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

In brief: Embryo biopsy and cryopreservation compromise pregnancy rates, limiting genetic testing in livestock and humans. This study shows that Y‑27632 improves post‑thaw embryo quality and increases pregnancy rates in biopsied sheep embryos. Abstract: Preimplantation 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 postbiopsy re-expansion rates (92.56%). An 8-hr postbiopsy 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.