key: cord-0887691-7xggimev authors: Goad, Jyoti; Rudolph, Joshua; Rajkovic, Aleksandar title: Female reproductive tract has low concentration of SARS-CoV2 receptors date: 2020-06-22 journal: bioRxiv DOI: 10.1101/2020.06.20.163097 sha: 50457af78448a976877ebb5995060f946c923dc3 doc_id: 887691 cord_uid: 7xggimev There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2. Our findings suggest that COVID-19 is unlikely to contribute to pregnancy-related adverse outcomes such as preterm birth, transmission of COVID-19 through breast milk, oogenesis and female fertility. SARS-CoV2 binds to angiotensin-converting enzyme 2 (ACE2) receptor on the host cells 74 through spike (S) protein on the surface of the virus (2, 5). In addition to ACE2, entry of the virus into the host cell is also mediated by proteases TMPRSS2 (5). In the absence of 76 TMPRSS2, SARS-CoV2 is known to use cathepsins, CTSB and CTSL as an alternate to enter 77 the host cells (5). These proteases are required for the priming of the S protein after it binds 78 to the ACE2 receptor for its entry into the host cell (5, 6). Recent analyses of existing single-cell sequencing datasets showed that the SARS-CoV2 80 receptor, ACE2 is expressed in various cell types of organs of the respiratory tract, with 81 relatively high expression in goblet cells and ciliated cells of nasal epithelium and club cells in 82 the lung (7). In addition to respiratory tract, additional single cell sequencing analyses of 83 cornea, ileum, colon, heart, and gallbladder besides the respiratory tract identified cells that 84 are susceptible to SARS-CoV2 infection (7). These findings may explain cardiovascular 85 inflammation, conjunctivitis and diarrhea as well as other symptoms among individuals 86 infected with SARS-CoV2. One of the major clinical concerns is the effect of SARS-CoV2 on pregnancy and fertility. Reports with data from a limited number of pregnant women suggest that SARS-CoV2 is 89 responsible for miscarriages, preterm birth, stillbirth, and fetal growth restrictions due to 90 placental abnormalities (8, 9) . However, the susceptibility of female reproductive organs to 91 SARS-CoV2 is poorly understood. We investigated the cell-specific presence of ACE2/TMPRSS2 receptor expression in the 93 female reproductive organs as a surrogate for their susceptibility to SARS-CoV2. We 94 examined the myometrium, uterus, ovary, fallopian tube, and breast single-cell RNA 95 sequencing datasets for cell specific expression of the SARS-CoV2 receptor, ACE2. Our study 96 gave us critical insights into the expression of SARS-CoV2 receptor and proteases TMPRSS2, 97 CTSB/L in the female reproductive tract. Our findings suggest that ovary, fallopian tube, 98 uterus, myometrium, and breast are unlikely to be direct targets for SARS-CoV2 entry. The published datasets can be found at: fallopian tube (GSE139079), breast (NCBI 103 GSE113197), ovary (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-8381/), and 104 uterus (GSE134355). We retained the cell clustering the same as described in the respective 105 manuscripts except for the uterus. For the uterus dataset, we filtered out cells expressing more 106 than 20% mitochondrial genes and used the standard Seurat pipeline to obtain the cell 107 clusters. Fresh tissue samples were collected, stored in ice-cold HBSS, and transported to the lab. The Single-cell RNA-seq data preprocessing and analysis for myometrium dataset 134 FASTQ files were analyzed using Cellranger (version 3.1; 10x Genomics). Raw count cell by 135 transcript matrices were imported into R and Seurat (version 3.0) and used for further analysis. For quality control, cells having fewer than 200 reads, greater than 2500 reads, or more than 137 seven percent mitochondrial gene expression were removed. The "sctransform" function was 138 utilized to integrate the Seurat object from the eight tissue samples. Any cells with more than 139 one percent expression of hemoglobin genes HBA2, HBA1, and HBB were removed. The raw 140 transcripts were normalized in each cell to transcripts per 10,000 UMI to remove the batch 141 effects and log2 transformed. The uniform manifold approximation and projection (UMAP) was 142 used for dimensionality reduction and clustering the cells. Expression of ACE2 or TMPRSS2 in the ovary 146 Normal ovarian function is essential for proper oogenesis and fertility. We investigated the 147 presence of the ACE2, TMPRSS2, CTSB, and CTSL in the human ovary cell types derived 148 from single-cell sequencing (10). Data processing and cluster annotation were performed as 149 previously described (10) (Fig. 1A , B). We found that ACE2 was expressed at a very low level 150 in less than 5% of stroma and perivascular cells of the ovarian cortex. We did not observe the 151 expression of TMPRSS2 in any of the eight distinct cell types in the ovary (Fig. 1C, D) . CTSB 152 and CTSL were found to be expressed in all eight ovarian cell types (Fig. 1C, D) . However, 153 we did not observe any cells in the ovary co-expressing ACE2/ CTSB or ACE2/CTSL ( Fig. 154 1D). Since ACE2 requires the co-expression of protease TMPRSS2 or CTSB/L to facilitate its 155 entry into the host cell by priming the S protein on its surface, our data suggest that SARS- CoV2 is unlikely to infect the ovarian cells and unlikely to affect oogenesis. Expression of ACE2 and proteases TMPRSS2, CTSB/L in the fallopian tube The fallopian tube is responsible for transporting the oocyte or fertilized egg to the uterus for 160 implantation. Inflammation and blockage of the fallopian tube can lead to infertility in women. We therefore analyzed SARS-CoV2 receptors expression in different cell types of the fallopian 162 tube. We analyzed the previously published single-cell dataset from the normal fallopian tube 163 (11). We performed the cluster annotation and filtering of the low-quality cells exactly as 164 previously described (11). Cell clusters and cluster annotations are shown in Fig. 2A in the myometrium express CTSB and CTSL (Fig. 3C, D) . Interestingly, we did not find co-191 expression of either CTSB or CTSL with ACE2 (Fig. 3D) . These findings indicate that SARS- CoV2 is unlikely to infect the smooth muscle cells in the myometrium, suggesting that COVID-193 19 infection is unlikely to cause myometrial inflammation and potentially preterm birth. fibroblasts, and macrophages (Fig. 4A, B) . Expression analysis of TMRSS2 revealed the 204 absence of TMPRSS2 RNA in all cell types of the uterus. We found very low expression of 205 ACE2 in approximately 5% of the stromal cells and 1% endothelial cells (Fig. 4C, D) . We also 206 assessed the expression of CTSB/L in the uterus dataset and found that both CTSB/L are 207 expressed in all cell types of the uterus (Fig. 4C, D) . However, none of these cell types co-208 expressed ACE2 with either CTSL or CTSB (Fig. 4D) . These findings suggest that it is unlikely 209 that uterus is susceptible to SARS-CoV2 infection in humans. Cell-specific expression of ACE2 and TMPRSS2 in breast epithelium We also wanted to investigate if the SARS-CoV2 can infect the mammary gland epithelium We found that while ACE2 was expressed in approximately 1% of stromal cells and 253 perivascular cells, TMPRSS2 was not expressed in any of the eight ovarian cell types. Furthermore, fallopian tube data showed very few ciliated cells, secretory cells, and leukocytes 255 that expressed ACE2. We did not find any cells co-expressing ACE2 and TMPRSS2 or 256 CTSB/L in either ovary or fallopian tube. Together these results suggest that SARS-CoV2 is 257 unlikely to affect female fertility. A recent study analyzing the previously published testes We also wanted to find out if breast cells are susceptible to the COVID-19 infection. 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Colors represent the expression level as shown in the scale bar