ROCK inhibitor Y-27632 improves embryo transfer pregnancy rates following cryopreservation of biopsied embryos

Pre-implantation genetic testing relies on embryo biopsy, yet biopsied embryos exhibit reduced cryotolerance and compromised post-thaw viability. In this study, we optimized biopsy parameters and explored cryoprotective strategies in sheep embryos. Trophectoderm biopsy at a position two-thirds distant from the inner cell mass yielded optimal cell numbers (21.96 ± 1.21 cells) while maintaining high post-biopsy re-expansion rates (92.56%). An 8-hour post-biopsy recovery period prior to vitrification was identified as optimal, achieving complete re-expansion with minimal apoptosis while avoiding progression to hatched stages unsuitable for transfer. Supplementation with 10 μM Y-27632 at different vitrification stages was assessed for effects on re-expansion, cell viability, apoptosis, and pregnancy rates after transfer. Transcriptomic profiling revealed that Y-27632 effectively suppressed lipid and sterol biosynthesis, shifting metabolism toward energy conservation pathways that enhance cryoresistance. Treated embryos also showed significantly reduced lipid droplet accumulation, confirming metabolic remodeling. Y-27632 administration, particularly before and after vitrification, markedly improved cryotolerance, developmental capacity, and pregnancy outcomes of biopsied embryos, increasing pregnancy rates from 24.3% to 37.2%. These findings establish an optimized biopsy and recovery protocol and identify molecular changes associated with the protective effects of Y-27632 by which Y-27632 enhances embryo cryotolerance, presenting a practical strategy to improve cryopreservation efficacy in livestock and human assisted reproduction.