Single-cell RNA sequencing of SARS–CoV-2 cell entry factors in the preconceptional human endometrium

Abstract STUDY QUESTION Are SARS-CoV-2 canonical cell entry machinery, consisting of ACE2, TMPRSS2, NRP1 and LY6E, or alternative potential cell entry machinery, consisting of BSG, ANPEP, CD209, CLEC4G, TMPRSS4, TMPRSS11A, FURIN, CTSB, CTSL and IFITM1, expressed in the human endometrium across the menstrual cycle? SUMMARY ANSWER Analysis of cell entry factors for SARS-CoV-2 by single-cell RNA-sequencing (scRNAseq) in the preconceptional human endometrium reveals low risk of infection. WHAT IS KNOWN ALREADY Gene expression datasets from bulk endometrial tissue show no significant expression of the SARS-CoV-2 receptor ACE2 and TMPRSS2. This is in contrast to reported expression of ACE2 at the single-cell level in the decidua and trophoblast cells at the maternal–fetal interface in early pregnancy, as well as vertical transmission of SARS-CoV-2 during pregnancy. STUDY DESIGN, SIZE, DURATION This analysis of SARS-CoV-2 cell entry machinery gene expression was conducted by scRNAseq in 73 181 human endometrial cells isolated from endometrial biopsies obtained from 27 donors across the menstrual cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS ScRNAseq examined the expression of genes encoding cell entry machinery for SARS-CoV-2. The raw data were from a previously published dataset. MAIN RESULTS AND THE ROLE OF CHANCE ScRNAseq analysis showed no significant expression of ACE2 in stromal or unciliated epithelial cells in any phase of the menstrual cycle. TMPRSS2 was expressed in epithelial cells during the early proliferative and mid-secretory phases. Interestingly, the expression of NRP1 was observed in both stromal and epithelial cells across all phases of the menstrual cycle, and LY6E was highly expressed in stromal cells. In the mid-secretory phase, coexpression of ACE2 and TMPRSS2 was detected in 0.07% of luminal epithelial cells. No cells simultaneously expressed ACE2, NRP1 and TMPRSS2 at the time of embryo implantation. Focusing on non-canonical cell entry machinery, BSG was highly expressed in all cell types across the menstrual cycle and may interact with CTSB or CTSL proteases, but viral infection using this machinery has not yet been confirmed. LARGE SCALE DATA All raw data in this study can be found at NCBI’s Gene Expression Omnibus (series accession code GSE111976) and Sequence Read Archive (accession code SRP135922). LIMITATIONS, REASONS FOR CAUTION Our findings at the single-cell level imply low efficiency of SARS-CoV-2 endometrial infection using c