Hum Reprod. 2026 Aug 20. pii: deag134. [Epub ahead of print]
Emily R Frost,
Dulama Richani,
Anne Poljak,
Ananya Vuyyuru,
Xiuhua Liao,
Elise Georgiou,
J M Binuri Gunasekara,
Bettina P Mihalas,
Irene E Sucquart,
Kaushiki Kadam,
Lindsay E Wu,
Robert B Gilchrist.
STUDY QUESTION: Which proteins underpin oocyte developmental competence, as modelled by oocytes of variable competence matured in vivo or matured in vitro under different conditions (capacitation in vitro maturation (CAPA-IVM) or standard IVM)?
SUMMARY ANSWER: Significant differences in the global proteome were observed in both oocytes and their corresponding cumulus cells depending on the mode of oocyte maturation, with key variations in eukaryotic translation, autophagy, and endocytosis pathways within oocytes, and changes in reactive oxygen species detoxification and serine biosynthesis in cumulus cells.
WHAT IS KNOWN ALREADY: Within the ovarian follicle, mammalian oocytes must acquire the necessary molecular machinery to support successful fertilization and embryonic development. Close contact with the surrounding cumulus cells ensures coordinated nuclear and cytoplasmic maturation of the oocyte, along with the accumulation of proteins stored within the oocyte in cytoplasmic lattices and endo-lysosomal vesicular assemblies.
STUDY DESIGN, SIZE, DURATION: This basic science study utilized a mouse model to assess proteomic changes across three oocyte competence models. Key proteins identified in mouse oocytes were also assessed in discarded immature human germinal vesicle (GV) oocytes and MII oocytes following rescue-IVM. Three oocyte maturation methods were tested: (i) in vivo maturation, (ii) CAPA-IVM, and (iii) standard IVM. In vivo maturation served as a positive control group, whereby metaphase II (MII) mature oocytes were collected from mice stimulated with pregnant mare serum gonadotropin (PMSG) and triggered with hCG, simulating full ovarian stimulation. For the IVM groups, immature cumulus-oocyte complexes (COCs) were collected from mildly stimulated (23 h PMSG) mice. For the standard IVM group, immature COCs were matured in media containing amphiregulin and epiregulin for 18 h. For the CAPA group, COCs were held for 24 h in pre-IVM conditions in the presence of c-type natriuretic peptide (CNP), oestradiol, insulin, and FSH, and then matured via IVM in media containing FSH, amphiregulin, and epiregulin. Four biological replicates were performed for mouse proteomics experiments; three biological replicates were performed for mouse immunocytochemistry experiments; and six replicates were performed for embryology experiments.
PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Four- to six-week-old C57BL/6JAusb mice were used for all mouse experiments. Embryology outcomes were used to confirm the variation in oocyte developmental competence between the three maturation groups. For the in vivo, CAPA and IVM groups, mature MII COCs were collected and separated into oocytes and cumulus cells. Oocytes and cumulus cells were subjected to mass spectrometry and bioinformatic analysis was performed using Proteome Discoverer and Ingenuity Pathway Analysis, with data validated by immunofluorescence. To assess conservation of proteins in human oocytes, 49 oocytes were collected from 36 patients following ART cycles and subject to immunofluorescence. Rescue-IVM was also performed with half of the human oocyte cohort to obtain MII oocytes.
MAIN RESULTS AND THE ROLE OF CHANCE: Proteomic profiling (at least two peptides per protein) identified around 1600 proteins in mouse oocytes and 3100 proteins in mouse cumulus cells across all three treatment groups. Differential expression analysis and pattern analysis collectively revealed a signature of proteins that were consistently differentially expressed between in vivo and in vitro oocyte maturation systems (log2FC of ± 1 and a P-value ≤ 0.05). These subsets of proteins were mapped to biological processes including eukaryotic translation, autophagy, and endocytosis pathways within oocytes. Orthogonal validation of clathrin, ribosomal protein L24, and eukaryotic initiation factor 2A supported the proteomic findings, and their expression was conserved in human GV and MII oocytes. Changes in reactive oxygen species detoxification and serine biosynthesis were observed in mouse cumulus cells, with fluorescence intensity changes in ferredoxin-1 and phosphoglycerate dehydrogenase supporting the dysregulation of cumulus cell processes during IVM.
LARGE SCALE DATA: The mass spectrometry data are available via ProteomeXchange with identifier PXD073269.
LIMITATIONS, REASONS FOR CAUTION: The foundational mechanisms of oocyte developmental competence remain elusive, particularly in humans where MII oocytes are heterogenous in quality within the same stimulation cycle and patient. Limitations in this study include the detection and proteome coverage of poorly expressed proteins, the inability to check molecular conservation in human CAPA-IVM and IVM oocytes, the use of fetuin in the mouse in vitro systems, and the potential source of keratin proteins in oocyte samples, and these factors must all be carefully considered. C57Bl6/J mice were used as the model species, allowing precise control over differing models of oocyte quality and capacity to analyse large numbers of oocytes. However, care is required when interpreting the significance of these findings in mice to mechanisms regulating human oocyte quality. Nonetheless, the in vivo stimulation and both IVM protocols used in this study are clinically relevant and developmentally matched. This study has also not addressed oocyte developmental competence in gonadotropin-free IVM oocytes, which is now a clinical reality.
WIDER IMPLICATIONS OF THE FINDINGS: This study adds to the wider literature indicating that oocytes matured in vitro, either through CAPA-IVM or IVM, are unable to achieve the developmental competence rates observed with in vivo stimulation. Through examination of the global proteome in oocytes, molecular pathways including eukaryotic translation, autophagy, and endocytosis were dysregulated in in vitro oocytes. Recent findings have revealed the critical role of these pathways to developmental competence in the context of in vivo development. In cumulus cells, changes in reactive oxygen species detoxification and serine biosynthesis were observed, adding to the extensive knowledge around metabolic activity in cumulus cells as a critical facet of oocyte quality. Combined, these data suggest that the necessary processes of protein storage and degradation in oocytes and metabolism in cumulus cells constitute important components of oocyte quality. These processes appear suboptimal in current IVM systems, providing a future research direction to optimize IVM protocols with consideration to these protein pathways.
FUNDING: This study was funded by a National Health and Medical Research Council Investigator Fellowship (APP1023210) awarded to R.B.G. and by a gift from Open Philanthropy.
DISCLOSURES: R.B.G. is a consultant to Dioseve Inc. L.E.W. is a co-founder, shareholder, director, and advisor of Jumpstart Fertility Inc. L.E.W. is also an advisor and shareholder in EdenRoc Sciences, the parent company of Metro Biotech NSW and Metro Biotech, and in Life Biosciences LLC and its daughter companies. His UNSW Industry Scientia position is partly funded by Proto Axiom. All other authors have no competing interests to disclose.
Keywords: assisted reproductive technologies; capacitation-IVM (CAPA-IVM); cumulus cells; developmental competence; folliculogenesis; in vitro maturation (IVM); mass spectrometry; proteomics