Evidence for the role of extracellular vesicles (EVs) in endometriosis pathophysiology suggests EVs may be a novel avenue for biomarker exploration. This study employed small particle flow cytometry to investigate plasma EVs with endometrial stromal cell markers and cervicovaginal fluid EVs with immunological receptors as diagnostic biomarkers of endometriosis.
Methods:
Blood and cervicovaginal fluid were collected from consenting patients undergoing surgery for suspected endometriosis. EVs were isolated from plasma (n = 15 per group) and cervicovaginal fluid (n = 20 per group), stained overnight with antibody probes, labelled with Aco-430 membrane stain then analysed by flow cytometry (Cytek Aurora). The proportions and antigen density of positive EVs were compared between people with endometriosis and symptomatic controls.
Results:
Measuring triple-positive EVs was deemed unsuitable given the variability in staining when multiplexing antibodies. Small proportions of antigen positive EVs were detected for all proteins investigated, however, no differences were observed for CD10+, CD90+, CD10+CD90+ and CD140b+ plasma EVs, or CCR2+, CD55+, CD59+ and CD82+ cervicovaginal fluid EVs.
Conclusions:
Although the EV populations did not distinguish endometriosis from symptomatic controls, this study provides valuable methodological insights into EV phenotyping to support future biomarker research and advance understanding of EV-mediated mechanisms in endometriosis.
NisenblatVBossuytPMMFarquharC, et al. Imaging modalities for the non-invasive diagnosis of endometriosis. Cochrane Database Syst Rev2016; 2(2): CD009591.
2.
AveryJCDeslandesAFregerSM, et al. Noninvasive diagnostic imaging for endometriosis part 1: a systematic review of recent developments in ultrasound, combination imaging, and artificial intelligence. Fertil Steril2024; 121(2): 164–188.
3.
HanlyCLeonardiMEathorneA, et al. The impact of diagnostic method on sense of control and powerlessness and social support in endometriosis patients-a retrospective cohort study. Acta Obstet Gynecol Scand2023; 102(10): 1390–1395.
4.
PatersonESJScheckSMcDowellS, et al. Retrospective review of endometriosis surgery at Te Whatu Ora: capital and coast. Aust N Z J Obstet Gynaecol2024; 64(3): 204–209.
5.
BrulportABourdonMVaimanD, et al. An integrated multi-tissue approach for endometriosis candidate biomarkers: a systematic review. Reprod Biol Endocrinol2024; 22(1): 21.
6.
ScheckSMHenryCBedfordN, et al. Non-invasive tests for endometriosis are here; How reliable are they, and what should we do with the results?Aust N Z J Obstet Gynaecol2024; 64(2): 168–170.
7.
WelshJAGoberdhanDCIO’DriscollL, et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles2024; 13(2): e12404.
8.
O’GradyTNjockM-SLionM, et al. Sorting and packaging of RNA into extracellular vesicles shape intracellular transcript levels. BMC Biol2022; 20(1): 72.
9.
ScheckSPatersonESJHenryCE.A promising future for endometriosis diagnosis and therapy: extracellular vesicles - a systematic review. Reprod Biol Endocrinol2022; 20(1): 174.
10.
CousinsFLGargettCE.Endometrial stem/progenitor cells and their role in the pathogenesis of endometriosis. Best Pract Res Clin Obstet Gynaecol2018; 50: 27–38.
11.
HsuCYHsiehTHLinHY, et al. Characterization and proteomic analysis of endometrial stromal cell–derived small extracellular vesicles. J Clin Endocrinol Metab2021; 106(5): 1516–1529.
12.
WangYDragovicRAGreavesE, et al. Macrophages and small extracellular vesicle mediated-intracellular communication in the peritoneal microenvironment: impact on endometriosis development. Front Reprod Health2023; 5: 1130849.
13.
Martínez-ZamoraMAArmengol-BadiaOQuintas-MarquèsL, et al. Macrophage phenotype induced by circulating small extracellular vesicles from women with endometriosis. Biomolecules2024; 14(7): 737.
14.
García-RomeroNCarrión-NavarroJEsteban-RubioS, et al. DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients. Oncotarget2017; 8(1): 1416–1428.
15.
MatsumotoJStewartTShengL, et al. Transmission of α-synuclein-containing erythrocyte-derived extracellular vesicles across the blood-brain barrier via adsorptive mediated transcytosis: another mechanism for initiation and progression of Parkinson’s disease?Acta Neuropathol Commun2017; 5(1): 71.
16.
ChenCCLiuLMaF, et al. Elucidation of exosome migration across the blood–brain barrier model in vitro. Cell Mol Bioeng2016; 9(4): 509–529.
17.
TaylorHSKotlyarAMFloresVA.Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet2021; 397(10276): 839–852.
18.
AldermanMHIIIYoderNTaylorHS.The systemic effects of endometriosis. Semin Reprod Med2017; 35(3): 263–270.
19.
KhalajKMillerJELingegowdaH, et al. Extracellular vesicles from endometriosis patients are characterized by a unique miRNA-lncRNA signature. JCI Insight2019; 4(18): e128846.
20.
PengCZhouYZhangD, et al. Identification of a serum exosome-derived lncRNA‒miRNA‒mRNA ceRNA network in patients with endometriosis. Clin Exp Obstet Gynecol2024; 51(2): 51.
21.
MunrosJMartinez-ZamoraMATassiesD, et al. Total circulating microparticle levels are increased in patients with deep infiltrating endometriosis. Hum Reprod2017; 32(2): 325–331.
22.
Carrillo TorresPMartínez-ZamoraMTàssiesD, et al. Impact of continuous estroprogestin treatment on circulating microparticle levels in deep endometriosis patients. Int J Mol Sci2023; 24(14): 11802.
23.
KajdosMSzymanskiJJerczynskaH, et al. Microvesicles released from ectopic endometrial foci as a potential biomarker of endometriosis. Ginekol Pol2023; 94(10): 780–791.
24.
WuYYuanWDingH, et al. Serum exosomal miRNA from endometriosis patients correlates with disease severity. Arch Gynecol Obstet2022; 305(1): 117–127.
25.
ZhangLLiHYuanM, et al. Serum exosomal MicroRNAs as potential circulating biomarkers for endometriosis. Dis Markers2020; 2020: 2456340.
26.
ShanSYangYJiangJ, et al. Extracellular vesicle-derived long non-coding RNA as circulating biomarkers for endometriosis. Reprod Biomed Online2022; 44(5): 923–933.
27.
LiWNHsiaoKYWangCA, et al. Extracellular vesicle-associated VEGF-C promotes lymphangiogenesis and immune cells infiltration in endometriosis. Proc Natl Acad Sci USA2020; 117(41): 25859–25868.
28.
QiuJJLinYYTangXY, et al. Extracellular vesicle-mediated transfer of the lncRNA-TC0101441 promotes endometriosis migration/invasion. Exp Cell Res2020; 388(1): 111815.
29.
QiuJJLinXJZhengTT, et al. The exosomal long noncoding RNA aHIF is upregulated in serum from patients with endometriosis and promotes angiogenesis in endometriosis. Reprod Sci2019; 26(12): 1590–1602.
30.
SunSGGuoJJQuXY, et al. The extracellular vesicular pseudogene LGMNP1 induces M2-like macrophage polarization by upregulating LGMN and serves as a novel promising predictive biomarker for ovarian endometriosis recurrence. Hum Reprod2022; 37(3): 447–465.
31.
ZhuYGaoLZhangJ, et al. Extracellular vesicle-packaged PKM2 from endometriotic stromal cells promotes endometrial collagen I deposition by inhibiting autophagy in endometriosis. Cell Signal2025; 127: 111523.
32.
ChenYWangKXuY, et al. Alteration of myeloid-derived suppressor cells, chronic inflammatory cytokines, and exosomal miRNA contribute to the peritoneal immune disorder of patients with endometriosis. Reprod Sci2019; 26(8): 1130–1138.
33.
JiSQiHYanL, et al. miR-4443 contained extracellular vesicles: a factor for endometriosis progression by PI3K/AKT/ACSS2 cascade in-vitro. Int J Nanomed2024; 19: 6085–6098.
34.
NazriHMImranMFischerR, et al. Characterization of exosomes in peritoneal fluid of endometriosis patients. Fertil Steril2020; 113(2): 364–373.e2.
35.
LeeSRLeeJCKimSH, et al. Altered composition of microbiota in women with ovarian endometrioma: microbiome analyses of extracellular vesicles in the peritoneal fluid. Int J Mol Sci2021; 22(9): 4608.
36.
ChengLSharplesRASciclunaBJ, et al. Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood. J Extracell Vesicles2014; 3(1): 23743.
37.
BeneckeLChiangDMEbnoetherE, et al. Isolation and analysis of tumor-derived extracellular vesicles from head and neck squamous cell carcinoma plasma by galectin-based glycan recognition particles. Int J Oncol2022; 61(5): 133.
38.
American Society for R. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril1997; 67(5): 817–821.
39.
PatersonESJScheckSMcDowellS, et al. Comparison of cervicovaginal fluid extracellular vesicles isolated from paired cervical brushes and vaginal swabs. J Extracell Biol2024; 3(5): e153.
40.
WelshJAJonesJC.Small particle fluorescence and light scatter calibration using FCMPASS software. Curr Protoc Cytom2020; 94(1): e79.
41.
Spherotech. STN-14 Rev B Determining PMT linearity in flow cytometers using the SpheroTM PMT quality control excel template. Spherotech Inc, https://www.spherotech.com/tech_SpheroTech_Note_14.html (2019, accessed 19 September 2024).
42.
KonradLKortumJNabhamR, et al. Composition of the Stroma in the Human Endometrium and Endometriosis. Reprod Sci2018; 25(7): 1106–1115.
43.
IwaseAKotaniTGotoM, et al. Possible involvement of CD10 in the development of endometriosis due to its inhibitory effects on CD44-dependent cell adhesion. Reprod Sci2014; 21(1): 82–88.
44.
ShippMAStefanoGBSwitzerSN, et al. CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide-induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation. Blood1991; 78(7): 1834–1841.
45.
BezmanNAKimCCSunJC, et al. Molecular definition of the identity and activation of natural killer cells. Nat Immunol2012; 13(10): 1000–1009.
46.
MasonJCYarwoodHTárnokA, et al. Human Thy-1 is cytokine-inducible on vascular endothelial cells and is a signaling molecule regulated by protein kinase C. J Immunol1996; 157(2): 874–883.
47.
TianYMaLGongM, et al. Protein profiling and sizing of extracellular vesicles from colorectal cancer patients via flow cytometry. ACS Nano2018; 12(1): 671–680.
48.
AibaidulaAZFainCEGarciaLC, et al. Spectral flow cytometry identifies distinct nonneoplastic plasma extracellular vesicle phenotype in glioblastoma patients. Neurooncol Adv2023; 5(1): vdad082.
49.
PalominoWATayadeCArgandoñaF, et al. The endometria of women with endometriosis exhibit dysfunctional expression of complement regulatory proteins during the mid secretory phase. J Reprod Immunol2018; 125: 1–7.
50.
ArabTMallickERHuangY, et al. Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms. J Extracell Vesicles2021; 10(6): e12079.
51.
van de WakkerSIBauzá-MartinezJRíos ArceoC, et al. Size matters: functional differences of small extracellular vesicle subpopulations in cardiac repair responses. J Extracell Vesicles2024; 13(1): e12396.
52.
AhmedEMasudMKWuethrichA, et al. Ultrasensitive multiplex detection of lung cancer EV-associated immune checkpoints using a mesoporous gold enhanced SERS biosensor. Nanoscale2025; 17(48): 28143–28154.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
