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Abstract Study question Could follicular fluid derived extracellular vesicles (ffEVs) impact human oocyte in vitro maturation (IVM)? Summary answer Supplementation of IVM 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 aggressive 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 facilitate 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 (n = 83) at University Hospital Zurich donated follicular fluid (n = 54 single follicles) and/or immature GV oocytes (n = 95). We aimed to: a) define differences in the protein cargo of ffEVs derived from follicles containing mature oocytes (MII-ffEVs, n = 10) versus (vs.) immature oocytes (GV-ffEVS, n = 5; metaphase I MI-ffEVs, n = 5), b) demonstrate the capacity of GV oocytes to uptake mature MII-ffEVs and c) determine the effect of MII-ffEVs supplementation on oocyte maturation. Participants/materials, setting, methods ffEVs were isolated by ultracentrifugation. The protein content of ffEVs was analysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n = 15) was assessed by confocal microscopy. GVs were cultured for rescue-IVM in a timelapse incubator with MII-ffEVs (n = 45 GVs) or without (n = 40 GVs) and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell proteomics and intracellular organelles appearance with transmission electron microscopy (TEM). Main results and the role of chance Successful isolation of ffEVs was confirmed by TEM and Western blotting analysis for characteristic protein markers of EVs (TSG101 and CD63). Mass spectrometry identified a total of 1340 proteins across ffEVs samples. Statistical analysis revealed differentially abundant proteins (DAPs) in ffEVs: 61 DAPs in the comparison MII-ffEVs vs. GV-ffEVs, 15 DAPs in MII-ffEVs vs. MI-ffEVs and 14 DAPs in MI-ffEVs vs GV-ffEVs (FDR0.2). Among the 61 DAPs, 7 proteins in higher abundance in MII-ffEVs (log2 fold change) were related to oocyte maturation, lipid accumulation, calcium binding and the coagulation cascade (e.g. FABP5, S100A9, F12, SERPINA10). Confocal microscopy confirmed the uptake of MII-ffEVs by GV oocytes after 24 hours of co-incubation. Supplementation of IVM culture with MII-ffEVs for 48 hours significantly increased the oocyte maturation rate compared to control by 22.8±9.4% (77.8% vs 55% mature oocytes respectively; P-value=0.0372). Single oocyte proteomics of IVM-matured oocytes revealed 59 DAPs (FDR 0.1) between ffEVs-supplemented and control groups. Among them, 37 DAPs were in higher abundance in ffEVs-supplemented mature oocytes (log2 FC) including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). TEM revealed differences in oocyte organelle distribution and appearance, particularly of endoplasmic reticulum (RE) and RE-mitochondria complexes. Limitations, reasons for caution This study utilised immature oocytes from stimulated cycles, therefore these results should be interpreted within the context of rescue-IVM potential. Wider implications of the findings Our findings pave the way for further mechanistic studies on the role of ffEVs in supporting oocyte maturation and inspire the development of clinical IVM supplements as well as novel complementary regimes for COS based on ffEVs or their associated specific cargo. Trial registration number N/A
Makieva et al. (Mon,) studied this question.