SARS-CoV-2 infection risk assessment in the endometrium: viral infection-related gene expression across the menstrual cycle

Abstract

Objective: To determine the susceptibility of the endometrium to infection by-and thereby potential damage from-SARS-CoV-2.

Design: Analysis of SARS-Cov-2 infection-related gene expression from endometrial transcriptomic data sets.

Setting: Infertility research department affiliated with a public hospital.

Patient(s): Gene expression data from five studies in 112 patients with normal endometrium collected throughout the menstrual cycle.

Intervention(s): None.

Main outcome measure(s): Gene expression and correlation between viral infectivity genes and age throughout the menstrual cycle.

Result(s): Gene expression was high for TMPRSS4, CTSL, CTSB, FURIN, MX1, and BSG; medium for TMPRSS2; and low for ACE2. ACE2, TMPRSS4, CTSB, CTSL, and MX1 expression increased toward the window of implantation. TMPRSS4 expression was positively correlated with ACE2, CTSB, CTSL, MX1, and FURIN during several cycle phases; TMPRSS2 was not statistically significantly altered across the cycle. ACE2, TMPRSS4, CTSB, CTSL, BSG, and MX1 expression increased with age, especially in early phases of the cycle.

Conclusion(s): Endometrial tissue is likely safe from SARS-CoV-2 cell entry based on ACE2 and TMPRSS2 expression, but susceptibility increases with age. Further, TMPRSS4, along with BSG-mediated viral entry into cells, could imply a susceptible environment for SARS-CoV-2 entry via different mechanisms. Additional studies are warranted to determine the true risk of endometrial infection by SARS-CoV-2 and implications for fertility treatments.

Keywords: ACE2; COVID-19; SARS-CoV-2; coronavirus; endometrial transcriptomics.

Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Endometrial data set integration and menstrual cycle clock. (A) Data set experiment effect and correction. Each point of the principal component analysis (PCA) plots represents endometrial gene expression of one sample and is colored by the endometrial transcriptomic data sets to which it belongs. Principal component 1 (PC1) and principal component 2 (PC2) explain the percentage of variability due to these components for each PCA. Integration of the transcriptomic studies showed a clear batch effect by the nature of each experiment (left PCA plot). After correction (right PCA plot), all genes in common between data sets were retained, amounting to a total of 13,437 genes, as no differentially expressed genes were detected between experiments. (B) Menstrual cycle effect. The samples of the integrated data sets are colored by the menstrual cycle phase to show how they are grouped by phases of the menstrual cycle. ESE = early secretory endometrium; LSE = late secretory endometrium; MSE = midsecretory endometrium; PF = proliferative phase.

Figure 2

Gene expression of viral infection-related genes throughout the menstrual cycle. (A) Landscape of expression changes. Genes were located depending on their relative expression against the whole set. Low, medium, and high expression thresholds correspond to 1% to 10%, 11% to 50%, and 51% to 100% categories of gene expression values of the entire integrated data set, respectively. Analysis of variance results for overall change of expression during cycle are shown for each gene. ∗P<.05; ∗∗∗P<.0001. (B) Molecular scheme of SARS-CoV-2 endometrial infection. ACE2, TMPRSS4, FURIN, and BSG are shown in plasma membrane of an endometrial cell (lower left figure). CTSL and CTSB are represented outside the cell. MX1 is shown in cytoplasm. Expression of viral genes in comparison to whole transcriptomic set is represented as arrows next to their names: up = highly expressed; down = lowly expressed. Viral genes are positioned in their schematized cell locations as stablished by GeneCards database > Localization section (release 4.14) (56). Only maximum confidence levels (5 and 4) for compartments-derived cell locations were used. Proteins were grouped considering the highest coactivation values between pairs of viral genes during the menstrual cycle, which are shown in the lower right table in the figure. Discontinuous arrow shows less evidence according to our results, given that only FURIN showed high activation with BSG and that further studies are needed to understand BSG-related mechanisms of SARS-CoV-2 entry. ESE = early secretory endometrium; LSE = late secretory endometrium; MSE = midsecretory endometrium; PF = proliferative phase.

Figure 3

Impact of age on viral-related infectivity gene expression throughout the menstrual cycle. (A) Effect of age on ACE2 expression. Gene expression is represented for ACE2 in each phase of the cycle according to the age of the sample analyzed. The range of age from patients involved in this study was 23 to 50 years. (B) Effect of age on viral gene expression. Pearson correlation R2 values are shown for each gene studied through of the phases of the menstrual cycle. Gray scale represents the magnitude of the correlation of increase or decrease in expression with age. High values are colored darker, and low values are colored lighter. ESE = early secretory endometrium; LSE = late secretory endometrium; MSE = midsecretory endometrium; PF = proliferative phase.

Supplemental Figure 1

Flowchart of the selection of transcriptomic studies evaluating the endometrium of control patients (without any known endometrial pathology). The selection of suitable individual transcriptomic studies at Gene Expression Omnibus (GEO) and the number of individual studies excluded and remaining after each filtering step are shown (n, number of studies; N, sample size; lncRNA, long noncoding RNA).

Supplemental Figure 2

Excluded samples from endometrial data sets. (A) An early secretory sample in the Bradley 2011 data set was grouped to proliferative samples at the transcriptomic level due to mistaken labeling, as progesterone levels for that sample were the same as the samples from the proliferative phase. Given the doubt, this sample was removed from further analysis. (B) After correcting a batch effect due to different sequencing protocols in the data set of Altmäe et al. (63), two outliers were detected due to different transcriptomic behaviors and consequently removed. ESE = early secretory endometrium; LSE = late secretory endometrium; MSE = midsecretory endometrium; PF = proliferative phase.

Supplemental Figure 3

Statistical tests for expression changes in viral genes throughout the menstrual cycle. (A) Analysis of variance P values and pairwise t-test P adjusted values are shown for each gene through the phases of the menstrual cycle. Pairwise t-test P values were adjusted by false discovery rate (FDR). Statistically significant values (FDR <0.05) are represented in red. (B) Fold change expression calculated for each gene from each phase to the phase immediately following in endometrial progression. Proliferative to early secretory, early secretory to midsecretory, and midsecretory to late secretory increases or decreases of expression are shown for each gene. ESE = early secretory endometrium; LSE = late secretory endometrium; MSE = midsecretory endometrium; PF = proliferative phase.

Supplemental Figure 4

Expression changes across menstrual cycle phases. Gene expression distributions of each viral infectivity related gene are shown for each phase of the menstrual cycle in figures A to H. Statistically significant pairwise t-test results evaluating expression changes between menstrual cycle stages is indicated as ∗P<.1; ∗∗P<.01; ∗∗∗P<.001. ESE = early secretory; LSE, late secretory; MSE = medium secretory; PF = proliferative phase.