Clinical Implications of Extracellular Vesicles in Endometriosis: A Systematic Review

Endometriosis (EM) is a gynecological condition affecting ten percent of women of reproductive age worldwide. It is characterized by the growth of endometrial tissue outside the uterus, which can lead to a number of symptoms including pelvic pain, infertility, and, in some cases, an increased risk of certain malignancies.[ 1 2 ] Women with EM are at an increased risk of developing ovarian cancer, with a lifetime risk of 1 in 56 compared to 1 in 75 in the general female population. While the risk of malignant transformation in superficial endometriotic lesions and deep infiltrating EM (DIE) lesions is minimal, ovarian endometriomas are considered to carry a higher risk of malignancy.[ 3 ] The disease is estrogen-dependent and involves a variety of complex pathogenic mechanisms, including genetic, immune, and environmental factors.[ 1 4 5 ] Some of the risk factors for EM include a short menstrual cycle, a family history of the disease, and specific genetic polymorphisms. In addition, modifiable factors such as smoking, a low body mass index, and low parity have been suggested to be associated with an increased risk of developing the disease.[ 6 ] The diagnosis of EM is often challenging in the early stages due to the nonspecific nature of the symptoms, which frequently include pelvic pain, dysmenorrhoea, gastrointestinal and urological problems. The presentation of symptoms associated with EM can vary depending on the specific phenotype manifesting. These include superficial peritoneal EM, ovarian endometrioma, and DIE. DIE is characterised by lesions infiltrating more than 5 mm into the peritoneal tissue and is frequently associated with increased pain severity.[ 7 ] In addition, there is often a little correlation between the severity of the disease and the severity of the symptoms.[ 8 ] Current treatments tend to focus on symptom management rather than seeking a cure. Proposed pathogenetic pathways include retrograde menstruation, immune dysregulation, hormonal imbalances, stem cell involvement, and alterations in epigenetic regulation. Recent studies have indicated a potential association between altered fetal developmental programming and the subsequent development of EM, emphasising the importance of understanding the disease’s pathogenesis to develop effective treatment strategies.[ 1 ] The diagnosis of EM is a complex process due to the overlapping symptoms with other conditions and the chronic nature of the disease. Traditionally, surgical methods such as laparoscopy have been the gold standard for diagnosis, contributing to delays of four to 11 years from symptom onset.[ 9 10 ] However, recent advancements have led to the development of noninvasive diagnostic techniques such as transvaginal ultrasonography and magnetic resonance imaging for accurate assessment.[ 11 ] The diagnostic reliability of imaging for EM is dependent on the specific subtype. Ultrasound has been demonstrated to be highly effective in the diagnosis of endometriomas. However, the sensitivity and specificity of transvaginal ultrasound for detecting DIE within the pelvic cavity vary between 53% and 93%.[ 7 ] Biomarkers such as CA125, brain-derived neurotrophic factor, interleukin-6, and MicroRNA (miRNA) are being investigated as potential diagnostic tools, offering a less invasive approach to detecting EM without the necessity for surgical intervention.[ 10 12 ] The combined use of clinical history, physical examination, imaging, and biomarkers plays a significant role in the diagnostic process, with the intention of reducing delays and improving accuracy in identifying EM.[ 10 ] The management of EM involves a range of approaches tailored to the patient’s symptoms and fertility desires. The objective of surgical intervention should extend beyond the simple resection of visible endometriotic lesions to prevent disease progression; it should also focus on restoring normal pelvic anatomy to improve the chances of a successful IVF outcome.[ 13 ] Although medical therapy is effective in managing pain associated with EM, there is limited evidence of its effectiveness in addressing infertility.[ 14 15 ] Surgical interventions, including laparoscopic procedures, are commonly used to alleviate the symptoms of EM. However, recurrence is common, with the risk increasing over time.[ 8 16 ] Exosomes are microsomal vesicles with a diameter of 30–160 nm produced by cells, and are responsible for the transmission of information from cells to the extracellular matrix. They carry a range of molecules, including proteins, long noncoding RNA (lncRNA), and DNA. Extracellular vesicles (EVs) play a vital role in the pathogenesis of EM, operating as intercellular communicators.[ 17 ] They carry molecular cargo involved in a variety of processes, including angiogenesis, immunomodulation, and fibrosis, which contribute to the development and progression of EM. EVs derived from EM lesions can be detected in the blood, offering potential for diagnosis and recurrence detection.[ 18 ] In this review, we will explore the current understanding of EVs in EM, with a particular focus on their clinical significance, including their involvement in diagnosing and treating the condition.

Twelve studies were included in the review. The initial search identified 192 articles, of which 25 duplicate records were removed. Another 129 papers were excluded after applying the inclusion criteria (original papers, concerning EVs from 2019 to 2024). Thirteen animal studies were excluded. One paper was excluded because it concerned only adenomiosis and not EM. An additional 12 reports were excluded as they did not relate to any clinical implementations. The process of study identification is illustrated in Figure 1 . Identification of studies via databases and register EVs associated with EM were isolated from tubal fluid,[ 19 ] plasma,[ 20 21 22 ] peritoneal fluid,[ 21 23 24 ] endometrial tissue,[ 25 26 ] ectopic endometrial tissue,[ 25 26 ] leukorrhea,[ 25 26 ] serum,[ 27 28 29 ] and menstrual blood-derived stem cells.[ 30 ] The EV’s isolation techniques differed among the studies. Ultracentrifugation was the most commonly used method. It was employed in five studies.[ 19 20 23 26 29 ] Commercial kits were used in three studies.[ 22 28 30 ] Further two reports employed centrifugation,[ 21 27 ] one gradient centrifugation,[ 19 ] and one differential centrifugation.[ 25 ] One of the studies applied magnetic separation[ 27 ] and one used size exclusion chromatography. Methods applied for the identification and characterisation of EVs varied between studies. Quantitative real-time polymerase chain reaction was the most commonly used method for the validation of EVs’ cargo. Other methods of identification included microarrays, flow cytometry, immunofluorescence antibodies, immunoblotting, and next-generation sequencing. The EVs’ cargo was examined for its potential diagnostic and therapeutic applications along with its role in the pathogenesis of EM. The most frequently examined components of EVs were miRNAs. These molecules, which are a class of small, noncoding RNA, function as key regulators of gene expression at the posttranscriptional level. They have been shown to play a vital role not only in the physiological processes within the human body but also in the pathogenesis of numerous diseases, including malignancies.[ 31 ] A variety of other markers have been identified in EVs, including lncRNAs, tRNA-derived fragments (tRFs), and tRNA-derived fragments (tiRNAs), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), CD63 and CD81, PRDX1, ANXA2, ITIH4, H2A type 2-C, and tubulin α-chain. Although these molecules may appear unrelated, they interact to form crucial signaling pathways. Among them, lncRNAs play a vital role in gene expression regulation, influencing cell differentiation, proliferation, and apoptosis.[ 28 ] TRFs participate in a wide range of cellular processes, including gene expression regulation, protein translation inhibition, and cell cycle control. Recent studies indicate that tRFs carried by EVs act as key regulatory elements, influencing cellular processes and mediating communication between cells. Likewise, tiRNAs have been linked to promoting cell migration and invasion.[ 32 ] Moreover, proteins such as VEGF are key players in angiogenesis, while MMP-9 is linked to extracellular matrix remodeling and increased proteolysis in ectopic endometrial lesions.[ 21 ] Ultimately, CD63 and CD81, which are exosomal tetraspanins, serve as important EV markers and may play a role in sorting cargo within vesicles.[ 33 ] Several studies discussed miRNA as a potential diagnostic marker for EM[ 20 22 26 29 ] and one study explored miRNA as a potential biomarker for evaluating the severity of ovarian EM.[ 27 ] Other articles listed exosomal tRF-Leu-AAG-001[ 25 ] and lncRNAs[ 28 ] as potential indicators for the diagnosis of EM. In one study, VEGF (+)/MMP-9 (+) microvesicles did not enable to differentiation between the test and control groups[ 21 ] and other reports have not addressed the diagnostic implications of EVs. One article discussed the potential therapeutic effect of exosomes isolated from menstrual blood-derived stem cells in EM treatment.[ 30 ] A number of studies have investigated the role of EVs in the pathogenesis of EM, with a particular focus on their involvement in cell communication, immunomodulation, promotion of proliferation, angiogenesis and inflammation.[ 19 20 21 23 25 26 27 29 30 ] The source of isolation and the cargo for selected EVs are illustrated in Figure 2 . Extracellular vesicles’ cargo identified in subjects with endometriosis from different sites The role of EVs in the pathogenesis of EM has been the subject of several articles. In particular, the investigation has focused on the role of Evs’ miRNA in the regulation of cell communication and signalling pathways.[ 19 20 27 29 ] The reports discussed the potential involvement of miRNAs in the mitogen-activated protein (MAP) and phosphatidylinositol 3-kinase (PI3K-AKT) signalling pathways, as well as in the tumour necrosis factor (TNF) and Toll-like receptor (TLR) pathways.[ 20 27 29 ] EVs have been demonstrated to play a major role in immune responses, particularly in the differentiation of T-helper (Th) cells. These vesicles have the capacity to influence Th1 and Th2 differentiation by carrying specific miRNAs that regulate key factors involved in this process. One of the studies demonstrated that miRNAs extracted from plasma-derived EVs promote the proliferation of mesenchymal stem cells and modulate the differentiation of Th1 and Th2 cells. This finding demonstrates the importance of EVs in immune responses and suggests that targeting these vesicles may offer novel therapeutic strategies. The content of EVs in terms of miRNAs dictates their capacity to either promote Th1 or Th2 polarization, thereby influencing cytokine production and immune responses.[ 20 ] Another study examined the role of tubal fluid miRNAs in the secretion and transport function of tubal epithelium, with the ultimate effect on female reproductive function.[ 19 ] Furthermore, the potential contribution of miRNAs isolated from peritoneal fluid to immunomodulation and cell proliferation in EM was discussed.[ 23 ] Finally, the role of Evs’ cargo in angiogenesis and inflammation was discussed in several reports, with particular reference to the participation of VEGF, MMP-9, tRFs, and miRNAs.[ 21 25 26 29 ] It has been shown that EVs contribute to angiogenesis by activating several growth factors, such as VEGF and fibroblast growth factor (FGF). This process is mediated through various molecules carried by EVs, including exosomal miRNA-15a-5p, which regulates gene expression, hypoxia-inducible factor, which is involved in the response to low oxygen conditions, and tRFs and tiRNAs, which can influence cellular processes related to stress and inflammation. These components work together to enhance angiogenesis, promoting the formation of new blood vessels, which is crucial for tissue repair and disease progression.[ 25 34 35 ] A number of studies have indicated the potential of EVs as a diagnostic tool for EM.[ 20 22 24 25 26 27 29 ] The majority of studies have focused on miRNA as a promising target[ 20 22 26 29 ] with one study in particular examining the possibility of using miRNA as a biomarker for assessing the severity of ovarian EM.[ 27 ] In one study, 30 plasma exosomal miRNA markers were sequenced using microarrays in order to identify and report on those associated with EM.[ 20 ] The results indicated that seven miRNAs were upregulated and 43 miRNAs were downregulated in patients with EM. In a separate report, a total of 45 miRNAs demonstrated significantly differential expression between the two groups. Of these, 26 were upregulated and 19 were downregulated in the EM samples in comparison to the control samples. Consequently, the article proposed that miRNAs such as miR-26b-5p, miR-215-5p, and miR-6795-3p may serve as potential biomarkers for evaluating the severity of ovarian EM.[ 27 ] An alternative report proposed utilising a panel of candidate miRNAs, which demonstrated diagnostic outcomes comparable to laparoscopy in differentiating between women with EM and a control group. The results produced a sensitivity of 0.96 and a specificity of 0.79 with positive predictive value 0.80 and negative predictive value 0.96, respectively.[ 22 ] Encouraging outcomes were also noted for miR-22-3p and miR-320a, which demonstrated significant upregulation in serum exosomes from patients with EM. The combination of miR-22-3p and miR-320a exhibited high sensitivity and specificity, thereby enhancing the efficiency of EM diagnosis. In addition, miR-22-3p was found to be associated with high-stage EM.[ 29 ] Finally, one article reported significantly higher expression of hsa-miR-202-3p and hsa-miR-202-5p in leukorrhea-derived exosomes from EM patients in comparison to the negative controls and proposed hsa-miR-202 as a valuable diagnostic biomarker.[ 26 ] Other discussed targets included leukorrhea-derived exosomal tRFs and serum-derived lncRNAs.[ 25 28 ] It was established that exosomal tRF-Leu-AAG-001, collected from vaginal discharge, exhibited high specificity and sensitivity for predicting the occurrence of EM.[ 25 ] A different study has identified 210 lncRNAs that were significantly dysregulated in the plasma of patients with EM. The combined expression level of RP3-399 L15.2 and CH507-513H4.6 was employed to differentiate between patients with EM and control participants, resulting in an 80.00% sensitivity and an 85.45% specificity. It was therefore proposed that these two lncRNAs have the potential to serve as diagnostic biomarkers for EM. Moreover, CH507-513H4.6 alone was identified to be a valuable diagnostic tool for detecting early-stage EM lesions.[ 28 ] Finally, one study has demonstrated that there are statistically significant higher levels of AnnexinV (+) microvesicles present in plasma samples of patients with EM. However, the usage of microvesicles with proangiogenic factors (VEGF and MMP-9) did not enable to differentiate between the test and control groups.[ 21 ] Other reports have not addressed the diagnostic implications of EVs. The majority of studies have not addressed the potential of EVs as therapeutic markers. Nevertheless, one study has proposed the utilisation of EVs as a potential strategy of improving the management of EM. Exosomes were isolated from menstrual blood-derived stem cells collected from healthy patients (NE-MenSCs) and employed for the treatment of endometriotic cells (E-MenSCs). The treatment with exosomes led to a significant reduction in the expression levels of markers associated with inflammation, proliferation, migration, and angiogenesis in E-MenSCs, which are altered in EM. In addition, apoptosis was induced in E-MenSCs. The study proposed that MenSCs-derived exosomes could be a superior treatment option for improving EM compared to conventional treatments.[ 30 ] The specific limitations of each study are listed in Table 1 . The most common limitation was the relatively small sample size. Seven of the twelve studies had a sample size of EM patients no >30.[ 19 20 24 25 26 29 30 ] Moreover, only two of the twelve studies reported a control group size of more than 25 participants.[ 22 28 ] Two articles encountered the issue of the impact of exclusion criteria on the number of study participants.[ 21 22 ] One study included only participants with endometriomas.[ 28 ] In addition, an important limitation of the present review is the absence of longitudinal analysis in the included studies. The available research has focused on comparing the composition of EVs in samples collected from different patient and control groups at a single time point, rather than tracking changes in EV levels over time. While one study investigated miRNA as a potential biomarker for assessing the severity of ovarian EM, this analysis was also based on cross-sectional comparisons rather than a longitudinal approach.[ 27 ] Future studies incorporating longitudinal designs could provide valuable insights into the dynamic role of EVs in the progression of EM.

EM is a chronic and frequently painful condition characterised by the growth of endometrial-like tissue outside the uterus. Current diagnostic techniques for EM tend to be invasive, mainly relying on laparoscopy. However, EVs have recently been identified as a potentially valuable non-invasive biomarker for diagnosing this condition. These vesicles contain a variety of molecular components, including proteins, lipids, and RNAs, which can provide insights into the pathological condition of the reproductive system. The utilisation of miRNAs and other particles derived from exosomes offers a potential noninvasive alternative, leading to an earlier diagnosis. While the potential of EVs as diagnostic tools for EM is promising, further research is necessary to confirm these results and develop standardised diagnostic protocols. Conceptualization: MSK, AW; design: MSK, AW; methodology: MSK, AW; definition of intellectualcontent: MSK, AW; literature search: MSK, AW; data acquisition: MSK, AW; data analysis: MSK, AW;manuscript preparation: MSK, AW; manuscript editing and manuscript review: MSK, AW; fundingacquisition: Nil. All authors have read and agreed to the final version of the manuscript. Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. There are no conflicts of interest.

Currently, diagnosis and management of EM present significant challenges. Conventional diagnostic techniques, such as laparoscopy, are invasive, difficult to access, and may result in delays in treatment. This highlights the urgent need for the development of non-invasive diagnostic techniques. Exosomes, which are microsomal vesicles released by cells, have the potential to become a highly promising diagnostic biomarker. They transport a wide range of molecular cargo, including proteins, lipids, and nucleic acids, which reflect the physiological and pathological states of their cells of origin. Given that exosomes can be detected in accessible body fluids such as blood, urine, and leukorrhea, they offer a promising opportunity for the development of less invasive diagnostic strategies. Despite the fact that the precise pathophysiology of EM remains incompletely understood, it is well-established that the disease is marked by the dysregulation of several critical signaling pathways, including the VEGF, TNF, and TLR pathways, along with MAP and the PTEN-PI3K pathways. The significance of these pathways stems from their role in regulating a number of key biological processes, including angiogenesis, cell proliferation, migration, and apoptosis. The potential role of EVs in the pathogenesis of EM has been the subject of several studies. EVs play a significant role in facilitating communication between cells in the endometrial microenvironment. Through this interaction, EVs can influence immunological responses, particularly the differentiation of Th1 and Th2 lymphocytes, potentially intensifying the inflammatory processes that characterize EM. In addition to this, EVs are involved in the promotion of angiogenesis by activating key growth factors such as FGF and VEGF. These observations suggest that while these pathways may appear separate, they are closely interconnected, with EVs acting as critical mediators that bridge these complex biological processes. The most frequently investigated components of EVs have been miRNAs, with several studies discussing their potential as diagnostic markers for EM. However, the identification of specific exosomal markers associated with EM remains a current area of research. The successful identification of such biomarkers could lead to the development of noninvasive diagnostic methods that contribute to the early detection and improved management of the disease. In addition, exosome analysis may provide insights into the underlying mechanisms leading to the disease and the diversity observed in EM phenotypes. As research in non-invasive diagnostic techniques progresses, exosomes may prove to be a crucial factor in improving the diagnosis of EM and may also contribute to a deeper understanding of other gynecological disorders. As the most common limitation in the studies reviewed was the limited number of participants, there is still a need for further research, including larger sample sizes.

A systematic review was performed based on the 2020 Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. The protocol for this systematic review was not prospectively registered. Three databases (PubMed, Medline, and Scopus) were searched on March 3, 2024 for keywords “endometriosis” and either “extracellular vesicles” or “EV” or “EVs” and either “treatment” or “monitoring” or “diagnosis”. Articles were screened by the two reviewers (MSK (Marcelina Sztyler-Krąkowska) and AW (Agnieszka Wąsowicz)) working independently at each stage of the screening. Articles were included if they were in English, published between 2019 and 2024, and if the studies concerned EVs in the pathogenesis of EM and their clinical implementation. Only original papers were included in the study. Animal studies were excluded. Articles only concerning adenomiosis and not EM were also excluded from the study. The studies were evaluated based on the following criteria: Number of EM patients, number of controls, EVs’ sample source, EVs’ isolation technique, EVs’ cargo, diagnostic and therapeutic implications, the role of EVs in EM pathogenesis, and study limitations. Data from the articles were extracted following the headings outlined in Table 1 , with the two authors (XX and YY) participating independently in both data extraction and compilation process. No automation tools were employed, and no additional data were required from the authors of the included studies. Key characteristics of studies of extracellular vesicles in endometriosis UC: Ultracentrifugation, SEC: Size exclusion chromatography, EVs: Extracellular vesicles, TNF: Tumour necrosis factor, TLR: Toll-like receptor, EM: Endometriosis, RT-qPCR: Real-time reverse transcription quantitative polymerase chain reaction, MAP: Mitogen-activated protein, PI3K-AKT: Phosphatidylinositol 3-kinase, lncRNAs: Long noncoding RNAs, tRFs: tRNA-derived fragments, tiRNAs: tRNA-derived fragments, VEGF: Vascular endothelial growth factor, MMP-9: Matrix metalloproteinase-9, miRNAs: micrornas To evaluate the risk of bias in the included studies, we employed the NHLBI’s Study Quality Assessment Tools. To ensure objectivity and minimize potential bias, each study was evaluated by two independent reviewers (XX and YY), who conducted their assessments separately. No automation tools were employed in the assessment process, ensuring thorough and consistent evaluation of the research. We declare that the AI tool DeepL Write ( https://www.deepl.com/en/write ) was utilized for grammar and language improvement in the preparation of this scientific article. The use of this tool was limited to refining the clarity, accuracy, and linguistic quality of the manuscript, without altering its substantive content or interpretations.