L26/O-313 Single-oocyte transcriptomics reveals redox- and mitochondria-related constraints during human rescue in vitro maturation and provides evidence for a modulating effect of antioxidant supplementation

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Abstract Study question What transcriptomic differences distinguish rescue in vitro–matured (r-IVM) human oocytes from those matured in vivo, and can antioxidant supplementation modulate the r-IVM molecular profile? Summary answer Rescue in vitro–matured oocytes retain meiotic transcriptional programmes but show redox and mitochondrial alterations, with antioxidant supplementation partially shifting the MII transcriptome towards in vivo. What is known already The possibility of maturing oocytes retrieved at immature stages could increase the number of usable gametes in IVF. However, despite longstanding efforts to optimise r-IVM, clinical outcomes remain suboptimal, reflecting the complexity of oocyte maturation. One explanation is that maturation involves not only meiotic progression but also profound cytoplasmic and transcriptomic changes that require tightly regulated physiological conditions. When these conditions are only partially reproduced, key cytoplasmic processes may be compromised. Oxidative stress and mitochondrial dysfunction have been proposed as potential contributors to poor outcomes, yet their molecular impact in human r-IVM oocytes remains incompletely characterised and warrants further investigation. Study design, size, duration This exploratory prospective study analysed 48 individual human oocytes at different stages of maturation from 16 young oocyte donors (25.1±3.1 years). Initially retrieved GV oocytes underwent r-IVM under control or antioxidant-supplemented conditions. Those reaching the MII stage (n = 11 per condition) were compared with in vivo–matured MII oocytes (n = 10). Oocytes that failed to mature and arrested at the GV (n = 8) or MI (n = 8) stage were also included in the comparative analysis. Participants/materials, setting, methods The study was conducted at a university-affiliated IVF centre and included oocytes donated for research. Oocytes undergoing rescue in vitro maturation under antioxidant-supplemented conditions were cultured with a defined cocktail comprising resveratrol (1.0µmol/L), retinoic acid (2.0µmol/L), melatonin (10µmol/L) and L-coenzyme Q10 (50µmol/L). Single-oocyte RNA sequencing was performed using a low-input SMART-Seq–based protocol. Differential gene expression analyses were conducted across the GV–MI–MII transition, with genes defined by log2 fold-change and Benjamini–Hochberg adjusted false discovery rate criteria. Main results and the role of chance Comparison of r-IVM MII with GV oocytes identified 2,788 differentially expressed genes (DEGs) under control conditions and 2,549 under antioxidant-supplemented conditions, indicating extensive transcriptomic reprogramming. In line with expectations, comparisons between r-IVM MII and MI oocytes identified fewer DEGs, with 253 under control conditions and 46 under antioxidant-supplemented conditions, consistent with a more limited transcriptional transition between meiotic stages.Core transcriptional programmes governing meiotic progression were preserved in r-IVM MII oocytes under both culture conditions and were broadly similar to those observed in in vivo–matured MII oocytes. Despite this similarity, r-IVM MII oocytes displayed a limited but consistent set of transcriptional differences compared with in vivo–matured MII oocytes, comprising 28 DEGs under control conditions and 32 under antioxidant-supplemented conditions. These differences predominantly involved genes related to oxidative stress regulation, mitochondrial function and extracellular matrix signalling, suggesting persistent cytoplasmic alterations rather than disruption of nuclear maturation. Antioxidant supplementation modulated 12 DEGs relative to control r-IVM oocytes, including downregulation of MAOB, and partially restored expression of genes such as LUM and PLN towards levels observed in in vivo–matured MII oocytes. Functional enrichment analysis identified antioxidant-specific transcriptomic changes affecting pathways related to detoxification, reactive oxygen species defence, MAPK signalling and ATP-dependent processes (Benjamini–Hochberg adjusted P < 0.05). Limitations, reasons for caution The study is limited by the sample size, which represents a common challenge in single-cell transcriptomic research due to the high per-sample cost of analysis and the limited availability of potentially viable oocytes donated for research. In addition, transcriptomic profiles cannot fully capture post-transcriptional regulation or functional competence. Wider implications of the findings To our knowledge, this is the first single-oocyte transcriptomic study assessing combined antioxidant supplementation during human rescue in vitro maturation. These findings indicate reduced r-IVM performance reflects cytoplasmic, not nuclear, defects and identify redox balance as a modifiable target to optimise protocols and improve clinical potential of otherwise discarded oocytes Trial registration number No

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