P-260 The zinc-finger transcription factors OVOL1/2 regulate blastocyst formation by promoting polarization

Abstract Study question In IVF, patients have multiple high-quality cleavage-stage embryos on D3, but both D3 embryo transfer and/or blastocyst culture fail. Reasons behind this remain unclear. Summary answer High-quality D3 embryos failed to form blastocysts are associated with insufficient activation of ZGA. Zinc-finger transcription factors OVOL1/2 regulate polarization by promoting actin formation. What is known already During mammalian preimplantation embryonic development, zygotic genome activation (ZGA) occurs as maternal transcripts are gradually consumed and degraded. During preimplantation embryonic development, compaction and polarization are the first critical morphological events that occur at the 8-cell stage. The apical domain is required for the ICM/TE lineage segregation during blastocyst development. OVOL is a zinc-finger transcription factor (TF) with C2-H2 zinc-finger domains that is evolutionarily conserved from metazoans to vertebrates. Vertebrates possess three Ovol genes (Ovol1-3), with Ovol1 and Ovol2 being the most extensively studied. Research has shown that OVOL1/2 promote the mesenchymal-to-epithelial transition (MET) in epithelial cells Study design, size, duration We collected human immature MI oocytes and matured them to MII oocytes through in vitro maturation. Zygotes were monitored using a time-lapse imaging system. Single blastomeres were biopsied from 8-cell that exhibited no visible abnormalities during the first three divisions and subjected to scRNA-seq. These embryos were subsequently cultured to the blastocyst stage. Participants/materials, setting, methods In total, the numbers of MI oocytes, MII oocytes, zygotes and biopsied 8-cell embryos were 129, 107, 84, and 26, respectively. Among the 26 biopsied 8-cell, 21 successfully developed into high-quality blastocysts (HQBs) with A- or B-grade ICM and/or TE, as assessed using Gardner’s scoring system, while the remaining 5 blastocyst formation failed (BFFs). C57BL6 background mouse strains were used in this study.Mice were maintained under specific pathogen free conditions in a controlled environment. Main results and the role of chance Among the 26 biopsied 8-cell, 21 successfully developed into high-quality blastocysts (HQBs), while the remaining embryos failed to form blastocysts (BFFs). Compared to HQBs, 100 genes were upregulated and 388 genes were downregulated in BFF embryos. Over 60% of the downregulated genes typically increase from the 4-cell to morula stages, suggesting insufficient activation of zygotic genome activation (ZGA) in BFFs. Interestingly, OVOL1/2 were significantly downregulated in BFFs, although their function in preimplantation embryos remains unclear. To investigate this, we co-injected siRNAs targeting both Ovol1 and Ovol2 into zygotes (low-dose (siO1 + 2-L) and high-dose (siO1 + 2-H)). Blastocyst formation was significantly reduced in siO1 + 2-L, while siO1 + 2-H were completely arrested at the morula stage. F-actin, phosphorylated Ezrin (pEzrin), and E-CAD were significantly reduced in siO1 + 2-L/H morulae, indicating impaired polarization and compation in Ovol1/2-knockdown embryos. Additionally, number of CDX2+ trophectoderm (TE) precursors was reduced, and TE functionality was compromised in siO1 + 2-L/H. Further investigation revealed that OVOL1/2 regulates embryo polarization through Arpc1b, supplementation with Arpc1b mRNA rescued blastocyst development in siO1 + 2-H. Collectively, we identified OVOL1/2 as key regulators of polarization, with insufficient activation leading to the failure of high-quality embryos to develop into blastocysts. Limitations, reasons for caution We only focused on two transcription factors, OVOL1/2, and how OVOL1/2 regulates actin formation remains to be studied. Wider implications of the findings This study provide a theoretical basis for the clinical diagnosis of High-quality D3 embryos failed to form blastocysts and possible directions for improving the success rate of ART. Trial registration number No