The SARS-CoV-2 receptor, angiotensin-converting enzyme 2, is required for human endometrial stromal cell decidualization†

Abstract

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 2 (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 have had maternal vascular malperfusion and decidual arteriopathy in their placentas. Here, we report that the ACE2 protein is expressed in both endometrial epithelial and stromal cells in the proliferative phase of the menstrual cycle, and the expression increases in stromal cells in the secretory phase. It was observed that the ACE2 mRNA and protein abundance increased during primary human endometrial stromal cell (HESC) decidualization. Furthermore, 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, the ACE2 protein was expressed in the uterine epithelial cells, and stromal cells increased through day 6 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.

SciScore for 10.1101/2020.06.23.168252: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	Consent: Human ethical approval and endometrial stromal cell isolation: Informed consent was obtained in accordance with a protocol approved by the Washington University in St. Louis Institutional Review Board (IRB ID #: 201612127). IACUC: Mice and hormone treatments: All mouse experimental procedures followed a protocol approved by the Washington University in St. Louis Institutional Animal Care and Use Committee (Protocol Number 20191079).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Human endometrial biopsies of healthy, reproductive-age women were collected during the proliferative phase (days 9 to 12) and secretory phase (days 14 to 26) of the menstrual cycle.

Table 2: Resources

Antibodies
Sentences	Resources
The PVDF membranes were washed, blocked for 1 hour in 5% non-fat milk in TBS-T (Bio-Rad, USA), and incubated with primary antibodies anti-ACE2 (1:1000, ab15348, Abcam) and anti-GAPDH (1:3000, #2118S Cell Signaling Technology, USA) in 5% BSA in TBS-T overnight at 4°C.	anti-ACE2 suggested: (Abcam Cat# ab15348, RRID:AB_301861) anti-GAPDH suggested: None
Subsequently, sections were blocked with 2.5% goat serum in PBS (Vector laboratories) for one hour at room temperature, and then incubated overnight at 4°C with anti-ACE2 antibody (1:200, ab15348, Abcam) or normal rabbit IgG (#2729, Cell Signaling Technology).	IgG ( #2729 , Cell Signaling Technology) . suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
CD1 wild-type mice (Charles River, Saint Louis, Missouri) were maintained on a 12-h light:12-h dark cycle.	CD1 suggested: None
Software and Algorithms
Sentences	Resources
The amplified cDNA was diluted to 10 ng/μl, and quantitative PCR was performed with primers specified in Table S1 and Fast Taqman 2X mastermix (Applied Biosystems/Life Technologies, Grand Island, NY) on a 7500 Fast Real-time PCR system (Applied Biosystems/Life Technologies)	Applied Biosystems/Life suggested: None
All data are presented as mean ± SEM. GraphPad Prism 8 software was used for all statistical analyses.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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The SARS-CoV-2 receptor, angiotensin-converting enzyme 2, is required for human endometrial stromal cell decidualization†

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