Abstract
Molnupiravir is a nucleoside analog antiviral that is authorized for use in the treatment of COVID-19. For its therapeutic action, molnupiravir is converted after ingestion to the active metabolite N4-hydroxycytidine, which is incorporated into the viral genome to cause lethal mutagenesis. Molnupiravir is not recommended for use during pregnancy, because preclinical animal studies suggest that it is hazardous to developing embryos. However, the mechanisms underlying the embryotoxicity of molnupiravir are currently unknown. To gain mechanistic insights into its embryotoxic action, the effects of molnupiravir and N4-hydroxycytidine were examined on the in vitro development of mouse preimplantation embryos. Molnupiravir did not prevent blastocyst formation even at concentrations that were much higher than the therapeutic plasma levels. By contrast, N4-hyroxycytidine exhibited potent toxicity, as it interfered with blastocyst formation and caused extensive cell death at concentrations below the therapeutic plasma levels. The adverse effects of N4-hydroxycytidine were dependent on the timing of exposure, such that treatment after the 8-cell stage, but not before it, caused embryotoxicity. Transcriptomic analysis of N4-hydroxycytidine-exposed embryos, together with the examination of eIF-2a protein phosphorylation level, suggested that N4-hydroxycytidine induced the integrated stress response. The adverse effects of N4-hydroxycytidine were significantly alleviated by the co-treatment with S-(4-nitrobenzyl)-6-thioinosine, suggesting that the embryotoxic potential of N4-hydroxycytidine requires the activity of nucleoside transporters. These findings show that the active metabolite of molnupiravir impairs preimplantation development at clinically relevant concentrations, providing mechanistic foundation for further studies on the embryotoxic potential of molnupiravir and other related nucleoside antivirals.