A fast and simple one-step oocyte vitrification and warming protocol: proof-of-concept validation in the mouse model

Oocyte vitrification is widely used in fertility preservation and assisted reproductive technologies (ART), but current protocols rely on sequential cryoprotectant exposure steps that are labor-intensive and operator-dependent. This study evaluated the feasibility of a simplified one-step vitrification and warming protocol using mouse metaphase II oocytes. An initial optimization phase using 716 oocytes identified the optimal exposure time to the newly developed vitrification and warming solutions. Subsequently, 876 oocytes were allocated to fresh controls, standard multi-step vitrification, or the optimized one-step protocol. Outcomes included post-warming survival, meiotic spindle integrity, chromosome alignment, reactive oxygen species (ROS) levels, mitochondrial distribution, intracytoplasmic sperm injection (ICSI) outcomes, blastocyst development, total blastocyst cell counts, and full-term development after embryo transfer. An one-step exposure time of 2 min achieved the highest post-warming survival (100%), comparable to the standard protocol (98.9%; p = 0.080). Immunofluorescence analysis demonstrated normal spindle morphology, chromosome alignment, oxidative stress levels, and mitochondrial distribution across groups. Survival after warming and ICSI, as well as blastocyst formation rates, did not differ significantly between vitrification methods and fresh controls (p > 0.05). Although blastocyst total cell numbers were slightly reduced in vitrified groups, full-term development rates were comparable among all groups, and offspring developed normally to adulthood. These findings demonstrate that one-step vitrification and warming can preserve oocyte developmental competence while substantially simplifying cryopreservation procedures, supporting its potential as a practical alternative to conventional vitrification protocols in ART.