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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\u2009&amp;lt;\u20090.001) while increasing the numbers of inner cell mass (P\u2009&amp;lt;\u20090.001), trophectoderm (P\u2009&amp;lt;\u20090.001), and total cell counts (P\u2009&amp;lt;\u20090.001). Melatonin protected against oxidative stress and restored mitochondrial dysfunction in blastocysts, as evident from increased mitochondrial activity (P\u2009&amp;lt;\u20090.05) and reduced levels of Ca2+ (P\u2009&amp;lt;\u20090.05) and reactive oxygen species (P\u2009&amp;lt;\u20090.05). 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\u221210 M melatonin to vitrification and warming solutions protects mouse blastocysts against the detrimental effects of oxidative stress and enhances the efficiency of cryopreservation.<\/jats:p>","DOI":"10.1093\/molehr\/gaaf054","type":"journal-article","created":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T13:05:23Z","timestamp":1762520723000},"source":"Crossref","is-referenced-by-count":0,"title":["Melatonin enhances vitrified mouse blastocyst development by mitigating oxidative stress and preserving mitochondrial function"],"prefix":"10.1093","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7569-4559","authenticated-orcid":false,"given":"Dan","family":"Zhou","sequence":"first","affiliation":[{"name":"Centre for Assisted Reproduction, Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University , Shanghai 200092,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiaoyu","family":"Chen","sequence":"additional","affiliation":[{"name":"Centre for Assisted Reproduction, Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University , Shanghai 200092,","place":["China"]},{"name":"Department of Reproductive Endocrinology, Women\u2019s Hospital, Zhejiang University School of Medicine , Hangzhou,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yunlong","family":"Bai","sequence":"additional","affiliation":[{"name":"Department of Bioinformatics, Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University , Fuzhou, 350122,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mingming","family":"Ye","sequence":"additional","affiliation":[{"name":"Centre for Assisted Reproduction, Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University , Shanghai 200092,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanping","family":"Jia","sequence":"additional","affiliation":[{"name":"Centre for Assisted Reproduction, Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University , Shanghai 200092,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinyu","family":"Chen","sequence":"additional","affiliation":[{"name":"The Second Affiliated School of Zhejiang Chinese Medical University , Hangzhou,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Orhan","family":"Bukulmez","sequence":"additional","affiliation":[{"name":"Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center , Dallas, TX,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7233-6177","authenticated-orcid":false,"given":"Li","family":"Meng","sequence":"additional","affiliation":[{"name":"IVF Laboratories, California IVF Fertility Center Sacramento , Sacramento, CA,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"William Shu Biu","family":"Yeung","sequence":"additional","affiliation":[{"name":"Shenzhen Key Laboratory of Fertility Regulation, Center of Assisted Reproduction and Embryology, University of Hong Kong\u2014Shenzhen Hospital , Shenzhen,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert J","family":"Norman","sequence":"additional","affiliation":[{"name":"Robinson Research Institute, School of Paediatrics and Reproductive Health, The University of Adelaide , Adelaide, SA,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5974-9900","authenticated-orcid":false,"given":"Wenqiang","family":"Liu","sequence":"additional","affiliation":[{"name":"Centre for Assisted Reproduction, Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University , Shanghai 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