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Proteins were reduced and alkylated by adding Tris(2-carboxyethyl)phosphine and 2-chloroacetamide to a final concentration of 2 mM and 15 mM, respectively. Then, the samples were incubated for 30 min at 30°C (700 rpm, and light-protected). After dilution with pure ethanol to reach a final concentration of 60% EtOH (v/v), the KingFisher Flex System (Thermo Fisher Scientific, Waltham, USA) was used to bind the proteins to the carboxylated magnetic beads (hydrophobic and hydrophilic) and wash them. The enzymatic digestion was performed by adding trypsin in 50 mM triethylammonium bicarbonate buffer (TEAB) and incubating the samples overnight at 37°C. The remaining peptides were extracted from beads with water, and the two elutions were combined and dried down. The digested samples were dissolved in aqueous 3% acetonitrile with 0.1% formic acid, and the peptide concentration was estimated with the Lunatic UV/Vis absorbance spectrometer (Unchained Lab).  Single-oocytes: The lysis buffer, consisting of 20 μl of 50 mM triethylammonium bicarbonate (TEAB) and 0.1% DDM (n-dodecyl-β-D-maltoside), was directly added to the oocytes, which were stored in 0.2 ml tubes. Samples were then snap-frozen in liquid nitrogen and heated at 95°C for 10 min. This freeze-heat cycle was repeated 3 times. Proteins were reduced and alkylated by adding Tris(2-carboxyethyl) phosphine and chloroacetamid to a final concentration of 5 mM and 15 mM, respectively. The samples were incubated for 30 min at 30 °C (700 rpm and light-protected). Then, digestion was done in buffered trypsin solution at pH 8 (10 mM Tris/2 mM CaCl2). Samples were enzymatically digested using 2 µl of 4 µg/µl trypsin overnight plus an additional incubation for 3 h the next day with 2 µl of 4 µg/µl Trypsin. Digestion was stopped by acidifying the samples. Peptide concentration was estimated using the Lunatic UV/Vis absorbance spectrometer (Unchained Lab). Consequently, samples were loaded on Evotip (Evosep) following the manufacturer’s instructions. Samples were analysed on an Evosep One LC coupled to a TIMS TOF Pro mass spectrometer (Bruker), acquiring the data in diaPASEF scans."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["ffEVs: The acquired data were processed using the Fragpipe v17 (Kong et al., 2017). The spectra were searched against the Homo sapiens (SwissProt) protein background database, using Acetyl (Protein N-term) and Oxidation (M) as variable modifications and Carbamidomethyl (C) as fixed modifications. The protein identification results were imported for further analysis in the software Scaffold v5.2.0 (Proteome Software Inc., Portland, USA). Single-oocytes: The protein identification and quantification were performed using FragPipe (Demichev et al., 2022). To filter and normalise the data, a set of functions implemented in the R package [prolfqua] was used (Wolski et al., 2023). The resulting protein matrix was filtered to consider proteins with a minimum of 2 peptides/protein."],"omics_type":["Proteomics"],"labhead":["Prof. Dr. Susanne Ulbrich"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["ETH Zürich, Department of Environmental Systems Science"],"species":["Homo Sapiens (human)"],"submitter_mail":["mara.saenz@usys.ethz.ch"],"publication":["41924634 Makieva S, Saenz-de-Juano MD, Almiñana C, Bauersachs S, Bernal-Ulloa S, Xie M, Velasco AG, Cervantes N, Sachs M, Ulbrich SE, Leeners B. Treatment of human oocytes with extracellular vesicles from follicular fluid during rescue &lt;i&gt;in vitro&lt;/i&gt; maturation enhances maturation rates and modulates oocyte proteome and ultrastructure. Hum Reprod Open. 2026 2026(2):hoag021 10.1093/hropen/hoag021"],"submitter_affiliation":["ETH Zurich"],"submitter_country":["Switzerland"],"pubmed_abstract":["<h4>Study question</h4>Could follicular fluid-derived extracellular vesicles (ffEVs) benefit human oocyte rescue <i>in vitro</i> maturation (rIVM)?<h4>Summary answer</h4>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.<h4>What is known already</h4>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.<h4>Study design size duration</h4>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.<h4>Participants/materials setting methods</h4>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.<h4>Main results and the role of chance</h4>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; <i>P</i>-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.<h4>Large-scale data</h4>Proteomics data are available via ProteomeXchange with identifier PXD073018.<h4>Limitations reasons for caution</h4>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.<h4>Wider implications of the findings</h4>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.<h4>Study funding/competing interests</h4>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."],"pubmed_title":["Treatment of human oocytes with extracellular vesicles from follicular fluid during rescue &lt;i&gt;in vitro&lt;/i&gt; maturation enhances maturation rates and modulates oocyte proteome and ultrastructure."],"pubmed_authors":["Makieva Sofia S, Saenz-de-Juano Mara D MD, Almiñana Carmen C, Bauersachs Stefan S, Bernal-Ulloa Sandra S, Xie Min M, Velasco Ana G AG, Cervantes Natalia N, Sachs Maike M, Ulbrich Susanne E SE, Leeners Brigitte B"],"additional_accession":[]},"is_claimable":false,"name":"Treatment of human oocytes with extracellular vesicles from follicular fluid during rescue in vitro maturation enhances maturation rates and modulates oocyte proteome and ultrastructure","description":"This project aimed to elucidate whether human follicular fluid–derived extracellular vesicles (ffEVs) enhance oocyte maturation during rescue in vitro maturation (rIVM). We characterised the protein cargo of ffEVs isolated from follicles containing oocytes at different maturation stages (GV, GVBD, and MII), identifying distinct proteomic signatures associated with follicular competence and oocyte maturity. Subsequently, single-cell proteomic profiling of IVM-matured oocytes supplemented with ffEVs demonstrated that ffEV treatment induces significant changes in oocyte protein abundance, including factors linked to oocyte maturation, endoplasmic reticulum function, steroid biosynthesis, and cytoskeletal organisation. These proteomic alterations were accompanied by changes in oocyte ultrastructure and organelle architecture.","dates":{"publication":"2026-04-06","submission":"2026-01-13"},"accession":"PXD073018","cross_references":{"TAXONOMY":["NEWT:1773","NEWT:6945","NEWT:3555","NEWT:38783","NEWT:8727","NEWT:1182590","NEWT:8726","NEWT:2","NEWT:157546","NEWT:10090","NEWT:935293","NEWT:749200","NEWT:35554","NEWT:4120","NEWT:5693","NEWT:9417","NEWT:347515","NEWT:8724","NEWT:51511","NEWT:1216979","NEWT:307972","NEWT:92867","NEWT:8723","NEWT:990346","NEWT:544496","NEWT:5334","NEWT:145953","NEWT:257309","NEWT:5180","NEWT:284812","NEWT:115104","NCBITaxon:1313","NEWT:1081927","NEWT:43330","NEWT:67825","NEWT:44544","NEWT:13076","NEWT:1249668","NEWT:373995","NEWT:544404","NEWT:3702","NEWT:8839","NEWT:317","NEWT:4232","NEWT:990119","NEWT:1736309","NEWT:4113","NEWT:7227","NEWT:11298","NEWT:7469","NEWT:885318","NEWT:171101","NEWT:4081","NEWT:876138","NEWT:554","NEWT:5691","NEWT:98334","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:627025","NEWT:400772","NEWT:3708","NEWT:106592","NEWT:237561","NEWT:9913","NEWT:10036","NEWT:4100","NEWT:7574","NEWT:1351","NEWT:1076","NEWT:6763","NEWT:7215","NEWT:803","NEWT:8030","NEWT:380394","NEWT:272563","NEWT:507601","NEWT:1639","NEWT:188229","NEWT:4909","NCBITaxon:79857","NEWT:95648","NEWT:746360","NEWT:6239","NEWT:1589","NEWT:135588","NEWT:470150","NEWT:135622","NEWT:216257","NEWT:6915","NEWT:9986","NEWT:101510","NEWT:95486","NEWT:3880","NEWT:58002","NEWT:9103","NEWT:4577","NEWT:5664","NEWT:2157","NEWT:146479","NEWT:1911079","NEWT:1000589","NEWT:145943","NEWT:1902","NEWT:85962","NEWT:160488","NEWT:317447","NEWT:3635","NEWT:7955","NCBITaxon:2","NEWT:235443","NEWT:1480154","NEWT:985076","NEWT:7959","NEWT:2261","NEWT:3197","NEWT:9615","NEWT:884019","NEWT:4565","NEWT:1264690","NEWT:169963","NCBITaxon:38727","NEWT:36329","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:9606","NEWT:367830","NEWT:157295","NEWT:243230","NEWT:931281","NEWT:373153","NEWT:7029","NEWT:915099","NEWT:1283300","NEWT:334747","NEWT:470","NCBITaxon:79824","NCBITaxon:4563","NEWT:3218","NEWT:84023","NEWT:5759","NEWT:9838","NCBITaxon:9615","NEWT:1736231","NEWT:1193501","NEWT:3055","NEWT:6287","NEWT:2242","NEWT:6326","NEWT:9796","NEWT:2762","NEWT:5476","NEWT:725","NEWT:1174673","NEWT:562","NEWT:260707","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1836","NEWT:1735272","NEWT:29760","NEWT:260705","NEWT:80863","NEWT:1148","NEWT:4932","NEWT:70448","NEWT:9825","NEWT:3603","NEWT:698936","NEWT:2759","NEWT:39946","NEWT:11676","NEWT:9823","NEWT:100226","NCBITaxon:6073","NEWT:4530","NEWT:4896","NEWT:6279","NEWT:7370","NEWT:573","NEWT:6282","NEWT:7091","NEWT:1134506"],"pubmed":["41924634"],"ORCID":["0000-0002-2860-2252"]}}