Treatment of human oocytes with extracellular vesicles from follicular fluid during rescue in vitro maturation enhances maturation rates and modulates oocyte proteome and ultrastructure

Abstract Study question Could follicular fluid-derived extracellular vesicles (ffEVs) benefit human oocyte rescue in vitro maturation (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 already known 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 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 (n=83) were recruited to donate 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 human follicles containing mature (MII-ffEVs, n=10) versus immature (GV-ffEVs, n=5; metaphase I MI-ffEVs, n=5) oocytes, b) demonstrate the capacity of human GV oocytes to uptake 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 rIVM 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 on transmission electron microscopy (TEM). 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 internalised MII-ffEVs, and their supplementation for 48 hours increased the oocyte maturation rate compared to control by 22.8±9.4% (77.8% vs 55% maturation rate respectively; p-value=0.0372). Proteomic analysis of ffEV-supplemented mature oocytes (n=6) revealed 56 differentially abundant proteins (DAPs) compared to not supplemented mature oocytes (n=5). Among them, 37 DAPs were in higher abundance in ffEVs- supplemented mature oocytes including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). Electron microscopy showed differences in oocyte organelle distribution and appearance, particularly that of endoplasmic reticulum (RE) and RE-mitochondria complexes. Functional enrichment analysis of differentially abundant proteins during ffEV-oocyte interaction revealed regulation of endoplasmic reticulum, steroid biosynthesis, and keratin organisation pathways. Large scale data N/A Limitations, reasons for caution This study utilised immature oocytes from COS cycles, therefore the results should be interpreted within the context of rIVM potential. Winder implications of the findings These results provide new insights into the role of ffEVs in enhancing oocyte maturation, offering potential improvements for clinical rIVM protocols and inspire the development of global IVM supplements based on ffEVs or associated specific cargo. Study funding/competing interest(s) This work was funded by an EMDO research fellowship and a FAN research grant (Fonds zur Förderung des akademischen Nachwuchses) from the University of Zurich. What does it mean for the patients Infertility rates are rising, with 17% of couples worldwide needing help to get pregnant, often through treatments like in vitro fertilisation (IVF). IVF usually involves using hormones to stimulate the ovaries to produce multiple eggs, which can be tough on a woman’s health, both physically and emotionally, and can be very expensive. In vitro oocyte maturation (IVM) is a gentler alternative, where eggs are matured outside the body, reducing risks and costs. However, IVM isn’t as effective as IVF yet, mainly because the current methods are not perfect. Our research is exploring a new approach to improve IVM by adding extracellular vesicles from follicular fluid to the egg culture. This could help the eggs mature better, leading to higher success rates and giving more options to couples struggling with infertility.