{"paper_id":"a81d94ee-ffa7-4dd8-88cd-0a0420a7558b","body_text":"Health Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nREVIEW PAPER \n \nEXTRACELLULAR VESICLES IN THE PATHOGENESIS OF ENDOMETRIOSIS: \nSCIENTOMETRIC ANALYSIS AND LITERATURE REVIEW \n \nAgnieszka Wąsowicz1(A,B,C,D,E,F), Marcelina Sztyler-Krąkowska2(A,B,C,D,E,F) \n1Central Clinical Hospital of Ministry of the Interior and Administration, Warsaw, Poland \n2Dr Anna Gostynska Wolski Hospital, Independent Public Health Care Institution, Warsaw, Poland  \n \nWąsowicz A, Sztyler -Krąkowska M. Extracellular vesicles in the pathogenesis of endometriosis: \nscientometric analysis and literature review. Health Prob Civil.  \nhttps://doi.org/10.5114/hpc.2025.148341 \n \nTables: 1 \nFigures: 2 \nReferences: 36 \nSubmitted: 2025 Jan 25 \nAccepted: 2025 March 6 \n \nAddress for correspondence:  Agnieszka Wąsowicz, Central Clinical Hospital of Ministry of the \nInterior and Administration, Wołoska 137, 02 -507 Warsaw, Poland, e -mail: \nagnieszkawasowicz99@gmail.com, phone: +48 47 722 20 00 \nORCID: Agnieszka Wąsowicz https://orcid.org/0009-0008-3256-793X, Marcelina Sztyler-Krąkowska \nhttps://orcid.org/0009-0004-4817-9537 \n \nCopyright: © John Paul II University in Biała Podlaska, Agnieszka Wąsowicz, Marcelina Sztyler -\nKrąkowska. This is an Open Access journal, all articles are distributed under the terms of the Creative \nCommons AttributionNonCommercial -ShareAlike 4.0 International (CC BY -NC-SA 4.0) License \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \n(https://creativecommons.org/licenses/by-nc-sa/4.0), allowing third parties to copy and redistribute the \nmaterial in any medium or format and to remix, transform, and build upon the material, provided the \noriginal work is properly cited and states its license. \n \nSummary \nPain and infertility are the most common symptoms of endometriosis  (EMS), a gynecological \ndisorder defined as the development of endometrial cells outside the uterus. Extracellular \nvesicles (EVs) are physiologically active granules that carry molecular content with both \ndiagnostic and therapeutic implications in intercellular communication. We performed a \nsystematic review based on the 2020 PRISMA guidelines to investigate the role of EVs in EMS. \nOn March 3th, 2024, the keywords “endometriosis”, “extracellular vesicles”, “EV”, or “EVs”, \nalong with the terms “treatment”, “monitoring”, or “diagnosis”, were used to search three \ndatabases (Medline, PubMed and Scopus). Articles published in English between 2019 and \n2024 were counted. 170 EMS patients from 14 studies were included in the analysis. Samples \nof EVs of various origins were investigated to find possible relevance in the pathophysiology \nand their diagnostic and therapeutic implications. The most often analyzed cargo were \nmicroRNAs. The possible contribution of EVs to pathophysiology has been examined in a \nnumber of studies, with an emphasis on their roles in inflammation, angiogenesis, \nimmunomodulation, cell communication, and proliferation. The reports indicated the \ninvolvement of EVs in numerous signaling pathways, including the MAP, WNT, TNF and \nPI3K-AKT pathways. \nKeywords: extracellular vesicles, endometriosis, EVs, diagnosis, treatment \n \n  \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nIntroduction \n \nAbout 10-15% of women of their reproductive years suffer from endometriosis (EMS), \na common gynecological illness marked by the growth of endometrial-like tissue exterior to the \ncavity of the uterus  [1]. The collection of accurate statistical data presents a challenge, as a \nsignificant portion of EMS cases remain undiagnosed for extended periods. EMS poses a great \nburden both for patients and the healthcare system. Women with EMS have significantly \nimpaired quality of life [2]. Furthermore, research showed elevated cancer risk among \nindividuals with th e disorder [3]. The diagnosis of EMS can be challenging due to the non -\nspecific nature of its symptoms. The main conditions that require a visit to a gynecologist are \nfertility issues ( EMS may result in the obstruction of the fallopian tubes and formation of \nantiphospholipid autoantibodies), menstrual cycle disorders, including dysmenorrhea (painful \nperiods), chronic, debilitating pelvic pain that typically intensifies prior to menstruation and \ndyspareunia (painful intercourse). Moreover, EMS may cause bleeding from the gastrointestinal \ntract and pain during defecation, as well as hematuria or postcoital bleeding [4,5]. \nThe underlying causes of EMS are not entirely clear. It is believed that EMS is \ninfluenced by a combination of both genetic and hormonal factors, as well as environmental \ninfluences. One of the most well-supported theories to explain the condition is that of retrograde \nmenstruation, whereby menstrual blood travels back through the fallopian tubes into the \nabdominal cavity, thereby endometrial cells are able to implant and grow away from the uterus \n[6-8]. Additionally, there is evidence to suggest that genet ic predisposition, immune system \ndysfunction, and hormonal imbalance play a significant role in the EMS pathogenesis. \nAccording to current findings, regulation of the inflammatory response is a pivotal factor in the \nonset and evolution of EMS, with immune dysfunction being a contributing element in the \ndevelopment of abnormal tissue growth [9-11]. \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nExtracellular vesicles (EVs) have emerged as potential, important contributors in the \npathogenesis of EMS and hold promise for both understanding the disease mechanisms and \ndeveloping diagnostic tools [12]. Extracellular vesicles, also known as exosomes and \nmicrovesicles, are small membrane -bound particles that are released by cells and can carry \nvarious biological molecules, including proteins, nucleic acids, and lipids [13,14]. The vesicles \nhave been found to be involved in intercellular communication and can transfer their cargo to \nrecipient cells, thereby influencing cellular functions. EVs in EMS have been shown to play a \nrole in the pathogenesis of the disease by facilitating the establishment and growth of \nendometriotic lesions [15]. \nUnderstanding the specific pathways and molecular cargo carried by EVs in EMS is \ncrucial for unraveling the mechanisms underlying the disease and identifying potential \ntherapeutic targets. Moreover, EVs have the potential to serve as a novel minimally invasive \ndiagnostic instrument, thereby reducing the necessity for laparoscopic procedures [16]. \n \nAim of the work \n \nIn the review, we aim at consolidating the current knowledge on EVs in EMS, with a \nfocus on their role in pathogenesis and relevant signaling pathways  to better understand its \nmechanisms and identify potential therapeutic targets. Since a reliable marker for the disease is \nstill lacking, EVs may offer a non-invasive diagnostic option, reducing reliance on laparoscopy \nand improving patients outcomes. \n  \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nMethods \n \nA systematic review was conducted based on the 2020 PRISMA guideline s [17]. A \ncomprehensive search was conducted on March 3th, 2024 across three major databases: \nPubMed, Medline and Scopus. The search terms included the following keywords: \n“endometriosis” and “extracellular vesicles ” or “EV” or “EVs” and either “treatment” or \n“monitoring” or “diagnosis”. The articles were independently reviewed by two researchers \n(A.W. and M.SK.) at every stage of the evaluation process. \n \nInclusion and exclusion criteria \n \nThe following inclusion and exclusion criteria were applied in order to identify relevant \narticles for the study. Firstly, articles must have been published in English. Secondly, the \npublication date range was set from 2019 to 2024. Thirdly, the articles were required to assess \nextracellular vesicle samples from EMS patients, whether or not they had controls. It was \nessential that the articles were original papers. Animal studies were excluded from the analysis. \nFinally, articles exclusively concerning adenomyosis and not EMS were also excluded. \n \nExtraction of data  \n \nThe authors (A.W. and M.SK.) performed independent evaluations of the reports, in \norder to compile and extract data. The following information was evaluated for each study: the \nnumber of patients and controls, the source of bodily fluids or tissue used to obtain EVs, the \ntechniques employed for isolating EVs, the specific cargo within EVs, the possible diagnostic, \ntherapeutic and pathogenetic implications of EMS, and any identified limitations. It is important \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nto note that no automation tools were applied during the process, and no supplementary data \nwas obtained from the authors of the selected studies. All extracted data is presented in Table \n1. \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nTable 1. Extracted and summarized data  \nStudy \nNumber of \nEMS patients \n(n=) \nControls \n(n=) \nEV's sample \nsource \nEV isolation \ntechnique EV cargo Pathogenesis Diagnostic \nimplications \nTherapeutic \nimplications Limitations \nAsl et al. [18] n=5 n=5 \nMenstrual \nblood-derived \nstem cells \nExocib kit \n(Cib Biotech \nCo) \nNot investigated \nCD63 and CD81 \nas exosome-\nspecific markers \n(flow cytometry ) \nAltered pathways: VEGF as a \nmajor mediator of physiologic and \npathologic angiogenesis, cyclin D1 \nis a cell-cycle regulator, MMP-2 \nand MMP-9 are markers of \nmigration and invasion, \ninflammatory factors like IL-6, IL-\n8, IL-1β, cox-2, NF-kb, HIF1α, and \nTNF-α, stemness factors like sox2, \nsall4, oct4, and Nanog, and finally, \nBCL-2 and Bax are apoptotic \ngenes. Additionally exosomes from \nthe research induced apoptosis in E-\nMenSCs. \nNot discussed \nPotential \ntherapeutic \neffect: proof \nthat \nmesenchymal \nstem cells can \ncure EMS by \nproducing \nexosomes, E-\nMenSCs' \nexpression \nlevels of \nmarkers linked \nto \ninflammation, \nproliferation, \nmigration, and \nangiogenesis are \ndecreased by \nexosomes from \npatients without \nEMS (NE-\nMenSCs). \nLimited numbers, in vitro \nonly, EVs cargo not \ninvestigated \nHsu et al. [19] n=3 n=3 \nEutopic (Eu) \nand ectopic \n(Ec) \nendometrial \nUltracentrifug\nation (UC) \nAnnexin A2 \n(ANXA2) \n36 proteins \nspecific to \nEVs-ANXA2 regulates the motility, \nproliferation, and angiogenic \npotential of ESCs via the \nextracellularly regulated kinase \nNot discussed \nNot discussed, \nbut potential \ntherapeutic \nimplications \nLimited numbers, controls are \nfrom patients with EMS. The \nstudy did not perform \npurification for non-sEV \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nstromal cells \n(ESCs) \nEcESCs-sEVs \ncomparing to \nEuESCs-sEVs \nIdentified by: \nWestern blotting, \nNTA and TEM. \n(ERK)/STAT3 pathway and \npathways related to adherens \njunctions, cdc42, wnt/β-catenin, \nactin cytoskeleton, and Rho family \nGTPases. \nprotein removal, and did not \ncompare sEV-enriched pellets' \nactivity to sEV-depleted \nfractions. \n \nWang et al. [20] n=6 \nn=6 \nendometria\nl stromal \ncells \n(ESCs) \nwithout \nany \nstimulation \nHuman \numbilical cord \nmesenchymal \nstem cells \n(UC-MSCs) \nUltracentrifug\nation (UC) \nNot investigated, \nidentification of \nEVs using \nelectron \nmicroscopy \nEV exposure significantly reduced \nESCs' expression of cyclin D1 and \nMMP-9, while EVs from UC-\nMSCs inhibited proliferation, \ninvasion, and expression of SF-1, \naromatase, and ERb. \nNot discussed \nUC-MSCs-\nderived EVs as \npotential \ntreatment option \nfor EMS \nLimited numbers, number of \nsamples not clearly stated \nZhou et al. [21] n=3 n=3 \nEutopic \nendometrial \nstromal cells \n(EuESC) of \nwomen with \nEMS-\nassociated \ninfertility and \nnormal \nendometrial \nstromal cells \n(NESC) of \nfertile women \nwithout EMS \nExoQuick-TC \nExosome \nIsolation Kit \n(SBI) \nA total of 49 \ndifferentially \nexpressed \nmiRNA, \nincluding 26 up-\nregulated and 23 \ndown-regulated in \nEuESC exosomes \nas compared with \nNESC exosomes \nIdentified by: \ntransmission \nelectron \nmicroscopy \n(TEM) \nHOXA10 and LIF identified as \npossible targets, \n(mRNA expression levels \nsignificantly decreased in EuESC \ncompared with NESC). \nIn addition, the predicated target \ngenes of these differentially \nexpressed exosomal miRNA were \nsignificantly enriched in 76 \npathways, including the MAPK and \nWnt signalling pathways. \nPotential \ndiagnostic \nimplications \nNot discussed RNA seq via RT-qPCR with \nno validation, limited numbers \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nQiu et al. [22] n=30 n=16 \nEndometriotic \ncyst stromal \ncells (ECSCs) \nand serum \nECSCs: Total \nExosome \nIsolation \nReagent \n(Thermo \nFisher \nScientific) \nSerum: \nExoQuick \nExosome \nPrecipitation \nSolution kit \nlncRNA aHIF \nExosomal aHIF modulates the \nproangiogenic behavior of \nHUVECs (human umbilical vein \nendothelial cells ) and stimulates \nEMS angiogenesis by activating \nVEGF-A, VEGF-D and FGF. \nSerum \nexosomal aHIF \nas a promising \nbiomarker for \nEMS \nExosomal aHIF \nas a potential \ntherapeutic \ntarget \nIn vitro only, endometriomas \nonly, ECSCs as the only cell \nmodel, proliferative phase \nsamples only, methods imply \nthat the fetal bovine serum \n(FBS) in the culture media \nwas not depleted of EVs, \npotentially introducing \ncontaminating EVs. \nHuang et al. [23] Not stated Not stated \nEctopic \nendometrial \ntissues of \nEMS patients \nand normal \nhuman serum \n(NHS) \nCentrifugation \nmiR-301a-3p \nidentified by \nTEM, NTA and \nwestern blot \nEMS derived exosomal miR-301a-\n3p mediated macrophage \npolarization via regulating PTEN-\nPI3K axis. \nNot discussed \nPotential \ntherapeutic \nimplications. \nDownregulation \nof miR-301a-3p \nreduces \nmacrophage \nactivity, thus \ninflammatory \nresponse in \nEMS. \nThe number of participants \nnot clearly stated as well as \nthe tissue source and methods. \nDifferent sources of EVs were \nused (tissue from lesions from \nEMS patients and serum from \ncontrols). It wasn't \ndemonstrated that EVs contain \nmiR-301-3a. \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nKhalaj et al. [24] n=6 \nNumber \nnot \nspecified \nEMS lesions, \nendometrium, \nperitoneal \nfluid and \nplasma, \nendometrial \nepithelial \ncarcinoma \n(EECC), \nHUVEC and \npatient-\nderived \nendometriotic \nepithelial \n(12Z) cell line. \nMiRCURY \nexosome \nisolation kit \n(#300,102; \nExiqon Inc) \nmiRNA(miR-206, \n–29c-3p,–381-3p, \n–100-5p, –193b-\n3p, –335-5p, –\n411-5p –139-3p, \n–let-7a-3p, –95-\n3p, –29b-3p, –\n495-3p, –136-3p, \n–887-3p,), \nlncRNA analyzed \nusing next-\ngeneration \nsequencing \n(NGS) validated \nusing quantitative \nPCR (qPCR) \nEVs from EMS lesions promote \nangiogenesis, cell growth and pro-\ninflammatory effects by IL 6, \nPDGF and macrophage-derived \nchemokine (MDC). Moreover they \ncarry cargo (miR–30d-5p, miR–\n27a-3p, and miR-375) that is \nspecific to EMS. \nDistinct EV \nmiRNA \nsignatures, miR-\n30d-5p, miR-\n27a-3p, and \nmiR-375, are \npotential \ndiagnostic \ntargets for EMS, \ndistinguishing \nbetween \npatients and \ncontrols, and \nmatched lesions. \nNot discussed \nLimited numbers. Study does \nnot specify number of control \nsample. \nWu et al. [25] \nn=13 \n(RNA seq n=3, \nRT-qPCR n=10) \nn=13 \n(RNA seq \nn=3, RT-\nqPCR \nn=10) \nCCM of the \ncontrol \nendometrium \nand paired \nendometrioma \nand \nendometrium \nprimary ESCs \n \nExoQuick-TC \nExosome \nIsolation Kit \n(SBI) \nATP6V1A, \nmiRNAs, mRNAs \n(seq), circRNAs, \ncirc_0026129, \nmiR-15a-5p \nvalidated with \nRT-qPCR \nElevated miRNA-15a-5p involved \nwith angiogenesis regulates VEGF-\nA hence contributing to the \npathogenesis of EMS. ATP6V1A is \nlikely involved with cell migration \nand growth. \nPossible \ndiagnostic \ntargets. Network \nanalysis of \nceRNAs \nidentified three \nkey components \n(circ_0026129, \nmiR-15a-5p and \nATP6V1A), \nATP6V1A \ncorrelates with \nseverity and \nendometrial \nresponsiveness. \nPotential \ntherapeutic \ntargets (AJUBA \nand miR-3187-\n3p. AJUBA as a \nnegative \nregulator of the \nHippo signaling \npathway, which \ninhibits \napoptosis and \ncell \nproliferation in \nEMS). \nSmall group of patients and \ncontrols. Low statistical power \nwith huge number of \ncomparisons and complex \ndisplaying calculations. \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nFeng et al. [26] n=5 \nn=6 \nThe \nsamples \nwere \ndivided \ninto two \ngroups \n(n=6): \ncontrol and \nHuc-\nMScs-exo \ntreatment \n(10 μg/ml) \ngroup \nCCM of \nprimary \numbilical cord \nderived MSCs \nExosome \nextraction kit \n(#E1310; \nBioruo) \nNo investigation \nHuc-MSCs-exo improve \nendometrial cell migration, increase \nN-cadherin and Vimentin \nexpression levels, and decrease E-\ncadherin expression at both mRNA \nand protein levels. \nNot discussed \nHuc-MScs-exo \nas potential \ntreatment option \nLimited numbers. The \numbilical cord (EV source) \nand EMS tissue from different \npatients. \nWu et al. [27] \nn=10 \n(n = 3 for RNA \nseq, \nn = 7 for RT-\nPCR validation) \nn=10 \n(n = 3 for \nRNA seq, \nn = 7 for \nRT-PCR \nvalidation) \nEMS group: \nESCs obtained \nfrom ovarian \nendometrioma\ns and eutopic \nendometrium \ncontrol group: \nESCs obtained \nfrom \nendometrium \nExoQuick-TC \nExosome \nIsolation Kit \n(SBI) \nmiRNA, mRNAs \n(seq), and \nlncRNA. \nRegulatory \nnetwork \nexpression MIB2 \nLOC105376166/\nmiR-214-3p and \nADCY3 \nLOC105371414/\nmiR-423-5p \nconfirmed using \nRT-qPCR \nExosomes derived from ESC by \ntransferring competing endogenous \nRNAs may play autocrine/paracrine \nroles and promote the pathogenesis \nof EMs. \nPossible use of \na panel of EV \nderived RNA \nprocessing for \ndiagnosis as a \nbiomarker. \nNot discussed \nbut potential \ntherapeutic \nimplications of \nlnc-RNA \napplication. \nSmall number of subjects with \nvery large number of \ncomparisons, selection bias \nand not the best method to \nverify the purification of \nexosomes. \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nLi et al. [28] n=26 n=25 \nNormal/ectopi\nc endometrial \ntissues and \nleucorrhea \nDifferential \ncentrifugation \ntRFs and tiRNAs \n(tRF-Leu-AAG-\n001) verified \nusing PCR \nThe tRFs and tiRNAs in ectopic \nexosomes are enriched in ten \npathways, with VEGF and Fc \nepsilon IR being the most \ninfluential. High expression of tRF-\nLeu-AAG-001 triggers mast cells to \nexpress inflammatory factors (IL-6, \nIL-10, IL-1β, TNF), promoting \ninflammation and angiogenesis. \nExosomal tRF-\nLeu-AAG-001 \ncould be a \npotential EMS \nbiomarker. \nNot discussed \nDample size of endometrial \ntissue not clearly stated, small \nsample size used for exosomal \nRNAs extraction and \ntRFs&tiRNAs sequencing. \nWu et al. [29] n=42 n=24 Serum \nCentrifugation\n, magnetic \nseparation \nmiRNA (miR-\n215-5p and miR-\n6795-3p, miR-\n26b-5p) validated \nwith RT-qPCR \nIdentified MiRNAs may be \ninvolved in MAP and PI3K-AKT \nsignaling pathways. \nThe possible \nbiomarkers \nmiR-26b-5p, \nmiR-215-5p, \nand miR-6795-\n3p can be \nemployed to \nassess the \ndegree of \novarian EMS. \nNot discussed No reports on miRNA chip \nanalysis methods \nHuang et al. [30] n=10 n=10 Plasma Ultracentrifug\nation (UC) \n50 DE-miRNAs \n(7 miRNAs \nupregulated and \n43 miRNAs \ndownregulated in \nthe EM patients) \nmiRNA \nsequenced using \nmicroarrays \nmiRNAs are associated with \npromoting mesenchymal cell \nproliferation, TNF and Toll-like \nreceptor signaling pathways, and \ndifferentiation of Th1 and Th2 \ncells. \nBlood exosomal \nmiRNAs as \npotential targets \nfor diagnosing \nEMS \nNot discussed, \nbut potential \ntherapeutic \ntargets \nLimited numbers \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nZhang et al. [31] \nn=9 (5 patients \nin morphology \nstudy, \n4 patients in \nmiRNA \nmicroarray \nanalysis) \nn=10 (5 \ncontrols in \nmorpholog\ny study, \n5 controls \nin miRNA \nmicroarray \nanalysis) \nTubal fluid \nGradient \ncentrifugation \nand \nultracentrifuga\ntion (UC) \nmiRNAs (miR-\n1273f, miR-5699-\n5p, miR-6087 and \nmiR-6747-5p) \nvalidated by \nquantitative real-\ntime PCR \nIdentified miRNAs may be \ninvolved in MAPK, Wnt, VEGF \nand ErbB signaling pathways \nplaying a crucial role in regulating \ntarget cell function, affecting cell \ncommunication between gametes \nand embryos and tubal epithelium \nsecretion. \nNot discussed Not discussed Limited numbers \n \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nMethodology for risk of bias assessment \n \nWe employed the NHLBI's Study Quality Assessment Tools to evaluate the included \nstudies' risk of bias. Each study was evaluated separately by two independent reviewers (A.W. \nand M.SK.) to maintain objectivity and reduce potential bias. No automation tools were \nconducted in the assessment process, ensuring a reliable and compr ehensive analysis of the \nresearch. \n \nLiterature review results \n \nSearch results \n \nA total of 14 articles were chosen for inclusion in the final review, following the initial \nscreening process which identified 192 articles. Of these, 129 articles did not align with the \nspecified inclusion criteria (covering the subject of extracellular vesicles, published in English \nbetween 2019 and 2024, original papers only). Following the removal of 25 duplicate records, \none article was excluded as its focus was exclusively on adenomyosis rather than EMS. \nAdditionally, eight further reports were excluded as they were not relevant to any pathogenesis \npathways. The identification process of relevant studies is illustrated in Figure 1. \n \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \n \nFigure 1. Data identification and screening following the 2020 PRISMA guidelines \n\n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nSource of extracellular vesicles \n \nThe most common EVs’ sample sources were eutopic and ectopic endometrial stromal \ncells (ESCs), which were examined in four of the studies [1 9,21,25,27] alongside with serum \n[22,23,29] and EMS lesions [23,24,28], as were analyzed in three of the articles each. Other \nEVs’ sources included menstrual blood -derived stem cells [1 8], human umbilical cord \nmesenchymal stem cells (UC -MSCs) [20,26], endometriotic cyst stromal cells (ECSCs) [2 2], \nendometrial biopsy [2 4,28], peritoneal fluid [2 4], leucorrhea [2 8], plasma [2 4,30] and tubal \nfluid [31].  \n \nIsolation and identification of extracellular vesicles \n \nA variety of techniques was employed to isolate EVs. The most common methods of \nextraction were ultracentrifugation (UC), used in five of the studies [1 9,20,21,30,31] and \ncommercial kits, which have been employed in a total of seven studies [18,21,22,24-27]. Other \ntechniques included gradient centrifugation [31], differential centrifugation [28] and magnetic \nseparation [29]. The following commercial kits were used: Exocib kit, ExoQuick-TC Exosome \nIsolation Kit (SBI), MiRCURY, Total Exosome Isolation Reagent (Thermo Fisher Scientific). \nIdentification of EVs was typically conducted through the use of transmission electron \nmicroscopy (TEM) and staining with exosome -specific markers, such as CD63 and CD81. \nAlternatively, Western blotting was employed to confirm the presence of EVs. The cargo within \nthe vesicles was then validated through the use of real -time quantitative PCR (RT -qPCR), \nqPCR, next-generation sequencing (NGS) or microarrays. \n  \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nCargo and function of extracellular vesicles \n \nEVs have been shown to transport special cargo and play a key role in the \npathophysiology of the disease by affecting processes such as angiogenesis, cell proliferation, \nand inflammatory response within the local microenvironment of ectopic, endometriotic \nlesions. Majority of studies have focused on analyzing the cargo of EVs, which is primarily \ncomposed of crucial inflammatory and angiogenic cytokines and micromolecules that are \nknown to be involved in disease progression. Some studies have demonstrated th e presence of \nEVs in specific samples without a thorough examination of their contents [1 8,20,23,26]. The \nmost frequently identified cargo of the EVs was miRNA, which was reported in a total of six \nstudies [2 1,25,27,29-31]. In addition, EVs were found to contain the following: lncRNA \n[22,27,30] mRNAs [25,27,30], circRNAs [25], sRNA [21,24] tRFs and tiRNAs [2 8], annexin \nA2 (ANXA2) [19]. \n  \nEVs role in the EMS pathogenesis \n \nThe investigation primarily concentrated on the role of EV-derived cargo in the process \nof inflammation, angiogenesis, cell migration and cell growth. They participate in the \ninflammatory response through the differentiation of Th1 and Th2 cells [30], the promotion of \nmacrophage M2 proliferation [2 3], or the activation of B cells [2 4]. The reports indicated the \npotential involvement of miRNAs in numerous regulatory pathways, such as the \nphosphatidylinositol 3-kinase (PI3K-AKT) signaling pathway [2 3,29], the mitogen-activated \nprotein (MAP) pathway [2 1,29,31], the WNT pathway [1 8,19,21,31], along with the tumor \nnecrosis factor (TNF) pathway [30]. \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nFurthermore, it has been demonstrated that EVs play a role in angiogenesis due to their \nability to activate various growth factors, including vascular endothelial growth factor (VEGF-\nA, VEGF-D) and fibroblast growth factor (FGF) via exosomal aHIF [22], miRNA-15a-5p [25] \nor tRFs and tiRNAs [2 8]. Additionally, annexin A2 ( sEVs-ANXA2), tRF-Leu-AAG-001 and \nother miRNAs have been proven to stimulate the growth of blood vessels [19,24,28,29,31]. \nA number of studies have shown the influence of EVs on cell growth and cell migration. \nIn patients with EMS, exosomes originated from Huc-MSCs were found to increase the levels \nof Vimentin and N-cadherin at the mRNA and protein levels, decrease E -cadherin expression \nand dramatically enhance the migration of uterine glandular epithelial cells [26]. Furthermore, \nATP6V1A is believed to play a role in cell migration and growth [2 5] and sEVs-ANXA2 has \nbeen shown to regulate the motility and proliferation potenti al of ESCs via the extracellular \nsignal-regulated kinases (ERK)/STAT3 pathway [19]. \nFurthermore, it has been demonstrated that numerous pathways are altered in EMS and \nmay potentially be treated with stem cells [18]. The cyclin D1 and MMP-9 expressions in ESCs \nwere significantly decreased by UC-MSCs-derived EVs, as were the SF-1, ERb and aromatase \nexpressions. Therefore, it is possible to achieve the inhibition of ESCs' proliferation and \ninvasion [ 20]. Ultimately, miRNAs regulate the functions of target cells, influencing \ncommunication between gametes and embryos, as well as the secretion and transport activities \nof the tubal epithelium. The processes consequently affect the health of the female reproductive \nsystem [31]. \nThe impact of various EVs and their cargo on pathogenic processes is illustrated in \nFigure 2. \n \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \n \nFigure 2. Visualization of EVs’ cargo and their role in the pathogenesis of EMS \n \nTherapeutic and diagnostic implications of extracellular vesicles \n \nTwo studies have indicated that EVs derived from menstrual blood stem cells and \nhuman umbilical cord mesenchymal stem cells (UC-MSCs) may represent a potential treatment \noption for EMS [18,20]. Exosomes have been demonstrated to inhibit the expression of markers \nassociated with inflammation, angiogenesis, proliferation and migration. One article suggested \nthe potential therapeutic implications of aHIF as a promising target [22]. Another study showed \nmiR-301a-3p overexpression in ectopic endometrial lesions, suggesting that downregulation of \nthe particular mRNA may result in a reduction of macrophage activity and, consequently, an \nattenuation of the inflammatory response observed in EMS [23]. A number of specific miRNAs \n[24,27,29,30], serum exosomal aHIF [22], ceRNAs [25] and exosomal tRF-Leu-AAG-001 [28] \nhave been identified as potential biomarkers for the diagnosis of EMS. Furthermore, ATP6V1A \n\n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \n[25] as well as serum miRNA [2 9] have been demonstrated to correlate with severity and \nendometrial responsiveness. \n \nLimitations \n \nThe specific limitations of each study are listed in (Figure 2). The most frequently \nobserved limitation was the relatively small sample size, with only four of the studies reporting \na number of EMS patients greater than ten individuals [22,25,28,29].  \nMoreover, the number of patients with EMS was not clearly stated in four additional \narticles [ 20,23,24,28]. Another common limitation was the absence of appropriate control \ngroups, as well as the use of different sources of EVs between EMS samples and controls \n[19,20,23,25,26]. A further limitation, observed in three of the studies, was the lack of \ninvestigation into EVs’ cargo [18,20,26]. As a result, the biological function of the EVs could \nnot be assigned to a specific molecular component. The authors also identified several other \nlimitations, including the potential for selection bias and the necessity for a more effective \nmethod for the collection and verification of exosome purification [27].  \n \nDiscussion \n \nEMS is a common yet disturbing condition for women. Current estimations suggest that \nbetween 10% and 15% of women at their childbearing age are affected by EMS, resulting in \napproximately 190 million individuals impacted [32]. \nThere is disagreement over the pathophysiology of EMS; three basic theories are \nprimarily accepted. The theories include: 1. Menstrual blood reflux to the pelvis; 2. Coelomic \nepithelium metaplasia; and 3. Endometrial tissue dissemination across the cardiovascular and \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nlymphatic systems. Moreover, others suggest that genetic factors and modifications in the \nimmune system may potentially contribute to the destructive progression of EMS [33-35]. \nAlthough the underlying pathophysiology remains largely unclear, significant progress \nhas been made in understanding progression of the disease. \nEMS is characterized by the dysregulation of various signaling pathways (for instance, \nthe vascular endothelial growth factor receptor (VEGF), mitogen -activated protein kinase \n(MAPK), tumor necrosis factor (TNF), ERK/STAT, WNT, and PTEN -PI3K), which are \nengaged in biological processes such as angiogenesis, proliferation, migration and apoptosis \n[19,23,36]. \nExtracellular vesicles, otherwise termed as EVs, hold molecular cargoes in the forms of \nproteins, lipids, and nucleic acids, including microRNAs that facilitate processes involved in \nthe development of EMS. \nEVs have been demonstrated to participate in the intercellular communication within \nthe endometrial microenvironment. The interaction can alter immune responses, such as Th1 \nand Th2 lymphocyte differentiation, M2 macrophage amplification, or B cell stimulation, and \nworsen the inflammation -induced state of EMS. Exosomes derived from mesenchymal stem \ncells of human umbilical cord (Huc -MSCs) and ATP6V1A are thought to be involved in cell \nmigration and proliferation in EMS patients. Furthermore, it has been dem onstrated that EVs \npromote angiogenesis by activating a number of growth factors, such as FGF and VEGF. \nTaking into consideration all the facts from the review, we may say that the seemingly distinct \npathways engage in intimate cross-talk, where EVs are the connecting binder. \nThe suggested role of EVs in the pathogenesis of EMS is outlined below. During \nretrograde menstruation, exosomes released by the endometrial tissue interact with shed cells \nof endometrium, facilitating their subsequent migration, adhesion, and implantation as well as \nimmune modulation. Furthermore, EVs from freshly implanted ectopic endometrial cells have \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nbeen shown to promote further inflammation, angiogenesis and cell proliferation, which in turn \nleads to the development of EMS lesions. However, it is a hypothesis and yet to be investigated. \nEven though it is thought that surgical intervention is the most effective method of \ndealing with the symptoms related to EMS, the outlook for the use of EVs in medical therapies \nand diagnostics appears to be very encouraging. EVs have proven to be a potential therapeutic \ntarget due to their multiple roles in disease processes. EMS patients treated with exosomes \ndisplayed reduced markers of inflammation, which supports the potential for them to be used \nin treatment for the disease. Furthermore, the presence of exosomes has been detected in various \nbody fluids, including peritoneal fluid, leukorrhea, and plasma; thus they may be useful as easily \ndetectable biomarkers. \n \nConclusions \n \nIn conclusion, there is an ongoing need for clinicians and scientists to collaborate in \norder to explore new pathways and to conduct studies on EMS therapies. Given the significant \nrole that EVs play in the pathogenesis of the disease, further research is required in order to \nexplore and validate their cargo as potential biomarkers, drug targets, or even treatments to \nultimately improve patient outcomes. \n \nDisclosures and acknowledgements \n \nThe authors declare no conflicts of interest with respect to the research, authorship, \nand/or publication of this article.  \nThis research received no specific grant from any funding agency in the public, \ncommercial, or not-for-profit sectors. \n\nHealth Problems of Civilization \neISSN: 2354-0265, ISSN: 2353-6942 \n \nThe authors declare that this scientific article was initially prepared with the assistance \nof the AI tool DeepL Write (https://www.deepl.com/en/write) for grammar and language \nimprovement. 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