{"paper_id":"56e6cd57-f465-4dc9-85a8-ae918ab8f4e4","body_text":"Endometriosis, the presence of extrauterine endometrial-like lesions, affects ∼5–10% of reproductive-aged females ( Zondervan  et al. , 2018 ) although rare premenarcheal manifestations and postmenopausal endometriosis have been described ( Gemmell  et al. , 2017 ). Globally, an estimated 190 million reproductive-aged women suffer from endometriosis ( Zondervan  et al. , 2020 ) although the true prevalence might be higher.\nWomen with endometriosis often experience severe menstrual and non-menstrual pain involving the lower abdomen, pelvis or lumbosacral region, deep dyspareunia, dyschezia, and dysuria ( Uimari  et al. , 2021 ) and infertility in 30–50% of cases ( Prescott  et al. , 2016 ). Patients with endometriosis suffer from chronic fatigue ( Ramin-Wright  et al. , 2018 ) and are at a higher risk of psychiatric disturbances (56.4%) than those without endometriosis (43.6%) ( Pope  et al. , 2015 ). Endometriosis is further associated with poorer health-related quality of life mean scores and work productivity as compared to asymptomatic controls (Nnoaham et al., 2011) resulting in an estimated €9.9 billion (equivalent to £8.4 billion) in societal cost in 2012 ( Simoens  et al. , 2012 )—a higher economic burden should be assumed today.\nEndometriotic lesions most commonly present as superficial peritoneal/serosal lesions of different colours and sizes, as ovarian endometriotic cysts or mostly fibrotic (deep) nodules >5 mm below the peritoneal surface ( Cornillie  et al. , 1990 ). The extent of pelvic endometriosis is widely described using the revised American Society for Reproductive Medicine (rASRM) classification system into stages I–IV based on direct observation of lesions during surgery ( Canis  et al. , 1997 ). Infrequently, extrapelvic locations, including abdominal surgical scars ( Wang  et al. , 2016 ), the umbilicus, diaphragm, thorax, pericardium, and lymph nodes ( Ceccaroni  et al. , 2012 ), have been described.\nTo date, the cause of endometriosis is not fully defined. Sampson’s theory of retrograde menstruation ( Sampson, 1927 ), does not explain why only some women develop endometriosis as retrograde menstruation is physiological ( Halme  et al. , 1984 ). Insufficient public and professional awareness, and the trivilisation of women’s pain ( Shah  et al. , 2010 ;  Samulowitz  et al. , 2018 ;  De Sanctis  et al. , 2020 ), absence of clinically relevant biomarkers ( May  et al. , 2010 ;  Liu  et al. , 2015 ;  Gupta  et al. , 2016 ;  Nisenblat  et al. , 2016a , b , c ), and the unspecific nature of endometriosis-associated symptoms ( Zondervan  et al. , 2018 ) are some of the challenges in diagnosing endometriosis. Previously, the gold standard for diagnosing endometriosis was diagnostic laparoscopy, which may have prevented earlier diagnosis of endometriosis. The latest ESHRE guideline on endometriosis no longer recommends diagnostic laparoscopy as the gold standard diagnostic tool, reserving it rather for women with negative MRI or ultrasound imaging, and/or where empirical treatment has failed ( Becker  et al. , 2022 ). Current endometriosis treatments are mainly symptomatic and rarely curative while also aiming for fertility preservation if required. All these factors contribute to the 6–8 years (average 6.7 years, measured in 16 clinical centres across 10 countries) waiting time between symptoms onset and endometriosis diagnosis (Nnoaham et al., 2011).\nExtracellular vesicles (EV) are nano-sized membrane-bound vesicles produced by almost all cells in the body. EVs comprise a heterogeneous population and over the years various terms have been used to describe these vesicles, including exosomes, ectosomes, microvesicles (MVs), and microparticles ( Fig. 1 ). The first investigations to describe EVs were two studies ( Harding  et al. , 1984 ;  Pan  et al. , 1985 ) that observed 50 nm vesicles released from maturing reticulocytes with transferrin receptors through the process of intraluminal budding of multivesicular endosomes that bind to the lipid cell membrane before their release into the circulation. These vesicles were named ‘exosomes’ by Rose Johnstone, although before the 1980s the term had been used to describe different phenomena such as ‘membrane fragments’ ( De Broe  et al. , 1977 ), platelet ‘dust’, or cellular debris ( Wolf, 1967 ). Ostensibly, the blood-derived pro-coagulant membrane-derived particles isolated by  Chargaff and West (1946)  were the platelet ‘dust’ isolated by  Wolf (1967) , therefore it could be argued that EVs were first observed in 1946.The term was even used to describe isolated 40–1000 nm ‘exosome complexes’ secreted by neoplastic cell lines with a 5′ nucleotidase activity ( Trams  et al. , 1981 ). Nevertheless, in 1996, these EVs gained renewed research interest with the exosome secretion of Epstein–Barr-virus-transformed B cells ( Raposo  et al. , 1996 ).\nThe diversity of extracellular vesicles produced by cells in the human body.  Compared to exosomes, MVs can be defined as EVs that are released directly from the plasma membrane and are on an average larger than exosomes, although their sizes could range from 30 nm to 1 µm ( Jeppesen  et al. , 2019 ). The terms ‘ectosomes’ and ‘microparticles’ are synonymously used to describe MVs. Like exosomes, MVs are essential for cellular communication and carry cargo such as mRNA, miRNA, lncRNA, and protein. Apoptotic bodies are released as cells undergo apoptotic cell disassembly, where the plasma membrane blebs and the apoptotic membrane protrudes and fragments. Therefore, different EV subtypes can be categorised according to their biogenesis, size, constituent protein, and isolation methods; however, the different criteria for EV subtypes often overlap, and can even contradict each other. This figure was created with BioRender.com. EV, extracellular vesicle; MV, microvesicle; mRNA, messenger RNA; miRNA, microRNA; lncRNA, long non-coding RNA.\nExosomes are small EVs [sEVs, defined as <200 nm ( Théry  et al. , 2018 )] of 30–150 nm in size. They are initially formed as an early endosome (EE) through the inward budding of the plasma membrane as it recycles receptors, proteins, and lipids back into the cell. As the EE matures into a late endosome (LE), it traffics and catalogues proteins from the Golgi apparatus, as well as genetic material from the cytoplasm, for degradation or transportation outside of the cell. The LE then forms the multivesicular body (MVB) with the invagination of the MVB membrane to form intraluminal vesicles (ILVs). When ILVs are released into the extracellular space via fusion of the MVB with the cell plasma membrane, they are defined as exosomes. Owing to their biogenesis, exosomes contain proteins involved in the MVB formation (e.g. ALIX, and TSG101), membrane trafficking (e.g. Rab GTPases and annexins), and are enriched in tetraspanins (e.g. CD9, CD63, and CD81) ( van Niel  et al. , 2006 ;  Zöller, 2009 ;  Raposo and Stoorvogel, 2013 ).\nHowever, there is no consensus on specific markers or specific separation methods for delineating different EV subtypes, beyond their size ( Fig. 1 ). There is no one separation method superior to the other, with one method sacrificing yield versus purity, and vice versa ( Théry  et al. , 2018 ;  Jia  et al. , 2022 ). The isolation method of choice will thus depend on the goal of the study, the amount and quality of sample biofluids, and the downstream analyses chosen. The studies included in this mini-review used differential ultracentrifugation (low purity containing protein complexes, medium yield, time consuming, suitable for large sample numbers), size exclusion chromatography (medium-to-high yield, high purity, suitable for low sample numbers), and ready-made isolation kits with precipitation buffers (high yield, low purity as contain polymers, suitable for large sample numbers) or magnetic capture (may be susceptible to charged particles in the sample) ( Xu  et al. , 2016 ;  Jia  et al. , 2022 ). The International Society for Extracellular Vesicles (ISEV) 2018 recommendations state that researchers should use the generic term EV unless the precise biogenesis can be captured using live imaging techniques ( Théry  et al. , 2018 ). ISEV also recommends further describing EV subtypes based on physical and biochemical characteristics and/or conditions/sources ( Théry  et al. , 2018 ). In this review, we utilise the term sEV, even though many of the studies we include may originally have referred to ‘exosomes’.\nsEVs are detected in a variety of bodily fluids ( van Niel  et al. , 2022 ). They transport cargo that can affect gene expression of target cells in remote parts of the body. Introduction of new receptors, proteins, or genetic material through sEV fusion with the target cells confer new cellular properties that mirror the parent cell’s expression ( Zomer  et al. , 2015 ). sEVs are implicated in various disease pathologies, including cancer ( Hoshino  et al. , 2015 ;  Zomer  et al. , 2015 ), diabetes ( Noren Hooten and Evans, 2020 ), cardiovascular disease ( Liu  et al. , 2021 ), neurodegenerative disease ( Vandendriessche  et al. , 2020 ) as well as in benign obstetric and gynaecological conditions such as pre-eclampsia ( Murugesan  et al. , 2022 ) and endometriosis ( Harp  et al. , 2016 ;  Nazri  et al. , 2020 ). A urinary sEV RNA-based diagnostic tool for prostate cancer ( Kretschmer  et al. , 2022 ), the ExoDx™ Prostate (IntelliScore) (EPI) test, has been used to aid clinical decisions for >50 000 patients so far and is included in the United States National Comprehensive Cancer Network guidelines for early prostate cancer detection.\nsEV cargos include the RNA species such as mRNA, piwi-interacting RNA, transfer RNA fragments, microRNA (miRNA), long non-coding RNA (lncRNA), and rRNA ( O’Brien  et al. , 2020 ). miRNAs are short, non-coding RNAs of ∼19–25 nucleotides in length, which are synthesised via DNA transcription into primary miRNAs, then processed in the cytoplasm into precursor miRNAs, and mature miRNAs ( O’Brien et al., 2018 ). miRNAs regulate post-transcriptional gene expression mainly via targeting mRNAs at the 3′ untranslated region to inhibit mRNA translation into protein or enhance mRNA instability, leading to its degradation ( O’Brien et al., 2018 ). miRNAs can also upregulate gene expression in certain microribonucleoprotein (miRNP) factors or cellular conditions ( Vasudevan, 2012 ) by controlling mRNA transcription rate and translation into proteins as they are shuttled between different subcellular compartments ( Makarova  et al. , 2016 ).\nWhile the role of miRNA as a diagnostic and therapeutic tool for endometriosis in itself has been an area of burgeoning research interest ( Agrawal  et al. , 2018 ), sEV-miRNA is better protected from degradation than free miRNA and thus conceivably the better biomarker candidate as it arguably better reflects the true pathophysiological state in the body ( Cheng  et al. , 2014 ).\nIn this mini-review, we have included all possible original articles that investigated sEV miRNAs in endometriosis regardless of sEV separation or characterisation methods ( Table 1 ). Literature searches were carried out on PubMed using the terms ‘endometriosis small extracellular vesicles miRna’, ‘endometriosis exosomes miRna’, and ‘endometriosis exosomes miRna’ and studies selected according to quality of hypothesis and methodological rigour in exploring the hypothesis. Review articles were excluded from this mini-review.\nStudies investigating the differential expression of sEV-miRNA.\nHuman samples :\nEndometriosis (stages I–IV, diagnosed via laparoscopy and confirmed with histology), n=25\nControl (tubal factor infertility), n=25\nMentioned about unbalanced menstrual cycle phases in the discussion but did not declare number of samples that were of proliferative, secretory, or menstrual phases.\nNTA:\nMean concentration:\n3.7 × 10 8  particles/ml.\nMean size:\n93±4.1 nm.\nVisualised via  TEM.\nWB :\nCD9 and CD63 positive.\nMicroarray analysis :\nEndometriosis, n=5\nControl, n=5\nfollowed by  qRT-PCR :\nEndometriosis, n=20\nControl, n=20\nHuman samples :\nEndometriosis (stages I–II, n=12; III–IV, n=30, diagnosed via laparoscopy), n=42\n*Proliferative phase samples, n=16, secretory phase samples, n=20, presumably unknown, n=6\nControl (benign ovarian teratoma, n = 17; simple ovarian cyst n = 7), n=24\n*Proliferative phase samples, n=14, secretory phase samples, n=11, total numbers do not match\nMagCapture™ Exosome Isolation Kit PS\n(FUJIFILM Wako, Japan)\nTEM:\n60–80 nm.\nNTA  not done.\nWB:\nCD9 and CD63 positive.\nMicroarray analysis :\nEndometriosis (stage I, n=1; stage III, n=2; stage IV, n=1), n=4\nControl, n=4\nfollowed by  qRT-PCR :\nEndometriosis, n=42\nControl, n=24\n↑ hsa-miR-26b-5p, hsa-miR-215-5p\n↓ hsa-miR-6795-3p\nSerum\nEutopic endometrium\nEndometriotic lesion\nEndometriosis mouse model\nHuman samples :\nEndometriosis (ovarian endometrioma, from ovarian cystectomy or oopherectomy and confirmed with histology) and control (hysterectomy for other pelvic masses):\n*Serum, n=20 each\nEutopic endometrium, n=unclear\nEndometriotic lesion, n=unclear\n*sEV source\n**Proliferative phase samples\nSamples from mice :\nEndometriosis mouse model, n=24, divided into 4 treatment groups\nTEM:\n90–120 nm.\nNTA  not done.\nWB:\nCD9 and CD63 positive.\nImmunohistochemistry  and  WB  for CCN2, α-SMA, and collagen α1.\nmiRNA extraction of all cell types and  qRT-PCR  for\nhsa-miR-214-3p.\nTransfection  of all cell types with hsa-miR-214-3p/mimics/NC and  qRT-PCR  for miRNA and CCN2.\nCo-culture  of transfected and untransfected cells followed by  qRT-PCR  for miRNA and CCN2.\nSerum sEV-miRNA:  qRT-PCR .\nEndometriosis mouse model:  sEV-miRNA uptake experiment ,  qRT-PCR .\nPlasma\nEutopic endometrium\nEndometriotic lesion PF\nHuman samples :\nEndometriosis (stages III–IV, diagnosed via laparoscopy):\nPlasma, n=6\nEutopic endometrium, n=6\nEndometriotic lesion, n=6\nPF, n=6\nControl (free from endometriosis, infertility, and gynaecological malignancies): No other information given\n*Matched, secretory phase samples\nTEM :\n30–150 nm.\nNTA  not done.\nWB:\nCD63 positive,\ncalnexin negative.\nsEV-miRNA sequencing  of all sample types followed by  qRT-PCR .\nProteomics experiments (out of review scope).\nsEV uptake experiments  by endometriotic lesion/eutopic endometrium-endothelial co-culture.\n↑ hsa-miR-27a-3p\n↓ hsa-miR-30d-5p, hsa-miR-375\nRegardless of sample types\nEutopic endometrium\nEndometriotic lesion\nHuman samples :\nEndometriosis (diagnosed via laparoscopy): eutopic endometrium, n=5; endometriotic lesion, n=5\nControl (n=5, with 4 patients with subserosal fibroids <3 cm): Eutopic endometrium, n=5\n*Secretory phase samples\nNTA :\nSize: 20–200 nm with peak size at 35 nm.\nVisualised via  TEM .\nsEV uptake experiment  by HUVECs.\nqRT-PCR  for hsa-miR-21-5p and hsa-miR-126-5p of miRNA from sEVs and cells followed by  sequencing  of total miRNA.\n↑ hsa-miR-21-5p\nBetween endometriotic lesion and eutopic endometrium\nHuman samples :\nEndometriosis (stages II–IV, infertile, diagnosed via laparoscopy), n=3\nControl (fertile, post-operative CIN II–III, malignancy excluded), n=3\n*Secretory phase samples\n**CIN: cervical intraepithelial neoplasia\nNTA :\nSize: 30–150 nm.\nVisualised via  TEM .\nFlow cytometry :\nCD63 and CD81 positive.\n↑ 26 miRNAs\n↓ 23 miRNAs\nEutopic endometrium\nEndometrioma\nHuman samples :\nEndometriosis (stages III–IV):\nEndometrioma, n=13\nEutopic endometrium, n=13\nControl (tubal factor infertility), n=13\n*Proliferative phase samples\nsEV-miRNA sequencing : Endometrioma, eutopic endometrium, control endometrium, n=3 each.\nqRT-PCR : Endometrioma, eutopic endometrium, control endometrium, n=10 each.\nDual-luciferase reporter gene assay .\nEutopic endometrium\nEndometriotic lesion\nEndometriosis mouse model\nHuman samples:\nEndometriosis (stage III–IV, ovarian endometrioma):\nPaired eutopic endometrium and *endometriotic lesion, n=24 *sEV source\nControl (laparoscopic myomectomy):\nEutopic endometrium, n=8\nSamples from mice:\nEndometriosis mouse model, n=12 divided into 3 treatment groups\n**No information about cycle phases\nTEM :\n50–100 nm.\nDynamic light scattering analysis and zeta potential analysis :\n∼65 nm particles (−20.1 mV).\nCell culture for hsa-miR-214-3p  transfection:\nPaired eutopic endometrium and endometriotic lesion, n=10, followed by  miRNA extraction and qRT-PCR for  hsa-miR-214-3p and GAPDH and  mRNA extraction and qRT-PCR  for CTGF, collagen αI and GAPDH detection.\nWB  for CTGF, αSMA, collagen αI:\nControl eutopic endometrium, n=2\nEndometriotic lesion, n=2\nIn situ \n \n hybridisation  with hsa-miR-214-3p probes:\nEndometriotic lesion, n=6\nControl eutopic endometrium, n=6 followed by qRT -PCR .\nImmunohistochemistry  for CTGF, αSMA, collagen αI of all human cell types.\nEndometriosis mouse model:  sEV-miRNA uptake experiments ,  qRT-PCR  and  histology .\nEutopic endometrium\nEndometriotic lesion\nEndometriosis mouse model\nHuman samples:\nEndometriosis (stage III–IV, ovarian endometrioma, diagnosed via laparoscopy, middle to late proliferative phase): Eutopic endometrium, *endometriotic lesion, n=24 *sEV source\nControl (tubal factor infertility): *Eutopic endometrium, n=20\n*sEV source\nSamples from mice:\nEndometriosis mouse model, n=24 divided into 2 treatment groups\n*Middle to late stage of proliferative phase\nNTA :\n∼ 80 nm.\nTEM :\n50–100 nm.\nWB :\nCD9, CD63, and HSP70 positive.\nCell culture and transfection of  hsa-miR-30c-5p / mimic/inhibitor±BCL9 and corresponding NC.\nmiRNA sequencing and microarray analysis  of eutopic and ectopic endometrial tissues:\nEndometriosis, n=5\nControl, n=5\nqRT-PCR of miRNA isolated from sEVs, tissues, and cells :\nEndometriotic lesion, n=24\nEutopic endometrium, n=20\n- Unclear number of sEV samples used\nsEV co-culture experiments  followed by  qRT-PCR.\nsEV transwell and wound healing assay.\nDual-luciferase reporter gene assay.\nEndometriosis mouse model:\nsEV uptake experiments  followed by  qRT-PCR , immunohistochemistry.\nEndometriotic lesion\nSerum\nHuman samples:\n*Endometriotic lesion from endometriosis patients, n=unclear\n*Normal human serum (no further information)\n*sEV source\n**No information about cycle phases\nNTA :\nEndometriosis sEV,\n83.68±16.85 nm\nNormal human serum sEV, 79.64±17.33 nm\nVisualised via  TEM .\nWB :\nCD63 and TSG101 positive. Calnexin negative.\nsEV treatment of macrophages , followed by  flow cytometry  for pro-repair (M2) marker and  WB  for PTEN and PI3K.\nTransfection of macrophages  with hsa-miR-301a-3p/mimic/negative control and then  sEV treatment  followed by  qRT-PCR, flow cytometry  for pro-repair (M2) marker, and  WB  for Arg-1, PTEN, and PI3K.\nHuman samples:\nEndometriosis (stages I, n=17; II, n=11; III, n=15; IV, n=11, diagnosed via laparoscopy), n=54\nControl (ovarian dermoid cyst, n=9; uterine leiomyoma, n=4, diagnosed via laparoscopy), n=13\n*Proliferative phase samples\nsEV-miRNA sequencing :\nEarly endometriosis, n=3\nAdvanced endometriosis, n=3\nControl, n=3\nfollowed by  qRT-PCR.\nPF\n(peritoneal macrophages)\nEndometriotic lesions\nHuman samples:\nEndometriosis (diagnosed via laparoscopy and confirmed with histology):\nPF, n=20\nEndometriotic lesions, n=unclear\nControl (uterine leiomyoma, tubal ligation):\nPF, n=20\n*sEV from peritoneal macrophages\n**No information about cycle phases\nNTA :\nMean concentration:\n4.1 × 10 8  particles/ml.\nMean size:\n105±3.9 nm.\nVisualised via  TEM.\nWB :\nCD9 and CD63 positive.\nCell migration and invasion assays  of sEV-treated endometriotic lesion.\nMicroarray analysis  of sEV-miRNA followed by  qRT-PCR.\nTransfection of endometriotic lesions  with hsa-miR-22-3p mimic/inhibitor/corresponding NC, followed by  qRT-PCR.\nDual-luciferase reporter gene assay.\nHuman samples:\nEndometriosis (stage III–IV, endometrioma, diagnosed via laparoscopy), n=22\nControl (including simple ovarian cyst, uterine leiomyoma, tubal ligation), n=25\n*Mentioned about that findings were regardless of secretory or proliferative phases in the results section but did not declare number of samples that were of proliferative or secretory.\nNTA :\nMedian size: 111.7 nm and the proportion of the main peak was 94.1%.\nVisualised via  TEM .\nWB :\nCD63, TSG101, and HSP70 positive. Calnexin negative.\nMicroarray analysis  of miRNA from sEVs and endometrial tissues from uterine aspirate fluid, followed by  qRT-PCR .\nsEV uptake experiments  with JNK activator anisomycin by M0 macrophages followed by  flow cytometry ,  WB,  and  MAPK phosphorylation antibody array .\nTranswell migration and invasion assay : Co-culture of endometrial tissues of control group and sEV-treated macrophages.\nTransfection of M0 macrophages  with hsa-miR-210-3p mimic/inhibitor/corresponding NC followed by  qRT-PCR  and  flow cytometry .\n↑ hsa-miR-210-3p\nRegardless of secretory or proliferative phases\nEutopic endometrium\nLeukorrhoea\nHuman samples:\nEndometriosis, n=11\nControl (hysteroscopic submucosal myomectomy, n=6; normal physical examinations, n=5), n=11\n*No information about cycle phases\nNTA :\n60–150 nm, with mean size of 95.5 nm.\nVisualised via  TEM.\nWB :\nCD63 and HSP70 positive.\nMicroarray analysis  of sEV-miRNA followed by  qRT-PCR :\nEndometriosis, n=6\nControl, n=5\nsEV-miRNA, small extracellular vesicle-microRNA; miRNA, microRNA; NTA, nanoparticle tracking analyses; TEM, transmission electron microscopy; WB, Western blot; qRT-PCR, quantitative real time PCR; PF, peritoneal fluid; TSG101, tumour susceptibility gene 101; HSP70, 70 kilodalton heat shock protein; CTGF, connective tissue growth factor—also known as CCN2; α-SMA, α-smooth muscle actin; NC, negative control; HUVEC, human umbilical vein endothelial cells; BCL9, B-cell lymphoma 9; PTEN, phosphatase and tensin homolog; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase.\n\nThe roles of sEV-miRNA have been proposed in several key mechanisms of endometriosis pathophysiology ( Table 1 ,  Figs 2  and  3 ), for instance in the immunomodulation of peritoneal macrophages (pMφ), proliferation and migration of ectopic endometriosis lesions ( Zhang  et al. , 2020b ,  2022 ;  Huang  et al. , 2022 ), and angiogenesis ( Harp  et al. , 2016 ;  Khalaj  et al. , 2019 ). Differential expression of sEV-miRNAs isolated from serum/plasma ( Khalaj  et al. , 2019 ;  Zhang  et al. , 2020a ,  2021 ;  Wu  et al. , 2022 ), leukorrhoea ( Zheng  et al. , 2023 ), eutopic endometrium ( Khalaj  et al. , 2019 ;  Zhou  et al. , 2020 ;  Wu  et al. , 2021 ;  Zheng  et al. , 2023 ), endometrioma ( Wu  et al. , 2021 ), endometriotic lesion ( Harp  et al. , 2016 ;  Wu  et al. , 2018 ;  Zhang  et al. , 2022 ), peritoneal fluid (PF) ( Chen  et al. , 2019 ;  Khalaj  et al. , 2019 ;  Zhang  et al. , 2020b ), and uterine aspirate ( Jiang  et al. , 2022 ) has been demonstrated in sEVs isolated from women with endometriosis compared to controls.\nRetrograde menstruation is only one part of the endometriosis pathophysiology.  (1) Retrograde menstruation brings endometrial cells into the peritoneal cavity. (2) sEVs are produced by all cells (endometrial cells, red blood cells, pMφ, andPMCs) in the peritoneal cavity. (3) sEVs containing miRNAs are taken up by endometrial cells, red blood cells, pMφ, and PMC, causing changes to recipient cells. (4) Uptake of sEVs and internalisation of miRNAs promotes proliferation, migration, and invasion of endometrial cells and of existing ectopic lesions, immunomodulation of macrophages, and potentially EMT changes of PMCs, although none of the studies in this review investigated the impact of sEV-miRNA on PMCs. PF-derived sEVs are likely to originate from a variety of cell types including endometrial cells, red blood cells, immune cells, ectopic lesions, and PMCs. This figure was created with BioRender.com. sEV, small extracellular vesicle; miRNA, microRNA; pMφ, peritoneal macrophage; PMC, peritoneal mesothelial cell; EMT, epithelial-to-mesenchymal transition; PF, peritoneal fluid; sEV-miRNA, small extracellular vesicle-microRNA.\nProposed sEV-miRNAs involved in the pathophysiology of endometriosis.  sEV-miRNAs involved in the pathophysiology of endometriosis through the proliferation, migration, and invasive potential of endometrial cells, immunomodulation, and angiogenesis (formation of blood vessels) to support explanted endometrial cells (ectopic lesions). Studies also investigated endometriosis-specific miRNAs and the signalling pathways involved. This figure was created with BioRender.com. sEV-miRNA, small extracellular vesicle-microRNA.\nRetrogradely spilt menstrual blood, cells, and tissue can be detected in the abdominal cavity of most women at the time of menstruation ( Halme  et al. , 1984 ). Therefore, an impaired immune response in combination with the increased proliferative and migratory activity of these cells may contribute to the establishment of endometriosis in some women ( Zondervan  et al. , 2018 ).\nSerum-derived sEVs from women with endometriosis contained higher hsa-miR-22-3p and hsa-miR-320a-3p levels compared to controls, with elevated hsa-miR-22-3p levels in stages III–IV versus stages I–II endometriosis ( Zhang  et al. , 2020a ). High hsa-miR-22-3p and hsa-miR-320a-3p levels were also correlated with high serum CA-125 levels in patients with endometriosis ( Zhang  et al. , 2020a ). Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis suggested mitogen-activated protein kinase (MAPK) activity, among others ( Zhang  et al. , 2020a ). Increased pMφ-derived sEV-hsa-miR-22-3p in patients compared to controls regulates the sirtuin 1/nuclear factor κB (SIRT1/NF-κB) signalling pathway leading to enhanced ectopic endometrial stromal cells (ESCs) proliferation, migration, and invasion ( Zhang  et al. , 2020b ).\nPro-repair phenotype polarisation and reduced phagocytic activity in Mφ exposed to lesion-derived sEVs were mediated by hsa-miR-301a-3p through phosphatidylinositol 3-kinase (PI3K) upregulation and tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) downregulation ( Huang  et al. , 2022 ). These findings echoed a previous study in an endometriosis mouse model which also revealed decreased total weight and volume of endometriotic lesions following treatment with ESC-derived sEVs ( Sun  et al. , 2019b ). Elevated hsa-miR-210-3p in uterine-aspirate-fluid-derived sEVs and eutopic endometrium of patients with endometriosis decreased CD80 +  Mφ (a surface marker of pro-inflammatory Mφ) by suppressing c-Jun N-terminal kinase (JNK, protein kinase in MAPK pathway) phosphorylation in Mφ, thus increasing pro-repair Mφ, which could escape immune surveillance. In support of these findings, sEV-treated Mφ increased proliferation, migration, and invasion of endometriosis ESCs compared to control ESCs in a transwell experiment ( Jiang  et al. , 2022 ). In another study, quantitative RT-PCR-verified endometriosis-specific serum-derived sEV-hsa-miR-26b-5p (downregulated), hsa-miR-215-5p (downregulated), and hsa-miR-6795-3p (upregulated) were found to be involved in the PI3K, MAPK, and protein kinase B (Akt) pathways in a KEGG analysis. Serum-derived sEV-hsa-miR-26b-5p expression was lower in rASRM stages III–IV versus stages I–II endometriosis ( Wu  et al. , 2022 ).\nIn a KEGG analysis of 49 differentially expressed sEV-miRNAs from eutopic endometrium of women with endometriosis-related infertility and control endometrium, the Wnt signalling pathway ( Zhou  et al. , 2020 ) was also implicated, in addition to MAPK, confirming previous findings ( Wu  et al. , 2022 ;  Zhang  et al. , 2022 ). Homeobox A10 (HOXA10) and/or leukaemia inhibitory factor (LIF)—targets of 12 miRNAs—were decreased in eutopic endometrium of women with endometriosis-related infertility compared to controls ( Zhou  et al. , 2020 ).\nHigher levels of leukorrhoea-derived sEV-hsa-miR-202-3p and hsa-miR-202-5p in patients with endometriosis compared to controls corresponded to levels in endometriosis eutopic endometrium ( Zheng  et al. , 2023 ).\nNeo-angiogenesis, the formation of new blood vessels from existing ones, is a process involved in many physiological (e.g. corpus luteum formation, endometrial proliferation) and pathological processes (e.g. cancer, chronic inflammation) ( Folkman, 2001 ). Retrogradely transplanted endometriotic cells and tissue require a blood supply for proliferation ( May and Becker, 2008 ).\nCultured human umbilical vein endothelial cells (HUVEC) treated with eutopic ESC-derived sEVs from patients with endometriosis exhibited more extensive tube formation compared to those treated with equivalent sEVs from controls ( Harp  et al. , 2016 ). In this study, ectopic lesion-derived sEVs contained elevated (pro-angiogenic) hsa-miR-21-5p compared to eutopic ESC-derived sEVs from controls. However, it is uncertain what assumptions can be made from this comparison without also comparing eutopic ESC-derived and possibly PF-derived sEVs from patients and controls. Although no specific sEV-miRNAs were investigated,  Sun  et al.  (2019a ) observed greater tube formation in endometriosis eutopic ESC-derived sEV-treated HUVECs compared to control eutopic ESC-derived sEVs. Treating HUVECs with plasma-derived sEVs from patients with endometriosis revealed similar findings ( Khalaj  et al. , 2019 ).  Sun  et al.  (2019a ) also observed increased neurite outgrowth induced by endometriosis eutopic ESC-derived sEVs compared to control.\nIn an RNA sequencing study of control and endometriosis eutopic ESC- and endometrioma-derived sEV-RNA, hsa-miR-15a-5p, involved in vascular endothelial growth factor A ( VEGF-A)  downregulation, was lower in the cell culture supernatant of patients with endometriosis versus control ( Wu  et al. , 2021 ). The relation between hsa-circ_0026129, hsa-miR-15a-5p, and  ATP6V1A  was uncovered in an sEV-competing endogenous RNA (cERNA) network analysis. This showed that increased sEV-hsa-circ_0026129 correlated with decreased hsa-miR-15a-5p and increased  ATP6V1A  expression in ovarian endometriomas. Dual luciferase reporter assays confirmed that hsa-circ_0026129 competitively binds to hsa-miR-15a-5p, which contributed to low hsa-miR-15a-5p levels thus increasing  ATP6V1A  expression in ovarian endometriomas compared to controls.  ATP6V1A  is an oncogene of endometrial cancer, which is linked to the severity of endometriosis and endometrial receptivity ( Wu  et al. , 2021 ).\nPF is rich in immune cells, prostaglandins, interleukin, cytokines, and growth factors that can influence the growth of endometrial lesions ( Koninckx, 1998 ), and owing to its proximity and direct contact with endometriotic lesions, investigating PF for endometriosis-specific sEVs seems a rational approach. Studying the PF could identify the different sEV populations from different cells (e.g. pMφ, red blood cells, endometrial cells, ectopic lesions, peritoneal mesothelial cells), and correlating PF-derived sEV-miRNAs in more accessible fluids (e.g. serum, urine, or saliva) may provide a comprehensive picture of sEV-miRNAs involved in endometriosis and their utility in diagnostics.\nIn total, four studies investigated PF-derived sEVs in endometriosis. Out of these studies, two groups investigated PF-derived sEV-proteins ( Khalaj  et al. , 2019 ;  Nazri  et al. , 2020 ). At present, three studies investigated PF-derived sEV-miRNAs in endometriosis ( Chen  et al. , 2019 ;  Khalaj  et al. , 2019 ) including one study reporting on pMφ-derived sEVs ( Zhang  et al. , 2020b ).\nIncreased PF-derived sEV-hsa-miR-130-3p levels, implicated in immune cell function, were found in patients with endometriosis versus controls. In stage III–IV endometriosis, increased PF-derived sEV-hsa-miR-451a and hsa-miR-486-5p were found ( Chen  et al. , 2019 ).  Khalaj  et al.  (2019)  attempted to compare the small RNA and proteome content of endometriotic lesion-derived sEVs versus patients’ matched eutopic endometrium and controls, as well as PF- and plasma-derived sEVs. Accounting for sample types, sEV-miRNAs unique to endometriosis include hsa-miR-30d-5p (upregulated), hsa-miR-27a-3p (downregulated), and hsa-miR-375 (downregulated). Using TargetScan (an online repository for miRNA biological target prediction), Khalaj  et al.  identified hsa-miR-30d-5p binding sites for thrombospondin-2, alpha-2-antiplasmin, and interferon regulatory factor 4, whereas hsa-miR-375 has binding sites for platelet-derived growth factor subunit A—all of which suggested angiogenesis and pro-inflammatory pathways ( Khalaj  et al. , 2019 ).\n\nWhile there is considerable potential for sEV-miRNAs to serve as diagnostic markers for endometriosis, so far only one study has reported the sensitivity and specificity of sEV-miRNAs in diagnosing endometriosis ( Zhang  et al. , 2020a ). For serum-derived sEV-hsa-miR-22-3p, the AUC was higher at 85.5% than the AUC of hsa-miR-320a-3p at 82.7%. Combining both miRNAs, the AUC was 88.3% ( Zhang  et al. , 2020a ), which is considered to be an encouraging AUC score in discriminating between patients with endometriosis and controls.\n\nFourteen studies had identified the differential expression of sEV-miRNAs in endometriosis ( Table 1 ), however, only three studies ( Wu  et al. , 2018 ;  Zhang  et al. , 2021 ,  2022 ) investigated the therapeutic potential of sEV-miRNA in endometriosis  in vivo , as described below.\nEndometriosis is a heterogeneous disease with a large range of phenotypes ( Zondervan  et al. , 2020 ). Although red lesions have been shown to express high levels of VEGF and other growth factors, and are therefore considered by some as the most active, fibrosis is a molecular hallmark and a frequently encountered clinical problem ( Burney, 2022 ). Therefore, non-surgical treatment and/or prevention of fibrosis is an urgently unmet clinical need.\nIn a study investigating liver fibrosis ( Chen  et al. , 2014 ), sEV-hsa-miR-214-3p decreased the expression of connective tissue growth factor (CTGF) consequently decreasing the fibrosis of hepatic stellate cells. Thus, hsa-miR-214-3p could be a promising therapeutic target for other fibrotic diseases, including endometriosis, by targeting CTGF. In an endometriosis rodent model (induced by i.p. injection of human endometriosis eutopic ESCs), fibrosis reduction in ectopic lesions was observed when treated with human ectopic ESC-derived sEVs transfected with hsa-miR-214-3p mimics, through CTGF and collagen reduction in ESCs and endometrial epithelial cells (EECs) (n = 3), compared to mice treated with control sEVs (n = 3) and PBS (n = 3) ( Wu  et al. , 2018 ).\nUsing a modified,  Burns  et al.  (2012)  protocol to induce endometriosis in nude mice (n = 24)—where one experimental group was treated with uterine tissue derived from CTGF-knock-out mice and the other with uterine tissue from wild-type mice—the above findings ( Wu  et al. , 2018 ) were confirmed ( Zhang  et al. , 2021 ). The endometriosis-induced mice (n = 24) were divided into four treatment groups (n = 6) of i.p. injections of saline, human serum-derived sEVs, hsa-miR-214-3p mimics, or sEV-hsa-miR-214-3p. This revealed increased fibrosis-related protein expression in endometriotic lesions of sEV-hsa-miR-214-3p-treated mice compared to other treatment groups. Additionally, no differences were seen in mice induced with CTGF-knock-out uterine tissue, confirming the sEV-hsa-miR-214-3p’s CTGF-dependent actions. Finally, hsa-miR-214-3p downregulation and CTGF upregulation in ectopic lesions and eutopic endometrium of patients with endometriosis compared to eutopic endometrium of controls ( Wu  et al. , 2018 ;  Zhang  et al. , 2021 ) correlated with lower serum sEV-hsa-miR-214-3p in patients with endometriosis ( Zhang  et al. , 2021 ).\nA hypothesis of endometriosis pathophysiology ( Nazri  et al. , 2020 ) had likened the formation of the endometriotic lesion to the ‘seed and soil’ hypothesis of cancer ( Paget, 1889 ), with sEVs causing increased invasive and migratory potential of retrogradely menstruated endometrial cells, similar to how tumour metastasis is instigated by sEVs ( Hoshino  et al. , 2015 ). In a study by  Zhang  et al.  (2022) , ectopic EECs from endometrioma patients produced sEVs with low hsa-miR-30c-5p, leading to BCL9 (B-cell lymphoma 9) overexpression. BCL9, a co-activator of the Wnt/β-catenin pathway has been implicated in breast cancer metastasis (Wang et al., 2021) and hence may shed light on the invasive and migratory potential of endometriotic lesions.\nIn contrast, EEC-derived sEVs from control patients transferred hsa-miR-30c-5p, which targets BCL9, thus suppressing the invasive and migratory potential of ectopic EECs ( Zhang  et al. , 2022 ). Blockage of the BCL9/Wnt/CD44 pathway by sEV-hsa-miR-30c-5p overexpression attenuated ectopic EEC invasive potential in an endometriosis nude mice model injected with sEVs pre-treated with hsa-miR-30c-5p mimic (n = 12, each). Notably, BCL9, Wnt1, β-catenin, c-myc, cyclin D1, CD44, vimentin, and N-cadherin expression was reduced, and E-cadherin increased, corresponding to increased hsa-miR-30c-5p levels. Indeed, attenuating the BCL9/Wnt/CD44 pathway via sEV-hsa-miR-30c-5p overexpression was associated with fewer ectopic nodules in the intestinal walls of nude mice with endometriosis compared to controls ( Zhang  et al. , 2022 ).\n\nEndometriosis is a common heterogeneous disease which lacks clinically reliable non-invasive diagnostic tools as well as disease-specific therapeutic approaches. In this review, we present the currently available literature on sEV-miRNA and its diagnostic and therapeutic potential in the disease. Their roles were studied in various biological fluids and tissues including PF, serum and plasma, leukorrhoea, uterine aspirate fluid, endometrioma as well as eutopic and ectopic endometriotic lesions. However, no studies investigated sEV-miRNAs from peritoneal mesothelial cells ( Fig. 2 ), and since they play a central role in endometriosis ( Koks  et al. , 2000 ;  Nair  et al. , 2008 ;  Griffith  et al. , 2010 ;  Knudtson  et al. , 2020 ) this is a clear gap in the field and one that warrants further investigation. Additionally, no groups have investigated saliva- or urine-derived sEV-miRNAs.\nOf the 14 studies included here, six focused on rASRM stages III–IV endometriosis ( Wu  et al. , 2018 ,  2021 ;  Khalaj  et al. , 2019 ;  Zhang  et al. , 2021 ,  2022 ;  Jiang  et al. , 2022 ). Four studies did not give information about the rASRM stages of endometriosis samples ( Harp  et al. , 2016 ;  Zhang  et al. , 2020b ;  Huang  et al. , 2022 ;  Zheng  et al. , 2023 ). One study investigated rASRM stages II–IV ( Zhou  et al. , 2020 ) and only three studies investigated all rASRM stages ( Chen  et al. , 2019 ;  Zhang  et al. , 2020a ;  Wu  et al. , 2022 ) although data analyses according to rASRM stage were not included in these studies. Investigating stages I–II endometriosis separately from stages III–IV is essential as increasing evidence shows that these two combined stages of endometriosis could represent different disease entities altogether, with different genetic and biological pathways underpinning the pathophysiology ( Painter  et al. , 2011 ;  Sapkota  et al. , 2017 ;  Uimari  et al. , 2017 ). In addition, testing sEV-miRNAs for their diagnostic potential would arguably be more relevant for superficial disease, which is difficult to visualise even using modern imaging technologies ( Nisenblat  et al. , 2016a ).\nAn additional problem is posed by the control samples used for some studies. Samples from women suffering from other gynaecological abnormalities, such as uterine leiomyomas, ovarian cysts, and ovarian teratomas, may confound results ( Table 1 ). For example, one study reported that sEV-miRNA from human uterine leiomyoma cells was associated with increased proliferation and angiogenesis of uterine leiomyomata ( Navarro  et al. , 2022 ). Similarly, sEVs from simple ovarian cysts and ovarian cancer have been used to stimulate lymphocytes to understand the differences in gene expression ( Li  et al. , 2017 ). Three studies had recruited patients with tubal factor infertility as controls ( Zhang  et al. , 2020a ,  2022 ;  Wu  et al. , 2021 ), although the current implications of this condition on sEV populations are not known. Arguably the most suitable controls in these studies would be those with pelvic pain and negative laparoscopic findings, i.e. symptomatic controls ( Tapmeier  et al. , 2020 ), although the difficulty of obtaining appropriate controls is widely recognised.\nNone of the studies investigated sEV-miRNA in all rASRM stages of endometriosis and all phases of the menstrual cycle, including menstruation. As endometriosis is an oestrogen-dependent disease, one could speculate that the changes in sEV cargo and miRNAs may correlate to changes in oestrogen and progesterone levels. Two studies acquired samples in proliferative and secretory phases ( Jiang  et al. , 2022 ;  Wu  et al. , 2022 ), four studies only in the proliferative phase ( Chen  et al. , 2019 ;  Wu  et al. , 2021 ;  Zhang  et al. , 2021 ,  2022 ), and three studies in the secretory phase ( Harp  et al. , 2016 ;  Khalaj  et al. , 2019 ;  Zhou  et al. , 2020 ). Five studies provided no information about the menstrual cycle phases of their samples ( Wu  et al. , 2018 ;  Zhang  et al. , 2020a , b ;  Huang  et al. , 2022 ; Zheng et al.; 2023). Therefore, regrettably, at the time of writing no research groups have yet analysed and compared sEV-miRNA and -protein in  all  menstrual cycle phases and  all  rASRM stages of patients with endometriosis and controls.\nOnly one study demonstrated the potential for sEV-miRNA as a diagnostic tool for endometriosis although not without the limitations discussed above ( Zhang  et al. , 2020a ). Three studies ( Wu  et al. , 2018 ;  Zhang  et al. , 2021 ,  2022 ) evaluated the potential therapeutic properties of sEV-miRNAs  in vivo . However, what remains to be investigated is whether treating endometriosis with sEV-has-miR-214-3p ( Wu  et al. , 2018 ;  Zhang  et al. , 2021 ) or has-miR-30c-5p ( Zhang  et al. , 2022 ) would translate to endometriosis symptom relief.\nMost importantly, none of these studies could justify using the term ‘sEVs’ or ‘exosomes’ as defined by the ISEV 2018 guidelines ( Théry  et al. , 2018 ). The recommendations stipulated the use of certain separation methods for specifc downstream analyses taking into account the biological fluid the sEVs are derived from, knowing that there is a trade-off between yield and purity. Compared to ultracentrifugation, differential centrifugation or size-exclusion chromatography, isolating sEVs using precipitation methods ( Harp  et al. , 2016 ;  Khalaj  et al. , 2019 ;  Zhou  et al. , 2020 ;  Wu  et al. , 2021 ;  Zhang  et al. , 2021 ) results in a high yield but risks sacrificing EV purity ( Gurunathan  et al. , 2019 ). To be able to use the term ‘sEVs’, studies must show quantiative analyses of particle yield, with a global concentration of particle yield and particle-to-protein or particle-to-lipid ratios, as well as demonstrating EV proteins from at least three categories (transmembrane, cytosolic, purity control proteins), and visualising the sEVs as single particles. Four studies did not report any quantitative analyses of the particles ( Table 1 ) ( Chen  et al. , 2019 ;  Khalaj  et al. , 2019 ;  Zhang  et al. , 2021 ;  Wu  et al. , 2022 ). Three studies neglected to perform experiments to confirm sEV proteins ( Harp  et al. , 2016 ;  Wu  et al. , 2018 ;  Chen  et al. , 2019 ). Note that  Chen  et al.  (2019)  did not provide any information about its EV characterisation methods and  Wu  et al.  (2021)  only mentioned the EV characterisation methods that they employed in their study but did not share the results. Therefore, none of the studies that performed sEV confirmation experiments satisfied the ISEV requirements. With promising results from these studies ( Table 1 ), it is unfortunate that these studies fall short of the minimum requirements of sEV separation methods.\nAny study must be independently replicated and validated in a separate cohort, ideally in large multi-national, multi-centre cohort studies. For such studies to be comparable, they should follow ISEV guidance with regards to sEV isolation ( Théry  et al. , 2018 ); in addition, studies in endometriosis ought to adhere to the WERF EPhect (World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project) protocols when collecting samples and data (Becker et al., 2014; Fassbender et al., 2014; Rahmioglu et al., 2014; Vitonis et al., 2014).\n\nEndometriosis is a chronic inflammatory disease with a significant impact on women, their families, and society. The lack of clinically usable biomarkers remains one of the obstacles to shortening waiting times between the first symptoms and endometriosis diagnosis. Currently available medical therapies are mainly aimed at hormonal suppression and disregard the heterogeneity of the biological phenotype and clinical need in women suffering from pain and infertility. sEV-miRNAs could be a promising tool to better understand endometriosis pathophysiology or a potential diagnostic and therapeutic tool. Unfortunately, research in this area is hindered by a lack of standardisation in separating and characterising sEVs and adherence to the WERF EPhect protocols.","source_license":"CC0","license_restricted":false}