key: cord-332178-0xyrmk5a
authors: Chadchan, Sangappa B.; Maurya, Vineet K.; Popli, Pooja; Kommagani, Ramakrishna
title: The SARS-CoV-2 receptor, Angiotensin converting enzyme 2 (ACE2) is required for human endometrial stromal cell decidualization
date: 2020-06-24
journal: bioRxiv
DOI: 10.1101/2020.06.23.168252
sha: 
doc_id: 332178
cord_uid: 0xyrmk5a

STUDY QUESTION Is SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE 2) expressed in the human endometrium during the menstrual cycle, and does it participate in endometrial decidualization? SUMMARY ANSWER ACE2 protein is highly expressed in human endometrial stromal cells during the secretory phase and is essential for human endometrial stromal cell decidualization. WHAT IS KNOWN ALREADY ACE2 is expressed in numerous human tissues including the lungs, heart, intestine, kidneys and placenta. ACE2 is also the receptor by which SARS-CoV-2 enters human cells. STUDY DESIGN, SIZE, DURATION Proliferative (n = 9) and secretory (n = 6) phase endometrium biopsies from healthy reproductive-age women and primary human endometrial stromal cells from proliferative phase endometrium were used in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS ACE2 expression and localization were examined by qRT-PCR, Western blot, and immunofluorescence in both human endometrial samples and mouse uterine tissue. The effect of ACE2 knockdown on morphological and molecular changes of human endometrial stromal cell decidualization were assessed. Ovariectomized mice were treated with estrogen or progesterone to determine the effects of these hormones on ACE2 expression. MAIN RESULTS AND THE ROLE OF CHANCE In human tissue, 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 (P < 0.0001) and protein abundance increased during primary human endometrial stromal cell (HESC) decidualization. HESCs transfected with ACE2-targeting siRNA were less able to decidualize than controls, as evidenced by a lack of morphology change and lower expression of the decidualization markers PRL and IGFBP1 (P < 0.05). In mice during pregnancy, ACE2 protein was expressed in uterine epithelial and stromal cells increased through day six of pregnancy. Finally, progesterone induced expression of Ace2 mRNA in mouse uteri more than vehicle or estrogen (P < 0.05). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Experiments assessing the function of ACE2 in human endometrial stromal cell decidualization were in vitro. Whether SARS-CoV-2 can enter human endometrial stromal cells and affect decidualization have not been assessed. WIDER IMPLICATIONS OF THE FINDINGS Expression of ACE2 in the endometrium allow SARS-CoV-2 to enter endometrial epithelial and stromal cells, which could impair in vivo decidualization, embryo implantation, and placentation. If so, women with COVID-19 may be at increased risk of early pregnancy loss. STUDY FUNDINGS/COMPETING INTEREST(S) This study was supported by National Institutes of Health / National Institute of Child Health and Human Development grants R01HD065435 and R00HD080742 to RK and Washington University School of Medicine start-up funds to RK. The authors declare that they have no conflicts of interest.

Although much of the focus during the severe acute respiratory syndrome coronavirus 2 76 (SARS-CoV-2)/coronavirus disease 2019 (COVID-19) pandemic has been on respiratory 77 symptoms, some reports suggest that SARS-CoV-2 and the related Middle East Respiratory 78

Syndrome Coronavirus can cause pregnancy complications such as pre-term birth and 79 miscarriages (Favre et al. 2020 ). Additionally, a few reports noted that pregnant women with 80 COVID-19 had maternal vascular malperfusion and decidual arteriopathy in their placentas 81 (Schwartz and Dhaliwal 2020; Shanes et al. 2020a) , and a recent clinical case study reported a 82 second-trimester miscarriage in a woman with COVID-19 (Baud et al. 2020 ). However, whether 83 SARS-CoV-2 infects the uterus has not been determined. 84

It seems likely that SARS-CoV-2 could infect the uterus because its receptor, Angiotensin 85

Converting Enzyme 2 (ACE2), is expressed fairly ubiquitously in human tissues such as the lungs, 86 heart, intestine, kidneys, and placenta (Hamming et al. 2004; Harmer et al. 2002; Riviere et al. 87 2005) . Moreover, ACE2 functions by cleaving the vasoconstrictor angiotensin II to the vasodilator 88 angiotensin (1-7). As a component of the renin-angiotensin system, ACE2 plays an important 89 role in regulating maternal blood pressure during pregnancy. ACE2 is expressed in the rat uterus 90 during mid-and late pregnancy (Merrill et al. 2002; Neves et al. 2008 ). In addition, ACE2 mRNA 91 expression was noted in the uterus of both rats (Brosnihan et al. 2012 During the secretory phase, the uterine stromal cells prepare for embryo implantation by 95 undergoing a progesterone-mediated differentiation process called decidualization. In this 96 process, the stromal cells divide, change from a fibroblastic to an epithelioid morphology, and 97 change their pattern of gene expression. Decidualization is essential for trophoblast invasion and 98 placentation (Carson et al. 2000; Norwitz et al. 2001; Wilcox et al. 1999) , and defects in this 99 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 101 determine whether ACE2 is expressed in endometrial stromal cells, is regulated by progesterone, 102 and is required for decidualization.

We first sought to determine whether ACE2 is expressed in the endometrium and whether 105 its expression differs according to the phase of the menstrual cycle. Thus, we obtained 106 endometrial biopsies from women during the proliferative or secretory phase of the menstrual 107 cycles and performed immunofluorescence with an ACE2-specific antibody. In the proliferative 108 phase, ACE2 was highly expressed in epithelial cells than in stromal cells (Fig. 1A) . However, in 109 the secretory phase, ACE2 expression was increased in the stromal cells (Fig. 1A) . Thus, we 110 wondered whether ACE2 expression increased during in vitro decidualization of human 111 endometrial stromal cells (HESCs). We isolated primary HESCs, exposed them to decidualizing 112 conditions, and confirmed that expression of the decidualization markers Prolactin (PRL) and 113

Insulin-like growth factor-binding protein-1 (IGFBP1) increased over six days. ACE2 mRNA also 114 increased over this time ( Fig. 2A) . Consistent with this finding, ACE2 protein abundance 115 increased during decidualization, as shown by both immunoblotting (Fig. 2B) and 116 immunofluorescence (Fig. 2C) . As expected, ACE2 protein predominantly localized in the 117 cytoplasm and cell membrane of decidualized HESCs. 118

Next, we wondered whether ACE2 was required for primary HESC decidualization. To 119 answer this question, we transfected HESCs with control or ACE2-targeting siRNAs and then 120 exposed the cells to decidualization conditions. HESCs transfected with control siRNA changed 121 from fibroblastic to epithelioid morphology (Fig. 3A) and had increased expression of the 122 decidualization markers PRL and IGFBP1 (Fig. 3B) . In contrast, HESCs transfected with ACE2-123 targeting siRNA did not show a morphology change over six days (Fig. 3A) and expressed 124 significantly less PRL, IGFBP1, and ACE2 than control cells ( Fig. 3B-C) . These results 125 demonstrate that ACE2 is essential for endometrial stromal cell decidualization. 126

Finally, we examined the expression of ACE2 in the endometrium during early pregnancy 127 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, 129 ACE2 localized to the cytoplasm and cell surface of epithelial and stromal cells. However, 130 beginning on day three, strong ACE2 staining was seen in the cytoplasm of stromal cells. This 131 staining was evident at least through day six, which is when robust decidualization occurs (Fig.  132   4) . Given this change in ACE2 abundance during pregnancy, we wondered whether ACE2 133 expression was regulated by steroid hormones. To test this, we ovariectomized six-week-old 134 mice, waited two weeks, treated the mice with either estrogen or progesterone for six hours, and 135 then collected the uteri (Fig. 5A) . Uteri from progesterone-treated mice expressed significantly 136 more Ace2 mRNA than uteri from vehicle-treated mice, which expressed significantly more Ace2 137 mRNA than uteri from estrogen-treated (Fig. 5B) . Consistent with this, immunofluorescence 138 revealed that uteri from progesterone-treated mice had significantly more ACE2 protein in stromal 139 cells than did uteri from vehicle-or estrogen-treated mice (Fig. 5C) . 140

Together, our findings suggest that ACE2 expression in the endometrial stroma is 141 promoted by progesterone in both humans and mice. Moreover, we show that ACE2 is required 142 for human stromal cell decidualization. Given the high ACE2 expression in the human 143 endometrium, SARS-CoV-2 may be able to enter endometrial stromal cells and elicit pathological 144 manifestations in women with COVID-19. If so, women with COVID-19 may be at increased risk 145 of early pregnancy loss. As more data become available, epidemiologists and obstetricians should 146 focus on this important issue and determine whether women who intend to get pregnant should 147 undergo additional health screenings during the COVID-19 pandemic.

Informed consent was obtained in accordance with a protocol approved by the Washington 151 Corporation, Carlsbad, USA) as described previously (Camden et al. 2017 

Protein extracts were prepared from HESCs as described previously (Oestreich et al. 2020) . with DAPI (cat. no. P36962 Thermo Scientific). Immunofluorescence images were captured on a 215 confocal microscope (Leica DMI 4000B). 216

HESCs were grown on poly-L-Lysine coated coverslips in 12-well plates and allowed to 218 decidualize for six days in EPC media as described above. Then, cells were fixed with 4% Sexually mature (8-week-old) CD1 females were mated to fertile wild-type males, and copulation 232 was confirmed by the presence of vaginal plug on the following morning, designated as 1 day 233 post-coital (dpc). Mice were euthanized, and uteri were collected on 1, 2, 3, 4, 5, and 6 dpc. To 234 determine the uterine estrogen or progesterone responses, six-week-old CD1 mice were 235 bilaterally ovariectomized, rested for two weeks to allow the endogenous ovarian-derived steroid 236 hormones to dissipate, and then subcutaneously injected with 100 μl sesame oil (vehicle control), 237 1 mg progesterone, or 100 ng estradiol (Sigma-Aldrich) in 100 μl sesame oil. Six hours later, mice 238 were euthanized, uterine tissues were collected and fixed in 4% paraformaldehyde, and RNA was 239 isolated and processed for qRT-PCR (Kommagani et al. 2016) . 240

A two-tailed paired Student t-test was used to analyze experiments with two experimental groups, 242 and analysis of variance by non-parametric alternatives was used for multiple comparisons to 243 analyze experiments containing more than two groups. P<0.05 was considered significant. All 244 data are presented as mean ± SEM. GraphPad Prism 8 software was used for all statistical 245 analyses.

We thank Dr. Deborah J. Frank (Department of Obstetrics and Gynecology, Washington 248

University) for assistance with manuscript editing. 249

RK conceived the project, supervised the work, analyzed the data, and wrote the manuscript. 

The authors have no conflicts of interest to declare. shown as mean ± SEM. The experiment was repeated three times; *P < 0.05, **P < 0.01, and 384 ***P < 0.001. 385 

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