Morphological and Mitochondrial Advantages of Ultrafast Freeze–Thaw in Oocyte Cryopreservation

Background: Cryopreservation of oocytes is central to assisted reproductive technologies (ART) and fertility preservation. Conventional vitrification (CVT), though widely used, involves prolonged exposure to high concentrations of cryoprotective agents (CPAs), potentially inducing cellular stress. Ultrafast freeze–thaw (UFFT) aims to minimize CPA exposure and achieve extreme cooling/warming rates, reducing cryodamage. Objective: To compare UFFT and CVT in mouse oocytes, focusing on spindle/polar body morphology, necrosis percentage via PI⁺ extracellular vesicles (EVs), and mitochondrial unfolded protein response (mtUPR) activation. Methods: Metaphase II oocytes (n = 100/group) were assigned to fresh control, CVT, or UFFT groups. Post-thaw morphology was assessed via light/polarized microscopy; necrosis was quantified as PI⁺ EV fraction by flow cytometry; mtUPR activation (Hsp60, Lonp1, Atf5, ClpP) was measured by qRT-PCR. Data were analyzed by ANOVA with Tukey’s post-hoc test. Results: UFFT oocytes exhibited significantly higher spindle integrity (91% ± 3%) and polar body retention (94% ± 3%) than CVT (65% ± 5% and 70% ± 4%, respectively; p < 0.001). PI⁺ EV percentage was reduced in UFFT (18.0% ± 3.0%) compared to CVT (34.0% ± 4.0%; p < 0.001). mtUPR gene expression was highest in CVT, intermediate in UFFT, and lowest in the fresh control. Differences between UFFT and control were not statistically significant (p > 0.05). Conclusion: UFFT preserved oocyte morphology and reduced membrane damage compared to CVT, without significantly elevating mitochondrial stress markers above control. These findings support UFFT as a promising alternative in ART, warranting further large-scale and clinical studies.