A one-step short rehydration protocol for warming is associated with live birth outcomes in frozen blastocyst transfers: a retrospective cohort study

We aimed to determine whether a one-step warming protocol for vitrified blastocysts achieves non-inferior efficacy and safety to those of the conventional multi-step method. This large-scale retrospective cohort study included 2,928 infertile patients undergoing single frozen–thawed blastocyst transfer between January 2021 and June 2025 at a single tertiary reproductive medicine center. A propensity score-matched subgroup of 1,314 patients (657 per group) with complete birth outcome data was analyzed. Following safety confirmation in a preliminary experiment using 128 donated blastocysts, a one-step warming protocol was clinically implemented in March 2024. Participants underwent either conventional three-step sucrose-based warming or one-step warming using 0.33 M sucrose solution lasting 2 min. Multivariable logistic regression was applied to adjust for potential confounders. The live birth rate was the primary outcome in the matched cohort; blastocyst survival, pregnancy-related outcomes, and neonatal outcomes, including birth weight and sex ratio, were also assessed. Blastocyst survival was 100% in both groups. In the overall cohort, the one-step warming group showed a significantly higher unadjusted ongoing pregnancy rate (35.6% vs. 31.7%; P = 0.029), despite less favorable baseline characteristics. In the propensity score-matched cohort, the one-step warming group demonstrated a significantly higher live birth rate (31.8% vs. 26.3%; P = 0.029), which remained significant even after adjustment (adjusted odds ratio = 1.360, 95% confidence interval: 1.058–1.750; P = 0.017). Neonatal outcomes were comparable between the groups. The one-step warming protocol simplifies laboratory workflow for vitrified blastocyst transfers. In this study, its use was associated with a higher live birth rate, suggesting that it may be a reliable and efficient option in clinical practice.