Shortened warming protocols preserve spindle integrity and mitochondrial mass less effectively than standard warming...

To assess the clinical applicability of shortened (SW) and ultra-shortened (USW) warming protocols for oocytes cryopreserved using standard vitrification (STD-V).

This prospective sibling study included 436 mature human oocytes from 47 patients, all vitrified using a single standard vitrification (STD-V) protocol. Experiment 1 compared the standard warming (STD-W) with the SW protocols (2 min), and Experiment 2 compared the SW protocol with USW protocol (1 min). Oocyte survival rates were assessed 1.5 h post-warming, and oocytes were immediately fixed. The analysis of meiotic spindle organization and chromosome alignment was assessed by detailed examination of individual optical sections and corresponding full Z-stack projections encompassing the entire spindle. Mitochondrial mass was quantified from Z-stack-based projections integrating fluorescence across all optical sections. We also performed a pooled analysis to directly compare STD-W, SW, and USW groups.

Survival rates were comparable across protocols. Both SW and USW protocols were associated with significantly lower odds of normal chromosomal distribution, spindle organization, and normal MII apparatus compared with the STD-W protocol, with no significant differences between SW and USW. Spindle length and pole width were increased in SW and USW versus STD-W. Mitochondrial mass was significantly reduced in SW compared with STD-W.

Although survival rates were comparable, our data indicate that SW and USW protocols are associated with a higher incidence of spindle and chromosomal abnormalities than the traditional approach. This may affect oocyte developmental competence, highlighting the need for further investigation regarding the clinical use of SW/USW protocols for STD-V-cryopreserved oocytes.