Table 1 Key characteristics of studies of extracellular vesicles in endometriosis

Chen 2019 [23]

Human

(n = 6)

Human (n = 3)

Peritoneal fluid

UC

No

Small RNA species (seq). RT-qPCR: miR-130-3p validated, miR-1908-5p not statistically significant

Not discussed, but miR-130-3p a potential diagnostic target

Not discussed

EVs contain miRNAs that may contribute to immunomodulation and cell proliferation in endometriosis

Limited numbers. Only validated 2 miRNAs via RT-qPCR. No description of UC methods

Feng 2020 [24]

Human

(n = 6)

None

CCM of primary umbilical cord derived MSCs

Exosome kit (#E1310; Bioruo)

Partially

No investigation

Not discussed

Not discussed

EVs from MSCs may enhance cell migration of endometrial cells

Limited numbers. EV source and endometriosis tissue from different patients

Harp 2016 [19]

Human

(n = 5)

Human

(n = 5)

CCM of ESCs derived from endometrial biopsies and endometriosis lesion biopsies

UC

Yes

miRNAs (seq). RT-qPCR validation of

miR-21-5p (p < 0.0001), miR-126-5p not significantly different

Not discussed, but miR-21-5p a potential diagnostic target

Not discussed

EVs from endometrium in endometriosis patients may promote angiogenesis

Limited numbers

Hsu 2021 [25]

Human

(n = 3)

Human (n = 3)

CCM of ESCs derived from endometrioma and endometrial biopsy

UC

Yes

Annexin A2 (ANXA2)

Not discussed, but potential diagnostic target

Not discussed

EVs from endometriosis lesions not found in endometrium may promote cell migration and angiogenesis

Limited numbers. May be specific to endometrioma only. Controls are endometrial biopsies from patients with endometriosis

Huang 2022 [26]

Not stated

Not stated

Endometrial tissue and normal human serum

UC

Partially

No investigation

Not discussed

Downregulation of miR-301a-3p reduces macrophage activity

miR-301a-3p inhibition reduced PI3K expression and promotes PTEN expression in macrophages

Overexpression of miR-301a-3p in endometriosis lesions increases macrophage activity

EVs from endometrial tissue increase M2 macrophage polarisation, through upregulation of PI3K and downregulation of PTEN

Study does not specify number of participants. Limited description of tissue source and methods. Compared tissue from lesions with serum from controls. Imply EV miR-301a-3p is the mechanism for EV induced changes in macrophage activity without demonstrating EVs contain miR-301-3a

Khalaj 2019 [18]

Human

(n = 6)

Number not specified

Endometriosis lesions, endometrial biopsy, peritoneal fluid and plasma

MiRCURY exosome isolation kit (#300,102; Exiqon Inc)

Partially

Small RNA species (seq). RT-qPCR validation of miR-27a, miR-200c, miR-200a-5p, miR-375 and let-7a. miR–30d-5p, miR-100, miR-136 and miR-193b, miR-206 not significantly different.

Potential diagnostic targets: Unique EV miRNA signatures differing between: endometrium from endometriosis patients and controls, plasma from endometriosis patients and controls, and matched endometrium and endometriosis lesions.

miR-30d-5p, miR-27a-3p and miR-375 network unique to endometriosis

Not discussed

Increased angiogenesis, cell growth and pro-inflammatory effects in presence of EVs from endometriosis lesions

Limited numbers. Control samples not clearly stated in methods

Lee 2021 [27]

Human

(n = 45)

Human (n = 45)

Peritoneal fluid

UC

Partially

Microbiota composition of EVs

Not discussed, but potential diagnostic targets

Not discussed

Microbiome of peritoneal fluid and/or genital tract may influence development of endometriosis

May be specific to women with endometrioma

Li 2020 [28]

Human

(serum n = 48)

Human (serum n = 21)

CCM of ESCs from endometrium and endometriosis lesions, serum

CCM: ExoQuick-TC Exosome Precipitation Solution (#EXOTC10A-1, SBI)

Serum: SEC

Yes

VEGF-C

Serum EV VEGF-C provided sensitivity of 81.3%, and specificity of 71.4%

Potential therapeutic target (e.g. with levatinib)

EV secreted VEGF-C induces lymphangiogenesis and pro inflammatory microenvironment to promote endometriosis progression

Number of participant samples used for CCM EV assays unclear

Li 2021 [29]

Human (n = 23)

Mouse

(n = 18)

Human (n = 10)

Mouse (n = 9)

CCM of M1 macrophages from endometrial biopsy

UC

Partially

Not investigated

Treatment of endometriosis with M1NVs (M1 macrophage derived EVs) safe in mouse model

M1NVs inhibit angiogenesis, migration and invasion of endometriosis, via reprogramming of M2 macrophage phenotype

EV cargo not investigated – mechanism of reprogramming unknown

Liu 2021 [30]

Human (n = 50)

Human (n = 50)

CCM of primary peritoneal macrophages isolated from peritoneal fluid

UC

Yes

lncRNA CHL1-AS1

Not discussed, but a potential diagnostic target

lncRNA CHl1-AS1 a potential therapeutic target

Peritoneal macrophage derived EVs downregulate endometrial cell apoptosis, and enhance proliferation and migration via lncRNA CHl1-AS1-miR-610 and MDM2 pathway

Munrós 2017 [31]

Human

N = 65

Human

N = 33

Plasma

No isolation method from plasma

No

Total circulating microparticle (cMP).

Circulating Microparticle Tissue Factor (cMP-TF)

cMP only elevated in patients with DIE or endometrioma

Not discussed, but potential target

Not discussed

Incorrect nomenclature.

No description of EV isolation methods or EV characterisation.

High likelihood of contamination from plasma lipids may affect cMP quantification and cMP specific TF analysis with ELISAs.

Muth 2015 [32]

Rhesus Macaque (n = 1)

Rhesus and pig tailed macaques (n = 2)

Cervicovaginal swab and lavage

UC

No

Not investigated

Cervicovaginal swab a potential source of EVs for diagnosis

Not discussed

EVs in endometriosis samples were less than healthy controls

Limited numbers. Limited reporting of EV isolation methodology.

Nazri 2020 [33]

Human

(n = 22)

Human

(n = 6)

Peritoneal fluid

SEC

Yes

Proteomics; PRDX1, ANXA2, ITIH4, H2A type 2-C and tubulin α-chain unique to endometriosis

Potential diagnostic target

Potential therapeutic target

Unique proteins in endometriosis samples

Limited numbers

Qiu 2019 [34]

Human

(n = 30)

Human

(n = 16)

Serum and

CCM of primary ESC isolated from endometriomas

Serum: ExoQuick Exosome Precipitation Solution kit (#EXOQ5A-1)

CCM: Total Exosome Isolation Reagent (Thermo Fisher Scientific)

Partially

lncRNA aHIF

aHIF is elevated in serum EVs from patients with endometriosis compared to controls

Potential therapeutic target

aHIF is transferred from endometrioma stromal cells via EVs to stimulate angiogenesis

Endometriomas only. Methods suggest FBS in culture media is not EV depleted, introducing contaminating EVs

Qiu 2020 [35]

Human (n = 29 serum; n = 10 endometrial cells)

Human (n = 16 serum; n = 10 endometrial cells)

Serum, CCM of primary ESCs isolated from endometriomas

Serum: ExoQuick Exosome Precipitation Solution kit (#EXOQ5A-1; SBI). CCM: Total EVs isolation reagent kit (#44,578,259; Thermo Fisher Scientific)

Yes

lncRNA TC0101441

Potential diagnostic Serum TC0101441 increases with stage

Potential therapeutic target

EV TC0101441 may promote invasion and migration by regulating N-cadherin, snail, slug and TCF8/ZEB1

Endometriomas only

Shan 2021 [36]

Human

(n = 5 for RNA seq, n = 80 for validation)

Human (n = 6 for RNA seq, n = 80 for validation)

Serum

ExoRNeasy Serum/Plasma Midi Kit (#76,214, Qiagen)

Yes

lncRNAs (seq)

Ten lncRNAs validated with RT-qPCR

Potential diagnostic marker RP3-399L15.2 AUC 0.86, Sn 67%, Sp 98%.

Using combination of RP3-399L15.2 and CH507-513H4.6 AUC 0.90, Sn 80%, Sp 85%.

Not discussed, but potential therapeutic targets

Not discussed

Endometriomas only

Sun 2018 [37]

Mouse (n = 10)

Mouse (n = 10)

ESC culture media

UC

Yes

Not investigated

Not discussed

Not discussed, potential therapeutic target

Endometriosis derived EVs induce M2 macrophage phenotype.

In vivo, treatment with endometriosis EVs increased lesions.

EV cargo not investigated, no proposed mechanism of action

Sun 2019 [38]

Human (n = 22)

Human (n = 6)

CCM of primary ESCs isolated from endometrium of patients with and without endometriosis

UC

Yes

Not investigated

Not discussed

Not discussed, potential therapeutic target

ESC EVs from patients with endometriosis induce angiogenesis, neurite outgrowth and inhibit neuron apoptosis

In vitro only

Sun 2021 [39]

Human (n = 52)

Human (n = 21)

CCM of ESCs from ovarian endometrial cyst walls,

and serum

ExoQuick EV precipitation solution kit (#EXOTC50A-1; SBI)

Yes

LGMNP1

Elevated serum EV LGMNP1 associated with infertility and recurrence. AUC for serum EV LGMNP1 to predict recurrence 0.869. Sn 93.3% and Sp 75.7%

Not discussed, potential therapeutic target

Endometriosis ESC EVs induce macrophage M2 phenotype via transfer of LGMNP1

Cyst wall derived ESCs only, may not be reproducible. Methods suggest FBS in culture media is not EV depleted, introducing contaminating EVs

Texido 2014 [40]

Human

(n = 14)

Human

(n = 13)

Fluid from simple cyst or endometrioma

ExoQuick Exosome Precipitation Solution (SBI)

Partially

Ectonucleotidases

Not discussed

Not discussed

Endometrioma EVs had increased ectonucleotidase activity compared to control

Cyst fluid only

Wu 2018 [41]

Human (n = 10)

None for EV experiments

CCM of primary ESCs from endometriomas

UC

Partially

miR-214

Not discussed, potential for diagnosis

Therapeutic potential of EV miR-214

MiR-214 enriched EVs down regulated CTGF and collagen (fibrosis) in a mouse model (n = 4)

Endometriomas only

Wu 2020 [42]

Human (n = 3 for seq, n = 7 for validation)

Human (n = 3 for seq, n = 7 for validation)

CCM of primary ESCs from paired endometriomas and endometrium, and endometrium from controls

ExoQuick-TC Exosome Isolation Kit (SBI)

Yes

lncRNA, miRNA and mRNAs (seq). Expression of regulatory networks LOC105376166/ miR-214-3p/ MIB2 and LOC105371414/ miR-423-5p/ADCY3 validated with RT-qPCR

Possible use of panel of EV derived RNA processing for diagnosis

Not discussed

Not discussed

Very large number of multiple comparisons with small subject numbers

Wu 2021 [43]

Human (n = 42)

Human (n = 24)

Serum

Beads/Precipitation

Partially

miRNAs (miRNA chip)

miR miR-26b-5p, miR-215-5p and miR-6795-3p validated with RT-qPCR

miR-26b-5p and miR-215-5p were decreased, and miR-6795-3p was increased in endometriosis, and related to stage

Not discussed

MiRNAs identified may be involved in MAP and PI3K-AKT signalling.

No reporting on miRNA chip analysis methods

Wu 2021 [44]

Human

(RNA seq n = 3,

RT-qPCR n = 10)

Human

(RNA seq n = 3, RT-qPCR n = 10)

CCM of primary ESCs from paired endometrioma and endometrium, and control endometrium

ExoQuick-TC Exosome Isolation Kit (SBI)

Yes

circRNAs, miRNAs, mRNAs (seq).

circ_0026129, miR-15a-5p and ATP6V1A validated with RT-qPCR

Network analysis of ceRNAs lead to identification of three central components different between subjects and controls (circ_0026129, miR-15a-5p and ATP6V1A), possible diagnostic targets

Potential diagnostic targets

Increased miRNA-15a-5p known to be involved with angiogenesis.

ATP6V1A likely involved with cell migration and growth.

Small numbers. Low statistical power with large number of comparisons and complex modelling calculations

Zhang 2020a [45]

Human (discovery cohort n = 5, validation n = 20)

Human (discovery cohort n = 5, validation n = 20)

Serum

UC

Yes

MiRNAs (microarray). RT-qPCR validation of miR-22-3p and miR-320a

miR-22-3p AUC was 0.855 (p < 0.01),

miR-320a AUC was 0.827 (p < 0.01), combined AUC was 0.883 (p < 0.01). MiR-22-3p associated with high stage.

Not discussed

miR-22-3p and miR-320a increased in endometriosis, may be associated with MAP and TNF signalling.

Limited numbers

Zhang 2020b [46]

Human (n = 20)

Human (n = 20)

CCM of primary macrophages isolated from peritoneal fluid

UC

Yes

MiRNAs (microarray). RT-qPCR validated increased miR-22-3p in patient EVs

Not discussed

MiR-22-3p/SIRT1/NFkB pathway could be novel therapeutic target

Endometriosis macrophage derived EVs promote proliferation, migration and invasion of ESCs compared to macrophage EVs from benign controls via delivery of miR-22-3p. MiR-22-3p activates NF-kB via SIRT1.

Number of biological replicates for cell culture assays unclear. Methods suggest FBS is not EV depleted, introducing contaminating EVs

Zhang 2021 [47]

Human (serum n = 20)

Human (serum n = 20)

CCM of primary ESCs derived from endometriomas and control endometrium, and serum

ExoQuick-TC Exosome Isolation Kit (SBI)

Partially

miR-214-3p

MiR-214-3p decreased in serum of endometriosis patients (p < 0.05).

EV miR-214-3p treatment reduced fibrosis in mice.

Downregulation of miR-214-3p increases fibrosis in vitro and in vivo via CCN2.

N for primary cultures unclear. Endometriomas only. Authors report serum miR-214-3p is from EVs however their methods indicate it is total plasma RNA, not EV specific.

Zhou 2020 [20]

Human

(n = 3)

Human

(n = 3)

CCM of primary ESCs from endometrium of women with endometriomas and infertility, and fertile controls

ExoQuick-TC Exosome Isolation Kit (SBI)

Yes

Small RNAs (seq)

Potential diagnostic

Not discussed

Target gene prediction identified HOXA10 and LIF as possible targets

Enriched pathways included MAPK and Wnt signalling

Low numbers, no validation of RNA seq via RT-qPCR