability to predict chorioamnionitis. Obviously, larger sample size is required to establish this protocol Cxcl1, Cxcl2 and Ccl2 in the uterus, fetal membranes and placenta, compared to PBS-treated mice (p<0.01). LPS treatment induced an influx of neutrophils, localised by Ly-6G expression, into the decidua, which were not present in PBS-treated mice. CONCLUSIONS: Ultrasound-guided intrauterine LPS injection reliably induces PTB in the mouse and mimics the local inflammatory and immune response observed in other, more invasive, models of inflammationinduced PTB. Notably, there was no PTB in the PBS control group. We believe that the development of this novel minimally invasive method of inflammation-induced PTB will be useful for future analysis of novel interventions to prevent PTB and improve neonatal outcome. -1β) , IL-6, IL-8, and granulocyte-monocyte colony stimulating factor (GM-CSF). IL-1β and IL-6 are associated with preterm birth and perinatal brain injury. IL-1β release is dependent on the activation of the ATP-gated ion channel P2RX7. Brilliant Blue G (BBG) dye is a specific inhibitor of the P2RX7 channel and prevents IL-1β release. Here, we sought to determine the role of P2RX7 in regulating acute endothelial response to lipopolysaccharide (LPS). METHODS: Human umbilical vein ECs (HUVECs) were cultured in vitro at 37°C, 5%CO 2 , and 95% humidity. HUVECs were activated with LPS (100,10,5,1, and 0.1ng/mL) for 3 h. Inhibition dose-response was performed with 100ng/mL LPS and BBG (100,10,5,1, and 0.1µM) and, after 3 h, cells and media were harvested for analysis. NOS1 and cytokine expression was assessed by qPCR and luminex assays. Caspase 3 activation and NOS1 expression was determined by immunohistochemistry.

Our results demonstrated a maximum cytokine and NOS1 response to LPS at 100 ng/mL. BBG attenuated these responses in a dose-dependent manner. qPCR analysis of cytokine expression showed a dramatic increase in IL-1β mRNA, which was reduced by 10 µM BBG. Luminex analysis of cytokines in supernatants showed that LPS stimulated a robust IL-8 response but only small increases in IL-1β, IL-6, and GM-CSF, all of which exhibited dose-dependent inhibition by BBG (Figure) . CONCLUSIONS: LPS leads to increases in cytokine expression and apoptosis. Inhibition with BBG shows a dose-dependent reduction in expression of NOS1, activated caspase 3, cytokins and apoptosis. While the role of P2RX7 in IL-1β release is well characterized, inhibition of IL-8 suggests a wider role for P2RX7 in immune pathways. Additional studies with extended LPS stimulation are needed to understand the role of P2RX7 in the temporal regulation of endothelial cell activation. * Figure(s) NF-κB controls the expression of contraction associated and inflammatory response genes and is activated in amnion and myometrium in labour. A strong association exists between inflammation/ infection and preterm labour. Furthermore, the presence of inflammation is an independent risk factor for adverse neonatal outcome. We have previously shown that 15-deoxy-D 12,14 PGJ2 (15dPGJ2) inhibits IL-1β induced NF-κB activation and COX-2 levels in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs), and pro-inflammatory cytokine production in PBMCs. In addition, when given to LPS treated mice, 15dPGJ2 is able to both delay preterm labour and increase pup survival.

We therefore examined the effect of 15dPGJ2 on contraction associated proteins (CAPs) and IL-1β induced pro-inflammatory cytokine expression in cultured amniocytes and myocytes. Amnion and myometrium were collected from women undergoing pre-labour elective caesarean section at term. Cells were cultured and treated with 15dPGJ2 or vehicle control, with or without IL-1β stimulation. NF-κB and AP-1 activity were measured by western analysis of phosphorylated p65 and c-Jun respectively. Changes in CAPs were determined by qRT-PCR, western analysis and ELISA. mRNA expression of pro-inflammatory cytokines was determined by qRT-PCR. RESULTS: 15dPGJ2 (32µM) inhibits IL-1β induced NF-κB and AP-1 activity in myocytes (p<0.05). Inhibition of the CAPs; cPLA2α (p<0.001), COX-2 (p<0.05), PGE2 (p<0.05) and pro-inflammatory cytokines IL-6 (p<0.01), TNF-α (p<0.001), CCL-2 (p<0.01), IL-8 (p<0.05) was demonstrated in myocytes.15dPGJ2 had similar inhibitory effects in amniocytes with reduction of IL-1β induced NF-κB (p<0.01), OTR (p<0.001), COX-2 (p<0.05), IL-6 (p<0.05), and CCL-2 (p<0.05) observed in treated cells.

In conclusion, 15dPGJ2 inhibits the expression of both contraction associated proteins and inflammatory response genes, and therefore holds promise as a potential therapeutic agent for the prevention of inflammation induced preterm labour. (1 mM x 15 min) , followed by exposure to heat-killed, FITC labeled GB37 (150 bacteria per cell x 3 h). Phagocytosis was measured fluorometrically. THP-1 cells were also exposed to live GB37 (10:1 bacteria per cell x 24 h) and supernatants were analyzed by ELISA for IL-8, IL-10, MCP-1, TNF-α, and IL-1β. Human gestational membrane (GM) transwells were treated on the choriodecidual side for 24 h with 0.5 e6 CFU GB37 and supernatants were analyzed by EIA for PGE 2 . Timed-pregnant female C57BL/6J mice were vaginally colonized on day13 of pregnancy (E13) with 10 9 CFU/ ml of GBS (10/84) or vehicle control. Dams were sacrificed on E15 and amniotic fluid collected for PGE 2 quantification. RESULTS: GB37 stimulated PGE 2 production into the choriodecidual chamber of GM transwell cultures. Furthermore, in mice, we observed increased PGE 2 in amniotic fluid following ascending GBS infection. PGE 2 significantly suppressed phagocytosis of heat killed GB37 in both THP-1 cells and PMs. PGE 2 significantly increased IL-8 and IL-1β production, and decreased MCP-1 and TNF-α production from infected macrophages. CONCLUSIONS: This study demonstrates increases in PGE 2 production in an in vitro and in vivo model of GBS CAM. In addition, we observed significant changes in human macrophage responses to GBS infection following treatment with PGE 2 . These findings suggest that PGE 2 signaling regulates innate immune defenses in the pathogenesis of CAM. In the current study, we sought to investigate mechanisms involved in disturbance of the circadian rhythms.

We utilized a mouse model of intrauterine inflammation to recapitulate elements of human disease with exposure to infectious pathogens in utero and long-term neurologic outcomes, using lipopolysaccharide (LPS). Pregnant CD1 mice were randomly allocated to either normal saline (NS) or LPS (50 mcg/mouse) intrauterine infusion at E17. Somatosensory cortex was derived from offspring male mice on postnatal days (PND) 30 and 90 at time-specific points in the day. We performed QPCR for message expression of circadian rhythm genes: CLOCK (circadian locomotor output cycles kaput), MTNR1A/1B (melatonin receptor 1A and B), GRIN1 (glutamate receptor subunit zeta-1, CRH (corticotropin releasing hormone), GABRA1 (gamma-aminobutyric acid receptor subunit alpha-1), DBP (D site of albumin promoter binding protein), PRNP (major prion protein) and PER2 (period circadian protein homolog 2). The protein products of these genes were utilized in ELISA for confirmation of changes in mRNA. In addition, melatonin levels in somatosensory cortex were investigated with ELISA. Student t-tests were run with significance set at p<0.05. RESULTS: On a protein level, ELISA revealed an increased level of melatonin in LPS-treated male mice between 2 and 4 pm, an abnormal finding not present in controls (p<0.05). *Figure(s) will be available online. CONCLUSIONS: Exposure to intrauterine inflammation leads to a long-term disruption of circadian rhythms which might be regulated by a life-long abnormal production and timing of melatonin. Our data may explain mechanisms of behavioral phenotype in children born preterm.

(Supported by NIH T32NS069351-05 (MT)) Bioinformatic analyses were carried out to elucidate regulated coding and long non-coding RNAs as well as micro-RNAs shared between and exclusive to term and infection-mediated preterm cervical ripening.

Analyses of coding transcripts demonstrate that infectionmediated preterm and term cervical ripening are governed via unique genes and pathways, which is in agreement with our lab's previous data. As expected, NFkB activation and regulation of prostaglandins pathways are highly upregulated in the infection model. Genes involved in intracellular signaling cascade and phosphorus metabolic processes were also enriched substantially in our infection model. Small RNASeq are underway and will identify commonly and exclusively regulated microRNAs in term and infection-mediated preterm cervical ripening. This will allow for integrative analyses with polyA RNASeq to identify targets of the microRNAs and disover gene regulatory networks in term and PTB. CONCLUSIONS: Next Generation Sequencing technologies elucidate numerous processes necessitating further study in infection-mediated preterm cervical ripening. Integration of small and polyA RNASequencing data sets is ongoing and will provide insight into the gene expression profiles regulating cervical remodeling in response to an infection and also at term. A better understanding of the pathways involved in each model of cervical ripening will allow for the development of detection methods and treatment strategies to prevent PTBs.

Role of Azithromycin in the Management of Preterm Premature Rupture of Membranes. Eduardo J Aguin, Aaron Kutinsky, Elizabeth S Langen. Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA.

The objective of our study is to examine the impact of substituting azithromycin for erythromycin in the treatment of preterm, premature rupture of membranes (PPROM).

We conducted this retrospective cohort study of patients whose pregnancies were complicated by PPROM between 24+0 and 33+6 weeks. Antibiotic regimens were decided based on availability of erythromycin in the pharmacy, patient allergies, and provider preference. Patients were divided into 3 groups. Group 1 Included patients who received ampicillin/amoxicillin plus erythromycin; group 2 included patients who received ampicillin/amoxicillin plus azithromycin; group 3 consisted of patients who received ampicillin/amoxicillin plus a combination of both erythromycin and azithromycin. Demographic variables studied were maternal age, race, history of previous preterm birth, and gestational age at the time of PPROM. Outcomes variables studied were latency interval, achievement of 34 weeks of gestation, composite neonatal morbidity (including fetal death, sepsis in < or =72 hours, intraventricular hemorrhage stage III or IV, and necrotizing enterocolitis stage II or III), maternal infection (chorioamniotis and or endometritis), and pathology reporting funisitis or necrotizing chorioamnionitis. ANOVA, linear regressions and Chi square test were used for analysis.

: 73 women with a diagnosis of PPROM identified between 2008 and 2013. Groups 1, 2, and 3 included, 29, 30, and 14 women respectively. There were no significant differences between the 3 groups in baseline characteristics with the exception of a greater number of White women in group 2. None of the outcomes studied showed a significant difference between groups (see table) . A linear regression model including all the demographic variables and outcomes showed non-significant difference using a P value of <0.05. CONCLUSIONS: This study did not identify a difference between the traditional latency antibiotics regimen including erythromycin and alternative regimens including azithromycin. Further study is needed to confirm this result. During cervical ripening, HA deposition is increased in the stromal matrix, epithelial pericellular matrix and in cervical mucus. HA depletion in the cervix results in inappropriate differentiation of the cervico-vaginal epithelia, and a striking increase in preterm birth rates in a mouse model of ascending vaginal infection. However it is unknown how HA depletion leads to impaired epithelia and mucus function. Here, we evaluate the role of HA and, its binding partners, CD44 and versican in epithelial and mucosal barrier function.

The conditional HA knockout mouse was utilized to determine potential mechanisms by which HA depletion affects epithelial and mucosal barrier function using in vitro permeability assays in human ectocervical cell line (ECT-1) +/-HA knockdown with siRNA and in vivo inoculation of fluorescently-labeled microparticles into the mouse vagina. The expression and contribution of HA-binding partners CD44 and versican was evaluated in wild type and CD44 null mice. RESULTS: HA knockdown with siRNA decreased HA production 60-70% and increased ECT-1 cell permeability significantly compared to control group. Compared to WT, the microparticles moved deeper into the mucosal layer in the HA KO, appearing closer to epithelia suggesting that HA within the cervico-vaginal mucus plays a role in mucosal barrier protection from pathogens. CD44 expression was confined to the nonmucosal epithelia while versican was expressed throughout the epithelia. In contrast to the HA KO, epithelial differentiation and mucus vacuole structures of the CD44 KO appear similar to WT. The HA binding proteoglycan versican is expressed in the epithelia similar to HA expression suggesting a role in epithelial function with HA. CONCLUSIONS: Hyaluronan secreted by the epithelia during late pregnancy plays key roles in barrier function that are critical for protection against pathogen invasion and resulting preterm birth. Both CD44 and versican are expressed in the cervical epithelia and may serve as bindingpartners with HA to regulate epithelial functions. Table. IL-10 levels are greater (55-65% of total cytokines) in non-pregnant and pregnant cervices compared with other cytokines (P<0.05). IL-4 levels were always lower (P<0.05). On GD 10, nearly all cytokines are lower compared to nonpregnant rats (P<0.05). From GD 10 to GD 20, levels of IL-17, IL-6, IL-8, IFN-r and TNF-α significantly increase (P<0.05). Conversely, levels of anti-inflammatory cytokines ( IL-4 and IL-10) decrease on GD 20 compared to GD 14. Thus, generally proinflammatory cytokines decrease in early GD then increase in the cervix during late GD, except for IL-1 and IL-2, whereas anti-inflammatory cytokines (IL-4 and IL-10) increase on GD 14 then decrease on GD 20.

The changes in cytokine levels during gestation reflect the physiological inflammatory responses which control cervical function during gestation, including cervical ripening. 

The results indicate that Nrf2 activation represents a key stress response in amnion mesenchyme cells to mitigate the adverse pro-inflammatory effects of thrombin on the fetal membranes. We suggest, therefore, that pharmacologic activation of Nrf2 may prevent the increased risk of preterm premature rupture of the membranes after subchorionic hemorrhage or bleeding during pregnancy. Women were divided into three groups based on whether they gained less than, more than, or met the IoM recommendation for weight gain. Outcomes including gestational age at delivery, mode of delivery, blood loss, infant weight, 5 minute Apgar, and umbilical artery pH, were evaluated. ANOVA and generalized linear models were used to analyze obstetrical outcomes between weight gain groups.

We identified 961 women with a BMI of >/= 40 at their first prenatal visit. The average weight of infants born to women who gained less than recommended (3254 g), was significantly less than those infants born to women whose weight was within goal (3401 g) and those of women gained more than recommended (3548g) (p<.02).

In addition, infants born to women that gained more than recommended were significantly larger than those infants born to mother who met goal (p=0.018). Infants born to those who gained less than the recommended amount delivered on average 3-4 days earlier than those born to mothers at goal or over goal (p<0.02). There was no difference in mode of delivery between the three groups. Women from each weight gain group were then separated by mode of delivery. After this separation, there were no significant differences in maternal blood loss, 5 minute APGAR, and umbilical artery pH *Figure(s) will be available online. A linear regression model accounting for starting BMI, maternal age, race, and ethnicity showed no difference in the trends of the above outcomes. CONCLUSIONS: Infant weight at delivery was the most important difference noted between the three groups of women, with women who gained more weight delivering significantly larger infants. Although women who gained less than the recommended weight delivered earlier than the other groups of women, this may not be clinically significant.

First Abnormal amniotic fluid (AF) volume is an obstetrical complication occurring in diabetic pregnancies. Normal AF volume is maintained by intramembranous (IM) transport of AF across the amnion; and aquaporins (AQPs) has been shown to modulate this IM transport. Recently, AQPs 1, 3, 8 and 9 have been described in human amnion and may play a role in amnion function. We hypothesized that alterations in amniotic fluid transport could be linked with abnormal expression of AQPs in human fetal membranes. Belonging to a family of 13 members (noted AQP0 to AQP12), aquaporins (AQPs) play a major role in regulating water homeostasis of the organism. To test this hypothesis, we analyzed the expression profiles of AQPs on human fetal membranes from diabetic pregnancies. METHODS: 129 amniotic membranes were collected at term from normal, type 1 (insulin dependent), type 2 (insulin independent) pregnancies and gestational diabetes.A differential AQPs expression profile (qualitative and quantitative) at transcriptional and protein level were established of these membranes. The links between these different levels of expression will correlate with the main clinical and biological data of the mother and newborn.

No qualitative modification could be determined in human fetal membranes in terms of AQPs profile between normal and diabetic pregnancies (just AQPs 1, 3, 8 and 9 found). Quantitative decreases of AQP3 and AQP9 mRNA and proteins expression were determined in fetal membranes obtained from diabetic patients (type 2 and gestational diabetes). These modifications were clinically and experimentally linked with insulin status. CONCLUSIONS: Our results showed that some AQPs (3 and 9) expressions were decreased in diabetic pregnancies. These modifications are clearly linked with clinical and experimental increase of insulin. This couple "AQPs and insulin" could be an interesting pathophysiological mechanism to explain abnormal amniotic fluid homeostasis during diabetic pregnancies.

The 

We recruited 50 laboring women at term at an urban academic hospital (06/2013-11/2013). A questionnaire adapted from prior publications (WHO) was used to assess the frequency and type of dietary fish intake and to screen for occupational and residential exposure. Medicaid insurance was used as a proxy for low SES. MB and PCB pairs were collected and analyzed for blood mercury levels using inductively coupled plasma/mass spectrometry. RESULTS: 50 survey questionnaires and 44 blood sample pairs were collected. 75% of patients surveyed had Medicaid insurance. Fish intake was infrequent and consisted of types primarily low in mercury. * Figure( s) will be available online.

No occupational or residential risk factors were noted. Results were available for 37 MB and 24 PCB samples. Sample clotting invalidated the rest. Only 3 out of 61 samples tested (1 MB, 2 PCB) had detectable levels of mercury (>3 ng/mL) and none were above acceptable range (>5.8 ng/mL). Surveys of patients with clotted samples were compared for selection bias and no statistically significant differences were noted. CONCLUSIONS: We found an overall low frequency of dietary fish consumption, and no significant MeHg contamination in this low SES obstetric population. Positive mercury results (n=3) did not correlate with type or frequency of fish intake or with SES. Medicaid patients trended towards lower fish consumption but we lacked sufficient power to show this association. Given the potential benefits of dietary fish, further research and education is warranted in this population. Further, whether these biomarkers vary by gestational age is not well understood. Our objective was to characterize protein inflammatory biomarkers in normal pregnancy by trimester and maternal weight. METHODS: Serial blood samples were collected from 29 healthy adult pregnant women throughout gestation at 3 week intervals. Maternal plasma C reactive protein, Interleukin (IL)-6, IL-8, IL-1B, IL-12, granulocyte colony stimulating factor, tumor necrosis factor-α, vascular endothelial growth factor (VEGF) and haptoglobin concentrations were measured by Luminex Bead Array (Millipore). Mean biomarker values were compared by trimester and between obese (BMI >35, n= 10) and non-obese subjects (BMI<35, n=19) using least square means with adjustment for race. RESULTS: There was no difference in any biomarker levels by trimester.

Controlling for maternal race, mean IL-6 and VEGF concentration were significantly higher in obese vs. non-obese subjects (Table 1 ). In contrast, haptoglobin (acute phase protein) was lower in the obese vs. non-obese subjects (Table 1) . Of note, this baseline alteration remained constant throughout the pregnancy. Other biomarkers evaluated were not significantly different by BMI after controlling for race. , 59 patients had an abnormal early GCT but were not diagnosed with GDM until standard screening (group B), 158 patients were diagnosed with GDM during standard screening after a normal early GCT (group C). There was no significant difference in the incidence of macrosomia (≥4,000gm) or mean birthweight when comparing groups. Group A and group B were significantly more likely to require antenatal medication to control blood glucose when compared to group C. *Figure(s) will be available online. CONCLUSIONS: Patients who are diagnosed with early onset GDM or who have evidence of early glucose intolerance are more likely to require antenatal medication to control blood glucose. However, these patients were not more likely to give birth to a macrosomic infant. The choroid plexuses are small brain ventricular organs producing cerebrospinal fluid (CSF) that constantly bathes the brain. These organs differentiate early, just after neural closure and have in recent years the plexus has been shown to have a much more active role in brain development than previously appreciated thereby it can influence both neurogenesis and neural migration by secreting factors into the CSF, regulating the internal environment of the growing brain. Still, much of choroid plexus developmental function is still unclear. Most previous studies on this organ have been undertaken in rodents but translation into humans is not straightforward since they have a different timing of brain maturation processes.

In these studies we have used a nonhuman primate, the baboon, with close brain development to humans. We have collected choroid plexus from three fetal gestational ages with or without 30% maternal nutritional restrictions (MNR). The transcriptome of the plexuses was determined by next generation sequencing and Ingenuity Pathway Analysis software was used to annotate functions and enrichment of pathways of changes in the transcriptome both during normal development and in relation to MNR.

The number of unique transcripts decreased during normal development and the majority of differentially expressed transcripts were down-regulated through development suggesting a more complex and active plexus earlier in fetal development. The functional annotation indicated changes across widespread biological functions in normal plexus development. In particular we find age-dependent regulation of genes associated with annotation categories: Gene Expression, Cardiovascular System Development, Nervous System Development and Molecular Transport. Longer durations of MNR resulted in more changes to the transcriptome and were mostly associated to carbohydrate and amino acid metabolism with methionine and histidine pathways significantly enriched. Particularly of significance to plexus function is that Na-K-2Cl co-transporter (SLC12A2), a key transporter for CSF secretion, was strongly down-regulated following MNR. CONCLUSIONS: Our observations support the idea that the choroid plexus has roles in shaping brain development and suggests that MNR during pregnancy could change important choroid plexus functions. Supported by NICHD HD 21350. Information about mothers' meal frequency and maternal diet was obtained from a validated self-reported food frequency questionnaire answered in mid-pregnancy. Using principal component analysis, three meal patterns were found; "main meal pattern", "snack pattern", and "evening meal pattern". Information on gestational age at delivery was obtained from the Medical Birth Registry of Norway and preterm delivery was defined as birth before 37 weeks of gestation. RESULTS: After adjustments for covariates (maternal age, pre pregnancy BMI, height, parity, total energy intake, maternal education, marital status, smoking, income and previously preterm delivery), there was a trend towards lower risk for preterm delivery when following a "main meal pattern", which included eating breakfast, lunch and dinner regularly, p for trend 0.025. After additional adjustments for meal frequency and fiber intake, glycemic load was associated with late preterm delivery in the whole group, hazard ratio for the highest vs. lowest quartile of 1.25 (95% CI 1.00 to 1.56). A positive trend for an increased risk of preterm delivery with increased glycemic load in older women (age > 35 years) was indicated (p = 0.089). High intake of dietary fibers was associated with a significantly reduced risk of preterm delivery in younger women (age <= 35 years) with hazard ratio for the highest vs. lowest quartile of 0.85 (95% CI 0.73 to 0.98), p-for trend 0.027. For obese women (BMI > 30) a trend of reduced risk of preterm delivery with increased fiber intake was indicated, p-for trend 0.059. CONCLUSIONS: Eating regular meals throughout the day were associated with a trend towards lower risk of preterm delivery. The significant association of various measures of glycemic properties with different subgroups of the study population and for different subgroups of preterm delivery indicate the importance of maternal diet for infant health and need further investigation to be fully understood. 

Being obese appears to protect against third and fourth degree lacerations independent of parity, race, birth weight and mode of delivery. We hypothesize that this may be because of perineal body differences among obese and nonobese women. These results lend themselves to future studies to help elucidate the etiologies of severe lacerations. Pre-gestational type 2 diabetes is associated with multiple maternal and fetal complications in human pregnancy and is characterized by increased vascular resistance in the maternal uterine circulation. The exact nature of higher vascular resistance remains unknown but may include insufficient structural remodeling and/or changes in the functional behavior of the uteroplacental vasculature.

Objectives of this study were to assess the impact of type 2 diabetes on:

(1) maternal and fetal pregnancy outcome;

(2) remodeling of maternal uterine vasculature; and (3) endothelial function of uterine radial arteries from pregnant rats. METHODS: Pregnant Goto Kakizaki (GK) rats (n = 10), a non-obese model of spontaneous type 2 diabetes, and Wistar control (WC) rats (n = 12) were used to assess the effect of type 2 diabetes on pregnancy outcome, vascular remodeling and function of maternal uterine vasculature.

Oral glucose tolerance tests (GTT, 2 g/kg) were performed on day 15 of pregnancy. Dimensions of the maternal vasculature were defined from photographs using Bersoft image software. Second order radial uterine arteries feeding the placenta were pressurized, pre-constricted with phenylephrine in the presence of L-NNA and indomethacin. The diameter responses to ACh (0.01-10 µM) were assessed in arteries of WC and GK rats. RESULTS: Levels of blood glucose during GTT were significantly higher in GK vs. WC rats (maximal 193 ± 16 vs. 87 ± 7 mg/dL). The number of fetuses was reduced from 15.4 ± 0.6 to 10.4 ± 0.6 and fetal resorption rate was increased from 0.4/rat to 2.2/rat in diabetes. Total fetal mass was much lower in GK (25.5 ± 1.2 g) vs. WC (36.9 ± 1.2 g) rats; placentas were heavier in GK (0.455 ± 0.01g) vs. WC (0.413 ± 0.01g). Main uterine artery length and axial elongation of the maternal mesometrial uterine vasculature was significantly reduced in diabetic pregnancy. ACh-induced NO-and prostacyclin-independent vasodilation of radial arteries was impaired in diabetic pregnancy (18 ± 8%, n = 5 vs. 54 ± 6%, n = 5 at 0.1 µM of ACh). CONCLUSIONS: Type 2 diabetes in pregnant Goto Kakizaki rats is associated with significant impairment of maternal vascular growth and reduced endothelium-mediated vasodilation. This vascular maladaptation most likely contributes to reduced maternal uterine blood flow and an increase in fetal demise in pregnancies complicated by type 2 diabetes. in the NPH group). There were no differences between the two groups with respect to maternal characteristics. The live birth rate was 95% in the IDet group (2 fetal demises at 10 and 20 weeks) and 100% in the NPH group. There were no significant differences between groups with respect to any of the perinatal outcomes examined (Table 1) . . This is not endorsed by the NICHD or ACOG due to the expected increase in prevalence of GDM (³18%) with no demonstrated improvement in outcome. We sought to determine the impact of using the 1-step method on the prevalence of GDM in a diverse racial population. METHODS: A retrospective cohort was performed using lab data from 1/2008-12/2013 of patients screened for GDM. The 1-step method was used by providers from 1/2011-10/2012 and GDM was diagnosed if ³1 of 3 values on the GTT was abnormal (fasting ≥92, 1 hour ³180, or 2 hour ³153mg/dL). In the remaining time, the 2-step method was used, beginning with a 50g 1-hour glucose challenge test (GCT); if ³130mg/ dL, a 100g 3-hour GTT followed. GDM was diagnosed if any value was ≥ 95, 180, 155, or 140 mg/dL at fasting, 1, 2, and 3 hours. The overall and race-specific prevalence of GDM was determined for the 1 and 2-step methods (using either ³1 or ³2 abnormal GTT values). RESULTS: 4,179 women had a screen; 1,600 underwent a 2-hour GTT and 142 (8.9%) were diagnosed with GDM. The 2-step method was used in 2,579 women: 592 (23.0%) had a positive GCT followed by a 3-hour GTT. GDM was diagnosed in 103 (4.0%) using ³2 abnormal values, and in 184 women (7.1%) using ³1 abnormal values. See Table 1 . 

In a diverse population, using the 75g 2-hour GTT is associated with a more than doubling in the overall and race-specific diagnosis of GDM. But, the prevalence does not reach the predicted 18% or more. It remains to be seen whether enhanced diagnosis of GDM is associated with improved pregnancy outcome.

in adverse pregnancy outcomes such as preterm birth (PTB) and small for gestational age (SGA), leading to programs aimed at rebalancing omega-6:omega-3 ratios using dietary or supplementary strategies. This pilot study aimed to examine the relationship between midpregnancy maternal omega-6 and omega-3 fatty acid status, and later PTB and SGA, using the Thousand Women Study cohort. METHODS: Maternal blood samples were obtained at 20 weeks gestation, plasma extracted and stored at -80 o C. Using a nested casecontrol design, these mid-trimester samples (11 preterm, 11 small for gestational age, and 11 controls) were analysed for essential fatty acids using HPLC-MS/MS. The relative proportions of omega-6 (AA, LA) and omega-3 (EPA, LNA) fatty acids were ascertained and intergroup differences sought using Mann-Whitney-U and Kruskall-Wallis tests. Further research is needed within interventional trials to ascertain whether these ratios are modified by dietary or supplementary intake and whether such strategies lead to a reduction in adverse pregnancy outcomes. 

Female Wistar rats were weaned to chow (C) or high energy, obesogenic diet (MO). One month before and in pregnancy 50% of females from both groups were treated with 20mg/kg/day Res orally and remained with the same diet until the end of lactation, providing two additional groups CRes and MORes. All females were mated at 120 days. Male and female OFF were weaned to C diet. At postnatal day 120 one male and female per litter were euthanized and retroperitoneal fat excised to determine fat cell size by histology. Fat cell size frequency was modeled with gamma distribution, defined by its shape (a) and scale (b) parameters which are related to data heterogeneity. RESULTS: Fat cell size was smaller in female compared to male. Fat cell size median (Fig. 1. A and B) and heterogeneity ( Fig.1. C and D) of male and female OFF were increased in MO, but returned to C level in MORes and were decreased in CRes vs C (Fig. 1. B and C) . Adiponectin and leptin are placental and adipocyte-secreted proteins involved in metabolic homeostasis; the adiponectin:leptin ratio (ALR) has been used as a proxy measure of insulin sensitivity. Vitamin D and the omega-3 fatty acid docosahexaenoic acid (DHA) have been reported to be insulin-sensitizing and to influence the adiponectin and the ALR in non-pregnant individuals.

We analyzed levels of adiponectin and leptin in maternal and umbilical cord blood in order to determine if DHA or eicosapentaenoic acid (EPA) supplementation increased insulin sensitivity or if an association exists between Vitamin D and measures of insulin sensitivity or resistance.

This study is a secondary analysis of a double-blinded, randomized controlled trial (the Mothers, Omega-3, & Mental Health Study) in which 126 women were randomized to receive either a DHArich fish oil supplement, an EPA-rich fish oil supplement, or a soy oil placebo. Maternal venous blood was collected at 12-20 weeks gestation (N=103) and 34-36 weeks gestation (N=103). Umbilical cord blood was collected at the time of delivery (N=94). We analyzed blood samples for adiponectin and leptin using ELISA. Vitamin D and DHA levels had been previously assayed. We performed a multivariate logistic regression to evaluate the association of Vitamin D with leptin and the ALR before and after supplementation, and in umbilical cord blood.

The ALR decreased from early to late pregnancy, consistent with increasing insulin resistance over gestation. Vitamin D was significantly associated with the adiponectin level at 12-20 weeks gestation (p=0.04), though it was not associated with the ALR. CONCLUSIONS: Although not associated with the ALR, the potential association of vitamin D with adiponectin, a marker of insulin sensitivity, warrants further investigation. Less is known about these hormone binding globulins in pregnancy and, to the best of our knowledge, no information exists to compare these binding globulins for any changes in lean vs obese pregnant women. METHODS: Pregnant women were enrolled and provided informed consent by 8 weeks of gestation and, with few exceptions, provided blood samples every two weeks starting at 8 weeks through gestation.

No subject provided less than 12 scheduled blood draws and the average was 14.xx draws per subject. Serum was assayed by ELISA for TBG and CBG and SHBG was assayed by chemiluminescence immunoassay. The longitudinal data was analyzed using 2-way mixed measures ANOVA with Bonferroni correction.

the course of gestation, as expected largely due to the effect of estrogen levels in pregnancy. Levels of the binding globulins were significantly different by habitus and interval, with lean patients having higher SHBG levels and lower TBG and CBG levels. CONCLUSIONS: Steroid and thyroid hormone levels in these same subjects are not different across gestation and suggest that these subtle significant differences in binding globulins have no great effect on circulating levels of their respective hormones.

Fetal Maternal obesity is a major problem in obstetrics: it impacts the outcome of the pregnancy per se and programs the offspring for adult metabolic disease. The placenta communicates the maternal and uterine environment to the fetus and mediates developmental programming. We have shown mitochondrial dysfunction, oxidative stress and inflammation in the placenta with maternal obesity. We hypothesized that placental lipid metabolism is dysregulated with increased maternal adiposity.

Placentas were collected at term from lean women (LN, pre-pregnancy BMI=22.0±0.4,) and obese women (OB, BMI=36.1±0.9) following C-section with no labor (n=20 placentas/group, 10 male and 10 female). Expression of total and phosphorylated (p)AMPK and AKT, transcription factor EB (TFEB) and of peroxisome proliferator-activated receptor gamma (PPARγ) were measured by Western Blot.

We found a 2-fold increase in the levels of pAMPK, an important metabolic regulator of fatty acid intake and oxidation, in male placentas of OB women compared to males of LN women (p<0.05).

Expression of TFEB and PPARγ, two transcription factors involved in lipid catabolism, was reduced by 70 and 85% respectively (p<0.05) in male placentas from OB compared to males of LN women. No differences in the levels of AKT and total AMPK were observed. A metabolomics analysis revealed consistent changes in lipid profiles only in male placentas from OB mothers compared to males of LN mothers (p<0.05): this included a 50% reduction in the levels of arachidonoyl carnitine (C20:4) and 2-fold increase in the levels of C16/C18 dihydroceramides. Similar changes in the content of these metabolites have been previously reported in plasma and adipose tissue of non-pregnant obese individuals and linked to metabolic syndrome and Type 2 Diabetes. There were sex differences in placental lipid profiles within the LN group: the content of C20 and C24 ceramides, C16 and C18 dihydroceramides, sphingosines (d18/d20), sphinganines (d18/d20) and several sphingomyelins were 25-50% reduced in male placentas compared to female placentas (p<0.05). CONCLUSIONS: Our study identifies sex differences in placental lipid profiles with maternal obesity. Placentas of males showed more metabolic perturbations associated with obesity and metabolic syndrome than those of female fetuses. The objective of this study was to use both fibrinogen levels and a thrombin generation (TG) assay to evaluate thrombotic tendency in obese compared to normal weight pregnant women longitudinally across gestation.

Nineteen healthy, non-smoking, normal weight (BMI 18.4 -24.9 kg/m2) and obese (BMI ≥ 30 kg/m2) women underwent venipuncture at 10-14 and 30-34 weeks of gestation. TG was evaluated in contact pathway inhibited, re-calcified plasma, initiated with 5pM tissue factor/20µM phospholipid in the presence of a fluorogenic substrate. TG was analyzed for the peak rate of formation, maximum level generated and total thrombin formed. Fibrinogen levels were also measured. Results are expressed as mean±SD. Student's paired and unpaired T-tests were used for statistical analysis. P < 0.05 was considered significant. RESULTS: Average BMI in the normal weight (n = 12) and obese (n = 7) group was 22 and 33.8 kg/m2, respectively. Maximum TG and fibrinogen were elevated in obese compared to normal weight women in early pregnancy ( 

In women complaining of RFM, maternal glucose administration has no effect on perceived fetal movement and does not reduce the need for admission or induction of labor due to persistence of complaints.

Maternal Obesity, Leptin, and Blood Loss at Delivery. 

With adjustment for established risk factors for PPH, BMI was not associated with EBL at VD. Leptin was not a predictor for EBL independent of BMI. We speculate that increased rates of PPH among obese women may be related to factors such as increased oxytocin usage and longer labor rather than an intrinsic abnormality such as impaired myometrial contractility.

Role In cancer cells high rates of glycolysis are regulated by activation of mTOR signaling, inhibition of microRNA(miR)-143 and upregulation of its target gene hexokinase 2 (HK-2). We hypothesized that activation of placental mTOR signaling inhibits miR-143 and results in activation of aerobic glycolysis in GDM placentae.

Placental tissue was collected at term after C-section (no labor) from women with GDM, treated with diet alone (A1GDM), treated with glyburide or insulin (A2GDM) (n=6/group). Control (CTRL) placentae were collected from healthy women and matched for pre-pregnancy BMI and gestational age (n=6). Expression of miR-143 and HK-2 was analyzed by RT-PCR and Western blot respectively. Mitochondrial respiration was measured by Seahorse XF Analyzer in syncytiotrophoblasts (ST) from CTRL and A2GDM placentae. RESULTS: No significant differences in fetal weight were observed, however, the placental weight was higher (p<0.04) in A2GDM (733±58g) compared to CTRL (587±48g) and A1GDM (706±76g). Therefore, the placental-fetal weight ratio was increased (p<0.03) in A2GDM vs. A1GDM and CTRL. The nutrient sensor AMPK Thr-172 was decreased in A2GDM but effectors of both mTORC1 (p70S6K Thr389 and 4EBP Thr37-46 ) and mTORC2 (AKT s473 ) were increased in A2GDM vs. A1GDM and CTRL (p<0.05). Expression of miR-143 was reduced by 50% while mRNA and protein expression of HK-2 were significantly increased in A2GDM vs. A1GDM or CTRL (p<0.05). Furthermore, mRNA and protein levels of glycolytic enzymes such as phosphofructokinase and lactate dehydrogenase and glucose transporter GLUT1 were increased (p<0.04) in A2GDM. Expression of mitochondrial complexes I, II, III and IV and transcription factors PPARg and PGC1a were decreased (p<0.05) in A2GDM. In vitro studies showed reduction in mitochondrial respiration in STs from A2GDM vs. CTRL, this was prevented by transfection with pre-miR-143 (p<0.05).

In conclusion, activation of mTOR signaling, decreased miR-143 expression, mitochondrial dysfunction and activation of aerobic glycolysis was found in A2GDM placentas. The preservation of mitochondrial respiration by miR-143 overexpression highlights the role of miR-143 in mediation of metabolic reprogramming towards glycolysis in A2GDM placentae. RESULTS: Compared to controls, IGF1, IGFBP1 and IGFBP2 gene expression was found to be significantly under-expressed in placentae of GDM group on diet (n=23) and in those on metformin (n=23) but not in those taking insulin (n=15). There was no difference in placental IGF2 expression. IGF1, IGFBP1 and 2 genes were significantly hypermethylated in women on diet only but not women on metformin or insulin *Figure(s) will be available online. CONCLUSIONS: Placental expression of IGFs and their binding proteins differ in pregnancies affected by GDM. Down regulation of IGF1 expression in GDM on diet and metformin group but not on insulin may suggest an endocrine role of insulin in normalising altered IGF1 expression in diabetic placenta. Significant hypermethylation in women only on diet but not on metformin or insulin raises the question of role of metformin and insulin in regulating methylation. Whether these alterations contribute to neonatal phenotype or accompany other pathogenetic mechanisms requires further investigation. We analyzed the differences in baseline characteristics between GDM diagnosed patients utilizing these two paradigms: ACOG 2-step approach of screening followed by diagnostic testing, the ADA recommendation of 1-step diagnostic testing.

A retrospective review of data from pregnant women (24-28wks) screened for GDM over two periods: 1) November 2011-May 2012; 2) November 2012-May 2013. Period 1: the 2-step approach employed a 1 hr 50 gm glucose followed by a 3-hr 100 gm glucose tolerance test (GTT) if the 1 hr screen was ³140 mg/dl. Period 2: a single abnormal 2hr 75gm GTT result was used for diagnosis. We compared: group means (maternal age, gestational age at delivery (GA), weight gain, weight at the start and end of pregnancy, HbA1C, neonatal ponderal indices (PI)), race, parity and treatment strategy (Diet/Glyburide/insulin) between GDM diagnosed patients from one approach to the other. We also performed univariate analysis comparing both 1-step and 2-step approaches for treatment strategy, HbA1C, estimated fetal weights (EFW), abdominal circumference (AC) and PI .

The mean maternal weights at the start and the end of pregnancy were significantly lower in the 1-step approach compared to the 2-step approach ((166.3, 213 .3 p=.0011) (186.13, 230.90 p=0.0014)) respectively although, the overall weight gain was similar. Parity, race, maternal age, gestational age at delivery, weight gain, HbA1C and ponderal index were not statistically significant. Univariate analysis comparing HbA1C level, parity (nulliparity vs multiparity), second and third trimester sonographic parameters (EFW, AC), ponderal index, treatment strategy (Diet, Glyburide and Insulin) between both approaches were statistically similar. CONCLUSIONS: Individuals diagnosed with GDM using the 1-step group began pregnancy with lower weights than those in the two step group, confirming more subtle degrees of dysglycemia are detected with the 1-step approach. Despite similar short term perinatal outcomes between the 1 and 2 step approaches, the long term benefits of prevention of dysmetabolism in those with subtle degrees of dysglycemia remains to be fully explored. (GD 30) . At GD 165 (0.9 gestation), placentas were collected and microvillous plasma membrane vesicles (MVM) were isolated. MVM System A and system L AA transport capacity was determined in vitro using radiolabeled substrates and rapid filtration techniques. In vivo transplacental AA transport was assessed by infusing a cocktail of nine 13 C or 2 H labeled essential amino acids (EAAs) as a bolus into the maternal circulation at the time of cesarean section. Fetal/maternal mean plasma enrichment ratio for each EAA was calculated. RESULTS: The activity of system A and system L was significantly reduced by 73% and 84% respectively (P<0.01) in MVM isolated from baboon placentas at GD 165 following MNR. Preliminary in vivo data suggests that the fetal/maternal plasma enrichment ratio for leucine, isoleucine, methionine, phenylalanine, threonine, tryptophan and lysine was reduced in MNR pregnancies. CONCLUSIONS: Placental transport has not been adequately examined in pregnancies subjected to undernutrition, and this is the first study to investigate placental AA transport in a nonhuman primate model of MNR. We provide direct evidence that the down-regulation of system A and system L activity in MVM isolated from the placenta of MNR baboons leads to decreased transplacental AA transport capacity and consequently reduced amino acid concentrations in the fetal circulation. This is likely a key mechanism linking maternal undernutrition to reduced fetal growth.

Sirtuin Sirtuin 1 (SIRT1) is a nutrient sensing protein deacetylase involved in glucose and insulin metabolism and nutrient transport. SIRT1 has been called a ''reverse thrifty gene'' that protects against metabolic diseases by directing the organism to limit energy consumption and expenditure. SIRT1 activators have been suggested as a potential treatment for Type 2 diabetes. We aimed to characterize SIRT1 expression in placentas of normal and GDM pregnancies and to determine the role of SIRT1 in the regulation of nutrient transport proteins in vitro. METHODS: Human term placentas were obtained at delivery from women whose pregnancies were normal (n=15) or complicated by GDM and controlled by diet (n=15). Gene expression for SIRT1, fatty acid transporter proteins FATP1-6, glucose transporter 4 (GLUT4) and amino acid transporters (SNAT1, 2, 4 and ASCT 1 and 2) were examined by real-time PCR and SIRT1 protein was localised by immunohistochemistry in human placental tissues. SIRT1 activity was inhibited in HTR-8 cells (first trimester trophoblast cell line) with vehicle, 2µM, 6µM or 10µM Ex-527 (SIRT1 inhibitor) treatment for 24 and 48 hours (n=3). RESULTS: SIRT1 mRNA expression was higher in villous placenta compared to other gestational tissues with no difference between placentas collected after labour vs elective Caesarean section. SIRT1 mRNA and protein expressions were down-regulated in term placentas from pregnancies complicated with GDM (p<0.0001). SIRT1 inhibition had no effect on cell viability in HTR-8 cells, but resulted in up-regulation of fatty acid transporter proteins FATP2 (p<0.01), FATP4 (p<0.05) and GLUT4 (p<0.01) after 24 hours and down-regulation of FATP6 (p<0.05), amino acid transporters SNAT2 (p<0.01) and ASCT1 (p<0.05) gene expressions after 48 hours. CONCLUSIONS: Our data implicate a role for SIRT1 in the pathogenesis of GDM and in the regulation and nutrient transporter proteins in human placenta.The need for improved medical treatments to both prevent and treat GDM is becoming increasingly apparent. Manipulation of SIRT1 may constitute a novel future approach for the treatment of GDM. Our study aimed to create a nomogram for expected physiologic changes in UP for PD throughout gestation. Our second objective was to determine changes that relate to the development of PE.

A retrospective cohort study of PD with UP collections during the pregnancy were analyzed using locally weighted scatterplot smoothing (LOESS) and logistic regression modeling. The primary outcome was total UP in 24 hours. Secondary outcomes included correlation of UP, initial pregnancy A1c, protein creatinine ratio, creatinine clearance and development of PE. RESULTS: A total of 150 patients were reviewed and 97, 28 and 6 of these had at least 1, 2 or three 24 hour UP collections, respectively. First trimester values were found in 42 (28%) of the patients. LOESS was used to demonstrate initial UP by gestational age (GA).

A total of 36 (24%) patients developed PE. Creatinine clearance did not correlate with UP and did not predict PE. The initial 24 hour UP and initial A1c univariately predicted PE, but when used together in a logistic regression model, neither was predictive when adjusted for the other. CONCLUSIONS: Proteinuria in pregnancy does not appear to significantly increase throughout pregnancy, or correlate with changes in creatinine clearance. Baseline UP appears increased for PD, but is not an independent predictor for PE. Our data does not support serial assessment of UP in PD, although other parameters, perhaps initial A1c, may be more valuable in prediction of PE.

Severe We evaluated the percentage of the population with a PTD and dichotomized the etiology of the PTD as indicated or spontaneous. Prepregnancy and first trimester maternal characteristics were compared between women with and without a PTD using chi-squared and Student's t-test as appropriate. RESULTS: One-hundred ten women with T1DM were identified. Of these, 20 had a PTD (18%) and 90 delivered at term (82%). The majority of these PTDs were indicated (14/20, 70%) and all (100%) of these were due to severe preeclampsia. The mean GA at delivery was 34±1 weeks. The remaining 6 patients (5% of total) had a spontaneous PTD with a mean GA at delivery of 34±2 weeks. Maternal age, body mass index, mode of insulin administration (continuous subcutaneous insulin infusion versus multiple daily injections), and hemoglobin A1c (HbA1c) did not differ between patients who had a PTD versus a delivery at term. Maternal age at diagnosis of T1DM was lower in those with a PTD (9±5 versus 14±7 years, p=0.008). There were no differences in maternal characteristics between the subset of patients with a PTD due to preeclampsia when compared to those who delivered at term.

The rate of spontaneous PTD in a cohort of pregnant patients with T1DM is lower than the baseline population risk. However, severe preeclampsia accounts for the majority of PTD and at a higher rate than expected. There do not appear to be any specific clinical predictors of preeclampsia thus patient signs and symptoms of this disease should be meticulously evaluated by the care provider. A future direction could include analysis of serum analytes to identify those at higher risk. There is little data on outcome in women who develop obesity during the pregnancy. Our goal was to determine whether obesity acquired during pregnancy increases the risk of adverse obstetrical outcomes.

A retrospective cohort of patients who presented with a singleton gestation in the 1 st trimester in 2013-2014 in a single practice. Patients were placed into 2 groups based on BMI at entry and at delivery. Group A, did not develop obesity; and group B, developed obesity during pregnancy; group C, started pregnancy obese. Outcomes were gestational diabetes [GDM], pregnancy related hypertensive disorders (PRH) (gestational hypertension and pre-eclampsia), Preterm birth, small for gestational age (SGA), cesarean delivery (CD), composite maternal (GDM, PRH, PTB, PPH, ICU admission and chorioamnionitis) and composite neonatal outcomes (SGA, NICU admission, IUFD, cord pH <7.2, and 5 minute APGAR <7). Groups were compared using chi square, fisher exact or student t-test where appropriate with p<0.05 used as significance. RESULTS: 377 women met criteria for inclusion. Patients who developed obesity gained the most weight. They were at increased risk of primary CD when compared to those who remained non obese and were at decreased risk for PRH and GDM than those who started off obese. 

This was a secondary analysis of a prospective cohort study examining angiogenic factors and the development of HDP. The dataset included singletons with a 1st visit <12 weeks and a 2nd visit at 24-28 weeks. We calculated the upper limit of MWG up to 28 weeks based on starting BMI and IOM guidelines. Actual weight gain was compared to the upper limit to determine whether women exceeded the guidelines. Data was compared with chi-squared and Wilcox rank sum as indicated and MVLR was used to determine the odds of developing each outcome adjusting for age, parity, race, and BMI category. RESULTS: Of 1,505 women studied, those who gained > IOM guidelines were more likely to be nulliparous (31 v 24.7%, p=0.015), white (66 v 52.2%, p<0.001), and less likely to be obese (15.1 vs 32%, p<0.001).

Adjusting for age, parity, race and obesity, women who gained > IOM guidelines weren't more likely to develop HDP or other APO; however they were more likely to require a cesarean delivery (table 1) . Additionally, when stratifying by BMI, overweight and obese women had significantly greater risk for HDP while normal BMI was protective. CONCLUSIONS: Women whose weight gain was >IOM recommendations by 28 weeks didn't have a higher risk of developing HDP. However, initial BMI did show a correlation with the development of HDP, regardless of weight gain. As such, preconception weight loss efforts will have the greatest potential impact on reducing the risk HDP.

Maternal Consumption of a high-fat Western-style diet (WSD) during pregnancy reduces uteroplacental blood flow and increases placental inflammation in our non-human primate (NHP) model of dietinduced obesity. Given the prevalence of maternal obesity in the human population, preconception dietary modification may be a feasible and cost-effective potential therapeutic intervention. We hypothesized that switching obese NHPs from a WSD to a control diet (CTR) prior to conception would improve maternal metabolic health, restore uterine blood flow and decrease placental inflammation. METHODS: Japanese macaques were fed either WSD (36% fat) for 4+ years (n=6), CTR (14% fat) diet (n=6) or were switched from a chronic WSD to CTR 3 months prior to breeding (REV, n=5). Doppler ultrasound was performed at gestational day (G) 90 and 129 (term=G175) and uterine artery (uta) blood volume was calculated from uta blood flow velocity and diameter (cQuta; ml/min/maternal weight in kg). Glucose tolerance test (GTT) was performed at G120. Placental inflammation was assessed by immunoassay of placental protein from tissue collected at G130 cesarean delivery. RESULTS: Maternal REV resulted in a significant reduction in body weight (Mean±SD; 18.5±3.4%, p<0.0005) and improvement in GTT area under the curve (pre-REV: 8457±1807 vs. G120: 5164±871, p<0.05).

We measured a non-significant reduction in cQuta with WSD which was restored beyond CTR at both G90 and G129 with REV. Placental IL-1b expression was decreased (p<0.05) by REV compared to WSD in addition to a trend in decreased RANTES levels. *Figure(s) will be available online. CONCLUSIONS: Our data demonstrate that switching obese NHPs from a WSD to a CTR diet prior to conception improves maternal phenotype, increases uta blood flow and decreases placental inflammation. This suggests that preconception dietary modification may be of benefit in the obese population.

Hypoxia . APGAR scores differed between cases and controls at one minute (p=0.03), but not at five minutes. There were no significant differences in need for induction of labor, shoulder dystocia, NICU admission, attendance of postpartum visits, permanent sterilization, birth weight, gestational age at delivery or route of delivery. CONCLUSIONS: Centering Pregnancy group prenatal care model can be used for select high-risk populations and can lead to improved obstetrics outcomes and increased postpartum diabetes testing and utilization of long-acting reversible contraception.

Increasing Obesity has been associated with sub-clinical hypothyroidism, and obesity during pregnancy is associated with large for gestational age infants. Excess gestational weight gain (GWG) is associated with adverse outcomes including gestational diabetes and newborn macrosomia. In this study we sought to determine if differences in maternal or cord free T3 (FT3), free T4 (FT4) and TSH levels are associated with increasing maternal obesity or excess GWG. METHODS: Maternal and cord blood was collected immediately following delivery, snap frozen and stored at -80°C until use. Serum FT3, FT4 and TSH was analyzed using ELISA. Subjects were categorized by pre-pregnancy or first trimester BMI into 5 categories: Normal weight (18.5-24.9, N=43); Overweight (25.0-29.9, N=51); Obese 1 (30.0-34.9, N=52); Obese 2 (35.0-39.9, N=37); Obese 3 (≥40.0, N=22). GWG was calculated using validated formulations adjusting for gestational age at delivery and accounting for initial BMI. Data was analyzed using SigmaPlot. Multiple linear regression analysis was performed, adjusting for maternal and gestational age. Beta-coefficients are reported for each TH [table 1 ]. Pearson coefficients were used to determine significant correlations between THs and birth weight. RESULTS: Both maternal and cord FT3 significantly increases with maternal obesity (table 1) , while maternal FT4 significantly decreases (table 1) . Cord FT4, as well as maternal and cord TSH remain unchanged by virtue of maternal obesity. There is a significant correlation between maternal and cord FT3 (r=0.407, p<0.001) and an inverse correlation between maternal FT4 and birth weight (r=-0.145, p=0.039). Maternal FT4 is significantly decreased with excess GWG compared with women with insufficient GWG across all BMI strata (0.86ng/dL vs. 0.95ng/dL, p=0.018). 

We report that maternal and cord THs are altered with increasing maternal obesity. We further these findings by revealing that maternal FT4 levels significantly decrease with excess GWG. We conclude that like maternal obesity, excess GWG during pregnancy deregulates the maternal thyroid axis. Given the modifiability of GWG relative to obesity during pregnancy, we speculate that these findings bear potential implications and would benefit from further study. T1DM) pregnancies are associated with the duration of T1DM, glycemic control and adverse pregnancy outcomes. The aim of this study was to compare pregnancy and perinatal outcomes in T1DM women with vascular complications (White's classes D (WD), R (WR) and F (WF). Only the last childbirth was included in the analyses. METHODS: 1086 consecutive T1DM women with a singleton childbirth between 1989-2011 were studied. 633 (58.3%) had vascular complications. WD patients (N=408) had DM onset <10 years of age, DM duration >20 years or background retinopathy, WR patients (N=119) had proliferative retinopathy with laser treatment before, during or immediately after pregnancy, WF patients (N=106) had diabetic nephropathy with proteinuria ³0.3 g/24h in each trimester. The same method for measurement of HbA 1c (Diamat, Bio-Rad Laboratories, Hercules, CA) was used during the whole study period. Three HbA 1c values were recorded: the first in the 1st trimester, at 20 weeks of gestation and the last value before delivery. ANOVA or Chi-square tests were used for statistical comparison. RESULTS: Maternal age did not differ between the groups. The age (mean±SD) at DM diagnosis was in WD 9.9±5.4, in WR 8.4±4.7 and in WF 9.3±4.6 years. The duration of DM was in WD 21.1±5.8, in WR 23.4±5.3 and in WF 20.8±5.8 years. The 1st trimester HbA 1c was 8.6±1.4% in WF, which differed (p<0.001) from HbA 1c in WD 7.6±1.1% and WR 7.7±1.2%. The HbA 1c values did not differ between the groups at midtrimester (WD 6.7±0.8%, WR 6.6±0.8%, WF 6.8±0.9%, p=0.59) or before delivery (WD 6.9±1.0%, WR 6.8±0.9%, WF 6.9±1.1%, p=0.90 

The rate of non-compliance with recommended follow-up testing for DM is high, even within a research protocol. Given this, testing with A1c and GTT prior to hospital discharge at the time of delivery can identify a population of patients at low risk, and allow focus of outpatient resources on achieving compliance in patients at higher risk. Adiponectin and leptin are adipose-secreted proteins that may be used as proxy measures of insulin sensitivity; increased adiponectin:leptin ratio (ALR) reflects enhanced insulin sensitivity. Our aim was to determine the association of maternal serum DHA levels with the adiponectin:leptin ratio (ALR).

This was a secondary analysis of a randomized controlled trial designed to determine if fish oil supplementation prevented pregnancy depressive symptoms. 126 women with singleton pregnancies were randomized to one of three arms: 1) an EPA-rich fish oil supplement (1060 mg EPA plus 274 mg DHA), 2) a DHA-rich fish oil supplement (900 mg DHA plus 180 mg EPA), or 3) a soy oil placebo. Maternal venous blood samples were collected at 12-20 weeks gestation (before supplementation) and at 34-36 weeks gestation after a 4 hour fast. We performed ELISA on stored plasma samples (N=115) to quantify leptin and adiponectin levels.

We investigated whether maternal serum DHA levels were associated with markers of insulin sensitivity. We performed a regression analysis to investigate the relationships between maternal DHA with adiponectin, leptin and the ALR, adjusting for vitamin D levels, maternal age, and maternal weight gain (known confounders of insulin sensitivity).

On multivariate analysis higher maternal serum DHA levels were associated with increased ALR before (p <0.02) and after (p <0.02) supplementation. As expected, early pregnancy BMI was positively associated with maternal leptin levels at baseline and in late pregnancy (all p < 0.001), and was inversely associated with the ALR (p < 0.001). Likewise, maternal weight gain was positively associated with higher maternal leptin levels (p < 0.002) and decreased ALR (P<0.02). See * Figure(s) will be available online. CONCLUSIONS: Maternal insulin sensitivity as measured by the ALR is positively-associated with higher maternal DHA levels. Future studies will be necessary to determine whether maternal DHA supplementation translates into improved insulin sensitivity. Mothers were bred at postnatal day (PND) 120 and ate pregnancy diet until weaning. OFF ate C diet from weaning. Male OFF were euthanized at 110, 450 and 650 PND -n = 5-7 males from different litters per group. Serum and liver were obtained. We determined offspring serum and liver malondialdehyde (MDA-spectometry) liver reactive oxygen species (ROS -spectrofluorometry) and superoxide dismutase activity (SOD -activity by xanthinoxidase). RESULTS: As MO OFF age, serum and liver MDA increased earlier (Figs 1A and B) as did ROS (Fig 1C) while liver SOD decreased earlier than in OFF of control mothers. *Figure(s) will be available online. Nonalcoholic fatty liver reflects hepatic insulin resistance and is a key feature of metabolic syndrome. There is little known about the impact of maternal nonalcoholic fatty liver disease on fetal growth. The purpose of this study was therefore to investigate the association of early elevated ALT with large for gestational age birthweight. METHODS: This is a secondary analysis from a nested case-control study of ALT values measured between 8-18 weeks and subsequent gestational diabetes (GDM). We included women with singleton gestations and without known diabetes, liver disease or moderate self-reported alcohol use during pregnancy. We used inverse probability weighting to standardize the population and minimize selection bias. We calculated population-based birthweight z scores and defined LGA as at or above the 90%ile. We compared baseline characteristics with either ANOVA, Fisher's exact test or Wilcoxon rank sum. Covariates were identified by univariate regression analysis. We then performed conditional logistic regression to evaluate the relationship between either ALT≥90%ile or logALT and LGA adjusting for maternal age, BMI, parity, GDM, smoking and maternal weight gain.

We identified 26 cases of LGA out of 323 subjects. The mean(SD) maternal BMI was higher in the LGA group compared to controls ( While there are many studies on maternal obesity on offspring (OFF) outcomes, few address milk composition. We evaluated effects of maternal obesity on milk composition and pup brain weight. METHODS: Rats were fed control diet (C= 5% fat) or obesogenic (MO= 25% fat) during growth (21-120 d), pregnancy and lactation. At lactation day (LD) 21 milk was obtained and assayed for water, protein, glucose, leptin and fat. Pup brain was weighed at post-natal day (PND) 21 and 36. Mean ± SE, t-student, * p <0.05, n = 7. RESULTS: Milk yield, water and carbohydrates were lower in MO compared to C; milk leptin and fat were higher in MO vs C and no changes were observed in protein content. OFF brain weight at PND 21 and male OFF at PND 36 were lower than C, but not in female OFF at 36. *Figure(s) will be available online.

Maternal milk at 21 dL. A) yield (ml), B) water (%), C) carbohydrates (%), D) protein (%), E) leptin (ng/ml), F) fat (%) and G) pups brain weight (g) at 21 PND (male and female combined) and H) at 36 PND. Mean ± EE; n=7, * p≤ 0.05 vs C. CONCLUSIONS: Maternal obesity has a major impact on milk quality and composition. We have previously published (1) in this model that at weaning (21 days) body weight is not changed but body fat is increased in MO offspring compared to C. Many authorities now state that "weight is a poor measure of growth in the context of developmental programming." Our data support that view. The high lipid composition in the milk in the MO mother likely adversely affect brain neonatal development. 

Network, Bethesda, MD, USA.

We investigated the relationship between smoking and adverse pregnancy outcomes including potential dose-response. METHODS: Secondary analysis of a multicenter trial of vitamin C/E for preeclampsia prevention in low-risk nulliparous women. Smoking status assessed at enrollment was categorized as currently smoking, quit after start of pregnancy, and never smoked/quit before start of pregnancy. Women were further analyzed by the number of cigarettes smoked/day: <1, 1-4, 5-9, ³10. Outcomes included a primary composite (severe pregnancy associated hypertension (PAH) or mild PAH with severe features), preeclampsia, PAH, abruption, preterm birth, low birth weight (LBW), and small for gestational age (SGA). Tests for trend and multivariable logistic regression adjusting for maternal age, randomization gestational age, race/ ethnicity, body mass index, education, vitamin use, previous pregnancy (< 20 weeks), alcohol use and treatment assignment were performed. RESULTS: 9,969 women were analyzed. Smoking prevalence was 15.6%. A significant trend in smoking status was found for: primary composite (p<0.01), early onset preeclampsia (p=0.02), PAH (p<0.01), preterm birth (p<0.01), LBW (p<0.01), and SGA (p<0.01). However, only LBW and SGA remained significant in multivariable analyses. Among smokers, a significant trend in number of cigarettes smoked/day was seen for preeclampsia and PAH. The objective of this study was to determine if Bayfront Health's new scheduling procedures have reduced the percentage of elective cesarean sections and vaginal deliveries between 37 0/7 and 38 6/7 weeks comparable to rates found in nationwide studies.

This was a retrospective cohort study that analyzed the indications for deliveries between November, 2011 through April, 2012 and July, 2013 through December, 2013 at gestational ages 37/0 weeks through 38/6 weeks. Medically indicated deliveries were determined by criteria set forth in the 39-Week Toolkit as outlined by ACOG, the National Quality Forum and the Joint Commission. Neonatal admissions were also analyzed. Chi square and t-test were used to assess the data. RESULTS: There were 545 patients in the study group (post-initiative) and 520 in the control group (pre-initiative). In the study group there were 4 patients versus 42 patients in the control group (p<0.0001) delivered between 37/0 and 38/6 weeks without an indication. There were 0 transfers to the NICU in the study group versus 5 in the control group (p<0.022) for non-medically indicated deliveries between 37/0 and 38/6 weeks. CONCLUSIONS: After implementation of the 39 Week Toolkit, nonmedically indicated early term deliveries at our hospital were significantly reduced. This indicates that implementation of standard scheduling procedures contributes to reduce early term elective deliveries. with α-agonists to maintain systolic blood pressure (SBP). The uterine vasculature is more sensitive to α-adrenergic vasoconstriction than the systemic vasculature, and this is greater in HTN women, thereby increasing the risk for decreased uteroplacental perfusion and fetal acidosis. We determined the incidence of maternal Hypot during CD with spinal RA, need for maternal PT with α-agonists, and occurrence of abnormal umbilical arterial blood gases and need for NICU care in a large inborn population. METHODS: Medical records of 1002 women undergoing CD with RA in 2011 and their neonates were reviewed. Hypot was defined as a SBP <100 mmHg or ³20% fall in SBP and was stratified by the α-agonist received (Hypot+ephedrine (E), Hypot+phenylephrine (PE), Hypot+E+PE) and no PT. Data were analyzed by C 2 , Mann Whitney U, or Kruskal-Wallis. RESULTS: 677 (68%) women with CD and RA had Hypot and were more likely (P<0.001) to have HTN (23% vs. 10%), delivery <37wk (16% v 6%) and higher pre-induction SBP vs. admission SBP (135±0.17[SD] vs 127±0.12 mmHg). 76% (n=516) of Hypot received PT; if SBP fell <100 mmHg, they were 4.5-fold more likely to receive PT (42% vs 9%) vs. a 20% fall in SBP. Neonates delivered of Hypot women were ~3-times more likely to require intensive care (11% vs. 4%, P<0.001). Moreover, umbilical artery pH was lower in neonates of Hypot women requiring E+PE vs. those delivered of nonHypot women (7.18±0.11 vs. 7.24±0.06, P<0.001). CONCLUSIONS: Women at-risk for Hypot after RA for CD are more likely to be HTN, deliver preterm, and have SBP fall to <100 mmHg at CD. Their neonates require NICU care, and if PT includes both pressors, they are at risk for abnormal umbilical artery blood gases at birth. The association between maternal Hypot, increased sensitivity to adrenergic agonists and need for PT increases the risk for fetal acidosis-hypoxia; thus, an alternative PT with less uteroplacental effects should be considered.

Cohort. Sara A Babcock Gilbert, 1 Amanda A Allshouse, 2 Reina Doyle, 2 Elaine Stickrath, 2 Kent Hayborne, 2 Torri D Metz. 2 1 Ob/Gyn, University of Colorado, Aurora, CO, USA; 2 Ob/Gyn, Denver Health Medical Center, Denver, CO, USA. INTRODUCTION: Based on data from the Consortium on Safe Labor (CSL), experts suggest that avoiding cesarean delivery (CD) prior to active phase labor (<6cm) or at least 2 hours of 2 nd stage could reduce the primary CD rate in the United States. We therefore aimed to determine if the low primary CD rate at our institution was primarily a result of fewer CDs for the indication of an arrest disorder.

We compared a retrospective cohort of primigravidas with a liveborn, cephalic, term singleton who delivered at Denver Health (DH) from Dec 2007 through May 2014 to women in the CSL cohort. Detailed data regarding labor course and indication for primary CD were extracted from the medical records of women undergoing primary CD at DH. The rates of primary CD by indication were compared to rates of primary CD in the CSL cohort (multicenter data collected prospectively from 2000-2008; Boyle et al 2013) using c 2 . RESULTS: Of 3,676 women included from DH, 575 (16%) had a primary CD vs. 17,531 (25%) of 69,485 in the CSL cohort (p<0.001). Among women at DH with a primary CD for an arrest disorder (n=415), only 25% underwent CD prior to 6 cm dilation vs. 42% in the CSL cohort. Among women who reached the 2 nd stage of labor (n=103), only 3% of women at DH underwent CD for arrest of descent prior to 2 hours vs. 17% in the CSL cohort. The rate of primary CD for an arrest disorder was lower at DH than in the CSL cohort (table; data are n (%)). However, the difference in rate of primary CD for all other indications provided a similar or greater contribution to the overall 9% reduction.

Denver Health (N=3,676)

Consortium for Safe Labor (N=69,485)

Arrest of dilation or descent 415 (11) 9,326 (13) <0.001

Nonreassuring fetal status 156 (4) 4,821 (7) <0.001

Elective 3 (0) 1,332 (2) <0.001

Other 1 (0) 2052 (3) <0.001

The lower rate of CD for arrest disorders at DH when compared to the CSL cohort may be attributed to fewer CDs prior to active labor and prior to 2 hours in the 2 nd stage. However, the reduction in CD rate was 2-3% for each indication suggesting that a comprehensive plan will be needed to reduce the overall rate of CD in the United States rather than targeting only CD for arrest disorders.

The The relative risks and benefits of selective serotonin reuptake inhibitor (SSRI) use during pregnancy are incompletely understood. While untreated depression increases the risk of adverse birth outcomes, recent studies suggest that SSRIs may not significantly improve outcomes and introduce teratologic risk. In addition, concomitant use of tobacco may synergistically increase potential negative effects, as has been found for other pregnancy exposures. Our goal was to determine if SSRI use for depression during pregnancy is associated with better birth outcomes than untreated depression, and whether tobacco use moderates any observed effects. METHODS: Study participants were 274 pregnant women, diagnosed with depression, recruited at entry to prenatal care. They were interviewed during pregnancy and medical records reviewed for infant morphometrics. 27% of participants were prescribed SSRIs throughout pregnancy; 42% were smokers.

For the full sample, the low birth weight rate was high at 16%, and birth outcomes (size parameters, gestational age) did not differ significantly between SSRI users and those with untreated depression. However, smoking status produced a differential pattern of effects. For non-smokers, SSRI use was associated with a 20% decrease in low birth weight (LBW) risk and a significant (p<.05) increase in birth length. However, among women who smoked during pregnancy, SSRI use predicted a 50% increased risk of LBW and an inch decrement in birth length, with a significant (p<.05) interaction effect for length in controlled regression analysis. CONCLUSIONS: While this study has limitations, findings preliminarily point to yet another risk of pregnancy smoking with a potentially synergistic effect in an already at-risk population. While further research is needed to confirm these findings and explore potential mechanisms and long term effects, these results suggest that smoking status should be considered when weighing the potential risks and benefits of SSRI use during pregnancy.

Improving The Joint Commission (JC) identified ineffective communication as a key source of medical errors in over 80% of events in the area of maternal injury or death in 2005. The JC, IOM and ACGME all recommend creation of patient safety programs and team training. Over the past 5 years, our Department submitted 24 events for Quality Reviews and 2 sentinel events in 2013. This study aims to improve teamwork culture in a Labor and Delivery (L&D) Unit through simulation-based interprofessional training at an Academic hospital without prior history of such programs. METHODS: Baseline teamwork attitudes (TA) were assessed by a survey validated by the Department of Defense's TeamSTEPPS (TS) program. A TS curriculum, incorporating teamwork-centered postpartum hemorrhage simulations, was developed and conducted for nursing (RN), residents (res), and attendings (Attd). The TA survey was repeated immediately after the training as well as 3 Mth (months) later. Acute decision making efficiency and outcome measures were followed for 6 Mths. RESULTS: 12 training sessions conducted over 2 days engaged 93 L&D RNs, 26 res & 17 Attd. Significant improvements were seen in NICU admissions for unplanned cesarean (2% from 7%, SE +/-3%) and maternal length of stay (2.45 from 3.61, SE +/-0.36). MD attitudes towards leadership importance increased by 6% and were sustained at 3 months. There were no significant differences in efficiency parameters including time from OR arrival to incision, incision to delivery, QBL or apgars. After 3 mth, >90% of both RN and MD staff indicated that they applied what they had learned in the training. 90% of providers expressed enhanced understanding of managing critical events. CONCLUSIONS: Decreased maternal length of stay and NICU admissions represent improved teamwork and overall nursing-physician communication. Adverse outcomes overall are rare events and require more time and ongoing periodic training to demonstrate significant changes. Simulation for teaching teamwork skills was well received by MD and RN staff. Leadership parameters particularly for physicians were impacted. Based on this program a similar program directed at emergency cesarean has been initiated at the County affiliate. This project may serve as a template for programs interested in improving teamwork culture on academic L&D units. The risk of fetal death for those remaining undelivered (FDRRe) has been shown to exceed the neonatal death rate (NDR) in low risk pregnancies after 37 wks gestational age (GA). We sought to determine at what GA the FDRRe exceeds the NDR in pregnancies with CHTN or DM where the risk of stillbirth is increased. METHODS: Linked birth and infant death data for non-anomalous singleton pregnancies between 2003-2005 from the U.S. NCHS were analyzed. Those with pre-existing maternal medical conditions (other than DM or CHTN), age ³35 years, and those delivered <34 wks GA were excluded. Data were stratified by DM, CHTN, or those without either diagnosis (low risk; LR). FDRRe was calculated as:(# fetal deaths that occurred at a given GA+all fetal deaths that occurred after that GA)/ (Total deliveries at a given GA+all remaining undelivered pregnancies).

The FDRRe for each group was compared to LR women and the NDR. RESULTS: 7,207,339 live births and 10,041 fetal deaths were examined. 2.6% were complicated by DM and 0.56% were complicated by CHTN.

The FDRRe was higher at each week of gestation for those with DM or CHTN compared to LR pregnancies. In pregnancies with CHTN, the FDRRe exceeded the NDR at all weeks of gestation. For those with DM the FDRRe exceeded the NDR between 34-35 wks gestation. CONCLUSIONS: Pregnancies complicated by DM or CHTN are at increased risk of fetal death, 2-3 times greater than LR pregnancies. The risk of stillbirth exceeded the NDR at 34 wks when pregnancy was complicated by CHTN and between 34-35 wks when complicated by DM. These data confirm the need for antenatal surveillance in these patients and suggest that prolongation of pregnancy complicated by CHTN or DM may be associated with excessive perinatal mortality. *Figure(s) will be available online. Intervals of <18 months between delivery of one pregnancy and conception of the next have been associated with adverse perinatal outcomes. While many short interpregnancy intervals (IPI) result from mistimed pregnancies, little is known about women's actual intent for birth spacing.

This was a secondary analysis of data from an RCT of a prenatal testing decision support guide. At the time of a questionnaire administered at 24-30 weeks, women were asked when they might like to have another baby, if at all. Women who thought they would want another baby within 2 years of delivery of the index pregnancy were considered as intending a short IPI. Demographic and attitudinal predictors were examined for their relationship with such intent, using univariate and multivariable modeling. Multiple imputation was employed to address missing data. RESULTS: Of 401 women who plan a future pregnancy, 161 (40%) desired a short IPI. Unadjusted and adjusted models are presented in the Table. Older age, nulliparity and planned index pregnancy remained as independent predictors of intent for short IPI. 

The pattern of women who desire short IPIs does not resemble the typical demographic of women at risk for unintended pregnancy. Interventions aimed at reducing short IPIs -and theoretically reducing adverse outcomes such as preterm birth -will need to focus both on women traditionally at high risk of mistimed pregnancy and on those who plan to space their births closely.

Born Appropriate for Gestational Age. Nadia Bardien, 1,3,5 Clare L Whitehead, 1,2,3 Stephen Tong, 1,2,3 Antoni Ugoni, 5 Susan McDonald, 5 Susan P Walker. . This study examined whether AGA fetuses that slow in growth are at increased risk of placental insufficiency. METHODS: Prospective longitudinal study of 48 pregnancies reaching term and a birthweight >10 th centile. We estimated fetal weight by ultrasound at 28 and 36 weeks, and recorded birthweight to determine the relative change in customised weight across two timepoints: 28-36 weeks and 28 weeks and birth. The relative change in weight centiles were correlated with fetoplacental Doppler findings performed at 36 weeks. We also examined whether a decline in growth trajectory in fetuses born AGA was associated with operative deliveries performed for non-reassuring fetal status.

The middle cerebral artery pulsatility index (lower readings reflecting cerebral vasodilation) showed a linear association with change in weight centiles between 28-36 weeks (p=0.03) and 28 weeks-birth (p=0.0003). The MCA PI 36 weeks was significantly higher among those with a relative weight centile fall <20%, compared to those with a fall of 20-30% (mean MCA-PI 1.94 vs 1.61; p<0.05), or >30% (mean MCA-PI 1.94 vs 1.55; p<0.01) *Figure(s) will be available online.

Of 42 who labored, operative delivery for suspected fetal compromise was performed in 9/17 (53%) cases where growth slowed, and 3/25 (12%) where growth trajectory was maintained (p=0.006). CONCLUSIONS: Slowing in growth across the third trimester among fetuses born AGA was associated with ultrasound and clinical features of placental insufficiency. Such fetuses may be under-recognised as a cohort at increased risk of stillbirth.

(18:1n9) and improved DHA levels in the fetal livers (p<0.05). HCALinduced fetal liver FA changes were accompanied by changes in gene expression related to FA metabolism (p<0.05) Figure 1 . *Figure(s) will be available online. CONCLUSIONS: In pregnant rats, HCAL-exposure was accompanied by significant changes in fetal FA metabolism, including reductions in fetal brain and liver PUFAs. Fetal liver FA changes were accompanied by altered expression of genes that regulate FA metabolism. Prenatal MET significantly attenuated some HCAL-induced changes in fetal liver (but not brain) FA profiles, supporting that MET regulates FA metabolism. Because these changes may predispose offspring to metabolic syndrome, future studies will investigate the long-term effects of prenatal MET exposure on FA/lipid metabolism in offspring.

Whole Fetal growth restriction and diabetes may be two phenotypes of genetically determined insulin resistance (IR). Evidence for the this fetal insulin hypothesis arises through the study of mutations in maturity-onset diabetes of the young (MODY). Having previously shown men who father an FGR pregnancy are more IR, we wished to investigate the hypothesis that this is due to inheritance of a genetic variant passed from father to offspring.

In this study we used whole exome sequencing (WES) of DNA from 10 growth restricted offspring, in order to identify novel and rare variants in recognised MODY genes that might be influencing fetal growth. Results were validated with Sanger sequencing or Taqman genotyping.

In 2 of the 10 offspring sampled, single point heterozygous mutations were identified in MODY recognised genes (HNF1β and NOTCH2). Both variants were rare and predicted to be pathogenic by SIFT and Polyphen. Inheritance was paternal in the case of HNF1β and IR was increased in this father. In a further, severely affected offspring, 2 rare heterozygous mutations in synergistically acting MODY genes (HNF1α and HNF4α) were identified. Inheritance was from both parents, the mother experiencing early onset pre-eclampsia and the father having increased IR and waist circumference. Mother/ Father HOMA-IR 0.6/ 1.2 0.6/ 0.8 0.6/ 0.9 0.6/ 0.9 CONCLUSIONS: Through WES, rare genetic variants in genes associated with MODY were discovered in 3/10 separate offspring affected by FGR. The same variant in NOTCH2 has recently been reported in a MODY subject. Whilst proof of cause is required they are implicated in the pathogenesis of growth restriction through their predicted pathogenicity, rareness and known biological mechanisms.

Fetal Epicardial Fat Thickness and Neonatal Adipokines, C-Peptide, and Birthweight. Daniel Jackson, 1 Steven Chernausek, 2 David Fields, 2 Christopher Aston, 2 April Teague, 2 Ravindu Gunatilake. Increased epicardial fat thickness (EFT) has been reported as a cardiometabolic risk factor in adults. We have previously demonstrated increased mid-trimester EFT in fetuses of diabetic (DM) mothers as compared to non-diabetic (non-DM) mothers. We sought to determine if this difference in EFT in fetuses of DM mothers predicted adiposity and insulin resistance at delivery.

We performed a secondary analysis of a prospective cohort of 28 non-DM and 36 DM pregnancies for which fetal cord serum was collected at delivery. EFT (mm) was measured in a standardized fashion in the mid-trimester along the free wall of the right ventricle as described previously. Cord serum leptin, adiponectin, c-peptide were measured via ELISA. The relationships between fetal EFT and cord serum markers of adiposity, birthweight (BW), and insulin resistance were analyzed using multivariate linear regression. RESULTS: Mean gestational age (GA), estimated fetal weight (EFW), and EFT varied significantly between DM and non-DM fetuses. EFT was significantly increased in DM vs non-DM fetuses when controlling for GA and EFW. 22 DM and 24 non-DM had cord serum measurements.

We assessed the relationship between EFT and leptin, adiponectin, c-peptide, and BW. In the total cohort and in those with DM, there was a trend between EFT and c-peptide and BW. In non-DM, EFT correlated with adiponectin, p= 0.05. CONCLUSIONS: The previously described association between maternal diabetes and EFT is again seen. There is a potential relationship between EFT and BW, and between EFT and cord leptin, adiponectin, and c-peptide, that merits further investigation with a larger sample. 4.62% glucose and 5.64% fructose plus unlimited fructose sodas before pregnancy; N=5). At 0.9 G fetuses were necropsied after exsanguination under general anesthesia. Maternal and fetal blood was measured for Glucose, INS, and C-peptide. Immunohistochemistry (IHC) was conducted for IgF1, glucagon, HNF4α, PDX, ki67 and caspase-3. *Figure(s) will be available online. INTRODUCTION: At birth, there is a shift in metabolic profile of the heart from glycolytic to oxidative. The regulatory mechanisms of this shift are very little understood and there is an urgent need for new information that will inform the treatment of hearts with common birth complications like pulmonary atelectasis where desaturated arterial blood leads to decreased contractile capacity. Meis1 is a transcription factor that maintains glycolysis in hematopoietic stem cells (HTCs) through its regulation of hypoxia-inducible factor 1α (HIF1α). If Meis1 is knocked down, the HTCs oxygen consumption through oxidative phosphorylation is increased. We hypothesized that fetal cardiac Meis1/HIF1α expression would decrease in fetal sheep cardiomyocytes with gestational age as the myocardium prepared to augment its workload at birth in a high oxygen environment. METHODS: RNA expression and protein levels of Meis1 and HIF1α was assayed in left ventricular cardiac tissue from fetal (90-95 and 135 days of gestation), neonatal (7-12 days) and adult sheep by qRT-PCR and Western blot, respectively. Metabolic measurements were performed on primary cultured cardiomyocytes (100 and 135 days of gestation) with the Seahorse metabolism analyzer. RESULTS: Meis1 expression decreased with fetal age (P<0.01, 90 vs. 135d, n=4-5) and decreased further postnatally (P<0.05, neonate vs. adult n=4-5) *Figure(s) will be available online.

(One-way ANOVA with Newman-Keuls post hoc test). HIF1α mirrored Meis1 levels and Western blot showed a decrease in protein and a change in Meis1 splice variants with ontogeny. Cardiomyocyte metabolic analysis showed an increase in oxygen consumption between 100 and 135 days of gestation.

The decrease in expression of Meis1 and HIF-1α during late gestational development and the increase of cardiomyocyte oxygen consumption are consistent with the hypothesis that Meis1 drives glycolysis in cardiomyocytes during fetal life and that a decrease in expression of Meis1 leads to metabolic changes that favor efficient oxidative phosphorylation. Meis1 will be a target for improving oxygen utilization in premature myocardium. Insulin-like growth factor-I (IGF-I) is the key regulator of fetal growth. IGF-I bioavailability is strongly influenced by IGF-binding protein-1 (IGFBP-1) phosphorylation, which increases affinity of IGFBP-1 for IGF-I resulting in decreased bioavailability. IUGR is associated with IGFBP-1 hyperphosphorylation in circulation and liver, the primary source of fetal IGFBP-1, suggesting that IGFBP-1 hyperphosphorylation plays an important role in the development of IUGR. However, the mechanisms underlying IGFBP-1 hyperphosphorylation in IUGR remain unknown. IUGR is characterized by decreased amino acid availability, which activates the amino acid response (AAR) and inhibits mechanistic target of rapamycin (mTOR) signaling. We hypothesized that inhibition of mTOR signaling and AAR activation increase IGFBP-1 secretion and phosphorylation in response to amino acid deprivation.

HepG2 cells, a model of fetal hepatocytes, were cultured with or without leucine (450/0 µM) and treated with mTOR inhibitor rapamycin (100 nM) or AAR inhibitor U0126 (10 µM) for 24 hours. Alternatively, cells were transfected with siRNA to alter mTOR/AAR signaling. 24 hours later cells were cultured with/without leucine for additional 72 hours. IGFBP-1 secretion and phosphorylation was determined using phosho-S101, S119 and S169 IGFBP-1 antibodies and mass spectrometry (MRM MS). ANOVA, Dunnet's Multiple Comparison Post-Test was used (n=3). RESULTS: Leucine deprivation alone or combined with mTOR inhibition decreased mTORC1 (p70-S6K(T389), -60%) and mTORC2 (pAkt(S473), -70%) activity while inducing IGFBP-1 secretion (+200%). mTOR inhibition increased IGFBP-1 phosphorylation (+200%) but was less pronounced than in leucine deprivation alone (+1100%). Activation of mTORC1+C2 attenuated leucine deprivation-induced IGFBP-1 secretion but not phosphorylation. Conversely, inhibition of AAR prevented both IGFBP-1 secretion and phosphorylation in response to leucine deprivation. CONCLUSIONS: mTOR and AAR independently regulate IGFBP-1 secretion and phosphorylation. We speculate that limited amino acid availability contributes to IUGR by increasing secretion and phosphorylation of IGFBP-1 mediated by mTOR inhibition and AAR activation in the fetal liver.

Model of Growth Restriction. Charlotte J Oyston, 1,2 Joanna L Stanley, 1,2 Jasmine F Plows, 1,2 Huan Zhao, 1,2 Philip N Baker. 1,2 1 Liggins Institute, University of Auckland, Auckland, New Zealand; 2 Gravida, National Research Centre for Growth and Development, Auckland, New Zealand. INTRODUCTION: Maternal obesity is associated with numerous pregnancy complications, including intrauterine growth restriction (IUGR) and alterations in uterine artery function. A reduction in utero-placental blood flow is an important cause of IUGR; obesity-mediated changes in uterine artery function may reduce placental perfusion, leading to IUGR. We hypothesise that in murine pregnancy, consumption of a high fat diet will reduce fetal growth, which will be associated with altered uterine artery vascular reactivity, in particular a reduction in endothelium dependent vasodilation. Sildenafil citrate, a phosphodiesterase type 5 inhibitor, potentiates vasodilation of blood vessels in the pelvis. We further hypothesise that treatment with sildenafil citrate during pregnancy will normalise uterine artery function and increase pup growth. METHODS: C57BL/6J female mice were fed normal diet (ND, 4% energy from fat) or a high fat diet (HFD, 45% energy from fat) for at least 6 weeks prior to mating and throughout pregnancy. Dams from both diet groups were randomised to treatment with sildenafil citrate from 12.5 days gestation or no treatment. At gestational day 18.5, fetal and placental growth was measured and uterine artery function assessed using wire myography. RESULTS: Consumption of HFD significantly reduced pup weight (ND control vs HFD control 1.09 ± 0.02 vs 0.89 ± 0.09 g; p=0.003), while sildenafil treatment was associated with an increase in fetal weight in both groups (ND control vs ND + SC: 1.09 ± 0.02 vs 1.16 ± 0.01 g, HFD control vs HFD +SC: 0.89 ± 0.09 vs 0.98 ± 0.03 g; p=0.03). There was no difference in maximum constriction of uterine arteries to phenylephrine. Maximum relaxation in response to endothelium dependent vasodilator acetylcholine was increased in the sildenafil treated groups (ND control vs SC: 71.2 ± 9.3 vs 106.1 ± 8%; HFD control vs SC: 92.1 ± 3.9 vs 98.8 ± 4.7%; p=0.05). There was no effect of diet or treatment on the EC50 for either constriction or relaxation. CONCLUSIONS: These results suggest that an alteration in uterine artery function is not the cause of growth restriction in this paradigm. Sildenafil citrate treatment increased vasodilation of the uterine artery, and had a beneficial effect on fetal growth. Further studies are needed to determine the precise mechanisms by which treatment increases fetal growth in this model. The relationship between birth weight centile and percent body fat (%BF) at delivery is not well understood. Our objective was to examine the correlation of neonatal %BF with birth weight centile for gestational age (GA).

We analyzed 206 neonates who underwent air displacement plethysmography (Pea Pod) to determine body composition within 72 hours of birth. Data was analyzed to determine the correlation between birth weight centile for GA and neonatal %BF using Pearson's correlation. Neonatal body fat composition comparisons were made between the less than 10th (SGA), the 10th to 90th (AGA), and greater than 90th (LGA) centile groups of infants using ANOVA. Linear regression was used to predict standard deviation (SD) intervals of %BF in the population. RESULTS: Mean birth weight in our sample was 3266g (SD=458g). Mean %BF was 13.6% (SD=4.5%). Birth weight centile for GA was significantly correlated to %BF (Pearson's r=0.53, 95%CI 0.43-0.63; p<.001).

LGA infants had mean weight of 4180g and mean %BF of 19.6% (95%CI 17.9-21.4%). AGA infants had mean weight of 3241g and mean %BF of 13.4% (95%CI 12.8-14.0%). SGA infants had a mean birth weight of 2549g and mean %BF of 9.6% (95%CI 7.7-11.2%). The mean difference of %BF among the groups were statistically different (p<.001). Our regression model demonstrated that a wide range of %BF is predicted to occur even within extremes of birth weight. For example, at the 10th centile birth weight for GA, average %BF is 10.4%, with one SD 6.5%-14.2% and two SD 2.7% to 18.0%. This compares to the 90th centile birth weight for GA, where average %BF is 16.8%, with one SD 13.0%-20.6% and two SD of the data falling between 9.2%-24.5%. * Figure(s) will be available online. CONCLUSIONS: Neonatal birth weight centile for GA correlates strongly with %BF. Despite this strong correlation, the SD intervals of %BF centiles predicted by birth weight centile are wide. Some overlap exists within one SD and a large amount of overlap exists within two SD of the distribution of %BF even at extremes of birth weight centile.

Association The determinants of newborn adiposity are poorly understood. The aim of this study was to determine the association of fetal biophysical and body composition measures across gestation with newborn adiposity. We hypothesized that a composite measure of fetal fat deposition would predict newborn body composition and account for a greater proportion of its variance than conventional fetal biophysical measures.

In a sample of 84 mother-child dyads, newborn percent fat mass was quantified by DXA (NB%FM). Fetal ultrasound was performed at 12, 20 and 30 weeks gestation for biometry (HC, AC, FL, EFW) and estimates of fetal fat mass (a composite measure of fetal arm and thigh % fat area and abdominal circumference). Multiple regression was used to determine the association of fetal biophysical and fat mass measures across gestation with newborn adiposity. Covariates included gestational age at birth, postnatal age at DXA scan, maternal age, parity, SES, race/ ethnicity, pre-pregnancy BMI, GWG, and obstetric complications. RESULTS: NB%FM was 13.7±6.2%. Fetal body composition but not biophysical characteristics predicted NB%FM. The final regression model (F=4.5, p=.001) predicted 46% of the variation in NB%FM. The estimate of fetal fat mass at 30 weeks gestation was the strongest predictor; each 1SD increase of the fetal body composition measure was independently associated with a 0.4SD increase in NB%FM. Post hoc analyses suggest this effect was driven primarily by fetal arm fat area (Beta=.30; p=0.014). *Figure(s) will be available online. CONCLUSIONS: A substantial proportion of the variance in newborn adiposity is accounted for by integrated measures of fetal body composition at 0.5 and 0.7 gestation. Excess fetal arm fat deposition may be a novel marker of increased risk for newborn adiposity. *Supported, in part, by NIH grants RO1 HD-065825 to SE, RO1 MH-091351 to CB and RO1 HD-060628 to PDW.

Left Early intrauterine evaluation of cardiac morphology and function in fetal congenital heart diseases is crucial for timely intervention, even in fetal life. Fetal heart function could be evaluated by Speckle Tracking Echocardiography (STE) other than traditional echocardiography (TE). Our aims were to compare cardiac parameters obtained with both STE and TE and to evaluate differences between right (RV) and left ventricle (LV) function, from 14 weeks of gestation (wg) until term in normal pregnancies.

We designed a prospective study at San Paolo Hospital (University of Milan). Consecutive normal fetuses from uneventful pregnancy, divided into 3 groups according to wg, were evaluated both with TE and STE, using convex probes (ranging from 1-8 MHz), Hitachi-Aloka Prosound Alpha10. In each fetus specific right (sQ RV ) and left (sQ LV ) cardiac outputs (sQ=[∏*(D/2) 2 *TAMXV*h*60]/Kg) were measured by TE; ejection fraction (EF=(VTD-VTS)/ VTD*100) and longitudinal strain (LS) were obtained with STE, from four-chambers view recorded and analyzed off-line by a specific software. Statistical analysis was performed with Anova, Tukey's and paired t-test.

Values obtained from 177 healthy fetuses were analysed. sQ RV was significantly higher than sQ LV in each group (p<0.0001). EF LV was significantly higher than EF RV in each group (A p<0.001; B and C p<0.0001). LS didn't show any differences between the two ventricles all along pregnancy. sQV and LS significantly increased in both ventricles from 14 until 29+6 wg (p<0.05), whereas EF significantly increased up to term (p<0.05).

Parameters Mean±SD group A 14-18+6 wg B 19-29+6 wg C 30-38 wg

CONCLUSIONS: Our data suggest a right prevalence of cardiac output in fetal life merely related to a higher volume flow through the right cardiac sections, with similar LS values in both ventricles. Eventually the higher EF LV all along gestation demonstrates that the left ventricle is more efficient than the right one, in order to sustain the dramatic increase of left cardiac output at birth. Ultrasonographic fetal heart evaluation from 12th week of pregnancy, nowadays possible with high resolution probes, allows an early diagnosis of congenital heart defects and a best understanding of their in-utero evolution. Moreover the study of fetal heart in the early period could suggest a possible relationship between some peculiarity of its function and the physiological phenomenon of nuchal translucency (NT) that takes place from 12 to 13+6 wg. The aim of the study was to evaluate normal fetal cardiac function at 12-13+6 wg, using both traditional echocardiography (TE) and Speckle Tracking Echocardiography (STE).

We designed a prospective study, at San Paolo Hospital (University of Milan). Normal fetuses with normal NT measurement, from uncomplicated pregnancies, were evaluated at 12 until 13+6 wg with a complete echocardiography (B-MODE, Color-Doppler, Pulsed-Doppler) in association with STE. We used convex probe (ranging from 1-8 MHz), Hitachi-Aloka Prosound Alpha10. In each fetus specific right (sQRV ) and left (sQLV) cardiac outputs (sQ=[∏*(D/2)2*TAMXV*h*60]/Kg ) were measured with TE; ejection fraction (EF=(VTD-VTS)/ VTD*100) and longitudinal strain (LS) were obtained with STE, from four-chambers view recorded and analyzed off-line by a specific software. Statistical analysis was performed with Anova, Tukey's and paired t-test.

We analyzed 38 fetuses. STE was feasible in 28/38 (74%) fetuses. sQV and EF were significantly higher in the right ventricle (RV) than in left one (LV) (sQ RV = 200 ± 115 ml/min/Kg vs sQ LV =119±52 ml/min/ Kg, p<0.001; EF RV =15±5% vs EF LV =11.4±4.3%, p<0.001). No statistical significant differences were observed between LS values in left and right ventricles (LS VD =5.3±2.3% vs LS VS =6.3±2.0%, p=ns).

The left ventricle is less efficient than the right one at 12 and 13+6 wg, determining a "functional" right ventricle overload, due to reduced EF LV and sQ LV . All pathological conditions leading to additional fetal ventricles overload could result in increased right atrium and venous system pressure and increased fluid extravasation in fetal retronuchal tissues, particularly relapsed in this period. The concomitance of these factors could determine an increased nuchal translucency.

Chronic Fetal Hypoxia Markedly Impairs the Fetal Brain Sparing Response To Acute Hypoxia. B J Allison, K L Brain, Y Niu, N Itani, K L Skeffington, T J Ashmore, D A Giussani. Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom. INTRODUCTION: It is widely believed that the chronically hypoxic fetus is more vulnerable to birth asphyxia. However, surprisingly, there is little evidence to support this. Until now, it hasn't been possible to record continuously from the fetal cardiovascular system throughout a chronically hypoxic pregnancy, which explains the paucity of knowledge.

We have created isobaric chambers to maintain sheep for the duration of pregnancy under significant chronic fetal hypoxia and a wireless data acquisition system to record continuous maternal and fetal cardiovascular data in free-moving animals. Here, we investigated for the first time the effect of chronic fetal hypoxia on the fetal in vivo real-time cardiovascular response to an acute hypoxic episode. METHODS: At 119+2 days of gestation (term is 145 days), 12 fetal sheep were surgically prepared with catheters and carotid and femoral flow probes. Five days later, all animals were exposed to an acute hypoxia (30 min, fetal PaO 2 20±0.5 to 10±1 mmHg, P<0.05). The day after, half of the animals (n=6) were subjected to chronic hypoxia (fetal PaO 2 21±1 to 12±1 mmHg, P<0.05) for 10 days, from 125 days of gestation and then returned to normoxia. The other half (n=6) were normoxic. At 136 days, the acute hypoxia (30 min, PaO 2 20±1 to 10±0.5 mmHg, P<0.05) was repeated in all animals. Cardiovascular data were recorded throughout.

In normoxic pregnancy, acute hypoxia triggered the fetal circulatory brain sparing response. This was characterised by redistribution of blood flow from the femoral (FBF) and towards the carotid (CBF) circulation, thereby increasing the CBF:FBF oxygen delivery ratio (Fig.1) . In contrast, the fetal brain sparing circulatory response to acute hypoxia was almost absent in chronically hypoxic fetuses (Fig.1) . CONCLUSIONS: Chronic fetal hypoxia markedly impairs the fetal brain sparing response to acute hypoxia. We provide in vivo evidence that the chronically hypoxic fetus is at greater risk of demise during additional challenges such as birth asphyxia. Supported by the British Heart Foundation. *Figure(s) will be available online. Prior studies in our lab have found that modest increases in maternal cortisol significantly increased the incidence of peripartum fetal death. Transcriptomic analysis of the hearts revealed that the increased incidence in fetal death could be due in part to changes in cardiac function, as there were significant changes in gene expression in pathways related to cardiac metabolism and cardiac K channels. Thus, we tested the hypothesis that chronic maternal hypercortisolemia impairs fetal cardiac function, resulting in decreased fetal aortic pressure, heart rate, and altered ECG in late gestation and during labor. METHODS: Four ewes carrying singleton fetuses were infused with 1mg/kg/d cortisol (CORT) from 115 days of gestation (d) to term. Four similar ewes were not treated (CON). At 120-124d, DSI PA-D70 PCTP radiotelemetry devices were implanted into the fetuses for continuous monitoring of ECG, heart rate (HR) and aortic and amniotic fluid pressures through delivery. DSI Ponemah 5.00 and DSI Dataquest Open A.R.T 4.31 software were used to analyze ECG, and calculate one hour means of HR, and mean aortic pressure (MAP). RESULTS: Fetal plasma cortisol was increased by maternal cortisol infusion. CORT fetuses delivered at 140±2d, and CON at 145±2d. At 130d and 140d gestation and 1h before birth, CORT fetuses had significantly lower HR and MAP compared to CON fetuses. CORT fetuses also had elongated QT intervals compared to CON.

Our results indicate that chronic maternal hypercortisolemia depresses fetal cardiac function, as indicated by lower MAP and slower HR with QT elongation during delivery. This suggests that these fetuses may have increased risk of arrhythmias secondary to decreased K channel expression, and heart failure secondary to defects in metabolism, as were revealed by our transcriptomic analysis. We observed no effect of 1,25(OH) 2 vitamin D on HUVEC tube formation, migration or proliferation in culture. These results contrast to our previous finding that vitamin D rescues the poor migration, proliferation and tubule formation exhibited by cord blood fetal endothelial progenitor cells from PE pregnancies. This difference might reflect heterogeneity among endothelial cell subtypes.

Responses in the Hypothalamus Exposed To Acute Hypoxia. Activation of these circuits may result in the generation of apoptotic and inflammatory mediators causing neurotoxicity. The hypothalamus contains a large number of NMDA receptors and might be susceptible to glutamatedependent neuronal damage after hypoxia. We have previously reported that hypoxic hypoxia (HH) up-regulates the expression of inflammatory and apoptotic markers in the frontal cortex and hippocampus. The administration of ketamine, an NMDA receptor antagonist, attenuates these responses. We propose that ketamine will have similar actions in the fetal hypothalamus. METHODS: Late gestation (123 ±3 days) ovine fetuses were chronically catheterized. Prior to the onset of HH (10 min), ketamine (3 mg/kg) was administered directly to the fetus as an intravenous bolus. Acute HH (30 min) was induced by intratracheal infusion of nitrogen gas to the ewe. Fetal hypothalami were collected 24 hrs after HH, mRNA extracted and hybridized to ovine Agilent 15.5k microarray. Significant differences in gene expression between the control and treatment groups (n=4/group) were determined with F-statistics using the limma package for R (P<0.05).

Differentially expressed genes were analyzed by gene ontology using Webgestalt, identifying statistically significant overrepresentation of biological processes (P<0.05, Benjamini correction).

The number of up-and downregulated genes by HH were 311 and 395, and by HH+ketamine were 381 and 243, respectively. The effect of ketamine on fetal HH hypothalamus was determined by analyzing the 84 overlap genes shared between HH upregulated and HH+ketamine downregulated groups. The enriched biological processes for the 84 overlap genes were involved in regulation of immune and inflammatory processes. CONCLUSIONS: Our results suggest that HH increased genomic expression in immune and inflammatory pathways in the fetal hypothalamus, and treatment with ketamine, significantly reduced these responses. Ketamine could be a potential therapeutic for pre-term infants in NICUs to attenuate neuronal damage as a consequence of hypoxic stress. Repetitive umbilical cord occlusions (UCO) with worsening acidemia induce fetal adaptive brain shut-down visible in electroencephalogram accompanied by cardiovascular decompensation evidenced by hypotensive blood pressure (BP) episodes during fetal heart rate (FHR) decelerations. The onset of this pattern is highly individual in time ranging from 10 min to 2 hours prior to reaching pH < 7.00. We hypothesized that fetuses have individual cardiovascular phenotype with regard to responses to rising acidemia with intermittent hypoxia, which would be reflected in individual temporal structure of FHR variability (fHRV) measure root mean square of successive differences of R-R intervals (RMSSD), a sensitive measure of vagal modulation of fHRV, which is known to increase with worsening acidemia. The algorithm uses continuous RMSSD to predict the temporal event when consistent hypotensive BP pattern emerges in an individual fetus. RMSSD was computed over 5 minute intervals for approx. 6 hours of each complete experiment and required 2.5 hours training time during baseline and mild UCO periods. This is similar to other machine learning methods. We then extracted change point information from the fluctuations in RMSSD time series to predict the individual hypotensive BP response. RESULTS: Our findings validated the hypothesis that the new algorithm can identify individual time points of BP responses to UCO with worsening acidemia. In the 26 out of 27 cases, the algorithm matched the expert prediction (Wang et al, PLoS One 2014) detecting the hypotensive BP response 17±36 min before its visible onset from RMSSD time series.

In one case, the algorithm failed due to multiple artefacts in HRV signal.

The novelty of the current work is that it permits individualized statistical-level predictions about pathological changes in BP that endanger fetal brain from RMSSD (which can be acquired in human fetus non-invasively). This method can be deployed online as bedside FHR monitoring for earlier prediction of incipient severe fetal acidemia. However, whether statins have beneficial or detrimental effects on fetal physiology in high risk pregnancy is completely unknown. Chronic fetal hypoxia is one of the most common consequences of complicated pregnancy. Here, we investigated the effects on fetal growth and fetal cardiac function of treatment with statins in human clinically relevant doses during hypoxic development in the chick embryo. This is the only animal model which permits isolation of the direct effects of statins on the fetus, independent of effects on the maternal and/or placental physiology. METHODS: Fertilised eggs were incubated under normoxia or hypoxia (14% O 2 ) from day 0 +/-pravastatin (1 mg.kg -1 ) or vehicle (water) treatment from day 13 of incubation (term is 21 days). At day 19, the embryo was euthanised by spinal transaction and weighed. Cardiac function was determined in a Langendorff preparation. A separate cohort of enbryos was perfusion fixed (4% paraformaldehyde at 2.66kPa) and the cardiac morphology established by stereology. RESULTS: Chronic fetal hypoxia led to fetal growth restriction (Fig.  1A) , depression of the left ventricular developed pressure (LVDP, Fig.  1B) , elevation of the LV end diastolic pressure (LVEDP, Fig. 1C ) and a reduction in the LV wall:lumen ratio (Fig. 1D) , indicating impaired systolic and diastolic function and significant dilated cardiomyopathy. Pravastatin in hypoxic embryos protected systolic and diastolic function but it did not prevent growth restriction or dilated cardiomyopathy. Pravastatin treatment in normoxic embryos also led to a dilated heart. CONCLUSIONS: We show isolated beneficial effects of statins on the fetal heart in compromised development. Statins may be attractive therapeutic candidates against fetal origins of heart disease as well as preeclampsia in human high risk pregnancy. However, statin treatment in normal pregnancy should be avoided. Supported by the British Heart Foundation *Figure(s) will be available online.

Sinusoidal Fetal Heart Rate Patterns. 

We describe 2 cases with different sinusoidal pattern morphology, and possible mechanisms. RESULTS: Case 1: The patient is a 27 yo G1 P0 at 37 5 / 7 weeks, with a classical, persistent sinusoidal pattern, with a sinusoidal frequency of about 3/min, and normal baseline rate. She related sudden cessation of fetal movement that day, and underwent Cesarean delivery. The newborn weighed 2976g, Apgar scores 2/5, umbilical artery pH 7.10 and BE -7 mM/l. The hemoglobin and hematocrit about were 3.7 g/dl and 12.9%. The Kleihauer-Betke test showed 5% fetal cells in maternal blood. Case 2:

The patient was a 28 yo G1 P0 at 41 0 / 7 weeks, without FHR variability and accelerations, mostly flat but with some blunted sinusoidal complexes, and late decelerations with intermittent uterine contractions. She related decreased fetal movement for "several days" before she sought care. At Cesarean delivery the newborn weighed 3316g, had Apgar scores of 1/5 and umbilical artery pH of 7.07 and BE -10.2 mM/l. The hemoglobin was 1.5g/dl. The Kleihauer-Betke showed 3.5% fetal cells in maternal blood.

We propose that the morphology of the sinusoidal pattern depends on the acuteness of onset of the anemia. Murata and coworkers (1985) have implicated arginine vasopressin (AVP) in the sinusoidal pattern. Sinusoidal FHR patterns and increased AVP blood levels were produced in fetal lambs by hemorrhage, and by AVP infusion into vagotomized or atropinized fetuses. These authors proposed that the direct effect of AVP on the sinus node may have affected calcium transfer, resulting in the pattern.

The less striking blunted pattern seen in the second case may reflect decreased AVP levels as the fetus adapts to its anemic state over several days. This is similar to the observation that fetuses' anemic due to Rh alloimmunization have the second type of pattern (Milio and coworkers, 1988) . Trans-abdominal fetal ECG (t-a fECG) sampling FHR at 900 Hz, combined with a complex signals bioinformatics approach, showed promise in a human cohort (Repro Sci 20, 3; F-092). Here we validate this finding in a retrospective human cohort study by comparing the performance of the same bioinformatics approach to predict pH and BE at birth in two cohorts with FHR sampled either at 4 Hz or at 900 Hz.

The open access intrapartum CTG data base (doi:10.1186/1471-2393-14-16) with n=552 subjects was used as 4 Hz FHR recording data set. The t-a fECG cohort of n=60 subjects was used as 900 Hz FHR data set. We have determined the goodness of fit (R 2 ) and root mean square error (RMSE) as the performance indicators of the model on each cohort.

The clinical characteristics of both cohorts were similar (gestational age 280±8 days; gender 50% male; birth body weight 3.5±0.5 kg; pH and BE at birth 7.24±0.1 and -6.3±3.7 mmol/L, respectively; 1' and 5' Apgar scores at birth 8.7±1.4 and 9.4±0.7, respectively). The 4 Hz FHR cohort rendered -for pH and BE -R 2 =0.26 and 0.2 and RMSE=0.087 and 3.44, respectively (cf. residual plots in Fig. 1 ). The 900 Hz FHR cohort rendered -for pH and BE -R 2 =0.9 and 0.77 and RMSE=0.03 and 1.70, respectively; i.e., lower FHR sampling rate increased the predicted error range ˜3 -4fold. *Figure(s) will be available online.

Intrapartum fetal monitoring fails to accurately detect fetal asphyxia, with the incidence of cerebral palsy virtually unchanged or increased in preterm births. We show that increasing FHR sampling rate to 900 Hz improves prediction of fetal pH and BE at birth. This should improve early identification of babies at risk of brain injury. The uterine artery (UA) must undergo a significant vasodilation to supply an adequate amount of maternal blood to the fetus for a successful pregnancy. The large conductance Ca 2+ -activated K + (BK Ca ) channel is highly expressed in vascular smooth muscle cells (SMCs) where it buffers depolarization and promotes vasodilation. BK Ca activity is increased in the UA during pregnancy and its inhibition reduces basal uterine artery blood flow. However, BK Ca expression is reduced in UA during pregnancy, thus, the mechanism by which the BK Ca channel is regulated in the UA during pregnancy is unclear. The BK Ca channel auxiliary g1-subunit increases channel activity in cerebral artery SMCs. We hypothesize that the g1-subunit enhances BK Ca channel activity in UA SMCs from pregnant mice to enhance vasodilation and increase uterine artery flow.

We used pressurized myography to assess vasodilation of UA during pregnancy and after iberiotoxin (IbTX) on UAs from wildtype (WT) and BK Ca knockout (BK Ca -/-) mice and arteries transfected with BK Ca -targeted shRNA. BK Ca channel knockdown was validated using shRNA in a mouse vascular smooth muscle cell line (MOVAS). BK Ca channels currents were studied using patch-clamp. RESULTS: Isolated UAs from day 14 and 18 pregnant mice (P18 and P14, respectively) have a greater sensitivity to IbTX compared to those from non-pregnant (NP) mice, an effect that was reduced in UAs isolated from both P18 BK Ca -/mice and P18 WT mice transfected with BK Ca -shRNA. Furthermore, a significant reduction in BK Ca protein expression and voltage-dependent whole-cell currents was observed in MOVAS cells transfected with BK Ca -shRNA. Overexpressing the g1-subunit increased BK Ca currents in MOVAS cells. We recorded BK Ca single-channel currents in freshly dissociated UA SMCs and observed three populations with distinctive voltage-dependent activation, thus in BK Ca channels from NP, P14 and P18 mice most of the channels showed a half-activation voltage around 0 mV, at 10 µM Ca 2+ , whereas in P14 and P18 some channels showed a 40-80 mV leftward shift of their voltage-dependent activation, similar to what is observed in the presence of b1-or g1-subunits. CONCLUSIONS: These findings suggest a novel mechanism involved in the pregnancy-dependent vasodilation of UA, where the association with different auxiliary subunits can modulate the BK Ca channel activity to regulate UA vasodilation.

Long Term Hypoxia (LTH) Modifies Hypothalamic Appetitive Neuropeptide Expression in the Late Gestation Ovine Fetus. D Myers, 1 K Singleton, 1 K Kaushal, 2 C Ducsay. 2 1 Ob/Gyn, Univ OK HSC, Oklahoma City, OK, USA; 2 Ctr Perinatal Biol, Loma Linda Univ SOM, Loma Linda, CA, USA. INTRODUCTION: Leptin plays a key neurotrophic role in governing appetitive neuropeptide development/expression during the neonatal period in rodents. In sheep and primates however, the appetitive neuropeptide neurons and circuitry develop primarily in utero. Therefore, exposure to excess leptin may alter expression of the appetitive neuropeptides: the anorexigenic peptides, proopiomelanocortin (POMC: αMSH precursor) and the cocaine and amphetamine regulated transcript (CART), and the orexigenic peptides, neuropeptide Y (NPY) and agouti related protein (AgRP). We reported that plasma leptin is elevated in late gestation LTH fetal sheep (AJP 291:R1406; 2006) . We therefore hypothesized that LTH alters expression of hypothalamic POMC, CART, NPY and AgRP in the late gestation sheep fetus.

Pregnant ewes were maintained at sea level (~300 m, normoxic control) or high altitude (LTH; 3,820 m) for ~100 days. At 138-141 days gestation, hypothalami were collected from LTH and control fetuses. POMC, CART, NPY, AgRP and the melanocortin receptor 4 mRNA (MC4R) were measured by real time PCR (qRT-PCR). Cyclophilin (CYCLO) was used as a housekeeping gene. Differences between LTH and control were determined by Student's t-test ( p<0.05). Data presented as mean±SEM (fg mRNA/100 ng RNA). RESULTS: CART, NPY and AgRP were not different between LTH and control fetal sheep, although a strong trend (p=0.07) was noted for NPY being elevated. POMC and MC4R were significantly decreased in LTH compared to control fetuses. CYCLO mRNA did not differ between groups. We aim to understand the effect of delayed cord clamping (DCC) on the acid-base status measured from the umbilical cord at birth. Additionally, antenatal corticosteroid (ACS) administration may have an influence, by preparing the fetal lung for this transition. Thus, we also investigated if ACS administration affects the relationship between DCC and cord gas parameters and Apgar scores. METHODS: Starting in 2008, DCC became routine practice at our institution. This is a retrospective cohort of all preterm (24 0/7 to 32 6/7 weeks) NICU admissions between 2008-2012 in which DCC was performed routinely. Outcomes of cord gas parameters (umbilical artery pH, umbilical artery HCO3, and umbilical artery base deficit), and 1-and 5-minute Apgar scores were compared with a historical control group without DCC (neonates born in [2006] [2007] . Data were further stratified by ACS use. Chi-squared, t-test, Mann Whitney U, ANOVA, and Kruskall-Wallace tests were used. RESULTS: 479 neonates were included in the analysis. Neonates in the DCC group were more likely to have undergone emergency cesarean (33% v 16%, P <.01) and had more exposure to ACS (97% vs 88%, P <.01). There was no difference in cord gas parameters or Apgar scores between the two groups (Table1). The duration of DCC had no effect on outcomes. There was no effect of ACS administration on umbilical artery cord gas parameters or Apgar scores. 

This data is the largest retrospective series on DCC in premature infants; and it shows that DCC has no effect on umbilical artery cord gas parameters or Apgar scores. Administration of ACS had no effect on these parameters in relation to DCC. Endothelial nitric oxide (NO) production is partly responsible for the maintenance of uterine vasodilatation during physiologic states of high circulating estrogen levels, e.g. pregnancy; however, they are reported to be low in preeclampsic cases. Estrogen receptors (ER-α/β), classical nuclear receptors, localize to the endothelial cells plasma membrane (3-5% of ERs) and are responsible for nongenomic responses. Endothelial Nitric Oxidize Synthase (eNOS) is under complex regulatory mechanisms; which correlate to its activity state. Estradiol-17β (E 2 β) induces eNOS activation to increase NO production, however, it is unknown if eNOS regulation is dependent on one or both ERs. We hypothesize that ER-α and ER-β localize to the plasma membrane of Uterine Artery Endothelial Cell (UAECs), are capable of changing eNOS phosphorylation and increase functional NO production. METHODS: UAECs cells were fixed and label with immunogold for visualization of caveolae, ER-α and ER-β using Transmission Electron Microscopy (TEM). UAECs were treated with: 1) vehicle or increasing concentrations of E 2 β (0.1-100nM; 0-30 minutes); 2) UAECs were pretreated with the nonspecific or specific ER inhibitors (ICI182,780, MPP or PHTPP) followed by E 2 β or specific ER agonists (DNP or PPT) to analyze the changes in eNOS stimulatory phosphorylation sites, Ser1177, Ser635 vs. inhibitory site Thr495 via Western blot analyzes or to quantify the UAECs total NO production (nitrate + nitrite). RESULTS: TEM revealed that UAECs have substantial numbers of caveolae structures on their plasma membrane. Immunogold labeling revealed that ER-a localize to the plasma membrane, cytosol and nucleus while the ER-β antibody lack specificity to yield a firm conclusion. E 2 β increased Ser1177, Ser635 phosphorylation and decreased phosphorylation Thr495 in eNOS in a concentration and time-dependent manner. The increase in NO production was observed with all agonists tested (E 2 β, DPN or PPT). NO production with E 2 β stimulation was not blocked when using ICI182,780 while partly being blocked when using combined ER specific antagonists (MPP/PHTPP). CONCLUSIONS: These data support the hypothesis 1) that ERs localize to the plasma membrane, cytosol and nucleus; 2) the existence of an ER-dependent and ER-independent mechanism that may be required for optimal NO production by UAECs during pregnancy . MBR showed no significant changes during pregnancy and did not show significant differences between pregnant and non-pregnant women.

The present study clearly demonstrated that the LSFG measurement of BOS was precisely correlated with decrease of resistance in retinal blood vessels, which was associated with physiological hemodynamic changes in pregnancy. Thus, LSFG may be useful, as a non-invasive, diagnostic tool to detect enigmatic vascular disorders such as preeclampsia. 

Main uterine arteries were isolated from late-pregnant (day 17.5) mice and myogenic reactivity was assessed using pressure myography. Agonist-mediated endothelium-derived vasodilator activity was assessed by wire myography. To examine the role of the endotheliumderived vasodilators, nitric oxide (NO) and prostacyclin in the regulation of endothelium-derived vasodilator activity, the NO synthase inhibitor, L-NAME (200 µmol/L) and cyclooxygenase inhibitor, indomethacin (1 µmol/L) were used. RESULTS: Myogenic reactivity increased significantly (n=8/group, P<0.05) in uterine arteries from Rln KO mice compared with wild-type (Rln WT ) animals. Treatment with L-NAME and indomethacin further increased myogenic reactivity, but the effect of L-NAME was significantly greater in arteries from Rln KO compared with Rln WT mice. In contrast, development of myogenic reactivity in the presence of indomethacin was significantly (n=8/group, P<0.05) less in Rln KO mice, suggesting that vasodilator prostanoid production was decreased in the former group. Agonist-mediated endothelium-derived vasodilation was compromised in Rln KO mice (n=7/group, P<0.05). In the presence of L-NAME, uterine arteries from Rln KO mice were significantly (P=0.01) less sensitive to agonist-mediated endothelium-derived vasodilation, suggesting reduced bioavailability of NO. The adverse changes in uterine artery function were accompanied by a reduction in fetal weights in Rln KO 758 ± 12 mg compared with Rln WT mice 845 ± 9 mg (n=10-15/group, P<0.05). CONCLUSIONS: Our data show increases in myogenic reactivity and impaired endothelium-derived vasodilation in uterine arteries of latepregnant Rln KO mice, which may contribute to reduced uteroplacental blood flow and fetal growth restriction. 

We report for the first time that PG may be associated with lower plasma lead, cadmium and total mercury but higher inorganic mercury when compared to not-PG cohort. Some of our urinary HM findings confirm a previous report The follicular phase and pregnancy are physiological states of elevated estrogen levels and uterine blood flow (UBF). Such pregnancy adaptations provide nutrients and oxygen to meet fetal growth demands. Leptin and its receptor (OB-Rb) are involved in nutritional status and reproductive processes (e.g. amenorrhea and infertility, hypertension, and cardiac disease). It is unknown, if OB-Rb is locally expressed in the uterine arteries (UA) or whether it is altered during these physiologic states. We hypothesize that UA endothelium (UAendo) and UA vascular smooth muscle (UAvsm), expression of OB-Rb is elevated in pregnancy versus nonpregnant sheep suggesting a role in local UBF regulation. METHODS: UA and systemic arteries (omental; OA and renal; RA) were obtained from nonpregnant, (luteal, n=4; follicular, n=4), and late pregnant (120-130d, term=147d) sheep. To study local UA adaptations we developed a unilateral sheep model of pregnancy via uterine horn isolation (nongravid) restricting pregnancy to one horn (gravid). Thus UAs were obtained from nongravid unilateral (n=7), gravid unilateral (n=7) and control bilateral pregnant (n=7) groups. OB-Rb protein expression was determined on endo and VSM by Western analysis.

No difference was observed between luteal, follicular, unilateral, and pregnant OB-Rb expression in OAendo, OAvsm, and RAvsm. Compared to follicular UAendo, OB-Rb was reduced (P<0.05) in UAendo from luteal (3.5-fold), nongravid (3.5-fold), gravid (9.5-fold), and control pregnant (6-fold) ewes. Compared to follicular UAvsm, OB-Rb was lower (P<0.05) in UAvsm from luteal (3.9-fold), nongravid (5-fold), gravid (12-fold), and control pregnant (undetectable) ewes. UAendo/ vsm OB-Rb in nongravid was reduced and interestingly less reduced than gravid and pregnant controls. Compared to unilateral pregnant and control pregnant groups OB-Rb was elevated (P<0.05) in the RAendo of follicular (1.8-fold and 4.5-fold, respectively) and luteal (1.6-fold and 4-fold, respectively) ewes. CONCLUSIONS: Contrary to our hypothesis OB-Rb is not upregulated in UAendo or UAvsm in pregnancy, but rather locally downregulated by placental progesterone during pregnancy. We observed higher OB-Rb in UAendo/UAvsm during the follicular phase suggesting a role in preparation of the uterus for the periovulatory period. (NIH HL49210, HD38843, HL87144, HL117341). Labetalol is one of the first-line antihypertensive medications commonly used during pregnancy and postpartum periods. The aim of this study was to compare the pharmacokinetics of oral labetalol during pregnancy and the postpartum period. METHODS: Ten pregnant subjects (mean age: 29 years) who were receiving oral labetalol at doses ranging from 100 to 400 mg, bid or tid for the treatment of persistent hypertension (SBP³150 mmHg) were recruited. Among the recruited subjects, 7 were studied both during pregnancy and the postpartum period (6 to 13 wks post-delivery), while 3 were studied only once. Plasma samples (n=12) were collected over one dosing interval (ie over 12 hours for bid or 8 hours for tid dosing). Plasma samples were subjected to liquid-liquid extraction with ethyl acetate followed by analysis by a sensitive and validated LC-MS/MS method. Pharmacokinetic parameters were calculated by non-compartment analysis with Phoenix WinNonlin. RESULTS: Pharmacokinetics of oral labetalol were not significantly different between pregnancy and the postpartum period. The exposure to labetalol defined as dose and body weight-normalized area under the curve (AUC) was 0.03± 0.02 vs. 0.03±0.03 µg·h/L/mg/kg during pregnancy and the postpartum period, respectively. The body weight-normalized oral clearance (CL/F) was 3.8±1.7 vs. 3.1±0.5 L/h/kg during pregnancy and the postpartum period, respectively. The terminal phase half-lives for labetalol in pregnancy and the postpartum period were similar (2.0 vs. 2.3 h). CONCLUSIONS: This study indicates that the pharmacokinetics of orally administered labetalol are similar between pregnant and postpartum women. Dosing recommendations therefore, do not need to be changed during pregnancy in women on this medication prior to pregnancy. Likewise, for women on labetalol during pregnancy, dosing recommendations do not need to be adjusted in the postpartum period. INTRODUCTION: Diltiazem is used in pregnant and non-pregnant individuals for renal protection and hypertension treatment. The impact of pregnancy on the pharmacokinetics of diltiazem is unclear. The aim of this study was to evaluate the impact of pregnancy on its pharmacokinetics.

We recruited 13 pregnant subjects (age: 15-49 years) who were taking diltiazem (dose range: 60 to 360 mg, bid or qd). Each subject was studied either during pregnancy, or in the postpartum period, or both. Seven subjects were studied in the 2 nd trimester, 6 in the 3 rd trimester, and 4 at 12 to 14 wks post-delivery, respectively. Blood samples (n=12) were collected over 1 dosing interval (12 or 24 h for bid or qd). Plasma samples were extracted and the concentrations of diltiazem and two active major metabolites, N-monodesmethyl-diltiazem and O-desacetyl-diltiazem were quantitated by a sensitive LC-MS/MS method. Pharmacokinetic parameters were calculated by non-compartment analysis using Phoenix WinNonlin.

During pregnancy, the systemic exposure to diltiazem, defined as mean dose-normalized area under the plasma concentration-time curve (AUC) was lower than that during the postpartum period (10 vs. 25 µg·h/L/ mg, P<0.01). The mean apparent oral clearance (CL/F) was higher during pregnancy than postpartum (100 vs. 40 L/h, P<0.01). The AUC ratios of the metabolites N-monodesmethyl-diltiazem or O-desacetyl-diltiazem to the parent diltiazem were higher during pregnancy than postpartum (0.60 vs. 0.46, 0.06 vs. 0.04, P<0.05). The apparent terminal elimination halflives of diltiazem were similar during pregnancy and postpartum. All the pharmacokinetic parameters during the 2 nd and 3 rd trimesters were similar. CONCLUSIONS: This study demonstrates that pregnancy increased the oral clearance of diltiazem. This suggests that dosing of diltiazem especially for renal protection during pregnancy should be adjusted to reflect the increased clearance of this drug. In the UK, sepsis in pregnancy is the leading cause of direct maternal deaths. The aim of this study was to evaluate the role of estrogen (E2) in conferring susceptibility to septic shock during pregnancy following an overwhelming immune response mediated by LPS. Previous work in our group showed that progesterone did not cause a heightened hypotensive effect on blood pressure. We hypothesised that administration of E2 may cause a significant cardiovascular collapse following LPS thus affecting maternal outcome. METHODS: Pressure transmitters were inserted into CD1 mice aorta capable of measuring mean arterial pressure (MAP) and heart rate (HR) prior to time-mating. A copulatory plug would indicate gestational day E0. All animals were kept on a 12:12 light/dark cycle regimen, with ad libitum access to food and water. E2 (20ug/mouse) or vehicle (peanut oil, PO) was supplemented subcutaneously (subc) daily on E14, E15 and E16. On E16 LPS (10ug/mouse) and was administered intraperitoneally (i.p.). MAP and HR were measured for 24 hours after these treatments. Latency to labour and pup survival were recorded. RESULTS: MAP and HR were altered after LPS injection in the E2 treated group. MAP in the first 5 hours post LPS was markedly decreased in comparison to the controls (102±3 mmHg for PO and 81±14 mmHg for E2). Following this, MAP increased significantly by 11 hours (96±5 for PO mmHg and 114±16 mmHg for E2) to only decrease again below baseline at 18 hours (100±2 for PO and 90±12). HR at baseline (684±6 bpm for PO and 599±40 bpm for E2) dropped over 24 hours (686±17 bpm for PO and 510±53 bpm for E2). Latency to labour was increased in the E2 group (21.25±5 hours for PO and 31.55±11 hours for E2). No pups survived in either group. CONCLUSIONS: Estrogen is an important hormone which is regulated in a multifaceted way. E2 supplementation in pregnancy resulted in dysregulated blood pressure and heart rate but no clear pattern was identified. Supplementation did not affect maternal mortality and therefore did not cause a worsened outcome. However, E2 appeared to sensitize the cardiovascular response following LPS by an unknown mechanism.

In particular, the decrease in heart rate would imply an inability to mount a chronotropic response following LPS.

susceptibility to infection in pregnancy are not well known. We propose to use CLP to study the impact of polymicrobial sepsis during pregnancy in CD1 mice. METHODS: Pressure transmitters were inserted into CD1 mice aorta capable of measuring mean arterial pressure (MAP) and heart rate (HR). Temperature (Tp) was measured by subcutaneous (subc) probes. All animals were kept on a 12:12 light/dark cycle regimen, with ad libitum access to food and water. All mice underwent CLP (70% ligation, 2x puncture with a 21G needle) or sham operation. A cohort of animals were time-mated prior to CLP, resulting in 4 experimental groups (nonpregnant: sham/CLP and pregnant: sham/CLP). A copulatory plug would indicate gestational day E0. CLP in pregnancy was induced on E16. MAP, HR and Tp were measured after this. Latency to labour and pup survival were recorded.

In the non-pregnant cohort MAP in the CLP group fell from 116±5 mmHg at baseline to 92±9 mmHg by 19 hours after the operation. The nadir occurred three hours from CLP (82±8 mmHg) which then gradually increased. Similar trends were also observed in systolic and diastolic arterial pressures. The heart rate in the non-pregnant CLP group decreased from baseline (612±8 bpm) to a nadir at 17 hours (464±63 pbm P<0.01, repeated measure ANOVA). During gestation, heart rate were significantly altered in particular during the late stages of severe sepsis. In all CLP groups weight loss occurred and activity was reduced in comparison to sham. A decrease of Tp below 30°C in the CLP-treated groups was most indicative of poor outcomes. CONCLUSIONS: These data will enable us to draw comparisons between CLP in pregnancy and non-pregnant controls. It will ascertain whether the haemodynamic and innate immune response to polymicrobial sepsis is altered during gestation, and the bearing this may have on maternal susceptibility to infection.

The After preeclampsia, the prevalence of heart failure stage B (HF-B) which precedes the clinical overt heart failure stage C, defined as structural heart disease without symptoms, is 25% in the first year after birth. It is not known whether HF-B dissolves or persists in the years thereafter. We hypothesize that a significant number of women with HF-B after birth (> 6 months) are still in their resolving period and will not have HF-B at least 2 years later.

In this prospective longitudinal study, we included 91 former PE women who underwent serial echocardiographic measurements. HF-B was diagnosed as left ventricular hypertrophy (left ventricular mass index (LVMi) >95 g/m2), concentric remodeling (RWT >0.42 and LVMi ≤95 g/m 2 ), systolic dysfunction (left ventricular ejection fraction <55%) or asymptomatic valvular disease.

The prevalence of HF-B in formerly PE women is 26% after birth and 22% at least 2 years later. On the one hand, HF-B resolved in 16% of women after birth. On the other hand, 12% of the women without HF-B after birth developed it in the next years (Fig 1) . At least 6 months after birth, 2/3 of women diagnosed with HF-B had reduced systolic function, while at least 2 years later, 2/3 of women with HF-B had diastolic dysfunction. *Figure(s) will be available online.

Although the prevalence of HF-B is high but comparable at both time points, some recover but some develop HF-B. Moreover, 6 months after birth HF-B is dominated by systolic dysfunction, while at least 2 years later by diastolic dysfunction.

Course Variable clinical presentations and misdiagnosis contribute to these causing maternal death and poor perinatal outcomes. We evaluated course and therapy during gestation. METHODS: After IRB approval, we obtained the medical records of women delivering at our institution over the past three years with an associated diagnosis of carotid or vertebral artery dissections. Five women were identified. Past history of dissection, connective tissue diagnosis, course during pregnancy, mode of delivery, postpartum complications and treatment were reviewed. RESULTS: Maternal ages ranged from 26-32 years, all patients were Caucasian and none were obese or hypertensive prior to or during the pregnancy. All women were diagnosed by MRI/MRA after presenting with symptoms. Four of five women had a history of either carotid or vertebral dissections and these women received prophylaxis with Low molecular weight heparin (LMWH) and/or aspirin (ASA). 75% of these women developed a new diagnosis of a vascular neck dissection despite therapy (one antepartum and two postpartum). Patient 3 developed a spontaneous right carotid dissection at 16 weeks and was treated with LMWH and ASA. There were no maternal deaths or long term neurologic sequelae. 

In our series the major risk factor for vascular dissection was having a prior history. Remarkably despite prophylaxis with LMWH and ASA recurrences were frequent. Although these are the mainstay of treatment, limited evidence exists on their effectiveness and further studies are warranted. Mode of delivery was also not necessarily protective of a recurrence. Vigilance should be maintained in recognizing symptoms including headache, neck pain or focal neurologic complaints which warrant immediate imaging and neurovascular consultation for prompt diagnosis and treatment.

Complications. 

Advanced Glycation End Products (AGE) and pregnancy complications (preeclampsia and obesity) and between AGE and cardiovascular disease (CVD). But AGE involvement in CVD later in life after pregnancy complications has not been well studied. The objective was to determine AGE levels later in life in mothers and offspring in existing mouse models of prepregnancy obesity (pPO) with/without sFlt1-induced preeclampsia (PE). METHODS: CD-1 female mice were fed either a standard (SF, n=4) or high-fat (HF, n=4) diet for 3 months prior to breeding. A separate cohort mice on SF (n=11) and HF (n=8) diets were injected on gestational day 8 (GA 8) with adenovirus-carrying soluble fms-like tyrosine kinase-1 (SFsFlt1, HFsFtl1 groups) or control virus carrying murine immunoglobulin G2αFc fragment (SFmFc, HFmFc groups). All groups after weaning were placed on SF diet. Serum AGE was measured using ELISA. Appropriate statistical methods were used for analyses (P£0.05). RESULTS: In the pPO model, AGE levels were significantly higher in SF dams (P=0.03) on GA 18. At 6 months (mo) postpartum (pp) AGEs were significantly elevated in HF group (P=0.04, Figure) . HF female offspring had significantly increased AGEs (P=0.05) at 6 mo of age. In the pPO+PE model, on GA 18 AGEs were significantly higher in HFsFlt (P=0.01), SFsFlt1 (P=0.009) and SFmFc dams (P=0.009) compared to HFmFc. At 6 mo pp HFmFc had a significant increase in AGE when compared to SFsFlt1 (P=0.007) and HFsFlt1 (P=0.004) mice. Male offspring at 6 mo of age in SFmFc (P=0.005) and HFmFc (P=0.003) groups had significantly higher AGE levels compared to SFsFlt1 mice. * Figure( s) will be available online. CONCLUSIONS: Depending on the pregnancy complication, timing in regards to pregnancy and gender of the offspring, we found direct and inverse correlations between the levels of AGE and the development of CVD. Our study confirms a well-established role for AGE as a marker for CVD and an emerging hypothesis that the accumulation of AGE in tissues decreases circulating levels of AGE. (HF-B) , which precedes the symptomatic stage C, is about 1 in 4 women. Low cardiac preload and high afterload may relate to both, PE and HF. We hypothesise that low cardiac preload and increased after load in the first two years after PE relate to the development of remote HF-B.

In this prospective cohort study, we included 107 formerly preeclamptic women who where admitted to cardiovascular (CV) risk assessment at least 6 months postpartum. We measured PV (ml/m 2 ) with the indicator dilution technique using I 125 -labelled albumin as proxy for cardiac preload. Total peripheral vascular resistance (TPVR in dynes/ sec/cm 5 ) was obtained by (80.mean arterial pressure (MAP, mmHg))/CO. At least 2 years later, women where re-invited for a follow up screening including a cardiac ultrasound to diagnose HF-B according to the AHA criteria as left ventricular hypertrophy, concentric remodeling, systolic dysfunction, or asymptomatic valve disease. RESULTS: 25/107 (23%) women had HF-B at follow up of which 2/3 had concentric remodeling indicating mainly diastolic dysfunction. Former PE women who developed HF-B at FU, had a lower PV at postpartum screening than women who did not develop HF-B (1551 vs 1473 ml . m 2 ; p <0.05) while TPVR was comparable in both groups. PV correlated with HF-B (r=-0.21, p trend 0.03) but not with TPVR (r=-0.06, p trend 0.54). CONCLUSIONS: Decreased PV, indicating reduced cardiac preload, relates to increased asymptomatic HF-B at follow up, while increased cardiac afterload as indicated by total peripheral vascular resistance did not relate to HF-B. INTRODUCTION: Strategies to prevent thrombosis associated with cesarean delivery (CD) vary widely based on risk factors. We sought to examine risk factors for venous thromboembolism (VTE) in order to identify the highest risk obstetric populations.

Secondary analysis of a multicenter prospective cohort that examined maternal and neonatal outcomes in women undergoing trial of labor after cesarean (TOLAC) or CD during a high CD era (1999-2002.) Women with postpartum VTE, including deep venous thrombosis (DVT) or pulmonary embolism (PE), were compared to women without VTE. Common risk factors were analyzed by logistic regression models to identify associations with postpartum VTE. RESULTS: 70,441 women were included in the analysis; 57,182 underwent CD and 13,259 had a successful vaginal birth after cesarean. 239 women were diagnosed with VTE; 81 women had a PE, 138 women had a DVT, and 20 women had both. The prevalence of VTE in this cohort was 0.34%, consistent with current literature. Advanced maternal age (AMA), African American race, diabetes, increasing BMI, CD, infection, and transfusion (also a surrogate for hemorrhage in this cohort) were significant risks. In multivariable regression models, AMA, African American race, morbid obesity, CD, infection, and transfusion were significantly associated with postpartum VTE. Morbid obesity, CD, infection, and hemorrhage were most strongly associated. Women with a FB>1L (+FB) had slower PP decline in sBP than women with a FB£1L (-FB). Change in dBP and pBP also differed by FB. dBP declined linearly for women with -FB, but increased slightly in those with +FB. Cases were 3.3 times more likely to have a +FB(p=0.013 There is a lack of evidence regarding the optimal timing of interventions during the management of PPH. This study seeks to determine whether the timing of implementation of secondary interventions makes a difference in maternal morbidity outcomes.

This analysis included all PPH that occurred at a highvolume tertiary care center during 2011. PPH was defined as vaginal and cesarean deliveries with estimated blood losses (EBL) of greater than 500ml and 1000ml, respectively. The EBL at which three different interventions were employed (dilation and curettage (D&C), balloon tamponade (BT), and interventional radiology (IR)) was determined and divided into quartiles. Outcomes including nadir hemoglobin, PRBC transfusion, ICU admission, and postpartum hysterectomy were compared according to the EBL quartile during which they occurred. RESULTS: 797 PPH (40% vaginal delivery, 49% cesarean delivery) occurred during the study period. The mean (standard deviation) EBL at which the D&C, BT, and IR, were initiated were 1654ml (737ml), 2012ml (748ml), and 2802ml (1334ml), respectively. Earlier BT groups were less likely to need a blood transfusion (test for trend, p=0.001) or ICU admission (test for trend, p = 0.003). Women who received BT earlier also had a higher nadir Hg (test for trend, p=0.012) and less risk of hysterectomy (test for trend, p=0.05). Lastly, women who received earlier IR (i.e., at an EBL less than the mean for IR) also were less likely to receive a hysterectomy (0% vs. 8.3%, p<0.049). CONCLUSIONS: Among women with PPH, earlier intervention with BT and IR was associated with a decrease in morbidity. The long-term maternal cardiovascular and metabolic implications associated with preeclampsia (PE) include risk of hypertension, heart disease and metabolic syndrome. The objective of this study was to investigate if a recent history of PE and/or lifetime risk of cardiovascular disease (CVD) is associated with detectable alterations in the circulating maternal proteome. METHODS: 6-month postpartum plasma from PE women (n=12) and women with uncomplicated obstetrical history (CTRL, n=12) were used for analysis. Depleted maternal plasma was analyzed by label-free liquid chromatography-mass spectrometry assay. Identified peptides were searched against the International Protein Index human database v3.87. Exponentially modified protein abundance indices were used for comparison. Results were analyzed using QIAGEN's Ingenuity® Pathway Analysis. Right-tailed Fisher's exact test was used to calculate a p-value. RESULTS: 113 eligible peptides were identified for analysis; 101 were common to both groups, 5 peptides were unique to CTRL and 7 unique to PE subjects. PE profiles were more strongly associated with markers of cardiovascular, hematological and inflammatory diseases compared to controls and mapped more significantly to cardiotoxicity functions.

Stratification of subjects by low (<39%) and high (³39%) lifetime risk of CVD rather than by diagnosis produced similar findings. Based on protein abundance index criteria, 5 PE-specific peptides were identified; of significant importance was F10, implicated in CVD. Comparison of low-risk CTRL (n=6) to low-risk PE groups (n=6) found that while similar for BMI, blood pressure and fasting lipid profiles at 6 months postpartum, PE peptide profiles continued to display stronger associations for CVD and cardiotoxicity functions. Within this cluster, the most abundant proteins were associated with thrombosis, vascular disease and infarction. CONCLUSIONS: Markers of CVD and dysfunction are evident in the maternal circulating proteome 6 months postpartum after PE. Moreover, modifications in circulating peptide profiles occur in previously PE patients who otherwise do not have clinically measurable cardiovascular risk factors. Our data highlights the need for early postpartum prevention programs in the PE population and identifies molecules that may be targeted for screening or therapeutic benefit.

Perinatal We sought to determine associations between perinatal post-traumatic stress (PPTS) symptoms and maternal mood, parenting, and infant feeding at 2 months postpartum. METHODS: Women intending to breastfeed were recruited in the 3 rd trimester for an ongoing longitudinal cohort study. Past or current major depressive disorder (MDD) or anxiety disorder (AD: PTSD, generalized anxiety disorder, obsessive compulsive disorder) was assessed via Structured Clinical Interview. We assessed PPTS symptoms, mood symptoms, parental stress, and emotions during feeding at 2 months postpartum using the Modified Perinatal Post-traumatic Stress Disorder Questionnaire (MPPQ), Edinburgh Postnatal Depression Scale (EPDS), Beck Depression Inventory (BDI), Spielberger State-Trait Anxiety Inventory (STAI), Parenting Stress Index (PSI), and the modified Differential Emotions Scale (mDES). We used multivariable linear regression to model the associations between MPPQ score and outcome, adjusting for SCID-verified active MDD or AD and interactions between active diagnosis and MPPQ score. RESULTS: In our sample 10/67 women had SCID-verified MDD or AD during pregnancy (ActiveDx), and 5/67 had MPPQ scores ³19, the established threshold. In multivariable models adjusting for ActiveDx, we found that MPPQ score was associated with higher EPDS (R 2 = .31, p<.001), BDI (R 2 =.34, p<.001), STAI (R 2 =.15, p<.01), and PSI scores (R 2 =.16, p<.01), and more negative emotions during feeding (R 2 =.14, p<.01) at 2 months postpartum. ActiveDx did not modify associations between MPPQ score and outcomes (all p for interaction >.05). *Figure(s) will be available online. CONCLUSIONS: PPTS symptoms are associated with maternal mood, parenting stress, and negative infant feeding experiences at 2 months postpartum, independent of preexisting MDD or AD. Psychological support for women with PPTS symptoms may improve outcomes for mothers and infants.

We analyzed participants in an ongoing longitudinal study of women intending to breastfeed. At baseline all participants underwent a Structured Clinical Interview (SCID) to determine history of anorexia, bulimia, or binge eating disorder (EDHx). At 2 months postpartum, we quantified current symptoms using the Eating Disorder Examination Questionnaire (EDE-Q4). We measured outcomes using the Beck Depression Inventory (BDI), the Edinburgh Postnatal Depression Scale (EPDS), the Parenting Stress Index -Short Form, the Breastfeeding Self-Efficacy Scale, and the modified Differential Emotions Scale (mDES). We used multivariable linear regression to model associations between outcomes and EDE-Q4, EDHx and interactions between EDHx and EDE-Q4. RESULTS: Of the 67 women, 1 had a SCID-verified current eating disorder and 10 had an EDHx. At 2 months postpartum, women with EDHx were more likely to meet clinical thresholds for depression indexed by BDI ≥ 17 (40 vs. 5%, Fisher's exact p<.01). Adjusting for EDHx, higher EDE-Q4 score was associated with higher BDI (R 2 = .44, p<.0001) and EPDS (R 2 =.22, p<.001) scores and reduced breastfeeding self-efficacy (R 2 =.12, p<.01). EDHx moderated other associations: among women without EDHx, higher EDE-Q4 score was associated with greater parenting stress (R 2 =.23, interaction p <.05), more negative emotions during feeding (R 2 =.36, interaction p<.003), and reduced breastfeeding intensity (R 2 =.16, p for interaction=.03). *Figure(s) will be available online. CONCLUSIONS: Maternal eating disorder symptoms were associated with depression symptoms, parenting stress, and negative breastfeeding outcomes. Parenting support may improve outcomes for mothers with disordered eating symptoms.

Is There is a significant improvement in glucose concentrations in women with gestational diabetes (GDM) immediately postpartum (PP). We assessed if these changes were related to improvement in insulin sensitivity (ISogtt) or insulin response (ISSI-2) and if the improvement was associated with changes in maternal lipids or cytokines. METHODS: This is a prospective observational study of women with GDM. Twenty-seven women with a singleton pregnancy were evaluated at 34-37 wks gestation and 2-3 days PP using a 75g 2 hour oral glucose tolerance test (OGTT). Women with pre-existing diabetes, infection, or requiring medication for glucose control PP were excluded. Maternal insulin and glucose were collected and ISogtt and ISSI-2 were calculated. Fasting lipid and cytokine profiles were obtained. The changes over time and correlation between changes in ISogtt/ISSI-2 and lipids/cytokines were assessed. RESULTS: A significant increase in ISogtt (3.7+1.8 vs 8.3+2.5, p<0.001) and ISSI-2 (2.6+1.1 vs 3.1+1.1, p=0.008) were observed PP. Lipids (cholesterol, triglycerides, VLDL, LDL) and leptin decreased PP while IL-6 and CRP increased. A significant correlation was observed between the change in leptin and ISSI-2 (r = -0.41, p=0.03). 

For women with GDM, there is a 120% increase in ISogtt and a 20% increase in ISSI-2 PP. The increase in ISSI-2 is interesting, as one would anticipate a decrease in ISSI-2 with an increase in ISogtt. We speculate there may be independent effects of pregnancy on ISogtt and ISSI-2. Further the significant increase in ISogtt postpartum may be related to placental hormones for e.g.TNF α which were not examined.

Immune Signatures of Women With Preterm Birth. Quentin J Baca, 1 Brice Gaudilliere, 1 Garry P Nolan, 2 Martin S Angst. 1 1 Anesthesia, Stanford University, Stanford, CA, USA; 2 Microbiology and Immunology, Stanford University, Stanford, CA, USA. INTRODUCTION: One in eight infants is born preterm, which accounts for a third of all infant death. While maternal immune traits rank high as a root cause of preterm birth, the nature of such traits is poorly defined. This study used mass cytometry to comprehensively map and interrogate the immune system of women with a history of term and preterm birth (Gaudilliere, Sci Transl Med 2014), and test the hypothesis that specific intracellular signaling responses in immune cell subsets are associated with preterm birth. METHODS: Blood samples from non-pregnant women (n=20) with a history of term (>37 GW) and preterm (<33 GW) birth were stained with 22 cell surface and 13 intracellular functional markers (STAT1, STAT3, STAT5, STAT6, CREB, NFkB, IkB, p38, MAPK, MAPKAP2, ERK, S6) at basal state and after stimulation with exogenous ligands (LPS, IL2, IL4, IL6, GM-CSF, INFa, TNFa) that activate intracellular signaling networks implicated in preterm birth. RESULTS: All samples have been banked and the assay has been validated in controls. Hierarchical clustering provided functional maps of the entire immune system and revealed cell-type specific signaling responses to exogenous ligands. *Figure(s) will be available online. Importantly, stimulation with ligands allowed the response sensitivity and magnitude of specific immune cell subsets to biologically relevant "stressors" to be inferred. Complete analysis will be available at the time of the conference and focus on immune signatures that uniquely characterize women with a history of preterm birth.

The outlined experimental approach allows for the comprehensive functional interrogation of the maternal immune system. Immune traits specific to women with preterm birth will allow women at risk to be identified and provide important clues regarding the biology that drives preterm birth. Tobacco use during pregnancy is associated with a pro-inflammatory state. We evaluated if smoking cessation during pregnancy could reverse the pro-inflammatory milieu associated with tobacco abuse.

This was a secondary analysis of a prospective, longitudinal multicenter study of women with a singleton gestation. Maternal serum was collected during each trimester. Cytokines (IL-1a, IL-1b, IL-2, IL-6, IL-8, IL-10, TNF α CRP and MMP-8) were measured using multiplex beadlyte assay on a Luminex IS-100. At each time point smoking status was validated by preset urine cotinine levels. We prospectively identified a cohort of women who reported smoking cessation during pregnancy, in comparison to patients who never smoked. Univariate and multivariate regression analyses were performed evaluating differences in cytokine levels as well as the change in cytokine levels across pregnancy, between study groups. Statistical analyses were performed using SAS 9.3

Of 393 patients who were followed during the study period, 27 had quit smoking during pregnancy, 279 never smoked, while the rest (n = 87) continued smoking throughout pregnancy. Demographics were similar among patients who had quit and those who never smoked. In comparison to patients who never smoked, patients who had quit smoking had a significantly higher pro-inflammatory statein the 1 st trimester, as noted by increased IL-1a (p = 0.025), TNF α (p = 0.024) and CRP (p = 0.022). Multivariate regression analyses showed significantly higher TNF-α levels (p = 0.004) even after smoking cessation, as compared to patients who never smoked, while adjusting for maternal age, BMI, race / ethnicity, education and income. In the second trimester a decreased IL-10 (p = 0.011) remained evident in those who had smoked versus nonsmokers.

In the third trimester, no significant differences were noted between these two groups. The change in serum cytokine levels between trimesters (1 st to 2 nd / 2 nd to 3 rd ) also did not differ between these two groups. CONCLUSIONS: Tobacco exposure affects the inflammatory physiology in pregnancy. For women who smoke this is evident in the 1 st and 2 nd trimesters. We could not identify a difference between these groups in the 3 rd trimester suggesting that cessation of tobacco use improves the inflammatory milieu in pregnancy.

Maternal ) are essential for normal pregnancy outcome. In vivo T reg depletion during pregnancy leads to autoimmunity, resulting in total pregnancy loss. There is a paucity of lymphocytes at the utero-placental interface (UPI) during normal pregnancy, thus the site of T reg action is unclear. METHODS: Low-dose (0.1ug) diphtheria toxin (DT) was given on day 11.5 post vaginal plug to pregnant female C57BL/6-FoxP3-DTR transgenic mice, in order to induce subtle depletion of maternal FoxP3+ T reg cells during mid-gestation. On day 17.5, fetal weight and gender were determined. In these syngeneic pregnancies, only the male pups harbor immunogenic antigen. The individual UPI were removed and subject to FACS and immunofluorescence microscopy.

In unperturbed mouse pregnancies, there is a paucity of immune cells in the UPI of male or female pups. Subtle T reg depletion during pregnancy resulted in effector T cell, NK cell, macrophage, and dendritic cell influx preferentially to UPIs of antigenic male fetuses, resulting in a reduction of fetal weights. Surprisingly, we also observed an increase of T reg numbers in the antigenic UPIs. Preliminary results indicate a local interaction between antigen-presenting cells, T cells, NK cells, and T reg within the previously depleted UPI. *Figure(s) will be available online. CONCLUSIONS: Systemic interruption of T reg tolerance during pregnancy allows for maternal attack on antigenic fetuses characterized by a cellular response dominated by active NK and T cells. In the unperturbed UPI, the absence of T reg suggests that maternal tolerance is mediated elsewhere. In disrupted maternal tolerance, the repopulation of T reg to the UPI provides insight into T reg cellular interactions that may contribute to the re-establishment of tolerance. The kinetics of the response suggest that a preformed pool of potential effector and regulatory cells the location of which requires further investigation to identify.

Immune Tolerance To Increased Immune Activation. Opinions are divided as to whether this represents a state of fetal specific tolerance or of a generalised suppression of the maternal immune system. We hypothesised that antigen-specific T cell responses are modulated by an inhibitory T cell phenotype and modified dendritic cell (DC) phenotype in a gestation dependent manner.

We analysed changes in surface markers of peripheral blood T cells, dendritic cells (DC) and natural killer cells (NK) measured by flow-cytometry, in combination with ex vivo antigen specific T cell responses measured by enzyme-linked immunospot technique (ELISpot).

We found that mid gestation is characterised by reduced antigen-specific T cell responses associated with 1) predominance of naïve and effector memory over other T cell subsets with reduced expression of MHC class II (HLA-DR) and co-stimulatory molecules (CD28); 2) up-regulation of inhibitory markers (PD-1) (p=0.0469); 3) increased TCRγ∆ subsets; 4) increased proportions of tolerant DC (CD3 -CD19 -CD11c + HLA-DR + CD14 + CD16 + CD123 + ILT4 + CD83 + HLA-G + ) (p=0.0313) and activated plasmacytoid DC (p=0.0481); and 5) increased CCR5 (p=0.0156) but reduced HLA-G (p=0.0156) expression on Tregs. Conversely antigen specific T cell responses normalised in late pregnancy and were associated with increased markers of T cell activation (CD38) (p=0.0313). However these changes occur with a simultaneous up-regulation of immune suppressive mechanisms including apoptosis (CD95) (p=0.0156), CD4 progesterone sensitivity (PIBF expression) and immune regulation (IL-10) with concurrent decreases in CD8 TCRγ∆ PIBF expression (p<0.0001) and cytokine producing CD56hi PIBF + NK cells (p=0.0313) through the course of pregnancy. CONCLUSIONS: Together, our data suggest that immune suppression dominates in the second trimester and that it is gradually reversed in the third trimester in association with immune activation as the end of pregnancy approaches.

The To evaluate whether bacteria-derived extracellular vesicles (EVs) excrete through the urinary tract and to compare the composition of bacteria-derived EVs in urine between women with and without pregnancy. METHODS: Seventy-three non-pregnant women, and 74 women with pregnancy (39 with normal delivery and 35 with preterm delivery) were enrolled from Dankook University and Ewha Medical Center, respectively. For metagenome analysis, DNA was extracted from urine EVs. 16S rDNA sequencing were conducted using high-throughput sequencing. 16S rDNA sequences were clustered to identify operational taxonomy units (OTUs), and taxonomy was assigned at phylum, class, order, and family levels. Using hierarchical clustering, samples were grouped in terms of the composition of the taxon.

Between pregnant and non-pregnant women, a total of 13 taxa show a significant difference in the microbial composition. In particular, Bacillus spp. is the most significantly enriched taxon in pregnant women (45.61%) compared with non-pregnant women (0.12%). On the other hand, Pseudomonas spp. are the most enriched taxon in non-pregnant women (13.23%) compared with pregnant women (4.09%). An OTU that is assigned to family Pseudomonadaceae is also significantly enriched in non-pregnant women. Interestingly, Methylobacterium spp. are enriched among both non-pregnant women and pregnant women with preterm delivery, whereas they rarely exist among pregnant women with normal delivery. As for the compositional difference between women with normal and preterm delivery, Ureaplasma spp. and family Veillonelaceae were more frequently detected among women with preterm delivery, compared with those with normal delivery. CONCLUSIONS: The composition of EVs derived from Bacillus spp. increased during pregnancy, whereas those from Pseudomonas spp. increased in non-pregnant women, suggesting that microbe-derived EVs play a key role in the maintenance of homeostasis during pregnancy. Recent studies suggest that NK cells possess some features of adaptive immunity including a certain type of immune cell memory, named trained immunity. During human pregnancy NK cells constitute the most abundant lymphocyte population (50-80%) found at the maternal fetal interface, the decidua. These NK cells (termed dNK) possess unique phenotypical and functional properties and are considered regulators of remodeling of the maternal fetal interface.

Here we investigate if the dNK cells have the ability to "remember" pregnancy. dNK cells were isolated from first trimester elective pregnancy terminations and the material was analyzed using RNA-seq.

We observed that dNK cells from parous women express NKG2C in significantly higher percentages as compared with NK cells from nulliparous women. Analysis of the RNA-seq results revealed an upregulation of various genes related to angiogenesis, tissue remodeling, TH1 to TH2 shift, immune modulation, growth factors production, and NK recruitment. For the most highly expressed genes we validated the results also by real-time PCR assay. CONCLUSIONS: Thus, we report for the first time that dNK cells display a trained immune response upon a second encounter with a semi-allogeneic fetus. This finding may give insight to the etiology of preeclampsia with increased risk in first pregnancy. was assessed by immunofluorescence, RT-PCR and Western Blot. Cells isolated from placenta of lean and obese women by sequential trypsin -DNase digestion and gradient centrifugation were used for primary culture. Modulation of TLR4 signaling by long chain saturated and unsaturated FA (500 µM, 24 hrs) was investigated in cultured placental cells. RESULTS: TLR4 was localized on both sides of the placental barrier (syncyciotrophoblast layer and fetal vascular endothelium) suggesting they can be activated by either maternal or fetal FAs. TLR4 protein and gene expression were 2 and 4 fold greater in placenta of obese vs lean women. The saturated palmitic (C16:0) and stearic (C18:0) acids but not the unsaturated oleic acid (C18:1) increased TLR4 in cultured placental cells. The stimulation was higher (9 vs 3-fold and 5 vs 2-fold, p<0.001 respectively) in cells from obese vs lean women. Following palmitic acid(PA) exposure, cells of obese women released more inflammatory cytokines IL-6 (8 vs 3-fold, p<0.001), IL-8 (14 vs 7-fold, p<0.001), TNF-alpha (5 vs 3-fold, p<0.001) than cells from lean women. This was concordant with a higher placental mRNA expression of IL-6 (5.4±2.5 vs 3.5±2.6 copies/ngRNA, p<0.05) and is a leading cause of fetal and maternal morbidity and mortality. CYP1A1 polymorphisms is associated with risk of spontaneous PTB (sPTB). As CYP1A1 can be activated via aryl hydrocarbon receptor (AhR), we hypothesize that altered transcription of placental CYP1A1 and AhR is associated with occurrences of sPTB. METHODS: Human placental tissues were obtained from normal term (NT; 39±1 weeks' gestation, n=10), sPTB (30 ± 4 weeks, n=5), and severe preeclamptic (sPE; 31 ± 3 weeks, n=5) subjects. Total RNAs were reversetranscribed (RT) into cDNAs. Real-time qPCR analysis was performed with Taqman gene expression assays using ACTB and GAPDH as internal controls. Equal amounts (250 copies/ng of total RNA) of a synthetic RNA transcript (Xeno) were added to the RT reaction serving as an external control for RT-qPCR analysis. RESULTS: Based on CYP1A1 mRNA levels, NT placentas were stratified into low CYP1A1 (NT-L; < 5 copies of CYP1A1 transcripts/ng RNA) and high CYP1A1 (NT-H; (> 50 copies of CYP1A1 transcripts/ng RNA). NT-L consisted of the majority (8 out of 10) of NT placentas studied. In comparison to NT-L, NT-H placentas displayed significant (P <0.05) upregulation of CYP1A1 (55-fold), CYP1B1 (3-fold), and AhR (1.8-fold) mRNA. Compared with NT-L, sPTB placentas exhibited significant (P <0.05) up-regulation of CYP1A1 (3-fold), significant (P <0.05) downregulation of AhR, and similar level of CYP1B1 mRNA. No significant difference in CYP1A1/B1 and AhR mRNA levels was detected between NT-L and sPE placentas. Moreover, the CYP1A1 mRNA levels in sPTB placentas were negatively correlated (Correlation Coefficient = -0.94, P=0.02) with gestational ages, ranging from 26 to 34 weeks; however, this correlation was not observed in NT placentas and gestational age matched sPE placentas. No significant correlation was observed between AhR, CYP1B1 and gestational ages in all groups. CONCLUSIONS: While CYP1A1 remains at a relatively low level in the majority of NT placentas, CYP1A1 can be robustly induced in a subset of NT placentas in association with AhR and CYP1B1 up-regulation. Data from this study also suggest that the altered transcriptional regulation of CYP1A1 and AhR observed in sPTB, but not in sPE, placentas may play an important role in inducing sPTB. NIH HD38843 and HL117341

Altered Ethanol increases intracellular Ca 2+ and cell death in several embryonic tissues. Therefore, we evaluated whether a calcium channel blocker can protect first trimester human trophoblasts from an adverse environment. METHODS: Human first trimester chorionic villous explants and the human trophoblast cell line HTR-8/SVneo (HTR) were cultured. Tissues and HTR cells were pre-treated with 12.5 -50 nM of the calcium channel blocker nifedipine for 1 hour before exposure to 50 mM ethanol for an additional hour. Intracellular Ca 2+ concentrations were monitored in real-time by epifluorescence microscopy using fluo4-AM. Apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). One-way ANOVA with post hoc t-test comparisons was used to identify significant differences (p<0.05) in Ca 2+ and apoptosis. RESULTS: Intracellular Ca 2+ concentrations increased synchronously in trophoblasts within 10 s of exposure to 50 mM ethanol from 96.9 ± 23.2 nM to 1230.8 ± 291.4 nM, followed by an increase in cell death within 1 h. The increase in intracellular Ca 2+ was completely blocked in cells pretreated with nifedipine for 1 h. Cell death increased significantly in both villous explants (from 15.6 ± 2.45% to 24.9 ± 0.84%) and HTR cells (from 4.3 ± 0.004% to 11.8 ± 0.001%) after exposure to ethanol (p<0.05). Treatment with nifedipine blocked ethanol-induced cell death. CONCLUSIONS: Ethanol-induced apoptosis in human first trimester trophoblast cells was dependent upon intracellular Ca 2+ signaling. We have previously demonstrated that increased apoptosis due to acute ethanol exposure requires both intracellular and extracellular Ca 2+ . This study shows that nifedipine, an L-type calcium channel blocker, can rescue first trimester trophoblast cells from an adverse environment. There is evidence suggesting that calcium channel blockers have direct positive effects on endothelial dysfunction, which is found in placenta-mediated disorders. Fetal alcohol syndrome is associated with intrauterine growth restriction, which is due in part to trophoblast cell death. Nifedipine could provide a novel intervention for women who are at risk for these obstetrical disorders. 

decidua may cooperate to drive the differentiation of pro-angiogenic neutrophils that aid in the uterine remodelling process by stabilising remodelled vessels. METHODS: PBN were treated with Decidual Conditioned Medium (DCM) or IL8 and TGF-b1 both with and without IL8 and TGF-β antagonists. Realtime PCR was performed to assess changes in angiogenic factors and in vitro invasion and tube formation assays were performed using uterine microvascular endothelial cells, (UtMEC), HTR8 and primary extravillous trophoblast. RESULTS: DCM-treatment strongly upregulates mRNA expression of several potent pro-angiogenic factors > 40-fold in comparison to matched SFM controls (CXCL2, IL8, CXCL3, IL6, and VEGF-A), and also downregulates expression of potent angiostatic factors (COL18A1, THBS1, ANGPTL1), this was abrogated by neutralisation of both IL8 and TGF-β signalling (p◊‹0.01, n=5). In in vitro tube assays either DCM or IL8 and TGFB priming of PBN led to a significant increase in an extensive organised network of tubes in both UtMEC and HTR8 cells as compared to controls (p◊‹0.01, n=5). Again both IL8 and TGF-β antagonists, alone and in combination, inhibited this effect (p◊‹0.01, n=5). CONCLUSIONS: IL8 and TGF-β from the decidua cooperate to drive neutrophil differentiation to an N2 like angiogenic phenotype that has potent angiogenic capability and induces the formation of stable well organised vascular networks. Future experiments will assess the effect of the angiogenic neutrophils on the acquisition of an endothelial phenotype by primary extravillous trophoblast.

Skewed X Chromosome Inactivation in Human Placental Tissue. Female mammals inactivate one of the two X chromosomes for a dosage compensation issue, which is known as X chromosome inactivation (XCI). The status of XCI varies by mammalian species or cell types. For example, in marsupials, the paternal X chromosome is inactive (imprinted) both in embryonic and extra-embryonic tissues, whereas in mice, XCI is random in embryonic tissues and imprinted in extra-embryonic tissues. It remains controversial whether imprinted XCI exists or not in human placental tissue in part because previous studies have analyzed a limited number of X-linked genes using resources containing various types of cells.

To assess the XCI status in human placental tissue, we performed a chromosome-wide allele-specific expression analysis using female cytotrophoblast cells (CTs) immunomagnetically purified from early placentas (7-9 weeks). We extracted RNA and genomic DNA from CTs and maternal peripheral blood cells, and Exome-and RNAsequencing were performed. The parental origin was determined using single nucleotides polymorphisms (SNPs), and the allelic expression ratio was calculated for each SNP.

The purity of CTs averaged 95% (97%-90%) in all 11 samples and the expression profiles were highly correlated with each other (R 2 > 0.96). On average, the allelic expression ratio from the maternal allele was 50.8±10.6% for autosomes and 66.2±17.3% for the X-chromosome (p < 0.0001). The allelic expression ratio of the X-chromosome was highly variable among samples (from 44.0±8.2 to 91.9±12.4%), but there was no sample with paternally-biased expression. We also identified many escape genes using samples with strong paternal XCI *Figure(s) will be available online. CONCLUSIONS: Our results suggest that XCI in human placental tissue is paternally skewed. We are accumulating more samples to reinforce our conclusion, and also planning to analyze allele-specific histone modification patterns to understand the regulatory mechanism of XCI.

From these studies, we will gain insight into the evolution of imprinted and random XCI in placental mammals. Increased angiotensin II activity and increased placental sflt-1 production are associated with preeclampsia. To better understand the relationship between sflt-1, the renin-angiotensin system, and pregnancy outcomes, we study a transgenic (TG) mouse model with an isolated 20% increase in maternal angiotensinogen (AGT) expression, which is sufficient to cause gestational hypertension and fetal growth restriction. TG dams have characteristic features of placental insufficiency, including elevated maternal serum sflt-1 and absent end-diastolic flow (AEDF) in fetal umbilical arteries near term. Others have suggested that AEDF may be a feature of reduced placenta capillary density. Therefore, we hypothesized that TG dams will have fewer fetoplacental capillaries compared with carefully age-matched wild-type (WT) controls. METHODS: Six TG and WT litters were compared at day 16.5 (term day 19.5). 24 randomly selected placentas from these six litters were used for analysis. The fetus was used to determine gender. Each placenta was weighed, measured, and cut into four unequal pieces, randomly oriented for paraffin embedding, and then sectioned for CD31 staining (abcam28364). Two low power microscopic fields (20x obj) from each section of the placental labyrinth were examined and the total number of complete capillary cross-sections per field were counted while blinded to maternal genotype. The mean capillary density was reported and the average per maternal genotype was used for statistical analysis.

The overall volume of the placental labyrinth in mm 3 was less in TG dams (p<0.05), which was especially evident when the fetus was male (146 mm 3 compared with WT 201 mm 3 ) (p=0.01). The long and short axis of the placentas were not different, but the mean thickness was significantly less in TG dams (2.5 mm) compared with WT placentas (3.0 mm) (p=0.002). The average capillary density per low power field was not significantly different per group, but tended to be less in male pups from TG dams (p=0.14 by ANOVA). CONCLUSIONS: Placentas from mice expressing 20% more AGT than WT controls tended to be thinner with a decreased overall capillary volume. This may explain why we observe AEDF and fetal growth restriction in this model. We suspect elevated local angiotensin II activity at the uteroplacental interface may inhibit angiogenesis, but this working hypothesis requires further study. with abnormal umbilical artery (UA) Doppler indices is unknown. In normal pregnancy branching of the placental villi occurs in late gestation, providing a low-resistance circuit for blood flow. The support structures for these villi are rich in collagens, fibrillin and elastin. We investigated the relationship between IUGR with abnormal UA Doppler indices and collagen, fibrillin and elastin expression in both singleton and multiple gestations. METHODS: Fetuses diagnosed with IUGR (weight <10%) and abnormal UA Doppler indices (increased systolic: diastolic ratio, absent or reversed end diastolic flow) were compared to fetuses with normal parameters. Placentas were collected from IUGR and control (normally-grown) subjects from both singleton and multiple pregnancies. For singletons IUGR (n=3) and controls (n=5) were unrelated. For multiple pregnancies, IUGR fetuses (n= 4) were compared to the normally-grown control fetuses (n=5) from the same pregnancies. At placental delivery, 1 central and 4 peripheral 3mm biopsies were collected and stored in RNAlater (Qiagen).

RNA was isolated. Collagens I-VI, fibrillin-1, and elastin-1 mRNA expression was determined with quantitative PCR. Statistical analysis (ANOVA) was performed to determine differences between IUGR and control fetuses in singleton and multiple pregnancies. RESULTS: In singleton pregnancies, IUGR fetuses had lower (P<0.01) mRNA expression for collagens I (83%), IV (56%), V (71%) and VI (67%). Fibrillin-1 and elastin-1 mRNA expression was reduced (69% and 81%, respectively). The gene expression in control fetuses from multiple pregnancies was similar to IUGR singletons. In multiple pregnancies, there was no difference in expression of collagens, fibrillin-1 and elastin-1 between IUGR and control fetuses. CONCLUSIONS: A reduction in extracellular matrix molecule expression is apparent in IUGR singletons compared to controls. This may reflect changes in the vascular growth which occurs late in gestation. These same differences in mRNA expression were not found when comparing the fetuses from multiple pregnancies. This may indicate that the 'normal' fetus is experiencing the same placental pathology as the IUGR fetus in multiple pregnancies.

What A lack of access to the developing placenta in vivo makes it difficult to reconcile in vitro data to in vivo conditions. In order to address this, we aimed to develop anatomically accurate computational models of the utero-placental circulation in early human pregnancy. METHODS: 3D micro-CT imaging, histology and measurements of uterine vessels in the literature were used to determine the anatomical parameters of first trimester uterine spiral arteries. These parameters were used to generate in silico models that can predict physiological shear stresses in the spiral arteries, as well as the resulting oxygenation of, and pressure experienced by the placenta. RESULTS: 1) Whilst upstream spiral artery flow generates around 100Pa of shear stress, in a partially remodeled spiral artery, our model predicts a low shear region of 0.1-5Pa extending ~7mm into the uterine tissue. This is important because our previous in vitro studies have shown low shear stress conditions in first trimester spiral arteries may promote trophoblast-induced remodeling of these vessels. Therefore, together these data suggest that both cellular interactions and the resulting structural changes to the vessels act in a cyclical manner to promote adequate spiral artery remodelling.

2) When our model of spiral artery blood flow was used to predict the penetration of blood into the intervillous space, we found significant flow penetrating up to 1.5-2cm. The further the flow penetration, the higher the model predictions of placental tissue oxygenation that is beneficial for the fetus, but the higher the pressure transmission to placental tissue that could potentially leading to pathological consequences. This highlights the fine balance that exists between these two parameters for optimal blood flow in pregnancy. CONCLUSIONS: Further development of these models will aid our understanding of normal utero-placental blood flow and allow the manipulation of ex vivo conditions to understand how aberrations in utero-placental hemodynamics affect placental function. INTRODUCTION: Impaired placentation is implicated in poor perinatal outcomes associated with Trisomy 21 (T21). Earlier studies revealed a role for abnormal cytotrophoblast (CTB) differentiation along the invasive pathway. As a followup, we investigated differences in global gene expression between T21 and euploid placentas. Caspase-2 (CASP2) expression was 8-fold higher in T21. In other studies, CASP2 is associated with apoptosis-related cytoskeleton degradation, oxidative stress-induced cell death, and cell cycle regulation. Little is known about its role in placental development. Therefore, this study aimed to further characterize CASP2 expression and investigate its role in T21 placentas.

We characterized CASP2 expression at RNA (qRT-PCR) and protein levels in samples from T21 (n=8) and euploid (n=4) agematched placentas. Apoptosis was investigated via the TUNEL assay. We also investigated CASP3, FAS/CD95, and Fas ligand (FasL) expression in the same samples. RESULTS: CASP2 was significantly overexpressed (protein and mRNA levels) in T21 placentas. Highest CASP2 expression was in villous cores, and in villous and extravillous CTBs CASP3 had the same expression pattern as CASP2. Using the TUNEL approach, we observed high variability in the number of apoptotic cells in biopsies from different regions of the same placenta and among different placentas. However, T21 placentas had more apoptotic cells, specifically in cell columns and basal plates. CASP2 co-immunolocalized with FAS/CD95 and FasL in TUNEL-positive extravillous CTBs, but not in the villous cores. *Figure(s) will be available online. CONCLUSIONS: T21 pregnancies are associated with poor placentation and apoptosis of invasive CTBs. Versus euploid controls, T21 placentas had higher CASP2 expression and TUNEL reactivity. This difference was most pronounced amongst invasive CTBs. Our results suggested a possible CASP2-related mechanism for altered CTB invasion in T21. This relationship will be further examined, at a functional level, in human trophoblast progenitor cells derived from T21 and euploid placentas.

Dynamic Expression of Notches in the Developing Mouse Placenta.

Heather I Levin, Amanda Adeleye, Carrie Shawber, Mark V Sauer, Jan K Kitajewski, Nataki C Douglas. Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA.

The uterine decidua supports early pregnancy prior to placenta formation. The decidua is the scaffold for the newly formed vascular network and maternal spiral arteries that are remodeled by embryo-derived trophoblasts (TBs) during placentation. Notch signaling is a fundamental regulator of angiogenesis and vascular remodeling. Deletion of Notch2 (N2) in the TBs of mice reduced TB invasion of maternal vessels resulting in poor placental perfusion, characteristic of preeclampsia (PE). Our goal was to identify Notch family proteins expressed during decidual angiogenesis and placenta formation.

The expression of Notch proteins, Notch1 (N1), N2, Notch3 (N3), and Notch4 (N4) and ligands, Jagged1 (Jag1) and Delta-like4 (Dll4) on CD31 + endothelial cells (ECs) and cytokeratin + TBs was characterized by immunohistochemistry. Uteri and placentas were analyzed at embryonic day (E) 6.5, E8.5 and E12.5. Images were captured by confocal microscopy. RESULTS: At E6.5, N1, N4 and Dll4 are expressed on CD31 + ECs of small maternal decidual vessels, with Dll4 expression restricted to the antimesometrial decidua. Jag1 is expressed on CD31 + ECs of large maternal decidual vessels. At E8.5, N1 and Jag1 expression is maintained on small and large vessels, respectively, while N2, N3, and N4 are expressed on decidual stromal cells. N2, N4, and Dll4 expression co-localizes with cyokeratin + TBs in the ectoplacental cone (EPC). At E12.5, N1 and Dll4 are expressed on CD31 + ECs of maternal decidual vessels and on fetal ECs in the labyrinth, while N3 and N4 are expressed on cells adjacent to CD31 + fetal ECs in the labyrinth. Jag1 is expressed on endothelium of large maternal decidual vessels and on cells adjacent to maternal canals in the labyrinth. N4 is expressed in the junctional zone, the placental structure derived from the EPC. * Figure( s) will be available online. CONCLUSIONS: These data support our hypotheses that Notch is required for proper EC/TB interactions during placentation and that abnormal Notch function leads to early miscarriages or PE. These studies serve as a framework to better understand Notch signaling in placentation and develop new mouse models of PE. 

Endothelin . Impairment of this process has been linked to pregnancy complications such as pre-eclampsia (PE). PE is also characterized by an increase in endothelin-1 (ET-1), a regulator of cell proliferation, migration and invasion. Therefore, we hypothesize that increased levels of ET-1 modify the expression of MMPs and their natural inhibitors (TIMPs) during the first trimester of pregnancy.

Primary human trophoblasts isolated from first trimester placentas (week 7 to 10 of gestation) were incubated in the absence or the presence of 10nM and 100nM ET-1. MMP-2, -14 and -15 and TIMP expression was determined by RT-qPCR and Western blotting. MMP-2 activity was assayed by zymography. RESULTS: ET-1 had a dose-dependent effect on MMP/TIMP expression after 24h, with 100nM ET-1 inducing the maximum response, downregulating MMP-2 (24%; p=0.025), MMP-14 (21%; p=0.054) and MMP-15 (26%; p=0.019) and up-regulating TIMP-3 (47%; p=0.01) and TIMP-4 (52%; p=0.03) at the mRNA level, as well as down-regulating MMP-2 (17%; p=0.004), MMP-14 (25%; p=0.055) and MMP-15 (26%; p=0.046) protein levels. CONCLUSIONS: ET-1 alters the balance between invasion promoting MMPs and invasion inhibiting TIMPs in human first trimester trophoblasts. Therefore, ET-1 might contribute to the impairment of trophoblast invasion observed in PE.

Impact of Combination Antiretroviral Therapy ( METHODS: Pregnant C57Bl/6 mice were exposed to human equivalent doses of cART throughout gestation (day 1-15). Controls included mice treated with vehicle or PI-sparing cART. On day 15 mice were euthanized, pregnancy loss, number, viability and weight of fetuses, and placenta weight were recorded.Placenta tissue and cardiac blood were collected for angiogenic factor analysis by q-PCR and ELISA. Some placentas were perfused with a casting agent into the arterial vasculature, and then scanned using a micro-CT. RESULTS: PI exposure resulted in significantly lower fetal and placental weight compared to control.PI exposure did not have a significant effect on the expression of pro-angiogenic factors such as VEGF, VEGF receptor 2 (Flk-1), or PlGF. But PI exposure significantly reduced the expression levels of VEGF receptor 1 (Flt-1), an anti-agiogenic factor. Levels of sFlt-1 directly correlated with fetal weight.Placental blood vessel imaging revealed a significant difference in the size and number of blood vessels in the PI exposed mice with increased number of arterioles with shorter length. CONCLUSIONS: Our findings suggest that PIs adversely affect angiogenesis during pregnancy. PI use was associated with a proangiogenic state characterised by reduced levels of sFlt-1 and greater branching of arterioles in the placenta.This state correlated with fetal growth restriction. These results address a gap in knowledge of HIV antiretroviral toxicity in pregnancy. The mouse model provides a useful tool to better understand the mechanisms that underlie pregnancy complications experienced by HIV+ women receiving cART. We are currently investigating the mechanisms involved in PI-induced downregulation of sFlt-1 and are extending our findings to HIV+ pregnant women. Understanding the impact of cART on angiogenesis during pregnancy may lead to better clinical management of HIV+ pregnant women. RESULTS: HTR8s, GD15 and GD20 rat placenta express the following OLN targeted receptors: 1) serotonin 5-HT 1A , 5-HT 1B , 5-HT 1D , 5-HT 2A , 5-HT 2B , 5-HT 5A and 5-HT 6 2) dopamine D 1 3) muscarinic M 1 , M 2 , M 3 , M 4 and M 5 4) adrenoreceptor a 1A , a 1B , a 2A , a 2B and a 2C 5) histamine H 1 .

HTR8s additionally express OLN targeted serotonin 5-HT 1E , 5-HT 2C , 5-HT 3 and dopamine D 2 , D 4 and D 5 receptors. Both GD15 and GD20 rat placenta express the serotonin 5-HT 7 receptor. The expression of the following receptors increased significantly (p<0.05) in GD20 relative to GD15 rat placenta: histamine H 1 (5.92-fold), muscarinic M 1 (4-fold), serotonin 5-HT 1D (1.7-fold), 5-HT 2A , (1.86-fold) and 5-HT 2B (5.13-fold). Additionally, adrenoreceptor a 1A expression significantly decreased (p<0.05) in GD20 rat placenta (4.64-fold). The expression of a key trophoblast invasion enzyme, matrix metalloproteinase 2 (MMP2), trended to increase by 21.44±0.03% in HTR8s treated with 1µM OLN. CONCLUSIONS: Comparison between receptor expression in HTR8s and rat placenta identifies which receptors are trophoblast and species specific. Additionally, comparing GD15 and GD20 rat placenta elucidates which receptors are likely important for placental function at various developmental stages. Furthermore, the presence of OLN receptors in the placenta suggests that OLN use during pregnancy can directly influence placental development/function but the specific trophoblast-mediated effects remain to be determined. this study was to investigate the expression patterns of some of these markers described in the mouse, to help identify trophoblast subtypes in the human placenta and possibly point to a trophoblast stem cell niche. METHODS: Following IRB approval, formalin-fixed paraffin-embedded blocks were retrieved from our and the department placental biobanks and consisted of fifteen normal human placental tissue samples from 5 weeks gestational age to term, and one ectopic tubal pregnancy at 6 weeks gestation. We performed immunohistochemistry (IHC), where validated antibodies were available; for human ELF5 and ASCL2, we performed in-situ hybridization (ISH), using probes from ACD-Bio. All antibodies and ISH probes were validated on positive control tissues.

The mouse trophoblast stem cell markers, CDX2 and ELF5, showed gestational age-dependent staining in the CTB layer of chorionic villi.

Week 5 samples showed the highest expression, with a dramatic decrease in staining after week 10. Unlike p63, which was uniformly expressed in all CTB, CDX2 and ELF5 showed a mosaic pattern, even in early specimens, with CTB facing the chorionic plate staining more strongly than CTB near the basal plate. ASCL2, a marker of ectoplacental cone and spongiotrophoblast in mouse, was expressed in all HLA-G+ cells, including Ki67+ proximal and p57+ distal column trophoblasts, and mature EVT at the basal plate. CONCLUSIONS: These data suggest that the continuous layer of CTB in early chorionic villi is not made of a uniform group of cells. Further studies will elucidate if CDX2+ CTB represents a true multipotent trophoblast stem cell in the placenta, giving rise to both STB and EVT. Future work will include staining for additional markers as well as double staining to evaluate co-expression in specific cellular compartments.

Signaling It is associated with defects in the cytotrophoblast (CTB) differentiation pathway leading to extravillous trophoblast (EVT), cells involved in uterine invasion. We used microarray analysis to compare gene expression differences between EGFR+ CTB and HLAG+ EVT, isolated from first trimester placental tissues. We identified STAT1 as a transcription factor significantly upregulated in EVT, and set out to evaluate its expression and function during CTB differentiation into EVT.

We isolated CTB from first trimester placental tissues by standard methods, and cultured them on fibronectin-coated plates for 4 days to differentiate them into HLAG+ EVT, in the presence or absence of IFN-g. We collected: 1) cells to evaluate proportion of EGFR and HLAG positivity by FACS; 2) lysates for western blot analysis and fixed cells on coverslips for staining, using antibodies to STAT1 and phosphorylated-STAT1 (p-STAT1); and 3) supernatants for MMP2 ELISA. Finally, we also fixed some first trimester villous tissue, some following a 30-minute treatment with IFN-g, and stained with the above antibodies. RESULTS: STAT1 upregulation during EVT differentiation was confirmed by western blot, with more intense STAT1 and pSTAT1 levels in samples cultured in medium containing IFN-g. Immunofluorescence staining of cultured CTB showed phospho-STAT1 only in HLA-G+ cells, and only following IFN-g treatment. Treatment with IFN-g significantly decreased MMP2 secretion, as previously shown; however, FACS analysis showed a 1.5-fold increase in proportion of HLA-G+ cells in CTB cultures differentiated in presence of IFN-g. Finally, in primary villous tissue, STAT1 and p-STAT1 were detected only in HLAG+ cells, with markedly increased staining for both following IFN-g treatment. CONCLUSIONS: IFN-g signaling through the STAT transcription factors has previously only been evaluated in context of EVT invasion and interaction with the maternal immune system. The above results suggest a possible involvement of STAT1 in differentiation of villous CTB into the EVT lineage. Additional studies, including overexpression and knockdown of STAT1, are required to further assess this hypothesis.

Role of High-Mobility Group A1 Protein in Human Trophoblast Invasion. Yuka Uchikura, Keiichi Matsubara, Yuko Matsubara, Miki Mori, Akihiro Nawa. Obstetrics and Gynecology, Ehime University, Toon, Ehime, Japan. INTRODUCTION: Extravillous trophoblast (EVT) cells can invade into uterine decidual spiral arterioles and mediate the remodeling of these vessels. Disturbed arterial remodeling can lead to the serious complications such as preeclampsia and fetal growth restriction. So far no specific function has been reported for high-mobility group A1 protein (HMGA1) in human trophoblast invasion. The aim of this study was to evaluate the effect of HMGA1 on trophoblast invasion.

We investigated HMGA1, HLA-G, MMP-2 and MMP-9 expressions in HTR-8/SVneo cells, JEG3 cells and Jar cells treated pregnancy-associated hormones such as human chorionic gonadotropin, progesterone and oestradiol using Western blotting, immunofluorescence analysis, and RT-PCR. Wound healing cell migration test and matrigel invasion test was also perfomed using HTR-8/SVneo cells. RESULTS: Pregnancy-associated hormones increased the expression of HMGA1 mRNA in the matrigel invasion assay. Especially, HMGA1 was strongly expressed in the invasive cells in immunofluorescence analysis. We also found cytoplasmic expression level of HMGA1 was increased in the migrating cell in wound healing cell migration test. CONCLUSIONS: It is suggested that HMGA1 might have a critical role in trophoblast invasion. The vascular exchange region of the human placenta, called the villus, brings fetal and maternal blood systems into close proximity to facilitate the transfer of gases and nutrients. Development begins at gestational age week 5 and vascular defects cause placental defects in up to 10% of all pregnancies, resulting in life long chronic disease in the mother and her child. We propose our lack of knowledge in human villus development is preventing advances in technologies to counter pregnancy complications. As most developmental processes involve transitory populations of cells that drive and organize developing cellular networks, I hypothesize a cellular analysis of the human placenta will identify populations of transitory cells that direct villus development. METHODS: Through a systems biology approach, I employed a 370 CD antigen screen to identify transitory populations of cells between week 6 or 10 that span the critical period of villus formation. I anticipate markers of progenitor cell subpopulations to be strongly expressed at week 6 and decrease by week 10, as the cells differentiate into complex villus structures. Villi samples were dissociated into two fractions: trophoblast and stromal enriched fractions. The high throughput flow cytometry assay was conducted on trophoblast fractions. Markers of dynamic populations are ranked by fold change differences and biological relevance for validation. RESULTS: Using the most stringent filtering criteria, 4 increasing and 6 decreasing developmentally regulated populations were confidently identified in both replicates of the screen. 74 dynamic markers were found to mark dynamic populations in only one replicate. We have confirmed two markers of transient, decreasing trophoblast cells are labeling independent populations. Targeted flow cytometry analysis confirmed distinct EpCAM+ and EpCAM-populations in week 5 placentas. A CDCP1 population was identified that does not co-express EGFR or EPCAM. In week 6 placentas, histological analysis revealed EpCAM+ cells were present in cytotrophoblasts (CTB) and columnar CTB but by week 8, EpCAM expression is reduced and restricted to columnar CTB, suggesting EpCAM becomes restricted to proliferative cells. CONCLUSIONS: This resource of CD antigen expression in early human villi development will be a valuable asset to studying placenta organogenesis. My work has developed strong evidence to support the existence of transient populations of cells in the human placenta. . CYP19 placental expression positively correlated with ERRy (p=0.012/R 2 =0.247). CYP19 expression was not significantly different in the whole IUGR group compared to C and the total IUGR group. However, when analyzing IUGR depending on severity, CYP19 expression was significantly higher (+33%) in the most severe IUGR (altered PI) vs C (p=0.036) *Figure(s) will be available online.

The higher ERRy expression, though not significant, is consistent with our previous data showing increased mtDNA in the same IUGR placentas, and possibly represents an adjustment to the impaired metabolism by increasing mt biogenesis. ERRy might also act on estradiol production through its interaction with CYP19. In vitro studies in other tissues suggest an estradiol protective role against oxidative stress. Interestingly, CYP19 levels are significantly higher in the most severe IUGR. We might speculate that the CYP19 alterations have an estrogenrelated protective action in more severe IUGR, which we showed to be characterized by increased mtDNA, a marker of oxidative stress. METHODS: Dual-perfused placental cotyledons (n=21) were prepared from C-section term deliveries. Following equilibration with 95% O2/5% CO2 (normoxia) the maternal side was sequentially perfused with Krebs solution with hypoxia (95% N2/5% CO2) and normoxia. Changes in perfusion pressure in the fetal system were monitored. 1-mM L-arginine (n=3), 1-mM D-arginine (n=2), 1-mM BQ123 (n=3) or normal saline (n=10) were administered to the fetal compartment prior to the initiation of hypoxia in the maternal side. 1-mM L-arginine (n=3) was also administered to the maternal system. Changes in the fetal-side perfusion pressure were compared in these groups. RESULTS: Hypoxia in the maternal side was associated with increased (mean delta 3.17+1.33 mm Hg) fetal-side perfusion pressure. L-arginine administration to the fetal system blunted the effect of hypoxia-induced fetal vascular hypertension (mean delta 0.40+0.6 mm Hg; p=0.006), while its introduction into maternal system had no vasodilatory effect (mean 1.93+0.35 mm Hg; p=0.15). D-arginine and BQ123 administration to the fetal compartment did not significantly change the hypoxic effect on the fetal vasculature (mean 2.1+0.71 and 2.23+0.9, respectively; p=0.7 and 0.3, respectively). CONCLUSIONS: 1) L-arginine, but not D-arginine or BQ123, reduces the fetal vasoconstriction suggesting that NO, but not endothelin, signaling is involved in fetal vasoconstriction; 2) L-arginine administration into maternal system did not reduce the vasoconstriction in the fetal side; 3) lack of maternal L-Arginine effects on the vasoconstriction suggests that either NO produced within the maternal compartment is not involved in regulation of fetal vascular reactivity or L-arginine transfer to the fetal side is insufficient to acutely relax chorionic vessels, or both. The fetus is largely dependent on the placenta for its lipid supply. Placentas of obese and diabetic women accumulate lipids due to increased fatty acid (FA) synthesis and esterification, which may alter fetal lipid exposure. Omega-3 polyunsaturated FA (N-3 PUFA) such as docosahexanoate (DHA) and eicosapentanoate (EPA) alter FA metabolism in hepatocytes, though their effect on placental tissue is largely unknown. The aim of this study is to investigate whether chronic N-3 PUFA supplementation during pregnancy affects lipid metabolism in placentas of obese and overweight women.

A secondary analysis from a RCT of n-3 PUFA supplementation during pregnancy in obese and overweight women (BMI: 33.4 ± 6.6 kg/m 2 , n=33) was performed. Women were recruited in early gestation (<16 weeks) and randomized to either placebo (wheat oil, n=16) or N-3 supplement (800mg DHA and 1200mg EPA/day, n=17) for the remainder of pregnancy. Fatty acids were measured in maternal plasma before supplementation and at 34-36 wks of gestation. Expression of candidate genes involved in FA oxidation (CPT1b, PPARα and AMPKa), lipid synthesis/accumulation (PPARγ, DGAT1, SCD1 and PLIN2) and FA uptake (CD36 and FABPpm) was analyzed by real time PCR in placental tissue collected at term delivery. RESULTS: Plasma concentration of EPA and DHA was significantly increased by 3 and 1.4 fold respectively in N-3 PUFA supplemented subjects (p<0.05) by the completion of the study. N3/N6 ratio in maternal plasma was increased in treated by 1.5-fold compared to placebo. The mRNA expression of placental PPARγ (r= -0.39, P=0.04) and its target genes DGAT1 (r= -0.43, P=0.02) and PLIN2 (r= -0.38, P=0.04) was significantly correlated with the maternal N3/N6 ratio. Expression of genes regulating FA oxidation or uptake, was not changed suggesting that n-3 PUFA supplementation alters the lipid distribution in placental tissue rather than FA uptake from maternal circulation. CONCLUSIONS: Our data suggest that a 1.5-fold increase in the N3/ N6 ratio during pregnancy in obese and overweight women is associated with inhibition of placental lipid accumulation pathways. This, in turn, may negatively regulate the levels of lipid substrates delivered to the fetus. 

We longitudinally assessed maternal serum apelin levels (ELISA) and hemodynamics (blood pressure, cardiac output, total peripheral resistance) between 20-34 weeks gestation in 18 women at high risk of placental dysfunction. Placental apelin staining was assessed by immunhistochemistry in placentas from uncomplicated pregnancies (n=6), preterm deliveries (n=6), pre-eclampsia (PET, n=8) and isolated intra-uterine growth restriction (IUGR, n=8). Placental apelin gene expression was assessed by quantitative PCR.

In the high risk cohort, 4 fetuses developed isolated IUGR and 6 women developed PET. We obtained a median of 5 (range 2-9) hemodynamic and apelin measurements per woman. Apelin levels throughout gestation were best fitted by a quadratic curve (figure). Mean apelin level between 20-26 weeks gestation correlated with total peripheral resistance (r=0.57, p=0.01) and showed a trend towards an inverse correlation with stroke volume (r=-0.42, p=0.08). Apelin serum levels were significantly lower in pregnancies complicated by IUGR than in uncomplicated pregnancies or in women with pre-eclampsia (p=0.009). Placental apelin gene expression was similar in IUGR, PET, preterm and term normal placentas. Apelin staining was seen both in syncitiotrophoblast and stroma of the placental villi. In IUGR placentas, apelin staining was strongly decreased in both compartments compared to normals. Pre-eclamptic placentas showed an intermediate staining level. * Figure( s) will be available online. CONCLUSIONS: Apelin levels mirror the cardiovascular changes seen in pregnancy and are therefore not its primary driver. Low apelin levels in IUGR may explain the decreased maternal cardiac output seen in these pregnancies. Furthermore, the renin-angiotensin system (RAS) is known to be activated in human preeclampsia. Angiotensin II receptor subtype 1 antagonist is efficacious antihypertensive agents, but are counter indicated during pregnancy due to nephrotoxicity in the fetus. We therefore investigated the effects of an angiotensin II receptor subtype 2 agonist (C21) on model mice expressing the preeclampsia phenotype. METHODS: Blastocysts that had been incubated with adenovirus vectors containing CD40L were transferred into the uterine horns of pseudopregnant ICR mice. A subcutaneous osmotic pump containing C21 or saline was placed in each mouse's side-dorsal region. Blood pressure (BP) was measured by the tail-cuff method and urine samples used for measuring albumin concentrations were collected via metabolism cages. On e17.5, the uteri were removed from the pregnant mice and, upon surgical delivery of the neonates, subjected to morphological investigation with the placentas. The neonates and placentas were weighed. RESULTS: C21 did not alter maternal BP or the weights of the neonates and placentas. However, relative to saline controls, albuminuria was attenuated significantly by C21. CONCLUSIONS: C21 improved maternal renal function without having significant effects on maternal BP or fetal growth. The aim of this study was to determine the levels of gamma-aminobutyric acid (GABA) receptor subunits (a 1 and β 2 ) in the hippocampus of rat models of preeclampsia and eclampsia as the subunits may be important in these disorders. METHODS: Sprague-Dawley rats (n = 5/group) were used: non-pregnant, pregnant controls and rats treated on gestation day (GD, 14) with low dose lipopolysaccharide (LPS, 1 ug/kg, Sigma Aldrich) to induce preeclampsialike conditions and rats treated with LPS (GD 14) + pentylenetetrazole (PTZ, 40 mg/kg, GD 20, Sigma) to induce seizures similar to eclampsia. The hippocampus from all rats was obtained 1 hour after PTZ treatment. Total RNA was extracted and reverse-transcribed into cDNA and RT-qPCR (QuantiTect Reverse Transcription Kit and QuantiNova SYBR Green PCR Kit, Qiagen, Alameda, CA) was performed as per Qiagen's protocol. The mRNA level for GABA subunits was estimated using the delta-delta Ct method compared to expression of HPRT. Immunohistochemistry (IHC) was performed using polyclonal antibodies to the subunits (Abcam, Cambridge, MA) by standard protocols. IHC was quantified (doubleblind x 2 observers) as grades of percent positive staining cells (scale = 1 to 3) x ratings of stain intensity (0 = no stain, 1 = light, 3 = strong) from slides (n=5/group) to obtain scores (scale = 0 to 9) using published IHC methods (J. Translational Med. 2014, 12:273) . Anova was used for statistical analyses. RESULTS: No significant differences in mRNA expression of GABA subunits for a1 or b 2 were found between groups by RT-qPCR (values = ca. 20 units per group, P>0.05). IHC reveals very low levels and no significant differences (p>0.05) of both a 1 and b 2 subunits of non-pregnant, pregnant and preeclampsia rats (scores = 0) but significantly higher levels of subunit expression in eclamptic-like rats (scores = 2.00±0.71, p< 0.001, a1 and 2.20±0.45, p<0.001, b 2 ). CONCLUSIONS: The lack of significance of gene expression for a 1 and β 2 subunits between groups as measured with RT-qPCR may be attributed to post-transcriptional regulation because IHC demonstrates high levels of the target subunits in eclampsia-like rats. The increase in subunits in the hippocampus by IHC indicates important roles in seizures associated with eclampsia. Aberrant UBF is found in pregnancy disorders such as preeclampsia with IUGR characterized by endothelial cell dysfunction. Endothelialderived vasodilators, PGI 2 and NO, regulate vasodilation by respectively modulating cAMP-and cGMP-mediated mechanisms in uterine artery endothelium. Uterine artery endothelial cells from pregnant ewes (P-UAEC) produce markedly more PGI 2 and NO in response to ATP via Ca 2+ mediated mechanisms which requires GJ protein Cx43 for normal pregnancy enhanced Ca 2+ responses to enhance NO production. cAMP signaling mechanisms acutely open GJ gating through the phosphorylation of GJ protein Cx43 serine (S)365, however, the role of cGMP in GJ function is unknown. Hypothesis: cAMP and cGMP will increase the phosphorylation states of Cx43 and also increase Ca 2+ response to ATP. METHODS: P-UAEC were treated with either 8-Bromo-cAMP or -cGMP (1uM/1mM) for 5min, 15min, 30min, 60min and 12hrs and analyzed by western blotting for the phosphorylation at Cx43 S365 (pCx43 S365) and S368 sites, respectively. For the Ca 2+ studies, P-UAEC were loaded with Fura-2 (Ca 2+ dye), stimulated by ATP (100uM) and imaged for 30min. 8-Bromo-cAMP or -cGMP (1uM/1mM) was added for 30min and restimulated with ATP and Ca 2+ bursts were measured. In GJ function studies, P-UAECs were analyzed using the scrape-loading/dye transfer technique.

We observed that both cAMP and cGMP increased (P<0.05) pCx43 S365 (5.5-fold and 5-fold, respectively), but only cGMP substantially increased the pCx43 S368 (5-fold). A significant cAMP potentiation of the ATP-induced Ca2+ response was noted, however this was specific because cGMP pretreatment did not potentiate ATP- .05)). There was no difference in the mean concentration or the median rate of change of maternal PlGF at any of the study time-points. The sensitivity/specificity in the first trimester for PlGF and sFlt-1 was 54.6/55.2% and 51.5/50%, respectively. These values did not improve as the pregnancy progressed.

In the third trimester, the positive predictive value of PlGF and sFlt-1 decreased to 26 % and 13%, respectively. The negative predictive value of PlGF reached 83% and that of sFlt-1 decreased to 69% in the third trimester. CONCLUSIONS: Prediction of preeclampsia with the use angiogenic factors in twin gestations was statistically possible but not with a positive predictive value and a negative predictive value that are clinically useful.

Derek Boeldt, Nauman Khurshid, Amanda Hankes, Ian Bird. Ob/Gyn, Univ Wisconsin, Madison, WI, USA. INTRODUCTION: Symptoms of preeclampsia (PE) including hypertension and vascular leakiness (edema/proteinuria) are associated with endothelial dysfunction, but the underlying cause is poorly understood. Growth factors and cytokines are possible mediators of acute endothelial dysfunction in PE through disruption of cell-cell junctions in an ovine model. VEGF-165 and TNFa are known to phosphorylate gap junction proteins at inhibitory residues and so limit cell-cell connectivity. Cell-cell connectivity is essential to maintain sustained Ca2+ signals in response to vasodilation-promoting agonists, which is necessary for the production of vasodilators such as nitric oxide. The resulting loss of vasodilation likely contributes to hypertension in PE. Others describe a possible role for VEGF-165 and TNFa in mediating tight junction breakdown. The resulting loss of monolayer integrity could contribute to the edema and proteinuria often observed in PE. We thus hypothesize that VEGF-165 and TNFa will both inhibit sustained Ca2+ responses and reduce monolayer integrity. < 30 (control, n = 6) , and pregnancies complicated with obesity (BMI > 30, n = 9). Arteries were pressurized and loaded with the Ca 2+ -sensitive dye fura 2 AM. Fura 2 fluorescence was assessed using a photomultiplier system. Bradykinin (BK)-induced vasodilation was evaluated in pre-constricted arteries before or after pretreatment with L-NNA and indomethacin. Changes in SMC [Ca 2+ ] i and the diameters were studied in response to NS309, a specific activator of SK/IK channels. RESULTS: BK-induced (0.3 -30 nM) vasodilation was associated with SMC [Ca 2+ ] i reduction and was significantly blunted in myometrial arteries of obese (OB) vs. normal weight (NW) women (45.9.± 14.5 vs. 85.4 ± 7.6% at 3 nM of BK). Vasodilation to BK was mostly preserved after inhibition of NO and prostacyclin in arteries of NW but was significantly inhibited in vessels from OB (62.2 ± 4.0 vs. 14.9 ± 8.2 % at 3 nM of BK). The degree of NS309-induced vasodilation was negatively correlated with BMI. CONCLUSIONS: BK-induced vasodilation was resistant to NO and prostacyclin inhibition demonstrating an important role of the EDHF system in the control of maternal myometrial vascular tone in normal weight women. Increased maternal adiposity in pregnancy resulted in blunted BK-induced dilation most likely due to impaired function of SK/ IK channels. Endothelial dysfunction in myometrial resistance arteries may be an important mechanism linking maternal obesity with increased risk for hypertension and preeclampsia in human pregnancy.

Melatonin INTRODUCTION: Preeclampsia remains a major cause of maternal and perinatal mortality and morbidity. It is thought to be, at least in part, due to the placental release of anti-angiogenic factors, such as soluble fmslike tyrosine kinase-1 (sFlt1), soluble endoglin (sEng) and activin A, that trigger maternal endothelial dysfunction, leading to the clinical features of preeclampsia including maternal hypertension and proteinuria. Current treatment is focussed on managing the hypertension to allow prolongation of pregnancy. There is no targeted therapy for the endothelial dysfunction. Melatonin and resveratrol are two antioxidants that offer promise as placental and endothelial targeted therapies and so as future treatments for preeclampsia. We aimed to assess the efficacy of resveratrol and melatonin in reducing placental oxidative stress, the placental production of antiangiogenic factors and in improving endothelial function. METHODS: Placental explants from normal term pregnancies were cultured at 1% oxygen and treated with either melatonin or resveratrol. 8-Isoprostane (a marker of oxidative stress), sFlt1 and sEng production was measured by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured with TNFα and with either melatonin or resveratrol. Endothelial activation was assessed by the measurement of ICAM1, VCAM1 and E-Selectin by flow cytometry and endothelin-1 levels by ELISA. RESULTS: Melatonin did not significantly affect placental 8-Isoprostane production or sEng production following exposure to hypoxia (P>0.05). Hypoxia-induced placental sFlt1 production was significantly decreased by Melatonin (hypoxia alone: 221.4±37.9 pg/mg tissue vs. hypoxia+melatonin: 159.2±22.4 pg/mg tissue). Melatonin did not significantly affect TNFα-induced increase in ICAM1, E-Selectin and Endothelin-1 in HUVECs, but reduced VCAM1 expression 3.5-fold (P=0.0001). Resveratrol reduced hypoxia-induced placental 8-isoprostane production 3-fold (P=0.0002) and halved hypoxia-induced sFlt1 production (P=0.01), but had no significant effect on placental sEng production. Resveratrol significantly reduced TNFα-induced endothelial activation markers ICAM1, VCAM1 and E-Selectin in HUVECs and the production of Endothelin-1 (P<0.05). CONCLUSIONS: Melatonin and Resveratrol reduce hypoxia-induced placental oxidative stress and sFlt1 production, and may improve TNFαinduced endothelial dysfunction. These data support the continued assessment of both agents as future therapies for pre-eclampsia. The glycocalyx (GC), located on the luminal surface of the vascular endothelium, has important functional and vasoprotective roles; e.g., as a selective barrier between endothelium and blood, and mediatior of nitric oxide mechanotransduction. Damage to the GC structure is implicated in reperfusion injury, atherosclerosis, and diabetes. We hypothesized that microvascular perfusion and GC structural integrity are reduced in postpartum women with a history of preeclampsia (PE).

We compared 5 women with prior PE to 9 women with prior uncomplicated pregnancy (NL), 6-24 months postpartum. All were primiparous, non-smokers, and non-lactating; groups were matched for age, days postpartum, body mass index, and menstrual cycle phase. The sublingual microvasculature was interrogated using an automated system from GlycoCheck. A sidestream-darkfield camera detects passing red blood cells (RBC) and software calculates the microvascular density and mean percentage of time vascular segments are perfused (% RBC). It also quantifies the dynamic lateral movement of RBC into the GC, defined as the perfused boundary region (PBR 

The effect of circulating factors on endothelial function was vascular bed specific. The bioavailability of NO and prostaglandins significantly impacted vasodilation in uterine arteries exposed to PE plasma but did not play a major role in the mesenteric vasculature. Understanding the pathophysiology is imperative to finding a potential preventative or therapeutic target for PE. dams versus wild-type (WT) controls) is sufficient to inhibit spiral artery growth, which leads to abnormal uteroplacental blood flow, placental damage, and fetal growth restriction. Since angiotensin II increases placental sflt-1 (soluble VEGF receptor) production, which may sequester spiral artery VEGF, we hypothesized that stimulating uterine VEGF expression may rescue the phenotype. Alternatively, reducing angiotensin II production with an ACE inhibitor may also rescue the phenotype. METHODS: Litter size and birthweight were measured in 21 WT and 23 TG dams without any intervention. Uterine vascular targeted transfections were performed in 12 WT and 12 TG virgin mice using either VEGF or luciferase plasmid conjugated to cationic microbubbles using our published protocols. Half of these mice were used to determine transfection efficiency. Uterine luciferase expression was monitored by intraperitoneal luciferin injection and bioluminescence; uterine VEGF expression was separately established by qRT-PCR. In addition, a separate set of dams were randomized to either 250 mg/liter of Captopril in drinking water for the first 10 days of gestation, or water without ACE inhibitor. We monitored daily water intake, maternal weight, newborn weight, and fetal kidney development in histologic sections evaluated by pathologist. RESULTS: VEGF and luciferase expression peaked at day 3 posttransfection and persisted through day 10 with detectable levels at term (day 19). Compared with WT controls, VEGF transfected WT dams had significantly larger litter sizes (8.1 +/-0.16 vs 10 +/-0.71, respectively) and similar birthweights (1.43 grams each). TG dams had significantly smaller pups than WT controls (1.26 grams), which persisted if transfected with luciferase (1.29 grams), but was ameliorated by VEGF delivery (1.41 grams). Captopril rescued male, but not female birthweight and did not appear to affect nephrogenesis. CONCLUSIONS: Ultrasound-mediated cavitation of cationic microbubbles conjugated to genes of interest provides a novel method to target molecular pathways like the VEGF/AGT axis, which plays a key role in uterine spiral artery remodelling. release of sFlt-1 and soluble endoglin (sEng) from placenta 2) endothelial dysfunction and 3) vasoconstriction. We performed functional studies in primary human tissues to examine whether proton pump inhibitors (PPIs) can counter these effects.

Functional studies were performed on: 1) primary trophoblast 2) HUVECs 3) uterine microvascular cells 4) human omental vessels and 5) placental explants obtained from women with severe PE. Five PPIs (including esomeprazole, rabeprazole, lansoprazole) were added at increasing doses (0-100µM), and the following examined: 1) sFlt-1/sEng production 2) endothelial dysfunction (modelled by adding TNFα or PE patient serum to endothelial cells) 3) vasodilation (whole vessels taken from the maternal omentum, and vasodilation measured by pressure myography). RESULTS: PPIs caused a dose-dependent reduction in mRNA expression of sFlt-1 variants (e15a and i13) and reduced secretion of sFlt-1 and sEng in all cell types. The decrease was potent: the top dose of esomeprazole decreased sFlt1 release from trophoblast by 50%, and sEng release by 90%. In contrast, the same doses of pravastatin had minimal or no effect on sFlt1/sEng production. Importantly, PPIs markedly reduced sFlt-1 production from placental explants obtained from women with severe PE. PPIs rescued both TNFα and PE serum induced endothelial dysfunction (quenching leukocyte adhesion and attenuating VCAM1 and endothelin-1 expression). Furthermore, PPIs blocked TNFα-induced disruption of endothelial tube formation (both HUVECs and uterine microvascular cells). Finally, esomeprazole dose-dependently promoted vasodilation in whole vessels obtained from the omentum. CONCLUSIONS: PPIs potently decrease sFlt-1/sEng release, quench endothelial dysfunction and promote vasodilation. Widely prescribed for gastric reflux during pregnancy, they are an exciting novel candidate therapeutic to treat severe preeclampsia.

was not observed in late-onset PE. Placental HtrA4 protein was secreted into the maternal circulation and the serum HtrA4 in normal pregnancy increased significantly between the first and second trimesters then remained constant for the remainder of the pregnancy. Women with earlyonset PE showed significantly higher serum HtrA4 levels compared to gestation-age-matched controls; again this was not seen in late-onset PE. High levels of HtrA4 disturbed endothelial cell capillary tube formation and increased monolayer permeability in a dose-dependent manner, and this was linked to alterations in junctional proteins and microtubule stability. CONCLUSIONS: HtrA4 is a novel placenta-specific serine protease that is specifically elevated in early-onset PE. High levels of HtrA4 in the maternal circulation may contribute to endothelial dysfunction and PE development. The glycocalyx is a gel-like matrix of glycoproteins, proteoglycans and plasma proteins that acts as a selective barrier between the blood and the endothelium. The breakdown of this vasoprotective layer leads to endothelial dysfunction and may increase cardiovascular risk. Glycocalyx damage can be detected by greater permeability of the glycocalyx to red blood cells (RBCs). We hypothesized that glycocalyx permeability would be increased in women with a history of preeclampsia.

Women with a history of preeclampsia or normotensive pregnancies who delivered between 1976 and 1982 were identified by diagnostic codes. Pregnancy history was confirmed by reviewing medical records for accepted clinical criteria (n=9/group). RBCs flowing through sublingual vessels were imaged using a sidestream darkfield camera.

The width of the portion of the glycocalyx that was permeable to RBCs (perfused boundary region, PBR) and the percentage of vessels that were filled with RBCs (RBC filling) were calculated by Glycocheck software. ANCOVA was performed to examine the effects of pregnancy history after adjusting for vessel diameter. RESULTS: Women were examined 35.2 ± 2.2 (mean ± SD) years after their first pregnancies, at 59.0 ± 3.0 years of age. PBR was significantly greater in women who had preeclampsia (mean difference in PBR: 0.15 µm, 95% CI: 0.07-0.22, p=0.004) among vessels with a median RBC column width of 9-19 µm. PBR was not different in smaller vessels. RBC filling was lower in women who had preeclampsia (4.45%, 3.6-5.3%, p=0.030). *Figure(s) will be available online. CONCLUSIONS: Women who had preeclampsia had glycocalyx damage and reduced microvascular perfusion several decades after pregnancy.

Evidence 

In this study we assessed the placental CRP stimulating ability of amniotic fluid (collected during 2 nd trimester from women who subsequently had normal or preeclamptic pregnancies) and the conditioned media from either preeclamptic or control placentas. Placentas were obtained from women with normal pregnancy and preeclampsia after C-section. Placental explants from normal pregnant women were cultured in RPMI1640 with or without amniotic fluids or placental explant culture conditioned media for 24 hours followed by Western blot to analyze the expression of CRP's. The mRNA levels of CRP's were analyzed by qPCR. RESULTS: Amniotic fluid from women who subsequently had normal pregnancy stimulated the up-regulation of CRP's in placenta and these effects were lower with amniotic fluid from women who subsequently developed preeclampsia. In contrast, conditioned media from normal term placental explant cultures failed to up-regulate CRP's, whereas conditioned media from preeclamptic placental explant cultures induced up-regulation of CRP's in normal term placentas. The mRNA levels of CRP's in placentas obtained from preeclamptic patients were significantly elevated compared to that of normal placentas. CONCLUSIONS: Data suggested that, during normal pregnancy the expression of CRP's at the feto-maternal interface is inducible early in pregnancy and therefore, normal pregnancy is characterized by low level of complement activation. In women who subsequently develop preeclampsia, complement activation is greater due to lack of increase in CRP's levels. However, during the 3 rd trimester, expression of CRP's in preeclamptic placenta is increased which was a late response and therefore, insufficient to adequately contain complement cascade.

Early There has been accumulating evidence that activation of the complement system is important in the pathogenesis of preeclampsia and that this effect is more pronounced in early-onset than in late-onset disease. Our aim was to determine the second-trimester amniotic fluid concentrations of complement split products in pregnancies subsequently affected by early-onset preeclampsia. We focused on amniotic fluid in order to better reflect processes operant at the fetal-maternal interface. METHODS: A cohort of 731 women with singleton non-anomalous pregnancies undergoing second trimester genetic amniocentesis was followed to delivery and analyzed as a nested case-control study. Cases of early-onset preeclampsia developing before 34 weeks' gestation (n=15) were compared with 47 uncomplicated term controls. Amniotic fluid collected at amniocentesis was tested for complement split products Bb, C4a, C3a, and C5a. RESULTS: Women who developed early-onset preeclampsia as compared with the term pregnant control group had significantly higher (p=0.04) median amniotic fluid C3a levels (318.7 ng/mL vs 254.5 ng/mL). Median amniotic fluid Bb levels were also significantly higher (p=0.03) in preeclamptic women than in normal pregnant women (1127 ng/mL vs 749 ng/mL). The median levels of C4a and C5a were not significantly different between the groups. CONCLUSIONS: Our data suggest that Complement activation in early pregnancy, as expressed by elevated C3a and Bb fragments measured in second trimester amniotic fluid, appears to be associated with the development of early-onset preeclampsia. We believe this to be the first prospective study to link complement activation in amniotic fluid in early pregnancy and the later development of preeclampsia.

Our findings provide evidence that immune dysregulation may precede the clinical manifestations of preeclampsia and that the alternative complement pathway, rather than the classical pathway, is principally involved. Decreased zinc levels in pregnancy are associated with adverse outcomes. Our theory is that hZIP1 zinc transporter expression is lower in pregnancies with preeclampsia, possibly contributing to the disease process and adverse outcomes. Our study compares hZIP1 expression in the placentas of women with preeclampsia to those without hypertensive diseases of pregnancy. METHODS: This retrospective cohort study was performed at the University of Illinois. The study participants were accrued through the pathology database of previously collected placentas from 2011-2012. Inclusion criteria were: 18-45 years of age and term gestation. Placental specimens were obtained from the membranes, superficial maternal surface, and full thickness of the placenta, incorporating the chorionic villi.

Microarray samples were made from the corresponding paraffin blocks. Immunohistochemistry was used to assess for hZIP1 protein expression using a previously well characterized avian antibody to human hZIP1 in each placental tissue core sample. Stain intensity scores were assessed on a 0-3 scale, with 0 being no staining and 3 being the highest intensity, compared between decidual cells in the same tissue samples. Statistical analysis was performed using chi square and t-test for categorical and continuous variables. p<0.05 was considered significant. RESULTS: 46 preeclamptic cases and 47 controls were included in the study. Preeclamptic patients were significantly younger; there were no differences in gravidity, parity and gestational age. In the preeclamptic women, there was higher use of magnesium sulfate and oral and IV antihypertensives. The placental weight was higher in the preeclampsia group, 490 g compared to 465 g. Decidual hZIP1 staining in preeclamptics had a score of 1.1 ± 1.3 (mean ± 1 S.D.), compared to controls, 1.0 ± 1.0, p=0.3. CONCLUSIONS: There was no difference in the expression of hZIP1 expression in the placentas of women with preeclampsia, compared to a control group. We were unable to show hZIP1 expression difference, suggesting it is not key in leading to adverse outcomes in women with preeclampsia. Calculator is one of the few internet-based tools available to assist clinicians in counseling patients at risk for preterm birth. Our aim was to survey these clinicians to assess awareness and utilization of the calculator.

The survey tool included a list of questions regarding the NICHD PTB Outcome Calculator. We collected responses from medical personnel in academic and community-based hospital systems. Survey questions assessed awareness level, frequency of use, knowledge of calculator variables and outcomes, and information regarding the database and its utility. Fisher's exact analysis was used to compare answers essential to the understanding of the calculator, with p < 0.05 significant.

We assessed the most commonly misunderstood aspects of the tool and compared answers amongst level of training and area of expertise. is not only an uncoupler of oxidative phosphorylation in the mitochondria, but also has been identified as a source of increased hydrogen peroxide and superoxide production, loss of mitochondrial membrane potential, increase in apoptosis, and depletion of mitochondrial glutathione, which is a potent antioxidant. The goal of this study was to determine whether the combination of chemotherapeutics with DNP would increase cell death in ovarian CSCs. METHODS: CSCs were isolated from the human epithelial ovarian cancer cell (EOC) line SKOV-3. Isolation was achieved utilizing the magnetic-activated cell sorting technique for CD44 + CD117 + cells via a CD44 or CD117 antibody coupled to magnetic beads. Isolated CSCs were treated with and without chemotherapy (taxotere (0.3 µM or cisplatin 50 µM) with or without increasing doses of DNP (0.125, 0.25, or 0.5 mM) for 72 hours followed by evaluation of proliferation utilizing the MTT cytotoxicity assay. RESULTS: CSCs isolated from SKOV-3 cells exhibited a 2.9, 10.9, and 2.8 fold increase in pluripotency markers Oct4, Nanog, and Sox2, respectively, as compared to SKOV-3 EOC cells (p<0.05). There was no effect on viability when CSCs were treated with either taxotere or cisplatin. Past studies have shown that BPA exposure in mice and monkeys disrupts meiotic activation and maturation in oocytes. Of concern, BPA has been identified in aspirates of ovarian follicular fluid collected from women with fertility issues, and higher urinary BPA levels in women undergoing assisted reproduction are associated with reduced numbers of oocytes retrieved and lower fertilization rates. We became interested in BPA because of its known ability to influence estrogen receptor (ER) signaling, the latter of which we have shown in as-yet unpublished studies to serve as a primary mechanism supporting the differentiation of oogonial stem cells ( Medicine 2012; 18:413-21] , was significantly enhanced by BPA in both a dose-and time-dependent manner. The peak response to BPA was detected at 72 hours of culture, with the highest dose of BPA leading to a 5-fold enhancement of IVD oocyte formation over control levels (P < 0.05). CONCLUSIONS: This is the first study we are aware of that ties endocrine disruptor exposure to alterations in the oogenic capacity of female germline stem cells in any species. We are currently assessing whether BPA interacts with ER signaling to activate the meiotic machinery in OSCs (e.g., Stra8, Sycp3) in a manner that resembles the response of OSCs to estrogen stimulation. (Support: NIH R37-AG012279)

Side-Population Trophoblasts Can Be Isolated From Human Placentae Throughout Gestation. Teena Gamage, Larry Chamley, Jo James. Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand.

The placenta is essential for fetal growth and survival in utero, and inadequate placental development is associated with pre-eclampsia and intrauterine growth restriction. Trophoblast stem cells isolated from mouse blastocysts have taught us a great deal about murine placental development, but a 'true' human trophoblast stem cell model has yet to be established. Hoechst low side-populations are characteristic of stem cells from a variety of tissues. We have previously isolated a Hoechst side-population of trophoblasts from first trimester placentae that are 98.5% pure and have a distinct gene expression profile from mature trophoblast populations, including the expression of genes associated with pluripotency and murine trophoblast stem cells. This work aimed to determine whether a similar population of cells are resident in term placentae. METHODS: 10g samples of term placenta underwent sequential trypsin and DNAse digest. The supernatant was collected, red blood cells lysed, and mesenchymal cells were depleted by plastic adhesion. Non-adherent cells were stained using Hoechst 33342 and a side-population identified by flow cytometry. Side-population cells were FACS sorted into Matrigel coated wells and dual immunofluorescence was used to determine the expression of the trophoblast marker cytokeratin-7 and either vimentin or HLA-G.

Side-population cells constitute a smaller proportion of total live cells in isolates prepared from term placentae (0.43% ±0.23% SE) in comparison to first trimester placentae (1.44% ±0.32 % SE). However average yields of 26742 side-population cells were able to be obtained from 10g of term placentae (n=12). Immunofluorescent staining demonstrated that 98.2% (±0.91% SE, n=4) of side-population cells obtained from term placentae were vimentin negative and cytokeratin positive, whereas and 98.4% (±1.23% SE, n=3) of side-population cells were HLA-G negative and cytokeratin positive (n=3).

We have isolated a >98% pure trophoblast sidepopulation from term placentae for the first time. The ability to isolate this potential stem cell population at term provides an exciting prospect for studying the underlying mechanisms of placental pathologies. 

We confirmed that Sox21 is highly expressed in undifferentiated TS cells and decreases upon removal of the factors that maintain them as stem cells (FGF4, heparin, and feeder conditioned medium). We then generated Sox21 over-expressing and knockdown stable TS cell lines and evaluated their differentiation profiles by qPCR using lineage specific primers. Sox21-overexpressing cells during differentiation showed an increased expression of both spongiotrophoblast and trophoblast giant cell (TGC) markers, while expression of labyrinthine markers, in particular Gcm1, were significantly reduced. By contrast, Sox21-knockdown cells showed reduction of spongiotrophoblast and TGC markers and enhanced expression of labyrinthine markers compared to controls. Finally, TS cells overexpressing Sox21 during differentiation showed a 2.5-fold increase in the number of invasive trophoblast giant cells compared to controls. CONCLUSIONS: Our results implicate Sox21 specifically in the regulation of spongiotrophoblast and giant cells differentiation and establish a new mechanism through which trophoblast sublineages are specified following FGF4 withdrawal. 

barrier and migrate along the olfactory neural route into the brain and cerebrospinal fluid. Our goal is to confirm this hypothesis by transnasally administrating Wharton's Jelly mesenchymal stem cells (WJ-MSC) to perinatal rats in a model of hypoxic-ischemic brain injury. Additionally we want to know how these cells may modulate the brain injury. METHODS: Wistar rat pups (P4), previously brain-damaged by combined hypoxic-ischemic and inflammatory insult, received WJ-MSC:

The heads of the rat pups were immobilized and 3 ml drops containing the cells (50'000 cells/ml) were placed on one nostril allowing it to be snorted. This procedure was repeated twice, alternating right to left nostril with an interval of one minute between administrations. The rat pups received a total of 600'000 cells. Animals were sacrificed 7 days after the application of the cells. Fixed brains were collected, embedded in paraffin or snap frozen and sectioned. Several immunohistochemical analyses followed. RESULTS: Transplanted cells were found in the layers of the olfactory bulb (OB), the cerebral cortex, thalamus and the hippocampus. The amount of cells was highest in the OB. Animals treated with transnasally delivered stem cells showed significantly decreased gliosis compared to untreated animals. CONCLUSIONS: Our data show that transnasal delivery of WJ-MSC to the newborn brain after perinatal brain damage is successful. The cells not only migrate the brain, but also decrease scar formation and improve neurogenesis. Therefore, the non-invasive intranasal delivery of stem cells to the brain may be the preferred method for stem cell treatment of perinatal brain damage and should be preferred in future clinical trials. Financial support by Cryosave Switzerland and The Eagle Foundation. from the umbilical cord might be ideal candidates to cure perinatal brain damage and other central nervous system disorders. Their secretome has been shown in vitro and in vivo to have beneficial effects on neurogenesis and neuroregeneration. Still, it is not clear if cell-to-cell contact may equally contribute to this positive effect. Therefore, the objective of this study is to elucidate through which of these two mechanisms neuroregeneration ought to be triggered the most in vitro.

The effect of WJ-MSC on the expression of neuroglial markers in neural progenitor cells (NPC) was assessed in vitro in conditioned medium and co-culture experiments by immunocytochemistry, real-time PCR and western blot. Furthermore, the differences between WJ-MSC derived from term or preterm deliveries were evaluated. Additionally the secretome of WJ-MSC was analyzed by mass spectroscopy and with a membrane-based antibody array. RESULTS: Over 500 secreted proteins were identified. Several of these proteins are involved in glio-and neurogenesis. Hippocampal NPC at passage 3 showed an increased expression of glial markers such as myelin basic protein (Mbp), galactocerebroside or glial fibrillary acidic protein (Gfap) after exposure to WJ-MSC-conditioned medium (CM) or after direct contact to WJ-MSC. Interestingly, WJ-MSC from term deliveries induced more strongly the expression of glial markers when compared to preterm. The co-culture had a more prominent effect on the expression of glial markers compared to CM, especially regarding the oligodendroglial markers. four different differentiation agents were added to drive cardiomyocyte differentiation. The first differentiation medium included Y27632, a ROCK inhibitor, CHIR99021, a GSK3 inhibitor that enables self-renewal of stem cells, and IWP-2, a Wnt signaling inhibitor, in RPMI/B-27 without insulin. The remaining three differentiation agents included 10 uM 5-azacytidine, 10 nM oxytocin (OT), and 100 nM OT, respectively. After 72 hours, all wells were replaced with complete medium. Control group wells received complete medium only. 14 days after differentiation, total RNA was extracted and expression of cardiomyocyte markers (NKX2-5, cTnT, cTnI, and ISL1) was analyzed by qRT-PCR. RESULTS: Treatment with OT yielded a dose-responsive increase in ISL1 and cTnI expression. 100 nM OT treatment resulted in significant 10-fold increase in ISL1 expression. Treatment with 10 nM OT resulted in a significant increase (2-fold) in cTnT expression. 10 nM OT and 100 nM OT treatments did not induce significant expression of NKX2-5. Treatment with conventional agents Y27632/CHIR99021/IWP-2 or 5-azacytidine did not result in significant increases in cardiomyocyte marker gene expression. Further, EDSC treated with 100 nM OT showed morphological changes suggestive of cardiomyocyte differentiation. T-eSF control (123 >1.5 fold) and IV-eSF endo vs. eSF control (134 >1.5 fold) included 50 genes in common, 72 unique to T-eSF endo vs. T-eSF control , and 84 unique to IV-eSF endo vs. eSF control . Pathway analysis showed in IV-eSF endo vs. eSF control increased proteolysis, decreased gene expression, and decreased lipid metabolism. T-eSF endo vs. T-eSF control uniquely showed increased cellular migration/recruitment/immune cell trafficking/ activation, and decreased cellular differentiation/development. T-eSF control and IV-eSF control but not T-eSF endo and IV-eSF endo responded to P4 with IGFBP1 secretion. CONCLUSIONS: Common DEG and P4-resistance in T-eSF endo and IV-eSF endo indicate changes derive from progenitors transmitted to differentiated lineage. Unique changes in T-eSF endo and IV-eSF endo T-eSF endo suggest the disease niche in endometriosis result in changes during eSF lineage commitment/differentiation/function contributing to the disease phenotype. Notably, the pro-inflammatory phenotype of T-eSF endo was not present in IV-eSF endo differentiated in vitro from eMSC endo . Support: U54HD 055764 (LCG). Zearalenone is a potent estrogenic mycotoxin that is broadly found in the diet of the US population. The purpose of this project was to assess whether alterations in placental hormone secretion by an environmentally-relevant concentration of zearalenone results from dysregulation of steroid metabolizing and syncytialization genes. METHODS: Human embryonic stem (hES, WA09/H9) cells were differentiated to trophoblasts using media supplemented with bone morphogenic protein 4 (BMP4). Cells were treated with vehicle, zearalenone (1 ng/ml), or 17β-estradiol (1ng/ml) as a positive estrogenic control. Media was collected on days 2, 4, 6, and 8 and RNA extracted from cells on day 8 for mRNA profiling. Progesterone and human chorionic gonadotropin (hCG) were measured by ELISA. Quantitative PCR was used to measure expression of trophoblast progenitors (CDX2) and steroidogenesis in trophoblasts (CYP11A1, hCGA, hCGB, syncytin). RPL13A was used as a reference gene. RESULTS: In cells exposed to BMP4 and zearalenone, progesterone and hCG levels in media were decreased 40% and 80%, respectively, compared to hES cells treated only with BMP4. This corresponded with reduced mRNA expression of CYP11A1, hCGA, and hCGB that was 24-37% of control cells. No significant difference in CDX2 or syncytin mRNA were observed with zearalenone treatment, although they tended to be reduced. Similar results were observed with 17β-estradiol treatment. CONCLUSIONS: Impaired secretion of progesterone and hCG from differentiating trophoblasts may result from down-regulation of steroidrelated genes following zearalenone exposure.

The Although uterine leiomyoma (UL) enlarges under hypoxic conditions like a cancer tissue, it has been reported that the hypoxic response mediated by the induction of hypoxia-inducible factor-1 (HIF-1) was lost in UL. In a previous study, none of HIF-1 expression was detected by immunohistochemistry or immunoblotting in human UL tissues obtained from surgical specimens. However, these specimens are constantly exposed to severe hypoxia in vivo; thus, an immediate response of UL against hypoxia has not been elucidated. To further investigate the immediate hypoxic response in UL, we measured the protein and mRNA expression levels of HIF-1α in primary cultured UL cells under hypoxic conditions. METHODS: The experimental protocol was approved by the Institutional Review Board of Chiba University, and a written informed consent was obtained from all the patients. We primarily cultured UL and adjacent myometrial cells (n=10), which were obtained from surgical specimens, under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions for 2, 4, 6, and 12 h, and then extracted total protein and RNA from the cells.

We detected protein levels of HIF-1α by immunoblotting using a specific antibody and measured mRNA levels of HIF-1α by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) using specific primers for HIF-1α.

: HIF-1α protein expression was not detected under normoxic conditions but was detected under hypoxic conditions in the UL cells. This expression increased in a time-dependent manner until 6 h but decreased at 12 h. In contrast, mRNA expression levels of HIF-1α decreased under hypoxic conditions in a time-dependent manner until 12 h. There was a discrepancy between protein and mRNA expression levels of HIF-1α against hypoxia in the UL cells.

We detected an immediate induction of HIF-1α protein, leading to decreased expression of HIF-1α mRNA in UL cells against hypoxia. HIF-1α protein expression is mainly regulated by protein degradation in the ubiquitin-proteasome system, suggesting that hypoxia induces HIF-1α by inhibiting the degradation of HIF-1 in proteasomes. The accumulation of HIF-1α protein may negatively regulate mRNA expression of HIF-1α at the transcriptional level in the primary cultured UL cells. Inflammatory pathways leading to disordered collagen deposition and changes in extracellular matrix play a role in the pathogenesis of both uterine fibroids and aortic aneurysms. However, there seems to be an opposing response to inflammatory markers in that collagen causes bulk and stiffness in fibroids, but weakness in vasculature. In addition, fibroids undergo smooth muscle cell proliferation while there is loss of smooth muscle cells at the site of aneurysms. If there is a systemic mechanism behind the different response, we would expect that women who develop fibroids would be at lower risk for aneurysm. Using a population-based design, we looked at the association of fibroids and aortic or peripheral aneurysm. METHODS: Using the Rochester Epidemiology Project recordslinkage system, we identified 6,759 reproductive aged women with a clinical diagnosis of fibroids between 1981 and 2010 in Olmsted County, Minnesota. We matched these women to referent women of similar age who did not have a diagnosis of fibroids. Index date was the date of fibroid diagnosis for each matched pair. A subset of 3,728 matched pairs was identified with women that did not undergo hysterectomy before index or for at least one year after index to reduce the effect that hysterectomy would have on outcomes. The risk of de novo aortic or peripheral aneurysms, defined by diagnostic codes, was estimated using Cox proportional hazards models which excluded women with aneurysms diagnosed before index. RESULTS: Over a median follow-up of 11.7 years, 140 women with fibroids and 161 referent women were diagnosed with de novo aortic or peripheral aneurysm (HR 0.80, 95% CI 0.64, 1.00). In the subset of women that did not undergo hysterectomy, the relative risk of aneurysm was significantly lower among women with fibroids compared to referent women (HR 0.73, 95% CI 0.53, 0.99).

CONCLUSIONS: Aneurysm appears to develop less frequently in women diagnosed with fibroids. Hysterectomy within one year does not drastically change that effect. Studying the mechanism behind these opposing responses to inflammatory pathways may contribute to treatment modalities. (SGI 2013) . Whether this is an effect of all fibroids or just fibroids severe enough to lead to hysterectomy is unknown. To investigate this question, we studied women with a diagnosis of fibroids who did not undergo hysterectomy compared with age-matched controls. METHODS: Using the Rochester Epidemiology Project records-linkage system, we identified 3,728 women diagnosed with fibroids between 1981 and 2010 in Olmsted County, Minnesota who did not undergo hysterectomy for at least one year after their fibroid diagnosis. These women with fibroids were matched to control women by age (±1 year) with no diagnosis of fibroids or hysterectomy during the same interval. The date of fibroid diagnosis was the index date. Using a case-control design, we estimated odds radios and 95% confidence intervals for preexisting CV risk factors before index date using conditional logistic regression models. We also assessed de novo CV disease after index using a Cox proportional hazards model with age as the time scale. Women with CV disease before index were excluded from the analysis of de novo disease. RESULTS: Fibroids were significantly associated with hyperlipidemia, obesity, and polycystic ovarian syndrome. Hypertension, metabolic syndrome, and diabetes did not significantly differ between women with and without fibroids. Over a median of 10.5 years, the risk of developing de novo stroke, myocardial infarction, or atherosclerosis did not significantly differ between women with fibroids and age-matched women without fibroids. availability and function of stem cells. We aim to identify the myometrial stem cell population in the Eker rat's uterus using surface markers and explore the role of hypoxia in their proliferation. METHODS: Uterine tissues from Eker rat were examined via double IHC to co-localize the putative myometrial stem cell markers Stro1/CD44 with established markers of undifferentiation. Then, the Eker rat uterus were digested to obtain single cell suspensions and compute the % of Stro1/ CD44 stem cells by flow cytometry. To determine the in vivo O 2 tension in this tissue, O 2 micronsensors were used and changes in hypoxia were also examined by injection of pimonidazole dye followed by IHC for the hypoxic markers CAIX and HIF-α.

In the Eker rat model, 85% of uterine fibroids occur in the cervix. We demonstrated the co-localization of Stro-1/CD44 with other stem cell markers:Oct-4, c-kit, nanog, validating their undifferentiated status and their distribution in Eker uterus. The IHC shows a significantly higher number of stem cells in the cervix compared to uterine horns, corroborating these results by flow cytometry (P<0.05). The quantification of in vivo oxygenation as well as pimonidazole, CAIX and HIF-α staining also demonstrated that the pO2 levels in cervix were significantly lower than in uterine horns(p£0.0014) confirming its hypoxic status. CONCLUSIONS: For the first time, we identified the myometrial stem cell niche by stem cell markers and we have established an association of hypoxic niches in uterine cervix which is the primary location of fibroids in Eker rat model.These observations could provide a putative mechanism by which myometrial stem cells regulate transformations into tumor initiating cells. We performed genome wide microarray (Affymetrix HGU133plus2) and RNA-seq (Illumina Hiseq) analysis and determined the MED12 mutation status for each sample RESULTS: Unsupervised clustering revealed a clear separation between myometrium and fibroid samples. MED12 mutated samples showed greatest divergence from the normal samples. This was reflected also in expression of individual genes, such as RAD51B and DCX. GnRH agonist treatment induced a sub-clustering within both the myometrium and fibroids but did not restore normal myometrial expression pattern. Interestingly, MED12 wild type fibroids displayed stronger changes in expression after GnRH treatment than MED12 mutated fibroids. In particular, likely target genes of polycomb transcription factor SUZ12 remained strongly up-regulated in MED12 mutated fibroids even after GnRH treatment. CONCLUSIONS: GnRH agonist treatment reveals distinct differences in the expression profiles of fibroid and myometrial tissue, albeit treatment did not induce a normal myometrial expression pattern. Specifically, MED12 status seemed to play a role in the strength of gene expressional changes induced by GnRH agonist treated fibroid tissues. Further analysis of these changes might lead to better understanding of both fibroid aetiology and individual differences in response to GnRH agonist treatment. Eventhough the safety profile of replication-incompetent adenoviruses is outstanding, there is still a need for minimizing any potential spread delivery beyond the fibroid lesion to avoid any possible adverse reactions.

The association of viral vector-based gene delivery with nanotechnology offers the possibility to develop more efficient as well as targeted gene therapy strategies. Magnetic nanoparticles (MNPs) complexed to Adenoviral vectors, in the presence of an external magnetic field, has been shown to greatly enhance targeted gene transfer into tumor cells ,such as intra-tumoral delivery of three feline cytokine genes in fibrosarcoma. These magnetic nanoparticles accelerate the transduction kinetics, a technique referred to as magnetofection. This approach has not been evaluated yet against human uterine fibroid tumor cells.

Magnetic nanoparticles formulations were complexed to a replication defective Adenovirus 5 with GFP as a reporter gene. The newly formed complexes were used to transduce immortalized human fibroid cells (DDcells) in vitro RESULTS: We have observed a 27% and 31% increase in transduction efficiency of DD cells at 2 different multiplicity of infection (MOI); MOI 1 and MOI 10 respectively, with magnetic nanoparticles compared to adenovirus-alone standard transduction strategy (P <0.01 at MOI 1 and P<0.006 at MOI 10). CONCLUSIONS: Our results demonstrate that combining magnetofection and Adenoviruses represents a promising strategy for highly targeted and efficient fibroid gene therapy. This combined approach will allow reduction of the total dose of injected viral particles to ablate a particular fibroid tumor size and thus improve safety without compromising efficiency.. Nanoparticle-enhanced adenovirus-based gene therapy might be a viable localized non-surgical treatment option for women with symptomatic uterine fibroids. A previous study reported that curcumin repressed proliferation in several tumor cells. However, curcumin's therapeutic potential is greatly limited by its low oral bioavailability. The aim of this study was to investigate the effect of native curcumin and nanocurcumin against a human uterine leiomyoma xenografted model in vivo.

The estrogen and progesterone pellets were implanted to immunodeficient NOD/SCID/gc-null (NOG) mice 2 days before the tissue transplantation. Human leiomyoma tumor tissues were collected from Japanese female patients at the time of hysterectomy and transplanted to these mice. The mice were oral administered with vehicle (corn oil), curcumin (30, 120 mg/kg/day) or nanocurcumin (30, 120 mg/kg/day) for 8 weeks after grafting. Then, tumors were evaluated for tissue morphology, collagen content and apoptosis. RESULTS: Tissue size increased by 1.23 times in the control group, but did not change in the curcumin and nanocurcumin group. Native curcumin and nanocurcumin group showed degeneration of the xenografted tissues. Reduction in the expression of muscle fiber was confirmed in the groups treated with curcumin (120 mg) and nanocurcumin (30 and 120 mg). Ki-67 expressions in the tissues were decreased in the curcumin group (120 mg) and the nanocurcumin groups (30 and 120 mg) compared with that in the vehicle group. In addition, TUNEL staining showed that apoptosis occurred following curcumin or nanocurcumin treatment for 8 weeks.

ERα, PR and aSMA expression levels were decreased by curcumin treatment at 120 mg, while 30 mg nanocurcumin treatment showed decrease in all protein levels. According to the results, the regressive effects of nanocurcumin on human uterine leiomyoma xenograft were much more intense than those of native curcumin. CONCLUSIONS: This study showed that both curcumin and nanocurcumin are potent inhibitors on uterine leiomyoma. The inhibitory effect of nanocurcumin was much greater than that of native curcumin.

Redox Dysregulation Promotes Activation of the Pro-Survival AKT Pathway in Uterine Leiomyomas. Overactive bladder syndrome (OBS) is a common health problem, mainly affect women in their reproductive and postmenopausal years. We have shown that vitamin D elicits a robust anti-inflammatory response in human myometrial cells (Thota et. al., 2013) . We hypothesize that vitamin D deficiency in a risk factor for OBS, which induces a heightened inflammatory microenvironment in the urinary bladder wall leading to hyper-excitability and premature contractions of the detrusor muscle. In the current study, we aimed to assess the effect of vitamin D on the expression profile of inflammation-mediating cytokines and chemokines in human detrusor smooth muscle (DSM) cells. METHODS: Primary human DSM cells established from normal female urinary bladder wall specimen were cultured in smooth muscle basal medium supplemented with 5% FBS, human epidermal growth factor (5 ng/ml), recombinant human fibroblast growth factor (5 mcg/ml), insulin (5 mcg/ml), 0.1% gentamicin sulfate, and amphotericin B. DSM cells were treated in triplicate with 100 nmol/L of 1, 25 dihydroxy vitamin D 3 dissolved in ethanol in the presence of 5% charcoal-treated FBS for 24 hours. Total RNA was extracted from the control and vitamin D (100 nmol/ L)-treated cells. The Complementary DNA (cDNA) prepared was subjected to superarray quantitative polymerase chain reaction (qPCR) using Acute Inflammation Response H96 array panels (Biorad). The qPCR data and cycle threshold (Ct) values were analyzed using software obtained from Biorad. A 2-fold change in gene expression compared to control is considered significant. RESULTS: In vitamin D treated cells versus control, superarray analysis revealed significant (P<0.05) expression downregulation of the chemokine (C-C motif) ligand (CCL)-2, the proinflammatory cytokine IL-13, alpha-2-macroglobulin (A2M), apolipoprotein A-I, coagulation factor II (thrombin) receptor-like 1, hemoglobin alpha-2, interleukin 6 signal transducer and elastase 'neutrophil expressed'. Interestingly, vitamin D treatment caused significant (P<0.05) upregulation of the anti-inflammatory cytokine IL-10 in treated versus untreated control detrusor muscle cells. CONCLUSIONS: Our results suggest that vitamin D exerts robust anti-inflammatory effect in female detrusor muscle cells. Further in vivo evaluation of vitamin D and its analogues in animal models of overactive bladder syndrome is warranted. Since patient race may significantly affect allele frequencies, only data from non-hispanic Caucasian women were included for genetic analysis. IL-4 (rs22432250) genotypes were determined by real-time PCR-based Taqman Allelic Discrimination (ABI). Data were analyzed by X 2 analysis. RESULTS: White controls had the expected risk allele frequency of 14-15%. The frequency was slightly less in primary PLV (11%), but significantly greater in secondary disease (25%); odds ratio 1.72 [1.2-2.5] (p-value=0.005). The prevalence of interstitial cystitis was increased in both primary and secondary PLV (32%, 27%, respectively) compared with controls (3%) (p<0.0001). Atopy was slightly more common in secondary PLV (39%) versus primary (27%) and controls (27%) (p<0.01).

The IL-4 promoter variant associated with interstitial cystitis and atopy may also play a role in secondary PLV. Future studies will explore the relationship between this mutation and CD4 T-cell Th2 subtype polarization in fresh vestibular biopsies from women with secondary PLV compared with primary disease and controls. were at increased risk for earlier FSI ( < 16 years). Adjusted odds ratios showed that total and externalizing behavior problems from age 5 years onward significantly increased the risk of earlier FSI for males. In females, externalizing problems from age 10 years increased the risk for earlier FSI. Internalizing problems at ages 8 and 10 were significantly associated with early FSI for boys but not girls. CONCLUSIONS: Externalizing behavior from as early as five in boys and 10 in girls is a significant risk factor for earlier age at FSI. Adolescent sexual health promotion should consider early intervention in children with behavior problems, particularly boys.

Unexpected 

and stromal fibroblasts (eSF). The objective of the current study was to evaluate transcriptomic effects of a non-toxic 10% UPG on co-cultured eEC and eSF to identify alternative mechanisms that may contribute to HIV infection risk. METHODS: An eEC/eSF co-culture system used previously to measure production of cytokines after treatment with either N9 or UPG was utilized here. eEC in co-culture (with monoculture controls) with eSF were treated with 10% UPG for 12.5h. RNA was collected for microarray analysis. For co-culture studies, only eEC were in direct contact with UPG; whereas eSF was affected by eEC paracrine effects. For monoculture, both cells were directly exposed to UPG. Data were analyzed by Genespring, and Ingenuity Pathways Analysis was used to analyze biofunctional pathways affected by differentially expressed genes (p<0.05, 1.5 fold change).

In monoculture, treatment with UPG resulted in activation of pathways associated with inflammation, immune cell recruitment, and vasculogenesis (positive Z score > 1.5) for both cell types. Interestingly, for co-cultured eEC and eSF, exposure to UPG resulted in down-regulation (negative Z score < -1.5) of genes associated with cellular differentiation, and increase in genes associated with tumor proliferation (positive Z score >1.5). The majority of genes associated with these changes centered on cytokines, angiogenic factors, and oncogenes. CONCLUSIONS: Exposure to UPG in monocultured cells stimulated a pro-inflammatory effect similar to reported effects of N9. However, in co-culture exposure to UPG actually promoted expression of tumorigenic genes. Together, these results suggest differential mechanisms by which UPG can affect the endometrial environment and this should be considered when testing for microbicide safety. Supported by AI083050 (LCG, WCG); F32HD074423 (JCC). . Of the patients with positive margins, 45.9 % of these patients were considered to be immuno-compromised due to factors such as chronic steroid use and smoking, chemotherapy or HIV positivity. The recurrence of abnormal pap smears within 2 years of an excisional procedure was 18.91% in the margin negative group and 28.54% in the margin positive group (P <0.05). The positive predictive value of cervical biopsy for determining CIN II on an excisional procedure was 85.2%. Of women with pregnancy outcome data, we found 19 patients with previous CIN II who had an excisional procedure and later had a pregnancy. Of these 19 patients, 7 patients had a preterm delivery; however 4 of these patients had a history of prior preterm delivery. CONCLUSIONS: Positive surgical margins on LEEP/cone biopsy is not uncommon and increases the risk for abnormal cervical cytology in the subsequent years post-procedure. Cervical biopsies when performed by trained physicians correlate well with final pathology on LEEP or cone. Risk factor assessment continues to be important in following patients. Proteomic profiles were compared between normal flora and BV, and also between those 24 (63.2%) subjects who successfully restored lactobacillar flora and 11 (28.9%) who remained with BV after metronidazole treatment.

Peptides spectra were searched against Uniprot database using Mascot. Gene ontology analysis and statistics were performed with Scaffold Q+ (version 4.3.4) RESULTS: From the total of 118 proteins identified and quantified, 20 (24.7%) were differently expressed in BV. Among those, 9 (45.0%) were involved in immune response to pathogens. Cathepsin G, Ig lambda-2 chain, neutrophil elastase, haptoglobin, protein S100-A8 and Ig heavy chain V-III were significantly overexpressed in BV, while cluster of Serpin B3, leukocyte elastase inhibitor and protein S100-A11 were more abundant in normal flora. No difference in the proteomic profile was observed between women with persistent BV and those who were successfully treated CONCLUSIONS: BV changes significantly the local proteomic profile, especially regarding immunity-related proteins. However, cervicovaginal proteome does not influence the response to treatment of BV. -125b, -1301, -130b, -148b, -16, -19a, -409, and -501) . IPA miR-target identification of these miRs revealed 289 experimentally validated miR-target relations. Pathway analysis showed reproductive system disease in the "top diseases and biofunctions" node (p-values = 1.56E-15 -6.29E-09). "Metabolic disease" was identified in "top networks". CONCLUSIONS: This study identified differentially expressed microRNAs in PCOS theca cells. Preliminary in silico pathway analysis shows the potential for functional relevance of these miRs (n=10) to PCOS and associated morbidities and will further be used for functional analyses.

to result in hyperandrogenism. We aim to determine whether alterations in the activity of these enzymes can be demonstrated by measuring the levels of cortisol and androgen metabolites in random urine samples of patients with PCOS. METHODS: Twenty-two patients who met the Rotterdam criteria for PCOS and 12 age-matched controls were enrolled in an IRB-approved case-control study. BMI, waist-to-hip circumference, and ethnicity of each patient were also documented. We collected a random urine sample from each patient and levels of urinary androgen and cortisol metabolites were measured using gas chromatography/mass spectrometry. RESULTS: Higher levels of urinary tetrahydrocortisone (THE), the metabolite of cortisone, were noted in patients with PCOS (p=0.03). There was also a statistically significant increase in the levels of urinary androsterone, the major metabolite of dihydrotestosterone, in patients with PCOS (p=0.04). CONCLUSIONS: There is a statistically significant increase in levels of urinary metabolites of cortisone and dihydrotestosterone, indicating the role of both 11 β-HSD-1 andDHT in the pathogenesis of PCOS. This change in enzymatic activity correlates with the hyperandrogenism observed in patients with PCOS and offers an alternative therapeutic target in the treatment of this complex disorder. Change in motility was assessed as a function of BMI. RESULTS: There is a positive correlation between increasing BMI and a beneficial effect of opiorphin; the benefit of opiorphin supplements on sperm motility and forward progression was greatest in the most obese men. When comparing the change in motility after addition of opiorphin, the obese men's sperm had a 1.12-fold increase in motility over the non-obese men. The obese men's forward progression had a 1.27-fold increase which was statistically significant. More than twice as many obese men with asthenozoospermia achieved normal motility (as defined by WHO) after addition of opiorphin compared to non-obese men with asthenozoospermia. CONCLUSIONS: These exciting preliminary data may offer treatment alternatives to obese men who suffer from infertility due to asthenozoospermia. The process of freezing / thawing causes serious structural and functional modifications in sperm samples. The presence of proteins in the seminal plasma is important for correction of these damages. Several tests have been proposed to predict the post-thaw sperm recovery, however the current methods are expensive and timeconsuming. The aim of this study was to assess the value of semen protein measurements through dipstick before freezing and its relation with postthaw recovery of live spermatozoa.

This was a prospective study including 149 men undergoing semen analysis due to male and/or female infertility. The seminal samples were analyzed according to the World Health Organization standards and protein concentrations were measured using a commercially available urinary dipstick. Samples were cryopreserved with Test Yolk Buffer-Gentamicine freezing medium under liquid nitrogen for an average of 120 days. The frozen aliquots were thawed at 37º for 10 minutes and analyzed using the same methods and protocols used pre-freezing.

The median live sperm recovery rates were 42%, 50% and 74%, respectively, in samples with protein concentrations of 0.15-1 g/L (-/+), 3 g/L (++) and ≥ 20 g/L (+++) in the fresh specimen dipstick analysis (P<0.05, Kruskal-Wallis ANOVA and Dunn's test). Accordingly, the probability of recovering at least 50% of the frozen spermatozoa increased progressively with higher protein concentrations in the fresh sample (chi-square for linear association = 7.17, P=0.007). CONCLUSIONS: In conclusion, seminal protein levels assessed with dipstick test correlate with the proportion of live spermatozoa recovered from cryopreserved samples. This simple, low cost test may add prognostic information to baseline semen analysis before banking. (n=7), esophageal stenosis (n=3). Their AMH levels tended to be lower (2.5, 0-6) than in those with milder disease (8.9, 1.6-4.2, p=0.07). AMH levels in males with DBA (3.5, 2.3-11.9), relatives (4.7, 0.1-13.5) and healthy controls were similar. CONCLUSIONS: Our findings suggest a defect in the production of AMH in post-pubertal males with FA and DC but not DBA, similar to the results in females. Larger, longitudinal studies are necessary to confirm these novel findings and evaluate the impact of reduced AMH levels in this population.

Vasomotor Symptoms, Sexual Function, and Quality of Life After Medicine issued an opinion stating that oocyte cryopreservation was no longer considered experimental. Frozen donor oocyte cycles offer several potential advantages, including lower associated cost, increased convenience, and decreased rates of cycle cancellation.

Studies have reported pregnancy rates using frozen donor oocytes comparable to non-cryopreserved oocytes. There are two methods to cryopreserve mammalian oocytes: slow-rate freezing and vitrification.

Questions still remain about the compared feasibility of these processes.

The purpose of this study is to compare our experience with frozen vitrified donor oocytes versus fresh donor oocytes.

The study was a retrospective review comparing vitrified donor oocytes and fresh donor oocytes at our institution from January of 2011 to May of 2013. Cases were included in which donors had provided oocytes for both fresh and frozen (vitrified prior to fertilization) cycles (N=32 donors). The study involved chart review and data extraction of donor egg cycles. A chi-squared test was performed to analyze the data.

Oocytes from screened egg donors

Oocytes fertilized for other reasons (preservation) RESULTS: The fertilization rates of fresh and vitrified oocytes were similar. The blastocyst formation rate (number of blastocysts per fertilized egg) was significantly higher in fresh compared to the vitrified oocytes. However, there was no statistical difference in the pregnancy rates (pregnancy rate per transfer) among the two groups. 

These results indicate that despite a lower blastulation rate, the pregnancy rate was not significantly different among fresh versus vitrified oocytes. Although pregnancy rates were not statistically different between the two groups, there was a trend towards decreased pregnancy rates when vitrified donor oocytes were used. This difference may be explained by the tendency to have a greater number of oocytes in a fresh donor oocyte cycles for utilization rather than an set number allocated in vitrified donor oocyte cycles. Published studies comparing COS initiated in the luteal phase to COS initiated in the follicular phase were included. With IRB approval, primary data from our center was also included. Case reports were excluded. Our primary outcome was number of oocytes retrieved. Secondary outcomes included duration of stimulation and oocyte fertilization rates. Meta-analysis data was synthesized with a DerSimonion-Laird random effects model. Results are reported as weighted mean difference (WMD) and 95% confidence interval. RESULTS: Four studies with a total of 238 women were included along with our center's primary data (Boots, n=43; Cakmak, n=125; Maman, n=18; Suikkari, n= 12; vonWolff, n=40) . No differences were noted in the number of oocytes retrieved from women undergoing COS initiated in the luteal phase versus the follicular phase (WMD: 0.02, 95% CI: -1.8-1.8) nor were any differences noted in the duration of COS (WMD: 0.06, 95% CI:-2.5-2.6) between the two groups. Oocyte fertilization rates were significantly higher in women initiating COS in the luteal phase (WMD: 0.16, 95% CI: 0.13-0.19). CONCLUSIONS: Initiating COS in the luteal phase does not compromise the number or quality of oocytes retrieved (based on fertilization rates), and duration of stimulation is no different between COS initiated in the luteal phase or the follicular phase. COS should be offered as soon as possible in women who need expedited fertility preservation. However, the mechanism whereby administration of α-GalCer leads to PTB and whether it causes an expansion of NKT cells in pregnant mice is unknown. A breakdown of maternal-fetal tolerance is considered a mechanism of disease in PTB. Maternal-fetal tolerance is maintained by a balance between anti-inflammatory and pro-inflammatory T cells. The aims of this study were to determine whether administration of α-GalCer (1) causes an expansion of NKT cells in pregnant mice and (2) alters the maternal T-cell repertoire. METHODS: Pregnant B6 mice were injected with 2 µg of α-GalCer (n=8) or DMSO (n=6) as a control at 16.5 days post-coitum. After 6 hours, mice were euthanized and the spleen, uterine-draining lymph nodes, and uterine and decidual tissues were harvested. Leukocytes were isolated for immunophenotyping. Within the NKT-cell gate (CD1dtetramer+DX5+) These include cells from the fetal skin, respiratory system, urinary and gastrointestinal tracts, along with populations of stem cells (AFS). AFS cells are characterized by their capacity for self-renewal and their ability to differentiate to toward lineages representative of all three germ layers including Lin-cells with a hematopoietic potential. Amniotic fluid may have also an immuno-priviledged status. We sought to characterize B cells population and their potential activation in different mice clones.

In the course of our experiments we performed allogeneic matings of C57BL/6, BALB/c and CBA females. The animals were sacrificed at day 14 of pregnancy and the AF was harvested by amniocentesis. CD19 + cells were isolated using MACS and the phenotype analysis was performed by flow cytometry.

In the first instance these experiments offered only low numbers of cells in the AF on the whole. But we were able to reveal the presence of CD19 + B-cells in the different mouse strains and furthermore, to define 16 to 50 % as IgM + , 4 to 20 % as IgD + and 3 to 20 % as both, IgM-and IgD-positive. CONCLUSIONS: Our data confirms that, despite an overall low number of total cells, the AF very well contains CD19 + B-cells. Moreover we hypothesize to even have found them in slightly different stages of development, referring to the double positive, as well as single expression of IgM and IgD surface markers. were euthanized after delivery and uterine-draining lymph nodes were processed for immunophenotyping. Additionally, neonates (1 week old) from F1SS (F2, n=21), F2SSS (F3, n=9), and control (n=19) mothers were euthanized, and their thymuses and spleens were processed for immunophenotyping. T-helper cells (Th1, Th2, Th9, and Th17), regulatory

IFNγ (CTLs) and IL17A (Tc17) were determined as proportions by flow cytometry. A p-value of £0.05 was considered significant. RESULTS: 1) F1SS and F2SSS mothers had fewer IL17+Tregs, Th1 and Th17 cells than control mothers; 2) F2SSS mothers had more Tregs and Th2 cells but fewer Tc17 cells than control mothers; 3) when comparing F1SS and F2SSS mothers, F2SSS mothers had more Tregs and Th2 cells but fewer Tc17 cells; 4) F3 neonates had fewer thymic Tregs and IL17+Tregs but more Tsups than control neonates; 5) no differences were seen between F2 and control neonates; 6) F3 neonates had fewer thymic IL17+Tregs than F2 neonates; 7) F2 neonates had more splenic CTLs, Tsups and Th1 cells than control neonates; 8) F3 neonates had more splenic Th2 cells but fewer Th17 cells than control neonates; and 9) when comparing F2 and F3 neonates, F3 neonates had more splenic Th2 cells but fewer Th17 cells. CONCLUSIONS: In mothers, prenatal stress reduces highly suppressive IL17+Tregs and effector T cells in the second generation, but these are partially restored in the third generation. Neonates born to third-generation stressed mothers have reduced IL17+Tregs but increased Tsups. These findings suggest that prenatal stress has adverse effects over generations in the T-cell repertoire of mothers and neonates.

The Pregnancy is an exceptional immunological phenomenon because tolerance is created towards the semi-allogeneic cells of the fetus. We do know that dysfunction of tolerance is associated with pregnancy disorders and that the maternal immune system memorizes fetal antigens thought to prevent disorders in subsequent pregnancies. This study aims to analyze the effects of pregnancy on memory T lymphocytes in order to reveal possible mechanisms responsible for tolerance towards the fetus. METHODS: Using flow cytometry, CD4+ and CD8+ memory T lymphocytes were analyzed in the peripheral blood of 14 healthy nulligravid and 9 healthy primigravid women. Effector memory (EM; CD45RO+CCR7-), central memory (CM; CD45RO+CCR7+), naïve (CD45RO-CCR7+) and effector (CD45RO-CCR7-) T lymphocytes were distinguished. Monoclonal antibody CD69 was used to detect their activation status. Comparison was done using Mann-Whitney U-test; p<0.05. RESULTS: In primigravidae, proportions of CD4+ EM cells were significantly higher (p=0.022) and proportions of CD4+ CM cells were significantly lower compared to nulligravidae (p=0.027). Significantly higher percentages of CD4+ CM cells were activated in primigravidae (p=0.003). Whereas CD4+ naïve cell proportions were lower (p=0.064), CD4+ effector cells were higher in primigravid women in comparison to nulligravid women (p=0.014). Percentages of CD8+ memory cell (sub) populations did not differ between the groups. CONCLUSIONS: In conclusion, pregnancy affects maternal CD4+ memory cells in peripheral blood. Presumably, conversion of CD4+ CM to EM cells occurs due to fetal antigen exposure. Also CD4+ naïve cells seem to proliferate during pregnancy. Most likely to memory and effector cells. Altogether, this study implies that memory cells play a role in maternal immune tolerance. TLR (1, 2, 3, 4, 5, 7/8, 9) ligands throughout pregnancy and postpartum. METHODS: A prospective cohort of 33 women with uncomplicated pregnancies had peripheral blood collected at 1)10-12wks, 2)at 26-28 wks, 3)at delivery, and 4)6wks postpartum. Peripheral blood mononuclear cells were isolated and stimulated with TLR ligands (see Table 1 ) for 18h, then preserved. Stained with fluorescent antibodies to identify DCs, and intracellular IL-6,IFNa, IL-12 and TNFa. 10-channel flow cytometry on a 3-laser LSR II instrument allowed identification of DCs and % expressing each cytokine. Repeated measures-adjusted ANOVA was used to evaluate the relationship between DC cytokine production at different timepoints. RESULTS: From 40 total comparisons (9 TLR ligands and control, by 4 measured cytokines), 11 demonstrated significant (p<0.05) differences across pregnancy and postpartum and are summarized in Table 1 . Broadly, TNFa and IL6 stimulated production tended to decrease across pregnancy while IL-12 and IFNa increased. Concerning B1 B cells, we observed a decrease in(CD20 + CD27 + CD43 + ) CD11bnumbers from the first to the third trimester of pregnancy. Unlike CD11b -, CD11b + B1 B cells were augmented in pregnant women.

We have shown that in response to pregnancy a significant suppression of auto-reactive B2 lymphopoiesis pathway occurs. In addition, IL10 producing B1 B cells are augmented during pregnancy. This well controlled mechanism may contribute to the process of pregnancy tolerance.

Stretch-Induced Changes in the Human Amnion. Uterine stretch plays an important physiological role in parturition. However, excessive uterine distension has been associated with preterm labour (PTL). Mechanical stretch has been shown to affect the fetal membranes, including the amnion though this has not been extensively investigation. Understanding the mechanisms behind these stretch-induced changes will allow the use of potential therapeutic targets to prevent PTL. METHODS: Human amnion epithelial cells, obtained from consenting women undergoing term elective caesarean section, were subjected to 11% mechanical stretch. In order to confirm stretch efficiency, COX-2 protein levels were investigated by western analysis. In addition, total RNA was extracted from these cells using an RNeasy kit (Qiagen, UK) and converted to cDNA. QPCR was carried out using a Rotor-Gene TM (Qiagen, UK) to investigate COX-2 and cytokine mRNA expression. An ELISA multiplex assay was used to detect levels of eotaxin-1, IL-8 and MCP-1 from cultured supernatant of amnion cells after 6 and 12 hours of 11% mechanical stretch. RESULTS: Following 11% mechanical stretch, COX-2 protein levels showed a distinct increase particularly after 6 and 12 hours. COX-2, IL-6, TNF-α, IL-1β and TGF-β mRNA levels all appeared to increase following 12 and 24 hours stretch (n=12). Observed concentrations in the supernatant showed statistically significant differences. Both MCP-1 and eotaxin-1 increased, 1.5 and 3.5 fold respectively, after 12 hours; MCP-1 and IL-8 demonstrated an increase, 2 and 0.5 fold respectively, after 24 hours (n=8). Results are given as fold increases due to stretch above control cells (** p<0.01).

The results obtained here closely mimic data from an in vivo macaque monkey model of stretch especially with respect to chemokine production. In addition, these results suggest that it is feasible to stretch human amnion epithelial cells after in vitro culture and that this would be a useful model to study the role of stretch in amnion during pregnancy and at the time of parturition. For our study, the exposure of interest was race and the primary outcome was the rate of cervical shortening (mm/week), which was obtained by dividing the difference in mm between the first and second cervical length measurements by the time difference. African American women were compared to other races, which included Caucasian and Hispanic. A subgroup analysis was also performed to determine if racial disparities in rate of cervical shortening exist among women who delivered preterm. Multiple regression was used to control for possible confounders. RESULTS: 2998 women with singleton pregnancies were included. 1874 were African American, 1124 were other races. African American women had a shorter mean CL, were more likely to have a CL < 25 mm, and to have a PTB, however they had similar rates of cervical shortening between the two exams compared to other races. *Figure(s) will be available online. CONCLUSIONS: African American women were more likely to have a CL < 25 mm at 22-24 weeks and 26-28 weeks and deliver preterm but they did not have a faster rate of cervical shortening compared to other races. Premature remodeling of the cervix may be a contributing factor to the complex mechanism that initiates preterm labor. Based upon evidence in rodents, the present study tested the hypothesis that increased presence of macrophages is associated with remodeling of the cervix in preterm and term birth. METHODS: Cervical biopsies were obtained from 26 women (~50 mm 2 , n=7-9/group) after vaginal delivery with preterm labor (PTL) and term labor (TL), prepartum at term not in labor, (TNIL), as well as from 3 nonpregnant women (NP). The study was approved by local Ethics Committee and the subjects gave their informed consent. Tissues were fixed, paraffin-embedded, sectioned, and processed to identify cell nuclei (CN) with methyl green counterstain and CD68 macrophages (Mϕ) by immunohistochemistry. CN and Mϕ were counted in photomicrographs over defined subepithelium and center subregions. Mϕ numbers were normalized to CN/volume to account for individual variations and tissue hypertrophy.

The morphology of cervix biopsies varied between individuals and within tissue subregions. In cervical biopsies, extravascular red blood cells were present in most tissue in PTL and TL groups, but absent in TNIL and NP women. Although cell numbers/volume varied between and within subregions, CN density was reduced in the cervix from pregnant women versus that in NP women. However, CN density was not different among TNIL, TL, and PTL groups. There were big individual differences in distribution of Mjs in cervical epithelium with no obvious differences between the groups. In NP women, Mjs were concentrated in subepithelium, especially near blood vessels, and sparsely distributed in the center region. By contrast, in the cervix from pregnant women, Mjs were more abundant throughout the biopsy tissue and present within blood vessels. Significantly increased numbers of Mϕ were found in women in PTL and TL groups vs that in NP women. CONCLUSIONS: Increased presence of macrophages is associated with remodeling of the cervix in preterm and term birth. Diverse patient histories, variations in morphology, and small biopsy area may limit accurate resolution of cellular characteristics that define processes that remodels the cervix with preterm birth and at term. We performed T2 Cube 3D FSE MRI scans of the pelvic region of each patient and generated anatomically correct FE models of the lower uterine segment and cervix. We modeled cervical tissue as a soft ground substance with embedded collagen fibers which sustain only tensile forces. We assigned mechanical properties to the cervix according to our group's

prior work. Fetal membranes were included in the models. For loading, we incremented the IUP over the physiological range (0 to 100 mm Hg).

Reducing the collagen stiffness parameter in our models produced a marked increase in the local tissue stretch levels and thus cervical shortening. Disabling adhesion between the fetal membranes and the decidua increased the maximum stretch levels in the cervix for both models by 50% or more, further decreasing cervical length. * Figure( s) will be available online. CONCLUSIONS: Cervical shortening is a complex function of multiple factors: geometry, cervical properties, and loading. Here we used FE techniques to show collagen properties are a critical factor influencing cervical shortening. Adhesion between the fetal membranes and cervix also affected cervical length, suggesting that detachment could play a significant role in cervical shortening. To analyze the specific contribution of choriodecidual leukocytes to MMP-9 secretion and activation in the intrauterine microenvironment, we examined intrauterine leukocytes. METHODS: Placentae and amniochorion samples were obtained from women at term gestation. Leukocytes were obtained from intervillous placental blood and from choriodecidua and maintained in culture up to 72 h. Gelatinase activity was determined by zymography and using specific substrates. MMPs in the culture media were measured by MultiAnalyte Profiling. Presence of a potential activator of pro-MMP-9 was monitored using a biotin-labeled pro-MMP-9. To characterize the activator of pro-MMP-9 in the culture media, a set of blocking monoclonal anti human MMPs were used. RESULTS: Leukocytes spontaneously secreted proMMP-9 after 24 h. Secretion increased significantly at 48 h, 3059.32 ± 621 in placental leukocytes and 5,310.2 ± 845 in choriodecidual cells (p<0.05). The active form of MMP-9 was appreciable at this time. Findings were confirmed with a specific MMP-9 activity assay and using the labeled pro-MMP-9. A significant increase in MMP-3 secretion was observed since 48 hours in culture (0.22 ± 0.14 pg/mg protein; p < 0.05) compared with 24 hours (0.0142 ± 0.0054 pg/mg protein) and up to 72 hours (0,600 ± 0.0978 pg/ mg protein; p <0.05). Activity of MMP-9 decreased significantly when adding an anti MMP-3 antibody to the culture media. CONCLUSIONS: Upon homing in the intrauterine environment, leukocytes are capable of producing different mediators involved in the degradation of the connective tissue. We have shown that placental blood and choriodecidual leukocytes from human term pregnancies are able to secrete large amounts of MMP-9 as well as the activator. We also demonstrated that MMP-3 plays a major role in such activation process. INTRODUCTION: Progesterone sustains pregnancy while local withdrawal of progesterone may mediate prepartum remodeling of the cervix. Whether a reduction in cells that directly respond to genomic actions of progesterone or a shift in progesterone receptor (PR) isoforms contribute to degradation of the extracellular matrix in the prepartum cervix is not known. Thus prepartum cervix remodeling, presumed related to loss of trophic effects of progesterone, was hypothesized to be mediated by a decline in the population of stromal cells that express PR.

The cervix from nonpregnant (NP; n=5), pregnant CD-1 (days 15-18 postbreeding), and postpartum mice (n=8-21/group) were fixed and paraffinized. Sections were processed with picrosirius red to assess extracellular collagen content and structure or by immunohistochemistry to identify PR (A+B isoforms, #3153, Cell Signaling Tech.) and counterstained with methyl green to assess cell nuclei/volume (CN), an indicator of tissue hypertrophy that varies with subregion morphology across sections, individuals, and groups (PMID24339918). Cervix sections from peripartum PGRBKO mice lacking the PRB isoform were similarly processed to count PRA cells. RESULTS: Extracellular collagen content and structure declined with pregnancy and before birth. The cervix hypertrophied with pregnancy because CN density declined in pregnant mice and more so before birth (p<0.05; D18<D15<NP). PR-stained cells were abundant in the cervix across all groups. Nearly half of all stromal cells stained for PR and numbers did not vary among groups of pregnant mice or relative to NP controls. Similar numbers of stromal cells stained for PR In sections from the cervix of pre-and postpartum PGRBKO mice. Thus, the prepartum cervix had a similar density of PR-stained stromal cells in CD-1 mice as in PGRBKO mice with only the PRA-isoform. CONCLUSIONS: Abundant PR-stained stromal cells in the cervix from pregnant, postpartum, and NP mice suggests that the number of PR-containing cells cannot account for loss of genomic responses to progesterone that may drive extracellular collagen degradation in the cervix prior to birth. Since transformation of cervix structure and timing of birth are similar in PGRBKO and CD-1 mice, the findings support the alternative possibility that PRA-mediated actions, rather than loss of PRB effects, contribute to the final common mechanism for remodeling in the process of parturition. 

Premature cervical softening is associated with cervical shortening and preterm birth. Yet, the biochemical mechanisms causing cervical softening are poorly understood. Animal models are commonly used to study cervical softening. However, softening in animal pregnancy may not reflect changes in the human cervix. Our objective was to develop a model for studying the effect of steroid hormones on mechanical strength of cervical tissue using a three-dimensional culture method and human cervical fibroblasts. METHODS: Fibroblasts were obtained from cervical biopsies of non-pregnant premenopausal women. Fibroblasts from passage 5 were seeded on cylindrical silk scaffolds using a previously described, three-dimensional (3D) culture protocol. In the present experiment, the cylindrical scaffolds were positioned end-to-end such that newly formed tissue filled the gap between the two scaffolds, essentially bonding the scaffolds together. At the end of the six week experiment, scaffolds were evaluated for H&E histology and mechanical strength. To measure mechanical strength, scaffolds were attached to a testing fixture and pulled apart. The peak force at failure was recorded, which measured the strength of the newly formed tissue filling the gap. Culture media included DMEM + 10% charcoal stripped FBS and three hormone conditions: 1) estradiol (E2) 10-8M alone, 2) estradiol 10-8M + progesterone (P4) 10-6M, and 3) vehicle control. Experiments were repeated in duplicate with cells from two different women. RESULTS: Cervical cells reliably synthesized new tissue that filled the gap between the two scaffolds. The presence of E2 without P4 was associated with increased mechanical strength compared with E2+P4 and vehicle control (p < .01). Histology correlated with mechanical testingimproved tissue synthesis was seen with E2 without P4 compared with E2+P4 and vehicle control. CONCLUSIONS: Exposure of scaffolds to E2 resulted in increased tissue production, which was associated increased tissue strength. P4 opposed this effect. Ongoing experiments are exploring the mechanism of these observed responses. This hormonally-responsive in vitro culture system could be useful for studying biochemical mechanisms of cervical softening during pregnancy. propagation is faster in stiffer tissues. We studied SWS in ex vivo cervices of rhesus monkeys to determine whether SWS estimates are sensitive enough to differentiate between ripened and unripened cervical tissues. METHODS: SWS were estimated in cervix specimens (n=29) from rhesus macaque monkeys, a subset of which were ripened with misoprostol (n=13). The cervix specimens were pinned to a piece of sound absorbing rubber and placed in a saline solution. A Siemens Acuson S2000 and 9L4 transducer, which was attached to a translation stage and aligned parallel to cervical canal, were used to generate and track shear waves in regions of interest within each specimen. SWS measurements were made at predetermined locations from distal to proximal ends of the specimen on both the anterior and posterior sides, with at least 5 replicate measures at each location. Displacements were calculated using the Loupas' routine and SWS were estimated with the RANSAC algorithm. SWS from middle location and mid-proximal location (the location midway between middle and proximal end) were used to compare SWS between ripened and unripened groups.

RESULTS: Preliminary data analysis shows that unripened cervix samples have higher SWS (some are very stiff) compared to ripened samples, especially in posterior side (7.22 ± 1.48 m/s vs. 5.84 ± 1.12 m/s at middle location, and 5.35 ± 4.61 m/s vs. 4.81 ± 2.22 m/s at mid-proximal location). The SWS changes with position in the normal nonpregnant cervix, and there are differences between anterior and posterior sides as found in analogous human studies. Monkey cervix tissue is stiffer than human cervix tissue which is consistent with our observation in nonlinear optical microscopy images that its collagen is more highly organized than seen in humans. CONCLUSIONS: SWS estimates appear promising for assessing cervical softness of rhesus monkeys and monkey studies may provide insight to human cervical studies.

Novel parturition, yet we do not have an understanding of the biology of cervical remodeling, and especially the role of progesterone (P 4 ) in controlling cervical stromal functions. This is due to lack of models of cervical stromal myofibroblasts, mechanically dynamic cells that provide structural support during pregnancy and remodeling during parturition. We have developed a novel, stable line of immortalized cervical fibroblasts that express PR receptors (PR-A and PR-B) in response to 17β-E 2 . METHODS: Cells explanted in culture in DMEM+10% FBS. After ~ 3 weeks, a homogeneous monolayer of fibroblasts was evident that was immunopositive for vimentin (mesenchymal marker) and negative for cytokeratin (epithelial marker) and CD31 (endothelial marker). Cells were subcultured and transduced with a lentiviral vector encoding human telomerase (hTERT) under puromycin selection. Cells were immunolabeled for hTERT and vimentin to verify transduction of stromal myofibroblasts. Clonal cell lines were prepared and immunostained for hTERT and vimentin. Parental and cloned cell lines were primed for 5-7 days with 17β-E 2 (10 -8 M) followed by extraction of total RNA and protein for estrogen receptor-α (ER-α) and PR expression for qRT-PCR and immunoblotting, respectively. RESULTS: In clonal hTERT CX cells, we detected uniform expression of nuclear hTERT, ER-α and PR expression. Interestingly, hTERT-transduced cervical cells exhibited a slower growth rate than wild-type fibroblasts, suggesting that hTERT expression leads to immortalization but not enhanced proliferation in these cells.

Cloning of stromal fibroblasts that express PR-A, PR-B in an estrogen-responsive manner provides a reliable and reproducible in vitro system to test the biochemical and mechanical properties of cervical cell biology in the context of inflammatory changes that lead to premature cervical remodeling. Moreover, the hormone-responsive nature of the cells will offer a novel cell model assess the mechanisms by which P 4 exerts its inhibitory actions on cervical ripening. 

The complex immunomodulatory effects of E 2 on cultured human cervical ectothelium appear to vary with the duration of exposure. The alteration in TLR2 and TLR4 expression levels at 10-minute duration might be correlated to non-transcriptional (non-nuclear) effects of E 2 , which appears within seconds and subsides within about two hours. Although no alteration in the expression of TLR 2&4 were detected at two and 18-hour incubation periods, the possible nuclear effects of E 2 in TLRs expression is yet to be understood. Prolonging the duration of exposure may clarify modulatory effects of E 2 through genomic mechanisms. Interim analysis of EIS data is ongoing. We hypothesize that it will be particularly useful in assessing women with short CL. So far two symptomatic women with CL£15mm have participated -both had positive FFN. Their impedance curves illustrate our anticipated results. Further assessment will determine the optimum predictive electrical frequencies for CI measurement and the most predictive cut-off values of CI. CONCLUSIONS: EIS is a promising tool -our study will rigorously assess its role in PTB prediction. We ultimately aim to design a randomised controlled intervention study based on screening for PTB by EIS. Nitric oxide relaxes myometrium in a dose dependent manner that is independent of the accumulation of cyclic guanosine 5'monophosphate (cGMP). We hypothesize that the direct S-nitrosation of myosin light regulatory polypeptide 9 (MYL9) by S-nitrosoglutathione affects smooth muscle contractile dynamics by altering myosinbased actin sliding velocities/actin-myosin ATPase activity as well as phosphorylation rate and steady state phosphorylation levels of MYL9. MYL9 is differentially regulated/S-nitrosated based upon the state of labor in women; therefore, a functional exploration of S-nitrosated MYL9 might provide insight into the regulation of muscle contraction and the initiation of labor.

(1) An actin motility assay determines the changes in myosin-based actin sliding velocities/actin-myosin ATPase activity in S-nitrosated and native MYL9. Smooth muscle myosin is adhered to a nitrocellulose coated slide and fluorescently-labeled actin filaments are visualized.

(2) An in vitro assay is used to determine whether the rate at which MYLK phosphorylates MYL9 (S19) is affected by prior MYL9 S-nitrosation (C109). The ADP-Glo™ Kinase Assay measures the ADP that is generated (as a result of ATP hydrolysis) when MYLK phosphorylates MYL9 using luciferase luminescence.

In order to investigate the functional relevance of MYL9 S-nitrosation in the myometrium we confirmed that our proteins of interest can be S-nitrosated in vivo using an endogenous nitric oxide donor (GSNO) under physiological conditions. We determined that MYL9 is S-nitrosated in telemorized human uterine smooth muscle cells when 100µM GSNO was applied to FBS-free media. We have also isolated SMM from human uterine smooth muscle for use the in the actin motility assay. Baseline actin velocities have been established and will be used as a reference when comparing the velocity of S-nitrosated MYL9. CONCLUSIONS: While there is a vast body of research dedicated to actin-mediated contraction in smooth muscle cells, the role of S-nitrosation in this process is unknown. This important regulatory mechanism could have a major influence in regards to the regulation of smooth muscle relaxation, as well as the induction of labor. My research focuses on a unique NO-signaling exception and uses advanced methodology to discover MYL9 S-nitrosation associated with human sPTL and the impact of this post-translational modification of smooth muscle mechanics. Human myometrial RNA subtraction studies previously revealed a set of genes that had not been associated with labor and pregnancy but had strong correlations with estrogen receptor alpha (ESR1). Pathway analyses using Ingenuity Pathway Analysis TM indicated a potential link via the RNA-bnding protein, embryonic lethal-abnormal vision or ELAV1.

We performed quantitative real time reverse transcription PCR (QRTPCR) to determine if ELAV1 gene expression changes with labor in the human myometrium. Human myometrial tissues from the upper cut edge of the lower uterine segment were obtained at term Caesarian section before labor (NL) and after labor (L) had commenced. Total RNA was extracted using guanidinium thiocyanate-acid-phenol method, treated with DNase, and reverse transcribed using Superscript (Invitrogen). QRTPCR was performed using primers (Sigma Aldrich) that spanned exon-intron junctions and reference gene 18S ribosomal RNA, with Sybr Green (Sensifast, Bioline) master mix.

Melt curve analyses showed only one product indicating PCR specificity. ELAV1 mRNA increased 5-fold with labor. CONCLUSIONS: Among the many known roles, ELAV1 has been implicated in regulation of stability and translation of genes in myogenesis, and shown to be essential for placental development in transgenic mice knockout studies. Together with

Characterization of TREK1 Currents in Freshly Isolated Myocytes. Chad L Cowles, Michael T Lee, Heather R Burkin, Iain LO Buxton. Pharmacology, University of Nevada School of Medicine, Reno, NV, USA. INTRODUCTION: Premature delivery of an underdeveloped fetus is the leading cause of newborn death worldwide. TREK-1 is a gestationally regulated K + channel which could significantly contribute to uterine quiescence until pregnancy reaches full term. Characterization of TREK-1

channel activity in freshly isolated myocytes from uterine tissue informs our understanding of how this stretch activated ion channel contributes to uterine relaxation. METHODS: Patch clamp electrophysiology was used to characterize TREK-1 currents in myocytes isolated from tissues from mothers who underwent cesarean section at different states of pregnancy (term/ preterm, laboring/non-laboring tissue). TREK-1 channel activity was also investigated in Human Telomerase Reverse Transcriptase (hTRT) pregnant uterine myocytes to understand the efficacy of a model cell system that has been developed in our laboratory. RESULTS: We found that TREK-1 channel activity was disparate in myocytes isolated from women undergoing cesarean section at different states of pregnancy. The gestational regulation of TREK-1 correlated with electrophysiological data based on the finding that TREK-1 channel activity was reduced in laboring versus non-laboring tissues. It was also found that the hTRT model cell system exhibits TREK-1 currents that are similar to freshly isolated myocytes. We are continuing to study how TREK-1 currents are altered in preterm versus term tissues. CONCLUSIONS: TREK-1 currents which cause hyperpolarization of the cell membrane could play a significant role in uterine quiescence to term. Our lab has also developed a model cell line that can be used to study ion channels in pregnant myometrium. In the future TREK-1 could be targeted for the development of tocolytics to prevent preterm labor.

Characterization The objective of this study was to determine the propagation speed from uterine electrophysiological signals and quantify the number of segments which contain contractile activity simultaneously and propagate across regions.

The data was recorded over the maternal abdomen with a 151 channel SARA system from 24 pregnant women between 37 to 40 weeks of GA. The uterine magnetomyographic (MMG) signals were scored for contractile patterns. Then, we split the sensor space into four quadrants (Fig. 1 ) and used the cross-correlation method to calculate the delay between pairwise combinations of quadrants. The propoagation speeds were calculated from the delays. Finally, we quantified the number of segments between pairwise combinations of quadrants wherein contractile activity occurs simultaneously and contains a delay.

The average propagation speed is within the range of 1.3-5.7 cm/s. Fig. 1 shows the propagation of activity between quadrants (Q1 -upper right; Q2 -upper left; Q3 -lower left; Q4 -lower right). *Figure(s) will be available online. The delay and speed on a 37 week GA recording are noted in the figure.

After the multiple pairwise test (99% CI), significant differences in speeds can be observed between certain vertical or horizontal combinations and the crossed pairwise combinations, e.g. between Q1 -Q4 (vertical), Q1 -Q3 (crossed) and Q2 -Q4 (crossed) quadrants. The number of segments that containing contractile activity in more than 5% of the sensors in a quadrant and having a detectable delay was significant higher in the lower abdominal pairwise combination of quadrant compared to others. CONCLUSIONS: The propagation speed values are within physiologically possible range. MMG signals provide us with high spatial-temporal infomation of the uterine contractile activity which will help us to optimize the limited sensor abdominal EMG recordings that are practical in clinical setting.

METHODS: PMCs were isolated from C57/Bl6 mice. [Ca 2+ ]i was measured by Fura-2 microscopy. External Ca 2+ entry was stimulated with the L-Type Calcium Channel (LTCC) agonist FPL64176 (1µM). Internal store Ca 2+ release was stimulated with thapsigargin (2µM). Channel-independent Ca 2+ entry was evaluated with the Ca 2+ ionophore, ionomycin (5µM). [Ca 2+ ]i with each treatment was quantified before and after addition of the H 2 S donor, NaHS (500µM). Ca 2+ stimulated P-MLC in PMCs after NaHS (0-1mM) was determined by western blot for P-MLC (ser19) normalized to total-MLC. In USM strips, tension change and P-MLC were assessed with NaHS (1mM) after initiating contraction in Ca 2+ free media with thapsigargin (5µM), ionomycin (10µM) plus Ca 2+ (15mM), or MLC phosphatase inhibitor, calyculin (0.2µM). Tension is quantified as area under the curve (g*s; AUC), and P-MLC/T-MLC by infrared fluorescence imaging. Data are mean±SEM, analyzed by Kruskal-Wallis and Mann-Whitney U test. RESULTS: In PMCs: LTCC dependent Ca 2+ entry (n=28) and internal store Ca 2+ release (n=55) were unaltered by NaHS. Channel-independent Ca 2+ entry is increased 3.5-fold by NaHS (n=26, p<0.0001). NaHS does not change Ca 2+ -dependent P-MLC in PMCs (n=5-9). In USM strips: NaHS inhibited Ca 2+ and calyculin-stimulated contractions by 6.2 and 1.6 fold, respectively (n=12, 11; p<0.005). In the same strips, NaHS increased P-MLC with Ca 2+ and calyculin by 1.8 and 1.2 fold respectively (n=12, 8; ns). The nitric oxide donor SNP (1mM) had no effect on calyculininduced contractions (n=6). CONCLUSIONS: NaHS profoundly inhibits myometrial contractions independent of intracellular Ca 2+ or Ca 2+ entry. NaHS relaxes USM strips without altering P-MLC, and the mechanism is distinct from nitric oxide. This non-classical tocolytic mechanism presents new opportunities for therapeutic targets to prevent preterm birth.

Myometrium. Carolina B Wandscheer, Craig C Ulrich, Heather Burkin. Department of Pharmacology, University of Nevada, Reno, NV, USA.

INTRODUCTION: Genes such as MMPs and TIMP2 appear to be involved in the process of parturition and the regulation of birth timing. MMP9(Gelatinase B) is an endopeptidase known to be involved in the breakdown of the extracellular matrix during cervical ripening and other physiological processes (Stygar et al.,2002) . TIMP2 is an endogenous MMP inhibitor that has been implicated in preterm premature rupture of membranes. CpG methylation in the gene promoter of MMP9 and body of the MMP9 inhibitor TIMP2 is associated with gestational age (Parets et al.,2013) . MMP9 activity is increased in the amniotic fluid of patients undergoing spontaneous rupture of membranes(term or preterm) and parturition(term or preterm) (Di Ferdinando et al.,2010; Maymon et al.,2000; Romero et al.,2002) . Secreted MMP9 has been implicated in initiating signaling events that regulate cellular contraction (Defawe et al.,2005; Kobayashi et al.,2014) and recent work indicates MMP2 and MMP9 inhibitors rapidly enhance the oxytocin-induced contractile response in rat myometrium (Yin et al.,2012) . We performed experiments to test the hypothesis that MMP9 activity is altered in preterm laboring myometrium where it may exert local effects on contractility. METHODS: Human myometrial tissue samples were collected under informed consent and with Institutional Review Board approval. TIMP-2 expression was assessed by semiquantitative western analysis and normalized to GAPDH expression. MMP9 expression and MMP-9 activity was assessed by western blot and gelatinase zymography. Data were compared by one-way ANOVA with p<0.01 considered significant. Localization in pregnant human myometrial tissue was assessed by immunofluorescent confocal microscopy. RESULTS: TIMP2 expression was lower in myometrial tissue from all pregnant groups(term in labor, term not in labor, preterm in labor, and preterm not in labor) compared to nonpregnant myometrial samples. Pro-MMP-9 and MMP-9 gelatinolytic activity was higher in preterm laboring myometrial tissue than in nonpregnant myometrial samples. Confocal microscopy showed MMP9 localized adjacent to myometrial cells in pregnant uterine tissue. CONCLUSIONS: Our preliminary data indicate MMP activity is increased and TIMP2 activity is low in myometrium from preterm laboring patients. The localization of MMP9 adjacent to myometrial cells is consistent with a potential role in regulation of myometrial contractility as well as ECM degradation. Future work will determine if MMP9 can affect contractility in human myometrial tissue. Other factors associated with neonatal morbidity were:African-American race,Hispanic ethnicity,obesity and pre-eclampsia,HELLP or eclampsia. CONCLUSIONS: In this study, women with high ratios of ROM duration-to-labor duration were at increased risk of neonatal morbidity. Further prospective research is needed to determine if the ratio can assist obstetricians in identifying laboring women at risk for neonatal morbidity.

Umbilical Cord Milking in Extremely Low Gestational Age Males Versus Females. Erin AS Clark, 1 Shrena Patel, 2 Christina E Rodriguez, 1 Torri D Metz, 3 Bradley A Yoder. 2 1 Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, UT, USA; 2 Pediatrics, University of Utah School of Medicine, Salt Lake City, UT, USA; 3 Obstetrics and Gynecology, Denver Health Medical Center, Denver, CO, USA. INTRODUCTION: Due to neurobiological differences between males and females, the efficacy of neuroprotective strategies in preterm infants may differ by neonatal sex. We evaluated the effect of umbilical cord milking on morbidity and survival in extremely low gestational age males versus females. METHODS: We implemented a prospective observational trial of umbilical cord milking in preterm neonates <30 weeks gestation starting 9/2011. Infants underwent umbilical cord milking 3 times over a duration <30 seconds at delivery. We compared the milked group to a retrospective cohort delivered between 1/2010 and 8/2011. The primary analysis showed a significant reduction in the composite outcome of severe intraventricular hemorrhage, necrotizing enterocolitis, and death prior to hospital discharge (22% vs. 39%; OR 0.54, 95% CI 0.31-0.93) (Patel et al., AJOG, 2014) . We conducted a secondary analysis evaluating the effect of umbilical cord milking on the same composite outcome in males versus females. Neonates with anomalies or aneuploidy were excluded from the analysis. A multivariable logistic regression model evaluated the association between umbilical cord milking and the composite outcome within sex strata. An interaction term tested heterogeneity across strata. RESULTS: We analyzed 187 males (n=101, milked group) and 150 females (n=77, milked group). In males, umbilical cord milking was associated with a significant decrease in composite outcome (OR 0.41, 95% CI 0.2-0.8; p=0.009). In females, umbilical cord milking was not associated with the composite outcome (OR 0.57, 95% CI 0.26-1.24). The p-value for the interaction term was 0.47, indicating no statistically significant difference in the effect of umbilical cord milking on the composite outcome between males and females when controlling for appropriate covariables, including gestational age, chorioamnionitis and placental abruption. CONCLUSIONS: The effect of umbilical cord milking on composite morbidity and survival in extremely low gestational age neonates is not different between males and females.

Maternal Smoking Effect on Stillbirth and Neonatal Demise After 32 Weeks. Sarah Crimmins, Stephen Contag. Obstetrics, Gynecology and Reproductive Sciences, University of Maryland, Baltimore, MD, USA. INTRODUCTION: Our objective is to define the effect smoking has on pregnancy in relation to risk of stillbirth and neonatal demise. METHODS: Retrospective cohort study for US birth certificate data from 2011. We included all singleton births between 32-42 weeks gestation. Exclusion criteria were gestational age outside of 32-42 weeks, congenital anomalies and multiple gestation. A population specific birthweight distribution was developed using the mean and standard deviation by week of gestation. Our primary outcomes were stillbirth, neonatal death, or adverse composite neonatal outcome (neonatal demise, admission to neonatal intensive care unit, neonatal seizures, or assisted ventilation for > 6 hours). RESULTS: 2.4 million singleton non-anomalous deliveries occurred in this cohort with 2,334 stillbirths, 2033 neonatal deaths, and 180,168 adverse neonatal outcomes. Smoking increased odds for low birth weight [table 1]. Stillbirth rates were stratified by maternal smoking status. There was no significant increase in the risk for stillbirth (OR 0.9, 0.8-1.0 95% CI, p=0.06, [figure] ). The attributable fraction for was higher for prematurity than for maternal smoking status for both stillbirth and neonatal demise [ In order to optimize management of neonates with gastroschisis we evaluated the risk of culture proven sepsis based upon the mode of delivery. Additionally, we investigated the effect of sepsis and mode of delivery on length of stay (LOS). METHODS: Records of neonates with gastroschisis and their mothers at two academic medical centers from February 1999 to December 2012 were reviewed. The mode of delivery was defined as: vaginally (V), cesarean section with labor (CS&L), and cesarean section (CS) without labor. Sepsis rates by mode of delivery were assessed. Log-binomial risk ratios (RR) and 95% confidence intervals (CI) were reported after adjustment for the presence of chlamydia, genital herpes and urinary tract infections (UTI); the effects of sepsis and mode of delivery on LOS were assessed using multiple linear regression. Figure( s) will be available online. CONCLUSIONS: A significant increase in risk of sepsis was observed for neonates with gastroschisis delivered by CS&L however their LOS was shorter compared to V; also, neonates delivered to mothers with chlamydia, genital herpes or UTI showed a significantly greater risk of sepsis.

Classical Cesarean: Effect on Subsequent Maternal Pregnancy Outcomes. Cassandra R Duffy, Cynthia Gyamfi-Bannerman. Department of Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA.

Little is known about how a classical cesarean affects future maternal outcomes. We compared adverse outcomes in a subsequent pregnancy after prior classical (PC) compared with prior low transverse (PL) cesarean. METHODS: This is a secondary analysis of an MFMU multicenter observational study of delivery after prior uterine scar. Women with singleton gestations and one prior cesarean, either PC or PL, were included. Those with other uterine scars were excluded. We compared adverse maternal outcomes between our two exposure groups (PC and PL) in a subsequent pregnancy. Multivariable log-binomial regression was used to estimate the risk of adverse maternal outcome associated with one prior classical cesarean. RESULTS: Of 28,112 women included, 27,310 (97.1%) had PL and 802 (2.9%) had PC. Women with PC were more likely to be African American, unmarried, smokers, carry government insurance, and have medical comorbidities. There were also significant differences in delivery type for the index pregnancy. For women with PL, 48.3% had a repeat cesarean, 37.8% had a failed VBAC, and 13.9% had a successful VBAC; for women with PC, 91.5% delivered by repeat cesarean. Unadjusted analyses showed significantly higher risk of intraoperative transfusion, endometritis, postoperative ileus, and the composite maternal outcome in those with PC. Marked variability exists in data addressing prevalence and significance of PT-GA in OBGYN literature with ethnicity rarely addressed. Our goal was to identify prevalence to guide management and resourceful testing in the era of personalized medicine for patients in hypercoagulable. METHODS: Pubmed, EMBASE, Medline and Cochrane library were interrogated for PT-GA and prevalence. Inclusion criteria were manuscripts defining the population, subject numbers, health status, and allele frequency. Carrier rate results were organized by continent and racio-ethnicity. RESULTS: 398 articles were identified and reviewed; 43 met inclusion criteria with a total 25, 689 subjects tested for PT GA. West and South African populations including Ethiopian Jews showed 0% prevalence. North African populations that were studied displayed rates between 0.7 in Egypt and 2.9% in Algeria and Morocco, likely due to admixtures in the population. In Asia, while Japan, Thailand, Singapore, China, and India have 0%, the Middle East displays varied rates of 0-6.7%. Middle Eastern Jews had rates of 1-4%, Sephardic Jews 5.5%, and Ashkenazi Jew 6.7%. There is a higher prevalence in Southern Europe (overall carrier rate 3%) than in Northern Europe (carrier rate 1.7%). Significantly higher rates were detected in Italy, France, Cyprus, Albania and Croatia of 4%. In Oceania, rates varied from 1.3-4.6%, but 0% in aboriginals. In North America, Mexicans, American Indians and African Americans showed 0% while the carrier rate was 2.5% among Caucasians in US. In South America, rates range from 0-1.6%; gene prevalence appeared in a select population considered caucasoid, though once again African Americans, Asians, and Amer-Indians displayed 0%.

The distribution of carrier rates supports the founder effect explaining divergence of Caucasoids and Mongoloids as well as

Africans from Non-Africans. This review of PT-GA prevalence can guide resourceful screening for identification of hereditary thrombophilia in populations of interest.

Respiratory Morbidity By Mode of Delivery at Term: All Things Are Not Equal at 39 Weeks. Victoria Fratto, Cande V Ananth, Cynthia Gyamfi-Bannerman. Department of Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA.

The risk of serious maternal and neonatal morbidity following trial of labor after prior cesarean is well documented. However, little data exists about more common neonatal outcomes, particularly at term. We analyzed the risk of neonatal respiratory morbidity at term, among infants of women with a prior cesarean, by intended and actual mode of delivery (MOD). METHODS: This is a secondary analysis of a Maternal-Fetal Medicine Units Network study of women with a prior cesarean. Women with nonanomalous, singleton gestations delivered at ³37 weeks were analyzed by intended mode of delivery: planned vaginal birth after cesarean (VBAC) vs planned repeat cesarean. A second analysis was performed by actual MOD: successful VBAC, failed VBAC, or planned repeat cesarean. The primary outcome was composite respiratory morbidity including respiratory distress syndrome (RDS) or transient tachypnea of the newborn (TTN). Multivariable log-Binomial regression was used to adjust for confounders. RESULTS: 44,055 women met inclusion criteria. 64.8% (n=28,546) delivered by planned repeat cesarean, while 35.2% (n=15,509) planned a VBAC. Of women who attempted VBAC, 26.6% (n=4,119) were unsuccessful and had an unplanned repeat cesarean. Overall, when analyzed by intended mode of delivery, respiratory morbidity was increased in infants delivered by planned repeat cesarean (RR 2.1, 95% CI 1.8, 2.5) (Figure) . When analyzed by actual mode of delivery, respiratory morbidity was lowest in infants delivered vaginally at 39 weeks (Table) .

Results were similar for RDS and TTN individually. CONCLUSIONS: Neonatal respiratory morbidity is increased after planned repeat cesarean compared to planned VBAC, even at term. 39 weeks remains the optimal gestational age for delivery. *Figure(s) will be available online. A major concern for pregnancies after prior myomectomy is risk of uterine rupture (UR). The true incidence of this complication is difficult to establish as most available evidence is described in case reports. Several authors suggest cesarean delivery (CD) at 36 -39 weeks to reduce the risk of UR. The aim of this study was to evaluate the incidence of UR and associated risk factors in women who experienced trial of labor after prior myomectomy (TOLAM) through a systematic review of the literature.

A systematic review of the literature was performed including all case series, with at least 5 cases, reporting outcomes of pregnancies after prior myomectomy. The terms 'myomectomy', 'pregnancy', trial of labor' and 'uterine rupture' were used in MEDLINE and OVID. RESULTS: 11 case series were identified that reported detailed data about TOLAM and the pregnancy outcomes, including 1034 pregnancies and 756 viable (³24weeks) deliveries. The incidence of UR was 0.93% (7/756): 0.47% (2/246) during TOLAM and 1.51% (5/330) before the onset of labor (Table) .Of the 7 uterine ruptures, 5 occurred before 36 weeks. CONCLUSIONS: UR after prior myomectomy usually occurs before 36 weeks and before the onset of labor. TOLAM is associated with a 0.47% risk of UR. Trial of labor should be offered to pregnant women as feasible and safe. The practice of scheduled cesarean delivery between 36-39 weeks may not prevent UR.

The Effect of Body Mass Index on Maternal and Neonatal Pertussis Antibody Levels in Pregnancy. Manisha Gandhi, Sridevi Devaraj, Haleh Sangi-Haghpeykar, Joan Mastrobattista. Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, USA. INTRODUCTION: As obesity has been linked with the potential to impair the immune response, we sought to determine if there is an association between maternal body mass index (BMI) and maternal and neonatal pertussis antibody concentrations after administration of the Tdap vaccine in pregnancy. METHODS: This is a nested cohort study of 123 women who received the Tdap vaccine during pregnancy. Women were stratified by BMI into three groups -normal, overweight, and obese based on first trimester or pre-pregnancy BMI. Maternal and umbilical cord serum samples were tested for the pertussis IgG antibody.

The mean maternal antibody concentration was 178.4 U/mL for normal BMI (n=29), 169.8 U/mL for overweight BMI (n=54), and 175.5 U/mL for obese BMI (n=40). No statistically significant differences were found in maternal and neonatal pertussis antibody levels between the three BMI strata. Seroprotection was achieved in 89.7% of neonates (26/29) in the normal BMI group compared with 87.0% of neonates (47/54) in the overweight BMI group and 97.5% of neonates (39/40) in the obese BMI group. There was no statistically significant difference between the thre BMI groups for achievement of neonatal seroprotection. CONCLUSIONS: Maternal BMI does not affect the maternal or neonatal antibody response to the Tdap vaccine in women who receive the vaccine in pregnancy. Maternal BMI is unlikely to affect the neonatal protective effects of the Tdap vaccine in pregnancy.

Does Prolonged labor course has been associated with adverse outcomes in women having a successful vaginal birth after cesarean (VBAC). However, the influence of rupture of membrane (ROM) duration on maternal morbidity is poorly understood. We hypothesized that the ratio of ROM: labor duration predicts maternal morbidity among VBAC cases. METHODS: Using MFMU Cesarean registry, our cohort comprised women with singleton, term pregnancies (³37 wks) who had a successful VBAC. Maternal morbidity was defined by at least one of the following: sepsis, chorioamnionitis, postpartum endometritis, intensive care admission, postpartum transfusion, uterine rupture or death. Ratio between ROM duration to labor duration was categorized into quartiles (<0.24;0.24-0.29;0.50-0.94;³0.95). Significant variables (P<0.1) were included in stepwise logistic regression model. RESULTS: Among 9,392 women with successful VBAC, 515 (5.5%) experienced maternal morbidity. Women with ratios in the upper quartiles were at increased risk of morbidity. Variables independently related to maternal morbidity were African American, Asian or Hispanic race/ethnicity; public insurance; BMI³25 and regional anesthesia. CONCLUSIONS: Among women having a VBAC, a ratio of ROM duration to labor duration of ³0.5 was associated with the highest odds for maternal adverse outcome. Prospective studies are needed to assess the value of this ratio in predicting maternal morbidity in women attempting VBAC. A massive transfusion protocol (MTP) is a structured system for delivery of blood products. MTP use has been shown to improve outcomes in a trauma population, but there is a paucity of data in the obstetric setting. We sought to compare maternal morbidity before and after implementation of a MTP at our institution.

We performed a retrospective cohort study of massive obstetric hemorrhage (4 or more units of packed red blood cells [pRBCs]) between 2007 and 2014 at our tertiary care hospital. Patients were compared before and after July 1, 2010, the date a MTP protocol was initiated. Our primary outcome was a composite maternal morbidity defined as unplanned peripartum hysterectomy, intensive care unit (ICU) admission, acute kidney injury (AKI, Creatinine ≥ 1.5 times baseline), mechanical ventilation or documented coagulopathy. Data was obtained via chart abstraction and blood bank transfusion records. RESULTS: There were 197 massive obstetric hemorrhages (83 pre-MTP and 114 post-MTP) during the study period. There was no difference in mean number of pRBC units between groups. In contrast, more women received fresh frozen plasma (FFP) post-implementation (60.5% vs 37.3%, p=0.001). Similarly, post-implementation patients were more likely to receive platelets (36.8% vs 21.7%, p=0.02). AKI was significantly more likely in the pre-implementation group (15.7% vs. 3.5%, p=0.003). There were no maternal deaths and no difference in composite maternal morbidity in the pre and post-implementation groups *Figure(s) will be available online. CONCLUSIONS: Implementation of a MTP in those with massive obstetric hemorrhage was associated with an increase in administration of FFP and platelets, a decreased incidence of acute kidney injury, but no difference in composite maternal morbidity. To examine differences in concentrations of lipids and cardiovascular indexes in women with and without spontaneous preterm delivery (sPTD).

In a multicenter observational cohort study women were included with a gestational age between 24 0 and 36 6 weeks of gestation in threatening preterm labor without known congenital heart disease, HIV, multiple pregnancy, uterine anomaly or congenital anomaly. Cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), glucose, and homocysteine were measured within 24 hours of delivery. Lipids and cardiovascular indexes were compared between the sPTD and a reference group. The reference group consisted of women with an uncomplicated pregnancy, who underwent an elective caesarean section.

In total 189 women with a sPTD and 29 references were included. In 165 women with a sPTD lipids and cardiovascular indexes were measured. Mean gestational age at delivery was 30.7±3.6 weeks in the sPTD group and 39.4±0.7 weeks in the reference group. Compared with the reference group, HDL, LDL, glucose and homocysteine levels were significantly higher in women with sPTD, while triglyceride levels were significantly lower in this group (table 1) .

Lipids and cardiovascular indexes, mmol/L sPTD (n=165) References (n=29) Cholesterol 6.0 (5.8-6.2) 5.6 (5. Glucose 6.2 (5.9-6.5)* 5.3 (4.6-6.0) Homocysteine 6.0 (5.5-6.6)* 4.7 (3.4-6.0)

Values as mean (95% CI) * p<0.05

Women with a preterm vaginal delivery had higher concentrations of cholesterol and LDL, compared with both the sPTD caesarean section and the reference group. Within the sPTD group no significant differences were observed regarding timing of corticosteroid administration.

This results indicate a perturbed maternal lipid profile shortly after delivery in women with spontaneous preterm delivery, even if levels were still within normal levels. However, these differences may be explained by the mode of delivery. INTRODUCTION: Outpatient therapeutic substitution for drug addiction is controversial in the pregnant patient. We hypothesize identification of pregnant women with positive urine drug screens and integrated addiction therapy in conjunction with therapeutic substitution would achieve substance abuse abstinence with improved perinatal outcomes. METHODS: Retrospective analysis of substance abuse in pregnant women from 1/1/2012-3/31/2013 who received prenatal care and delivered at Charleston Area Medical Center (CAMC). Variables analyzed from urine drug data linked to addiction intervention program.

abdominal or bowel trauma as a result of delivery in any of the cases. We experienced one neonatal death in the cesarean delivery group, in whom care was withdrawn due to the burden of other fetal anomalies. CONCLUSIONS: Omphaloceles commonly occur with other fetal anomalies and genetic conditions. Cesarean delivery did not appear to provide additional benefit over vaginal delivery at our institution during this time, despite its recommendation in a few clinical circumstances. We encourage consideration of this practice at other institutions to evaluate for the potential for vaginal delivery where it might not otherwise be considered.

Maternal INTRODUCTION: Maternal obesity is a known risk factor for cesarean delivery (CD), while increasing height has been associated with lower rates of CD. Height may be protective against cesarean delivery, even in the setting of obesity. This study aims to quantify the protective effect of maternal height against CD. METHODS: This is a secondary analysis of a multicenter trial of fetal pulse oximetry among 5,341 nulliparous women in spontaneous and induced labor of singleton gestations ³36 weeks gestation. Congenital anomalies, and missing values in height, pre-pregnancy weight or delivery weight were excluded. CD rates were calculated by pre-pregnancy BMI (<20, 20-24.9, 25-29.9, 30-34.9, ³35) stratified by height in inches (<64, 64-68, >68). For comparisons, BMI 20-24.9 and height 64-68 in. were used as reference. Multivariable regression modeling was used to control for confounders. Similar analyses were also performed using BMI at delivery. RESULTS: Complete data were available for analysis in 5,131 women. The overall rate of CD was 26.8%. Decreasing rates of CD occurred with increasing height at BMI <20, 20-24.9 and 25-29.9 (Table; p=0.02, p<0.0001, p=0.03 respectively). In multivariate analysis, when compared to the reference group the adjusted odds ratio (aOR) decreased with increasing height for BMI 25-29.9, BMI 30-34.9 and BMI ³35 women; the aORs were significant for short and average height women at these BMIs (Table) . Similar results were observed for BMI at delivery (Table) . When height and BMI were assessed as continuous variables, the estimated aORs (not shown) approximated well to those shown. *Figure(s) will be available online. CONCLUSIONS: Increasing maternal height was associated with a decreasing risk of CD at all BMIs. The increasing rates of CD among obese women are attenuated by increasing height, with a lower aOR in tall, obese women than in short, average weight women. These results may be useful in the antenatal counseling of obese women and may also be useful in the development of a prospective labor prediction model.

The Etiology of Periventricular Leukomalacia in Southern Japan: INTRODUCTION: Periventricular leukomalacia (PVL) is a major risk factor for cerebral palsy, especially in preterm infants. The aim of this study was to determine its prevalence and perinatal risk factors in a population-based study in southern Japan where the perinatal mortality rate is low. METHODS: This population-based study examined perinatal mortality and poor neurological outcome in our district (about 10,000 deliveries annually). From among the 143,900 cases registered between 2000 and 2013, 407 were stillbirths (>22 weeks of gestation) and 173 were neonatal deaths (<28 days of life), and there were 2 cases of maternal deaths. There were 292 registered as at high risk of poor neurological outcome, including 62 cases of PVL diagnosed by ultrasonography, computed tomography, or magnetic resonance imaging. We analyzed the type of PVL and perinatal factors associated with PVL.

The crude perinatal mortality rate over the study period was 4.0 per 1,000 deliveries. Of the 292 cases of poor neurological outcome, 62 (21%) were diagnosed as PVL: 28 in 2000-2006 and 34 in 2007-2014. The types of PVL were as follows: cystic (n=50), volume loss (n=9), combined (n=1), and unknown (n=2). The prevalence of PVL before 35 weeks in the population was 52/2,187 (2.4%). There were 40 cases (65%) concentrated between 27 and 32 weeks. Associated risk factors were premature rupture of membrane (n=14), bag formation (n=9), chorioamnionitis (n=7), previous vertical cesarean section (n=4), maternal hemorrhage (n=2), diabetes mellitus or gestational diabetes mellitus (n=2), and hypothyroidism (n=2). Fetal factors were nonreassuring fetal status (n=14), monochorionic twins (n=10), and intrauterine growth restriction (n=4). Post-intraventricular hemorrhage was seen at 22-30 weeks, infection at 24-33 weeks, monochorionic twins at 24-35 weeks, and congenital anomalies at only 38-39 weeks. CONCLUSIONS: The prevalence of PVL was 2.4% in preterm infants born at 22-34 weeks, which is consistent with previous reports. Infection and cardiac dysfunction are possible causes of PVL in some preterm infants. Oligohydramnios is an indication for delivery at term based on the premise it represents fetal compromise. If true, perinatal outcomes of women induced for oligohydramnios may compare unfavorably to those with normal fluid. We compared the perinatal outcomes of patients with oligohydramnios induced at term to a group of patients with normal amniotic fluid induced at term.

We performed a single site, retrospective cohort study from 2000-2014 using a perinatal database. Patients were identified who had a formal amniotic fluid assessment after 34 weeks and underwent induction of labor (IOL) at term. Fetal anomalies or demise, malpresentation, multiple gestation, and ROM were excluded. Oligohydramnios was defined by amniotic fluid index ≤5cm and when available, single deepest pocket £2cm. Patients with oligohydramnios were compared to controls with normal fluid with a similar balance of potential confounders using propensity score matching (R software). RESULTS: From a pool of 3768 undergoing IOL, a total of 173 patients with oligohydramnios were compared to 519 patients with normal fluid. Groups were similar in baseline characteristics. There were no significant differences in the perinatal outcomes between the two groups. The cesarean rate (34/173 vs. 110/519, p=0.75) and cesarean rate for nonreassuring fetal status (8/173 vs 21/519, p=0.82) were not significantly different. The prevalence of NICU admission was unchanged (11/173 vs 33/519, p>0.9). There were no differences in five minute APGAR scores < seven (4/173 vs 8/519, p=0.51) or meconium stained fluid (18/173 vs 41/519, p=0.34). 

CONCLUSIONS: Patients induced with oligohydramnios had no demonstrable differences in delivery mode or neonatal outcomes compared to well matched patients with known normal amniotic fluid induced at term.

Timing of Antenatal Corticosteroids and Neonatal Respiratory Outcomes. Heather I Levin, Christian Benjamin-Boamah, Jhenette Lauder, Moeun Son, Cande V Ananth, Cynthia Gyamfi-Bannerman.

Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA. INTRODUCTION: Previous work suggests that 80% of women who receive antenatal corticosteroids (ACS) deliver at >7 days from administration of ACS. This practice of non-ideal administration potentially diminishes the benefit to the neonate. Therefore, we chose to assess both the proportion of women who received steroids and delivered at >7 days, and the potential effect of this practice on the neonate. METHODS: This is a retrospective cohort study at a single institution between 2006 and 2011. The study included singleton non-anomalous pregnancies between 24 and 34 weeks gestation which received a course of ACS, remained pregnant for ³24 hours following the first ACS dose, and delivered at <35 weeks gestation. An ideally timed course was defined as delivery ≤7 days from the first ACS dose. A non-ideal course was defined as delivery more than 7 days following the first dose. The primary outcome was neonatal respiratory distress syndrome (RDS), confirmed by chest x-ray or chart review when the x-ray was not available. Secondary outcomes included ventilator support, surfactant use, and neonatal demise (NND). Associations between ACS timing and neonatal outcomes were examined through logistic regression models before and after adjustment for confounders. RESULTS: A total of 456 women were included, of whom 51.8% (n=236) had ideal ACS. Among the 220 (48.2%) women that received non-ideal ACS, 29.5% (n=65) received rescue steroids. Mean GA was 30.3 weeks (± 3.2 weeks) for ideal ACS and 31.0 weeks (±3.0 weeks) for non-ideal ACS. As shown in the table, the group with ideal timing had a significantly lower risk of RDS (73.6% vs 81.7%). The risk of ventilator support, surfactant use or death was similar between groups. *Figure(s) will be available online. CONCLUSIONS: In our cohort over 50% of patient had ideally timed steroids. Ideal timing of the first course of ACS was associated with a reduction in RDS. These findings suggest that the timing of the first course of ACS remains important, even in the setting of rescue steroids. 

A secondary analysis of prospective case-control multicenter study of SPTB. This analysis was limited to SPTB cases only, and included all singleton infants delivered £34.0 weeks gestation. Each woman was assessed for the presence of ³1 possible underlying SPTB etiologies: inflammation/infection, maternal stress, decidual hemorrhage, uterine over-distention, cervical insufficiency, placental dysfunction, premature rupture of the membranes (PROM), maternal comorbidities, and association with familial factors. Rates of composite severe neonatal morbidity (grade III/IV IVH, PVL, severe NEC, BPD, and/or death) were compared for each phenotype. We used multivariable regression to examine factors associated with composite severe neonatal morbidity. RESULTS: 1030 women/infant pairs met inclusion criteria. 210 neonates (20%) had major mobidity; baseline characteristics were similar between those with and without major neonatal morbidity. Neonates with major morbidity were delivered earlier (26.1 vs. 31.0 weeks, p<0.001), and were more likely to be delivered following decidual hemorrhage or cervical insufficiency (Table) . 

The gestational age at delivery, not the presumed etiology of SPTB, remains the best predictor of initial neonatal outcomes.

Maternal proposed as a marker of obstetric care quality. We examined the patterns and predictors of postoperative complications in women undergoing cesarean delivery (CD).

We identified a population-based cohort of women in the US that underwent CD between 2006-2012 using a commercial hospitalization database. Maternal complications included a composite of hemorrhage, infection, thromboembolism, and operative injuries. Multivariable regression models were developed to account for patient, obstetric, and hospital risk factors related to CD complications. Between-hospital variability in rates of a composite of CD complications was calculated using generalized linear mixed-models. RESULTS: Among 1,339,397 women that underwent CD, 6.4% (n=85,838) experienced a complication. The most frequent complications were hemorrhage, transfusion, length of stay >7 days, and endometritis. Complication rates were 1.3-fold (95% confidence interval 1.2, 1.3) higher among black (8.8%) than white (5.7%) women. Complications were more frequent in teaching compared to nonteaching hospitals (8.1% vs. 5.4%, P<0.001). Complications were strongly associated with the presence of preexisting or pregnancy related maternal illness as well as obstetrical conditions. Between-hospital variation in CD complications is displayed in Figure 1 . *Figure(s) will be available online.

Each hospital was ranked in ascending order on the x-axis by the mean probability of CD complication over the study period. 5.9% of hospitals had complication rates lower than the 5%ile of complication rates, and 4.8% of hospitals had complication rates higher than the 95%ile of complication rates. CONCLUSIONS: CD complication rates were strongly related to patient and obstetric factors. Complication rates vary significantly across hospitals, but 90% of hospitals had complication rates between 3 and 13%. CD complication rates may be a quality metric of limited utility, although risk assessment tools may provide an opportunity to reduce maternal morbidity. 

Among women with Hgb ≥7 and <10mg/dL following CD, transfusion is associated with higher rates of wound complications after controlling for a number of important confounders. The impact of transfusion on wound healing warrants further investigation to establish evidence based guidelines for transfusion in the postpartum patient. Prior studies have shown that for cesarean (CD) labeled "emergent," decision-to-incision time <30min does not improve neonatal acidemia. However, the effect of skin incision to delivery (surgical speed, SS) on umbilical artery (UA) pH has not been previously evaluated, particularly as it relates to primary versus repeat cesareans. Our objective was to determine whether SS was associated with neonatal acidemia for patients who require emergent CD for umbilical cord prolapse. METHODS: This is a secondary analysis of a multicenter, prospective observational study designed to study maternal and fetal outcomes after a prior uterine scar. In the current analysis, women undergoing emergent CD for cord prolapse were dichotomized into those with a skin incision to fetal delivery time £2min (I-D£2) and >2min (I-D>2) based on the mode (2 min). The primary outcome was UA pH<7. Secondary outcomes were UA pH and SS. The relationships between SS and outcomes were evaluated using Chi-squared, Wilcoxon Rank Sum, and log-linear regression.

: 314 women had an emergent cesarean delivery for cord prolapse. The median SS for the cohort was 3min (IQR 2-5min). Median SS was 3min (IQR 2-5min) for primary and 5min (IQR 3-10min) for repeat CD (p<0.01). 125 (39.8%) had SS£2 min. The groups were similar with respect to age, race, BMI, and preexisting maternal disease. 44 (35.2%) with SS£2 and 53 (28%) with SS>2 had UA pH<7 (p=0.18). SS was not associated with acidemia after controlling for confounders. The median cord pH was similar for both groups (p=0.63). * Figure(s) will be available online. The incidence of HIE was similar for both groups (p=0.77). CONCLUSIONS: Among women who require emergent cesarean delivery for cord prolapse, surgical speed is not associated with neonatal acidemia. The median SS was 3min for this cohort. This may indicate appropriate triage of a "true" emergency and suggest that a goal of 3min between skin incision and delivery is appropriate in the setting of cord prolapse. After adjusting for confounders including chorioamnionitis and preeclampsia, transfusion remained associated with pulmonary edema and ICU admission. CONCLUSIONS: Transfusion was associated with an increased risk for complications including pulmonary edema, endometritis, wound complications, pulmonary embolism, and ICU admission. A restrictive approach to blood transfusion warrants further study in the obstetric population.

Trends Over Time and Perinatal Outcomes in Dichorionic Versus Trichorionic Triplet Gestations. Danielle Peress, Lynn Yee, Alan Peaceman. Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA. INTRODUCTION: Monozygotic twinning has increased with growing use of assisted reproductive technology (ART). Inadequate data examine this phenomenon in triplet pregnancies. We aimed to evaluate trends in the proportions and outcomes of dichorionic-triamniotic (DT) compared to trichorionic-triamniotic (TT) triplet gestations.

Retrospective cohort of all patients with a triplet gestation identified by first trimester ultrasound (US) and delivered at a large academic center between 2005-14. Chorionicity/amnionicity were determined by US and confirmed on placental pathology. Chi square, Fisher's exact and ANOVA tests were used. RESULTS: 158 triplet pregnancies were identified. 44 (28%) were DT and 113 (72%) were TT. There were no differences in maternal demographic characteristics including age, race, parity or BMI. 152 (96%) conceived by ART. A greater proportion of DT compared to TT triplets conceived by IVF (85% v 59%, p=0.003). There was a significant increase in the proportion of DT pregnancies over time. DT sets were more likely to deliver as a singleton or triplet gestation. While a greater proportion of DT triplets experienced adverse events, these differences did not reach statistical significance (table) . There were no differences in maternal outcomes including preeclampsia or hemorrhage (data not shown).

Perinatal outcomes for dichorionic and trichorionic triplet gestations 

The proportion of triplet gestations that are DT more than doubled from 2005-14 at this institution. Major differences in maternal or neonatal outcomes were not identified. Differences in neonatal morbidity may be apparent in a larger sample size.

Clinician Awareness of NICHD Preterm Birth Outcome Calculator. Calculator is one of the few internet-based tools available to assist clinicians in counseling patients at risk for preterm birth. Our aim was to survey these clinicians to assess awareness and utilization of the calculator.

The survey tool included a list of questions regarding the NICHD PTB Outcome Calculator. We collected responses from medical personnel in academic and community-based hospital systems. Survey questions assessed awareness level, frequency of use, knowledge of calculator variables and outcomes, and information regarding the database and its utility. Fisher's exact analysis was used to compare answers essential to the understanding of the calculator, with p < 0.05 significant.

We assessed the most commonly misunderstood aspects of the tool and compared answers amongst level of training and area of expertise. RESULTS: There were a total of 141 participants, (40% resident, 30% Ob/ Gyn attending, 8% MFM attending, 8% MFM fellow and 9% midwife). 97% reported caring for patients at risk for preterm birth. Only 42% reported awareness of the calculator. Of those aware of the tool, 75% had visited the website, with 82% of those visiting the site actually having used the calculator. MFM physicians versus generalists were more likely to have heard of the calculator (75% vs. 32%) and used it (75% vs. 17%);

p<0.05. Ob/Gyn residents had similar familiarity with the calculator as attending generalists. Of those having used the tool, 25% knew the 5 calculator variables, with only 19% knowing the 3 estimated outcomes.

Interestingly, a majority of users misunderstood database components, with only 1/3 of users actually aware that the data are not predictive of individual outcomes and are not updated annually. CONCLUSIONS: A majority of clinicians who care for patients with threatened preterm birth are either unaware or have limited understanding of the NICHD Preterm Birth Outcome calculator, offering a potential area of improvement in the counseling of such at-risk patients.

Moved to Friday (Tdap) immunization for all pregnant women in the third trimester 1 . We sought to evaluate adherence to these recommendations in patients who presented to an obstetric triage unit. METHODS: A survey was given to patients presenting to the obstetric triage unit in a large academic urban hospital. Patients were asked if they had received a Pertussis (Tdap) immunization. If a patient had not received an immunization, she was given a list of multiple reasons for why she had not received an immunization. The answers included 1) a failure to be advised by physician 2) an inability to pay for the immunization 3) a belief that vaccines are harmful to the fetus 4) a belief that vaccines are not beneficial and 5) other. The rate of immunization in this population was determined and reasons for not receiving the immunization were recorded. RESULTS: Sixty-eight out of 277 patients (24.%) had received the recommend immunization. One hundred and fifty two out of 209 (72.7%) of those who did not receive the vaccine, the most common reason indicated was that they were never told about it. The second most common reason was concerns about safety of receiving the vaccine while pregnant (6.7%, 14/209). CONCLUSIONS: Physician failure to adhere to established recommendations for immunization with Tdap during pregnancy is the primary reason for low immunization rates. To determine the risk of poor maternal weight gain and adverse pregnancy outcomes in patients requiring pharmacotherapy for nausea and vomiting in pregnancies with twin gestations.

A retrospective cohort study of all patients who presented with twin gestations between 2005-2013 to a single obstetrical practice was performed. The rates of poor maternal weight gain and adverse pregnancy outcomes were compared between patients who required a prescription medication for nausea and vomiting during pregnancy and those who did not. Poor maternal weight gain was defined as patients who failed to meet the recommended IOM guidelines based on pre-pregnancy BMI categories. Odd ratios with 95% confidence intervals were calculated for all outcomes. RESULTS: 642 twin pregnancies were included in the cohort. Patients who required a prescription medication for nausea and vomiting during pregnancy were just as likely to gain the appropriate minimum weight per week as patients who did not require pharmacotherapy (OR 0.595, 95% CI 0.336-1.055). Patients who required pharmacotherapy also had no increase in spontaneous preterm birth less than 37 weeks gestation (OR 1.318, 95% CI 0.742-2.343) or intrauterine growth restriction less than the 10% (OR 0.598, 95%CI 0.334-1.072) compared to patients who did not require pharmacotherapy. 

Patient with nausea and vomiting requiring prescription medication with twin gestations are not more likely to have inappropriate weight gain or adverse pregnancy outcomes than patients who do not require prescription medication. To determine if using twin gestation specific thyroid stimulating hormone (TSH) reference ranges identifies patients at increased risk of adverse pregnancy outcomes.

A retrospective cohort study of all patients who presented with twin gestations between 2005-2013 to a single obstetrical practice was performed. All patients had a TSH drawn at their first prenatal visit.

Patients who were not diagnosed with or treated for hypothyroidism were included. Two standard deviations above the mean has been previously used to diagnose patients with hypothyroidism. Patients with an initial TSH drawn in the first trimester that was greater than two standard deviations above the mean for this cohort were identified as having elevated hypothyroidism. Adverse pregnancy outcomes in patients with an elevated TSH were compared to patients with a normal TSH. Odds ratios with 95% confidence intervals or Pearson's chi square tests were calculated for all outcomes. RESULTS: 428 patients with twin gestations met inclusion criteria. The mean TSH in the first trimester was 1.05 uIU/mL with a standard deviation of 0.96uIU/mL. Using these twin gestation trimester specific reference ranges identified 15 additional patients with an elevated TSH. There was no difference in the risks of spontaneous preterm birth, intrauterine growth restriction, pre-eclampsia, or intrauterine fetal demise in these patients compared with patients with a TSH less than 2SD above the mean. 

METHODS: 10 ovine pregnancies were instrumented with fetal vascular catheters at 117±1d GA (term~145d); 5 days postoperatively fetuses underwent a 10 day exposure to normoxia (n=5) or hypoxia (n=5, ↓ fP a O 2 21±1 to 12±1mmHg). Fetal heart rate (FHR) was sampled in 6x5 min blocks at a consistent time of day. FHR power spectra in active sleep were compared before and after exposure to normoxia or chronic hypoxia. RESULTS: Advancing gestation in normoxic pregnancy decreased FHR and increased the SDANN, an index of total FHRV (Fig.1) . Advancing gestation in hypoxic pregnancy led to a greater fall in FHR, a fall in the LF/HF ratio (index of sympathetic dominance), an increase in the RMSSD (an established parasympathetic index) and a loss of total HRV. CONCLUSIONS: Chronic significant fetal hypoxia is associated with a loss of total power and increased parasympathetic dominance in the FHRV power spectra. British Heart Foundation, Action Medical Research *Figure(s) will be available online.

Neonatal Outcomes After Uterine Rupture. Devin D Smith, Cynthia Gyamfi-Bannerman. Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA.

Little is known about the most common adverse neonatal outcomes (ANO) after uterine rupture (UR), an important element of patient counseling. Thus, we sought to describe common ANO following UR, comparing them to women with failed trial of labor after cesarean (TOLAC) without UR. METHODS: This is a secondary analysis of a multicenter observational study of cesarean delivery (CD). Included were women with nonanomalous singleton gestations and 1 prior low transverse CD who attempted TOLAC. The primary outcome was a neonatal composite including death, respiratory complications, hypoglycemia, sepsis, 5-min Apgar<3, need for resuscitation, encephalopathy (HIE), pH<7, neonatal intensive care unit (NICU) admission, and length of stay (LOS). The primary outcome and most common ANO were compared in neonates after failed TOLAC with UR to those without UR. Chi-squared, Fisher's exact test, Student's t-test and Wilcoxon rank sum were used in bivariate comparisons where appropriate. A log-linear regression model was fit to adjust for confounders. RESULTS: Of 3575 included women, UR occurred in 84 cases (0.02%). After UR, 54% (n=45) had a pH<7, 39% (n=33) were admitted to the NICU, and 17% (n=14) had a prolonged LOS or required ventilator support. Seizures, abnormal neurologic diagnoses or HIE occurred in 5% (n=6). There was 1 neonatal death. Neonates in the UR group were more likely to have seizures, HIE, and a 5-min Apgar<3 [Table] . The rate of the primary outcome was similar in both groups (UR 67% v. no UR 57%, p=0.06). After adjusting for confounders, no clinically significant difference was found (RR 1.16, 95%CI 0.99, 1.35). However, when analyzed individually, there were significant increases in ANO in the UR group. Although increased rates of death were not statistically significant, the relative risk of death occurring after UR was greatly increased [Table] . CONCLUSIONS: Adverse outcomes such as HIE and seizures are more common after UR. These data, along with the data regarding common ANO associated with rupture, fill a void in the counseling of patients desiring TOLAC. To determine obstetrical factors associated with a tendency to fetal umbilical artery acidosis.

Umbilical cord blood analysis is routinely performed on all deliveries in our health system. We obtained the obstetrical electronic records of over 8000 term singleton neonates delivered in 2013. We calculated the lowest quartile for the umbilical artery ph(pHA) and compared with demographic, medical, obstetrical and intrapartum factors. We used Chi-square, student T test and regression analysis as indicated. RESULTS: There were 5448 valid pHA results with a mean+SD of 7.27+0.064 and the lowest quartile was 7.24. Factors significantly associated with the lowest quartile pHA included higher maternal BMI, tobacco smoke, induction agents, pre-gestational diabetes, preeclampsia, Labetalol use, Magnesuim sulfate use, PROM, shoulder dystocia, longer 2nd stage of labor and abnormal fetal heart rate patterns; whilst cesarean and cefalozin administration were protective. Regression analysis confirmed smoking(p=0.001,), magnesium sulfate use(p=0.005), cesarean section(p=0.001), abnormal fetal heart rate(p=0.000), and second stage of labor(p=0.000) as independent predictors. Small-for-gestational-age (SGA) neonates are at risk for adverse perinatal outcomes. When delivery is indicated, it remains unclear whether cesarean delivery improves neonatal outcomes. Even less is known regarding outcomes for SGA neonates that experience trial of labor after cesarean delivery (TOLAC). We hypothesized that women who attempt TOLAC and deliver SGA neonates have comparable neonatal outcomes to those who undergo repeat cesarean without labor.

This is a secondary analysis of a multicenter prospective observational study. Women with nonanomalous, singleton gestations and one prior cesarean who delivered SGA neonates were included. Twin gestations and antenatal stillbirths were excluded. Women with SGA neonates attempting TOLAC regardless of ultimate mode of delivery were compared to women with SGA neonates who elected to undergo repeat cesarean without TOLAC.The primary outcome was a composite neonatal morbidity including neonatal death, respiratory complications, hypoglycemia, sepsis, NICU admission, and hospital stay >5 days. The association between TOLAC and neonatal morbidity was estimated using log-linear regression adjusting for possible confounders. RESULTS: 1009 patients were identified. 258 (25.6%) underwent repeat cesarean and 751 (74.4%) attempted TOLAC, of which 536 (71.4%) were successful and 215 (28.6%) failed. There were significant differences between women who attempted TOLAC and those who elected repeat cesarean. *Figure(s) will be available online.

On unadjusted analyses, the composite adverse outcome occurred in similar frequencies with both groups (69.9% vs. 70.2%, p=0.94). Controlling for age, race, body mass index (BMI), smoking, baseline disease, prior vaginal birth after cesarean (VBAC), antenatal corticosteroids, and preterm gestational age at delivery, the composite adverse outcome occurred in similar frequencies with both groups (aRR 1.00, 95% CI 0.91-1.11, p=0.95). CONCLUSIONS: Women who undergo TOLAC and deliver SGA neonates have similar neonatal outcomes to those who deliver by repeat cesarean without trial of labor. We conclude that TOLAC is an acceptable option for women with a history of prior cesarean and SGA neonates. The effects of smoking status on adverse perinatal outcomes is well-known. However, to what extent smoking cessation early in pregnancy is related to risk reduction of major smoking-related complications is less clear. Aim: To assess associations between self-reported smoking status in pregnancy and major perinatal outcomes. METHODS: Setting: Single-centre study of women who delivered at University Hospital Southampton, United Kingdom. Comparisons: Women who reported, at booking, to have never smoked, stopped >1 year prior to conceiving, stopped <1 year prior to conceiving, stopped when pregnancy was confirmed, continued smoking up to 10 cigarettes/day, continued smoking up to 10-20 cigarettes/day, and continued smoking over 20 cigarettes/day. Outcomes measures: Percentage of preterm birth <37 weeks gestational age, small-for-gestational age (SGA) <p10, SGA <p5 and stillbirths. Mean differences in birth weight, birth weight centile, head circumference, placental weight, placental-to-fetal weight ratio, with adjustments for fetal gender, maternal height, parity by logistic regression models. RESULTS: 39,715 (53.3%) of women were never smokers, 14,946 (20.1%) stopped smoking prior to pregnancy, 6,455 (8.7%) stopped smoking when pregnancy was confirmed, and 13,333 (17.9%) continued smoking 10 cigarettes/day or more. We observed no difference between non-smokers and women who quit when pregnancy was confirmed for preterm birth (6.6% vs 6.2%, P=0.25), SGA (9.5% vs 10.0%; P=0.15) and stillbirth (0.5% vs 0.3%; P=0.14) In contrast, when compared with non-smokers, of those women who continued to smoke during pregnancy, a significantly greater proportion experienced preterm birth (9.8%, p<0.001), SGA (19.7%; P<0.001), and stillbirth (0.6%; P=0.010). In addition, when compared to mothers who continue to smoke in pregnancy, women who stop smoking have infants with a higher birth weight, birth weight percentile, head circumference, placental weight and placentalto-fetal weight ration (all P<0.001).

In comparison to women who continue smoking throughout pregnancy, women who stopped smoking early in pregnancy have a reduced in risk of preterm birth, SGA infants, and stillbirth that is equivalent to the level of risk of never smokers. Our findings hold great promise for the potential benefit of smoking cessation to improve perinatal outcome. Stepwise logistic regression was used to determine outcomes related to uterine rupture. RESULTS: Of almost 8 million births, 1,925 were complicated by uterine rupture(Absolute Risk=2.43/10,000 births). For maternal outcomes, the model retained 4/5 variables analyzed, with maternal transfusion(~15%) being the most common and hysterectomy having the highest odds(~100 fold). For neonatal outcomes, the model retained 3/11 variables, with neonatal seizures being the most frequent(~35%) & with highest odds(~20 fold). 

A randomized control trial to compare a plain bath wipe vs. one with 2% CHX in 20 labor triage patients (10 per wipe group). Following informed consent, patients were randomized using sealed opaque envelopes. A pre-wipe swab was collected from the EPS. The area was wiped front-to-back using 3 wipes, akin to a clean catch urine collection, and allowed to dry. The post-wipe specimen was collected. Swabs were placed in a BD BBL Port-A-Cul envelope and sent to micro lab for aerobic and anaerobic bacterial culture. Lab personnel were blinded to type of wipe used and collection order of each patient's swabs. QBB was assessed on a 5 point scale (5=heavy, 4=moderate, 3=light, 2=very light, 1=no growth). Results were analyzed using student's paired t-test and Fisher's exact test. RESULTS: 19 of 20 patients had moderate to heavy growth on the EPS pre-wipe (9 of 10 in CHX group; 10 of 10 in plain group) p=1. All 10 patients in the CHX group had a decrease in the QBB, where as only 4 of 10 patients in the plain group had a decrease (100% vs. 40%, p=0.01). 

We prospectively investigated 132 pregnant women with CMV IgM positive from 2008 to 2013. IgM antibody was measured in occasion of maternal screening or suspicion of intrauterine infection. IgG AI was measured in the same sera used in measurement of IgM, when IgM was positive. The urine of 132 neonates was examined via PCR to confirm the congenital infection. Usefulness of IgG AI for detection the congenital infection according to IgG AI values and the relationship between IgG AI and IgM were investigated. This study was approved by the institutional Review Board, and written informed consent was obtained from all participants.

The pregnant women were measured IgM and IgG AI at 13.4±4.6 weeks of gestation. Total of 12 newborn were congenitally infected. The prevalence of infected newborn according to IgG AI value was higher in <35% (8/25) when compared with in 35-50% (0/24) and in ≥50% (3/108). In women measured IgG AI at £14 weeks of gestation, the prevalence of infected newborn was higher in <AI of 35% group. In contrast, there was no difference between the three groups in women at ³15 weeks of gestation. No correlation was seen between IgM levels and IgG AI. CONCLUSIONS: IgG AI was useful marker to detect high risk woman for the congenital infection when it was measured at £14 weeks of gestation. during pregnancy is well documented, little is known of the epidemiology and impact of these infections when they are in association with extended spectrum beta-lactamase (ESBL) producing organisms. ESBL are enzymes produced by gram-negative bacilli that result in resistance against most antibiotics. Carbapenem is the standard treatment.

maternal line represents the first generation that is not directly exposed. We characterised nephron number and cardio-renal phenotype of F3 offspring born to normally grown and growth restricted (F1) mothers or fathers. METHODS: Late gestation rat uteroplacental insufficiency was induced (Restricted) or sham (Control) surgery. To generate F3 paternal line offspring, F1 Control and Restricted males were mated with normal females and F2 Control and Restricted males were then mated with normal females. F3 maternal line offspring were similarly generated. F3 body weights were measured (birth to 12 months). Nephron number was quantified using unbiased stereology at day 35. Renal excretions, creatinine clearance (both 24h) and blood pressure were measured at 6 (maternal and paternal lines) and 12 (maternal line only) months. Data were analysed by t-test within a gender and line. RESULTS: Although F1 offspring were born small, F2 and F3 offspring (maternal and paternal lines) had normal birth weights. F3 growth trajectories (birth to 12 months) were not different between groups and lines. F3 male and female nephron endowment and blood pressure was not different between groups for paternal and maternal lines. F3 maternal line renal function was normal at 6 months. However, at 12 months, renal dysfunction emerged in males (proteinuria) and females (increased urinary creatinine), despite normal creatinine clearance (eGFR). The paternal line F3 offspring had evidence of impaired eGFR (reduced creatinine clearance) at 6 months. CONCLUSIONS: F3 offspring, born to F1 growth restricted mothers or fathers are not programmed to be of low birth weight but developed renal dysfunction in the absence of obesity. The proteinuria that emerged with aging in F3 maternal line males was in the absence of nephron deficits, hypertension and reduced eGFR, suggesting tubulointerstitial injury. In contrast, F3 paternal line offspring had glomerular dysfunction (reduced eGFR), indicative of glomerular filtration deficits. Our findings provide novel evidence of transgenerational transmission of renal dysfunction to F3 offspring from both maternal and paternal lines.

Visceral INTRODUCTION: Antenatal exposure to glucocorticoids (GC) is associated with significant cardiometabolic alterations. Adipose tissuederived inflammatory mediators are thought to participate in the regulation of blood pressure in obese and diabetic humans. Our working hypothesis states that GC alters the development of white adipose tissue (WAT) which will contribute to the development of metabolic alterations in the adult offspring. The aim of this study is to ascertain if visceral fat ablation can ameliorate the effects of antenatal GC on blood pressure and arterial stiffening. METHODS: Pregnant sheep were treated with two IM doses of betamethasone (Beta, 0.17 mg/kg) or vehicle (V) 24-h apart at 80 days gestation and allowed to deliver at term. Visceral fat was removed in a subset of Beta sheep (FRB) at 2 mo of age. At 1 y of age animals were chronically catheterized Beta (n=15), V (n=12) and FRB n=12) and blood pressure measured for a minimum of 2 d. ASI was calculated using the bisector regression method (Sym_Slope) on two individual data sets of 1400 points/sheep/day. Data are presented as Mean±SEM and were analyzed by ANOVA and two sample t test.

We confirmed that Beta significantly increases BP in both female and male adult sheep (MAP rose from 93±0.8 to 99±0.6 and from 94.4±0.8 to 101±0.8; SBP from 112±1.2 to 121±0.6 and from 113±0.7 to 122±1.0 in females and males respectively. ASI changed from 1.09±0.009 to 1.08±0.009 and from 1.10±0.007 to 1.03±0.02 (P<0.05 by ANOVA). As shown in the figure fat ablation significantly ameliorated the effect of Beta on BP and ASI. *Figure(s) will be available online.

The findings of this study are: 1) we confirm that prenatal exposure to a single course of GC at 0.55 gestation has programming effects on the regulation of arterial blood pressure and 2) the data suggests that alterations in visceral fat depot play an important role in the development of hypertension. In addition to the increases in blood pressure we report the novel finding of a change of the stiffening properties of the arterial tree. The Sym_Slope was higher in Beta animals and improved with visceral fat ablation. HL 68728 and HD 04784.

RESULTS: Fig. 1 shows NT increased in all six locations to differing extents. The increased staining in liver was most obvious around the central vein. No difference were observed between males and females. *Figure(s) will be available online. Fig. 1 Nitrotyrosine immunohistochemistry Kidney C -Kidney cortex, Kidney M -medulla. FC -frontal cortex, GM -gray matter, WM -white matter. Micrographs top row CTR fetuses, middle row IUGR fetuses; bottom row percentage fraction of section stained pooled data for both sexes as there were no differences between sexes; n= 7 -15 per group. * p < 0.05 CTR vs IUGR. Mean + SEM. CONCLUSIONS: IUGR resulting from MNR and, hence, decreased fetal nutrient supply has a widespread effect on fetal tissue OS. There was no difference between male and female fetuses. If these changes persist into adult life they may constitute a mechanism whereby poor fetal growth and intrauterine nutrition predisposes to widespread later life organ dysfunction (HD 21350).

In The Developmental Origins of Health and Disease hypothesis holds that embryonic and fetal adaptation to suboptimal uterine environments, including preimplantation embryo culture, can predispose a series of metabolic diseases in adulthood including cardiovascular disease, diabetes and hypertension. It is believed that embryo culture in vitro, routinely performed during in vitro fertilization (IVF), is associated with epigenetic changes at imprinted loci. Further, an increase in imprinted disorders has been documented in IVF children. However, it is unknown if epigenetic changes are limited to imprinted loci or are a widespread phenomenon, affecting other non-imprinted loci. Previously we observed downregulation of Glut3 mRNA levels in placentae of IVF fetuses, and here we investigated if DNA methylation differences were present at this locus in placenta of IVF or naturally conceived mice fetuses. METHODS: Blastocysts (CF1 x B6D2F1/J) generated by IVF and cultured in KSOM with amino acids, or produced by natural mating and flushed from uterus (FB group) were transferred to pseudopregnant recipients. At E18.5 placentae were removed for assessment of Glut3 mRNA and protein levels. Additionally, DNA was extracted for methylation via bisulfite sequencing of up to 922 bp 5' of the Glut3 transcription start site (TSS). RESULTS: At E18.5, Glut3 mRNA was downregulated in IVF placentae.

Further there was reduced DNA methylation at the level of the first intron of GLUT3. CONCLUSIONS: The process of IVF is associated with epigenetic changes at the level of Glut3, a non-imprinted gene. These findings indicate that epigenetic changes following in vitro culture may be widespread and affect genes important for placenta development and nutrient transport to the fetus; further they provide an explanation of how the embryo and fetus adapt to suboptimal uterine environments and may be predisposed to chronic disease in adulthood.

Maternal INTRODUCTION: Seasonal variation in term birthweights (3-6%) have been reported around the world. This affect may be related to latitude and ambient temperature, but the etiology is unknown. Accumulating evidence suggests an altered CD4 Th1>Th2 immune response to pregnancy may play a key role in the development of placental insufficiency. Since seasonal allergies (atopy) and maternal body mass index (BMI) are known to affect cell-mediated immunity with a Th2 bias, we hypothesized obese women with allergic rhinitis may have less placental insufficiency and larger babies at term.

Retrospective chart review of 4552 Caucasian normal term deliveries at our institution from 2006-2009. Metrics included birthweight, the month of delivery, maternal race, age, body mass index, fetal gender, concurrent pregnancy history of allergic rhinitis +/-antihistamine use, smoking history, and socioeconomic indicators (marital status, Medicaid). Data were analyzed by ANOVA with post-hoc corrections and regression modeling. RESULTS: Single women on Medicaid (n=406) had slightly smaller babies (3449 +/-481 gms) than those with private insurance (3533 +/-541) (p=0.01). Smoking was also associated with a 3% decrease in birthweight (3445 versus 3533 in "never" cohort) (p=0.02). Interestingly this affect was most pronounced in male progeny (p=0.007 vs p=0.37 for females).

In contrast maternal pre-pregnancy obesity (28% of cases) led to larger babies (3667 +/-439 vs lean mothers' 3479 +/-476) (p=0.001). Atopy history had no affect. We observed peak term birthweights in the Fall with the largest babies in December (3583 gms) and smallest in August (3460 gms) (p=0.002). Multivariate analysis revealed pre-pregnancy BMI was most closely correlated with this variance with heavier mothers in the Winter delivering the largest babies in the Fall. CONCLUSIONS: Normal term birthweights are affected by a number of variables, which include maternal pre-pregnancy BMI. Our data suggest seasonal variation in birthweight may be related to maternal BMI despite a number of significant covariates.

Use Livestock farming uses genetic selection of animal characteristics related to efficient production of meat, dairy and wool. However, the phenotypic signatures are not merely related to information hidden in DNA structure, but reflect many other life events. The metabolome is the set of small molecules (<2000 Da) synthesised through biochemical reactions within the living organism. As a downstream product of genetic predisposition and environmental factors, metabolomics may allow more accurate determination of the future growth and development of individual animals. Aim: to identify plasma metabolic signatures which discriminate between lambs that grow well and those that grow poorly. Accurate prediction of lambs that will grow faster to slaughter would enable better use of feed resources, enhance farm efficiency and thus provide commercial benefit to the producer. METHODS: Ram EDTA plasma samples from two separate flocks were collected at docking (n 1 =79, d 1 =22 days; n 2 =77, d 2 =19 days) and weaning (n 1 =63, d 1 =98 days; n 2 =60, d 2 =88 days). Metabolite profiling was conducted using gas chromatography-MS. Chromatographic data was divided into discovery and validation steps, separated according to the flock. In each group, the metabolome of low weight lambs (below 20 th centile of slaughter weight) were compared to the remaining animals. RESULTS: 87 metabolites were identified in plasma samples. Two organic acids and one amino acid, were found in both discovery and validation steps to significantly differ (p<0.05) between low and high slaughter weight animals in the samples collected at weaning. There were no common metabolites, between flock 1 and 2, able to predict lamb growth to slaughter (p>0.05) at the docking stage. CONCLUSIONS: Poor lamb growth was reflected in their plasma metabolome in blood collected at weaning. Causal relationship between the identified metabolites and growth will be further investigated. The current study suggests that the use of biomarkers could improve sheep farming profitability by identification of slow-growing animals at an early stage. Smoking is associated with changes in the composition of the maternal microbiome and there is now considerable evidence to suggest that the establishment of the infant microbiome may play an important role in the development of obesity. Therefore we hypothesized that fetal and neonatal exposure to nicotine, the major addictive component of cigarette smoke, would result in gut microbial community differences in postnatal life. METHODS: Female rats were randomized to receive daily injections of saline (N=20) or nicotine (1.0 mg/kg/d; N=20) from 2 weeks prior to mating until weaning. To examine the effect of nicotine exposure on the composition of the gut microbiome, fecal samples were collected at 3 (weaning), 12 and 26 weeks. Pair-end sequencing was done on the V3 variable region of the 16S rRNA gene on an Illumina MiSeq. Data was processed through a bioinformatics pipeline (Farncombe Genomics Facility, McMaster University) and was analyzed by QIIME to determine changes in the community composition and the diversity of the gut microbiome in nicotine-exposed and control offspring. RESULTS: At the phyla level, fetal and neonatal exposure to nicotine resulted in alterations in the proportion of both Firmicutes and Bacteriodetes at 26 weeks of age. There were significant changes in a number of operational taxonomic units (OTUs) at all time points; these differences were observed in both male and female nicotine-exposed offspring. A number of OTUs belonging to the Lachnospiraceae family were increased in the nicotine-exposed offspring, suggesting increased energy extraction in these animals.However, there were no changes in beta diversity between treated and control offspring suggesting that nicotine-induced differences in the gut microbiota represents proportional alterations rather than an effect on the overall species richness in the gut. CONCLUSIONS: Maternal nicotine exposure resulted in differences in the microbial communities within the gut of the offspring; an effect that persisted into adulthood. Since changes in gut microbiota have been associated with increased weight gain and adiposity, these data suggest that alterations in the gut microbiome as a result of maternal nicotine exposure may explain, in part, the increased risk of obesity in children born to mothers who smoke.

Inhibition of MicroRNA-122 Improves Cholesterol Elimination in Female IUGR Rats Fed a High Fat Diet. Erin K Zinkhan, Baifeng Yu, Lisa Joss-Moore. Pediatric Neonatology, University of Utah, Salt Lake City, UT, USA. INTRODUCTION: Fetal intrauterine growth restriction (IUGR) often occurs in the context of a maternal high saturated fat diet (HFD). The combination of IUGR and a HFD (IUGR+HFD) persistently increases cholesterol levels in humans and rodent models. One pathway regulating cholesterol levels involves hepatic conversion of cholesterol to bile acids for elimination from the body. The rate-limiting step in the formation of bile acids is the enzyme Cyp7a1. Cy7a1 is inhibited by microRNA-122 (miR-122). Compared to non-IUGR HFD rats, we previously demonstrated that IUGR+HFD increases hepatic cholesterol, decreases hepatic Cyp7a1 protein, and increases miR-122 in juvenile female, but not male, rats. However, the importance of increased miR-122 in IUGR+HFD induced decreased cholesterol elimination from the body remains unknown. We hypothesized that in vivo inhibition of miR-122 in female juvenile IUGR+HFD rats would decrease hepatic cholesterol and miR-122 and increase hepatic Cyp7a1 protein abundance. METHODS: Adult female rats were fed a HFD prior to mating through gestation and weaning. IUGR was induced by uterine artery ligation at E19 of a 21 day gestation. At postnatal day 21 (D21) IUGR+HFD female rats were injected with either a miR-122 inhibitor or saline control and fed a HFD for an additional 5 days. At D26, we measured hepatic cholesterol with a colorimetric kit, Cyp7a1 protein abundance with western blot, and miR-122 with real-time RT-PCR. RESULTS: Compared to saline injected IUGR+HFD rats, miR-122 inhibitor injected IUGR+HFD rats had decreased miR-122 (p<0.05), increased Cyp7a1 protein (p<0.05), and non-significantly decreased hepatic cholesterol (p=0.1).

We conclude that miR-122 inhibition in IUGR+HFD female rats normalizes miR-122 and Cyp7a1 protein. We speculate that miR-122 is a causal link between IUGR+HFD and decreased cholesterol conversion to bile acids for elimination from the body. Implications of this study include potential utility of miR-122 inhibitors for improving cholesterol elimination from the body. Our next step is to determine the impact of miR-122 inhibition on long-term cholesterol physiology and potential toxicity. We previously identified differential gene expression in fetuses of obese compared to lean pregnant women in the second trimester, including significant upregulation of the gene Apolipoprotein D (APOD). APOD is specific to the central nervous system; its expression is increased in the setting of oxidative stress. We sought to investigate these findings in a mouse model of maternal diet-induced obesity. METHODS: Female C57BL/6J mice were fed either a 60% fat high-fat diet (HFD), or a 10% fat control diet (CD). Embryos were retrieved on embryonic day 17.5 and weights and crown-rump lengths (CRLs) were recorded. RNA was extracted from 10 male and 10 female embryonic brains per diet group. Quantitative RT-PCR was performed for brain apod, using gapdh and hprt as endogenous controls. Differences in apod expression were estimated with the ∆∆CT method. Mann Whitney testing was used to determine significant differences between groups; p£0.05 was considered significant. RESULTS: Embryos of obese dams weighed significantly less than those of CD dams, with males more affected than females (males 0.82 vs. 1.08 ±0.05 g, p=0.003; females 0.85 vs. 0.93 ± 0.06 grams, p=0.38). The CRL was significantly reduced in embryos of obese dams, males were again more affected than females (males 19.42 vs 21.76 ± 0.43 mm, p=0.003; females 19.5 vs 20.48 ± 0.53 mm, p=0.31). Apod expression was slightly increased in male embryos and decreased in female embryos of obese compared to control dams (p=0.09, Fig 1) . Within the HFD group, apod expression was significantly higher in male compared to female embryos (p=0.05). *Figure(s) will be available online. CONCLUSIONS: Maternal obesity is associated with significantly smaller male embryo size. Brain gene expression of apod is higher in male compared to female embryos of obese dams. This may reflect sexspecific differences in oxidative stress in the embryonic brain. Future evaluation of the effects of maternal obesity on fetal neurodevelopment should consider fetal sex.

of MRKH as only infrequent mutations in WNT4, LHX1, and HNF1B have been identified. The purpose of this study was to identify candidate genes in MRKH patients. METHODS: DNA of 88 MRKH patients, parents, and controls was studied using whole exome sequencing (WES). Since previous studies have shown repetitive deletions in chromosome 16p11.2 and 17q12 in multiple MRKH patients, these regions were given top priority along with variants seen in two or more affected individuals that had frameshift, nonsense, or splice site variants. Variants were confirmed by Sanger sequencing. RESULTS: A total of 45 variants were seen in at least two MRKH patients including five nonsense, 17 splice site, and 23 frameshift. These variants are currently being confirmed by Sanger sequencing. A heterozygous frameshift variant (4bp deletion) in ZNHIT3 on 17q12 was identified and confirmed in one MRKH female and her mother who had bradycardia, valvular heart disease, scoliosis, and rib abnormalities. ZNHIT3 has been reported to interact with retinoid receptors, defects of which may impair Mullerian development in mice. All protein coding exons and splice sites of ZNHIT3 are being sequenced in additional MRKH patients. CONCLUSIONS: If additional variants in this gene are identified, this will provide strong evidence for involvement of ZNHIT3 in mullerian development. Our findings indicate that next generation DNA sequencing may provide new clues for genes involved in the molecular basis of Mullerian development. Parental separation is known to increase the risk of ill-health in children and could also have long term health impact. Previous research has indicated associations between experiences of parental separation during childhood and inflammation in adulthood as well as elevated risk of pregnancy hypertension. This study explored childhood experience of parental separation and serum levels of interleukin (IL)-6 at preterm and term delivery in relation to current stress. METHODS: Blood samples were collected during labor from 23 women with preterm delivery and 36 women with term delivery with no pre-eclampsia or other systemic disease and IL-6 was quantified using Bio-Plex. Women filled out a questionnaire, including questions on family situation during childhood and if parents lived together or divorced/never lived together. Participants were also asked to rate their current experience of stress, using a visual analogue scale (VAS). A linear regression model was conducted with the variables Preterm/Term delivery, Family situation (intact family/separated parents) and current stress. RESULTS: Experiences of parental separation did not differ between preterm and term groups of women. In the preterm group five women reported having separated parents, whereas 14 women in the term group reported having separated parents. The mean stress did not differ between preterm and term women. The linear regression model showed a significant association between family situation and labor IL-6 (β=.31, p=.017). Preterm delivery was negatively associated with IL-6 (β=.-25 p=.046). Current stress was not associated with labor IL-6. *Figure(s) will be available online.

The results of this explorative study showed an association between childhood experience of parental separation and levels of pro inflammatory cytokines during delivery, occurring either preterm or term. This is in line with previous studies showing the importance of family relations during childhood on adult health. Further studies are needed to evaluate the importance of these findings regarding pregnancy outcomes. The objective of this study is to assess the correlation of unconjugated bisphenol A (uBPA) and bisphenol A-glucuronide (BPA-G) levels with pregnancy outcomes. METHODS: As part of an IRB approved prospective cohort study, twenty-four women presenting for care at the University of Michigan Medical Center were recruited. Patients had singleton gestations with spontaneous conception, and were greater than or equal to 18 years of age. Blood was drawn between 8 and 14 weeks gestation for evaluation of maternal BPA levels, which were measured using approaches described in a recent National Institute of Environmental Health Sciences round robin study (Vandenberg et. al., Environmental Health 2014, 13: 25) . Pregnancy outcome data were abstracted from the electronic medical record. Linear regression analysis was used to correlate uBPA and BPA-G levels with pregnancy outcomes.

In a linear regression analysis there was no significant correlation of uBPA or BPA-G levels with gestational age at delivery, birth weight in grams, birth weight percentile, maternal weight gain in pregnancy, or maternal BMI at the start or the end of pregnancy. 

Few studies have documented levels of BPA in the maternal circulation at the beginning of pregnancy. This study found no significant correlation of pregnancy outcomes with early pregnancy BPA levels. Larger cohort studies are needed to validate or refute concerns over the impact of maternal exposure to bisphenol A on pregnancy outcomes. We have shown that MPR during pregnancy reduces sperm concentration and impairs male reproductive capacity 1 . GRTH/ DDX25 is a testis-specific member of the DEAD-box family of RNA helicases, essential for completion of spermatogenesis 2 . Males lacking GRTH are sterile with absence of sperm 3 . However it is unknown whether GRTH/DDX25 expression can be affected by developmental programming by poor maternal nutrition. We investigated the effects of MPR during pregnancy on GRTH/DDX25 expression.

We studied male rat OFF whose mothers ate control (C) (20% casein) or restricted (R) (10% casein) isocaloric diet in pregnancy. After birth all rats ate C diet. At postnatal day (PND) 110 male OFF were

angiogenesis-related functions. Vitamin D significantly rescues these functions. These findings may have implications for vascular function of infants exposed to a diabetic intrauterine environment. INTRODUCTION: Transient UCO causes cerebral asphyxia and reoxygenation, which might influence fetal development and lead to clinical disorders later in life. UCO triggers the fetal hemodynamic response characterized by an increase in blood pressure (BP) and a decrease in heart rate (HR). This will allow a redistribution of the combined ventricular output (CVO) for survival. However, this response is temporary and fetal death can occur if the stimulus continues. We have previously reported that ketamine, a noncompetitive N-Methyl-Daspartate receptor antagonist, can modify the fetal hemodynamic response to ventilatory hypoxia and cerebral ischemia-reperfusion. We propose that treatment with ketamine will enhance the fetal hemodynamic response when exposed to UCO. METHODS: Fetal sheep were chronically catheterized at gestational day 125 (n=5-11/group), and an extravascular occluder was placed around the umbilical cord. Ketamine (3mg/kg) was administered intravenously to the fetus 10 min prior to the insult. UCO was induced by infusing saline into the occluder to limit the umbilical vein blood flow until fetal P a O 2 levels were reduced by 50%. Maternal and fetal blood gases, fetal HR, and BP were recorded throughout the experiments. Data was analyzed as a three-way ANOVA (factors: stimulus and treatment) with time as repeated measurements. RESULTS: UCO produced an initial increase on fetal BP in both control and ketamine groups (P=0.018 time), followed by a decrease in the control group whereas values remained higher with ketamine. HR values decreased after UCO (P=0.041 stimulus*time) in both groups, but the reduction was higher at the beginning of the experiment in control compared to ketamine groups. Fetal P a CO 2 levels increased after UCO (P<0.01 stimulus*time) but values were higher in the control versus ketamine groups. UCO significantly decreased fetal pH values (P<0.01 stimulus*time) with a greater effect on the control versus ketamine group. CONCLUSIONS: Ketamine improved the hemodynamic fetal response and partially reduced the levels of P a CO 2 after UCO. Higher fetal BP and HR values, compared to the control group, suggests a rise in the CVO leading to an increase in fetal blood flow, better oxygenation, and a higher pH. Ketamine might play an important role in reducing fetal mortality and morbidity during UCO episodes in human pregnancy.

The Hypoxia ischaemia (HI) due to neonatal asphyxia is the most common cause of acute mortality and chronic neurological disability. There is no effective means to repair the resultant damaged brain. The aim of this study was to investigate whether different maternal diets have effects on a clinically relevant model of perinatal HI in mice. METHODS: E2 mice were randomly assigned into 3 groups: a baseline diet group fed a 2.27% fat diet, a control group fed a 10.2% fat diet or a docosahexaenoic acid (DHA) enriched group fed on the 10.2% fat diet supplemented with 1.5% DHA as measured in relation to the total fat content of the diet. The same diets were fed to the pups after birth. Subsequently on the expected day of delivery (E18), pups were delivered either by vaginally (naïve group) or by caesarean section. This was further divided into a normoxic caesarean section (CS) group or with 15 min of hypoxic ischaemia caesarean section (HI group). HI was induced by placing half of the uterine horn, containing the pups, in a 37°C saline bath for 15 min. Maternal blood was collected at E18. Pups were sacrificed at different time points and the whole brain was collected.

The survival rates of mice pups at 1h after delivery were baseline diet CS and HI groups: 98.3% and 53.4%, control diet CS and HI groups: 95.5% and 31%, DHA enriched diet CS and HI groups: 98.4% and 37.9%, respectively. Fatty acid composition of experimental diets (Table 1 ) and in vivo samples (Table 2 ) are shown below. CONCLUSIONS: These data demonstrate that DHA transferred into the pup brain from the maternal diet. However, DHA supplementation did not improve the outcome of pups which suffered HIE. Moreover, a 10% high fat diet may increase inflammation in animals, potentiating the effects of HI. We will test this hypothesis in future experiments.

The if there are intra-uterine environment changes, fetus secretes CTL to adapt to the changes and hypersecretion of CTL may lead fetal growth restriction. The aim of current study was to investigate the reaction of CTL against intra-uterine inflammation and fetal circulatory insufficiency with using fetal sheep. METHODS: We used Suffolk sheep (n=15) and divided into sham group (n=6), inflammation group (n=5) and inflammation + hemorrhage group (n=4). Inflammation group was administered G-CSF and LPS to induce inflammation. Inflammation + hemorrhage group was withdrawn a 40 % of fetal blood after G-CSF / LPS to induce circulatory insufficiency. We monitored fetal ACTH and CTL at baseline, before LPS, before hemorrhage, 20min, 1, 6 and 24 hrs, respectively. RESULTS: There were significant differences in ACTH at 20 min and at 1 hour only in inflammation + hemorrhage group. Additionally, there were significant differences in CTL at before hemorrhage and 1 hour in inflammation group, and at before hemorrhage, 20 min and 6 hours in inflammation + hemorrhage group. CONCLUSIONS: This data showed that fetus secreted ACTH and CTL against hemorrhage. However, only CTL was increased against inflammation. This might indicate that inflammation directly lead the increase of CTL without the cascade of hypothalamic-pituitary-adrenal system.

DNA methylation is an epigenetic mechanism that can impact placental function, and we have undertaken an epigenome-wide association study to identify novel variation in DNA methylation associated with IUGR. METHODS: Genome-wide DNA methylation was profiled using the Infinium HumanMethylation450 BeadChip (Illumina; San Diego, CA) in 192 term infant placental samples collected as part of the Rhode Island Child Health Study (RICHS) cohort. All SNP associated, crosshybridizing, or sex associated probes were eliminated yielding ~351,000 sites for analysis. We compared samples from IUGR (n= 17) versus normal pregnancies (n= 175) following a reference-free correction to account for different cell proportions and inclusion of infant gender, maternal age, and gestational age as potential confounding variables. In addition, differentially methylation regions (DMRs) were identified using the R package DMRcate. Array results were validated by pyrosequencing, and qRT-PCR was used to determine correlations between gene methylation and expression. RESULTS: DMR identification yielded several regions with variable methylation levels between infants from IUGR and normal pregnancies. Among these was an approximately 2000bp window within the CRHR2 gene which acts as a responsive element to the master regulator of the HPA axis, corticotropin-releasing hormone. Differences in methylation were also determined at the single base pair resolution which yielded significantly different (FDR < 0.05) methylation levels at a CpG site within the 5'UTR of KRAS, an important regulatory element in cellular propagation and growth. CONCLUSIONS: In this study, we have identified candidate sites that due to altered methylation levels, may be playing a role in fetal growth restriction. The identified targets include genes not previously associated with a clinical risk of IUGR. Adequate placental delivery of long chain fatty acids (LCPUFA) is essential for normal fetal development and is regulated by placental uptake and metabolism. AMP-activated protein kinase (AMPK) is a key nutrient sensor in the cell and its phosphorylation is associated with fatty acid uptake in adipose tissue and skeletal muscle. Effect of AMPK stimulation on placental fatty acid uptake has not been reported. We hypothesize that AMPK activation in vitro will increase LCPUFA uptake and fatty acid transporters in human placental explants. METHODS: Explants were isolated from term placental tissue of lean (N=10) and obese (N=8) women with informed consent following caesarean delivery. Explants were incubated for 24hr with the AMPK activators resveratrol (100µM) and AICAR (1mM) and their respective vehicles. Following treatment, the uptake of 14 C-labeled palmitate (PA) oleate (OA), arachidonic acid (AA) and docosahexanoic acid (DHA) was measured. Expression of the placental fatty acid transporter genes CD36, FABPpm and FATP4 was assessed by QPCR. P value <0.05 vs vehicle control by t-test was considered statistically significant. RESULTS: Both resveratrol and AICAR treatment stimulated phosphorylation of AMPKα (catalytic subunit) in the explants. Resveratrol decreased AA and DHA uptake by ~50% (P<0.0001) compared to control while PA and OA uptake was unaffected (N=18). AICAR inhibited the uptake of PA (by 13%, P=0.04), OA (22%, P=0.001) and DHA (30%, P=0.0005) (N=13). Placental CD36 mRNA expression was increased following resveratrol (P=0.01) and AICAR (P=0.001) treatment, while the expression of FABPpm was not significantly altered. AICAR (P=0.001), but not resveratrol (P=0.10), increased FATP4 mRNA. Neither maternal obesity nor fetal sex altered these findings. Preliminary results show that resveratrol decreased the oxidation of 3 H-PA by ~50% in placental explants (N=3). CONCLUSIONS: Activation of AMPK led to a decrease in fatty acid uptake in term placental explants, contrary to our hypothesis. This lower uptake was associated with higher expression of the fatty acid transporters, CD36 and FATP4, suggesting that decreases in placental fatty acid transporter expression are not responsible for the decrease in LCPUFA uptake. Suppression of fatty acid oxidation, instead, may drive lower rates of uptake.

Determinants INTRODUCTION: Accumulation of intracellular cholesterol is highly cytotoxic, necessitating an adaptive regulatory mechanism. ABCA1 and ABCG1 are two critical mediators of cholesterol efflux, both transcriptionally activated by the nuclear receptor LXR and expressed in the human placenta. However, mechanisms regulating the cholesterol efflux function of these transporters to their target molecules (ABCA1 to ApoA1 and ABCG1 to HDL) has not been well delineated in the placenta.

Because the placenta uniquely interfaces with circulations of two distinct organisms, we hypothesized that these proteins may preferentially direct cholesterol transport to the maternal or fetal circulation. We aimed to localize ABCA1 and ABCG1 within the placenta and to elicit conditions that stimulate the efflux activity of these proteins in primary human trophoblasts (PHTs).

We performed immunohistochemistry to localize ABCA1 and ABCG1 in paraffin-embedded sections of human placental biopsies.

We also compared the effect of the LXR ligand T0901317 on efflux of radiolabeled [ 3 H]cholesterol to specific acceptor molecules in media of cultured PHT cells. RESULTS: ABCA1 is primarily expressed on the apical surface of synctiotrophoblast, adjacent to maternal blood in the intervillous space.

In contrast, ABCG1 is most abundant in the endothelial cells adjacent to fetal vessels. *Figure(s) will be available online.

In PHTs, T0901317 stimulated increased efflux of cholesterol to the ABCA1 target ApoA1, with no effect on efflux to the ABCG1 transporter target, HDL. CONCLUSIONS: Our findings suggest that ABCA1 is the predominant LXR-driven cholesterol efflux protein in trophoblasts. The localization of ABCA1 to the apical membrane of syncytiotrophoblasts indicates that it may preferentially mediate cholesterol efflux to the maternal circulation, while ABCG1, localized to placental endothelium, may mediate efflux to the fetal circulation. deficiency during pregnancy in mice is associated with intrauterine growth restriction (IUGR) and altered metabolism. We examined the effect of maternal Mg deficiency on placental nutrient transporter expression and nutrient uptake by placenta cells. METHODS: SW dams were fed diets with 100%Mg(control) or 10%Mg(MgD) from gestational day 6(GD6) until euthanasia(GD18) to produce moderate Mg deficiency. Expression of placental nutrient transporters was assessed by qRT-PCR. The effect of various Mg concentrations (25%-100%) on placental nutrient uptake was determined using 3 H-glucose and 14 C-MEAIB with BeWo placental cells. In vivo data were analyzed using Student's t-tests; in vitro data were analyzed by ANOVA/Bonferroni. RESULTS: MgD altered placental expression of genes associated with nutrient transport. MgD significantly decreased Slc7a5 gene expression (Table, p<0 .05), reduced Slc38a2, Slc38a4 and Glut-1 expression (not significantly) and increased Slc38a1 and Slc27a4 expression (not significantly). Amino acid and glucose uptake by BeWo placental cells were significantly impaired by Mg deficiency (Fig, p<0 .05). The formation of oxysterols, the products of cholesterol oxidation, is promoted by oxidative stress and hypoxia, common insults germane to FGR. Indeed, levels of oxidized LDL (oxLDL) particles are increased in the maternal, fetal and placental compartments of pregnancies affected by FGR. We have previously shown that in primary human tropholbasts (PHTs), the oxysterol 25-hydroxy cholesterol (25OHC) inhibits the transcription factor SREBP2, resulting in reduced expression of LDLR and HMGCR, key mediators of cholesterol uptake and synthesis, respectively. These transcriptional changes were associated with increasing rates of cell death. We aimed to further delineate the effect of cholesterol oxidation on placental lipid trafficking.

We used flow cytometry to measure the uptake of flourescently labeled LDL particles (DiI-LDL) in PHTs exposed to 10 µM of the oxysterols 7-keto cholesterol (7KC) and 25OHC, and to 100 mg/mL of oxLDL. To probe mechanisms of toxicity and determine if oxysterols promoted programmed cell death, we used Western immunoblotting to compare levels of cleaved PARP, a marker of apoptosis. (n=3 for all experiments)

RESULTS: 25OHC and 7KC caused a >95% inhibition of LDL uptake, and oxLDL reduced uptake of LDL by over 50%. 25OHC also caused a dose-responsive increase in levels of cleaved PARP. *Figure(s) will be available online. CONCLUSIONS: Products of cholesterol oxidation inhibit lipid uptake and promote programmed cell death in trophoblasts. These preliminary studies into determinants of placental lipid homeostasis and trophoblast toxicity support the need for further investigation into the influence of cholesterol oxidation on placental biology.

Changes Circulating RNA in the maternal bloodstream provides a means to examine dynamic changes of the fetus and the placenta in developing hypoxia.

Our aim was to determine whether changes in circulating hypoxiaassociated RNA corresponded to changes seen in placental explants and cytotrophoblasts under hypoxic conditions.

We performed a prospective cohort study of women with pregnancies complicated by preterm fetal growth restriction (FGR) requiring delivery before 36 weeks (n=14). We compared this to a group of gestation-matched controls (n=18). We quantified the expression of hypoxia-induced genes in the maternal blood using digital PCR (dPCR).

We compared the expression of these genes to placental explants (n=3) and primary cytotrophoblast cells (n=3) cultured under hypoxic conditions with quantitative PCR (qPCR) and dPCR.

We examined the expression of 4 key hypoxia-associated genes (HIF1A, LDHA, ADM, EPAS1) whose expression we have previously shown to be altered in response to states of hypoxia in pregnancy. We found that in placental explants HIF1A, LDHA and ADM showed significantly increased expression at 1% compared to 8% oxygen. In cytotrophoblast there was significantly increased LDH expression using qPCR and dPCR.

In patient blood samples we found LDH expression was significantly increased in pregnancies complicated by FGR measured by dPCR. *Figure(s) will be available online. Mean and S.E.M are graphed. CONCLUSIONS: The measurement of circulating RNA in the mother's blood stream represents a novel diagnostic approach to the problem of FGR. We have shown that changes in the expression of hypoxia-associated genes likely mirror the changes in placental tissues. ) at the expense of PPV (7.1%;CI 3.5-13). This was improved further by combination of UTAD with PMA(NPV 99.8%); LR-were 0.37 & 0.14 respectively. CONCLUSIONS: In an at risk population a normal UTAD and PMA effectively exclude severe early onset FGR. After normal assessment at 22-24 weeks, the risk of preterm FGR is very low (risk<1:500) and there is little benefit in serial ultrasound assessments. FGR requiring late pre-term delivery cannot be adequately excluded and repeat ultrasound examination at 35-36 weeks is required. A combination of PMA with UTAD is an effective way of reducing the number of 3rd trimester scans; we propose an algorithm for ultrasound assessment of high risk women that reduces scan frequency without compromising FGR detection. The aim of this study was to evaluate the usefulness of gestation corrected hyperuricemia to predict the recurrence of preeclampsia on subsequent pregnancy.

The retrospective study of 64 women who had previous preeclampsia and checked serum uric acid was analyzed. Gestation corrected hyperuricemia (GCH) was defined as being one standard deviation above the gestation-specific mean. And we used uric acid z-scores ([serum uric acid value -gestation specific mean] / standard deviation of the population) to account for gestation-specific alterations in uric acid and tested this as a continuous variable. P<0.05 was considered as significant. RESULTS: Of 64 women, seventeen had the development of recurrent preeclampsia (26.6%). 

admission, IUFD, cord pH <7.2, and 5 minute APGAR <7). Groups were compared using chi square, fisher exact or student t-test where appropriate with p<0.05 used as significance. RESULTS: 377 pregnancies were included. Group B was more likely than group A and group C to have PRH, adverse outcomes and delivered earlier. They delivered later than group D. Group C was more likely than group A to have PRH, and less likely than group D to have PRH, adverse outcomes and delivered later. 

Pre Patients were divided based on the highest 1 st trimester blood pressure. Group A; normal blood pressure (<120/80), group B; pre-hypertension (120-139/80-89) and group C; chronic hypertension (³140/90). Outcomes were pregnancy related hypertensive disorders (PRH) (gestational hypertension (gHTN) and pre-eclampsia (PEC)), Preterm birth, small for gestational age (SGA), intrauterine fetal demise (IUFD), composite maternal (GDM, PRH, PTB, PPH, ICU admission and chorioamnionitis) and composite neonatal outcomes (SGA, NICU admission, IUFD, cord pH <7.2, and 5 minute APGAR <7). Groups were compared using chi square, fisher exact or student t-test where appropriate with p<0.05 used as significance. RESULTS: There were 377 patients (table) . Compared to group A, group B was more likely to develop PRH, have an adverse outcome and deliver earlier. There were no differences between group B and group C. CONCLUSIONS: Pre-hypertension in the first trimester increases the risk for pregnancy related hypertensive disorders, adverse obstetrical outcomes and deliver earlier. 1 st trimester pre-hypertension should be considered a risk factor for pregnancy complications.

Pre-Hypertension in Early Pregnancy, What Is the Significance? Jonathan Rosner, Margaret Dziadosz, Terri-Ann Bennett, Cara Dolin, Allyson Herbst, Sarah Lee, Ashley S Roman. OB/GYN, NYULMC, NYC, NY, USA. INTRODUCTION: Determine the risk of adverse pregnancy outcomes in patients with pre-hypertension in early pregnancy (<20 weeks GA).

A retrospective cohort study of patients with a singleton gestation between 2013 and 2014 of a single practice. Patients were divided based on the highest blood pressure in early pregnancy. Group A; normal blood pressure (<120/80), group B; pre-hypertension (120-139/80-89) and group C; chronic hypertension (³140/90). Outcomes were pregnancy related hypertensive disorders (PRH) (gestational hypertension (gHTN) and pre-eclampsia (PEC)), Preterm birth, small for gestational age (SGA), intrauterine fetal demise (IUFD), composite maternal (GDM, PRH, PTB, PPH, ICU admission and chorioamnionitis) and composite neonatal outcomes (SGA, NICU admission, IUFD, cord pH <7.2, and 5 minute APGAR <7). Groups were compared using chi square, fisher exact or student t-test where appropriate with p<0.05 used as significance. RESULTS: There were 377 patients (table) . Compared to group A, group B was more likely to develop PRH, have an adverse outcome and deliver earlier. There were no differences between group B and group C. 

Maternal Resveratrol (Res) Administration During Pregnancy (P) Improves Anxiety Type Behavior in Male Offspring (OFF) Rats of Protein Restricted Mothers. Luis A Reyes-Castro

2 Elena Zambrano. 1 1 Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán

INTRODUCTION: Mullerian Aplasia (MA), also known as Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, is a congenital disorder of the Mullerian duct system characterized by the absence of the uterus and vagina. It is also commonly associated with renal agenesis, skeletal disorders, cardiac anomalies, and hearing loss. MRKH affects 1/5000 females and is the second most common cause of primary amenorrhea in adolescent girls. Currently, little is known about the genetic etiology euthanized and epidiymal and vas deferens sperm obtained to determine sperm concentration

RESULTS: At PND 110, sperm concentration (Fig 1A) and testicular DDX25 expression (Fig 1B) were reduced in R vs C OFF. At PND 110 FR was similar in R and C, but lower in R vs C at PND 450 (Fig 1C). ) sperm concentration, B) DDX25 mRNA expression, C) Fertility rate. Mean ± SEM, n=6, p<0.05 * vs C. CONCLUSIONS: MPR in pregnancy decreased male OFF testicular DDX25 expression representing a potential mechanism for the decline of sperm count and eventual loss of fertility. References: 1)

Expression. Toral Parikh, 1 Joe Devaney, 2 Brennan Harmon, 2 Shannon Sullivan, 1 Veronica Gomez-Lobo. 1,2 1 Obstetrics and Gynecology, Washington Hospital Center, Washington, DC, USA; 2 Genetics, Childrens National Hospital, Washington, DC, USA. Uterine tissues express efflux transporters, P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP, encoded by ABCG2 gene), but their role in pregnancy is unexplored. We hypothesize that P-gp and BCRP are involved in the differentiation of pregnant uterine muscle (myometrium) and that mechanical and hormonal stimuli regulate myometrial expression of ABCB1/P-gp and ABCG2/BCRP during gestation. METHODS: To test this hypothesis we measured the expression of P-gp and BCRP in the rat myometrium across gestation. Myometrial samples was collected from pregnant, laboring and post-partum (PP) Wistar rats (n=4-6/gestational day, GD), total RNA and protein was isolated for gene/ protein expression analysis using real-time PCR or Western blotting and localized with immunohistochemistry. To examine the effects of elevated progesterone (P4) during late gestation, rats were injected with P4 (16mg/ kg) or vehicle starting from GD20. The myometrial tissue from control and P4-treated rats were collected on GD21, 22, 23 (L), GD24 (only P4treated) and P-gp and BCRP expression was studied. The influence of early hormone withdrawal on P-gp and BCRP expression was examined by treatment of pregnant rats with P4 receptor antagonist RU486. We also used unilaterally pregnant rats to study ABCB1 and ABCG2 gene induction by static mechanical stretch. Pre-pregnancy and pre-delivery weights were used to calculate weight gain within or above IOM recommendations by BMI category. Racial categories were non-Hispanic white, non-Hispanic black, Hispanic, and Asian-American. Perinatal outcomes included GDM, preeclampsia, low birthweight, macrosomia (birthweight >4,500g), and nulliparous, term, singleton, vertex cesarean delivery. We fit multivariable logistic regressions for each outcome, controlling for maternal age, education, public insurance status, prenatal care initiation, and parity.

Fetal Origins of Heart and Vascular Disease: The Role of Xanthine Oxidase. C Beck, N Itani, K L Skeffington, D A Giussani. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom. INTRODUCTION : Chronic fetal hypoxia is one of the most common problems in high-risk pregnancy. We have reported that chronic hypoxia triggers a fetal origin of cardiovascular dysfunction secondary to the generation of oxidative stress. Accordingly, hypoxic pregnancy in rats induced oxidative stress in the fetal heart and vessels and maternal treatment with the antioxidant vitamin C during hypoxic pregnancy prevented these effects (Giussani et al. PLoS ONE 7 (2): e31017, 2012). However, the source of fetal oxidative stress in hypoxic pregnancy is unknown. Here, we tested the hypothesis that xanthine oxidase (XO)derived reactive oxygen species trigger a fetal origin of cardiovascular dysfunction during hypoxic development using the chick embryo. METHODS: Fertilized eggs were randomly divided and incubated under normoxia or hypoxia (14% O 2 ) from day 0 (term is 21 days). From day 13 of incubation, the XO inhibitor allopurinol (5 mg/kg -1 ) or vehicle (4M NaOH, 0.2ml) was injected daily into the air cell for six consecutive days. At day 19, the embryos were euthanised, weighed and measured and blood was obtained for haematocrit. Second order femoral arteries were isolated for in vitro wire myography and tested with acetylcholine (Ach) ± LNAME. Basal cardiac function was assessed in isolated hearts under Langendorff preparation. Comparison were assessed by Two-way ANOVA+Tukey test. INTRODUCTION: Mean birth weight increased from the 1950s until the 1990s. However a recent large, American study utilizing birth certificate data and a study of over 68,000 term singleton births at our institution showed a reversal of this trend over the last decade. We sought to determine if ultrasound estimates of fetal growth followed the same trend in this cohort. METHODS: A retrospective cohort study of women that had a full term singleton delivery between 2003 and 2013 who underwent a routine mid-trimester ultrasound between 16 and 24 weeks at Magee-Women's Hospital (MWH) were identified. Data were accrued from the Magee Obstetric Medical and Infant Database. Linear regression was utilized to evaluate the mean change per calendar year in head circumference(HC), biparietal diameter(BPD), abdominal circumference(AC)and femur length(FL). Models were then adjusted for maternal age, race, BMI, smoking, parity, fetal sex and gestational age at ultrasound. RESULTS: There were 102,259 deliveries at MWH from 2003-2013 and 46,028 ultrasounds from this cohort that met inclusion criteria. Mean gestational age at the time of ultrasound was 19.2±1.2 weeks. Although mean birth weight decreased over time(3458 g to 3395 g, P< .001) fetal growth measures assessed at the mid-trimester increased. There were statistically significant increases in mean HC (adjusted β=0.047 mm/yr, P<.001), AC (adjusted β=0.044 mm/yr,P<.001) and FL (adjusted β=0.016 mm/yr, P<.001) per calendar year. FL and AC showed the greatest yearly increase in mean ultrasound measure in the adjusted models. *Figure(s) will be available online. CONCLUSIONS: Despite a significant reduction in birth weight,fetal biometric measurements in the mid-trimester have increased over the past decade. This discrepancy suggests that the decline in birth weight is rooted in diminished intrauterine growth in the third trimester. Given the developmental origins of disease evidence that reduced growth in the second half of pregnancy is linked to offspring disease risk, the long-term health consequences of this finding warrants further study.

Fetal Growth Restriction in Isolated Single Umbilical Artery Singleton Pregnancies. Antonia P Francis, 1 Zainab Al-Ibraheemi, 1 Astrid Mondaca, 2 Ana Mrkaic, 1 Dawnette Lewis. 1 1 Obstetrics and Gynecology, Mount Sinai Roosevelt Hospital, New York, NY, USA; 2 Obstetrics and Gynecology, Bayfront Health, St. Petersburg, FL, USA. INTRODUCTION: To determine if isolated single umbilical artery (SUA) singleton pregnancy is associated with fetal growth restriction (FGR) and congenital cardiac abnormalities. METHODS: This is an IRB-approved, retrospective cohort study of pregnant women identified with isolated SUA during routine anatomy survey at Mount Sinai Roosevelt and Mount Sinai St. Luke's between January 2003 and December 2013. We collected ultrasound biometric values, serial EFWs from sonograms performed every two to four weeks, fetal echocardiography results, and actual birth weights. Fetal growth restriction was defined as EFW ≤ tenth percentile. RESULTS: Of 59,032 deliveries occurring during this time period, 376 (0.64%) were diagnosed with SUA at anatomy survey; of these, 332 (88.29%) were isolated SUA. Prior to delivery, 10 cases out of 332 (3%) were identified with FGR; 9 (90%) of these were small for gestational age (SGA) at time of birth. Two out of 332 (0.6%) had an abnormal fetal echocardiogram. CONCLUSIONS: Fetuses with isolated SUA are at increased risk of fetal growth restriction. At our institution, antenatal ultrasounds were able to identify as growth-restricted 90% of isolated-SUA fetuses that were SGA at birth. A very small percentage of these fetuses had cardiac abnormalities. Increased surveillance for fetuses with isolated SUA is recommended.

Maternal Metformin Attenuates Diet-Induced Changes in Fetal Liver Fatty Acid Metabolism. Kemoy Harris, 1 Neeraj Desai, 2 Madhu Gupta, 3 Burton Rochelson, 1 Christine Metz. 3 1 Maternal Fetal Medicine, Manhasset, NY, USA; 2 Maternal Fetal Medicine, Winnie Palmer Hospital, Orlando, FL, USA; 3 Immunology and Inflammation, Feinstein Institute for Medical Research, Manhasset, NY, USA. INTRODUCTION: Metformin(MET) mitigates hepatic lipid accumulation and improves lipid metabolism in the setting of diabetes/ metabolic syndrome. Because MET crosses the placenta, we examined the effect of maternal MET on fetal liver and brain fatty acid(FA) metabolism using a rat model of gestational metabolic syndrome. METHODS: Female Wistar rats (6wks old) were fed normal(NORM) or high fat/high sugar(HCAL) diets for 5wks. After mating with NORM males, half of HCAL dams received MET(300mg/kg,PO,daily); dams (6-8/group) continued their diets through euthanasia on gestation day 19. Fetal livers and brains were analyzed for triglyceride(TG) FAs and phospholipid(PL) FAs, respectively. Fetal livers were analyzed for FA metabolism-related gene expression. Data were analyzed using ANOVA/ Bonferroni. RESULTS: Prenatal HCAL exposure significantly decreased polyunsaturated FAs(PUFAs: 20:4 {arachidonic acid/AA}, 22:4n6, and 22:5n6) in fetal brains (p<0.05). Fetal livers showed significantly increased saturated FA(18:0) and monounsaturated FAs(MUFAs: 18:1n9, 18:1n7) and decreased PUFAs(18:2, 20:4{AA}, 22:6n3 {docosahexaenoic acid/ DHA}); exposure to MET significantly attenuated HCAL-induced MUFAs METHODS: Vaginal fluid was obtained from 43 women with BV, 52 with VVC and 77 with no vulvovaginal disorders. Diagnosis was based on microscopy, Nugent score, culture for Candida and clinical signs. Vaginal fluid concentrations of D-and L-lactic acid, alpha amylase, secretory leukocyte protease inhibitor (SLPI), neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP)-8 and hyaluronidase were measured by ELISA. RESULTS: Vaginal fluid alpha amylase was correlated with the vaginal concentration of D-(p=0.0020) but not with L-lactic acid (p=0.2746). Correlations were observed between alpha amylase and SLPI (p<0.0001), hyaluronidase (p=0.0003), NGAL (p=0.0020) and MMP-8 (p = 0.0049). The median vaginal fluid alpha amylase level was 1.94 mU/ml in control woman, 1.45 in women with VVC and 1.07 in women with BV (p=0.1704). CONCLUSIONS: Since D-lactic acid is produced primarily by Lactobacilli while L-lactic acid is produced by bacteria and host epithelial cells, the results suggest that the more alpha amylase is present the higher is the concentration of vaginal Lactobacilli. NGAL and SLPI have anti-bacterial properties while hyaluronidase and MMP-8, by promoting degradation of the extracellular matrix, increase glycogen availability. Their association with alpha amylase levels suggests that they are inducible in the vaginal epithelia by Lactobacilli or by products of glycogen metabolism. Exogenous alpha amylase supplementation may be a useful adjunct to promote growth of lactobacilli in women with BV.

Jossimara Polettini, 1 Camila Marconi, 1 Larissa D Marcolino, 1 Nathalia M Noda-Nicolau, 1 Moises D Lima, 2 Jacques Ravel, 3 Marcia G Silva. 1 1 Department of Pathology, Botucatu Medical School, UNESP, Univ. Estadual Paulista, Botucatu, SP, Brazil; 2 Department of Gynecology and Obstetrics, Medical Sciences Center, Paraiba Federal University, Joao Pessoa, PB, Brazil; 3 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA. INTRODUCTION: Vaginal predominance of Lactobacillus sp. represents a healthy state, in part by local immune induction. The shift to anaerobic bacteria predominance characterizes an abnormal vaginal flora, which triggers a local immune response imbalance. Natural antimicrobials, as beta defensins (HBDs), are involved in vaginal primary defense. We evaluated HBD1, 2, 3 and 4 according to bacterial community in the vagina based on the microbiome characterization METHODS: In this cross-sectional study, specimens of vaginal swab and lavage fluid were collected from 93 reproductive-age and nonpregnant women at primary medical care units in João Pessoa, PB, Brazil. The studied groups were defined according to the type of vaginal bacterial communities defined by high throughput sequencing of V3-V4 region of 16S rRNA gene. HBD 1-4 concentrations were measured in vaginal lavage fluid by enzyme-linked immunoassay, and compared by Kruskal-Wallis test, p < 0.05 significance. RESULTS: Eighteen samples were classified into bacteria community I (Lactobacillus crispatus predominance) and constituted the studied group 1. Groups 2 and 3 were established by 45 and 24 women presenting communities III (L. iners predominance) and IV (not dominated by Lactobacillus sp.), respectively. Only 2 and 1 samples represented communities II (predominance of L.gasseri) and V (predominance of L. jensenii), respectively. HBDs were evaluated in 87 samples. HBD1, 2 and 4 levels were not statistically different among the three studied groups, while HBD3 concentration was statistically lower in the group 3 compared to the group 2 (p=0.01). CONCLUSIONS: The studied population showed diversity in bacteria communities that had no effect on HBD1, 2 and 4 vaginal levels. The no Lactobacilli-dominated flora can trigger an anaerobic environment, which inhibits the HBD3 activity. As HBD3 is the most responsive defensin to microbial, the lower expression of HBD3 impairs the immune defense, enabling anaerobic bacteria proliferation. Identifying such mechanisms will contribute to the understanding of immune response deficiency according to diverse vaginal microbiota

Characterization of the Endometrial Microbiome. Shvetha M Zarek, 1 Jeffrey H Kim, 2 Matthew T Connell, 1 Torie C Plowden, 1 James H Segars, 1 Alan H DeCherney, 1 Erin F Wolff. 1 1 Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA; 2 Radiant Genomics, Inc, Emeryville, CA, USA. INTRODUCTION : Clinical dogma has held that the endometrium is a sterile environment, with infections occurring only in the pathologic setting. The advent of microbiome sequencing has revolutionized our ability to detect unidentified microorganisms. Our objective was to characterize the endometrial microbiome in healthy reproductive aged women using DNA sequencing. METHODS: In a prospective observational study, standard endometrial sampling via biopsy pipelle was performed on eleven healthy, reproductive aged volunteers who denied a history of infertility or gynecologic disease. Each sample was processed using a commercially available total DNA preparation kit (MoBio Inc. PowerSOIL TM max). 16s Illumina-compatible ribosomal RNA amplicons were generated from a hypervariable region of the 16s gene from each sample, in triplicate, using standard PCR cycling conditions. Each amplicon was quantitated via gel electrophoresis and Picogreen TM fluorometry prior to pooling at equimolar concentrations for paired-end (PE) 300bp Illumina sequencing. RESULTS: Quantitative Insights Into Microbial Ecology (QIIME) was used to perform phylogenetic analyses including beta-diversity and rarefaction analysis which revealed that the endometrium contains a core, conserved microbiome that is distinct from matched vaginal environments as well as from published vaginal microbiome profiles. At a phylum level, three dominant contributors to endometrial microbiome were identified: Bacteroidetes, Firmicutes, and Actinobacteria. Unweighted UniFrac TM analysis demonstrated that taxonomic composition was consistent between samples and did not vary by menstrual cycle phase. Most interestingly, the primary driver of endometrial composition was a previously uncharacterized Actinobacterium. CONCLUSIONS: Here we demonstrate for the first time that healthy endometrium is not a sterile environment, and report a straight-forward method to sample the endometrium without vaginal micriobiome contamination. We have identified a novel bacterium, which has never before been found that is the primary driver of microbial diversity in the endometrium. Ongoing studies are underway to sequence endometrium in the setting of infertility, which could become an important part of clinical testing in the future. NICHD, NIH

Initiation. Ilana Ramer, Tomi T Kanninen, Giovanni Sisti, Steven S Witkin, Steven D Spandorfer. Obstetrics and Gynecology and Perelman Institute for Reproductive Medicine, Weill Cornell Medical College, New York, NY, USA. INTRODUCTION: Components of the IGF system, IGF-1, IGF-2 and IGF binding protein-1 (IGFBP-1), enhance in vitro embryo quality and implantation rates in both animal models and in human IVF. We evaluated whether differences in serum levels of these components in women prior to initiation of an IVF cycle would be predictive of subsequent IVF outcome. METHODS: In this retrospective study sera from women obtained at day 2 of their IVF cycle (at baseline before stimulation) were assayed for IGF-1, IGF-2 and IGFBP-1 by ELISA. Samples from 54 women with a live birth, 38 with a transient biochemical pregnancy, 45 with a spontaneous abortion, 54 who did not become pregnant and 35 who had an ectopic pregnancy were available for analysis. Associations between the assays and outcome were evaluated by the Kruskall Wallis test. RESULTS: Median concentrations of IGF-1 were 29.1 ng/ml in women with a live birth, 25.1 in women with a biochemical pregnancy, 21.2 in spontaneous abortion, 18.7 when not pregnant and 4.2 in those with an ectopic pregnancy. Conversely, median levels of IGF-2 were reduced in women with a live birth (0.30 ng/ml) as opposed to 0.36, 0.41, 0.43 and 0.39 ng/ml in women with a biochemical pregnancy, spontaneous Saturday Posters abortion, not pregnant or having an ectopic pregnancy, respectively. Median IGFBP-1 concentrations were markedly elevated in women with a live birth (23.6 ng/ml) compared to 0.5, 14.1, 14.0 and 9.5 ng/ ml in women with a biochemical pregnancy, spontaneous abortion, not pregnant or with an ectopic pregnancy. Differences between groups on each test were highly significant (p<0.0001). CONCLUSIONS: Maternal serum levels of IGF-1, IGF-2 and IGFBP-1 at the start of an IVF cycle and prior to embryo transfer are correlated with the likelihood of a live birth vs. other outcomes. While there were no differences in the number of oocytes retrieved, oocyte quality or fertilization rates between groups it remains uncertain whether alterations in maternal IGF system components caused subtle changes in oocyte quality or early post-fertilization events. The role of maternal IGF system components in promoting optimal embryo implantation is another possible explanation for our observations. (VDR) . Polymorphisms in the VDR gene were correlated with cell proliferation and immune function but to date few studies have investigated the presence or absence of correlation between the VDR polymorphisms and fertility. Since female VDR knockout mice exhibit defects in follicular maturation we sought to investigate VDR polymorphisms frequency (BsmI, FokI and TaqI) in normal (fertilityproven) and in women undergoing fertility treatments.

The study cohort comprised 47 infertile women and 58 fertility-proven healthy control subjects from the general population. Genotypic analyses were done by the RFLP. The Qui-square and Odds ratio (GraphPAD, Prism) were used to compute the statistical difference between groups (p<0.05). RESULTS: The genotipic distribution for TaqI and FokI SNPs did not differ significantly between fertile and infertile subjects (p=0.6 and p=0,32 respesctively). The frequency of BsmI SNP, BB, Bb and bb were 19%, 33% and 48% in fertile patients and 23%, 34%, 43% in infertile patients respectively (p= 0,91), however the alleles frequency B and b were 36%, 64% in fertile patients and 61%, 39% in infertile patients respectively (p= 0,007). CONCLUSIONS: We aim to complete the study with a total of 300 women, however, our preliminary results detected an association between BsmI polymorphism and female fertility in the VDR gene. INTRODUCTION: AMH correlates with ovarian reserve and response to controlled ovarian stimulation. Accumulating data suggests that serum AMH level may also predict ART outcomes. However, AMH is elevated in PCOS women and it is unknown whether it may predict ART outcomes in this population. METHODS: Retrospective cohort study of 129 PCOS women (Rotterdam criteria) undergoing their first fresh IVF/ICSI cycle. Women were divided into 3 groups according to <25 th (low), 25 to 75 th (average), or >75 th (high) percentile of serum AMH concentration. Cycle stimulation parameters and reproductive outcomes were compared between groups. RESULTS: Women in the low AMH group were older than those in the average or high AMH (p<0.05), and required greater gonadotropin dose for stimulation compared to the high AMH group (p<0.05). Women with high AMH had greater testosterone level compared to women in the low or average AMH groups. No differences were noted between groups in terms of maximal E2, oocytes retrieved and fertilization rate. However, low AMH women had significantly greater live birth rates (p<0.05) and showed a trend towards greater clinical pregnancy rates compared to women in the average and high AMH groups (p=0.09). IR strongly correlates with BMI in a population of non-hispanic caucasian women; specifically a BMI less than 29 kg/m 2 is highly predictive of non-IR. They would likely not benefit from insulin sensitizers, and have a very low risk of gestational diabetes and metabolic syndrome. Thus the etiology of PCOS in the "lean PCOS" patient appears very different from the hyperinsulinemic, hyperandrogenism of Stein-Leventhal PCOS. INTRODUCTION: Polycystic ovarian syndrome (PCOS) is one of the most common reproductive disorders in women. PCOS is generally diagnosed by anovulation, hyper-androgenism, and/or the presence of multiple cysts within the ovaries. The etiology of this condition is poorly understood with its highly variable phenotypic presentation likely secondary to variations in genotypic expression. Long term risks of PCOS include insulin resistance and endometrial hyperplasia or cancer. Through this is a pilot study, we compared miRNA expression, small non-coding RNAs (19 -25 nucleotides) involved in gene expression, in patients with PCOS and healthy controls. METHODS: 12 adult women diagnosed with PCOS and managed without the use of any hormone modulating medications (>6 months) were recruited along with 12 aged-matched healthy controls. Patients had no other known medical co-morbidities that would confound the results obtained. Serum miRNA was isolated and miRNA PCR arrays (Nanostring nCounter Analysis System), and analyzed for changes in 800 commonly studied miRNAs. Background was designated as the mean of the negative controls plus 1 standard deviation and only significant data (p<0.05) was considered. Confirmatory tests performed using real-time PCR. RESULTS: After correcting for multiple testing using False Discovery Rate (FDR), two miRNAs survived (miR-16 and miR-181a). miR-181a had an elevated level in the healthy controls versus PCOS patients with a fold change of 1.8 (p-value = 3.5 x 10 -4 ) and miR-16 was elevated in PCOS patients versus controls (fold change = 1.6; p-value = 9.1 x 10 -4 ). If we did not use an FDR, 12 different miRNAs had a p-value < 0.05. Four of the miRNAs showed a lower value in PCOS patients versus controls and nine showed an elevated value in PCOS patients versus controls. CONCLUSIONS: These two miRNAs may serve as potential biomarkers for PCOS and may present targets for molecular therapy. miR-181a is known for its tumor suppressive properties which may explain the increased susceptibility of PCOS patients to endometrial hyperplasia and cancer. Similarly miR-16 is involved with glucose metabolism, with its up-regulation involved in insulin resistance in PCOS patients. We plan to validate these biomarkers in additional patients with analysis of endometrial expression.

Increased Fibulin-1 Plasma Levels in Polycystic Ovary Syndrome (PCOS) Patients: A Possible Contribution To the Link Between PCOS and Cardiovascular Risk. Elisa Scarinci, Anna Tropea, Francesca Moro, Federica Romani, Ornella Alesiani, Antonio Lanzone, Rosanna Apa. Obstetrics and Gynaecology, Università Cattolica del Sacro Cuore, Rome, Italy. INTRODUCTION: Fibulin-1 is an extracellular matrix (ECM) glycoprotein that interacts with several ECM proteins including elastic fibers and basement membranes. Its specific function is unknown, but in the last years fibulin1 is emerging as a new factor in the development or progression of cardiovascular diseases. In particular, high plasma levels of fibulin-1 have been detected in patients with type 2 diabetes (DM2) and correlate with hypertension and other cardiovascular risk markers, such as plasma N-terminal pro B-type natriuretic peptide and arterial stiffness. Several studies also reported a correlation with atherosclerosis and systemic inflammation, being fibulin-1 able to bind fibrinogen. Based on these findings, the purpose of our study was to investigate a possible relation between fibulin-1 and polycystic ovary syndrome (PCOS). PCOS is a heterogeneous condition characterized by several symptoms and clinical signs related to reproductive and cardiometabolic disorders. Interestingly, it is often associated with insulin resistance, DM2, obesity, dyslipidemia, hypertension, oxidative stress and inflammation, well-known cardiovascular risk factors. To this aim we investigated fibulin-1 plasma levels in euglycemic women with PCOS and in healthy age-and body mass index-matched controls who attended to Unit of human reproductive pathophysiology, Università Cattolica del Sacro Cuore, Rome. METHODS: Plasma was collected using EDTA, or heparin as an anticoagulant, and quantitative determination of human fibulin-1 was assayed by ELISA Kit.

Our preliminary results demonstrated that women with PCOS showed significantly increased plasma concentration of fibulin-1 when compared with control patients. CONCLUSIONS: These results could contribute to explain the hypothesized increased cardiovascular risk and vascular damage in patients with this syndrome. A better understanding of the cellular and molecular mechanisms involved in cardiometabolic disorders associated with PCOS is mandatory to identify new therapeutic strategies to eventually prevent the progression of cardiovascular diseases in these patients.

METHODS: We conducted a secondary analysis using data from a population-based cohort study of 1,359 women, aged 20-44, diagnosed with invasive breast cancer in the three-county Seattle region, 2004-2010. A subset of women (n=458) from the original cohort was recontacted and reinterviewed, collecting detailed data related to oncofertility treatment and reproductive history. Statistical analysis was completed using Wilcoxon, Fisher's exact, or chi square. RESULTS: Of the original cohort who provided oncofertility information, 4.9% (22/454) sought oncofertility treatment or counseling. 18.2% had gonadotropin injections, 22.7% had oocyte cryopreservation, 27.3% had embryo cryopreservation, and 13.6% had an oncofertility consultation but did not pursue treatment. There were 366 women with complete information regarding parity at cancer diagnosis; of these, 47 were nulliparous and 319 parous. The nulliparous and parous groups were similarly aged (median 40.9 and 41.4 years, p=0.24) and did not differ for racial (p=0.40) or ethnic (p=0.64) distribution. More women in the nulliparous group tended to have Stage I disease, however this was not statistically significant (74.5% vs 53.3%, p=0.06). Triple negative receptor tumor type did not differ between the groups (29.8% vs. 20.8%, p=0.19) . Oncofertility treatment was pursued more often by nulliparous women than parous women, 17% (8/47) versus 1% (3/317), respectively, p<0.001. More nulliparous women became pregnant following a breast cancer diagnosis than parous women, 15% (7/47) versus 2% (6/319), respectively, p<0.001. CONCLUSIONS: Overall, the number of women who sought oncofertility treatment or counseling in this cohort was low, and the underlying reasons for this warrant further investigation. Parity status at the time of breast cancer diagnosis significantly influenced pursuit of oncofertility treatment and subsequent pregnancy attainment in young women with breast cancer. INTRODUCTION: Fertility preservation is being offered clinically for many women with cancer. In treating these women, many clinicians adjust traditional ART protocols to meet time restrictions the patient and her treating oncologist may be working with or to avoid estrogen elevations in estrogen-sensitive cancers. While innovative, the published data on the outcomes of such practices are lacking. Our objective was to address this knowledge gap through evaluation of our own center's experience. METHODS: This was a retrospective cohort study of fertility preservation cycles for women with cancer performed in our center between '01 and '13. We included cases involving either oocyte or embryo cryopreservation. Our exposures of interest included controlled ovarian stimulation (COS) starting in the luteal phase and use of letrozole. Our primary outcome was number of mature oocytes retrieved. Secondary outcomes included duration of COS (days) and peak estradiol (E2) levels (pg/ml). Analyses were performed using appropriate bivariate statistics in SPSS. RESULTS: 46 women were identified. 10 of these women took letrozole during stimulation and 5 of the women started their stimulation in the luteal phase. 6 women had both adjuvants. Women receiving letrozole had significantly lower peak E2 levels compared to women who did not (549.0 ± 292.5 vs. 2246.4 ± 1052.3, p<0.001) while number of mature oocytes and days of COS were no different (11.3 ± 6 vs. 12.1 ± 6.3, p=0.7; 10.5 ± 2.5 vs. 9.6 ± 2.4, p=0.2). Outcomes for women who initiated stimulation in the luteal phase of their cycle were no different than women who started stimulation in the follicular phase per routine. (mature oocytes: 11.2 ± 6.3 vs. 12.5 ± 5.7 p=0.6; Duration of COS: 10.2 ± 2.6 vs. 9.9 ± 2.3, p=0.7; peak E2: 1765.7 ± 0.2 vs. 1263.4 ± 1482.1, p=0.3 ). CONCLUSIONS: Adjuvants including letrozole during COS and initiation of COS in the luteal phase yield excellent results for women with cancer undergoing fertility preservation. 

We find that maternal carriage of HY restricting HLA class II alleles decreases the chance of live birth among women with SRPL after the birth of a boy. The present study extends our previous findings from first pregnancy after referral to cumulative outcome. In addition to the confirmation of HLA-DRB1*15 and HLA-DQB1*05:01/02 as prognostically negative for SRPL after the birth of a boy, we report the same for HLA-DRB1*07. Our findings support the hypothesis that an aberrant immune response to the semi-allogenic fetus by the mother's immune system may play an important role in SRPL following the birth of a boy. We hypothesized that the administration of rosiglitazone, a PPARγ agonist, in mice may rescue NKT activation-induced PTB. METHODS: Pregnant B6 mice were injected with α-GalCer (2µg) followed by a rosiglitazone (10mg/kg) injection during late gestation (n=14). Control mice were injected with α-GalCer, rosiglitazone, or DMSO (n=10-20 each). Following injection, mice were monitored via video camera to determine gestational length and stillbirth rate. Additionally, pregnant mice were euthanized 4h (n=6-10 each) and 22h (n=8-10 each) post-injection of rosiglitazone, including the corresponding controls. Activation of T cells, neutrophils, DCs, and M1 and M2 macrophages were determined by flow cytometry in the liver, uterine, and decidual tissues. Expression of cytokines/chemokines and PPARγ targets was determined by RT-PCR in decidual and uterine tissues. Serum cytokine and chemokine concentrations were determined by multiplex arrays. RESULTS: Administration of rosiglitazone to mice injected with α-GalCer (1) rescued NKT activation-induced PTB, increasing the gestational length and improving the stillbirth rate;

(2) reduced T-cell activation in the uterine tissues and liver; (3) reduced the activation of dendritic cells and neutrophils in the decidual and uterine tissues; (4) reduced the number of infiltrating IL10-producing M2 and M1-like macrophages in the decidual tissues; (5) down-regulated the expression of Ccl2, Ccl1, Ccl12, and TNF by decidual tissues; (6) down-regulated the expression of Ccl2 in the uterine tissues; (7) up-regulated the expression of Fabp4 in the uterine tissues; and (7) increased the systemic concentrations of IL10, IL17, IL5, and VEGF. CONCLUSIONS: Rosiglitazone rescues NKT activation-induced PTB by rapidly reducing the activation of T cells, M1 and M2 macrophages, and neutrophils at the maternal-fetal interface and by suppressing the local and systemic pro-inflammatory response in the mother. These findings demonstrate that the PPAR-γ pathway is a new molecular target for future preventative methods for PTB. (CD4+IFNγ+), Th2 (CD4+IL10+), Th9 (CD4+IL9+), Th17 (CD4+IL17+), and Tc17 (CD8+IL7+) cells were determined by flow cytometry. Plasma progesterone, estradiol, and cytokine concentrations were determined using ELISA or multiplex assay. RESULTS: 1) Administration of hCG increased the number of M2-like macrophages (p=0.035) and reduced the number of M1-like macrophages (p=0.043) in the decidual tissues; 2) administration of hCG increased the proportion, but not the number, of decidual Th17 cells (p=0.015); 3) administration of hCG diminished the number of Th9 (p=0.043) and Tc17 (p=0.05) cells; 4) plasma IL1β concentrations were higher in mice injected with hCG than in controls (p=0.043); and 5) no differences were observed in plasma progesterone and estradiol concentrations between mice injected with hCG or PBS. CONCLUSIONS: Administration of hCG in pregnant mice creates an anti-inflammatory micro-environment at the maternal-fetal interface by reducing M1-like macrophages and increasing M2-like macrophagesan M1-M2 polarization. Administration of hCG in pregnant mice also prompts an anti-inflammatory response in the maternal circulation by reducing pro-inflammatory Th9 and Tc17 cells. These events were not associated with plasma progesterone and estradiol concentrations. These results provide further information regarding the immune mechanism whereby hCG maintains pregnancy and prevents preterm birth. hypothesised that the fetal skin would produce LL-37 in response to inflammatory stimuli, and that this might contribute to intrauterine and fetal inflammation. The aim of this project was to determine if fetal skin expressed LL-37 and to explore the response to the inflammatory stimulus lipopolysaccharide (LPS). METHODS: Skin samples were collected from the torsos of 16 human fetuses after medical termination of pregnancy between 13 and 20 weeks gestation, and frozen for immunohistochemistry. Human fetal keratinocytes were cultured using an explant outgrowth technique, and treated with LPS 1 mg/ml or untreated as control. Cells were harvested at 3 and 24 hours. Fetal ovine skin was obtained from date-mated Australian Merino ewes (term = 148+/-2 days) bred to have singleton pregnancies. Animals were injected with 10 mg intra-amniotic lipopolysaccharide (LPS) or control 5, 12, 24 or 48 hours before delivery at 124 days. RNA was extracted from cultured human keratinocytes and ovine skin, and cathelicidin gene (CAMP) expression analysed by Taqman PCR. RESULTS: hCAP/LL-37 was immunolocalised in the epidermis of human fetal skin at all gestations examined. LPS stimulation of human fetal keratinocytes resulted in downregulation of CAMP (p<0.05) despite increasing other inflammatory chemokines such as IL-8. SMAP-29 (sheep cathelicidin orthologue) transcription was also decreased after 24 hours of intrauterine LPS treatment (p<0.05). CONCLUSIONS: The unexpected reduction in cathelicidin transcription by fetal skin in response to LPS may contribute to preterm babies' vulnerability to skin infections. Further study could help identify whether cathelicidin could be a useful therapy to prevent newborn skin infections.

Labor: An Influx of CD8 T EM Cells at the Maternal-Fetal Interface. Yi Xu, 1 Roberto Romero, 1 Tippi C Mackenzie, 2 Nardhy Gomez-Lopez. T cells reside at the maternal-fetal interface (decidua basalis and decidua parietalis) throughout pregnancy. An influx of memory-like CD45RO+CD4+ T cells at the maternal-fetal interface is associated with the process of spontaneous labor at term (Gomez-Lopez, et al., Am J Reprod Immunol 2013) . However, it is unknown whether these T cells express markers of central or effector memory, and whether this T-cell repertoire is altered at the maternal-fetal interface of women who undergo spontaneous preterm labor/birth. METHODS: Decidual basalis and decidua parietalis were isolated from women who delivered at term with (sTL, n=23) and without (TNL, n=13) spontaneous labor, and preterm with (sPTL, n=21) and without (PTNL, n=10) spontaneous labor. Leukocytes were isolated by enzymatic dissociation from decidual tissues. Naïve (CD45RA+CCR7+), central memory (CD45RA-CCR7+, T CM ), effector memory (CD45RA-CCR7-, T EM ) and CD45RA+ effector (CD45RA+CCR7-) T-cell populations were determined by flow cytometry, within the CD3+CD4+ or CD3+CD8+ gate. A p-value of 0.05 was considered significant. RESULTS: 1) A differential T-cell distribution was observed in the decidua basalis compared to the decidua parietalis; (2) the proportion of CD4 T EM cells in the decidua basalis was higher in the sTL than in the TNL group, while the proportion of naïve CD4 T cells was lower; (3) the proportion of CD8 T EM cells in the decidua basalis was higher in women with sPTL than in those in sTL, while the proportion of naïve CD4 and CD8 T cells was lower; and (4) the proportion of CD8 T EM cells in the decidua parietalis was lower in women with sPTL than in the PTNL group. CONCLUSIONS: Spontaneous term labor is characterized by an influx of CD4 T EM cells, whereas spontaneous preterm labor is characterized by an influx ofCD8 T EM cells at the maternal-fetal interface. These results suggest that spontaneous preterm labor is associated with the infiltration of cytotoxic T cells that previously encountered and responded to a specific antigen (i.e., the paternal-fetal antigen). Our findings provide further evidence that maternal-fetal rejection is one of the mechanisms of disease in the pathogenesis of spontaneous preterm labor. Preterm neonates are at an increased risk for shortterm complications due to the immaturity of multiple organ systems, and are highly susceptible to infection. It was recently shown that the neonatal host defense against infection is maintained by a temporal presence of immunosuppressive CD71+ erythroid cells (Shokrollah et al., Nature 2013) . The aims of this study were to investigate (1) whether there are fewer CD71+ erythroid cells in the cord blood of premature newborns than in term newborns, and (2) whether these cells are present in the maternal periphery and reduced in women who undergo preterm labor/birth. METHODS: Maternal peripheral blood and umbilical cord blood were collected from women who delivered: 1) preterm with labor (PTL, n=19), 2) preterm without labor (PTNL, n=11), 3) at term with labor (TL, n=45), and 4) at term without labor (TNL, n=21). Mononuclear cells were isolated from these samples by density gradient centrifugation using the Ficoll Paque Plus medium. CD71+ erythroid cells were identified as CD235a+CD71+ within the CD3-gate by flow cytometry as proportions and numbers. Acquisitions and analyses were performed on a BD LSRII flow cytometer system, using FACSDiva™ v6.0 software. A p-value of £0.05 was considered significant. RESULTS: 1) The proportion of CD71+ erythroid cells was 100-fold higher in cord blood than in maternal blood; 2) fewer CD71+ erythroid cells were found in cord blood from women who underwent PTL than from women who delivered PTNL; 3) fewer CD71+ erythroid cells were seen in cord blood from women who underwent TL than from women who delivered TNL; 4) no differences were seen in cord blood CD71+ erythroid cells between women who underwent PTL and women who delivered TL; and 5) fewer CD71+ erythroid cells were seen in maternal blood from women who underwent PTL than from women who delivered PTNL. CONCLUSIONS: Term and preterm labor are characterized by a reduction of CD71+ erythroid cells in umbilical cord blood. Preterm labor is also characterized by a reduction of CD71+ erythroid cells in the maternal circulation. These novel findings suggest that a premature reduction of CD71+ erythroid cells in the cord blood and maternal blood may contribute to the process of preterm labor. Cervical insufficiency is a risk factor for preterm birth characterized by premature dilation of the cervix in the absence of uterine contractions. Organization of fibrillar collagen in the extracellular matrix determines the biomechanical properties of the cervix. Collagen fibrillogenisis (synthesis, processing and assembly) is a complex process that is regulated in part by proteoglycans such as decorin. The focus of this study was to determine if decorin regulates collagen fibrillogenisis and ultimately cervical function during pregnancy. METHODS: Decorin transcripts and protein were analyzed. Decorin null mice were assessed. The ultrastructure of collagen was analyzed through transmission electron microscopy. Biomechanical testing was conducted to assess stiffness and strength. RESULTS: Decorin mRNA and protein expression were constant in nonpregnant (NP) and pregnant cervix. Protein expression was restricted to stroma. Decorin null females had normal parturition and cervical collagen fibril ultrastructure appeared similar to wild type cervix before (gestation day 15) and during (day 18) cervical ripening. Unexpectedly and in contrast to the decorin null late pregnant cervix, the collagen ultrastructure in the NP and gestation day 6 decorin null cervices was abnormal with irregular shaped fibrils of variable size interspersed between normal fibrils. Consistent with the timing of defects in collagen ultrastructure, the NP and early pregnant (day 6) decorin null cervices were mechanically compromised with reduced strength and stiffness. Consistent with normal collagen ultrastructure in late pregnancy, biomechanical parameters were normal in decorin null cervices on gestation day 15 and 18. CONCLUSIONS: Decorin regulates cervical collagen architecture and consequently its biomechanical properties differently during different stages of reproduction. Specifically, this study demonstrates a role for decorin in guiding collagen fibrillogenesis in the NP and early pregnant cervix. This timing corresponds with the activity of the collagen cross-link forming enzyme, lysyl oxidase (LOX). Ongoing experiments will address the interaction of decorin with LOX in regulating collagen fibrillogenesis and test the hypothesis that lack of decorin in early pregnancy may be a risk factor for cervical insufficiency.

Pregnancy Outcomes in Primiparous Women With a Shortened Cervix: Cerclage Vs. Expectant Management. Marc Parrish, Maitreyi Salpekar, Gene Lee. Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, KS, USA. INTRODUCTION: There is minimal data regarding pregnancy outcomes in primigravida or nulliparous women with a shortened cervix who have undergone cerclage placement. Many previous studies evaluated cerclage efficacy in women with a shortened cervix and a history of preterm birth. Although some of the investigations evaluated all women with a shortened cervix, none exclusively evaluated the outcomes of those women who were defined as primigravida or nulliparous without a pregnancy lasting beyond 14 weeks. We sought to determine if these subsets of women would benefit from cerclage placement in the presence of a shortened cervix. METHODS: This investigation is a retrospective cohort study involving data collected between 01/01/2008-06/01/2014 in women whose cervical length measured < 25 mm prior to 24 weeks gestation. Additional inclusion criteria involved any pregnant, primigravida women and/or nulliparous women who had never carried a pregnancy beyond 14 weeks gestational age. Our primary outcome involved comparison of those women who received cerclage vs. expectant management, and the length of time from diagnosis of short cervix < 25 mm and delivery between the two groups. Secondary outcomes include evaluation of preterm delivery rates at certain GAs and outcomes based on a range of cervical lengths. We also compared outcomes in women receiving vaginal or IM progesterone to women who had a cerclage or those who were being expectantly managed.

In primigravida women or those women with a history of pregnancy loss occurring at <14 weeks, who were observed to have a short cervix between 14-24 weeks GA: placement of a cerclage led to a shorter period of pregnancy latency when compared to expectant management or progesterone therapy (Cerclage 85 days vs. Expectant management 116 days; p=0.02) (Cerclage vs. progesterone 112 days; p=0.04). These same subsets of women had a higher risk for delivery prior to 32 weeks if a cerclage was placed compared to those who were expectantly managed (R.R 6.7 [1.45-30.6]) and when compared to those women who had vaginal or IM progesterone intervention (R.R. 3.7 [1.0 to 13.4]). CONCLUSIONS: Our study demonstrates no benefit of cerclage placement in primigravida women or nulliparous women who have never carried a pregnancy beyond 14 weeks gestational age. Until further investigations can be performed with a larger sample size, our data would indicate that cerclage placement should be discouraged in these subsets of women. Recently, the role of inflammatory cells that migrate into the amniochorion environment has been proposed as a mechanisms of induction of connective tissue degradation of these structures, leading to rupture. Objective. The aim of the present study was to develop an in vitro model of the ACM rupture event that emulates the actual rupture sequence. METHODS: Full-thickness human ACMs were obtained after delivery by elective C/S from women at 37-40 weeks of gestation, with no evidence of active labor. The membranes were cultured in a two compartment experimental model in which amnion was facing the upper compartment and chorion to the lower chamber. The ACMs were subjected to a mechanical stress of 5.1 g/cm 2 along all incubation period. Membranes were treated with bacterial collagenase to develop a reference model of the kinetics of rupture of the membranes. Other tissues were coincubated with preparations of leukocytes isolated from the choriodecidual compartment of ACM obtained from women with labor, and without labor. The ACMs were observed during 48 hours after the stimulus until they were broken. We quantified active matrix metalloproteinase (MMP-9) in every experiment. RESULTS: Ten different experiments in duplicates revealed that all membranes were ruptured after 6 h in the presence of bacterial collagenase. Treatment with leukocytes isolated from women in labor using concentrations from 1 to 5 million cells per well resulted in the rupture of the membranes in a range from 24 to 48 h (n=18). Cells obtained from non-laboring tissues failed to induce rupture of the membranes after 48 h of incubation. Higher concentration of active MMP-9 was associated to tissues in which rupture was observed. CONCLUSIONS: It was possible to develop an in vitro model of the rupture of the membranes using co-incubation with leukocytes isolated from membranes obtained from women with active spontaneous labor. This is the first model of rupture of the membranes including a potential mediator of the degradation of the connective tissue. This model will allow the analysis of the sequential events leading to the rupture of the membranes. Gilner, Stephanie Pierce, Thomas Price, Chad Grotegut. Obstetrics & Gynecology, Duke University, Durham, NC, USA. INTRODUCTION: We encountered a patient with a non-contractile uterus. She had a germ line mutation of a T-type calcium (Ca 2+ ) channel protein (Cav3.1), but no other evident clinical/genetic abnormalities. We hypothesized that a T-type Ca 2+ channel plays a critical role in uterine contractility, which may be useful in development of a uterine-specific tocolytic therapy. We used a preferential T-type Ca 2+ channel antagonist to assess the role of these channels in murine uterine contractility. METHODS: Mibefradil (Mib) is a Ca 2+ channel antagonist with high affinity/specificity for low-voltage-activated T-type Ca 2+ channels. Uterine tissue from non-pregnant and term pregnant wild-type C57BL/6J mice was suspended in a tissue bath to measure contraction force. The effect of Mib on spontaneous and oxytocin-induced uterine contractility was expressed as percentage of baseline contractility. Dose response curves were constructed using nonlinear regression and the best-fit curves compared between treatment groups (4 mice/group). Figure( s) will be available online. CONCLUSIONS: Mibefradil, a preferential T-type Ca 2+ channel antagonist, decreases spontaneous uterine contractility in a non-pregnant and pregnant murine model. Furthermore, Mibefradil markedly decreases oxytocin-induced contractility in pregnant tissue. In vivo work is needed to determine the effect of Mibefradil on preterm labor.

The gene expressions of both P-gp and BCRP increased throughout gestation, peaked at around GD19-21 ( 31.7 and 9.1 fold increased, respectively), dropped on GD23L and 1 day PP. Treatment with the P4 receptor antagonist RU486 significantly decreased ABCB1 and ABCG2 mRNA levels, whereas artificial maintenance of elevated P4 prevented the fall of ABCB1 and ABCG2 genes. Moreover, both genes were up-regulated only in gravid but not in the empty horns of unilaterally pregnant rats. Protein expression levels of BCRP but not P-gp paralleled the transcript levels. CONCLUSIONS: Our data demonstrate significant changes of P-gp and BCRP in rat myometrium through pregnancy; their exact role in the myometrial differentiation is still under investigation. Our lab has defined the endoplasmic reticulum (ER) stress response and it related unfolded protein response (UPR) to be a critical players in regulating gestational length. An integrated ER stress response maintains uterine quiescence by disabling the contractile architecture in a caspase-3 (CASP3)-dependent manner 1,2 . In contrast at term the induction of the unfolded protein response blocks both ER stress and CASP3 activation allowing the pregnant uterus to regain its contractile potential. More recently the ER stress and the UPR have been implicated in autoimmune and inflammatory diseases. Therefore the goal of this study was to assess if the markers of ER stress and the UPR are present in the circulating peripheral blood mononuclear cells, (PBMCs) across gestation. As in co-operation with endocrine factors, circulating PBMCs maintain a functional cross-talk with the maternal-embryo interface, therefore we speculated the PBMCs may recapitulate the ER stress-UPR profile observed in the pregnant uterus. METHODS: Blood and uterine tissues were obtained from timed pregnant CD-1 mice on gestation day (E)6-19. PBMCs were isolated from heparinized blood by Histopaque-Ficoll density gradient separation. Uterine cytosol and nuclear protein extracts were prepared as described previously 1 . The markers of ERSR were evaluated by immunoblot analysis. RESULTS: Immunoblot analyses revealed that DDIT3, a marker of ER stress response and CASP3 activation maintained a similar gestationally regulated profile in the PBMCs as that observed in the pregnant uterus with dramatic increases found between E6-E13 which declined towards term (E19). Similarly the chaperone protein GRP78 a marker of adaptive UPR was found to increase later in gestation at E12 -E18 in both the pregnant mouse uterus and the circulating PBMC's. CONCLUSIONS: Taken together, we have identified a conserved stressresponse in both the isolated PBMC's and gravid uterus across gestation in pregnant mice. We believe that a gestational profile of PBMC-ERSR across gestation might serve as a readout of normal progression of pregnancy and has the potential to act as a potential predictive marker of preterm labor. Acute cAMP elevation has previously been shown to promote relaxation in contracting human myometrium tissue strips in vitro, prompting interest in the use of cAMP agonists in preventing preterm labour. However, we have previously shown cAMP analog 8-bromo-cAMP (8-Br-cAMP) can promote spontaneous contractions after 24 h exposure. We hypothesised 8-Br-cAMP-mediated cAMP elevation promoted contractions due to the absence of P4. METHODS: Myometrial tissue obtained from non-labouring women undergoing caesarean section at term with written consent (n=16) were incubated in serum-free DMEM under tension for 24 h with 1 mM 8-Br-cAMP or H 2 O vehicle and either 10 µM P4 or ethanol vehicle. Contractile activity (spontaneous and oxytocin-induced, 1 nM), expressed as mean integral tension (MIT), was measured in an isometric organ bath. PKA activity was determined using a Peptag PKA activity assay kit. Western blot determined differences in labour-associated and cAMP signalling protein levels. All data presented represent n=8. Mean ± SEM data were analysed using Wilcoxon test or paired t-test (significance at P<0.05). RESULTS: 24 h exposure to 8-bromo-cAMP alone increased spontaneous contractions (+850.6 ± 410.7 % vs vehicle, P<0.01) and PKA activity (+102.7 ± 37.8 % vs vehicle, P<0.01). This effect of 8-Br-cAMP on spontaneous contractions was reduced by P4 when it was present both during the 24 h pre-treatment period and in the organ bath (-47.6 ± 12.9 % vs 8-Br-cAMP alone, P<0.05); co-treatment for 24 h alone was not sufficient to block contractions promoted by 8-Br-cAMP. P4 exposure did not alter PKA activity. COX-2 protein abundance was reduced by P4 treatment alone (-24.2 ± 18.2 % vs vehicle) and in co-treatment with 8-Br-cAMP (-36.1 ± 14.2 % vs vehicle), but to only borderline significance (P=0.106 and P=0.055, respectively) in tissues where P4 was present beyond the 24 h incubation period. CONCLUSIONS: P4 reduces 8-Br-cAMP-associated increase in spontaneous contractions but only when constantly present in the extracellular environment and without altering PKA activity. Further studies into the effect of P4 concentration and duration of exposure, as well as its effects on sub-cellular cAMP changes, are needed to determine if P4 and cAMP signalling interact to create the balance between myometrial quiescence and labour onset.

HoxA13 Is a Master Regulator of Regional Gene Expression and Contractile Function in the Pregnant Human Myometrium. INTRODUCTION: Bipedalism, a key evolutionary step that separates humans from most other animal species, results in significant physiologic challenges, particularly with respect to the maintenance of pregnancy and induction of parturition. Thus it was necessary to establish a contracted lower uterine segment (LUS) and a relaxed fundus (FUN) during pregnancy in order to prevent pressure on the cervix from the fetal head, due to gravity. With the onset of labor, this regionalization of myometrial function needed to be reversed, with the FUN becoming contractile and the LUS relaxing to allow descent of the fetus, dilation of the cervix and expulsion of the fetus through the birth canal. We propose that this regionalization of myometrium phenotype requires differential regulation of gene expression in the LUS and FUN. METHODS: RNA sequencing, real-time PCR and immunoblotting, chromatin immunoprecipitation, RNA run-on and in vitro contraction assays were performed. RESULTS: Myometrial cells from LUS and FUN of 6 patients undergoing elective Caesarean section before the onset of labour were cultured for <3 passages. HoxA13, PTGIS and POSTN genes were detected by RNA sequencing to be more highly expressed in LUS than FUN cells. Realtime PCR and immunoblotting assays confirmed LUS cells expressed 5-, 4-and 2-fold HoxA13, PTGIS and POSTN mRNA levels, and 15-, 9-and 4-fold protein levels over FUN cells. HoxA13, PTGIS and POSTN mRNA levels were detected at statistically higher levels in LUS than in FUN of paired term-non-laboring myometrium. HoxA13 induced 8-30 fold increases in PTGIS and POSTN mRNA and protein levels,

while HoxA13 knockdown resulted in 60-70% suppression of PTGIS and POSTN expression in myometrial cells. HoxA13 induced 50-200% increases in Histone3 acetylation in PTGIS and POSTN promoters and thereby induced ~5-fold increases in transcription initiation rates of these genes. Cell contraction assays showed that pre-labor LUS myocytes or HoxA13-overexpressed cells exhibited stronger contraction. CONCLUSIONS: In term-non-labor myometrium, LUS is more contractile and expresses higher levels of PTGIS and POSTN than FUN. This pregnancy-maintaining regionalization of myometrium is medicated by HoxA13. Voltage gated potassium channels play an important role in regulating the transmembrane potential of smooth muscle cells. We have previously report the presence of voltage gated potassium channel family K V 7 in myometrial smooth muscle from pregnant women at term. The aim of this study is to demonstrate functional channels in freshly isolated myometrial smooth muscle cells and to study the effect of channel modulation on the transmembrane potential. METHODS: We obtained, with written consent, myometrial tissue from pregnant women (>37 weeks', n=28) undergoing elective caesarean section. The tissue was enzymatically digested and the cells dislodged by trituration. Conventional whole cell patch clamp was used to study currents. Briefly, a holding potential of -70 mV was applied and the current responses to -70 to +40 mV command potential (1 s) were recorded in the presence and absence of the K V 7 activators retigabine and ML-213 and the K V 7 blocker XE911. For the study of transmembrane potential, whole cell current clamp was employed. Vm was recorded in physiological salt solution and after addition of XE911. RESULTS: Native K V currents were present in 93% of the cells studied. Retigabine (5 µM) enhanced the voltage activated current amplitude in 21% of cells at +40 mV (614±93 pA retigabine vs 452±81pA control, n=6), whereas addition of the K V 7 channel inhibitor XE911 (10 µM) partially depressed the current amplitude in 88% of cells tested (239+/-44pA, n=7). Moreover, the K V 7.2 and K V 7.4 activator, ML-213, elicited a current at 510 nM (n=3). The native membrane potential in myometrial smooth muscle cells was -43±1mV. Addition of XE911 (10 µM) depolarised the membrane to -33±4mV (n=3). CONCLUSIONS: These data demonstrate the presence of functional native K V 7 currents in human myometrial smooth muscle cells. K V 7 activators increase the current amplitudes in some cells, whereas the K V 7 blocker XE911 attenuates them. We speculate that K V 7 currents are basally active and that this could explain the absence of further current activation by retigabine in 79% of all cells. The cell membrane depolarised in response to XE911, in tissue this response would lead to smooth muscle contraction, supporting our hypothesis that K V 7 channels are potential tocolytic targets. Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Our previous study has shown that human myometrium expresses cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principal enzymes responsible for hydrogen sulfide (H 2 S) generation, and therefore produces H 2 S. The expression levels of CSE and CBS in pregnant myometrium are down-regulated after onset of labour. We hypothesized that H 2 S might be involved in the transformation of uterus from quiescent state to contractile state during pregnancy. METHODS: The myometrial tissues were obtained from pregnant women who were in labor (TL) or not in labor (TNL) at term. The output of cytokines was determined by ELISA. Western blot analysis was used to determine the levels of uterine activation proteins (UAPs). Uterine smooth muscle cells (USMCs) were isolated from TNL myomtrium tissues and cultured in vitro.

The levels of CSE and CBS inversely correlated to the expression of connexin43 (CX43), oxytocin receptor (OTR), prostaglandin F 2α receptor (PTGFR) and prostaglandin endoperoxide synthase2 (PTGS2) as well as the levels of proinflammatory cytokines including IL1 and IL6 in human pregnant myometrium. H 2 S donor NaHS and H 2 S precursor L-cysteine inhibited the expression of CX43, OTR, PTGS2 and PTGFR in cultured USMCs in dose-dependent and time-dependent manner. H 2 S also dose-dependently suppressed IL1, IL6 and TNFa output, while IL1, IL6 and TNFa increased the expression of UAPs including CX43, OTR, PTGS2 and PTGFR. The effects of L-cysteine were not occurred in the cells transfected with CSE and CBS siRNA. Glibenclamide, an inhibitor of ATP-sensitive potassium (K ATP ) channels, reversed the effects of H 2 S on UAPs and cytokines. H 2 S activated PI3K and ERK1/2 signaling whereas inhibited NF-κB signaling via K ATP channels pathway. Blockage of PI3K and ERK1/2 signaling reversed the effects of H 2 S. CONCLUSIONS: H 2 S generated via CSE and CBS suppresses inflammation and expression UAPs in human pregnant myometrium. The effects of H 2 S is mediated by K ATP channels and dependent on NF-κB, PI3K and ERK1/2 signaling pathways. H 2 S produced within myometrium contributes to maintenance of quiescent state of uterus during pregnancy.

INTRODUCTION: Proper regulation of uterine contractility is essential for a successful pregnancy. Uterine contractions are directly controlled by the membrane potential of myometrial smooth muscle cells (MSMCs). Depolarization of the MSMC resting membrane potential (RMP) induces activation of Ca 2+ channels, action potential generation, and influx of Ca 2+ resulting in the activation of contractile machinery. Throughout pregnancy the RMP of MSMCs becomes more depolarized as the uterus transitions to a more contractile state. MSMC RMP is modulated by the pregnancy hormones estrogen and progesterone. However, the mechanism by which these pregnancy hormones modulate RMP is unknown. Previously, we have identified expression and activity of a Na + leak channel (NALCN) in human MSMCs. NALCN is known to reduce the RMP in other tissue types. We hypothesized that estrogen and progesterone may modulate MSMC RMP by regulating NALCN expression throughout pregnancy. METHODS: NALCN expression was measured in the mouse uterus by immunoblot, and in the human uterus by qRT-PCR. Additionally, the effects of estrogen and progesterone on NALCN expression was assessed by treating cultured human MSMCs with these hormones, and assaying NALCN expression by immunoblot. RESULTS: In the mouse uterus, NALCN protein expression decreased mid-pregnancy (days 14 -18) and increased near parturition (day 19). Furthermore, NALCN mRNA expression in human uterine tissue was higher in laboring samples compared to non-laboring samples, correlating with the previously described decrease in the RMP. Additionally, treatment of cultured human MSMCs with pregnancy hormones showed a modulation of NALCN expression. CONCLUSIONS: NALCN expression is regulated by pregnancy through the effects of estrogen and progesterone, and may serve as the mechanism by which these hormones affect myometrial membrane potential.

Oksana Shynlova, 1,2 Anna Dorogin, 1 Xuesen Dong, 3 Stephen Lye. The molecular mechanisms regulating preparation of the uterus for labor are not fully understood. We have previously reported that mechanical stretch of the uterine wall by the growing fetus plays a direct role in the regulation of major components of the extracellular matrix in late pregnant rat myometrium. As was shown by others, periostin (POSTN), a matricellular protein involved in the process of fibrosis and myofibroblast differentiation, is expressed in tissues subjected to mechanical stress. However, the expression of POSTN in human or animal myometrium during gestation and its response to uterine stretch has not yet been investigated. METHODS: Using (1) primary human myometrial cells (HMCs) derived from the lower uterine segment (LUS) and fundus (F), (2) human myometrium collected before and during labour from LUS and (3) a unilaterally tubal-ligated rat model in combination with Real-time PCR, immunoblot and immunohistochemical staining, we examined gestational variations and the effect of mechanical stretch on POSTN expression. RESULTS: Using HMCs, we found that under control conditions POSTN mRNA and protein levels in cells derived from LUS were higher as compared to cells from F of the same term non-laboring patient. When cells from LUS were subjected to static mechanical stretch for 24 hs, POSTN transcript levels were significantly up-regulated but only in HMCs also expressing high levels of desmin, a marker of muscle cell terminal differentiation. Cells derived from F did not react to mechanical stretch. In rat myometrium expression of the POSTN gene was significantly upregulated at late gestation (day 17-21) and during term labour (day 23) as compared to early gestation. Importantly, most changes in POSTN gene expression occurred specifically in the gravid horn but not in the empty horn of unilaterally pregnant rats, indicating the effect of gravidity. Immunostaining for POSTN was highly elevated in both muscle layers of late pregnant rat myometrium compared to early gestation and was more pronounced in the gravid uterine horn. CONCLUSIONS: We identified the differential expression of POSTN by HMCs in the uterine F vs LUS and established its spatiotemporal expression patterns in the pregnant rat myometrium, but its function during gestation is largely unknown. Additional studies are in progress to examine the role of POSTN in myometrial differentiation. The association between antenatal infection and preterm labor is widely accepted. However, little is known about the intracellular calcium regulation in uterine smooth muscle which is a key component of contractility. Here, we hypothesize that bacterial endotoxin, lipopolysaccharide (LPS) and pro-inflammatory cytokines, such as IL-1β and TNFα, alter calcium homeostatic mechanisms and may, in turn, augment myometrial contractility. METHODS: Primary uterine smooth muscle cells (UtSMCs; prepared by nonezymatic dispersion from a nonpregnant donor) were obtained from Lonza (USA). Cells were grown to 80% confluence and then serum starved for 24 hours prior to exposure. UtSMCs were then treated with LPS (1 mcg/ml), IL-1β and TNFα (10 ng/ml) for 48 hours. Intracellular Ca 2+ ([Ca 2+ ] i ) responses to oxytocin (500 nM), was measured in UtSMCs loaded with the Ca 2+ indicator fura-2 and perfused with Hank's balanced salt solution containing 2 mM extracellular Ca 2+ . Additionally, expression of calcium regulatory proteins in cytokine exposed UtSMCs was assessed by western blot analysis.

In untreated UtSMCs, oxytocin acutely elevates peak [Ca 2+ ] i response and is followed by decay. Cells exposed to pro-inflammatory cytokines had a significantly increased peak [Ca 2+ ] i response to oxytocin when compared to untreated cells. Additionally, pro-inflammatory cytokines appeared to slow decay of the [Ca 2+ ] i response to baseline, indicating reduced sarco-endoplasmic reticulum calcium ATPase (SERCA2) function. Western blot analysis showed increased expression of calcium regulatory proteins such as transient receptor potential cation channel 3 (TRPC3), stromal interaction molecule 1 (STIM1), SERCA2 and phospholamban in UtSMCs treated with cytokines. Notably, SERCA2/ phospholamban ratio is decreased, reflecting decreased SERCA2 function. CONCLUSIONS: These data demonstrate the altered intracellular calcium regulation in UtSMCs in response to inflammation. This is an important mechanism in the cascade of events leading to dysfunctional contractility in antenatal infection-related preterm labor. The switch from a quiescent phenotype to a contractile state during pregnancy likely involves the integration of multiple signaling networks generated from both hormonal and mechanical signals. Identifying how these networks change and interact is crucial to understanding the biochemical processes involved in the induction of labor. We have conducted global phosphoproteomic studies to identify and quantitate the specific phosphorylation changes that occur in pregnant human myometrial cells in response to acute progesterone and/ or mechanical stretch. METHODS: 18% biaxial stretch experiment were performed in the presence or absence of 600 nM acute progesterone treatment to elucidate signaling pathways activated by hormonal and mechanical distention in pregnant human uterine smooth muscle cells. Protein samples were extracted and prepared for mass spectrometry based analysis. Samples were digested with a trypsin/Lys-C mixture and phosphopeptides were enriched using immobilized metal affinity columns. Enriched samples were differentially TMT labeled and quantitated using reverse phase liquid chromatography coupled to tandem mass spectrometry. RESULTS: Biaxial stretch of human uterine smooth muscle cells confirmed an increase in phosphorylation of ERK2, as well as revealed changes in the upstream signaling molecules MEK, and the ERK scaffolding protein AHNAK. We observed a statistically significant increase in phosphorylation of AHNAK at Serine 4890 and Serine 5752. These sites have been shown in other smooth muscle models to be regulated by CDK5 and involved in shifts of phenotype and cell cycle regulation. CONCLUSIONS: Identifying the global signaling changes and interactions that occur in response to myometrial stretch will improve our understanding the biochemical processes involved in the induction of term and preterm labor. Future work will focus on confirming the stretched induced phosphorylation changes in ERK and AHNAK signaling networks. We have shown that H 2 S is a potent channel-independent uterine tocolytic. To define its mechanism further, we hypothesized that H 2 S reduces intracellular Ca 2+ and P-MLC. We performed pharmacologic studies to discern H 2 S signaling in primary myometrial cells (PMCs) in vitro and uterine smooth muscle (USM) strips in physiologic organ bath.

RESULTS: Inclusion criteria met by 458 patients with a mean age of 25.6 years and mean delivery of 38 weeks. Ninety-two (20.1%) women tested positive on urine drug screens for substances. Substances found in decreasing frequency: marijuana-73 (15.9%), opiates-23 (5.0%), buprenorphine-9 (2.0%), cocaine-6 (1.3%), benzodiazepines-5 (1.1%), methadone-3 (1.0%), and amphetamines-1 (0.2%). Half smoked, (228; 49.8%), and alcohol detected in 17 (3.7%) women. Of the women who used drugs, nineteen (20.1%) used more than one substance. Addictions counseling with therapeutic substitution was offered to all women with positive screens. Thirty-nine (42.4%) achieved drug abstinence at delivery. Rates of Neonatal Abstinence Scores (NAS) were significantly lower for women achieving abstinence (1;2.6%) verses those still using drugs (16; 30.2%), p=0.001. Opiate abstinence was achieved in 9/23 patients (39.1%) with an estimated savings of over $300,000 in abstinence therapy ($34,000 per neonate) in NICU costs alone. CONCLUSIONS: We present the first study using outpatient management with therapeutic substitution coupled with integrated addictions therapy for positive urine drug screening. Significant cost and decreased positive NAS scores resulted from this approach. These findings support outpatient therapeutic substitution with integrated addictions' therapy programs as means to achieve abstinence in pregnant patients.

Ultrasonographic Evaluation for Intra-Abdominal Fluid After Cesarean Section. Claire Hoppenot, Joan Tankou, Sabrina Stair, Dana Gossett. Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA. INTRODUCTION: Hemorrhage is the second leading cause of maternal mortality in the United States and is most often due to atony. Cesarean section increases the risk of hemorrhage and introduces the possibility of occult intra-abdominal bleeding. Focused assessment with sonography in trauma (FAST) scans are used in the trauma setting to assess for intraabdominal bleeding to triage patients for immediate surgical intervention. But unlike trauma patients, stable post-operative patients have been noted to have intra-abdominal fluid on CT studies. If seen on ultrasound (US), there is no known baseline for how much fluid is normal. The purpose of this study is to evaluate how much fluid can be seen on a FAST scan in a stable patient after a non-emergent c-section. METHODS: This prospective observational study was approved by the Institutional Review Board. Patients were approached while waiting for non-emergent c-sections. In recovery, a modified FAST scan was performed by 1 of 3 trained providers. The scan included a right upper quadrant (RUQ) view of the liver and kidney, a left upper quadrant (LUQ) view of the spleen and kidney, and a pelvic view from above the surgical bandage. A scan was considered positive if any echolucent fluid was visualized, and each view was noted separately. Patient demographic, operative and post-operative information were gathered from electronic medical records. RESULTS: One hundred patients underwent the modified FAST scan. Sixty-five patients (65%) were scheduled repeat cesareans, 22 (22%) were breech, and 13 (13%) had another indication for c-section (5 elective, 3 prior myomectomy, 2 macrosomia, 2 placenta previa and 1 obstructing fibroids). Average operative estimated blood loss was 787 mL ± 328. The mean time of the scan was 73 ± 36 minutes after the end of surgery. Ninety-eight (98%) of the RUQ scans were negative, 1 (1%) positive and 1 (1%) inconclusive. Ninety-six (96%) of LUQ scans were negative and 4 (4%) were inconclusive. All suprapubic scans were inconclusive due to the enlarged postpartum uterus. CONCLUSIONS: In our series of stable patients after c-section, US was feasible in 97.5% of upper quadrant scans and overwhelmingly negative. Upper abdominal fluid seen on US in a patient with signs of bleeding cannot be dismissed as normal post-operative fluid, and intra-abdominal bleeding must be seriously considered. . Whether melatonin has protective effects on the fetus independent of placental effects in risky development is unknown. Here, we isolated the direct effects of melatonin on fetal cardiac structure and function during chronic hypoxia in the chick embryo, the only model that permits direct investigation of the fetus independent of effects on the maternal and placental physiology. METHODS: Fertilised eggs incubated under normoxia or hypoxia (14% O 2 ) from day 0 were treated with melatonin (1 mg.kg -1 ) or saline vehicle +/-luzindole (non-selective melatonin receptor antagonist, 10 uM) from day 13 of incubation (equivalent to ca. 25 wks of human pregnancy). At day 19, the embryo was euthanised by spinal transection. Cardiac function was assessed in a Langendorff preparation. A separate cohort of animals was perfusion fixed (4% paraformaldehyde at 2.66kPa). The presence of significant omphaloceles is considered by some as a contraindication to vaginal delivery. We sought to evaluate the impact of the delivery method in cases of known fetal omphaloceles at our institution and to evaluate the effect of delivery method on neonatal outcomes. METHODS: We reviewed all cases at a single academic institution from 2002-2014 of sonographically diagnosed fetal omphaloceles that ended in delivery of a viable neonate with omphalocele confirmed at time of delivery. We evaluated route of delivery, as well as neonatal outcomes including omphalocele repair, associated fetal anomalies, and NICU course. RESULTS: A total of 34 cases are included in our analysis. Of these 34 cases, 16 underwent a trial of labor and 10 delivered vaginally. Of the 10 delivered vaginally, 4 were reported giant omphaloceles wth a significant portion of fetal liver outside of the fetal abdomen. Indications for cesarean delivery in those that labored included non-reassuring fetal status (3) and arrest of labor (3). Scheduled cesarean delivery was performed in the other 18 cases. Of these, 12 of these were for routine obstetric indications including repeat elective cesarean delivery, malpresentaton, and placenta previa. Notably, 6 cases were cesarean deliveries with the specified indication being the presence of a fetal omphalocele, including one classical cesarean delivery. Associated genetic and fetal anomalies were seen in a number of cases. There was no evidence of neonatal CONCLUSIONS: Many extremely preterm infants develop severe morbidity. Understanding how maternal hypertension influences outcome can aid in counseling families of extremely preterm infants. We started the fetal heart rate (FHR) monitoring network system in our region. We have 7 primary and 2 secondarylevel institutions where 3000 women deliver every year. This system enables the attending physicians at secondary institutions to evaluate multiple intrapartum FHR monitorings simultaneously from the primary institutions. The purpose of this study is to evaluate the effect of this system on perinatal outcome. METHODS: The FHR monitoring network system started in July 2012. We collected data from January 2011 to June 2012 (period 1) and from July 2012 to December 2013 (period 2) in all the 9 institutions. We compared the incidence of low umbilical blood pH (<7.10) or intrapartum fetal death (FD), and of cesarean section due to non-reassuring FHR between 2 periods. Fetal asphyxia not preventable with FHR were excluded. RESULTS: We had 4250 deliveries in period 1 and 4886 in period 2. Incidence of low pH or FD was 13 and 11, and of cesarean section was 260(6.1%) and 265(5.4%), which did not reach statistically significant. However, in 3 primary institutions that use the network more frequently (>15%), incidence of low pH or FD decreased from 9 to 2 (p<0.03), while the cesarean section rate did not change. CONCLUSIONS: FHR monitoring network system could reduce the incidence of fetal acidemia without increasing cesarean section rate. Although neonatal encephalopathy (NE) due to perinatal asphyxia accounts for a notable proportion of brain injury and poor neurodevelopmental outcome (NDO), the causal pathway remains largely unexplained, and the contribution of the placenta and its associated histopathology is unclear. We sought to examine the histologic changes that occur in the placenta of neonates with NE and relate this to NDO METHODS: This is a retrospective cohort study of neonates ³36wks gestation born at Parkland Hospital from 01/2006-11/2011 with NE. Umbilical artery blood gases were examined and placental histology reviewed and validated by a Pediatric pathologist blinded to clinical outcomes (Placenta. Apr 2005;26:S114-S117). Abnormal NDO was defined as death or Bayley III score <85 at 18-24 mon of age. RESULTS: 120 of 86,274 neonates (1.4/1000 LB) ³36wks gestation had perinatal acidosis and NE as determined by standardized neurological examination. 47 had mild NE and received no treatment, while 73 had moderate (n=70, 96%) or severe (n=3, 4%) NE and received systemic hypothermia therapy. 9 (12%) neonates died. All survivors receiving hypothermia had a Bayley III assessment at 22±7(SD) mon of age. The presence of "significant" placental pathology, chorioamnionitis with or without fetal response, and patchy/diffuse chronic villitis were independently associated with the severity of NE (P<0.001). Univariate logistic regression revealed that only "significant" placental pathology (OR 3.5, 95% CI:1.1-11.4), and specifically, patchy/diffuse chronic villitis (OR 9.29, 95% CI:1.11, 77.73), predicted abnormal NDO. CONCLUSIONS: 'Significant' placental pathology is associated with the severity of NE and abnormal NDO in neonates receiving systemic hypothermia, suggesting the placenta might contribute to the inflammatory response seen with NE, modify placental perfusion or mirror the inflammatory changes in the brain, resulting in abnormal NDO.

Obesity and the Risks of Vaginal Birth After Cesarean. Natalia C Miranda, Ruofan Yao, Lauren Plante. Obstetrics and Gynecology, Drexel University, Philadelphia, PA, USA. INTRODUCTION: There are few reports regarding the impact of obesity on outcomes of trial of labor after cesarean section (TOLAC). Most of the available reports took place, when prostaglandins were still routinely used to induce women attempting TOLAC. Therefore, we provide an updated evaluation on the outcomes of TOLAC among obese women using a more recent database. METHODS: This is a retrospective cohort analysis using the Texas vital records database from 2006 -2011. We categorized pre-pregnancy BMI according to WHO guidelines. Analysis was limited to obese women (BMI³30) with non-anomalous pregnancies that delivered between 24 and 42 weeks gestation who had reported one or two prior cesarean deliveries. We compared social-demographic information between women who attempted TOLAC versus elective cesarean delivery (ECD). Logistic regression was used to estimate the risk of adverse maternal and neonatal outcomes between the two comparison groups stratified by obesity class. The results were corrected for possible confounding variables. RESULTS: A total of 91,376 deliveries were included in the analysis. The overall rate of TOLAC was 10%. The mean age at delivery was similar between TOLAC and ECD group at 38 weeks. Minority women and women with lower education are more likely to attempt TOLAC. Among women in the TOLAC group, the cesarean delivery rate was higher for women with severe obesity. However, there are no significant differences in adverse maternal outcomes between TOLAC and ECD groups for all BMI categories, including risk for transfusion, ICU admission, unplanned operations or hysterectomy. TOLAC increases the risk of low 5 and 10 minute APGAR scores among all neonates, except those born to class III obese women. The risk of neonatal death is 2.6 times higher among class II obese women in the TOLAC group compared to ECD group. However this effect was not appreciated for class I or class III group and may be due to random variation. The overall neonatal composite score was not different between the two groups for all obesity classes. CONCLUSIONS: Obesity does not appear to increase the risks associated with TOLAC compared to ECD except for low 5 and 10 minute APGARs. In the 10 years since Hibbard et al published their findings of the risks of TOLAC among obese women using data collected prior to 2002, new management guidelines may have improved maternal and neonatal outcomes among this group of women. This is a study using a population based database, further studies are required to confirm these findings.

CONCLUSIONS: The use of twin gestation trimester specific TSH reference ranges to identify patients with elevated TSH does not identify patients at an increased risk of adverse pregnancy outcomes.

The Effect of Hypothyroidism on Outcomes in Twin Pregnancies. Jonathan Rosner, 1 Andrei Rebarber, 2 Daniel Saltzman, 2 Chad Klauser, 2 Nathan Fox, 2 Simi Gupta. 2 1 OB/GYN, NYULMC, New York, NY, USA; 2 MFM, Ichan Mount Sinai School of Medicine, NYC, NY, USA. INTRODUCTION: To assess the impact of maternal hypothyroidism on adverse pregnancy outcomes in patients with twin gestations. METHODS: A retrospective cohort study of all patients who presented with twin gestations between 2005-2013 to a single obstetrical practice was performed. Patients who were diagnosed with hypothyroidism prior to pregnancy were identified. Patients were followed with serial thyroid function tests and treated appropriately. The rates of adverse pregnancy outcomes were compared between patients with and without hypothyroidism with p<0.05 used for significance. RESULTS: 696 twin pregnancies were included in the cohort. 99 patients were diagnosed with hypothyroidism prior to pregnancy. Patients with hypothyroidism were more likely to have assisted reproduction, but otherwise there was no difference in age, parity, body mass index, or ethnicity. Patients with hypothyroidism had no increased risk spontaneous preterm birth < 37 weeks gestation(OR 0.916, 95% CI 0.5710-1.4680), intrauterine growth restriction (OR 0.724, 95%CI 0.4640-1.1290), hypertensive disorders ( OR 0.688 95%CI 0.3720-1.1690) or composite adverse outcomes (pre-eclampsia, gestational hypertension, spontaneous preterm birth, small for gestational age, intrauterine fetal demise and spontaneous abortion) (OR 0.641 95%CI 0.4020-1.0210) compared to patients who did not have hypothyroidism. 

The presence of treated maternal hypothyroidism in twin gestations does not confer an increased risk of adverse pregnancy outcomes when compared to controls.

The Vaginal bleeding in the second and third trimesters, but not the first trimester, is associated with a higher risk of preterm birth in patients with twin pregnancies. We postulate that unexplained vaginal bleeding in the second and third trimester in twin pregnancies can represent a form of placental dysfunction that can lead to preterm birth through several pathways. Twin pregnancies with second or third trimester vaginal bleeding should be followed closely in pregnancy.

Klippel- Suboptimal treatment of these infections is common, however, based on initial data, it is unclear whether this leads to adverse outcomes.

Comparing The optimal screening approach for tuberculosis in pregnancy is unknown. This study demonstrates the completion rates of and agreement between an interferon gamma release assay (T-SPOT. TB) and the tuberculosis skin test (TST) in detecting latent tuberculosis in pregnant women. METHODS: This was a cross-sectional analysis of an observational cohort study of 141 pregnant women enrolled between August 2013 and July 2014 from an urban obstetrics practice with a large immigrant population. Women with a history of a positive TST result were excluded. Demographic and clinical risk factors for tuberculosis were assessed. Enrolled women underwent a T-SPOT.TB test (Marlborough; Oxford Immunotec) and placement of TST (Toronto; Sanofi Pasteur). Participants were instructed to return in 48-72 hours for TST interpretation. We calculated the completion rate and frequency of a positive result for each test, as well as the concordance between the T-SPOT.TB and TST using the kappa statistic. RESULTS: Among the 141 women, the median age was 28 years, 75% were either Latina or African-American, 44% were born in a country with a high TB prevalence, and 52% had received the BCG vaccine. Seven women (5%) screened positive for tuberculosis; there were a total of 3 positive T-SPOT.TB results and 6 positive TST results. All women with positive screens were immigrants from countries with high TB prevalence.None of the women with positive screens had received the BCG vaccine. The completion rate for the T-SPOT.TB was 98%, while the completion rate for the TST was 63%. The concordance of the two tests was 96.3%. 

We demonstrated a high correlation between the T-SPOT.TB and the TST in pregnant women, and a high completion rate for the T-SPOT.TB compared to the TST. These findings suggest that the T-SPOB.TB is the preferred screening method in pregnancy. Targeted screening of women from countries with a high prevalence of tuberculosis may be warranted during prenatal care.

Transplacental We studied the lung histology of 2-day-old neonatal mice and 3-week-old infant mice following intrauterine exposure to F. nucleatum, respectively, as well as the lung of a stillborn baby who died due to in utero exposure to F. nucleatum. RESULTS: Both the neonatal mice and the stillborn baby showed pulmonary hemorrhage and filamentous bacteria in the airway. Infant mice exposed to F. nucleatum in utero demonstrated lung inflammation and impaired lung development. The lung inflammation was characterized by patchy distributions of thickened alveolar septa, which were 43% thicker than those of the saline-control mice, and accompanied by increased cell infiltrates, which were positive for leukocyte common antigen. Furthermore, 29% of infant mice exposed to F. nucleatum showed impaired lung development, with alveolar numbers decreased by 39% and pulmonary vascular density decreased by 30% as compared to the saline control. CONCLUSIONS: We conclude that transplacental F. nucleatum infection causes neonatal lung injury and results in chronic lung disease during infancy in our murine model.These results suggest transplacental F. nucleatum infection may be a previously unrecognized but potentially treatable cause of chronic lung disease in preterm infants. Augmentation of labor was associated with bacteremia (aOR 2.11, CI 1.11-4.03, p=0.02) while amniotomy appeared to be protective (aOR 0.57, CI 0.35-0.92, p=0.02). Hospitalization for more than 1 day prior to delivery also predicted bacteremia (aOR 2.05, CI 1.07-3.95) *Figure(s) will be available online. CONCLUSIONS: When considering a variety of demographic and clinical variables, fever during labor and hospitalization for greater than one day are associated with an increased risk of bacteremia. Ensuring blood cultures are obtained on women with fevers during labor or those admitted more than 24 hours prior to delivery could maximize identification of mothers at risk for sepsis and potentially severe morbidity due to bacteremia. RESULTS: HCMV infection triggered a rapid immune activation in the decidua, significantly affecting decidual cytokine -chemokine expression pattern, with predominant induction of interferon (IFN)-γ and IP-10. Decidual tissue innate response was induced early upon virustissue contact by a conserved structural virion component, and was not affected by neutralizing HCMV antibodies. Of note, IFN-γ induction was distinctive to the decidual tissue, and was not observed in concomitantlyinfected placental villi. Importantly, functional analysis of conditioned media recovered from infected tissues revealed the direct effect of the viral-induced decidual environment on antiviral protection along with leukocyte attraction and induction of apoptosis. CONCLUSIONS: These studies, in a clinically-relevant surrogate human model, provide a novel insight into the first-line tissue response, mediating both protection and damage in the maternal-fetal interface, which could determine the outcome of infection.

Effects 

In offspring from normoxic pregnancy, ageing impaired diastolic dysfunction (Fig. 1a) , decreased coronary flow rate (Fig. 1b) , increased cardiac sympathetic dominance (Fig.1c) and limited myocardial mitochondrial oxygen consumption (Fig. 1d) . Offspring from hypoxic pregnancy showed accelerated cardiac ageing already at 4 months of age ( Fig. 1 a-d) . Ageing in offspring of hypoxic pregnancy further increased diastolic dysfunction and further decreased coronary flow rate, but did not further affect sympathetic dominance or myocardial mitochondrial oxygen consumption. by which exposure to GDM increases rates of offspring obesity and CVD. Leptin is elevated in mothers with GDM. We hypothesize that high levels of leptin present in mothers with GDM are responsible for alterations in metabolism and blood pressure observed in offspring of mothers with GDM.

We utilized two mouse models of hyperleptinemia: 1) wild type (WT) mothers (normal maternal leptin), were compared to Lepr db/+ mothers (very high maternal leptin), and 2) saline infused mothers (normal maternal leptin), were compared to leptin infused mothers (moderate maternal leptin). Two male and two female WT offspring were kept from each mother and half of these offspring were placed on a high fat diet (HFD) at 20 weeks of age. Offspring were weighed weekly and sacrificed at 6.5 months of age. Glucose tolerance, heart lipid accumulation, fibrosis and cardiac gene expression were evaluated in all offspring. Blood pressure, vascular function, and pancreatic vascularization were evaluated in male offspring from WT and db/+ mothers. RESULTS: Offspring from db/+ mothers weighed less (p<0.05) than WT offspring on both a normal and HFD. This effect continued in offspring from leptin treated mothers compared to saline treated mothers but was less pronounced. Offspring from db/+ mothers had better (p<0.05) glucose tolerance than WT offspring on a normal diet, though this difference was completely lost on a HFD. There were no differences in heart lipid accumulation or fibrosis. Cardiac gene expression was not altered forGlut4, Fabp3, Pgc1α and Insr. No differences were found in blood pressure or vascular vasoconstriction. There was no significant difference in vasodilation on a normal diet, though male offspring from db/+ mothers, were if anything, more responsive (p= 0.08) to insulin. Analysis of pancreatic vascularization is ongoing. CONCLUSIONS: These data indicate that a high maternal leptin environment does not cause the obesity and CVD observed in offspring of GDM pregnancies, and may even protect offspring from damaging effects of a GDM environment. Research funded by the American Heart Association and American Diabetes Association. (PCBs) are environmental contaminants which impart detrimental metabolic health consequences on exposed individuals. Further, PCBs are lipophilic and stable, which permits placental transfer to fetuses of exposed mothers. The liver is the main site of detoxification of PCBs so we set out to measure hepatic gene expression in pregnant ICR(CD1) mice exposed to PCBs, as well as their offspring. METHODS: Female mice were gavaged once every other week with 1 µmole/kg PCB 126 or vehicle beginning at mating. In one cohort, mice were sacrificed and fetuses removed on embryonic day 18. In the second cohort offspring were aged to 4 months prior to tissue collection. Livers were extracted from dams, fetuses, and offspring and snap frozen. RNA was isolated and analyzed via Nanostring Technology for cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyp1a1) and cytochrome P450, family 1, subfamily B, polypeptide 1 (Cyp1b1) (metabolize PCBs and upstream markers of oxidative stress production), NAD(P) H dehydrogenase [quinone] 1 (NQO1) and nuclear factor (erythroidderived 2)-like 2 (Nrf2) (antioxidant response), and autophagy-related protein 7 (Atg7) and C/EBP homologous protein (CHOP) (markers of endoplasmic reticulum stress). Student's t-test was used to analyze data between maternal PCB exposure and control mice. RESULTS: Dams and cesarean pups exposed to PCBs displayed increased Cyp1a1, Cyp1b1, NQO1, and Nrf2 (p<0.05) compared to those exposed to vehicle. Interestingly, 4 month old offspring had no measurable PCBs in liver; however these mice displayed increased expression of CYP1a1, Atg7, and CHOP (p<0.05). CONCLUSIONS: These results provide evidence for early deleterious effects of maternal PCB exposure on fetus and offspring hepatic gene expression. Further, persistent elevation of Cyp1a1 in 4 month offspring is suggestive of epigenetic changes to PCB exposure in utero and future studies will be aimed at examining these alterations. 

In 1,028 pregnancies in the Generation R Study we assessed fetal circulation using pulsed wave Doppler examinations between 28 and 34 weeks of gestation. To test associations between fetal gender and fetal circulation measurements linear regression models were used adjusting for fetal weight, gestational age and fetal heart rate. RESULTS: A higher pulsatility index of the ductus venosus was observed in male fetuses compared to female fetuses with a lower E/A ratio of the tricuspid and bicuspid valves. This was mainly determined by significant differences in the E wave of the tricuspid and bicuspid valves. A lower peak systolic velocity was seen of the pulmonary artery with a similar trend regarding peak systolic velocity of the aorta ascendens when comparing male fetuses to female fetuses. CONCLUSIONS: Male fetuses exhibit a higher afterload and higher enddiastolic preload compared to female fetuses. This is in agreement with a higher systemic blood pressure. Both cardiac and arterial compliance are reduced in male fetuses, consistent with a stiffer arterial vasculature and less elastic heart. These results might be related to gender specific differences in CVD in adulthood. Previous studies demonstrated the ability of Sildenafil to increase fetal growth in mouse models of fetal growth restriction (FGR), supporting the development of a randomized control trial in women with severe FGR. It is vital, however, to determine any potential long-term effects of treatment on the health of offspring. The aim of this study was to determine the effect of Sildenafil treatment on the metabolic and cardiovascular health of adult mouse offspring. METHODS: Pregnant C57Bl6/J mice were randomized to receive Sildenafil (0.2mg/ml in drinking water) or vehicle from gestational day 12.5 until pups were weaned at 21 days of age. Four male mice per litter were then randomized to either chow (4% calories from fat) or a high fat diet (HF, 45% calories from fat). Body weight was measured from birth to 90 days of age (P90). At P90 an oral glucose tolerance test was performed and systolic blood pressure measured. Mesenteric artery function, an indicator of cardiovascular health, was also assessed.

There was no effect of Sildenafil treatment, compared with vehicle, on birth weight (1.5 ± 0.1 vs. 1.4 ± 0.1 g; p>0.05) or body weight at P90 (26.9 ± 0.1 vs. 27.6 ± 0.3 g; p>0.05). Consumption of a HF diet significantly increased body weight at P90 (30.7 ± 0.1 vs. 28.5 ± 0.9 g, Sildenafil vs. vehicle; p<0.05, two-way ANOVA), but there was no effect of Sildenafil on this measure (p>0.05). Mice fed a HF diet demonstrated impaired glucose tolerance (p=0.03) but there was no effect of Sildenafil treatment (p=0.62) on this measure. There was no effect of Sildenafil (p=0.67) or diet (p=0.49) on systolic blood pressure. There was no change in maximal endothelium-dependent relaxation in mesenteric arteries from vehicle vs. sildenafil treated mice fed chow (69.5 ± 10.7 vs. 68.8 ± 4.2 % relaxation) or a HF diet (67.4 ± 9.3 vs. 66.5 ± 7.2 % relaxation). CONCLUSIONS: Sildenafil citrate treatment during pregnancy and lactation had no effect on the subsequent growth, metabolic or cardiovascular health of adult offspring, suggesting that this treatment has no adverse effects on fetal / adult growth and development. Further studies, in growth-restricted offspring, are planned to confirm these results.

Sex Previous studies showed NO donors stimulated renal production of cGMP which reduces sodium and fluid reabsorption. We have shown that that prenatal exposure to clinically relevant doses of glucocorticoids at the time of nephrogenesis leads to impaired Na + excretion in adult male but not female sheep. Since 50% to 70% of sodium is reabsorbed in the renal proximal tubule (RPT), the present study was to determine if there are sex related differences in the effect of the NO donor (SNP) and cGMP on Na + uptake by RPTC from prenatal Beta exposed male and female sheep METHODS: RPTC were isolated from 1-yr-old male and female vehicleor Beta exposed offspring (exposure at 0.6gestation) and cultured for 7-10 days. The fluorescence dye sodium green was employed to determine cytoplasmic Na + uptake in RPTC exposed to different Na + concentrations in the presence of the Na + /K + ATPase inhibitor ouabain.

In males after either SNAP or cGMP stimulation, the slopes of the Na + uptake lines were significantly less in RPTC from vehicle than from Beta animals(1.03±0.09 vehicle vs 2.35± 0.32 Beta SNAP; 0.78±0.06 vehicle vs 2.21±0.20 Beta cGMP p<0.001 for both) i.e. cells from vehicle animals took up less Na + . Contrary to the situation in RPTC from males, there was no effect of Beta exposure on the slopes of the Na + uptake lines in RPTC from females after SNP or cGMP stimulation. CONCLUSIONS: These data indicate that there are sex differences in in the effects of prenatal steroid exposure on responses to SNP and cGMP-induced inhibition of Na + uptakeby RPTC. The inhibition of Na + uptake is suppressed to an equal degree in cells from female beta exposed animals but inhibition of uptake is greatly reduced in cells from Beta exposed males. This suggests that impairment of renal NO effects may explain some of the deficit in Na + excretion seen in males but not in females exposed to Beta in fetal life. Supported by NIH grants HD 17644 and HD 47584. Here, we aimt to study in mice the interactions between obesity in pregnant dams, and MET treatment on placental expression of selected inflammatory genes important in the placental response to lipids. METHODS: Female C57/BL6J mice were fed control diet (C, 7% kcal fat) or high-fat diet (HF, 45% kcal fat) 6 weeks prior to mating through pregnancy. A subset of pregnant C and HF dams were given MET in drinking water (250mg/kg bodyweight/day) throughout pregnancy, generating 4 dam groups: C diet without treatment (C), C diet with metformin (C+MET), HF diet without treatment (HF) and HF diet with metformin (HF+MET). Dams were killed on day 16 of pregnancy and the placentas were collected. RNA was extracted from the placentas and converted to cDNA. Expression of inflammatory genes interleukin-1 beta (IL-1b), tumour necrosis factor (TNF), chemokine C-C motif ligand 2 (CCL-2) and Toll-like receptors 2 and 4 (TLR-2 and TLR-4) was measured using quantitative real-time RT-PCR. Mean differences between groups were assessed by one-way ANOVA. RESULTS: Feeding a HF diet 6 weeks preconception resulted in dams that were heavier and fatter at the time of pregnancy vs C-fed animals. The obese condition resulted in increased mRNA expression of CCL-2 in the HF dams vs C dams irrespective of MET treatment (P=0.004). Further, MET treatment was associated with increased IL-1b expression (P=0.043) in the HF+MET and C+MET dams vs corresponding HF and C dams. Likewise, TNF-a mRNA expression (P=0.034) increased in the HF+MET vs HF but not in the C-fed dams. No effects of both the HF diet and MET were found on placental expression of TLR-2 and TLR-4. CONCLUSIONS: Both HF diet induced obesity in pregnant mouse dams and MET treatment modulate the expression of certain inflammatory genes in the placenta, indicating interactions between the maternal environment and the placental inflammatory response.

Renal year old offspring of mothers exposed our undernutrition regimen have impaired cardiac function indicated by increased 3D sphericity index, a measure of diastolic dysfunction. Impaired Titin function causes diastolic dysfunction associated with preserved ejection fraction a common cause of heart failure. We hypothesized that IUGR results in development of hearts with less contractile power due to decrease in the N2B isoform. We determined the effect of IUGR on the isoform mRNA ratio and mRNA and protein for RBM20. IUGR decreased the percentage N2B and RBM 20 mRNA and protein (Fig 1) CONCLUSIONS: The N2B titin isoform decreases associated with decreased RBM20 in IUGR left atria. N2B is expressed less in the systolic failing heart and recent studies have shown less RBM20 in patients with end-stage heart failure. We hypothesize that decreased contractile strength in the hearts of IUGR offspring would predispose to cardiac dysfunction. We propose to examine abundance of N2B, N2BA and RBM20 in all four heart chambers at different stages of gestation. NICHD HD21350 We have shown that maternal low protein in P and/or lactation (L) affect adult male rat risk assessment behavior (1) potentially related to increased maternal reactive oxygen species (ROS). We hypothesized that maternal treatment with the antioxidant Res in P and L improves OFF anxiety behavior. METHODS: Pregnant Wistar rats, were assigned to Control (C-20% casein) or a restricted (R-10% casein) isocaloric diet in P and L. Half C and R mothers received 20 mg/kg Res orally in P and L (Cres and Rres). Litters were adjusted to 12 pups. From postnatal day (PND) 21 male pups were fed with C diet until the end of the experiment. At PND 106 -107, eight male OFF were tested in the elevated plus maze (EPM) and Open Field and activity recorded by video analysis. At PND 110 serum corticosterone and ACTH were determined and amygdala obtained to quantify Malondialdehyde (MDA-spectrophotometry). Analysis by ANOVA. RESULTS: R male OFF made fewer open arm and center zone entries vs C and, both entries increased in Rres (Fig 1A and B) . Serum corticosterone and ACTH were higher in R vs C, and decreased in Rres (Fig 1C and D) . MDA amygdala was similar among groups ( Fig 1E) . (MLP) in P and L impairs OFF associative learning and motivation. Increased OS may play a key role in these negative outcomes. We hypothesized that maternal anti-oxidant treatment with res in P and L improves OFF spacial learning and memory. METHODS: Pregnant Wistar rats, ate Control (C-20% casein) or restricted (R-10% casein) isocaloric diet in P and L. Half C and R mother received 20 mg/kg Res orally in P and L (Cres and Rres). At 19 days gestation (dG), half of mothers were euthanized to obtain fetal brain and determine fetal brain malondialdehyde (MDA). Remaining rats delivered at term. From postnatal day (PND) 21 male pups were fed C diet until the end of the experiment. At PND 108, eight male OFF were tested in the Morris water maze (MWM). RESULTS: At 19dG, male fetal brain MDA was higher in R and lower in Cres and Rres vs C. After 24 h of learning (spatial memory) the escape latency to locate the target platform was higher in R vs C; however the latency decreased in Rres. Number of entries, time spent and distance recovered into target zone were lower in R OFF (Fig 1C, D, E) ; Rres increased entries and target area time spent. Total distance was similar among groups. *Figure(s) will be available online. Fig1. A) Fetal brain MDA at 19dG; MWM test: B) Latency (sec) to locate platform area, C) number of entries, D) time spent and E) distance recovered into target zone during 24h retention test. M ± SEM, n=8 from different litters; p<0.05 * vs C, different letter for R vs Rres and C vs Cres. CONCLUSIONS: Res improved markers of learning and spatial memory, as well as oxidative damage indicating a correlation between MLP induced adverse outcomes behavior and brain ROS. As the prevalence of obesity among pregnant women continues to rise, an increasing number of children are exposed to an 'obese intrauterine environment'. Maternal obesity increases the risk of offspring obesity, due in part to altered development of appetite regulatory neurons in hypothalamic arcuate nucleus (ARC). The ARC contains two populations of neurons, derived from neural stem cell (NSC) progenitors, which have opposing actions on food intake: orexigenic (AgRP; agouti-related protein) and anorexigenic (POMC; proopiomelanocortin). The development of these neurons from is regulated by bHLH neuroproliferative factor (Hes1) which promotes NSC proliferation and inhibits downstream bHLH neurodifferentiation factors (Mash1, Ngn3). Ngn3 further promotes the development of anorexigenic POMC neurons, while inhibiting AgRP expression. We hypothesized that (1) maternal obesity impacts fetal hypothalamic ARC development, increasing expression of appetite (AgRP) versus satiety (POMC) neurons, and (2) these changes are mediated by Hes1 and Mash1/Ngn3. METHODS: Female mice were fed either control (10% k/cal) or high fat (45% k/cal) diet to create maternal obesity (MO) prior to mating. Diets continued throughout pregnancy and lactation. Newborns were delivered spontaneously, males sacrificed at day one of life, and brains collected. Hypothalamus was dissected and protein expression analyzed (Western Blot) for Hes1 (neuroproliferation), Mash1 and Ngn3 (neurodifferentiation), and AgRP and POMC expression. RESULTS: At 1 day of age, MO males were heavier (1.55±0.09 vs 1.31±0.01 g; P<0.05) than Controls. MO males had decreased hypothalamic expression of Hes1 (0.6-fold; P<0.05) and Ngn3 (0.8-fold; P<0.05) with increased expression of Mash1 (2.8-fold; P<0.05). Consistent with these changes, expression of AgRP was increased (1.5-fold; P<0.05) and POMC was reduced (0.7-fold; P<0.05). CONCLUSIONS: MO offspring appetite regulation is biased toward orexigenic, and away from anorexigenic neurons, resulting in excess appetite, reduced satiety and development of obesity. The putative underlying mechanism involves reduced Hes1, which promotes premature neuronal differentiation as evident by increased Mash1. Reduced Ngn3 expression impairs anorexigenic POMC neuronal development, resulting in increased AgRP expression. Early modifications of the maternal nutrient environment may prevent altered development of appetite regulation. Among them, stress has been linked to increased risk of emotional and behavioral disorders such as depression in offspring, which was likely mediated by glucocorticoids (GCs).

Pregnant rats were exposed to dexamethasone (DEX) (0.1 mg/kg/day) in the last week of pregnancy. The behavior of offspring was assessed at 9 weeks after birth. Some offspring was mated to produce a second generation. Hippocampus of offspring was collected for determining the expression of corticotrophin-releasing hormone (CRH), CRH receptors (CRHRs) and methylation status of CRH and CRHR1. The effects of GCs on CRH and CRHR1 were confirmed in the model of cultured hippocampal slices in vitro. RESULTS: Prenatal DEX exposure induced depression-like behavior and increased the expression of CRH and CRHR1 but not CRHR2 in hippocampus. Intrahippocampal administration of CRHR1 antagonist antalarmin alleviated depression-like behavior in offspring upon prenatal DEX exposure. Intrahippocampal injection of lentiviral vectors expressing CRHR1 in adult rats resulted in depression-like behavior, confirming that increased CRHR1 expression would lead to depressionlike behavior. Prenatal DEX exposure decreased global methylation and caused demethylation at several CpG sites of CRHR1 promoter, but did not affect the methylation status of CRH promoter in hippocampus. In the model of cultured hippocampal slices, either DEX or corticosterone treatment caused an increase in CRH and CRHR1 expression. The stimulatory effects of GCs on CRHR1 expression was reversed by DNA methyltransferase agonist and DNA methyltransferase agonist decreased CRHR1 expression in cultured hippocampal slices. Furthermore, we found that increased depression-like behavior and expression of hippocampal CRH and CRHR1 as well as DNA global demethylation were also evident in the second generation offsping who was delivered by prenatal DEXexposed female offspring. CONCLUSIONS: Prenatal DEX exposure results in persistent changes in both depression-like behavior and hippocampal CRH and CRHR1 expression, which can be transmitted to the next generation mainly through maternal line. The depression-like behavior induced by prenatal DEX exposure is at least in part mediated by increased CRH and CRHR1 expression in hippocampus and epigenetic mechanism is involved in CRHR1 expression.

Expression and Characterization of TRPV1 in Healthy Versus Diseased Fallopian Tubes. Lindsey Borgia, 1 Deborah Tinnemore, 2 Richard Burney, 1,2 Avedis Kazanjian. The complex molecular landscape of this microenvironment is not well understood. Transient Receptor Protein Subfamily V Member 1 (TRPV1) is a cation channel activated by a wide variety of stimuli and implicated in numerous biological processes throughout the body. Our objective was to characterize expression of TRPV1 in healthy and diseased fallopian tube. METHODS: Mid-isthmic oviductal segments were obtained at the time of laparoscopic tubal sterilization from twenty-two women. Tubal segments were processed for quantitative polymerase chain reaction and immunohistochemistry (IHC) to measure gene expression and localize protein expression. Archival smaples from patients with hydrosalpinx were utilized for protein expression with IHC. RESULTS: TRPV1 mRNA expression was unchanged between the different phases of the menstural cycle in healthy fallopian tubes. IHC analysis of protein expression in the tubal epithelium demonstrated similar levels in both secretory and ciliated cells. Intrestingly, a marked increased in protein expression was observed in tubal peg cells. Protein expression was maintained in hydrosalpinx with loss of distinct expression patterns compared to healthy epithelium. CONCLUSIONS: This is the first report of the expression pattern of TRPV1 in the fallopian tube and provides a basis for the study of dysregulation in pathologic conditions such as hydrosalpinx. The hormone independent expression of TRPV1 in the luminal epithelium with increased expression in peg cells suggests a potential role in secretory activity and anterograde transport. Further studies are needed to examine the significance surrounding the loss of marked expression of TRPV1 in peg cells in the setting of hydrosalpinx. , and intermedin (IMD), play significant roles in facilitating pregnancy-associated vascular adaptation. CGRP, AM, and IMD share common receptor components-calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1, 2 and 3 in different combination in exerting their biological functions: CRLR-RAMP1 for CGRP, RAMP2 and 3 for AM, and all three for IMD. The present study was designed to determine the proximity association of CRLR with RAMPs and the influence of CGRP family peptides on the interactions of the receptor components on the cell surface of rat mesenteric artery smooth muscle cells (RMASMCs). METHODS: RMASMCs were grown on Lab Tek chamber slides, and treated (2 minutes) with CGRP, AM, and IMD at the concentration of 10nM. Proximity ligation assay (PLA) was performed by using a DuolinkII Fluoresence kit (Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. Fixed cells were incubated with primary antibodies to CRLR in combination with RAMP1, 2, or 3, PLA probes, followed by ligation and amplification of the PLA probe. The images were observed under fluorescence microscope and the red spots were counted using Cellsense Digital Imaging software in 3 -5 randomly selected fields. RESULTS: (1) Using PLA technology, we demonstrated that RAMP1, 2, and 3 are each closely associated with CRLR on the cell surface of RMASM cells; and no significant differences were noted in their basal association.(2) CGRP treatment stimulates the complex formation of CRLR with RAMP1 (p=0.0052) and RAMP2 but not with RAMP3 when compared to controls; AM increases the complex formation of CRLR with RAMP3 (p=0.0045) but not with RAMP1 or 2, when compared with controls; and IMD enhances the complex formation of CRLR with RAMP1 and 3 (p=0.006) but not with RAMP2. CONCLUSIONS: Cell surface association of CRLR with all three RAMPs exists with similar degree of associations in resting RMASM cells. CGRP family peptides deferentially enhance proximal association of CRLR with RAMPs on cell surface. These findings suggest the potential use of PLA technology for monitoring the functional CRLR/RAMP dimerization and receptor/peptide interaction in the reproductive and vascular actions of CGRP family peptides. Animal studies indicate that several reproductive disorders are influenced by adverse factors during fetal and neonatal development 1 . We have shown that MPR during P and L reduces sperm concentration 2 . It is suggested that early life nutritional adversity is associated with increased OS 3 . OS damages spermatogenic cells, the spermatogenic process and sperm function 4 . We therefore investigated MPR effects MPR in P and L on sperm OS, function fertility. METHODS: We studied male rat OFF whose mothers ate control (CC) (20% casein) or restricted (RR) (10% casein) isocaloric diet in P and L. After birth all rats ate C diet. At postnatal day (PND) 110 were rats were euthanized and epidiymal and vas deferens sperm obtained to measure: 1) OS by quantifying reactive oxygen species (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). Reproductive capacity was evaluated by fertility rate. Data are M ± SEM; analysis by t-test; n=6 per group, p<0.05. RESULTS: At PND 110, RR sperm had higher ROS levels and decreased GPx activity and sperm concentration vs C without changes in MDA ( Figure 1A-D) . Sperm viability and motility were similar. Fertility rate was unaffected in RR ( Figure 1E ). *Figure(s) will be available online. (Dodic, Clin Sci 1999) influence development of the ANS with negative implications for stress susceptibility and cardiovascular health in later life. We hypothesized that prenatal stress (PS) alters development of the fetal ANS determined by fetal heart rate variability (FHRV) and baroreceptor reflex (BR) responses. METHODS: Seven pregnant ewes underwent isolation stress twice weekly for 3h between 0.2 and 0.67 gestation (g). Eight non-stressed pregnant ewes were used as controls. After chronic fetal instrumentation, electrocardiogram was recorded and FHRV calculated in the time (SDNN/ RMSSD) and frequency domain (LF/HF) at 0.75 and 0.87g, i.e. 12 and 30 days after PS. BR sensitivity was derived from blood pressure (BP) responses to phenylephrine (PE) and sodium nitroprusside (SNP). RESULTS: Baseline fetal BP was lower in previously stressed fetuses (SF) at 0.75 (40±1 vs. 35±2 mmHg, mean±SEM) and 0.87g (51± 2 vs.43±1 mmHg) and fetal heart rate (HR) was lower at 0.87g (178±5 vs. 164±4 bpm) (p<0.05). SDNN was higher in SF at 0.75g reflecting higher autonomic tone (p<0.05). RMSSD and HF which reflect parasympathetic activity were lower in SF at 0.87g (p<0.05). Since LF which reflects sympathetic activity was also reduced at 0.87g (p<0.05), LF/HF ratio as a marker of sympathovagal balance was lower in SF (p<0.05) implicating a shift towards parasympathetic activity. PS did not affect BR sensitivity. However, range of BR regulation in response to PE was lower in SF at 0.75 and 0.87g (p<0.05, Fig. 1) . PS resets the BR as shown by a left shift of the BR response curve meaning that HR was lower for any given pressure (p<0.05, Fig. 1 ). CONCLUSIONS: Higher autonomic tone after PS implicates early maturation of ANS. Sympathovagal balance and range of BP regulation were still altered 30 days after PS suggesting prolonged effects on fetal autonomic HR and BP regulation. *Figure(s) will be available online. (GDM) is associated with longterm cardiovascular and metabolic diseases in offspring. However, the mechanisms are not well understood. We explored whether fetal exposure to a diabetic environment is associated with endothelial progenitor cell dysfunction, and whether vitamin D can reverse the impairment. METHODS: This was a nested case-control study. Nineteen women with uncomplicated pregnancies and 18 women with GDM were recruited. Time to first appearance of endothelial colony forming cell (ECFCs) colonies and number of colonies formed were determined. Angiogenesisrelated functions of ECFCs in vitro were tested in the presence or absence of vitamin D. RESULTS: Fetal ECFCs from GDM pregnancies formed fewer colonies in culture (GDM: 2.94 ± 1.66, uncomplicated pregnancies: 5.83 ± 3.41, P=0.04) and displayed increased population-doubling times (GDM: 4.17 ± 1.23 days, uncomplicated pregnancies: 2.31 ± 0.43 days, P=0.02) compared to uncomplicated pregnancies. Fetal ECFCs exposed to GDM in pregnancy or to hyperglycemia in vitro exhibited less migration (GDM: 54.72% ± 9.25%, uncomplicated pregnancies: 64.6% ± 7.63%, P=0.04; control (5.5 mM)= 1 vs. 7 mM glucose: 0.94 ± 0.06, P=0.04; 11 mM glucose: 0.88 ± 0.07, P=0.006; 15 mM glucose: 0.87 ± 0.09, P=0.009) and less tubule formation (GDM: 3.28E+07 mm ± 7.75E+06 mm, n=15; uncomplicated pregnancies: 3.95E+07 mm ± 9.63E+06 mm, P=0.03; control (5.5 mM) =1, 7 mM glucose: 0.79 ± 0.16, P=0.045; 11 mM glucose: 0.77 ± 0.06, P=0.001; 15 mM glucose: 0.69 ± 0.22, P=0.03) than control. Vitamin D significantly improved the dysfunction of fetal ECFCs from pregnancies complicated by GDM or after exposure of healthy ECFCs to hyperglycemia. CONCLUSIONS: Fetal ECFCs from GDM pregnancies or ECFCs exposed to hyperglycemia in vitro exhibit impaired quantity and

Adami, Clark Johnson, Irina Burd, Ernest Graham, Ernest Graham. Gynecology and Obstetrics, Johns Hopkins Hospital, Baltimore, MD, USA. INTRODUCTION: Chorioamnionitis is associated with increased risk of cerebral palsy and neurologic morbidity. Hypoxic-ischemic encephalopathy (HIE) is a significant risk factor for similar injuries. We sought to evaluate the association between presence of chorioamnionitis and acute neonatal neurologic injury as seen on MRI.

We performed a retrospective cohort study of all neonates with clinically suspected HIE admitted to the Johns Hopkins NICU from January 2007 to September 2014 for whole body cooling. The cohort was stratified based on presence of clinically diagnosed chorioamnionitis at delivery. MRI imaging, performed between 2-23 days of life, was evaluated for evidence of brain inflammation or edema. RESULTS: During this time period, 155 neonates were admitted to our NICU for whole body cooling with suspected HIE. Of these, 137 had brain MRIs performed near delivery. Results regarding presence of clinical or histologic chorioamnionitis and presence of abnormality as noted on MRI are noted in Table 1 There is increasing evidence of cerebral side effects such as delayed myelination but knowledge of impact on functional brain development is sparse. We examined the influence of GC on maturation of cerebral processing of somatosensory evoked potentials (SEP) in fetal sheep, the animal model in which antenatal GC therapy has been developed. Cortical SEP reflect information processing in the cortical network. Additionally, early components are influenced by afferent and subcortical transmission of the stimuli. METHODS: Chronically instrumented pregnant sheep received 2x110µg/ kg BM i.m. 24h apart (n=12) or saline (n=9) at 105, 112 and 119 days gestation (dG, term 150 dG). This dose is equivalent to the clinically used 2x8mg BM administered to a 70kg woman. Trigeminal SEP were evoked prior to, 4h and 24h after each injection and 7d after the last treatment course. RESULTS: SEP peaks were already detectable at 0.7 gestation. The early and late cortical responses N20 and N200 were the most reproducible peaks (Fig. 1) . Latencies of these peaks decreased over the examination period (p<0.05, Fig. 1 ). BM did not have an acute effect on SEP amplitudes and latencies 4h and 24h following administration. Chronically, BM delayed the latency decrease of the N200 but not of the N20 by two weeks (p<0.05, Fig. 1 ). CONCLUSIONS: Cerebral processing of sensory stimuli is present in the third trimester. BM delays maturation of late but not early cortical SEP responses suggesting that BM affects maturation of sensory processing in the cortical network rather than maturation of afferent pathways. *Figure(s) will be available online. Fig. 1 Example of the developmental changes of the N20 and N200 between 105 and 126dG (left) and effects of BM on development of the N200 latency (right) (*** p<0.001 compared to the next younger age, $$ p<0.01 compared to controls). (GCS)is an enzyme complex which metabolises glycine with production of 70%of cytoplasmis 1carbon units.Mutation of GCS-encoding in humans are associated with neural tube defects(NTDs)and non ketotic hyperglycinemia(NKH),an autosomal recessive disease,characterized by respiratory problems and seizures.50%of children with NKH show brain anomalies such as hydrocephalus.We studied a mouse model of GCS deficit with NTDs in 40%of mutants and hydrocephalus,identified at 18.5 in 20%of mutants without NTDs.The aim of this project was to study the brain development in this model using ultrasound(US)to clarify the stage of onset and pathogenesis of hydrocephalus METHODS: We used a mouse model carrying a gene-trap vector in the glycine decarboxylase gene.Pregnant mice were scanned under anaesthesia using 40and50MHz probes(Vevo2100)to acquire 3D volumes of the embryos'brain at E12.5,E16.5,E18.5.Post mortem examination included brain histology and genotyping by PCR of DNA.3Dvolumes were analysed blind to genotype by 1operator to evaluate brain structures RESULTS: US study was completed in 37/40embryos(92%):11wild type,13heterozygous,13mutants from 6litters.6/13(46%)mutants had exencephaly evident on scan in all cases.On scan all embryos without NTDs showed normal 4th ventricle,its choroid plexus and cerebellum,at E16.5andE18.5.In 2mutants without NTDs,an enlargement of the 3rd and the lateral ventricles was observed to develop at E16.5(n=1)and at E18.5(n=1);in these embryos the 4th ventricle was normal.These findings were confirmed by histology * Figure(s) 

The study group consisted of 36 healthy fetuses with gestational age (GA) ranging from 27 to 38 weeks. Fetal AER, VER and No-Stimulus(NS) data was recorded on the same day for every visit using a151-channel SARA system. Interfering cardiac signals were removed and fBS was determined according to Nijhuis criteria for each recording and classified in to quiet, active and transition states. AER and VER were further scored for latency and amplitude. RESULTS: Overall 46 AER, 50 VER and 49 NS recordings were analyzed. The frequency of observing the fetus in active sleep and in a transition between quiet and active sleep states increased with GA (Fig  1) , whereas the frequency of quiet sleep decreased with GA. *Figure(s) will be available online.

Under stimulation, the distribution of states did not seem to change significantly. Fig. 2 shows the distribution of the states and evoked responses. A positive response rate to auditory stimulus was higher in state quiet sleep(82%) and transition (100%) states than in state active sleep (56%). For visual stimulation, the positive response rate was also higher in quiet sleep (89%) and transition (90%) compared to active sleep state (75%). CONCLUSIONS: Neither auditory nor the visual stimulation has an effect on state distribution. On the other hand, fetal evoked response to auditory and visual stimulus is closely connected to fBS. Response rate is higher for quiet sleep and transition state as compared to active sleep state for both type of stimulations.

Role of Vagus Nerve in Fetal Glucosensing and Neuroinflammation. M Cao, 1 S Kuthiala, 1 T Shafiee, 1 H L Liu, 1 P Burns, 2 A Desrochers, 2 G Fecteau, 2 M G Frasch. We hypothesized that vagotomy (Vx) will result in hyperglycemia and this will be correlated to a higher degree of neuroinflammation. METHODS: Near-term fetal sheep were surgically prepared with vascular catheters and bilateral Vx without (n=11) or with vagus nerve stimulation (VNS) electrodes (n=3). At 72h post-operative recovery, n=11 animals served as controls; n=13 received lipopolysaccharide (LPS) (LPS group) to induce inflammation; Vx and Vx+VNS animals also received LPS (Vx+LPS and Vx+VNS+LPS groups). Fetal arterial blood was sampled during surgery before and after Vx, on each post-operative day, baseline (0 hours) and seven selected time points (3-48h) to profile inflammation (ELISA IL-6, pg/mL), insulin (ELISA, ng/mL) and glucose levels (mg/ dL). 54h post LPS, necropsy was performed and neuroinflammation quantified as Iba1+ brain microglia immunofluorescence. Results are reported for p<0.05. RESULTS: Surgery, Vx and post-operative period had no effects on glucose averaging 17±5 (mean±SEM). Overall, glucose levels in Vx+LPS were higher vs. control and LPS groups at all time points and VNS normalized them; LPS alone had no effect on glucose or insulin levels, so we report mean values for control and LPS groups. At baseline, Vx+LPS glucose levels were 21±5 vs. 16±3; insulin values were similar. At 3h post LPS, IL-6 peaked 481±310 vs. 1±1 at baseline and Vx+LPS glucose was 20±4 vs. 15±3. Vx+LPS insulin levels increased at 0.6±0.4 vs. 0.2±0.1. At 48h post LPS, Vx+LPS glucose was 22±6 vs. 17±3, while insulin values returned to baseline. In Vx+LPS, higher glucose levels at 54h correlated to higher degree of neuroinflammation in hippocampal regions CA23 and dentate gyrus as well as thalamus (r=0.92, r=0.95, r=0.84 (p=0.07)). CONCLUSIONS: Complete withdrawal of vagal innervation results in a 72h delayed onset of sustained hyperglycemia for at least 54h. Under conditions of moderate fetal inflammation, this is related to a transient hyperinsulinemia and higher levels of brain inflammation. This suggests an important role of vagus nerve in both glusosensing and neuroinflammation and warrants further investigation to help prevent perinatal brain injury. We developed an in vivo -in vitro animal model to study the mechanisms of the developmental programming of lipopolysaccharide (LPS) induced neuroinflammation with focus on microglia phenotype. Near-term fetal sheep were exposed in vivo to low dose LPS (second hit control, SHC) to mimic subclinical FN; naïve microglia were exposed in vivo to NaCl (naïve control, NC) and then to LPS in vitro (naïve LPS, NL). SHC were then exposed to LPS in vitro (second hit LPS, SHL). We reported that in such paradigm LPS second hit results in amplification of microglial cytokine (IL-1β) production in vitro (Cao et al., IJDN, in press) .

To deeper explore the microglial phenotype we sequenced the whole microglial transcriptome at high throughput. First, we identified differentially expressed genes in NL microglia. Second, we compared NC to SHC. Third, we evaluated the difference in response between NL and SHL microglia. RESULTS: Up regulation of inflammatory pathways NFKB, PIK3-Akt and JAK-Stat in NL microglia was accompanied by a down regulation of metabolic pathways. FBP was up regulated in SH microglia and was unique to these samples (log 2 = 4.057 and padj = 9.40 x 10 -2 ) confirming recent observations in neonatal murine microglia that FBP may regulate We performed whole transcriptome sequencing at high throughput of microglia primary cultures (Figure 2 ). We then performed 3 differential analyses: we compared (1) NL versus NC;(2) NC to SHC and (3) the difference in response between NL and SHL. For downstream analysis see Figure 2 . RESULTS: Our method allowed a fast screening of the complete fetal sheep transcriptome at low cost. A poorly covered reference genome limited our approach, however, this will improve with time. Our 3 differential analyses allowed discovering significant genes, such as HMOX1 and FBP and pathways involved in FN. Clustering genes by hierarchical clustering, by functionality and by ontology allowed to filter out candidate genes having an effect on FN. Notably, our comparative approach permits inferences even with a single replicate. This allowed us to quantify the genomic response of SH compared to NL. CONCLUSIONS: Fetal sheep microglial transcriptome approach allows studying mechanisms of FN to identify mechanisms and potential therapeutic targets. Future research should aim to perform bisulfite sequencing to complete our analysis of the transcriptome by epigenetic studies.

Gangliosidosis (Sandhoff disease) in Cortical Brain Tissue. Once established, dendritic initiation never occurs again on these neurons -with one remarkable exception discovered years ago in Tay-Sachs disease. "Ectopic dendritogenesis" refers to a phenomenon, unique to lysosomal diseases, in which dendritic initiation occurs in specific mature neurons exhibiting ganglioside accumulation. We hypothesize that morphologically mature pyramidal neurons are capable of re-initiating the growth of new dendrites. Our goal is to use a model of ectopic dendritogeneis to determine the genetic mechanisms underlying normal dendritic initiation.

We performed whole transcriptome analysis on cerebral cortical tissue from cat models of two lysosomal storage diseases: α-mannosidosis and Sandhoff disease (GM2 gangliosidosis) which show 20% and 90% ectopic dendridogenesis on cortical pyramidal neurons, respectively. RNA libraries were prepared for directional RNAsequencing. 100bp single end reads of 6 samples (2 α-mannosidosis, 2 Sandhoff disease, 2 healthy controls cat brain cortical tissue) were sequenced in one multiplexed lane using an Illumina HiSeq2000 platform. RESULTS: Because the UCSC reference genome database for domestic cat has relatively low depth coverage (12x), we aligned sequenced reads with human genome and identified several differently expressed genes, including those related to growth factors and the tyrosine kinase system, GM2 ganglioside synthesis/degradation enzymes, GM2 activator protein, iron metabolism proteins (transferrin and lactotransferrin), and dendrite spine development proteins (semaphorin3A, TAOK2, intersectins, septin7) and their signal transduction elements. To assess the robustness of our human/cat homology, we validated 17 differentially expressed genes by q-PCR. CONCLUSIONS: Our findings are the first steps to understanding the underlying mechanism of control of dendritic initiation. Such discovery will not only provide insights into normal brain development, but could also lead to therapeutic discoveries for lysosomal diseases and other neurodegenerative disorders.

Model. after IR on GD18, molecular changes in the fetal brain was the focus of this investigation.

During surgery, Fetal ECG was used to monitor the fetal condition at late preterm (near-term birth). Response to IR, P53 which showed a significant increase on binding to promoter site of various genes. Vaginal LPS administration increases phosphorylation of JNK-p53 in the fetal brain response to IR. The vulnerability of brain hemorrhage in response to IR was increased in the V plus IR group. Moreover, a known p53 inhibitor, PFT-alpha, blocked P53-dependent various protein phosphorylation. Thus, pretreatment with PFT-α may serve to rescue fetuses from cerebral hemorrhage response to IR with vaginal LPS. CONCLUSIONS: Maternal inflammation increased the vulnerability of fetal brain hemorrhage may be via a P53-dependent protein phosphorylation. One effect of DHA is the up-regulation of galanin via the induction of leptin, a hormone that modulates food intake. To assess the involvement of galanin in neurogenesis, we evaluated concentrations of galanin and factors affecting neural development in amniotic fluid. METHODS: Amniotic fluid was obtained from 523 women undergoing an amniocentesis at 15 to 19 weeks gestation. Aliquots were assayed by ELISA for concentrations of galanin, neurotrophin (NT)-3 and -4, brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), leptin, interleukin (IL)-6, IL-8, extracellular matrix metalloproteinase inducer (EMMPRIN) and the 70kDa heat shock protein (hsp70). Intraamniotic levels of fatty acids were determined by gas chromatography. Correlations between assays were analyzed by the Spearman rank correlation test. RESULTS: Positive correlations were observed between galanin and arachidonic acid (r=0.3930, p=0.0027), DHA (r=0.3644, p=0.0058), oleic acid (r=0.3341, p=0.01), leptin (r=0.7205, p<0.0001), and neurotrophin 4 (r=0.2811, p=0.0375). Conversely, negative correlations were seen between galanin and gestational age at delivery (r=-0.2280, p=0.0002), EMMPRIN (r=-0.3107, p=0.0187), NT-3 (r=-0.3003,p=0.0381) and birthweight (r=-0.1310, P=0.0378). No associations were found between galanin and maternal age, body mass index, gestational age at time of amniocentesis, gravidity, parity, meconium at delivery, palmitic acid, stearic acid, linoleic acid, MMP-8, NGF, BDNF, IL-6, IL-8 and Hsp70. CONCLUSIONS: We propose that good nutrition (high intake of DHA and arachidonic acid) induces leptin (promotes fat intake) which then induces galanin to enhance neural cell survival and neurogenesis. In addition, galanin inhibits production of EMMPRIN which disrupts the blood-brain barrier and causes inflammation, and NT-3 which induces apoptosis of neural cells. The findings support the importance of a diet high in DHA and AA supplementation during pregnancy. However, whether brain weights are similarly reduced, or preserved by "brain sparing" mechanisms, and whether energy levels are depleted leading to membrane failure and overt injury, remain unknown. Our purpose was to determine the extent to which MNR in guinea pigs as a causative factor for fetal growth restriction (FGR) impacts brain growth,

the degree of "brain sparing", and measures of overt injury by quantifying brain weight, brain to liver weight ratios, and cellular necrosis/apoptosis. METHODS: Guinea pig sows were fed ad libitum (Control) or 70% of the control diet pre-pregnancy switching to 90% at mid-pregnancy (MNR). Animals were necropsied near-term for fetal growth measures, and fetal brains were immersion-fixed for later assessment of necrotic cell injury using standard H&E criteria and apoptotic cell injury using the TUNEL assay method. Significance was assumed for p<0.05. RESULTS: Nine control (31 fetuses) and twelve MNR (42 fetuses) sows were necropsied with MNR fetal weights decreased 28%. Select AGAcontrol (n=18) and FGR-MNR (n=18) fetuses underwent full necropsy with FGR-MNR weights decreased 37%, brain weights decreased 12% and brain to liver weight ratios increased 48%. Overall, low levels of necrotic cells were observed, with no differences between groups. While low levels of apoptotic cells were also observed, averaging 0.75/HPF and 1.18/HPF (40x) for AGA-control and FGR-MNR animals, respectively, FGR-MNR levels were higher in the periventricular white matter (2x), the hippocampus CA1 (3x), CA4 (1.7x) and dentate gyrus (3x) CONCLUSIONS: MNR in guinea pigs results in FGR with small livers relative to brain weights, likely indicating blood flow redistribution, characteristic of asymmetrical FGR. These fetuses have reduced brain weights, but with substantial "brain sparing", and with no increased necrotic cell injury indicating that the threshold for membrane failure with energy depletion has likely not been reached. However, apoptotic indices were increased primarily in hippocampal regions that may involve alterations in the balance of pro-and anti-apoptotic factors and underlie risk for increased cognitive impairment seen in FGR offspring. pro-inflammatory mediators. We aimed to characterize periventricular white matter inflammation and neuronal injury after experimental CDI and contrast this with Ureaplasma intra-amniotic infection (IAI), which has been associated with cerebral white matter damage. METHODS: Chronically instrumented rhesus macaques received intra-amniotic inoculation with U.parvum (serovar 1; 10 7 cfu/mL) at 127±1dGA (term=167d; n=5). Age matched controls received vehicle only (n=6). Three animals with catheters placed between the maternalfetal membranes received repeated choriodecidual inoculation (10 7 -10 9 cfu/mL). Fetal brains collected after cesarean section at 153±4dGA were processed for histopathological assessment of inflammatory cell activation (GFAP, Iba1) and cell death (Casp-3). Levels of inflammatory mediators and Ureaplasma colony counts were determined in serial samples of amniotic fluid. RESULTS: CDI resulted in negative AF bacterial cultures and absence of labor-like contractions. In contrast, IAI resulted in a robust fetal inflammatory response and onset of contractions. Fetal brains from IAI animals showed a significant increase in periventricular microglial activation (Iba1 positive) compared to control (p=0.001). Despite the lack of microbial invasion into the amniotic cavity, CDI fetal brains showed a further marked increase in periventricular Iba1 staining compared to IAI animals. Brain and CSF cultures were negative for ureaplasma in all animals.

The current data suggests that inflammatory processes initiated in the maternal-fetal membranes are associated with brain inflammation even prior to microbial invasion of the amniotic cavity. The fetus may be at risk of inflammatory brain injury at an earlier stage of ascending infection than previously thought. The utilization of these unique non-human primate CDI and IAI models of preterm birth will aid the elucidation of mechanistic pathways that result in poor neonatal outcomes, with the aim of developing new therapeutic approaches. Vx+LPS and Vx+VNS+LPS groups (Fig. 1) .

From 24h onward (post 2 nd LPS dose), IL-6 in the Vx+VNS+LPS was undetectable. In parallel, Iba1+ cell intensity in ileum was lower in Vx+VNS+LPS vs. LPS group but not different from controls. CONCLUSIONS: VNS reduced the levels of systemic and regional inflammation to control values. Therapeutic potential of VNS for early postnatal treatment of babies at risk of sepsis with ensuing organ injury should be considered.

Stromal Derived Factor-1α Is Key To Treating Neonatal Hypoxic-Ischemic Encephalopathy. Miki Mori, Keiichi Matsubara, Yuko Matsubara, Yuka Uchikura, Toshiaki Yasuoka, Akihiro Nawa. Gyneclogic Oncology, Ehime University Hospital, Toon, Ehime, Japan. INTRODUCTION: With perinatal medical progress, the survival rate of the newborn baby was improved, but in late years, cerebral palsy is increasing with the increase of preterm birth.Hypoxic ischemia (HI) is known as a main cause of the cerebral palsy; however, the effective medical therapy has not been established. Stromal derived factor-1α(SDF-1α)is reported to regulate the migration of cells derived from bone marrow into the ischemic lesion of damaged tissues. In this study, we evaluated the effect of SDF-1α on brain damage of neonatal rats.

Left common carotid arteries of seven-day-old Wistar rat pups were ligated with 6-0 silk threads. The pups were subsequently exposed to hypoxic gas mixture (8% oxygen, 92% nitrogen) for 120 min. The pups were divided into 4 groups, 1) control, 2) HI and saline intracranial injection, 3) HI and SDF-1α (60µg/kg) intracranial injection, 4) HI and SDF-1α (600µg/kg) intracranial injection, and 5) SDF-1α intracranial injection only. Seven days later, brain damage of pups was evaluated with magnetic resonance imaging.11 to 15 days later, neurocognitive outcomes were evaluated using the Morris water maze (WM). Four weeks later, Motor coordination ability was assessed using the accelerating rotarod assay. After the rotarod test, the pups were sacrificed and the brains were dissected. Brain injury was assessed using TTC staining.

: WM showed that there was an improvement of spatial perception ability in the SDF-1α (600 mg/kg) injection group. On the other hand, meaningful improvement of the motor coordination ability was not observed by the rotarod assay. Furthermore, the size of brain injury was not changed by SDF-1α. CONCLUSIONS: In neonatal HI encephalopathy, it is possible that SDF-1α might be influential on the spatial perception ability without physical improvement of the brain injury. Interleukin-1 beta (IL-1β) is a known cytokine mediator of perinatal brain injury with exposure to intrauterine inflammation. However, whether the maternal or fetal contribution is essential to IL-1β-mediated perinatal brain injury remains unclear. The aim of this study was to utilize the embryo transfer technique of KO IL-1β mice to elucidate fetal/maternal contribution to perinatal brain injury. METHODS: CD-1 dams, both wild type (WT) and knockout for IL-1β (mt), were naturally mated or received embryo transfer with either pups who were wildtype (WT) or mutant for IL-1β (mt). There were four groups (maternal genotype/embryo genotype): WT/WT (n=5), WT/mt (n=8), mt/WT (n=9), mt/mt (n=6). Each group was treated with normal saline (NS) or LPS (50 mcg/mouse) intrauterine infusion at E17. Fetal brains were harvested and RNA extracted from offspring mice at 6h post injection. Microarray analysis was performed using the Partek, Spotfire, and Ingenuity Pathway Analysis. A one-way ANOVA was conducted with indentification of differentially expressed genes (DEG) showing an a priori p<0.05 and a fold-change greater than 1.5. Confirmatory pilot qPCR and immunocytochemical analysis of cortical fetal neurons were performed.

The microarray results are shown in Table 1 . LPS significantly increased the DEGs in the WT/mt group. In contrast, the mt/WT cohort had the greatest reduction in DEGs. Gene ontology analysis showed a significant increase in genes implicated in neuronal death, neuronal death regulation, apoptosis, apoptosis regulation and brain development. The mutant/WT group only had a higher prevalence of genes involved in neuronal differentiation (n=9). Pilot qPCR analysis showed a significant change in IL-36 expression. Immunocytochemistry showed altered dendrite formation in primary cortical neuron cultures.

The results suggest that maternal genotype has a profound effect on neonatal response to intrauterine inflammation. Maternal absence of IL-1 attenuates the fetal response and blunts the inflammatory downstream effects. Processing of acoustic stimuli in the auditory cortex was determined in 6 fetuses. Cortical auditory evoked potentials (cAEP) were induced at 105±1, 112±1, 119±1 and 126±1 days gestation (dG, term 150 dG) using a tone of 500Hz, 100dB SPL and 50ms duration with inter-stimuli intervals of 0.8-1.2sec applied at the maternal abdominal wall for 5min. To induce a fetal arousal, VAS were applied to 9 sheep at 120±2, 127±2 and 130±1dG using an electronic artificial larynx (12 stimuli for 30sec with a peak frequency of 250-750Hz producing an intrauterine sound pressure of 125-35dB). The cochlea was ablated in 3 fetuses to discriminate the role of the peripheral auditory and proprioceptive system in perception of VAS. Fetal arousal was detected by spectral analysis of the EEG. RESULTS: cAEP were already detectable in 5/6 fetuses at 110dG. Latency of cAEP peaks decreased continuously until 125dG and amplitudes started to increase at 125dG reflecting cortical maturation (p<0.05). During NREM sleep, cortical arousal in response to VAS was firstly detectable at 127dG revealed by fast activation of the EEG within 32ms. At 131dG, arousal prolonged or induced a sleep state change. At this age, unspecific changes of cortical activity without a fast arousal response developed during REM sleep. Bilateral cochlea ablation prevented fetal arousal. CONCLUSIONS: Cortical processing of acoustic stimuli begins in the early third trimester and precedes arousability of the fetus which starts in the middle of the third trimester. The fetus is especially sensitive to acoustic but not vibratory stimuli. (n=104). GSEA identified greater enrichment of neuroinflammatory processes in the LPS-MgSO 4 cohort than the LPS-PBS cohort, most prominently in the Tnfr1 network. The LPS-PBS cohort was significantly enriched in extracellular matrix genes. In this group, the network downstream of Tgfb1 was most enriched (z-score, 5.6, p<0.05). Tgfb1-regulated genes were also enriched after MgSO 4 (z-score, 6.2). qPCR for genes in the networks for Tnfr1 or Tgfb1 confirmed the pathway analysis in pilot studies comparing PBS-PBS and LPS-PBS (Tlr4 and Il6, p<0.05).

This transcription expression analysis confirms that BMTZ plays a critical anti-inflammatory role in a model of fetal neuroinflammation and offers potential mechanisms for the neuroprotective benefit of combination therapy.

Effects Previous studies have shown that prematurity and inflammation can cause defects of neurogenesis. Protective effects of sex steroids such as progesterone and estrogen have not been well studied in the perinatal brain. In this study we looked specifically at the deleterious effects of inflammation on neurogenesis, and the possible protective effects of progesterone. METHODS: Timed pregnant mice (n = 8) were given intraperitoneal (IP) injections of 5-Bromo-2'-deoxyuridine, a thymidine analog (Brdu) (20mg/kg) and progesterone (42mg/kg) or vehicle on embryonic day 17. Two hours later, mice were given IP LPS (100ug/kg) or vehicle. C-section was performed on embryonic day 18. Two-color immunofluorescence was performed with primary antibodies Tbr2 and Iba1. Cells counts were obtained from images spanning cortex from the ventricular to pial surface. Statistical analysis was determined using Anova and Tukey-Kramer. RESULTS: Microglia were abundant in progenitor zones of the developing cortex and contacted intermediate neural progenitor (INP) cells. After LPS exposure, microglia displayed a more activated phenotype and cell density was increased compared to sham or progesterone only controls (p < 0.0001). Tbr2+ INP cell density decreased after LPS exposure compared to control (p=0.0022). However, progesterone pretreatment prior to LPS, statistically increased Tbr2+ INPs compared to progesterone treatment alone (p=0.0022) and was not different from control INPs *Figure(s) will be available online. CONCLUSIONS: LPS induced systemic inflammation reduces prenatal neurogenesis. Progesterone treatment prior to inducing systemic inflammation is associated with increased neurogenesis compared to progesterone alone and was equal to controls. Progesterone may protect the preterm brain from defects of neurogenesis induced by inflammation.

Emilie Vander Haar, Cynthia Gyamfi-Bannerman, Jaclyn Coletta-Lucas. Ob/Gyn, Columbia University Medical Center, New York, NY, USA.

To determine whether chorioamnionitis is associated with cerebral palsy.

We conducted a secondary analysis of the NICHD MFMU multicenter, randomized controlled trial of magnesium for cerebral palsy (CP) prevention. The current study included non-anomalous singleton gestations for which there was complete information on CP and chorioamnionitis (COA). We excluded multiples and those with missing outcome data. Our primary exposure was COA, defined by clinical diagnosis of COA and a maternal fever >100F; our secondary exposure was culture-proven sepsis (CPS). Our primary outcome was diagnosis of CP and our secondary outcome either CP or death (CP+death). CP was defined as two of the following: delay in motor milestones, abnormalities on neuromotor exam, aberrations in primitive reflexes or diagnosis of spastic hemiplegia or diplegia. We conducted bivariate analyses on the exposures and outcomes and fit a log-linear regression model, adjusting for possible confounders to assess factors related to CP. RESULTS: 1858 patients were included; 218 (11.73%) had COA, and 312(16.79%) had CPS. Only 21.1% of infants of women diagnosed with COA had CPS. Gestational age (GA) at delivery was significantly lower in infants born to mothers with COA (28+5/7 wks vs 29+6/7 wks, p<0.001) compared with those without COA. Women with COA were more likely to deliver via cesarean and deliver infants with a 5-min Apgar score less than 7. There were no significant differences in CP (13.58% vs 11.65% p=0.5973) or CP+death (11.45% vs 11.76% p=0.9041) for infants born to mothers with or without COA. After adjusting for confounders there remained no difference in CP in either group. However, infants diagnosed with CPS were more likely to develop CP (33.33% vs 16.04% p=<0.001) as well as CP+death (41.57% vs 14.36%, p=<0.0001). After adjusting for confounders, CPS was still associated with a higher risk of CP+death (table 1) . *Figure(s) will be available online. CONCLUSIONS: Exposure to COA did not influence rates of development of CP, however a diagnosis of CPS was associated with moderate to severe CP or death. Neonatal sepsis, rather than chorioamnionitis, should warrant close surveillance for neurologic sequelae. Our objectives were to quantify mRNA and protein levels of 5 AQPs (AQP1, AQP3, AQP8, AQP9 and AQP11) in ovine amnion during control conditions, determine changes in AQP expression profile when AFV and IMA rate were experimentally altered, and analyze the relationships of AQP expression levels with IMA rate and AFV. We hypothesized that individual AQP levels would correlate positively with IMA rate and AFV.

We measured AFV and IMA rate in fetal sheep during 4 experimental conditions: 1) control, 2) urine drainage, 3) urine drainage with replacement of Ringer's solution, and 4) intra-amniotic infusion of Ringer's. After 2 days, the amnion was collected for analysis. AQP mRNA levels were determined by real time RT-qPCR, corrected for efficiency, and normalized to the geometric mean of 2 internal references. Protein levels were determined by Western Immunoblot. Statistical analysis was performed using ANOVA or regression RESULTS: Under control conditions, AQP1, 3, 8, 9 and 11 in the amnion were differentially expressed with AQP1, 8 and 11 mRNA levels 8-23 fold higher than AQP9, the least expressed. * Figure(s) will be available online.The relative quantities of AQP1, 3, 8, 9 and 11 mRNA and protein were unchanged in the 3 experimental conditions as compared to control. AFV and IMA rate decreased during urine drainage and increased with intraamniotic infusion. The combined data showed that AFV was positively correlated with IMA rate (P<0.01). However, neither AFV nor IMA rate correlated with AQP1, 3, 8, 9 or 11 mRNA or protein levels. CONCLUSIONS: Five AQPs are differentially expressed in the ovine amnion. Further, AQP1, 3, 8, 9 and 11 mRNA and protein levels are not altered when AFV and IMA rate are experimentally modified. These findings do not support a significant role of AQPs in the regulation of AFV or IMA rate under these conditions.

Increased 

Obesity in pregnancy is associated with increased fetal growth and adiposity and is linked to the development of metabolic syndrome in the adult offspring. Fetal growth is dependent on nutrient supply, hence related to placental transport of nutrients. Placental fatty acid transport proteins (FATPs) and fatty acid translocase (FAT) are integral transmembrane proteins responsible for cellular uptake of fatty acids. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is a lipid sensor that regulates the expression of FAT/FATPs. We hypothesized that maternal obesity in mice is associated with an increased placental expression of FAT/FATPs and lipid accumulation in the fetal liver. METHODS: C57/BL6J female mice were fed either a control (C; n=10) or an obesogenic (OB; n=10) high fat-high sugar diet for 4-weeks before mating and throughout pregnancy. At E18.5 animals were sacrificed, fetal and placental tissues collected. Trophoblast plasma membranes (TPM) were isolated from pooled placental homogenates (H) for each litter. TPM purity was determined by alkaline phosphatase enrichment. Protein expression of FAT/FATPs (TPM) and PPAR-γ (H) was determined by immunoblotting. Sections of fetal livers were stained for Red Oil O and lipid droplets were quantified using ImageJ software. Statistical significance was determined by Student's t-test. RESULTS: TPM alkaline phosphatase enrichment was similar between C and OB groups (10-fold vs. 11-fold). TPM protein expression of FAT and FATP2 was not different between C and OB groups. In contrast, TPM FATP6 expression increased 36% (p<0.05) in OB compared to C placentas. PPAR-γ expression was unchanged between groups. Preliminary data indicated a ~10-fold increase in lipid droplets in the livers of fetuses from OB compared to C group (n=3/group; p<0.05). CONCLUSIONS: Our results show that FAT, FATP2 and 6 proteins are expressed in TPM from mouse placentas. TPM FATP6 expression was increased in obese dams and was associated with increased lipid deposition in the fetal liver. Up-regulation of placental FATP6 in maternal obesity may contribute to increased delivery of fatty acids to the fetus and accumulation of lipids in the fetal liver. Intrauterine growth restriction (IUGR) affects approximately 8% of pregnancies and is associated with peri-and postnatal adverse health outcomes including metabolic syndromes, obesity, and cardiovascular disease. The most frequent cause of IUGR is placental dysfunction, making this tissue a prime candidate for further investigation of molecular level alterations that may be linked to fetal growth restriction.

acquired for each sequence. An ROI identified all placental tissue in the image slice and the mean signal of the difference images was extracted. Data analysis was performed in Matlab fitting a single parameter model of diffusion.

There was a wide range in delivery gestations, from 224-284 days, and birthweight centiles from 1-64. Perfusion under air and oxygen breathing was (mean ± SD (range)); 178± 53 (122.8 -289.8) and 187± 55 (108.9 -268.8) ml/min/100ml, respectively. There was no significant difference between placental flow under air or oxygen-breathing (P = 0.7322, paired t-test). CONCLUSIONS: Mean placental perfusion was consistent with prior measures obtained at 0.5 and 1.5T. Placental perfusion did not change significantly with an oxygen challenge. Hence, R1 and R2* changes under maternal hyperoxia are likely to be reflective of changes in pO 2 and SO 2 , supporting the usefulness of BOLD and OE-MRI imaging to determine oxygenation as a measure of placental function. The range of pregnancy outcomes in the study suggest this result is not limited to healthy pregnancy.

Alterations of Mitochondrial Content in Obese Placentas. C Mando, 1 C Novielli, 1 G M Anelli, 1 V Clivio, 1,2 M Cardellicchio, 2 I Cetin. Obese women (OB) have increased risk for gestational problems and impaired fetal growth, with programming effects on intrauterine growth and future health. Indeed, we recently showed abnormal placental (P) biometry in OB, leading to decreased P efficiency. Chronic mild inflammation and mitochondrial (mt) dysfunction have been reported in many tissues, but little is known about the obesity effect on P mt. Here, we studied P mtDNA levels, accounting for the cell mt content, in OB and normal weight (NW) women METHODS: P collection at elective cesarean section from 10 NW (BMI 18-25) and 13 OB (BMI³30) with no diabetes or other maternal diseases.Maternal decidua was discarded, villi quickly frozen. mtDNA measured by RealTimePCR relative quantification (CytB/RNAseP) RESULTS: In our study population mtDNA was significantly increased in P villi from OB placentas vs NW (331±104 vs 170±51; p=0.000) *Figure(s) will be available online. mtDNA levels were significantly related to BMI (R 2 =0.54, p=0.000) OB weight gain was significantly lower vs NW (7.8±3.4 vs 11.5±3.3; p=0.03) while maternal-gestational age, P and fetal weight, maternal Hb were not significantly different. No mtDNA differences depending on fetal sex CONCLUSIONS: We previously reported altered mt levels in pregnancies characterized by P insufficiency, presenting increased inflammation/oxidative stress (OxS). Here we found increased mt content in our study population of OB-non diabetic women, suggesting a P functional impairment depending on maternal BMI. This might account for increased OxS, related to inflammation, due to the lipotoxic effect of high intracellular fatty acids in OB. Higher mtDNA may reflect a P compensatory response to obesity, which in several tissues have been associated with impairment of mt function. Further data are needed to confirm our results

S-240 Almost half of women of reproductive age are either overweight or obese. A high maternal dietary fat and salt intake can affect placental function, thereby increasing the incidence of fetal growth restriction (FGR). FGR is associated with increased perinatal morbidity as well as increased incidence of cardiovascular disease, hypertension and diabetes in adult life. Metabolomic profiling has the potential to reveal altered maternal and fetal metabolic pathways in a diet-induced FGR rat model. The present study therefore examined the effects of maternal high fat and/or salt on placental metabolism. METHODS: Virgin Sprague Dawley rats were assigned to one of 4 dietary groups: control (CD; n=8; fat 10% kcals); high salt (SD; n=6; salt 4%, fat 10%); high fat (HF; n=8; fat 45%) and HF and salt (HFSD; n=6; salt 4%, fat 45%) 21 days prior to mating. At gestational day 18, dams were culled, and fetus and placenta weighed. Tissues were snap-frozen for metabolic profiling of placentas using gas-and liquid-chromatography mass spectrometry. RESULTS: A maternal HF intake significantly reduced fetal (HF 3.68±0.04 vs. CD 3.99±0.07 g; p<0.05) and placental weight (HF 0.50±0.1 vs. CD 0.64±0.02 g; p<0.05). There was no effect of high salt on fetal weight when compared to controls (p>0.05), but placental weight was significantly reduced in males only (SD 0.61±0.01 vs. CD 0.67±0.03 g; p<0.05). A significant decrease in the abundance of palmitoleic acid (p<0.001) and an increase in the levels of longer-chain polyunsaturated fatty acids (PUFA; p<0.001) were observed in HF and HFSD placentas. A non-significant trend in pentadecanoic acid and 4-aminobutyric acid metabolism alteration due to high salt intake was observed. CONCLUSIONS: A maternal HF diet altered placental metabolic profiles, with increased amounts of longer-chain PUFA observed. It was not determined, however, whether changes in essential fatty acid metabolism took place in maternal or placental tissues. Further studies are required to fully elucidate the impact of a maternal HF diet on the availability of PUFAs to the placenta and/or fetus. This, and future studies, are important in providing a better understanding of maternal obesity and its impact on fetal growth.

Maternal We previously demonstrated that lung development in female rat pups is compromised by in utero exposure to MTS, while male rat pups are unaffected. Lung development is also influenced by essential fatty acids. We demonstrated that circulating fetal essential fatty acids were altered by placental insufficiency in a sex-specific manner. Essential fatty acid transfer across the placenta is regulated by fatty acid transport proteins 1 (SLC27A1), 2 (SLC27A2) and 4 (SLC27A4), as well as endothelial lipase (LIPG). We hypothesize that in utero MTS exposure sex-specifically alters mRNA transcript levels of SLC27A1, SLC27A2, SLC27A4 and LIPG in the rat placenta. METHODS: Pregnant rats were exposed to tobacco smoke (MTS) or room air (Control) from embryonic day 11 to term. Placentas from male and female MTS and control pups were collected at delivery. Real-time RT-PCR was used to measure mRNA transcript levels of SLC27A1, SLC27A2, SLC27A4 and LIPG. RESULTS: Male placental mRNA levels of SLC27A1 and SLC27A2 were 55±16%* and 38±45%*, respectively, of the female placental levels. Basal SLC27A4 and LIPG mRNA levels were similar between male and female placenta. In male placenta, MTS decreased LIPG mRNA to 52±27%* relative to male control placenta. In female placenta, MTS increased SLC27A1 to 211±88%* and SLC27A4 to 155±41%* mRNA relative to female control placenta. (*p<0.05). CONCLUSIONS: Basal mRNA levels of fatty acid transporters differs between placenta of male and female pups. In addition to basal differences in expression, MTS also alters mRNA levels of fatty acid transporters in a sex-dependent manner in rat placenta. We speculate that MTSinduced sex-specific differences in lung development may be a function of differences in maternal-fetal transport of essential fatty acids in male and female offspring.

To examine the molecular and cellular mechanisms of magnesium sulphate in regulating placental function using in vitro models of the human placenta.

and 48h post treatment with Lipopolysaccharide (LPS) (10-1000ng/ml) and MgSO 4 (100nM-1mM)by MTT assay. To validate the MTT results we investigated cell nuclear morphology stained with DAPI to determine the numbers of pyknotic nuclei. Quantitative real time PCR was also used to evaluated STOX1 and TNF-α mRNA expression. RESULTS: This study demonstrated a dose dependant effect of MgSO 4 on cytotrophoblast viability. Treatment of BeWo cells with 200ng/ml LPS had a statistically significant effect on cell viability with only 30% viable cells evident after 48h. Interestingly, we found that pre-treatment of these cells with 1mM MgSO 4 resulted in an increase in cell viability following administration of 200ng/ml LPS to57% compared to the controls. These data show that MgSO 4 has the ability toprotect placental cells against the detrimental effects of LPS exposure in vitro. Furthermore, fluorescent DAPI nuclear staining was used to validate the protective effect of MgSO 4 by showing a reduced number of pyknotic nuclei in cells pre-treated with MgSO 4 . Finally we found that MgSO 4 treatment reduced the mRNA expression of STOX1 and TNF-α in cytotrophoblasts.

This study provides evidence that MgSO4 is protective against LPS induced inflammation potentially by modulating STOX1 and TNFα expression in the placenta.

Inhibition of Placental Mammalian Target of Rapamycin Complex 2 (mTORC2) Signaling in Human IUGR. Chen YY, 1,5 FJ Rosario, 1 TL Powell, 2 MB Gupta, 3 T Jansson. Intrauterine growth restriction (IUGR) is associated with decreased placental nutrient transport, ER stress and altered placental signaling, which contributes to placental insufficiency. Placental insulin/ IGF-I and mTORC1 signaling has been reported to be inhibited in IUGR, however mTORC2 signaling, which responds to growth factors and controls cell metabolism, cell survival, and the organization of the actin cytoskeleton, has been much less studied. We hypothesized that placental mTORC2 signaling is inhibited in human IUGR. METHODS: IUGR was defined as a birth weight < 3rd percentile for gestational age (GA) and placentas obtained from appropriate-forgestational age (defined as a birth weight between the 25th to 75th percentiles) served as controls. Villous tissue was collected from IUGR (n=25, birth weight = 1804 ± 548 (SD) g, GA = 35.7 ± 3.1 (SD) weeks, range 27.9 -40.6 weeks) and Controls (n=19, birth weight = 2493 ± 1029 (SD) g, GA = 33.9 ± 4.2 (SD) weeks, range 27 -40 weeks) and frozen. Subsequently placental homogenates were prepared and mTORC1 and mTORC2 activity was determined by Western blotting analysis using wellestablished functional readouts for these signaling pathways. Differences between the two groups were analyzed statistically by t test.

Thr-37/46 was decreased by 53 % (p<0.05) in IUGR placentas, despite increased total expression of 4EBP1 (+119 %, p<0.05). In addition, the phosphorylation of AKT at Ser 473, a residue specifically phosphorylated by mTORC2, was reduced by 42 % in IUGR placentas (P<0.05).

We confirm previous reports of inhibited placental mTORC1 signaling in IUGR and we show that IUGR is also associated with inhibition of placental mTORC2. Because mTORC2 is a positive regulator of trophoblast amino acid transport, mTORC2 inhibition may contribute to decreased placental amino acid transfer in IUGR. INTRODUCTION: Triclosan (TCS) is a potential endocrine disruptor, which is widely used in personal care products and can be detected in human body fluids. It has been shown that triclosan can interfere with steroid biosynthesis and metabolism. 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is a high-affinity cortisol-inactivating enzyme which is highly expressed in human placental syncytiotrophoblasts, thus ensuring the normal development of fetus in the presence of high levels of maternal cortisol in pregnancy. However, effect of triclosan on 11β-HSD2 and apoptosis and whether these two events are correlated in human placental syncytiotrophoblasts remains unexplored. METHODS: Primary human placenta syncytiotrophoblasts were isolated from placenta collected at c section without labor at term. After syncytialization, the level of 11β-HSD2 was examined after treatment with triclosan from 0.001µM to 10µM for 24h. We also examined the levels of proteins associated with apoptosis, including caspase3, Bcl-2 and Bax. In order to explain the relationship between inhibition of 11β-HSD2 and apoptosis induced by triclosan, pre-incubation (1 h) of the cells with apoptosis inhibitor Z-VAD-FMK (30µM) followed by co-incubation (24 h) with Z-VAD-FMK (30µM) and triclosan (0.1µM) was carried out. RESULTS: Triclosan inhibited 11β-HSD2 mRNA, protein and activity levels in a concentration-dependent manner from 0.001µM to 10µM. Triclosan reduced cell viability and induced apoptosis in human placenta syncytiotrophoblasts as demonstrated by observations ofincreased DNA fragmentation and apoptosis-associated proteins such as Bax and cleaved-caspase3, and decreased pro-caspase3 and Bcl-2. Blocking the apoptosis induced by triclosan using pan-caspase inhibitor Z-VAD-FMK, the inhibition by 11β-HSD2 was significantly attenuated. CONCLUSIONS: Triclosan may induce apoptosis in human placental syncytiotrophoblasts thereby reducing the level of 11β-HSD2. These findings raise the possibility that over-exposure to triclosan could impair the placenta glucocorticoid barrier in pregnancy thus influencing the development of the fetus.

Cholecystokinin Expression and Localization in Human Placenta. Yamilette Y Borja, 1 Robert L Boudreaux, 1 Kirk P Conrad. RNA was isolated from frozen tissue for synthesis of cDNA and RT-PCR. Fixed tissue was embedded in paraffin, and used for immunohistochemical localization of CCK. RESULTS: Expression of CCK and one of its receptors (CCK1R) was identified by RT-PCR in villous and basal plate, as well as in choriocarcinoma cell lines. CCK protein was localized to syncytiotrophoblast and extravillous trophoblast cells using two polyclonal antibodies recognizing different epitopes. Extravillous trophoblast localization of CCK was verified by staining adjacent sections using cytokeratin antibody.

These findings suggest that CCK mRNA and protein are expressed by human trophoblast. Though we also observed mRNA expression of CCK1R, we could not detect CCK1R by immunohistochemistry, most likely due to insufficient number of receptors. The functional role of placental CCK and CCK1R in normal pregnancy and preeclampsia is presently unknown, and requires further study. Trophoblast CCK may be secreted serving an endocrine, paracrine, or autocrine function-all possibilities that we are currently investigating.

Abstract Withdrawn We have shown that increases in T levels in pregnant rats similar to that in preeclampsia induce hypertension, endothelial dysfunction, placental insufficiency and fetal growth restriction leading to the development of hypertension in adult offspring and is more pronounced in males than females. Since successful placental development is crucial for optimal health of the embryo/fetus into adulthood, we tested whether elevated maternal T induce sex-specific alterations in genetic pathways involved in angiogenesis and vascular development in the placentas. METHODS: Pregnant SD rats were injected with vehicle or T propionate (0.5 mg/kg/day from gestation day 15-19 to increase plasma T levels twofold, similar to that observed in preeclampsia. Global gene expression in placentas from T-exposed rats was compared with controls at 20 days of gestation using the Affimetrix Rat 1.0 microarray chip, and results confirmed with real-time PCR. RESULTS: Elevated T levels induced significant differences in 675 genes with 429 being up-regulated and 246 down-regulated in male T placentas compared to male control placentas ( ³1.2-fold; P<0.05). The web based gene set enrichment analysis (WebGestalt) analysis revealed that in male T placentas key genes involved in the vasculature development (46 genes), blood vessel morphogenesis (40 genes), and angiogenesis (36 genes) were dysregulated. Elevated T levels in the female placentas, differently expressed 419 genes with 272 being up-regulated and 147 down-regulated compared to female control placentas (³1.2-fold; P<0.05). WebGestalt analysis revealed a small set of genes related to angiogenesis (20 genes) and fat cell differentiation (12 genes) were differentially expressed in female T placentas compared to controls. Interestingly, T dysregulates unique sets of angiogenesis genes in male (7 genes) and female (23 genes) placentas. CONCLUSIONS: These results suggest a novel role for T in dysregulating genes involved in vascular development and angiogenesis in placenta in a sex-specific manner, which may contribute for fetal growth restriction and long term gender-specific cardiovascular dysfunction. We sought to characterize the initiation of placental hormonal production in singleton and multifetal pregnancies as defined by the production of estradiol (E2) and progesterone (P4). METHODS: Oocyte donation charts from 1/04 to 4/13 were reviewed to identify programmed recipient cycles with live birth. Patients were treated with E2 (monitored step-wise program) and P4 50mg daily. Outcome measures were serial E2 and P4 with mean values calculated for cycle days 28 (baseline) through 60. Singleton (single gestational sac w/single live birth) vs twins (two gestational sacs w/two live births) data were evaluated. Baseline mean value was compared to each daily mean cycle day value using an unpaired t-test (p<0.05 was considered statistically significant). RESULTS: 622 cycles met inclusion criteria. E2 and P4 remained low but continued to increase daily. E2 was significantly elevated on Day 36 in both groups when compared to Day 28 baseline. P4 was significantly elevated on Day 47 in singletons and Day 45 for twins. When comparing the two groups, E2 significantly differed on Day 45 while P4 was not consistently different. * Figure( s) will be available online. CONCLUSIONS: Endogenous placental E2 production occurs on day 36 in singleton and twin gestations while twin placental P4 production occurs earlier (day 45 vs 47). This study demonstrates that the luteoplacental shift occurs earlier than previously reported and also suggests that increased placental mass (ie twin pregnancies) results in increased placental steroidogenesis.

The The human placental growth hormone variant (22kDa hGH-V) is secreted continuously from the syncytiotrophoblast layer of the placenta during pregnancy, and is thought to play a key role in maternal adaptation to pregnancy. Studies have demonstrated that maternal serum hGH-V is associated with fetal growth. hGH-V has been shown to induce severe insulin resistance in non-pregnant transgenic mice and has been proposed as a likely candidate to mediate the insulin resistance of pregnancy. However, the effects of hGH-V administration on outcomes related to maternal and fetal growth and metabolic outcomes have yet to be investigated. We examined the dose response relationship for hGH-V administration in a mouse model of normal pregnancy. METHODS: Pregnant C57BL/6J mice were randomized to receive vehicle or hGH-V (1, 2, 5 mg/kg per day) by osmotic pump from gestational days 12.5-18.5. Maternal weight and food intake was monitored and blood samples taken at day 12.5, 15.5 and 18.5 for analysis of maternal serum IGF-1. At day 18.5 fetal and placental weights were recorded and crown/rump length measured. Maternal fasting plasma glucose and insulin concentrations were measured and tissue weights recorded.

There was no effect of hGH-V treatment on maternal food intake, body weight or fasting glucose levels. In addition, no changes in fetal or placental weights were observed between the treatment groups. hGH-V treatment did not affect the weight of maternal liver, spleen, or pancreas. However, hGH-V at a dose of 2mg/kg significantly increased gonadal fat (162.8 ± 13.03 vs. 245.6 ± 26.58mg; p<0.01) and perirenal fat (34.4 ± 2.77 vs. 51.0 ± 4.09; p<0.05). In addition, a small but statistically significant increase in kidney weight was observed in the 5mg/kg group (133.7 ± 3.34 154.3 ± 9.41mg; p<0.05). Maternal serum IGF-1 and fasting insulin levels are currently being analysed. CONCLUSIONS: hGH-V had no detrimental effects on maternal or fetal growth or placental weight during normal pregnancy in mice, despite significant increases in maternal adipose deposition. Although maternal glycemia was not affected by hGH-V, analysis of maternal plasma IGF-1 and fasting insulin levels are ongoing to examine insulin sensitivity. If indicated, future studies will investigate the effect of hGH-V in a model of fetal growth restriction. Imbalances in the intrauterine cytokine milieu around the time of implantation and invasion may play a causative role in disorders associated with early pregnancy failure, and are also associated with the abnormal trophoblast development seen in gestational trophoblastic disease. This study aimed to evaluate the effect of glucocorticoids on the placenta level of cytokines of the interleukin system and correlate this with the pregnancy and placenta outcome. METHODS: Dexamethasone acetate (DEX) was administered via drinking water at two doses 0.25 and 1 mg/ml. Control rats had access to dexamethasone-free drinking water. Whole placenta were snapfrozen in liguid nitrogen and stored at -80ºC. They were thawed and homogenized and the supernatant was used for ELISA to evaluate amounts of rat placental TNFα, IL-1α, IL-1β, IL-2, IL-4, IL-6 and IL-10 using EbioScience kits; statistical analysis was done using SPSS software. RESULTS: DEX ingestion was associated with a significant reduction of mean maternal and placental weights in the experimental groups with no change in placental efficiency and fetal body weights. No significant differences were detected in TNFα between the control and experimental groups. IL-1α levels were higher in the experimental group compared to control at 21 dg using the high DEX dose. IL-1β levels were lower at 16 and higher at 19 dg in the experimental group with high DEX dose. With IL-2, the levels increased at 19 dg in the experimental group using low DEX dose. IL-4 and IL-10 levels were significantly higher at 16 dg in the experimental group using the high DEX dose. IL-6 levels were lower at 19 and 21 dg in the experimental group of the high dexamethasone dose compared to control. CONCLUSIONS: Dexamethasone administration is associated with induction of intrauterine growth restriction. In this experiment dexamethasone affected placenta growth and altered the expression of the pro-inflammatory and ant-inflammatory cytokines. is an enigmatic inflammatory condition of the placenta associated with fetal growth restriction (FGR) and stillbirth. It occurs in 5-15% of third trimester pregnancies. The cellular profile of VUE has been documented in live births, but has not been quantified or described in stillbirths. Moreover, little is known about the cytokine profile of the lesions thus full characterisation remains to be described. We hypothesised that the immune cells present in VUE lesions in stillbirth would be similar to live birth. We also proposed that the cytokines being produced by the cells in VUE lesions would indicate a cell-mediated immune response.

Placental tissue was obtained from pregnancies ending in stillbirth whose cause of death after post-mortem was recorded as VUE (n=5). Immunostaining for immune cell markers: CD45, CD163, CD4, CD8 and neutrophil elastase, and cytokines: interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 and transforming growth factor β (TGFβ) was performed on serial sections. Inflammatory foci were identified, serial images taken and compared to areas devoid of lesions, sections from FGR infants (live born (n=12) and stillborn (n=12)) and healthy pregnancies (n=12). RESULTS: VUE lesions comprised 32% leukocytes: of these 37.5% were macrophages, 37.5% CD4 T cells, 22% CD8 T cells and 3% neutrophils. Significant pan-placental differences were seen in the number of leukocytes in pathological conditions, macrophages in stillbirth, CD4 T cells in VUE (p<0.0001) and CD8 T cells in VUE (p=0.002). Elevated levels of cytokines IL-2 (T cell) and IL-12 (macrophage) were shown in VUE lesions compared to non-lesion areas and healthy placenta. IL-4 was present in all placentas though distribution differed between trophoblast (controls) and lymphocytes (VUE). IL-6, IL-10 and TGFβ were rarely noted in any tissue sections.

The cell profile of VUE lesions tallies to a proposed host-versus-graft immune response. The cytokine profile in VUE lesions is indicative of a localised Th1-type response. This is in contrast to the reported skew towards humoral immunity in healthy pregnancy. The pan-placental differences in cell population and imbalance of Th1/Th2 immune responses may be significant when studying the effects of VUE on placental function. HCV is the most common cause of chronic hepatitis in in the Western world. In pregnant women the prevalence is estimated between 1-8%, in the U.S. alone >40,000 births annually are affected.

Rates of vertical transmission range between 3-6% with odds 90% higher in HIV co-infection. Prevention of vertical transmission is still not possible due to lack of an approved therapy for use in pregnancy or an effective vaccine. HCV infection is an independent risk factor for preterm delivery, perinatal mortality and other complications of pregnancy.In this study, we define the mechanisms mediating recognition of HCV by primary trophoblast cells and the innate response involved in control of viral infection at the MFI. METHODS: Villous explants and primary cytotrophoblast cells were isolated from elective terminations of normal pregnancies (1 st /2 nd trimester). Trophoblast cells were phenotyped by Flow cytometry and Immunohistochemistry. HCV uptake was determined by confocal microscopy. Antiviral responses were analysed by qRT-PCR after stimulation with an HCV-specific pathogen-associated molecular pattern. Cytokine/chemokine production was detected by ELISA. Apoptosis and NK chemotaxis was assessed by Flow cytometry. RESULTS: Our data suggest that trophoblast cells express many receptors involved in HCV-binding/entry and that trophoblast cells uptake HCV and respond to HCV-RNA sensing with production of Type I/III interferons. We also demonstrate that HCV-RNA sensing affects of trophoblast cells viability and induce secretion of chemokines that recruit and alter the phenotype of decidual NK cells. CONCLUSIONS: These data suggest that the placenta is as a highly active immune organ that coordinates the response to HCV at the MFI. These results provide novel insights into mechanisms associated with viral protection and will guide approaches to intervene therapeutically. 

Our results of 1,25(OH) 2 D 3 suppression of IL-6, IL-8, and TNFα production induced by oxidative stress in placental TCs suggest that inhibition of cytokine production by 1,25(OH)<sub style="font-family: 'Times New Roman';">2D<sub style="font-family: 'Times New Roman';">3 could be a mechanism of vitamin D antiinflammatory effects during pregnancy. These data provide evidence that vitamin D has the ability to inhibit oxidative stress induced inflammatory response in placental TCs.

Axl INTRODUCTION: Current methods utilized to study signal transduction pathways in the human placenta lack the capacity to simultaneously examine cellular phenotype, morphology and spatial arrangement of cells within intact placental structures. Interferon-gamma (IFN-g) is a proinflammatory cytokine expressed at the human maternal-fetal interface, but its role(s) in normal pregnancy are currently unclear. IFN-g signal transduction is mediated through the JAK-STAT1 pathway. To identify the cells within the human placenta that respond to IFN-g, we developed placental whole mount immunofluorescence (WMIF) to examine JAK-STAT1 signaling. METHODS: Human placental explants from 1 st trimester (7-12 weeks) and term (39 weeks) placentas were cultured in vitro +/-IFN-g, and subjected to WMIF in conjunction with confocal microscopy. Cellular responsiveness to IFN-g was determined by examining expression of activated, phosphorylated STAT1 (pSTAT1). Furthermore, placentas were enzymatically dissociated to generate single cell suspensions, which were treated in vitro with IFN-g and subsequently analyzed for pSTAT1 expression by multicolor flow cytometry. RESULTS: All of the major cell populations within the human placenta could be distinguished by WMIF. Staining for pSTAT1 was not detected in untreated placentas, with the exception of sporadic expression in extravillous trophoblast (EVT). Differential pSTAT1 expression in response to IFN-g was observed in 1 st trimester placental explants: staining for pSTAT1 was detected in immune cells, fibroblasts and EVTs, but not in syncytiotrophoblast, villous cytotrophoblast or blood vessel endothelial cells. Similar results were observed by flow cytometry. Furthermore, comparable patterns of pSTAT1 staining were observed in term explants exposed to IFN-g.

Our study demonstrates that WMIF can be successfully used to examine cellular signaling cascades within intact human placental villi. The results suggest that IFN-g responses through the JAK-STAT1 pathway are regulated in a differentiation-dependent manner in human trophoblast cells. Thus, IFN-g may have important roles in regulating EVT differentiation and/or functions. (Th1) and T-helper 2 (Th2) cytokines has been implicated in this latter process in normal pregnancies. In pregnancies complicated by preeclampsia and intrauterine growth restriction the Th1/Th2 cytokine balance may be altered (as indicated by elevated IL-2 and TNF-α expression in these conditions). In non-gestational tissues, myostatin (a member of the TGF-β superfamily) has been implicated in the regulation of cytokine expression, however, its role in regulating placental cytokine production is yet to be described. Thus, the aim of this study was to establish the effect of exogenous myostatin on the release of IL-2 from first trimester placental explants incubated in vitro.

Brisbane and Women's Hospital, and the University of Queensland approved the collection of first trimester placental tissues from women undergoing terminations of pregnancy for psychosocial reasons. Placental explants (10 weeks gestation) were maintained at oxygen tensions of 1% (hypoxic) 3% (physiologically relevant) in an automated PROOX 110-scaled hypoxia chamber (BioSpherics, USA) and 21% oxygen (standard culture conditions) ± 1mg/ml myostatin treatment (Fitzgerald Industries, USA). The Bio-Rad Bio-Plex 200 instrument and Bio-Plex Manager v6.0 software were used to determine the concentrations of Interleukin-2 (IL-2). RESULTS: Following treatment with myostatin, the concentration of IL-2 in placental explant-condition media was significantly decreased (p<0.0005) when compared to controls (at all oxygen tensions tested). IL-2 concentration in placental explant-condition media was inversely correlated with oxygen tension (1% >3% >21%, p <0.05) CONCLUSIONS: We postulate myostatin to be a key regulator of placental formation, development and function. The findings obtained in this study are consistent with the hypothesis that myostatin may regulates cytokine production in the placenta, potentially facilitating in the switch in Th1 / Th2 cytokine formation required for successful pregnancies.

Dendrimer-Based N-Acetyl-L-Cysteine Therapy Modulates Placental Immune Activation. 

While CD4+ T-cell counts did not change, CD8+ T cells were increased with LPS compared to treatment and control groups (p<0.01 for LPS vs. LPS+DNAC and LPS vs. NS+DNAC; Figure) . LPS induced an increase in phosphorylation of both NF-κB and STAT3 (p<0.05). DNAC significantly inhibited the LPS-induced phosphorylation of NF-κB and STAT3 in the placenta (p<0.05). LPS exposure increased the expression of cytokines in the placenta which was ameliorated by DNAC (IL-1β and IL-6: p<0.05; TNF-α: p<0.01). CONCLUSIONS: DNAC reduced activation of innate immune pathways in the placenta and prevented CD8+ T cell accumulation following LPS exposure. The transcription factors NF-κB and STAT3 showed decreased activation. This decrease was concomitant with reductions in cytokine expression. Together, these data suggest that targeted therapy exerts anti-inflammatory effects through modulation of the NF-κB and STAT3. *Figure(s) will be available online. . This variant alters antigen processing, a critical role in shaping the immune response that may contribute to PE. However, the mechanism of immune pathophysiology in PE is unclear.

The genotype and protein expression of ERAP2 and human leukocyte antigen-C (HLA-C) in choriocarcinoma cell lines (BeWo, JEG-3, JAr, and T3M-3) were determined by allelic discrimination qPCR, Western blot analysis and immunocytochemistry. Additionally, ERAP2 and HLA-C expression profiling of untreated versus gamma-interferon (IFN-γ, 20 ng/ml)-treated cells was performed. Lastly, to determine if there is a relationship between the ERAP2 variant and HLA-C expression level on immune response, a cellular immune activation assay was developed using cord blood (CB) lymphocyte response assessed by flow cytometry. RESULTS: BeWo and JEG-3 cells are homozygous for the major T allele of SNP rs2549782 and for the major G allele of SNP rs2248374 that causes non-sense mediated decay of the ERAP2 mRNA, thus lack ERAP2 protein expression. In these cells post-IFN-γ treatment, HLA-C was elevated 2-fold (p=0.0037) and 3-fold (p=0.0446) respectively but ERAP2 remained undetectable. On the other hand, JAr and T3M-3 are heterozygous for both SNPs allowing potential expression of the ERAP2 and ERAP2-v. The difference between these cells is T3M-3 up-regulated both ERAP2 and HLA-C by IFN-γ. However, JAr cells had increased only ERAP2 expression by 2-fold (p=0.003) post-IFN-γ treatment but HLA-C expression remained untraceable. Interestingly, preliminary observation of the cellular immune activation assay revealed that only T3M-3 cells with ERAP2 expression and HLA-C caused immune activation of CD3-CD69+ cells by 3-folds compared to the other choriocarcinoma cell lines. The data suggests the presence of ERAP2-v and HLA-C in T3M-3 have contributed to the activation of immune response. The low level of immune activation in BeWo, JEG-3, and JAr may be due to lacking either ERAP2-v or HLA-C. CONCLUSIONS: Our observations suggest a possible immune mechanism for PE related to ERAP2-v and HLA-C expression, both of which are required for the activation of an immune response to trophoblast cells. Recently it has become apparent that, under certain circumstances, B cells are also able to produce a range of cytokines, which in turn influence activation or tolerance of other immune cells. The aim of this work was to evaluate the cytokine milieu produced by B cells during pregnancy in mice.

We used an established mouse model of pregnancy disturbances in which DBA/2J mated CBA/J females display immunologically mediated pregnancy loss while CBA/J females mated with BALB/c males show normal pregnancies. As control, CBA/J nonpregnant mice were included. CD19 + B cells were isolated from maternal spleen. CD 19+ cells were then stimulated with LPS, anti-CD40, anti-IgM or combinations of these. Cytokine expression was evaluated by flow cytometry or in the supernatant by ELISA-based methods or Bio-Plex system.

We observed a significantly lower production of the proinflammatory cytokines TNFα and IL-12, as well as GM-CSF by B cells from healthy pregnant mice compared to those from non-pregnant controls. Surprisingly, the potent anti-inflammatory cytokine, IL-10, was also detected in lower levels in the supernatant of B cells from mice with normal pregnancy compared to those of non-pregnant controls.Remarkably, this phenomenon was absent in mice with immunologically mediated pregnancy loss. CONCLUSIONS: Our data show that the expression of both, pro as well as anti-inflammatory cytokines by murine B cells is reduced in normal pregnancy, indicating a possible involvement of B cell originated cytokines in the pathology of early pregnancy losses. To study whether rejection of the fetus could be induced by anti-fetal immunityin women with unexplained recurrent miscarriage, characterized by activation of the classical complement system.

A case-control study was performed at an university hospital. Products of conception of 34 women with three or more unexplained consecutive miscarriages within 20 weeks of gestation with the same partner (case group) and two control groups, in the first group 21 women with one spontaneous miscarriage and no history of complicated pregnancy(ies) were included (sporadic miscarriage group), and the second control group consisted of 34 women who underwent an elective abortion for psychosocial reasons (elective abortion group). Immunohistochemical staining for C4d was performed on products of conception embedded in paraffin. Two experienced observers, blinded to the patients' clinical data, independently evaluated the slides. Positivity for C4d was scored semi-quantitatively. RESULTS: C4d deposition was present in products of conception of 18 out of 34 women with unexplained recurrent miscarriage (51.4%), compared to 7 out of 21 women with a sporadic miscarriage (33.3%), and 7 out of 34 women with an elective abortion (20.6%) (p=0.020). CONCLUSIONS: This study shows that C4d, a classical complement activation marker, is increased at the fetal-maternal interface in women with unexplained recurrent miscarriage, this is indicative for anti-fetal immunity in these women. Further knowledge of this pathogenic mechanism may lead to the development of new treatment strategies for this group of patients. . This study aimed to expand this strategy by using a much wider-based screening approach to the same end. METHODS: Plasma miRNAs (n=752) from pregnant women (5 with PET, 5 with PET and small-for-gestational-age (SGA) foetuses, and 10 gestation-matched controls) were profiled using locked nucleic acid qPCR. MiRNA expression levels were normalised to mean total miRNA expression levels. Data normality was assessed using Shapiro-Wilk tests and unpaired t-tests were used to compare miRNA profiles between groups. In silico miRNA target pathway predictions were performed using Mirpath. RESULTS: Comparison between complicated vs. uncomplicated pregnancies revealed 38 miRNAs whose levels differed significantly between PET/PET+SGA cases and controls; 23 of these were elevated in PET and 15 were reduced (P<0.05). Of the former, 9 belonged to the placenta-specific chromosome 19 miRNA cluster (C19MC). Unsupervised hierarchical clustering and principal components analysis of miRNA profiles showed that PET samples partially segregated according to whether the pregnancies were normal or complicated by PET (PET+SGA and pure PET samples did not segregate). However, miR-1 was specifically lower in PET+SGA samples relative to pure PET and controls. In silico pathway analysis of C19MC miRNAs showed partial pathway convergence, particularly in those associated with cell signal transduction and immunomodulation (e.g. TGF-β signalling). By contrast, limited convergence was observed with non-placental-specific miRNAs of these, the vasopressin-regulated water reabsorption pathway was the most significant predicted target. CONCLUSIONS: To our knowledge, this is the largest study profiling patient-specific circulatory miRNA signatures in PET. Our preliminary screen's identification of discriminatory miRNAs (additional to CM19C species) highlights the potential of circulatory miRNA signatures as noninvasive biomarkers for the prediction of PET. with clinic blood pressure (CBP) during pregnancy to determine if these measurements are inversely related to infant birth weight. The aim of this study was to clarify this issue. METHODS: 605 Japanese women were included in this prospective cohort study. Exposures were initial CBP, made before 20 weeks' gestation, and HBP made within 1 week of CBP. Outcome was infant's birth weight, categorized and ranked as follows: ³3500 g, 3000-3499 g, 2500-2999 g, and <2500 g. First, CBP and HBP were included into the proportional odds model separately. Second, both CBP and HBP were included in the model simultaneously to compare the effect size among CBP and HBP. RESULTS: Associations between diastolic blood pressure (DBP) measured in the clinic and at home and infant birth weight when each DBPs were included separately were described in figure1. When both clinic and home DBP were included simultaneously, the adjusted ORs per 1 standard deviation(SD) increase in each DBPs were 1.09 (95%CI: 0.89-1.34) and 1.27 (95%CI: 1.03-1.57), respectively. Neither systolic BP measurement was associated with infant birth weight. CONCLUSIONS: HBP before 20 weeks' gestation was more strongly associated with infant birth weight than CBP. HBP may be worth measuring during pregnancy. *Figure(s) will be available online. Undetected fetal growth restriction (FGR) is strongly associated with stillbirth. Serial ultrasound assessment of growth has been suggested to increase detection, but is resource intensive with each woman requiring 3-4 3 rd trimester ultrasound assessments. Uterine artery Doppler (UTAD) and placental morphometric assessment (PMA) have been suggested as adjuncts to fetal growth measurement, but do not have adequate individual specificity as primary screening tools for FGR. In this study we examine whether combining these tools in high-risk populations can reduce scan frequency with accurate detection of early onset FGR. METHODS: Data was obtained from a prospective cohort attending a high-risk antenatal clinic (Manchester Placenta Clinic). 582 women at high risk of FGR (previous small gestational age (SGA) delivery (266) or extreme placental serum markers (316)) were assessed at 22-24 weeks 5 5 The absence or presence of GCH was not associated with the recurrence of preeclampsia on subsequent pregnancy (p=0.267). And gestation-specific uric acid z-score as a continuous variable did not show any association with the prediction of preeclampsia on subsequent pregnancy (p=0.427). GCH was associated with the small for gestational age (p=0.010).

Clinical characteristic and biochemical data at first delivery according to the recurrence of preeclampsia on subsequent pregnancy together afflict about 25% of first pregnancies. However, interventions to prevent these pregnancy complications cannot be applied because we are not able to identify which nulliparous women are at risk. We aimed to identify women at risk for these four complications in the Adelaide and Auckland cohorts of the SCOPE study using clinical, lifestyle and genetic markers. METHODS: Detailed clinical and lifestyle variables were recorded at 15 and 20 weeks gestation and DNA was extracted from peripheral blood from mother, father, baby trios and single nucleotide polymorphisms (SNPs) were genotyped. SNPs were selected on the basis of identified roles of the genes in which they occur in fundamental processes in placentation and maternal adaptation to pregnancy. Pregnancy outcomes were recorded after delivery. A three tier model for each complication was developed using logistic regression with the first tier aimed at high sensitivity and the second aimed at high specificity after which the two models were integrated. Correction for FDR was undertaken. At the first tier women were deemed to be at risk or at low risk. At risk pregnancies were assessed in the second tier to stratify participants into high or moderate risk. Figure( s) will be available online. CONCLUSIONS: Home BP was immediately elevated just after the earthquake and gradually decreased over a month as we previously reported among hypertensive patients. Because we only analyzed BP who could keep their equipment, the BP change just after the earthquake might be underestimated.

Application of Intrinsic Subtype Classification To Microarray Data From a Set of Women With Mild and Severe Pre-Eclampsia. Satoshi Mizuno, 1,2 Hidekazu Nishigori, 1 Takashi Sugiyama, 1 Soichi Ogishima, 1 Jun Nakaya, 1 Nobuo Yaegashi. 1 1 Gynecology and Obstetrics, School of Medicine, Tohoku Univ., Sendai, Miyagi, Japan; 2 Medical Informatics, School of Medicine, Tohoku Univ., Sendai, Miyagi, Japan; 3 Bioclinical Informatics, Medical megabank, Tohoku Univ., Sendai, Miyagi, Japan. INTRODUCTION: In the previous studies, various subtypes of preeclampsia (PE) have been defined on the basis of the clinical features of patients. In addition, varied pathological backgrounds and symptoms have been observed in each of these subtypes. Thus, an intrinsic subtype classification (ISC) of PE that is based on these different backgrounds is required. ISC is a method to predict the RNA expression in different patient subtypes that are accompanied by different pathophysiologies.In the present study, we applied ISC to microarray data from pregnant women with PE.

We performed ISC of microarray data sets obtained from the placenta of women both with and without PE. All data were obtained from the GEO database. First, we defined three groups to apply ISC. The first group contained women without PE and those with mild PE. The second group contained women without PE and those with severe PE. The third group contained women with mild PE and those with severe PE. We applied ISC using the following steps: (1) Selection of "intrinsic" genes that varied significantly from the arrays of the other groups but had relatively less variation among arrays within the same group. (2) Class prediction, which was performed using prediction analysis for microarrays (PAM), for drawing dendrograms with expression profiles of the selected intrinsic genes. The dendrograms represented the distances of the predicted classes. RESULTS: ISC could differentiate women without PE from those with severe PE in the first group. Similar results were observed in the second group. In addition, ISC could clearly differentiate women with mild PE from those with severe PE in the third group. Moreover, at least two subtypes were predicted in both the mild and severe PE groups by drawing a dendrogram of the intrinsic gene expression patterns. *Figure(s) will be available online. CONCLUSIONS: In conclusion, ISC may be a useful method to predict PE subtypes with varied expression profiles.

The CONCLUSIONS: PWV is a marker of vascular compliance and is a likely indicator of the degree of maternal vascular adaptation to pregnancy. PWV in this high risk cohort was significantly associated with an increased risk of preterm delivery indicated by maternal and/or fetal disease.

not known what the relationship is between CPR and different types of PS. We hypothesized that subtypes of PS differ in CPR trend and prevalence of DV in the 2 nd -3 rd trimester.

We retrospectively studied 81 singleton pregnancies between 2011-2013 in a tertiary center in the Netherlands, divided into three subgroups: preeclampsia (PE), as defined by ISSHP criteria (n=13), intrauterine growth restriction (IUGR) defined as birth weight <10 th percentile (n=55) and PE+IUGR (n=13). Dopplers of the MCA and UA were carried out between 19-38 weeks. After delivery the placenta was histologically examined for various abnormalities including DV. RESULTS: Compared to the population mean, the MCA-PI was lower, the UA-PI was higher and CPR was lower across all subgroups, especially in PE+IUGR and between 24-28 weeks. IUGR CPR followed the trend for the population mean, while in PE and PE+IUGR CPR showed a reversed trend across gestational age. *Figure(s) will be available online. Compared to IUGR, CPR was lower in PE (p=0.004) and PE+IUGR (p<0.001). Similarly, compared to IUGR gestational age at delivery was lower in PE (30w5d, p=0.008) and PE + IUGR (30w4d, p=0.006) and there was higher prevalence of DV in PE (62%, p=0.005) and PE+IUGR (62%, p=0.005) compared to IUGR (22%). CONCLUSIONS: PE and PE+IUGR have reversed CPR trend and more decidual vasculopathy compared to IUGR, suggesting that preeclampsia and IUGR have different pathophysiology. Moreover, CPR assessment at 24-28 weeks may play a role in identifying women who may benefit from individualized surveillance and interventions to reduce their high a priori risk of perinatal complications. . PS is a characterized by placental insufficiency and abnormal release of trophoblast products into maternal circulation, such as PAPP-A and β-hCG. These markers, used in first trimester screening for Down syndrome, have also been used in various models to predict PS later in pregnancy. It is unclear how these placental biomarkers relate to different subtypes of PS. We hypothesized that subtypes of PS differ in first trimester levels of PAPP-A and β-hCG.

We retrospectively compared clinical outcome and first trimester biomarkers from control uncomplicated pregnancies (n=513) and pregnancies complicated by placental syndrome (n=218), further subdivided into: preeclampsia (PE, n=44), intrauterine growth restriction (IUGR=birth weight <10 th percentile, n=62), PE+IUGR (n=5), gestational hypertension (GH, n=101), and HELLP syndrome (n=6). RESULTS: MoM PAPP-A was significantly lower in PE, IUGR and PE+IUGR groups (0.73, 0.69 and 0.57, resp.), but not in the GH and HELLP groups (0.92 and 1.17, resp.), compared to controls (0.99). Free b-hCG was not significantly altered in any of these groups. Using logistic regression, MoM PAPP-A had an adjusted OR of 0.30 (CI 0.13-0.75) for PE and 0.28 (CI 0.12-0.66) for IUGR. Using multivariate regression, MoM PAPP-A correlated negatively to mean BP, systolic BP, diastolic BP, and positively to neonatal weight, but not to proteinuria, platelet count or liver enzymes. CONCLUSIONS: A lower first trimester PAPP-A, but not b-hCG, is related to the later development of placental syndrome (PE and/or IUGR). GH and HELLP syndrome are not likely to be caused by placental dysfunction and should not be considered as placental syndromes. The prediction and management of preeclampsia should take into consideration the heterogeneous nature of the disease and stratify for placental function tests, such as PAPP-A. and we have reported that severe ICP and twin pregnancy are major risk factors for PET (Annual meeting of SGI 2014, abstract # 662).The mechanism by which ICP leads to PET is still unknown but it has been suggested that elevated serum total bile acid (TBA) plays a major role in the pathophysiology of PET. Since early onset ICP is considered to be associated with increased incidence of maternal and fetal complications, we hypothesized that early onset ICP (<32 weeks) may also result in an increased incidence of PET. The aim of the study was to compare the incidence of PET among women with early onset (<32 weeks) and late onset (>32 weeks) ICP.

Between 2008-2013 we had 78 women with ICP, defined as presence of pruritus, elevated liver enzymes, elevated TBA and no evidence for PET or other liver disorders. The onset of disease was defined as the time pruritus and elevated liver enzymes were first reported. All women were treated with deoxycholic acid and labor was induced at 37 weeks. RESULTS: Twenty six women developed early onset ICP and 52 had late ICP. There were no significant differences in the age, gravity, ICP severity and TBA level. Interestingly, the time period from onset of ICP to diagnosis of PET was shorter in the late onset ICP compared to early onset (16±7 vs.29±7 days, respectively, p<0.05). The incidence of PET and severe PET among women with early and late onset ICP, both for singleton and twins, was similar ( CONCLUSIONS: Although our sample size is relatively small, the data suggest that once TBA accumulates in the serum it may result in PET later in pregnancy unrelated to onset time of ICP. Therefore, physicians should be aware of the risk of women with both early and late onset ICP to have PET 2-4 weeks following onset of ICP.

Prehypertension in Early Versus Late Pregnancy. J Rosner, M Dziadosz, T Bennett, K Uquillas, M Gutierrez, A Pham, A Roman. OB/ GYN, NYULMC, NYC, NY, USA. INTRODUCTION: Determine the risk of adverse pregnancy outcomes in patients with early (<20 weeks GA) versus late (>20 weeks GA) prehypertension in pregnancy. METHODS: A retrospective cohort study of patients with a singleton gestation in 2013-2014 by a single practice. Patients were divided on the timing of a prehypertensive blood pressure. Group A; normal (<120/80), group B; pre-hypertension (120-139/80-89) at <20 weeks GA, group C; prehypertension >20 weeks GA and group D; chronic hypertension (³140/90). Outcomes were pregnancy related hypertensive disorders (PRH) (gestational hypertension (gHTN) and pre-eclampsia (PEC)), Preterm birth, small for gestational age (SGA), intrauterine fetal demise (IUFD), composite maternal (GDM, PRH, PTB, PPH, ICU admission and chorioamnionitis) and composite neonatal outcomes (SGA, NICU 

Pre-hypertension in early pregnancy increases the risk for pregnancy related hypertensive disorders, adverse obstetrical outcomes and earlier delivery. Pre-hypertension <20 weeks should be considered a risk factor for pregnancy complications.

Mode We examined mode of delivery (MOD) among women undergoing induction of labor (IOL) secondary to severe preeclampsia (pre-e) before 34 weeks, considering the effect of intrauterine growth restriction (IUGR) on successful vaginal delivery (VD).

We performed a retrospective cohort study of women undergoing IOL for severe pre-e between 25-34 weeks at our tertiary care institution from 2002-2011. We examined MOD among women in our cohort, comparing pregnancies with IUGR to those without. IUGR was defined as estimated fetal weight less than the 10 th %. Exclusion criteria included multiple gestation, intrauterine fetal demise, placenta previa, non-cephalic presentation, and history of cesarean delivery (CD). The chi square test was used to compare proportions and p <0.05 was considered statistically significant. RESULTS: Our cohort of women with severe pre-e at 25-34 weeks gestation who underwent IOL included 137 women. Among this cohort, 24 women (17.5%) had IUGR. There was no difference in rates of VD with or without IUGR (57.5% vs 54.2%, p=0.76). There was also no difference in rates of VD among nulliparous women with and without IUGR (50.0% vs 48.7%, p=0.93). There were no statistically significant differences in MOD by presence or absence of IUGR when stratified by GA at delivery (Tabe 1). There were no observed differences in key neonatal outcomes (Apgars, cord pH, discharge status, birth weight) or maternal events (PPH, atony, abruption, seizure, length of stay, readmission, mortality) by ultimate MOD and presence or absence of IUGR. Most common indications for CD were fetal intolerance 28% and failed induction 7.1%. Worsening pre-e was an indication for CD in only 2.1% of cases. *Figure(s) will be available online. CONCLUSIONS: The proportion of women induced for severe pre-e before 34 weeks who ultimately deliver vaginally does not vary significantly by the presence or absence of IUGR. Our data suggests that IUGR is not associated with a decreased chance of successful VD or worse neonatal or maternal outcomes in preterm women with severe pre-e.

The Association of HtrA1 Serum Levels With Different Types of Preeclampsia. Sonia Teoh, 1,2 Yao Wang, 1,2 Ying Li, 1,2 Qi Chen, 3 Guiying Nie. proteases are a family of serine proteases conserved from bacteria to mammals, and are known to have important functions in protecting cells from stress conditions. There are four mammalian HtrA paralogs (HtrA1-4). HtrA1 is involved in apoptosis and anoikis, and is proposed to function as a tumour suppressor. It has also been implicated in the development of other diseases such as arthritis, Alzheimer's disease, age-related macular degeneration, and cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy. We have previously shown that HtrA1 is highly enriched in the placenta compared to other tissues, and that in women HtrA1 is expressed in placental and endometrial cells at the placental-maternal interface during the first trimester of pregnancy. HtrA1 has recently been reported to be upregulated in preeclampsia (PE), a pregnancy-specific systemic disorder primarily involving hypertension and proteinuria, typically occurring after 20 weeks of gestation in previously normotensive women. However, it is unclear whether HtrA1 is secreted into the maternal circulation and if serum HtrA1 is altered in preeclamptic pregnancies.

In this study, we developed a specific sandwich enzyme linked immunosorbent assay (ELISA) suitable for the detection of serum HtrA1. RESULTS: Using this assay, we discovered that serum HtrA1 levels increase progressively throughout gestation in normal pregnancies. However, this trend was perturbed in women with PE. Compared to gestation-age matched normal pregnancies, HtrA1 serum levels are significantly increased in early-onset PE, which occurs before 34 weeks of gestation, but significantly reduced in late-onset PE, which occurs after 34 weeks of gestation. CONCLUSIONS: This is the first report to show a clear increase of HtrA1 in the maternal circulation during normal pregnancy, consistent with HtrA1 being highly expressed in the placenta. This study also highlights the complex regulation of HtrA1 in different subsets of PE. TEG measures the viscoelasticity of clot formation and allows global assessment of hemostatic function. Therefore TEG is a standard test for diagnosis management and treatment of acute bleeding and coagulopathies. The aim of this study is to describe the coagulopathy of patients with AFE and PPH by using TEG. METHODS: This is multicenter retrospective study. We compared blood clot formation analysis by TEG in women with a diagnosis of AFE and compared it to women with PPH. All women in both groups had clinical or laboratory findings of Disseminated Intravascular Coagulation (DIC). TEG parameters were compared between AFE and PPH groups by using Mann-whitney U nonparametric test, p<0.05 was considered significant. RESULTS: A cohort of 5 women with AFE and 7 women with PPH were included in the analysis. No significant demographical and obstetrical

parameters were observed between the groups(P>0.05). No significant differences in numbers of blood products needed for resuscitation (P>0.05). At onset of acute events, all TEG parameters showed a significant more severe results among women with AFE when compared to women with PPH(P<0.05). *Figure(s) will be available online. CONCLUSIONS: TEG reading in women with AFE shows total failure of clot formation. Based on our study, patients with AFE present more severe coagulopathy when compared to obstetrical DIC. We suggest that TEG is unique in AFE and can be used for its diagnosis of AFE.