Validation and clinical study of single-step vitrification combined with single-step warming of human blastocysts

This study assesses the feasibility of single-step vitrification and warming (SSV/SSW) for human blastocysts. Previous animal study models have shown possibilities of shorter cryopreservation methodologies by cryoprotectant and temperature adjustments. The main research question investigates whether SSV/SSW can serve as an effective alternative to stepwise vitrification (SWV) and warming procedures, ensuring comparable embryological and clinical outcomes.

The study was conducted at a single center. Validation of SSV combined with SSW assessing survival, re-expansion, and development after 24 h was performed using research blastocysts. Cryoprotectant (CPA) levels were measured in three different SSW sucrose solutions after SSV or SWV. We introduced the valid method in the clinic and report laboratory and clinical outcomes of 333 SWV/SSW against 568 SSV/SSW blastocyst transfers.

During validation, SSV at 37 °C, compared to SWV, combined with 1 M sucrose SSW demonstrated comparable collapsing (89.6% vs. 98.1%) and re-expansion outcomes (97.9% vs. 88.7%). Extracellular CPA analysis (79.3 mg/L vs. 127.3 mg/L; p < 0.001) indicated significantly lower intracellular uptake following SSV compared to SWV. Clinical data showed significantly lower rates of collapsing (67.8% vs. 92.5%, p < 0.001) and re-expansion (72.0% vs. 89.5%, p < 0.001) after SSV compared to SWV, but similar rates of ongoing pregnancy (25.9% vs. 26.1%) and secondary clinical outcomes.

SSV combined with SSW is a feasible, operationally efficient alternative to SWV and warming procedures open to further optimize the collapse status before or during vitrification. The method reduces handling time, CPA exposure, and operator-related errors while maintaining ongoing pregnancy rates.