Simplified warming protocol for vitrified human embryos: a comprehensive analysis across various developmental stages and morphological qualities

Purpose: To determine whether a simplified warming protocol for vitrified human embryos is effective regardless of embryo characteristics such as developmental stage, morphological quality, and day of blastulation. Methods : This is a retrospective cohort study conducted at a single IVF center. A total of 1501 single vitrified-warmed embryo transfer cycles, involving 1351 blastocyst- and 150 cleavage-stage embryos, performed from January to December 2024, were analyzed. Post-warming survival rate and embryo transfer outcomes were compared between a conventional three-step warming protocol (thawing solution [TS] for 1 minute, dilution solution [DS] for 2 minutes, and washing solution [WS] for 3 minutes, n = 741) and simplified warming protocols, including a two-step (TS for 1 minute, followed by DS for 2 minutes, n = 461) and a one-step (TS for 1 minute, n = 298). The analysis primarily focused on blastocyst-stage embryos, for which detailed subgroup analyses were performed based on developmental stage, morphological quality, and day of blastulation. In addition, a separate analysis was conducted for cleavage-stage embryos. Results For blastocyst-stage embryos, post-warming survival rates were excellent and comparable among the three-step, two-step, and one-step protocols (100%, 99.3%, 99.6%; p = 0.1112). There were also no statistically significant differences in the fetal heartbeat confirmation rates (47.7%, 46.2%, 43.9%; p = 0.5765). Furthermore, this equivalence was consistently observed across all subgroups, including those based on developmental stage (full-blastocyst: 43.2%, 39.6%, 33.7%, p = 0.3111; expanded-blastocyst: 49.6%, 49.5%, 49.1%, p = 0.9950), morphological quality (poor-grade: 27.5%, 23.5%, 23.4%, p = 0.7636; good-grade: 52.1%, 51.6%, 48.4%, p = 0.6390), and day of blastulation (day 5: 52.7%, 52.9%, 50.3%, p = 0.8234; day 6: 34.4%, 28.5%, 27.4%, p = 0.4088). For cleavage-stage embryos, all protocols resulted in 100% post-warming survival with comparable fetal heartbeat confirmation rates (11.1%, 12.5%, 10.8%; p = 0.9717). Conclusions A simplified one- or two-step warming protocol is a safe and effective protocol for warming of vitrified human embryos. This approach is applicable to a variety of embryos, regardless of their characteristics.