Extracellular vesicles and their content in the context of polycystic ovarian syndrome and endometriosis: a review

It is well established that EVs released from the endometrium of patients with endometriosis are different compared to control patients without endometriosis [ 153 – 156 ]. The prevailing hypothesis suggests that EVs potentiate the migration and implantation of endometrial cells during retrograde menstruation with a distinct immune contribution, leading to inhibited clearing of invading endometrial cells, in a similar fashion to cancer cells EVs [ 49 , 106 ]. Consequently, endometriosis lesions can affect multiple organs and EVs secreted from them can be found in several biological fluids like FF, peritoneal fluid, uterine cavity fluids, and serum (Table 2 ). Table 2 Potential biomarkers found in extracellular vesicles (exosome-like vesicles) of different biological origins from patients with different stages of endometriosis Types of molecule Potential biomarker(s) Biological origin Comparision EV isolation method EV characterization techniques Detection technique Sample size Endometriosis stage Total number differentially expressed Reference Year of publication Complete reference miRNAs ↑ miR-342-5p, miR-130b-3p, miR-210-3p ↓ miR-132-5p, miR-335-3p Uterin cavity fluid Endo vs Control Exosome isolation kit (Echobiotech) TEM, NTA, WB RNASeq 4 endo, 4 ctrl All patients III-IV 7 upregulated 2 downregulated PMID: 36,551,866 2022 Jiang Y, Chai X, Chen S, Chen Z, Tian H, Liu M, Wu X. Exosomes from the Uterine Cavity Mediate Immune Dysregulation via Inhibiting the JNK Signal Pathway in Endometriosis. Biomedicines. 2022 Dec 2;10(12):3110. https://doi.org/10.3390/biomedicines10123110 . PMID: 36,551,866; PMCID: PMC9775046 miRNAs ↑ miR-6795-3p, miR-146b-3p, miR-32-3p ↓ miR-128–1-5p, miR-215-5p, miR-26b-5p Serum Endo vs Control Exosome binding enhancer and magnetic beads TEM, WB Microarray 4 endo, 4 ctrl 1 patients I-II, 3 patients III-IV 26 upregulated 19 downregulated PMID: 34,542,679 2022 Wu Y, Yuan W, Ding H, Wu X. Serum exosomal miRNA from endometriosis patients correlates with disease severity. Arch Gynecol Obstet. 2022 Jan;305(1):117–127. https://doi.org/10.1007/s00404-021-06227-z . Epub 2021 Sep 20. PMID: 34,542,679; PMCID: PMC8782809 proteins ↑ RAN, FTH1, UBB ↓ HEL70, MMP2, HEL-S-1 Endometrium and endometriotic lesions Endo vs Control Ultracentrifugation TEM, NTA, WB LS-MS/MS 6 endo, 10 ctrl Not reported 3 upregulated 6 downregulated PMID: 35,557,941 2022 Abudula M, Fan X, Zhang J, Li J, Zhou X, Chen Y. Ectopic Endometrial Cell-Derived Exosomal Moesin Induces Eutopic Endometrial Cell Migration, Enhances Angiogenesis and Cytosolic Inflammation in Lesions Contributes to Endometriosis Progression. Front Cell Dev Biol. 2022 Apr 26;10:824,075. https://doi.org/10.3389/fcell.2022.824075 . PMID: 35,557,941; PMCID: PMC9086167 circRNAs, miRNAs and mRNAs qRT-PCR: ↑ circ_002612309 (ectopic vs ctrl/eutopic) ↓ miR-15a-5p (eutopic/ectopic vs ctrl) ↑ ATP6V1A (eutopic/ectopic vs ctrl) Endometrium and endometriotic lesions Overlapping DEGs between Ectopic vs Ctrl, Eutopic vs Ctrl and Ectopic vs Eutopic ExoQuick-TC (System Biosciences) TEM, NTA, WB RNASeq 3 endo (3 eutopic, 3 ectopic samples), 3 ctrl All patients III-IV circRNAs: 2915 upregulated 640 downregulated miRNAs: 17 upregulated 9 downregulated mRNAs: 550 upregulated 136 downregulated PMID: 33,901,012 2021 Wu J, Fang X, Huang H, Huang W, Wang L, Xia X. Construction and topological analysis of an endometriosis-related exosomal circRNA-miRNA-mRNA regulatory network. Aging (Albany NY). 2021 Apr 26;13(9):12,607–12630. https://doi.org/10.18632/aging.202937 . Epub 2021 Apr 26. PMID: 33,901,012; PMCID: PMC8148458 miRNAs ↑ miR-615-3p, miR-6873-3p, miR-3195 ↓ miR-1273 h-3p, miR-4262, miR-1269a Eutopic endometrium from patients with endometriosis and normal endometrium Endometriotic lesions vs Ctrl ExoQuick-TC (System Biosciences) TEM, FC, NTA RNASeq 3 endo, 3 ctrl All patients II-IV 26 upregulated 23 downregulated PMID: 32,593,507 2020 Zhou W, Lian Y, Jiang J, Wang L, Ren L, Li Y, Yan X, Chen Q. Differential expression of microRNA in exosomes derived from endometrial stromal cells of women with endometriosis-associated infertility. Reprod Biomed Online. 2020 Aug;41(2):170–181. https://doi.org/10.1016/j.rbmo.2020.04.010 . Epub 2020 May 3. PMID: 32,593,507 miRNAs ↑ miR-197-5p, miR-22-3p, miR-320a ↓ miR-134-5p, miR-3141, miR-4499 Serum Endo vs Control Differential centrifugation TEM, NTA, WB Microarray 5 endo, 5 ctrl Not reported 18 upregulated 6 downregulated PMID: 32,076,458 2020 Zhang L, Li H, Yuan M, Li D, Sun C, Wang G. Serum Exosomal MicroRNAs as Potential Circulating Biomarkers for Endometriosis. Dis Markers. 2020 Jan 23;2020:2,456,340. https://doi.org/10.1155/2020/2456340 . PMID: 32,076,458; PMCID: PMC7008302 proteins PRDX1, H2A type 2-C, ANXA2, ITIH4 and tubulin a-chain Peritoneal fluid Endo I/II vs Endo III/IV vs Ctrl Exo-spin size-exclusion chromatography columns (Cell Guidance Systems) TEM, NTA, WB LC–MS/MS 16 endo I/II, 6 endo III/IV and 6 ctrl Patients split I/II and III/IV 5 proteins exclusively found in endo PMID: 32,106,990 2020 Nazri HM, Imran M, Fischer R, Heilig R, Manek S, Dragovic RA, Kessler BM, Zondervan KT, Tapmeier TT, Becker CM. Characterization of exosomes in peritoneal fluid of endometriosis patients. Fertil Steril. 2020 Feb;113(2):364–373.e2. https://doi.org/10.1016/j.fertnstert.2019.09.032 . PMID: 32,106,990; PMCID: PMC7057257 miRNAs ↑ miR-1908-5p, miR-130b-5p, miR-4488 ↓ miR-6508-3p, miR-145-5p, miR-365b-3p Peritoneal fluid Early endo, late endo vs Ctrl Ultracentrifugation None RNASeq 3 early stage endo, 3 advanced stage endo and 3 ctrl Patients I-IV early vs ctrl: 130 upregulated 43 downregulated late vs ctrl: 87 upregulated 63 downregulated PMID: 30,453,861 2019 Chen Y, Wang K, Xu Y, Guo P, Hong B, Cao Y, Wei Z, Xue R, Wang C, Jiang H. Alteration of Myeloid-Derived Suppressor Cells, Chronic Inflammatory Cytokines, and Exosomal miRNA Contribute to the Peritoneal Immune Disorder of Patients With Endometriosis. Reprod Sci. 2019 Aug;26(8):1130–1138. https://doi.org/10.1177/1933719118808923 . Epub 2018 Nov 19. PMID: 30,453,861 miRNAs ↑ miR-27a-3p ↓ miR-375, miR-30d-5p (endometrium, lesions and plasma) Endometrium, endometriotic lesions and plasma Eutopic vs Ectopic, Ectopic vs Ctrl and Eutopic vs Ctrl miRCURY exosome isolation kit (Qiagen) TEM, WB RNASeq 6 endometrium, 6 endometriotic lesions and 6 plasma All patients III-IV Plasma: 21 DE Ectopic/Eutopic samples: 14 DE PMID: 31,534,048 2019 Khalaj K, Miller JE, Lingegowda H, Fazleabas AT, Young SL, Lessey BA, Koti M, Tayade C. Extracellular vesicles from endometriosis patients are characterized by a unique miRNA-lncRNA signature. JCI Insight. 2019 Sep 19;4(18):e128846. https://doi.org/10.1172/jci.insight.128846 . PMID: 31,534,048; PMCID: PMC6795291 lncRNA ↑ circulating exosomal aHIF and exosomal ectopic endometrium Endometrium, endometriotic lesions and plasma Eutopic vs Ectopic, Ectopic vs Ctrl and Eutopic vs Ctrl Total Exosome Isolation kit (Invitrogen) for cell culture and ExoQuick exosome precipitation solution kit for serum TEM, WB qRT-PCR 30 endo and 16 ctrl All patients III-IV Single gene assessment PMID: 30,808,247 2019 Qiu JJ, Lin XJ, Zheng TT, Tang XY, Zhang Y, Hua KQ. The Exosomal Long Noncoding RNA aHIF is Upregulated in Serum From Patients With Endometriosis and Promotes Angiogenesis in Endometriosis. Reprod Sci. 2019 Dec;26(12):1590–1602. https://doi.org/10.1177/1933719119831775 . Epub 2019 Feb 26. PMID: 30,808,247 miRNAs qRT-PCR: ↑ miR-21-5p Endometrium and endometriotic lesions Endometriotic lesions vs Ctrl Total Exosome Isolation kit (Invitrogen) TEM, NTA Targeted qRT-PCR 5 endo (5 eutopic, 5 ectopic samples), 5 ctrl Not reported Non applicable PMID: 26,841,879 2016 Harp D, Driss A, Mehrabi S, Chowdhury I, Xu W, Liu D, Garcia-Barrio M, Taylor RN, Gold B, Jefferson S, Sidell N, Thompson W. Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro. Cell Tissue Res. 2016 Jul;365(1):187–96. https://doi.org/10.1007/s00441-016-2358-1 . Epub 2016 Feb 3. PMID: 26,841,879; PMCID: PMC4917586 Potential biomarkers found in extracellular vesicles (exosome-like vesicles) of different biological origins from patients with different stages of endometriosis ↑ miR-342-5p, miR-130b-3p, miR-210-3p ↓ miR-132-5p, miR-335-3p 7 upregulated 2 downregulated ↑ miR-6795-3p, miR-146b-3p, miR-32-3p ↓ miR-128–1-5p, miR-215-5p, miR-26b-5p 26 upregulated 19 downregulated ↑ RAN, FTH1, UBB ↓ HEL70, MMP2, HEL-S-1 3 upregulated 6 downregulated qRT-PCR: ↑ circ_002612309 (ectopic vs ctrl/eutopic) ↓ miR-15a-5p (eutopic/ectopic vs ctrl) ↑ ATP6V1A (eutopic/ectopic vs ctrl) circRNAs: 2915 upregulated 640 downregulated miRNAs: 17 upregulated 9 downregulated mRNAs: 550 upregulated 136 downregulated ↑ miR-615-3p, miR-6873-3p, miR-3195 ↓ miR-1273 h-3p, miR-4262, miR-1269a 26 upregulated 23 downregulated ↑ miR-197-5p, miR-22-3p, miR-320a ↓ miR-134-5p, miR-3141, miR-4499 18 upregulated 6 downregulated ↑ miR-1908-5p, miR-130b-5p, miR-4488 ↓ miR-6508-3p, miR-145-5p, miR-365b-3p early vs ctrl: 130 upregulated 43 downregulated late vs ctrl: 87 upregulated 63 downregulated ↑ miR-27a-3p ↓ miR-375, miR-30d-5p (endometrium, lesions and plasma) Plasma: 21 DE Ectopic/Eutopic samples: 14 DE qRT-PCR: ↑ miR-21-5p Research aimed at elucidating the secretion and molecular contents of EVs in endometriosis typically involves isolating primary endometrial stromal cells (ESCs) from both ectopic and eutopic endometrial tissue. These cells are then used to establish cultures and to collect EVs secreted from spent culture media. Several studies used RNAseq of the EV miRNA cargo and showed a different exosomal miRNA expression in endometriosis lesions compared to control biopsies [ 155 – 159 ]. Indeed, on top of the identified differentially expressed sncRNAs and mRNAs identified, Wu et al. established a regulatory network based on the expression of circRNAs, miRNAs and mRNAs. They then validated the expression of the key players identified, namely an up-regulation of circ_0026112309 and ATP6V1A , and a down-regulation of miR-15a-5p in samples from patients with endometriosis compared to controls [ 158 ]. Zhou et al. also investigated the miRNA content of exosomes isolated from moderate to severe lesions (stage III/IV), compared to controls using RNAseq. They identified 26 upregulated and 23 downregulated miRNAs [ 156 ]. There have also been reports profiling the differences between early-stage (stage I/II) and advanced-stage (stage III/IV) lesions compared to healthy endometrium and they identified a similar number of differentially expressed miRNAs in in these groups, compared to controls [ 160 ]. Other groups have also investigated specific miRNAs found in EVs secreted from endometriosis lesions, such as miR-21-5p, a pro-angiogenic miRNA [ 155 ], however, this miRNA alone is not specific to one pathology and is reported to be altered in various pathologies [ 161 , 162 ]. Different biological fluids have also been used to isolate EVs and investigated their content in the context of endometriosis. Indeed, Jiang et al. isolated EV miRNAs from uterine cavity fluid and identified 7 upregulated and 2 downregulated miRNAs cited in Table 2 [ 163 ]. In addition to sncRNAs, proteomic studies have been conducted on peritoneal fluid exosomes from patients with endometriosis and controls [ 164 ] and directly from endometriosis lesions [ 157 ]. In the peritoneal fluid, five proteins were exclusively found in EVs from patients with endometriosis: PRDX1, H2A type 2-C, ANXA2, ITIH4 and tubulin α-chain [ 164 ]. From endometriosis lesions, 3 upregulated (RAN, FTH1 and UBB) and 6 downregulated (top 3: HEL70, MMP2, HEL-S-1) proteins were identified [ 157 ]. Qui et al. specifically studied the lncRNA, aHIF, in circulating exosomes and those secreted from ectopic endometrium. Exosomal aHIF was found to be upregulated in patients with endometriosis [ 159 ]. Another study also investigated miRNAs and lncRNAs in endometriosis and showed an increased number of proteins associated with the immune system, metabolic processes, and coagulation pathways compared to healthy fertile patients; thus demonstrating the influence of this specific condition on EVs protein cargo [ 154 ]. Furthermore, EV content from plasma or serum of patients with endometriosis may also contribute to our understanding of its pathology. Using microarray, Wu et al. identified 26 upregulated and 19 downregulated miRNAs in the serum of patients with endometriosis compared to controls. Whereas, Zhang et al. identified 19 upregulated and 6 downregulated miRNAs. Functional studies on EVs from patients with endometriosis indicated a significant impact on other cells important in the physiopathology of the disease. Endometrial stromal cells (ESCs) and epithelial cells (ESC) from patients with and without endometriosis were isolated and cultured with human umbilical vein endothelial cells (HUVECs) to investigate the angiogenic potential of EVs isolated from endometriosis ESCs [ 155 ] or EECs [ 154 ]. Treatment of HUVECs with EVs isolated from endometriosis ESCs showed an increased ability to form branches and promote tube formation [ 155 ]. Moreover, Sun et al. showed that exosomes derived from endometriosis lesions can be internalized by both HUVECs and dorsal root ganglion (DRG) neurons; and they enhanced neuroangiogenic activities of these cells [ 165 ]. miR-130b was the only miRNA upregulated in two studies [ 160 , 163 ]. This miRNA has been reported to be important in human and bovine granulosa cell viability and proliferation [ 166 ]. They also showed that an inhibition of miR-130b expression during oocyte in vitro maturation led to reduced maturation rate and blastocyst formation [ 167 ]. However, miR-130 is implicated in a plethora of cellular mechanisms [ 168 – 171 ], therefore, the specificity and sensitivity of this potential marker would need to be assessed to consider it as a clinically useful biomarker of endometriosis. The role of inflammation in the physiopathology of endometriosis has been of great interest, not only with the goal of improving our understanding of the biological mechanisms underlying the pathology, but also for developing more targeted treatments. Studies compiled in Table 2 highlighted a strong inflammatory component in patients with endometriosis, compared to controls [ 154 , 160 , 172 ]. Indeed, EVs isolated from immortalized endometriotic epithelial cells showed increased expression of granulocyte colony-stimulating factor (G-CSF) and TNF-α, when cultured with endothelial cells [ 154 ]. Furthermore, it has been shown that exosomal miR-22-3p derived from peritoneal macrophages was able to increase proliferation, migration, and invasion of ectopic endometrial stromal cells through SIRT1/NF-кB signaling [ 172 ]. Moreover, macrophage polarization is modified through PI3K upregulation and PTEN downregulation when treated with lesion-derived exosomal miR-301a-3p [ 173 ]. Exosomes isolated from the uterine cavity exhibited potential mutual influence with immune cells on endometriosis lesions, suggesting a global immune dysregulation is involved in endometriosis pathophysiology [ 163 ]. EVs carry a variety of pro- and anti-inflammatory mediators that can contribute and actively participate in the disease [ 174 ]. Moreover, recent reviews have reported modulatory functions of EVs on immune cells, including lymphocyte T, Natural Killer (NK)-cells, dendritic cells, and macrophages [ 175 , 176 ]. Furthermore, EVs derived from endometriosis lesions are able to induce an increased expression of IL-1β, IL-18 and TNF-α cytokines, among others [ 157 ]. In addition to identifying miRNAs associated with inflammatory pathways, Chen et al. showed an increase in chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, monocyte chemoattractant protein 1 (MCP-1), MCP-3 and hepatocyte growth factor (HGF). They also showed an increase in monocytic myeloid-derived suppressor cells and T-reg cells in the peritoneal fluid of patients with endometriosis [ 160 ]. Altogether, these recent advancements improve our understanding of the pathophysiology of endometriosis and highlight the important contribution of inflammation to the disease. This opens the door to the development of potential EV therapies targeting inflammation to alleviate endometriosis symptoms and inhibit lesions growth. The diagnostic and prognostic potential of EVs has led to an increase in EV research in the past decade [ 42 ]. A recent review collected the research and advancements made in past years on the differences found in EV cargo between patients with and without endometriosis and their potential therapeutic effects [ 49 ]. As described, a great number of biomolecules have been shown to be differentially expressed in samples from patients with endometriosis, compared to control. However, only a few have the potential to become a clinical biomarker and/or to be used as part of a therapeutic strategy. In that sense, a recent review investigating the role of EV-miRNAs in endometriosis compiled 14 studies that identified differentially expressed miRNAs, highlighting the great potential of these molecules as biomarkers and therapies [ 153 ]. Of note, the study conducted by Khalaj et al. showed a unique miRNA-lncRNA signature in EVs, including exosomes, isolated from eutopic and ectopic endometriosis lesions as well as peripheral blood [ 154 ]. They identified 14 miRNAs differentially expressed between EVs isolated from ectopic endometriosis lesions and eutopic endometrium, compared with control endometrium from normal healthy fertile patients, and 21 miRNAs differentially expressed in plasma-derived EVs [ 154 ]. Three miRNAs were differentially expressed in both patient plasma- and tissue-derived EVs, making them potential diagnostic markers (miR-375, miR-27a-3p and miR-30d-5p). Pathway union analysis revealed that these miRNAs are associated with lysine degradation, hippo signaling pathway, protein processing in endoplasmic reticulum, and viral carcinogenesis [ 154 ]. Zhang et al. also showed that miR-223p and miR-320a-39 were elevated in serum-derived EVs from patients with endometriosis, compared to controls [ 172 ]. Wu et al. utilizing qRT-PCR, confirmed these sequencing results and demonstrated that miR-26b-5p, miR-215-5p and miR-6795-3p were differentially expressed in serum-derived EVs from patients with endometriosis compared to controls [ 177 ]. As for diagnostic purposes, only a subset of studies has reported on the sensitivity and specificity of EV biomarkers. These include: vascular endothelial growth factor C (VEGF-C) (sensitivity 81.3%/specificity 71.4%) [ 178 ], lncRNA RP3-399L15.2 (sensitivity 67%/specificity 98%), a combination of lncRNAs RP3-399L15.2 and CH507-513H4.6 (sensitivity 80%/specificity 85%) [ 179 ], a combination of miR-320a and miR-22-3p (sensitivity 80%/specificity 80%) [ 180 ] and pseudogene LGMNP1 (sensitivity 93%/specificity 76%) [ 49 , 181 ]. These studies are a promising start towards utilizing EVs and their cargo as biomarkers for endometriosis. With further optimization and reduction in the cost, more studies will be possible to assess the utility and performance of these molecules, individually, or in a multi-analyte approach, as diagnostic biomarkers. Further promising developments involve utilizing EVs as potential therapies for endometriosis. It has been demonstrated that EVs can inhibit angiogenesis, migration, and invasion of endometriosis in mouse models [ 182 , 183 ], and the specific EV-derived miR-214-3p downregulates fibrosis in a mouse model [ 184 ]. Another study showed that EV-derived miR-301a-3p is overexpressed in endometriosis lesions compared with serum from healthy controls. They also showed that downregulation of this miRNA in EVs influenced macrophage polarity by increasing the number of M2 macrophages and reducing the phagocytosis capacity [ 49 , 173 ]. Moreover, normal endometrial epithelial cells-derived exosomes have been used to deliver miRNA-30c to endometriosis-associated ectopic endometrial epithelial cells in vitro , and they suppress their invasion and migration activity [ 153 , 185 ]. The investigation of EVs within the context of endometriosis has provided valuable insights in the physiopathology of the condition, their role in mediating inflammation, and their potential use as biomarkers or treatments.

The intricate interplay between EVs and the cellular components of ovarian follicles has a crucial role in folliculogenesis, oocyte maturation, and overall ovarian function. The exploration of EVs in the context of PCOS and endometriosis has unveiled a multifaceted landscape of intercellular communication that can potentially be used as biomarkers and/or novel therapies. The significance of this study lies in its comprehensive analysis of the role of EVs in these conditions, highlighting their potential as both diagnostic and therapeutic targets. By providing a detailed examination of EV cargo, including miRNAs, proteins, and lipids, this review offers valuable insights into the molecular mechanisms underlying PCOS and endometriosis. This review concatenated the studies investigating biofluid-derived EVs from women with two prevalent gynecological disorders, PCOS and endometriosis. All studies showed that miRNAs are the most abundant sncRNAs in their analyses, regardless of the tissues analyzed (FF, endometrial biopsy/endometriosis lesions, etc.) [ 37 , 154 ]. For PCOS, we included 9 articles using different isolation and sequencing methods for FFEVs, investigating different sncRNAs, miRNAs being the most studied of them. Only two miRNAs were common between studies; miR-379 being downregulated in two studies [ 39 , 122 ] and miR-200 being upregulated in one [ 98 ] and downregulated in another [ 121 ]. For endometriosis, we included 11 studies that used different isolation and sequencing methods, but also on different biological sources, including cultured primary cells isolated from endometriosis lesions, peritoneal fluid, uterine cavity fluid and serum. There was one miRNA common to two studies, miR-130b being upregulated [ 160 , 163 ]. PCOS and endometriosis are distinct clinical entities, however they share several pathophysiological mechanisms, including hormonal imbalances, chronic inflammation, and metabolic disturbances. Interestingly, when comparing both gynecological disorders, miRNAs from the miR-30 and miR-15 families were common to PCOS and endometriosis, namely miR-30a and miR-15b in PCOS and miR-30d and miR-15a in endometriosis. The miR-30 family miRNA has been reviewed in the past and has shown to be implicated in the reproductive system and several inflammatory disorders [ 186 , 187 ]. On the other hand, miR-15 levels in FF have been correlated with poor ovarian response, decreased granulosa cell proliferation and promotion of apoptosis [ 188 ]. This review included studies on gynecological disorders compared to control patients, however the definition of the compared control group is often limited by the fact that the patient did not present with that specific condition, but are undergoing IVF treatment for infertility which maybe stemming from other gynecological abnormalities, potentially confounding the results. Specifically regarding endometriosis, if the patient did not have a diagnostic laparoscopic procedure, it cannot be certain that they do not have this condition as it can often be associated with minimal symptoms or asymptomatic. With respect to PCOS, there is a spectrum of cases from more mild cases to more severe. Additionally, the dynamic nature of these conditions necessitates longitudinal studies to capture the temporal changes in EV composition and function, offering a more comprehensive understanding of their involvement in disease progression in the menstrual cycle. Moreover, since most studies include patients with a severe form of the pathology, future studies will also need to assess the performance of any potential diagnostic biomarkers in medium to mild presentations. The majority of the studies cited here on endometriosis used the revised American Society of Reproductive Medicine (rASRM) classification system [ 189 ], but not all of them. Another technical limitation for most of the published studies is the low numbers of samples explained by the relatively high costs of FF harvesting, EV isolation and analysis, and sequencing costs. To further increase the complexity, EV isolation can require specialized equipment not practical for clinical settings and several methodological variations in isolation and characterization techniques pose challenges in achieving standardized and reproducible results. However, implementing the ISEV guidelines is crucial to achieve a standardization of the characterization of EV subtypes. However, this can be sample and cost prohibitive when dealing with patient samples [ 21 ]. In conclusion, the study of EVs in ovarian follicles and their implications in PCOS and endometriosis not only deepens our understanding of reproductive physiology and pathology, but also opens avenues for potential diagnostic and therapeutic advancement for these conditions. Future research directions should focus on refining methodologies, standardizing, and validating protocols, and establishing a consensus on EV nomenclature and characterization criteria. Further clinical studies need to be performed and validated to unlock the potential of EVs as biomarkers or for therapeutics.

Extracellular vesicles (EVs) are evolutionary conserved and heterogeneous nano-sized spherical bodies composed of a lipid bilayer and are released by cells into the extracellular space [ 1 ]. They participate in intracellular communication by transporting a wide variety of bioactive molecules including nucleic acids, proteins, and lipids, both locally and systemically [ 2 ]. EVs can be further subdivided into apoptotic bodies, microvesicles and exosomes, and are characterized by their biogenesis, release pathways, size, content, and functions [ 1 , 3 – 5 ]. Apoptotic bodies, with diameters of 500–5000 nm, are produced by cells undergoing apoptosis and contain intact organelles and other cytoplasmic components [ 1 ]. They are known to communicate with immune cells to aid in the clearance of inflammation [ 6 , 7 ]. Microvesicles are 100 nm-1000 nm derived vesicles formed by outward budding of the cell membrane through the action of cytoskeletal components and fusion machinery including SNAREs and tethering factors [ 1 , 3 – 5 , 8 , 9 ]. Because of their origin, their protein content closely reflects the plasma membrane and includes heat shock proteins, integrins and post-translationally modified proteins [ 10 , 11 ]. Exosomes are a unique class of EVs based on their size (30–150 nm), formation, secretion, and contents. They are formed through endosome inward budding and are packaged and transported in multivesicular bodies to incorporate the cell membrane before being secreted by the cell or sent to the lysosome for degradation [ 1 , 12 ]. The formation of multivesicular bodies and exosome formation is regulated through either the “endosomal sorting complex required for transport” (ESCRT) pathway [ 13 – 15 ] or an ESCRT-independent mechanism mediated by a sphingomyelinase enzyme [ 16 – 18 ]. Despite sharing common markers like tetraspanins CD63, CD9, and CD81 with other vesicles, exosomes require detailed analysis for accurate identification [ 19 , 20 ]. Because of the overlap in sizes, protein markers, and contents, a multistep characterization is essential to assess exosomes [ 1 ], ideally following the International Society for Extracellular Vesicles guidelines [ 21 ]. Exosomes have been shown to play an important role in intercellular communication, to serve as disease biomarkers, and to have potential in targeted drug delivery due to their stability and the ability for them to be bioengineered to target and bind to specific cell types [ 22 – 31 ]. In brief, release and uptake of EVs greatly depends on biological factors such as: source and recipient cell type, physiological state, and the microenvironment. Moreover, a significant aspect of the research on EVs lies in the extensive variation of isolation techniques and cell origins. Consequently, it is critical to appropriately isolate, enrich and characterize the EV population prior to conducting further experiments for biomarker discovery or mechanistic studies. Folliculogenesis is tightly controlled by hormonal and intrafollicular signalling and events during the menstrual cycle [ 32 , 33 ]. Ovarian follicles undergo a series of developmental stages, from primordial follicles to mature antral follicles. Oocyte development is a highly orchestrated process involving the endocrine system, supportive follicular somatic cells (granulosa cells—GCs, and cumulus cells—CC), and the oocyte [ 32 ]. EVs represent one route of this crucial intercellular communication (Fig. 1 ) [ 2 , 34 ]. EVs have been found in the follicular fluid of patients undergoing in vitro fertilization (IVF) and represent a great opportunity to better understand key follicular development events and deepen our knowledge of the signaling pathways, and may help discover potential EV biomarkers of oocyte quality [ 35 – 42 ]. This review will focus on the roles of EVs in common gynecological diseases, specifically polycystic ovarian syndrome (PCOS) and endometriosis, and their potential clinical implications. Fig. 1 Schematic of extracellular vesicle signalling in the ovarian follicle (adapted from Kalluri and Lebleu, 2020 [ 190 ]) Schematic of extracellular vesicle signalling in the ovarian follicle (adapted from Kalluri and Lebleu, 2020 [ 190 ]) As reviewed by Shrivastava and Conigliaro in 2022, PCOS is a complex, multifactorial, and commonly encountered endocrine disorder affecting 6–15% of women of reproductive age, characterized by a combination of hormonal imbalances, menstrual irregularities, and metabolic disturbances [ 43 , 44 ]. Patients with PCOS often exhibit metabolic disorders, including insulin resistance and increased androgen production, especially in theca cells; leading to accelerated apoptosis of granulosa cells and disrupted folliculogenesis [ 39 , 40 , 43 ]. Consequently, these disruptions manifest as anovulatory cycles, and increased immature follicles, all which contribute to infertility [ 45 ]. The pathogenesis of PCOS is multifaceted and influenced by multiple genetic, environmental, and hormonal factors. Obesity, particularly visceral fat accumulation, which is more common in patients with PCOS, can induce chronic inflammation and exacerbate PCOS symptoms [ 43 , 45 ]. In the past decades, EVs have emerged as potential key players in PCOS pathophysiology and have been touted for their potential diagnostic and therapeutic applications. Endometriosis, another widespread gynecological condition, reviewed by Chapron et al. in 2019, marked by the growth of endometrial-like tissue outside the uterine cavity [ 46 ]. This abnormal growth can lead to a wide range of symptoms, including dysmenorrhea, dyspareunia, and infertility. While retrograde menstruation is a primary hypothesis, other factors including inflammatory factors, hormone imbalance, genetic and epigenetic factors as well as environmental and lifestyle choices may be contributing to the disease. However, the exact cause of endometriosis remains elusive [ 46 ]. Endometriosis is strongly associated with infertility because the disease can adversely affect the ovary, the oocyte, and the endometrium primarily due to chronic and systemic inflammation [ 46 – 48 ]. Endometriosis diagnosis remains challenging due to the heterogeneity of the disease and currently, diagnosis relies on imaging either through transvaginal ultrasound or magnetic resonance imaging (MRI), with the gold standard being laparoscopic surgery and biopsy [ 46 ]. Available therapies manage the symptoms and not directly the cause, such as pain management using non-steroidal anti-inflammatory drugs (NSAIDs), and/or hormonal treatments like oral contraceptives, progestins, and gonadotropin-releasing hormone analogues (GnRHa) [ 46 ]. Surgical options range from conservative laparoscopic lesion excision or ablation to bilateral oophorectomy with or without hysterectomy [ 46 ]. Amidst the quest for enhanced diagnostic tools, EVs have emerged as potential biomarkers for endometriosis because lesions have been shown to release EVs into circulation and may contain a specific signature reflecting the disease state [ 49 ]. This review aims to explore the roles of EVs in PCOS and endometriosis, shedding light on their potential clinical implications and paving the way for future research and therapeutic strategies.