key: cord-0688999-vg8hx0qf authors: Henarejos-Castillo, Ismael; Sebastian-Leon, Patricia; Devesa-Peiro, Almudena; Pellicer, Antonio; Diaz-Gimeno, Patricia title: SARS-CoV-2 infection risk assessment in the endometrium: Viral infection-related gene expression across the menstrual cycle date: 2020-06-17 journal: Fertil Steril DOI: 10.1016/j.fertnstert.2020.06.026 sha: 25df791a58e5f0923899b5cae1873e89350283fa doc_id: 688999 cord_uid: vg8hx0qf STRUCTURED ABSTRACT Objective To determine 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 datasets. 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 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. The rapid international spread of novel coronavirus disease 2019 (1) has resulted in 5,103,006 confirmed cases and 333,401deaths worldwide as of May 23, 2020 (2) . COVID-19 is caused by SARS-coronavirus 2 (SARS-CoV-2) infection in the lower respiratory tract (3) , but the mechanisms underlying infection remain poorly understood (4) . The rapid spread of SARS-CoV-2, genetically closely related to severe acute respiratory syndrome coronavirus (SARS-CoV-1) (5), has resulted in a global health emergency with long-term consequences on economics markets, nutritional habits, and mental and physical wellbeing (5) (6) (7) (8) (9) . Further, as information is still emerging about its health consequences, assisted reproductive treatments (ARTs) have been delayed due to fear of the unknown impact of SARS-Cov-2 on fertility (10, 11) . Fertility is compromised by age, and the longer treatments are delayed, the less likely that successful outcomes will be achieved (12) . Furthermore, effects of SARS-CoV-2 infection increase in severity with host age (13) (14) (15) , meaning that women of more advanced reproductive age undergoing ART could be at higher risk of infection. It is therefore critical to determine exactly how the virus affects reproductive physiology. Although studies to date are discordant, vertical transmission during pregnancy seems to occur infrequently, ranging from 0 to 11% (11, (16) (17) (18) . To date there is no information about how infection affects early implantation and development. The ability of SARS-CoV-2 to damage tissue is determined by its capacity to enter and infect cells in that tissue (4) . The SARS-CoV-2 entry point on the cell is angiotensin-converting enzyme 2 (ACE2) (19, 20) , which plays a key role in the renin-angiotensin system, cleaving angiotensin II to angiotensin 1-7. The system is disrupted after SARS-CoV-2 gains cell entry and down-regulates the expression of ACE2, leading to up-regulation of the pro-inflammatory response by angiotensin II (21) (22) (23) . ACE2 exhibits moderately increased expression with age (24) , which could explain disease severity in older people. Another path of entry, using Basigin (BSG) as receptor instead of ACE2, has been proposed (25) . To enter the cell, SARS-CoV-2 uses its spike S protein to bind to ACE2, which leads to fusion with the cell membrane and endocytosis (4, 20, 26) . TMPRSS2, a transmembrane protease, cleaves the S protein (27) . Cleavage is necessary for the virus to bind to ACE2 and spread through the infected host (19) . Other proteases are under investigation as possible implications in SARS-CoV-2 infectivity related to S protein cleaving. TMPRSS4 increased virus infectivity on its own, at least in gut epithelial cells (28) , while cathepsins B and L (CTSB and CTSL, respectively) had residual cleaving activity of viral S protein in TMPRSS2-cells (19) . FURIN is another protease predicted to cleave S protein and presents alongside ACE2 in epithelial layers of several oral mucosal tissues (29, 30) . MX dynamin-like GTPase 1 (MX1) regulates neutrophil infiltration, favoring infection through protein S modification by neutrophil elastase (31) . While the clinical presentation of COVID-19 ranges from mild respiratory symptoms to severe progressive pneumonia, gastrointestinal symptoms, fecal shedding, multiorgan failure, and even death (32, 33) , few studies have focused on the virus' effect on fertility, damage to reproductive tissues, and concerns regarding the use of reproductive treatments. Leydig and Sertoli cells in the testis (34) (35) (36) (37) , oocytes (38) , and ovarian tissue (37) are likely to experience damage due to their medium-high expression of the ACE2 receptor. The endometrium is crucial for human reproduction and embryo implantation; however, studies of the effect of SARS-CoV-2 infection on menstrual cycle progression have not been performed. Delineating the virus's impact on the tissue is important for determining risk to ARTs, given that a healthy endometrium is needed for embryo implantation and growth. The endometrium is a complex tissue subjected to a cycle of cell death and renewal approximately every 28 days (39) . Numerous transcriptomic studies have sought to understand gene expression changes throughout the menstrual cycle (40) and most of these datasets are available in public repositories, such as the Gene Expression Omnibus (GEO) database (41 A thorough literature search identified proteins related to the SARS-CoV-2 disease-causing mechanism, including genes related to cell entry, genes with implications in health and fertility, among others. The search for relevant genes associated with viral infectivity was made chronologically up to May 10, 2020. Keywords searched in PubMed (44) included all possible combinations between "SARS-CoV-2", "COVID-19", "coronavirus", "cell entry mechanisms", "long-term implications", and "fertility". Public transcriptomic datasets were used to analyze expression of SARS-CoV-2 infectivity-related genes throughout the menstrual cycle. Endometrial transcriptomic experiments for control patients (without any known endometrial pathology) were systematically searched in the GEO database (41) with no restrictions on publication date or language and according to PRISMA guidelines (45) . Keywords were: uter* OR endometr*, filtered by "homo sapiens". Experiments were selected if: -RNA was extracted directly from human endometrial biopsies. -Endometrial biopsies were collected at different times during the menstrual cycle. -Cycle phase at time of biopsy was available for all samples. -Endometrial gene expression was evaluated by microarray or RNA sequencing using Affymetrix, Illumina, or Agilent platforms. -Raw gene expression data were freely available to download from GEO. Transcriptomic raw data were downloaded from the GEO database and processed according to the standards of the technology used (microarrays or RNA-seq) using limma R-package (version 3.34.9) (46) . Expression data from each experiment were annotated with biomaRt (version 2.30.0) (47), logtransformed, and quantile normalized using limma (46) . Principal components analysis (PCA) was done using the prcomp() function and scores were displayed using the ggplot2 R-package (48) to look for possible batch effects and were corrected if using linear models. Outliers were deleted from posterior analysis if there was a distinct transcriptional behavior according to PCA. To integrate the included endometrial transcriptomic experiments, several steps were followed as recommended by Tajti et al. (49) : After being independently normalized, selected studies were joined in a unique dataset and batch effects were corrected using linear models (limma R package; 43). A relative expression value of low, medium, and high expression were established. The thresholds correspond to 1% -10%, 11% -50% and 51% -100% categories of gene expression values of the entire integrated dataset. Pairwise differential expression analysis between experiments was carried out using limma to identify genes with expression differences. P-values were corrected using false discovery rates (FDR) (50) and genes differentially expressed between experiments were removed from the analysis (FDR < 0.05). An analysis of variance (ANOVA) was performed for each selected gene related to SARS-CoV-2 infectivity to assess which genes showed significant differences between endometrial phases. ANOVA was followed by a pairwise t-test to determine significant differences between phases. Fold changes in gene expression from phase to phase were also calculated. Mean expression and confidence intervals for each gene in each phase were represented using ggplot2 R-package (version 3.0.0; 45). Pearson's correlation (51) was used to assess co-expression between each pair of selected proteins related to SARS-CoV-2 infectivity and to analyze the effect From a total of 694 studies retrieved from GEO, our search identified five unique studies that evaluated endometrial gene expression in women with normal endometrium, comprising 112 samples (Table 1, see Supplemental Fig. 1 for detailed filtering steps). All studies were analyzed separately for correction of possible batch effects and three samples were excluded from analysis (details in Supplemental Fig. 2) . A unique combined dataset with a population of 109 patients was obtained, comprising 29 samples in the proliferative phase, 29 in the early secretory phase, 43 in the medium secretory phase, and eight in the late secretory phase ( Table 1 ). The 109 samples were grouped depending on the experiment rather than by the menstrual cycle phase (Fig. 1A, left) . To make datasets comparable after the integration of all samples, this batch effect was removed (Fig. 1A, right) . The resulting gene expression samples showed a behavior based on menstrual cycle phases rather than study nature (Fig. 1B ). The gene expression landscapes for each viral gene across the cycle are shown in Fig. 2B . TMPRSS4, CTSL, CTSB, FURIN, MX1, and BSG were highly expressed throughout the cycle, while TMPRSS2 was moderately expressed and ACE2 was low ( Fig. 2A) . Gene expression of viral proteins depended on menstrual cycle phase. Calculated P-values and adjusted P-values are presented in Supplemental Fig. 3A . All genes except TMPRSS2 (P = 0.053) had significant changes in expression across the menstrual cycle ( Fig. 2A) . Specifically, the genes most affected by menstrual cycle progression (P < 0.0001) were ACE2, which increased expression from early secretory to mid secretory; TMPRSS4, whose expression increased from proliferative to mid secretory and from early secretory to mid secretory (P < 0.0001); CTSL and CTSB, which increased from early secretory to mid secretory (P < 0.0001); TMPRSS2 with decreased expression from proliferative to early secretory (P < 0.05); and BSG and FURIN which increased from proliferative to mid secretory (P < 0.01) ( Fig. 2A) . All genes, including TMPRSS2, showed increased expression from early secretory to mid secretory, as indicated in Supplemental Correlations between genes showed activations and repressions between them throughout the menstrual cycle (Supplemental Table 1 (42) . ACE2 is also reduced in critical tissues such as lungs (24), however, we cannot suggest that low levels of ACE2 expression imply no effect of the virus on the tissue. Further studies are needed to elucidate the effect of decreased ACE2 in the endometrium. Interestingly, ACE2 increased (FC = 2.47) from the early secretory to mid secretory phases, implying an increase in ACE2 in the window of implantation and a high risk of viral infectivity at this stage of the menstrual cycle. Several possible mechanisms of SARS-CoV-2 cell entry have been proposed (19, 28) . TMPRSS2, the most-reported protease involved with SARS-CoV-2 infectivity alongside ACE2, had medium endometrial expression in our study. However, there was no correlation between TMPRSS2, ACE2, and the rest of the genes studied. These results imply that the endometrium should be safe against SARS-CoV-2 infectivity mediated by TMPRSS2, though the expression of other proteases associated with S protein cleavage show a different landscape than TMPRSS2. TMPRSS4 significantly changes its expression throughout the menstrual cycle, showing a significant increase in the mid secretory phase. This protein also showed an interesting correlation with other genes, including an increase alongside ACE2 in the early secretory phase. While TMPRSS2 is implicated in cell entry, the fact that S proteins could be targeted by TMPRSS4 and that TMPRSS4 increased infectivity in gut epithelial cells (26, 28) We also investigated the effect of age on viral gene expression throughout the menstrual cycle. In a recent study in lungs the ACE2 expression was detected as highly variable among individuals ranging from 0.17 transcripts per million to 14.5 transcripts per million in the same demographic group and highlighted a skewness of expression that increased significantly with age (24) . Our results showed a positive correlation between age and ACE2 from the proliferative to mid secretory phases, especially in the early secretory phase, meaning that the endometrium in older women (to age 50) could be more susceptible to SARS-CoV-2 infection. TMPRSS2 decreased with age, particularly in the mid and late secretory phases, which may mean that cell-mediated entry of the virus is lowered with increased age. However, TMPRSS4 increased in expression from proliferative to early secretory similar to CTSB, CTSL, and MX1. These results, in conjunction with ACE2 expression changes, highlighted the same behavior related to age than in lungs (24) Due to it is a study integrating datasets from GEO, this approach is limited by the study design defined previously. This also means that results depend on the experimental transcriptomic procedures of prior reported results and the sample cohort included in each study. However, the inclusion criteria for the selected studies have been very careful and uniform and raw data has been preprocessed controlling undesired effects. Likewise, the virus differentially affects individuals due to each person's genetic profile (31, 61) . Further prospective research is needed to determine the mechanisms of SARS-CoV-2 infection in the endometrium and how it could affect the fertility of each patient. While TMPRSS2 expression implies a safe environment against infection, the roles of proteases and neutrophil regulating proteins TMPRSS4, CTSB, CTSL, FURIN, and MX1 could imply susceptibility of infection especially during the early secretory and mid secretory phases. Additionally, the high expression of BSG could imply an alternative mechanism independent of ACE2 for endometrial viral infection. Our findings show that viral gene expression increases with age, suggesting that the endometrium of older women undergoing ARTs is at higher risk of viral infection. A careful approach is advised, with precautions implemented when resuming ARTs. 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