Melatonin Enhances Vitrified Mouse Blastocyst Development by Mitigating Oxidative Stress and Preserving Mitochondrial Function

Abstract This study aimed to investigate the benefits of melatonin supplementation during blastocyst vitrification and thawing process and explore underlying mechanisms to prevent apoptotic event. 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 analyze effects of optimized melatonin supplementation. Additionally, transcriptome analysis in blastocysts by Smart-seq2 was performed to investigate the underlying transcriptional mechanism. The 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 increased the numbers of inner cell mass (ICM) (P < 0.001), trophectoderm (TE) (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 ROS (P < 0.05). Importantly, melatonin alleviated cryodamage and preserved blastocyst ultrastructure, and rebalanced altered gene expression induced by the vitrification and warming. These results are suggestive that adding 10−10 M melatonin in vitrification and warming solutions protected mouse blastocysts against the detrimental effects of oxidative stress and enhanced the efficiency of cryopreservation.