SARS-CoV-2 susceptibility and vulnerability of human blastocysts during in vitro preimplantation development.

Human preimplantation embryos express the SARS-CoV-2 entry factors ACE2, TMPRSS2 and BSG (CD147) on trophectoderm (TE) cells, suggesting susceptibility to infection. Likewise, SARS-CoV-2-pseudotyped reporter viruses induced cytopathic effects in blastocysts, while the zona pellucida (ZP) appeared to confer partial protection. The impact of authentic SARS-CoV-2 infection on embryonic lineage specification and developmental competence has not been established. Here, we exposed cryopreserved human embryos, donated under ethics approval and informed consent, to authentic SARS-CoV-2 (B.1.617.2, Delta variant). While embryos with an intact ZP remained uninfected, laser-assisted hatching rendered 19/21 (90.5%) of embryos susceptible to infection, as confirmed by detection of viral dsRNA. Moreover, SARS-CoV-2 infection had a pronounced effect on blastocyst development with a possibly fatal outcome. Compared to mock-treated embryos (n = 47), infected blastocysts (n = 48) showed a significantly reduced surface area (p = 0.0003), reduced total cell number (p = 0.0139), and reduced NANOG+ epiblast cell numbers (p = 0.0054) 24 hours post-exposure, while the number of GATA4+ primitive endoderm cells remained unaffected (p = 0.5255). Intriguingly, despite viral replication being largely confined to the trophectoderm (TE), this selective epiblast reduction suggests a disruption of TE-derived paracrine signaling required for epiblast specification. Finally, prolonged viral replication resulted in embryonic lysis and death. Digital transcriptomic profiling (mock, n = 29 embryos; SARS-CoV-2, n = 28 embryos; each in n = 5 minipools) revealed upregulation of innate antiviral and inflammatory genes, including IFIH1, IRF7, NOD2, and TNFRSF1A, as well as the recently identified SARS-CoV-2 entry factor TMEM106B, alongside altered developmental signatures concerning genome activation, pluripotency, and lineage-specific programs. These findings show that ZP disruption makes human blastocysts susceptible to productive SARS-CoV-2 infection, with adverse consequences for lineage allocation and direct implications for biosafety and virological counselling in medically assisted reproduction practice.