Résumé
Presence of SARS-CoV-2, the virus responsible for COVID-19, has been reported in numerous organs and tissues of infected patients, including the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood. While cases of intrauterine transmission between expectant mother and fetus have been documented, the impact of SARS-CoV-2 infection on early embryogenesis and establishment of a pregnancy are not known. This prompted us to ask if SARS-CoV-2 can infect embryos, since such an event could impact embryo viability and affect a subsequent pregnancy. We used a three-pronged approach to investigate this possibility: 1) Using RNA-seq and immunofluorescence, we learned that ACE2 and TMPRSS2, two factors required on target cells for SARS-CoV-2 entry, are coexpressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos;2) Using fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, we observed robust infection of trophectoderm cells, and this permissiveness could be attenuated with blocking antibodies targeting S or ACE2;and 3) Exposing human blastocysts to live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.
Résumé
Objective: To determine whether human pre-implantation embryos have the potential to be infected by SARS-CoV-2, the virus responsible for COVID-19. Design: Assessment of expression levels of SARS-CoV-2 entry mediators in human embryo biopsies by RNAseq analysis, and infection of cultured embryos with SARS-CoV-2 Spike glycoprotein pseudotyped reporter virions expressing green fluorescent protein (GFP). Materials and Methods: Trophectoderm biopsies from blastocyst-stage embryos (n=24) were processed for RNAseq using a commercial kit and sequenced;results were analyzed for expression of factors implicated in SARS-CoV-2 cellular entry. For viral infection experiments, blastocyst-stage embryos (n=94) were hatched from zonas mechanically, and infected by spinoculation with GFP-reporter virions pseudotyped with the SARS-CoV-2 Spike glycoprotein (required for SARS-CoV-2 entry). Embryos were subsequently monitored for fluorescence at 24-48 hours post-infection. Various control conditions were used as specified in the ‘results’ section. A mixed population of euploid, aneuploid, and untested embryos used in the study were from IVF treatment, donated to research by signed informed consent. The project was approved by an Institutional Review Board. Results: Cells collected from blastocyst-stage embryos robustly expressed the canonical SARS-CoV-2 entry receptor ACE2 and the putative activator protease TMPRSS2, in addition to other reported entry factors. Embryos exposed to reporter virions pseudotyped with SARS-CoV-2 Spike glycoprotein displayed robust GFP signal, often in numerous cells with cytoplasmic localization. Specificity was confirmed by the absences of fluorescence in embryos treated with virions lacking the Spike glycoprotein (“bald” virus), or when embryos were spinoculated with media alone in the absence of virus. Embryos exposed to Spike glycoprotein-positive reporter virus in the presence of neutralizing anti Spike- or anti-ACE2-blocking antibodies exhibited negligible GFP signal, while control monoclonal IgG antibody-treated embryos maintained GFP expression. These results implicated the canonical Spike-ACE2 axis in the viral entry. Lastly, embryos exposed to reporter virions pseudotyped with Spike glycoprotein of SARS-CoV-1 (which also enters cells via ACE2) displayed GFP fluorescence, while embryos exposed to reporter viruses pseudotyped with Spike glycoprotein of MERS (which utilizes Dipeptidyl Peptidase IV (DPP4) instead of ACE2) resulted in no fluorescence. Conclusions: Our results indicate that cells present in pre-implantation embryos are permissive to the canonical Spike-ACE2 viral entry mechanism utilized by SARS-CoV-2. These results encourage further investigation into the potential of SARS-CoV-2 infection in human embryos and may have wider implications in natural conception and ART practice.