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

## TL;DR

Adding extracellular vesicles from follicular fluid to immature human oocyte cultures improves maturation rates and changes oocyte proteins and structure.

## Contribution

Demonstrates that follicular fluid-derived extracellular vesicles can enhance human oocyte maturation in vitro and alter oocyte proteome and ultrastructure.

## Key findings

- Supplementing rIVM culture with follicular fluid-derived extracellular vesicles increased oocyte maturation rates by 22.8%.
- ffEVs were internalized by GV oocytes and altered the abundance of 56 proteins, including Hyaluronan Synthase 1.
- Electron microscopy revealed changes in oocyte organelle distribution, especially endoplasmic reticulum and mitochondria complexes.

## Abstract

Could follicular fluid-derived extracellular vesicles (ffEVs) benefit human oocyte rescue in vitro maturation (rIVM)?

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.

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.

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: (i) 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, (ii) demonstrate the capacity of human GV oocytes to uptake MII-ffEVs and (iii) determine the effect of MII-ffEVs supplementation on oocyte maturation.

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 the appearance of intracellular organelles upon transmission electron microscopy (TEM) analysis.

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 48 h 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 = 5) revealed 56 differentially abundant proteins (DAPs) compared to non-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. 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 reticulum, steroid biosynthesis, and keratin organization pathways.

Proteomics data are available via ProteomeXchange with identifier PXD073018.

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 48 h.

These results provide new insights into the role of ffEVs in enhancing oocyte maturation, offering potential improvements for clinical rIVM protocols, and inspiring the development of global IVM supplements based on ffEVs or associated specific cargo.

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. The authors declare no competing interests.