Treatment of human oocytes with extracellular vesicles from follicular fluid during rescue in vitro maturation enhances maturation rates and modulates oocyte proteome and ultrastructure
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2026
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STUDY QUESTION: Could follicular fluid-derived extracellular vesicles (ffEVs) benefit human oocyte rescue in vitro matura tion (rIVM)? SUMMARY ANSWER: Supplementation of rIVM culture with ffEVs isolated from mature follicles enhanced oocyte maturation rates by >20%, inducing changes in oocyte protein profile and organelle distribution. WHAT IS KNOWN ALREADY: IVM involves the culture of immature germinal vesicle (GV) oocytes under set laboratory conditions to allow for their transition to mature metaphase II (MII) stage, which is confirmed by the extrusion of the first polar body. Efficient IVM could circumvent controlled ovarian stimulation (COS), reduce the cost and broaden the repertoire of infertility treatments. Animal studies suggest that extracellular vesicles (EVs), membranous nanosized vesicles containing different molecular content (e.g. nucleic acids, proteins) and present in the ovarian follicular fluid, could enhance oocyte maturation. The uptake of ffEVs by bovine, equine, and feline oocytes, but not human, has been demonstrated. STUDY DESIGN, SIZE, DURATION: Women undergoing transvaginal oocyte retrieval after COS (n¼83) were recruited to donate fol licular fluid (n¼54 single follicles) and/or immature GV oocytes (n¼95). We aimed to: (i) define differences in the protein cargo of ffEVs derived from human follicles containing mature (MII-ffEVs, n¼10) versus immature (GV-ffEVs, n¼5; metaphase I MI-ffEVs, n¼5) oocytes, (ii) demonstrate the capacity of human GV oocytes to uptake MII-ffEVs and (iii) determine the effect of MII-ffEVs sup plementation on oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: ffEVs were isolated by ultracentrifugation. The protein content of ffEVs was ana lysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n¼15) was assessed by confocal micros copy. GVs were cultured for rIVM in a timelapse incubator with MII-ffEVs (n¼45 GVs) or without (n¼40 GVs), and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell pro teomics and the appearance of intracellular organelles upon transmission electron microscopy (TEM) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 1340 proteins in ffEVs, with proteins such as F12, IGKV1-39, FREM2, and C1QC being significantly enriched in MII-ffEVs. GV oocytes internalized MII-ffEVs, and their supplementation for 48h increased the oocyte maturation rate compared to control by 22.8±9.4% (77.8% vs 55% maturation rate respectively; P-value¼0.0372). Proteomic analysis of ffEV-supplemented mature oocytes (n¼5) revealed 56 differentially abundant proteins (DAPs) compared to non- supplemented mature oocytes (n¼5). Among them, 37 DAPs were in higher abundance in ffEVs-supplemented mature oocytes, in cluding Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation. Electron microscopy showed differences in oocyte organelle distribution and appearance, particularly that of endoplasmic reticulum (ER) and ER–mitochondria complexes. Functional enrichment analysis of differentially abundant proteins during ffEV–oocyte interaction revealed regulation of endoplasmic reticu lum, steroid biosynthesis, and keratin organization pathways. LARGE-SCALE DATA: Proteomics data are available via ProteomeXchange with identifier PXD073018. LIMITATIONS, REASONS FOR CAUTION: This study utilized immature oocytes from COS cycles; therefore, the results should be interpreted within the context of rIVM potential. The employed oocytes were vitrified and warmed, and the rIVM was performed for 48h.
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2026 (2)
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Oxford University Press
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Follicular fluid; Extracellular vesicles; In vitro maturation; Human oocyte; Single-cell proteomic analysis
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