Optimizing Temperature Regulation in the Initial Thawing Step to Enhance Viability and Survival Rates of Early Cleavage-Stage Human Embryos in IVF-ET Programs

Background: Embryo cryopreservation is an integral to assisted reproductive technology (ART), with cleavage-stage and blastocyst-stage, embryo frozen thawed cycles offer distinct advantages. This study evaluates the impact of thawing modifications to improve survival rates. Significance of freezing early cleavage stage embryos at day2, culture them after thawing to the blastocyst stage to select the best embryo for transferring to improve reproductive outcomes mainly the live birth rates (LBR), and also to enable sequential embryo transfer (Seq-FET) in patients suffering from unexplained repeated implantation failures. Methods: A total number of patients 500 (aged 22–38 years) were chosen to undergo frozen embryo transfer (FET) and were categorized into two groups including cleavage-stage (n=250) and blastocyst-stage (n=250) vitrification groups. In the cleavage stage group, the embryos were vitrified on Day2, upon thaw these embryos were cultured to the blastocyst stage prior to embryo transfer. In the blastocyst stage group, the embryos were directly frozen and, post thaw these were cultured for 2 to 3 hours to allow re-expansion prior to embryo transfer. Post-thaw survival rates, beta-human chorionic gonadotropin (β-hCG) positivity, clinical pregnancy rates, ongoing pregnancy rates, and live birth rates were assessed. The standard 37°C thawing protocol was compared to a modified protocol with a gradual temperature reduction to 35°C to see its impact on embryo survival rates. Results: Cleavage-stage embryos had a higher survival rate (99.2±0.85) than blastocyst-stage embryos (96.97±1.21). β-hCG positivity was highest in cleavage-stage embryo (66.4±2.0), followed by sequential frozen thawed transfers (62.0±1.8) and direct blastocyst-stage frozen thawed cycles transfers (54.8±2.3). Clinical pregnancy rates were 64.8% (cleavage), 58.0% (sequential), and 51.6% (blastocyst). Ongoing pregnancy rates were highest in cleavage-stage (61.2%) and sequential transfers (56.0%), while blastocyst transfers had the lowest rate (42.8%). Take-home baby rates followed a similar trend (cleavage: 59.2%, sequential: 52.0%, blastocyst: 38.8%). Sequential transfers showed higher cumulative implantation potential, with 30.83% twin pregnancies and 20% twin deliveries. Conclusion: Cleavage-stage vitrification and sequential transfer enhance implantation, ongoing pregnancy, and live birth rates, making them viable strategies for improving ART outcomes. The modified thawing protocol (35°C) further optimizes embryo survival and implantation, presenting a novel approach to enhance reproductive success.