Extracellular vesicles in endometrial-related diseases: role, potential and challenges

Endometriosis

Endometriosis is a benign and hormone-dependent disease caused by ectopic deposition of endometrial tissue in the abdominal cavity and outside the uterus. The main clinical manifestations are infertility, dysmenorrhea, irregular menstruation, and discomfort during sexual intercourse (Asghari et al., 2018). Studies have shown that women with endometriosis have significant differences in long chain non-coding RNAs, miRNAs, and proteins involved in histone modification, angiogenesis, and immunomodulation contained in endometrial cell exosomes compared to normal women, and it has also been experimentally demonstrated that exosomes and their vectors play important roles in angiogenesis, neuromodulation, immunomodulation, and endometrial stromal cell invasion (Freger, Leonardi & Foster, 2021; Khalaj et al., 2019; Zhou et al., 2020; Wu et al., 2021). Thus, it is suggested that exosomes play an important role in the pathophysiology of endometriosis.

EVs enhance cell proliferation, migration and invasion

Exosomal miR-22-3p, an important regulator of cell proliferation, was significantly increased in the exosomes of abdominal macrophages from patients with endostosis, and the functional analysis of miR-22-3p by knockdown and overexpression of miRNAs showed that the upregulation of exosomal miR-22-3p induced an increase in the expression of nuclear factor-κB (NF-κB) (Zhang et al., 2020a). The results of this study showed that exosomal miR-22-3p in abdominal macrophages may enhance the proliferation, migration and invasion of Ec-ESCs by regulating the SIRT1/NF-κB signalling pathway.

EVs are associated with immune response and chronic inflammation

In addition, endometriosis is an inflammatory disease whose pathogenesis has been linked to peritoneal macrophages, and exosomes have been implicated as key regulators of several inflammatory diseases. A study found elevated levels of small heat shock protein (sHsp)-22 in exosomes of patients with EMS, and the level of heat shock protein expression was positively correlated with the expression of markers of cytotoxic immune response, i.e., perforin and granzyme B, suggesting a link between exosomes and immune imbalance in EMS (Wyciszkiewicz et al., 2019). A study by Chen et al. (2019) concluded that exosomal miRNAs derived from myeloid-derived suppressor cells (MDSCs) may contribute to the development of EMS by suppressing the immune function of the organism, suggesting that exosomes play an important role in the process of immune imbalance in EMS.

Sun et al. (2019b) injected EMS-exosomes derived from ectopic stromal cells into mice with endometriosis model, while the control group received con-exosomes from mice without endometriosis. Finally, the peritoneal macrophages were evaluated and the ectopic lesions were counted and measured. It was found that after the treatment of EMS-exosome, the macrophages polarized into m2-like phenotype, and their phagocytosis decreased. Mice in the EMS-exosome group showed a significant increase in total lesion weight and volume and enhanced m2-like macrophage infiltration in the EMS-exosome group compared with the con-exosome group. This emphasizes the importance of EMS-exosomes in the pathogenesis of endometriosis. Huang et al. (2022) found that the endo-heteroplasia-derived exosome miR-301a-3p may induce M2-type macrophage polarisation by up-regulating PI3K expression and down-regulating PTEN expression through the phosphatase and tensin homolog (PTEN)-PI3K axis. The above studies suggest that exosomes transferred from ESCs to macrophages contribute to immune dysfunction by inducing the conversion of macrophages to an M2-like phenotype and attenuating their phagocytic capacity. Also, exosomes derived from ESCs promote infiltration of M2-like macrophages and increase the size and weight of ectopic lesions in mice. Figure 2 summarizes the information transfer among ESCs, macrophages, and MSCs through EVs. The above experiments, although they may not fully mimic human physiology, may, to a certain extent, also indicate that extracellular vesicles identifying macrophage targets and altering macrophage polarisation type and function in therapy will have a positive impact on the treatment of endometriosis.

Infertility

Intrauterine adhesion

Intrauterine adhesions, also known as Asherman’s syndrome, are primarily fibrous tissue that forms after damage to the endometrium and can arise as a result of gynecologic surgery or as a complication of pregnancy. With the rise of cesarean sections and endometrial surgery, Asherman’s syndrome has become a growing problem. Due to partial or complete occlusion of the patient’s uterine cavity, this in turn can lead to a variety of complications such as decreased menstruation, amenorrhoea, incomplete pregnancy or recurrent miscarriages (March, 2011). Modern research has found that exosomes can be used to treat intrauterine adhesions.

Endometrial cancer

MIR210HG acts as an oncogene to promote proliferation and metastasis in endometrial cancer cells and regulates malignant development of endometrial cancer cells partly through spongy miR-337-3p and miR-137, thus regulating the level of high mobility group protein A 2 (HMGA2) (Fan et al., 2021). Another study found that circRNAs in extracellular vesicles isolated from sera of patients with endometrial cancer play an important role, and that the main pathway for these circRNAs is the isolation of cancer-mediated miRNAs. circ 0109046 and circ 0002577 reached a greater than 2-fold fold fold change when using real-time quantitative PCR (Xu et al., 2018).

Endometrial cancer is the second most common cancer in the female reproductive system and the sixth most common cancer in women (Sung et al., 2021). Abnormal uterine bleeding is the most common symptom of endometrial cancer (Amant et al., 2005). Cancer cells interact closely with neighboring stromal cells and jointly promote disease through bidirectional communication. A large number of studies have found that exosomes play a communication role between cancer cells and neighboring fibroblasts. Exosome-derived miRNAs regulate the adhesion, invasion, and angiogenic capacity of endometrial cells and modulate endometrial cell proliferation and apoptosis. Yan et al. (2014) found for the first time that miR-302 cluster could directly target Cyclin D1 and indirectly regulate the expression of cell cycle protein-dependent kinase 1 gene, thereby inhibiting the proliferation and migration of endometrial cell lines Ishikawa and HEC-1B, and blocking the cell cycle at G2/M phase, inhibiting proliferation and differentiation as well as decreasing the tumourigenicity of cancer cells. A large number of studies have found that exosomes play a role in communication between cancer cells and neighbouring fibroblasts. Exosome miR-320a secreted by cancer-associated fibroblasts is directly transferred to endometrial cancer cells and can inhibit cancer cell proliferation (Zhang et al., 2020b).

Maida et al. (2016) isolated endometrial fibroblasts from normal endometrial tissues and endometrial cancer tissues and examined the intercellular transfer of exosomes originating from the endometrial cancer cell line Ishikawa, and showed that exosomes secreted by endometrial cancer cells were accepted by endometrial fibroblasts, and these exosomes were able to transport functional small RNAs to fibroblasts, demonstrating for the first time that endometrial cancer cells communicate with neighbouring fibroblasts by transferring small regulatory RNAs via exosomes, and suggesting that tumor cells may regulate the tumor microenvironment through the contents of exosomes which are mainly miRNAs. Srivastava et al. (2018) have searched for hsa-miR-200c-3p from exosomes of urinary origin for the diagnosis of endometrial cancer. Therefore using liquid biopsy technique, uterine lavage fluid to search for exosomes as an early marker for endometrial cancer is more potential development. Roman-Canal et al. (2019) successfully extracted EVs from the peritoneal lavage fluid of endometrial cancer patients and healthy individuals, and then analysed the miRNA content of these EVs in detail. The results of the study showed that there was a significant imbalance in the expression levels of 114 miRNAs in the diseased group compared to the control group. Accordingly, they hypothesised that such EV-associated miRNA signature profiles may be a key source of biomarkers for early cancer detection. Li et al. (2019) found that miR-148b could be transferred from cancer-associated fibroblasts (CAFs) to endometrial cancer cells via exosomes and inhibit endometrial cancer metastasis by directly binding to its downstream target gene, DNA methyltransferase 1 (DNMT1), thus acting as an oncogene. In endometrial cancer, DNMT1 plays a potential role in enhancing cancer cell metastasis by inducing epithelial-mesenchymal transition (EMT). Therefore, down-regulation of miR-148b induces EMT in endometrial cancer cells, thereby alleviating the inhibitory effect of DNMT1. However, it has also been found that exosomal nuclear enriched abundant transcript 1 (NEAT1) from cancer-associated fibroblasts promotes endometrial cancer progression through the miR-26a/b-5p-mediated STAT3/YKL-40 pathway (Fan et al., 2021). Shi et al. (2020) purified exosomes from endometrial cancer cell culture medium and found that endometrial cancer cells could secrete exosomes containing miR-133a, confirming that miR-133a may regulate the down-regulation of the FOXL2 (forkhead box L2) gene in endometrial cancer tissues thereby inhibiting tumor cell proliferation and increasing apoptosis.

The exosomes in plasma also play a key role in the pathophysiology of endometrial carcinoma. Plasma derived exocrine miR-15a-5p is a promising and effective diagnostic biomarker for early detection of endometrial carcinoma (Zhou et al., 2021). Another study discovered that as EC progressed, the plasma exosomal lectin galactoside-binding soluble 3 binding protein (LGALS3BP) rose indicating that human umbilical vein endothelial cells (HUVEC) function through the activation of the PI3K/AKT/VEGFA signaling pathway both in vitro and in vivo, and that highly contained exosomal LGALS3BP contributed to EC cell migration and proliferation. It also suggested that plasma exosomes containing LGALS3BP contributed to EC growth and angiogenesis during EC progression, offering a new insight into EC diagnosis and prognosis (Song et al., 2021).

There are also several studies that have identified tumor-associated macrophages (TAM) as a major regulator of endometrial cancer (EC) development, and TAM can crosstalk with cancer cells by transferring exosomes carrying microRNAs (miRNAs) (Wang et al., 2022). Exosomes from CD45ROCD8 T cells have been isolated to examine their regulatory role in endometrial cancer. miR-765 was found to be the most severely down-regulated miRNA in the UCEC genes, and these exosomes limit endometrial cancer disease progression by regulating the miR-765/PLP2 axis (Fan et al., 2021).

Therapeutically, exosomes can be used as drug carriers to participate in tumor therapy, and targeting exo-miRNAs to treat tumors is a novel and effective therapeutic modality. Studies have shown that a single miRNA can target hundreds of mRNAs at the same time, affecting cellular biological functions by regulating the post-transcriptional activity of multiple target mRNAs, suggesting the feasibility of targeting miRNAs as a therapeutic tool (Shah et al., 2016). Song et al. (2022) analysed SERPINA5 expression in EC patients using The Cancer Genome Atlas (TCGA) database. Compared with EC patients without distant metastases and the normal population, SERPINA5 expression was significantly lower in EC patients with distant metastases (P < 0.01), while overexpression of SERPINA5 or high exosomal SERPINA5 levels inhibited the metastatic potential of EC by suppressing the activation of integrin β1/FAK signalling pathway. It is suggested that exosomes play an important role in tumor immunity.

Endometritis

Chronic endometritis is a persistent inflammatory disease characterized by plasma cell infiltration in the endometrial stromal area (Kimura et al., 2019), with clinical manifestations including amenorrhoea, scanty menstruation, infertility, or recurrent miscarriages (Kelleher et al., 2018), which seriously affects women’s health and fertility. MiR-27a-3p and miR-124-3p have been found to be up-regulated in endometrium and serum affected by chronic endometritis, suggesting that miR-27a-3p and miR-124-3p may represent non-invasive markers of CE and could be used in the future to assess endometrial quality in IVF (Di Pietro et al., 2018).

The role of endometrial epithelium-derived exosomes in endometritis

In one study, 118 differentially expressed miRNAs were found in the uterine fluid secretions of healthy cows and patients with endometritis, of which 52 miRNAs were down regulated and 66 were up regulated. In addition, compared with IVF embryos co-incubated with healthy exosomes, IVF embryos co-incubated with endometritis exosomes significantly reduced the blastocyst formation rate. Therefore, the exosomes miRNA may be the cause of infertility due to endometritis (Wang et al., 2019). miR-218 in endometrial epithelial cell (EEC)-derived exosomes has also been implicated in the pathogenesis of bovine endometritis. Reduced levels of miR-218 in EEC-derived exosomes, when transferred to placental trophoblast cells, affects embryonic development and inhibits migration of placental trophoblast cells by targeting secreted curl related protein 2 (Wang et al., 2021). Exosomes released by endometrial epithelial cells, which contain miR-218, exhibit a powerful anti-inflammatory potential by effectively blocking the activity of a series of immunomodulatory factors and chemokines. These exosomes act as natural delivery vehicles that can precisely deliver miR-218 into the uterine microenvironment and its neighbouring target cells, thereby finely regulating the local immune response. Specifically, miR-218 maintains the internal immune homeostasis of the uterus by inhibiting the expression of various key immune molecules, including interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and chemokines, such as macrophage inflammatory protein-1α (MIP-1α) and MIP-1β, and plays its central role as an central role of immunosuppressive factors (Wang et al., 2020). This process not only reveals the important role of exosomes in intercellular communication, but also provides new molecular targets and therapeutic ideas for the regulation of endometrial inflammation. Another study demonstrated that overexpression of miR-193a-3p in bovine endometrial epithelial cells (BENDs) significantly increased the expression levels of LPS-induced pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. Therefore, it is hypothesised that inhibition of miR-193a-3p may be a potential molecular target for the future treatment of endometritis (Yin et al., 2021). However, the role of miR-193a-3p in endometritis has not been fully explored, and the clinical validation of its therapeutic effect on endometritis is currently very much open, and the extension of the results from specific cell lines or experimental conditions to clinical practice should be done with caution, and the mechanism of EVs on endometritis will need to be validated in further basic and clinical studies in the future.

EVs from different MSC sources in the treatment of endometritis

MSC-derived exosomes are an important paracrine product that has been used in the treatment of inflammatory diseases due to their immunomodulatory functions and tissue repair capabilities similar to those of MSCs. An injectable hydrogel scaffold with interleukin-1β (IL-1β)-activated MSC-derived exosomes has been demonstrated for the treatment of endometritis (Zhao et al., 2023). In addition to this, exosomes from umbilical cord MSCs (hUCMSCs-Exo) play key roles in tissue repair, including anti-inflammatory, antioxidant, implantation-supporting, and trophic functions (Samsonraj et al., 2017). A previous study reported that treatment of cows with bovine adipose tissue-derived stem cells (ADSCs) suppressed levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in endometrial epithelial cells, thereby inhibiting lipopolysaccharide (LPS)-induced inflammation, demonstrating the role of ADSC-Exos in the endometrial cells via the miR-21/TLR4/NF-κB signalling pathway. Endometrial cells overexpress miR-21 through the miR-21/TLR4/NF-κB signalling pathway, thereby decreasing the expression of TLR4, reducing the phosphorylation of p65, a downstream effector of TLR4, and exerting anti-inflammatory effects (Wang, Li & Yu, 2023). It suggests that ADSC-Exos has a positive effect on the treatment of chronic endometritis. ADSCs-derived Exos inhibited LPS-induced endometrial cell inflammation and mouse models (Lu et al., 2021). Table 1 summarises the source and mechanism of action of extracellular vesicles in endometrial-related diseases.

Technical challenges

Extracellular vesicles hold great promise in unfolding as novel biomarkers of disease, therapeutics, and drug delivery vectors; However some reports describe conflicting results, even from extracellular vesicles of the same cell type. For example, MSC-derived exosomes have been shown to both inhibit and promote tumourgrowth (Bruno et al., 2013; Zhu et al., 2012). Secondly, there are still many technical challenges regarding how to effectively utilise these small molecule messengers for future applications. Despite the clinical potential of EV, the lack of sensitive EV preparation and analysis techniques poses a barrier to clinical translation (Konoshenko et al., 2018). There is an urgent need for efficient and large-scale clinical-grade preparation processes, effective isolation techniques, suitable storage conditions, precise modification strategies, rigorous purification steps, and precise target delivery methods (Sil et al., 2020). Multiple approaches have been used to assess the biodistribution of EVs in vivo, including fluorescent labelling of lipids and proteins, immunofluorescence, bioluminescence, Pet, sPeCt, Mri and Ct imaging59 (Van Niel et al., 2022). All these methods have limitations in tracking the distribution of EVs, so new methods with large dynamic range of temporal and spatial resolution will be needed in the future to overcome these limitations.In addition, methodological issues and the heterogeneity of EV components have hindered the progress of EV validation trials and the development of EV-based diagnostic and therapeuticproducts (González et al., 2024). Because of the inherent complexity, size heterogeneity, and natural variation of exosomes throughout the assembly process, the application of exosomes as reliable therapeutic vectors necessitates a scalable manufacturing process that produces exosomes in a rapid, cost-effective, and reproducible manner (Rezaie, Feghhi & Etemadi, 2022).