Melatonin enhances vitrified mouse blastocyst development by mitigating oxidative stress and preserving mitochondrial...

This study aimed to investigate the benefits of melatonin supplementation during the blastocyst vitrification and thawing process and explore underlying mechanisms to prevent apoptotic events. We evaluated blastocysts in three groups: fresh blastocysts (Control), non-melatonin-treated vitrification (VT), and melatonin-treated vitrification (MVT). We compared their developmental potential and oxidative stress levels to analyse effects of optimized melatonin supplementation. Additionally, transcriptome analysis in blastocysts by Smart-seq2 was performed to investigate the underlying transcriptional mechanism. Antioxidant enzyme and mitochondrial function protein expression were investigated by immunofluorescence staining. Our results showed that supplementation of melatonin to the vitrification and warming solution significantly reduced the apoptotic cell proportion (P < 0.001) while increasing the numbers of inner cell mass (P < 0.001), trophectoderm (P < 0.001), and total cell counts (P < 0.001). Melatonin protected against oxidative stress and restored mitochondrial dysfunction in blastocysts, as evident from increased mitochondrial activity (P < 0.05) and reduced levels of Ca2+ (P < 0.05) and reactive oxygen species (P < 0.05). Importantly, melatonin alleviated cryodamage and preserved blastocyst ultrastructure, and rebalanced altered gene expression induced by the vitrification and warming. These results suggest that adding 10-10 M melatonin to vitrification and warming solutions protects mouse blastocysts against the detrimental effects of oxidative stress and enhances the efficiency of cryopreservation.