Short-Term Post-Warming In Vitro Culture (<2 h vs. 2–4 h) Differentially Affects Vitrified Blastocyst Outcomes Depending on Transfer Number and Blastocyst Quality

Background: Despite the wide application of vitrification-warming in assisted reproduction, the impact of post-warming in-vitro culture duration on the developmental potential of vitrified blastocysts remains controversial. Therefore, clarifying the optimal duration is crucial for better clinical outcomes. This study aimed to evaluate the effect of different short-term post-warming culture durations (<2 h vs. 2–4 h) on clinical outcomes and to explore optimal strategies based on transfer number and blastocyst quality. Methods: This retrospective analysis included 1600 vitrified blastocyst warming-transfer cycles performed between 2021 and 2023, which were grouped according to post-warming culture duration (860 cycles: <2 h; 740 cycles: 2–4 h). Further subgrouping was conducted according to transfer number and blastocyst quality. Baseline data, clinical outcomes (such as implantation, clinical pregnancy, live birth and early abortion rates), and neonatal outcomes were compared. Statistical analyses were performed using the chi-square test for categorical variables and the Mann-Whitney U test for continuous variables, with Bonferroni correction for multi-group comparisons. A p value < 0.05 was considered statistically significant. Results: There was no difference in baseline data between groups (p > 0.05). In single blastocyst transfers, the 2–4 h culture group demonstrated higher implantation rate (75.45% vs. 47.04%, p < 0.001), clinical pregnancy rate (75.45% vs. 47.04%, p < 0.001) and live birth rate (75.12% vs. 46.30%, p < 0.001). In double blastocyst transfers, the <2 h culture group had a higher live birth rate (46.74% vs. 35.34%, p < 0.05). Overall, the 2–4 h group exhibited a higher live birth rate compared to the <2 h culture group (67.97% vs. 46.40%, p < 0.001), and the D5 high-quality group had the highest live birth rate compared to other quality-related groups (61.93%, p < 0.001). For neonatal outcomes, the 2–4 h group displayed a higher singleton delivery rate (91.44% vs. 86.01%) and a lower multiple delivery rate compared to the <2 h culture group (p = 0.008), while other indicators showed no differences (p > 0.05). Conclusion: The optimal post-warming culture duration may vary according to transfer number and blastocyst quality. Specifically, 2–4 h of culture appears beneficial for single blastocyst transfer, while <2 h may be preferable for double transfers. D5 high-quality blastocysts demonstrate optimal developmental potential. Culture duration does not compromise core neonatal health outcomes. These observations provide a basis for individualized transfer protocols to optimize clinical outcomes.