The SARS-CoV-2 receptor, Angiotensin converting enzyme 2 (ACE2) is required for human endometrial stromal cell decidualization

Abstract

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.

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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