CELL-LEVEL EXPRESSION OF SARS-COV-2 CELL ENTRY FACTORS IN HUMAN ENDOMETRIUM DURING THE PRECONCEPTION PERIOD

Vilella, Felipe; Wang, Wanxin; Moreno, Inmaculada; Quake, Stephen R.; Simon, Carlos

Objective: ACE2 enzyme serves as SARS-CoV-2 human receptor through binding of the viral S protein and subsequent trimming of S protein between S1 and S2 units by host serine proteases as TMPRSS2, CTSB or CTSL. Here, we aim to investigate the expression of the different cell entry proteins involved in SARS-CoV-2 infection in the different cell types of the human endometrium throughout the menstrual cycle using single-cell RNAseq (scRNAseq). Design: Gene expression patterns for SARS-CoV-2 entry molecules were analyzed by scRNAseq in a total of 73,181 endometrial single cells obtained from endometrial biopsies from 19 reproductive-age women across the full menstrual cycle (Fluidigm C1: 2,149 cells) and 10 women from the same cohort (10x: 71,032 cells). For two women, both C1 and 10x data were collected as anchors for comparison. Materials and Methods: After tissue dissociation, single cell capture was performed on Fluidigm C1 system (n= 2,149 cells) or Chromium 10x system (Chromium Next GEM Chip G, 10x Genomics) (n= 71,032 cells) followed by reverse-transcription, cDNA generation and library construction. Barcoded libraries were sequenced in pair-end reads on Nextseq (Illumina) for the C1 dataset or Novaseq (Illumina) for the 10x dataset. Data pre-processing, quality filtering, and statistical analyses were performed using custom Python, R, and Java scripts. Results: Expression analysis across the menstrual cycle showed no significant expression of ACE2 in stromal or unciliated epithelial cells in any cycle phase. TMPRSS2 was expressed more highly in glandular epithelial cells during the early proliferative phase and towards the end of the cycle. Interestingly, expression of CTSB and CTSL was observed in both stromal and epithelial cells across all phases of the menstrual cycle, with CTSB the more abundant of the two. All four genes were simultaneously expressed in less than 0.7% of glandular epithelial cells. Expression analysis during the secretory phase did not detect significant expression of ACE2 (less than 2% of epithelial or stromal cells). TMPRSS2 showed mild expression in about 12% of unciliated epithelial cells. In contrast, CTSB and CTSLwere highly expressed in ∼80% and ∼40% of cells during the mid-late secretory phase, aligning with what we detected via Fluidigm. In addition, while CTSB was highly expressed in both epithelial and stromal cells, CTSL was more highly expressed in stromal cells across the menstrual cycle. Conclusions: Percentages of endometrial cells expressing ACE2, TMPRSS2, CTSB, or CTSL were <2%, ∼12%, ∼80%, and ∼40%, respectively, with <0.7% of cells expressing all four. This finding implies low efficiency of SARS-CoV-2 infection in the endometrium before embryo implantation, providing information to assess preconception endometrial infection risk in COVID-19 asymptomatic carriers. FV & WW contributed equally.