Single-step warming for blastocysts: a clinically validated alternative to conventional multi-step protocols

STUDY QUESTION: Can a single-step warming protocol for vitrified blastocysts provide comparable or superior outcomes to a conventional multi-step warming approach in a clinical IVF setting? SUMMARY ANSWER: Single-step warming resulted in higher blastocyst survival, intactness, and transfer rates compared to the conventional multi-step protocol, with comparable ongoing pregnancy rates and a consistent trend toward lower miscarriage rates across all subgroups. WHAT IS KNOWN ALREADY: While vitrification has become the gold standard for embryo cryopreservation, the warming process remains critical to embryo survival and implantation potential. Traditional warming relies on multi-step dilution protocols to minimize osmotic stress. Preliminary studies have suggested that single-step rehydration protocols may be equally effective, but data on clinical validation remain scarce. STUDY DESIGN, SIZE, DURATION: A three-phase validation study was conducted at a single university-based IVF centre including (i) risk analysis, (ii) preclinical validation (n = 246 blastocysts), and (iii) a clinical comparison of outcomes over one year (March 2024-March 2025) between single-step warming (n = 1925 cycles) and the conventional multi-step protocol (n = 1744 cycles, March 2023-March 2024). PARTICIPANTS/MATERIALS, SETTING, METHODS: In the preclinical phase, vitrified surplus and PGT blastocysts were rewarmed using the single-step protocol and compared to historical controls. Survival (≥50% intact cells), intactness (100% intact), and transfer suitability were assessed. Single-step warmed blastocysts were monitored for 24 h post-warming in a time-lapse incubator, and viability was further evaluated using live/dead fluorescent staining to quantify cell damage. In the clinical phase, outcomes (survival, transfer rate, pregnancy, ongoing pregnancy, miscarriage) were retrospectively compared across protocols, stratified by PGT status and day of vitrification. MAIN RESULTS AND THE ROLE OF CHANCE: In the preclinical phase, blastocyst survival was ≥50% in 99.1% of cases after single-step warming versus 96.8% after multi-step warming (P = 0.0924). Fully intact blastocysts were significantly more frequent in the single-step group (85.4% vs. 76.3%, P = 0.0058), and transfer suitability at 2 h post-warming was also higher (96.7% vs. 91.2%, P = 0.0076). Time-lapse monitoring confirmed a high re-expansion rate (93.9%) with a mean re-expansion time of 3.3 ± 2.7 h. Fluorescent viability staining showed that 90.5% of blastocysts exhibited no or minimal cell damage. In the clinical cohort, significantly higher survival rates were seen in the single-step group across multiple subgroups, including non-PGT Day 5 (98.5% vs. 96.1%, P = 0.0003) and PGT Day 5 (100% vs. 98.2%, P = 0.0407) blastocysts. Fully intact rates were significantly higher in all subgroups and transfer rates were significantly higher in the single-step group for non-PGT Day 6 (97.2% vs. 92.8%, P = 0.0060) and PGT Day 5 cycles (100% vs. 97.8%, P = 0.0209). While some early pregnancy outcomes (e.g. clinical pregnancy in non-PGT Day 5) favoured the multi-step protocol (40.5% vs. 35.7%, P = 0.0286), ongoing pregnancy rates were not significantly different in any subgroup. Miscarriage rates showed a consistent trend in favour of single-step warming but did not reach statistical significance. LIMITATIONS, REASONS FOR CAUTION: This was a single-centre study, and the control group was retrospective. WIDER IMPLICATIONS OF THE FINDINGS: The single-step warming protocol is a clinically validated, safe, and efficient alternative to conventional multi-step warming. Its implementation may improve workflow and streamline blastocyst handling without compromising clinical outcomes. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received. The authors declare no conflicts of interest apart from KT, who has received travel support related to the manuscript from Fuijifilm Irvine Scientific, in the form of payment to her institution. TRIAL REGISTRATION NUMBER: N/A.