key: cord-0964058-kccrlsj8
authors: Stanley, Kate E.; Thomas, Elizabeth; Leaver, Megan; Wells, Dagan
title: CORONAVIRUS DISEASE (COVID-19): TRANSCRIPTOMIC AND PROTEOMIC INVESTIGATION OF CELLS OF THE MALE AND FEMALE REPRODUCTIVE SYSTEMS TO EVALUATE SARS-COV-2 INFECTION RISK
date: 2020-09-30
journal: Fertility and Sterility
DOI: 10.1016/j.fertnstert.2020.08.1020
sha: d9fd72b70b2b68ca7fb44065720aacb2bdcc5721
doc_id: 964058
cord_uid: kccrlsj8

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DESIGN: Transcriptomic and proteomic analysis. MATERIALS AND METHODS: Publicly available single cell RNA sequencing (scRNAseq) data in human testis and non-human primate ovary was analyzed, as well as bulk RNA and proteomic data in human testis and ovary. Additionally, novel RNA sequencing data from 18 samples of human cumulus cells was generated. Tissue-and cell-type specific risk of viral infection was predicted based on the co-expression of host receptor angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) which are required for viral binding and cleavage, respectively. Expression of receptor basigin (BSG) and cysteine protease cathepsins L (CTSL) were also assessed as they are hypothesized to facilitate viral entry.

RESULTS: Based on scRNAseq data in non-human primate ovarian tissue, ACE2 and TMPRSS2 co-localize in a sub-population of oocytes in antral follicles (62% of cells, Pearson correlation¼0.37), but to a lesser extent in less mature oocytes (Pearson correlation¼0.13-.21) and not at all in ovarian somatic cells. While ACE2 transcripts were detected in human cumulus cells (mean 24.03 transcripts per million), TMPRSS2 expression was absent in 15/18 samples and low in the others (0.13 TPM). Consistent with these findings, bulk RNA and protein data showed ACE2 expression in ovarian tissue, but no TMPRSS2 expression. In testicular cells, including sperm, co-expression of ACE2 and TMPRSS2 was not detected (Pearson correlation¼-0.01). Bulk RNA and protein data revealed ACE2 but no TMPRSS2 expression. BSG was more broadly expressed in testis than ACE2 and was co-expressed with CSTL in early (78.7%) and late (90.8%) primary spermatocytes.

CONCLUSIONS: Given that known COVID-19 symptoms are associated with tissues co-expressing ACE2 and TMPRSS2 (e.g. lung, heart, kidney, gut), it is conceivable that reproductive function could be effected if constituent cells co-express these genes. Based on these results, testicular cells including sperm are not at risk of ACE2-TMPRSS2-mediated viral entry, however low levels of SARS-Cov-2 in human semen have been reported and may suggest alternative routes of entry. The cells predicted to have the greatest susceptibility to infection are antral oocytes which are either ovulated or atrophy within several days of appearance each cycle and are therefore unlikely to have sustained impact on female fertility if infected. Moreover, the lack of ACE2-TMPRSS2 expression in cumulus cells may act as a barrier to infection. Therefore, procedures in which cumulus cell-enclosed oocytes are collected and fertilized outside the female reproductive tract (e.g. IVF) may not pose a risk. IVF success rates decline with increasing female age and it is therefore imperative that patients are allowed to access fertility treatments if safe to do so. The results presented here are indicative, but ovarian pathology data from women of reproductive age with COVID-19 at time of death is needed for definitive confirmation. OBJECTIVE: SEPT12, a member of the Septin gene family, has expression restricted to the testis and is critical for normal spermatogenesis. Septin genes code for polymerizing guanosine triphosphate (GTP) binding proteins whose homologs have been conserved throughout evolution from yeast to humans (1) . Roles assigned to Septin genes include cell division, cytoskeletal organization, and membrane-remodeling events including the epithelialmesenchymal transition in metastatic cancers (2) . A study that investigated azoospermia and teratozoospermia in a mouse knockout model demonstrated oocytes fertilized via ICSI with sperm targeted SEPT12 antisense alleles resulted in embryo arrest by the morula stage (3) . The application of RNA sequencing to elucidate the expression profile of SEPT12 in human preimplantation embryos may unlock insights into the transcriptional events of early embryogenesis.

DESIGN: Prospective cohort study on human, donated embryos. MATERIALS AND METHODS: The study included patients who donated fresh embryos at the blastocyst stage during an IVF cycle between January, 2016 and June, 2016. Embryos were biopsied, and approximately 2-4 cells were removed for preimplantation genetic testing for aneuploidy (PGT-A) by next generation sequencing (NGS) using the ReproSeq assay to assess copy number variants (CNVs). The remaining cells of the embryo were designated for RNA Sequencing. Read counts per gene were summed across embryo cohorts and normalized using the median of ratios. Differential gene expression between embryo cohorts was calculated using DESeq2, in order to estimate variance-mean dependence and evaluate differential gene expression using a negative binomial distribution. A likelihood ratio test was used to account for heterogeneity due to patient, batch, and ploidy and growth status (arrested/ongoing). The adjusted threshold for significance was p<0.05.

RESULTS: 43 blastocysts underwent PGT-A assessment and RNA sequencing. 36 showed expression of SEPT12, 6 of the 7 blastocysts that failed to show SEPT12 expression had poor trophectoderm morphology grade. The expression of SEPT12 was further examined in 15 embryos, 9 were enriched (>90%) for trophectoderm cells (TE) and 6 enriched (>80%) for inner cell mass cells (ICM). SEPT12 expression was significantly higher in TE cells than ICM cells, where P<0.0001.

CONCLUSIONS: Septins were first discovered nearly fifty years ago however their function remains poorly understood (4) . The importance of this gene family has been indicated by the conservation of the functional domains throughout evolution. SEPT12 has been shown to be critically important in spermatogenesis. This study is the first to characterize SEPT12 expression in the human embryo. Our data supports the findings that wild type SEPT12 expression was preferentially associated with blastocyst formation compared to arrest at the morula stage for embryos that contained the antisense SEPT12 allele (3). Our current studies are focused on characterizing SEPT12 expression in cleavage and morula stage human embryos to further elucidate this gene's role in early embryogenesis.

The septin family of GTPases: architecture and dynamics

The pathophysiology of the septin gene family

SEPT12 deficiency causes sperm nucleus damage and developmental arrest of preimplantation embryos

Genetic control of the cell division cycle in yeast

IV Genes controlling bud emergence and cytokinesis