key: cord-1039486-8jc7vdvh authors: Chadchan, Sangappa B; Popli, Pooja; Maurya, Vineet K; Kommagani, Ramakrishna title: The SARS-CoV-2 receptor, Angiotensin converting enzyme 2 (ACE2) is required for human endometrial stromal cell decidualization date: 2020-11-18 journal: Biol Reprod DOI: 10.1093/biolre/ioaa211 sha: 763186f51d781f913fa88f78074989ab72613369 doc_id: 1039486 cord_uid: 8jc7vdvh The coronavirus disease 2019 (COVID-19) first appeared in December 2019 and rapidly spread throughout the world. The SARS-CoV-2 virus enters the host cells by binding to the angiotensin-converting enzyme (ACE2). Although much of the focus is on respiratory symptoms, recent reports suggest that SARS-CoV-2 can cause pregnancy complications such as pre-term birth and miscarriages; and women with COVID-19 had maternal vascular malperfusion and decidual arteriopathy in their placentas. Here, we report that ACE2 protein is expressed in both endometrial epithelial and stromal cells in the proliferative phase of the menstrual cycle, and expression increases in stromal cells in the secretory phase. The ACE2 mRNA and protein abundance increased during primary human endometrial stromal cell (HESC) decidualization. Further, HESCs transfected with ACE2-targeting siRNA impaired the full decidualization response, as evidenced by a lack of morphology change and lower expression of the decidualization markers PRL and IGFBP1. Additionally, in mice during pregnancy, ACE2 protein was expressed in uterine epithelial, and stromal cells increased through day six of pregnancy. Finally, progesterone induced Ace2 mRNA expression in mouse uteri more than vehicle or estrogen. These data establish a role for ACE2 in endometrial physiology, suggesting that SARS-CoV-2 may be able to enter endometrial stromal cells and elicit pathological manifestations in women with COVID-19 including an increased risk of early pregnancy loss. Syndrome Coronavirus can cause pregnancy complications such as pre-term birth and miscarriages [1] . Additionally, a few reports noted that pregnant women with COVID-19 had maternal vascular malperfusion and decidual arteriopathy in their placentas [2, 3] , and a recent clinical case study reported a second-trimester miscarriage in a woman with COVID-19 [4] . However, whether SARS-CoV-2 infects the uterus has not been determined. It seems likely that SARS-CoV-2 could infect the uterus because its receptor, Angiotensin Converting Enzyme 2 (ACE2), is expressed fairly ubiquitously in human tissues such as the lungs, heart, intestine, kidneys, and placenta [5] [6] [7] . Moreover, ACE2 functions by cleaving the vasoconstrictor angiotensin II to the vasodilator angiotensin (1-7). As a component of the renin-angiotensin system, ACE2 plays an important role in regulating maternal blood pressure during pregnancy. ACE2 is expressed in the rat uterus during mid-and late pregnancy [8, 9] . In addition, ACE2 mRNA expression was noted in the uterus of both rats [10] and humans [11] , in which its expression may be higher in the secretory phase than in the proliferative phase of the menstrual cycle [11] . During the secretory phase, the uterine stromal cells prepare for embryo implantation by undergoing a progesterone-mediated differentiation process called decidualization. In this process, the stromal cells divide, change from a fibroblastic to an epithelioid morphology, and change their pattern of gene expression. Decidualization is essential for trophoblast invasion and placentation [12] [13] [14] , and defects in this process may underlie early pregnancy loss in some women. Given the important function of the uterine stroma and the possibility that SARS-CoV-2 could infect the uterus, our goal here was to determine whether ACE2 is expressed in endometrial stromal cells, is regulated by progesterone, and is required for decidualization. We first sought to determine whether ACE2 is expressed in the endometrium and whether its expression differs according to the phase of the menstrual cycle. Thus, we obtained endometrial biopsies from women during the proliferative or secretory phase of the menstrual cycles and performed immunohistochemistry with an ACE2-specific antibody. In the proliferative phase, ACE2 was more abundant in epithelial cells than in stromal cells ( Fig. 1A-B) . However, in the secretory phase, ACE2 expression was increased in the stromal cells ( Fig. 1A-B and S2A ). To ensure antibody specificity, isotype control was included with staining procedure (Fig. 1C ). Further, we carried out the western blotting and found that ACE2 was elevated around 2fold in the secretory phase endometrium compared to proliferative phase endometrium (Fig. 1D) . Interestingly, the TMPRSS2 also expressed in the epithelial cells and stromal cells of proliferative as well as secretory phase endometrium ( Fig. S1A-B and S2B) . Given the elevated expression of ACE2 in stroma of secretory phase endometrium, we wondered whether ACE2 expression increased during in vitro decidualization of human endometrial stromal cells (HESCs). We isolated primary HESCs, exposed them to decidualizing conditions, and confirmed that expression of the decidualization markers Prolactin (PRL) and Insulin-like growth factor-binding protein-1 (IGFBP1) increased over six days. ACE2 mRNA also increased over this time ( Fig. 2A) . Consistent with this finding, ACE2 protein abundance increased during decidualization, as shown by both immunoblotting (Fig. 2B) and immunofluorescence (Fig. 2C) . As expected, ACE2 protein predominantly localized in the cytoplasm and cell membrane of decidualized HESCs. Next, we wondered whether ACE2 was required for primary HESCs decidualization. To answer this question, we transfected HESCs with control or ACE2-targeting siRNAs and then exposed the cells to decidualization conditions. HESCs transfected with control siRNA changed from fibroblastic to epithelioid morphology ( Fig. 3A) and had increased expression of the decidualization markers PRL and IGFBP1 (Fig. 3B) . In contrast, HESCs transfected with ACE2targeting siRNA did not show a morphology change over six days (Fig. 3A) and expressed significantly less PRL, IGFBP1, and ACE2 than control cells (Fig. 3B) . Further, quantification of secreted Prolactin (PRL) from cultured media revealed a significant decrease in PRL protein levels at day 6 in HESCs with ACE2 knockdown (Fig. 3C) . As expected, siRNA against ACE2 effectively downregulated ACE2 protein levels at day 6 ( Fig. 3D) . These results suggest a functional role for ACE2 in human endometrial stromal cell decidualization. Finally, we examined the expression of ACE2 in the endometrium during early pregnancy in mice. We mated female wild-type mice with males of proven fertility and then stained their uteri with an ACE2-specific antibody at different days in early pregnancy. In days one through four, ACE2 localized to the cytoplasm and cell surface of epithelial and stromal cells. However, beginning on day three, strong ACE2 staining was seen in the cytoplasm of stromal cells. This staining was evident at least through day six, which is when robust decidualization occurs ( Fig. 4 and S3) . Given this change in ACE2 abundance during pregnancy, we wondered whether ACE2 expression was regulated by steroid hormones. To test this, we ovariectomized six-week-old mice, waited two weeks, treated the mice with either estrogen or progesterone for six hours, and then collected the uteri (Fig. 5A) . Uteri from progesterone-treated mice expressed significantly more Ace2 mRNA than uteri from vehicletreated mice, which expressed significantly more Ace2 mRNA than uteri from estrogen-treated ( Fig. 5B) . Consistent with this, immunofluorescence revealed that uteri from progesteronetreated mice had significantly more ACE2 protein in stromal cells than did uteri from vehicle-or estrogen-treated mice ( Fig. 5C and S4) . Together, our findings suggest that ACE2 expression in the endometrial stroma is promoted by progesterone in both humans and mice. Moreover, we show that knockdown of expression in the human endometrium, SARS-CoV-2 may be able to enter endometrial stromal cells and elicit pathological manifestations in women with COVID-19. If so, women with COVID-19 may be at increased risk of early pregnancy loss. As more data become available, epidemiologists and obstetricians should focus on this important issue and determine whether women who intend to get pregnant should undergo additional health screenings during the COVID-19 pandemic. Informed consent was obtained in accordance with a protocol approved by the Washington HESCs isolated from the proliferative phase of the menstrual cycle were grown in a six-well culture plate to 60%-70% confluence and transfected with 60 pmol of non-targeting siRNA (D- for gene specific primers. [15, 17, 18] . Protein extracts were prepared from human endometrial biopsies or HESCs as described Then, blots were probed with anti-Rabbit IgG conjugated with horseradish peroxidase (1 µg/5000 µL, #7074, Cell Signaling Technology) in 5% BSA in TBS-T for 1 hour at room temperature. Signal was detected by using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA), and blot images were collected with a Bio-Rad ChemiDoc imaging system [17] and quantification of the blots were conducted by densitometry using Image Lab software. The human endometrial biopsy tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned (5 μm in 100 μl sesame oil. Six hours later, mice were euthanized, uterine tissues were collected and fixed in 4% paraformaldehyde, and RNA was isolated and processed for qRT-PCR [17] . A two-tailed paired Student t-test was used to analyze experiments with two experimental groups, and analysis of variance by non-parametric alternatives was used for multiple comparisons to analyze experiments containing more than two groups. P<0.05 was considered Representative cross-sectional images of uteri stained for ACE2 (green) and DNA (blue); LE, luminal epithelium; S, stroma; G, glands, scale bar: 100 μm. -nCoV epidemic: what about pregnancies? Lancet, 2020 INFECTIONS IN PREGNANCY WITH COVID-19 AND OTHER RESPIRATORY RNA VIRUS DISEASES ARE RARELY, IF EVER, TRANSMITTED TO THE FETUS: EXPERIENCES WITH CORONAVIRUSES, HPIV, hMPV RSV, AND INFLUENZA Placental pathology in COVID-19. medRxiv Second-Trimester Miscarriage in a Pregnant Woman With SARS-CoV-2 Infection Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis Angiotensin-converting enzyme 2 (ACE2) and ACE activities display tissuespecific sensitivity to undernutrition-programmed hypertension in the adult rat Quantitative mRNA expression profiling of ACE 2, a novel homologue of angiotensin converting enzyme Angiotensin-(1-7) in normal and preeclamptic pregnancy ACE2 and ANG-(1-7) in the rat uterus during early and late gestation Decidualized pseudopregnant rat uterus shows marked reduction in Ang II and Ang-(1-7) levels The vasoactive peptide angiotensin-(1-7), its receptor Mas and the angiotensin-converting enzyme type 2 are expressed in the human endometrium Embryo implantation Implantation and the survival of early pregnancy Time of implantation of the conceptus and loss of pregnancy Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization Acceleration of the glycolytic flux by steroid receptor coactivator-2 is essential for endometrial decidualization We thank Dr. Deborah J. Frank (Department of Obstetrics and Gynecology, Washington University) for assistance with manuscript editing.