Carnosine enhances mouse oocyte vitrification outcomes with associated redox and transcriptional changes

Oocyte vitrification is widely used for fertility preservation, but cryopreservation-associated oxidative stress and mitochondrial dysfunction can impair subsequent embryonic development. This study investigated whether carnosine supplementation improves the developmental competence of vitrified mouse oocytes. Metaphase II oocytes were vitrified and warmed with 0, 100 or 500 µM carnosine, followed by intracytoplasmic sperm injection and embryo culture. Fresh oocytes served as controls. Reactive oxygen species (ROS), mitochondrial membrane potential, and NANOG-positive epiblast (EPI) cell counts were assessed in the 100 µM group and controls. NADH/FAD imaging was performed descriptively in one independent experiment. Proteomic and transcriptomic analyses were performed on resulting 2-cell embryos. Carnosine at both concentrations significantly restored 2 cell formation rates (80% and 81% vs. 70% in controls) and blastocyst formation (59% and 56% vs. 40%). At 100 µM, carnosine significantly reduced ROS fluorescence and increased NANOG-positive EPI cell counts compared with conventional vitrification. JC-1 ratios were numerically higher with carnosine, suggesting a potential improvement in mitochondrial membrane potential. Descriptive NADH/FAD imaging showed higher autofluorescence signals with carnosine. Proteomics of late 2-cell embryos identified carnosine-associated protein changes, including upregulation of the mRNA export scaffold Thoc2, the actin-based motor Myo1c, and downregulation of stress chaperone Clusterin. Transcriptomics identified 1026 differentially expressed genes, enriched in transcriptional regulation; the changes of cell fate determination-related Tfap2c were validated by RT-PCR. Carnosine supplementation improved preimplantation development following mouse oocyte vitrification and was associated with reduced oxidative stress and increased EPI cell numbers. Mitochondrial observations and molecular profiling suggest potential mechanisms. These findings support further evaluation of carnosine as an additive for oocyte cryopreservation.