Extracellular vesicles in reproductive biology and disorders: a comprehensive review

In the past few years, EVs have attracted growing interest concerning their function in reproductive system diseases, including PCOS, endometriosis, male infertility, and RPL. The subsequent sections will discuss the role of EVs in diverse reproductive disorders, with a specific emphasis on their involvement in the immune system ( Figure 5 ). This figure illustrates the process of EV generation and their contents, including proteins, RNA, and lipids. The lower part of the image specifically details the role of EVs in four reproductive system-related diseases: Polycystic Ovary Syndrome (PCOS), endometriosis, male infertility, and recurrent pregnancy loss. In these conditions, EVs influence cellular communication by carrying specific biomolecules, thereby contributing to disease progression and pathogenesis. Created with BioRender.com . EVs exert multifaceted regulatory roles in polycystic ovary syndrome (PCOS) pathophysiology by delivering specific miRNAs that orchestrate critical molecular pathways. These vesicles modulate energy supply through miR-34a-5p, which suppresses lactate dehydrogenase A to impair glycolysis in granulosa cells, while miR-143-3p disrupts Smad1/5/8 signaling pathway by targeting BMPR1A, promoting granulosa cell apoptosis ( 99 , 100 ). Within the ovarian microenvironment, miR-379 inhibits granulosa cell proliferation via phosphoinositide-dependent kinase 1 upregulation and impedes M2 macrophage polarization, contributing to follicular developmental arrest ( 20 ). In PCOS mouse model, serum-derived miR-128-3p was down-regulated, which promotes granulosa cells ferroptosis ( 101 ). Notably, mesenchymal stem cell-derived EVs (e.g., BMSCs-Exo) counteract ovarian dysfunction by delivering therapeutic miRNAs that attenuate CD31 overexpression, normalize aberrant angiogenesis, and inhibit NF-κB-mediated inflammation in granulosa cells ( 102 ). Clinically, circulating EV miRNAs such as miR-143-5p and miR-34a-5p exhibit strong correlations with gamma-linolenic acid, serving as dynamic biomarkers for inflammatory monitoring in PCOS ( 103 ). EVs serve as effective drug delivery carriers, enabling the targeted delivery of therapeutic agents to specific cells. For instance, the delivery of anti-inflammatory drugs and insulin sensitizers through EVs can significantly ameliorate the inflammatory state and enhance insulin sensitivity in patients with PCOS, ultimately leading to improved treatment outcomes ( 104 – 106 ). Collectively, these insights underscore EV miRNAs as central mediators in PCOS pathogenesis, offering a dual promise for precision diagnostics and targeted therapies. Endometriosis is a common gynecological condition marked by the presence of endometrial tissue situated beyond the uterus. EVs are crucial in the pathophysiological mechanisms linked to endometriosis. Specifically, EVs containing miRNAs and growth factors facilitate cell migration and invasion. For instance, endometrial cells transfer miR-15a-5p through EVs, which ultimately contributes to the development of ectopic lesions ( 107 ). Furthermore, lncRNA carried by EVs, such as CHL1-AS1, can inhibit cell migration and proliferation ( 108 ). Additionally, EVs carrying IL-10 can inhibit NK cell activity, further facilitating the development of ectopic lesion ( 109 ). EV protein markers in blood can be leveraged for early diagnosis and monitoring of endometriosis. Research has shown that the expression levels of miR-22-3p and miR-320a in peripheral blood are positively correlated with the severity of endometriosis, establishing their potential as reliable biomarkers for the condition ( 110 ). Lastly, EV-mediated gene therapy may represent a novel therapeutic strategy. EVs demonstrate significant therapeutic potential for endometriosis management, as their capacity to transport bioactive cargo to designated cellular or tissue targets enables their employment as precise drug delivery vehicles and instruments for targeted therapeutic interventions ( 21 ). Male infertility is a multifaceted condition influenced by numerous factors related to spermatogenesis and sperm maturation. Testicular sertoli cells and germ cells transfer miRNAs and proteins through EVs, influencing the development of male germ cells. For instance, miR-34b is transferred via EVs and regulates the sperm motility and count ( 111 ). In addition, EVs that carry antioxidant enzymes help regulate oxidative stress within sperm cells, thereby protecting them from damage ( 112 ). EVs originating from Sertoli cells have demonstrated the ability to prevent spermatogonial stem cell apoptosis by transferring miRNAs like miR-10b, resulting in the downregulation of KLF4 expression ( 113 ). Mesenchymal Stem-Cell derived EVs can contribute to attenuating cell injuries through specific miRNAs, such as miR-19a, miR-21-5p, and miR-144 ( 114 ). The possible role of these miRNAs in alleviating sperm damage caused by chlamydia indicates potential therapeutic use for EVs. Furthermore, the detection of EV miRNAs in semen may serve as valuable biomarkers for male infertility. It has demonstrated that semen miRNA levels correlate positively with sperm quality and male fertility, underscoring their potential as biomarkers for assessing these reproductive parameters ( 22 ). In treatment, EV delivery of antioxidants or miRNAs can ameliorate the oxidative stress in sperm, consequently enhancing sperm quality and fertility. Administration of superoxide dismutase (SOD) or miR-126-5p via EVs has been shown to maintain sperm viability and morphology, ultimately contributing to improved male fertility ( 115 , 116 ). Recurrent pregnancy loss (RPL) is a prevalent complication during pregnancy characterized by a complex pathogenesis, in which immune factors play a pivotal role. In recent years, the immune regulatory functions of EVs in RPL have garnered significant attention. miRNAs, proteins, and lipids carried by EVs, which are essential for maintaining immune balance at the maternal-fetal interface ( 117 ). Decidua-derived EVs have been shown to be essential modulators of T-cell differentiation and function through the delivery of specific miRNAs, thereby facilitating the development of immune tolerance ( 118 , 119 ). This immunoregulatory mechanism extends to their capacity to activate macrophages and dendritic cells while maintaining inflammatory homeostasis - a critical function for protecting the embryo from pathological immune response ( 120 ). Stem cell-derived EVs have demonstrated notable immunosuppressive and anti-inflammatory properties. A landmark study by Xiang et al. employed ultracentrifugation to isolate EVs from bone marrow mesenchymal stem cell cultures, which were then administered to pregnant mice with a history of RPL. The intervention resulted in significantly improved pregnancy outcomes, as evidenced by increased serum levels of the anti-inflammatory cytokines IL-4 and IL-10, along with concomitant reductions in proinflammatory mediators TNF-α and IFN-γ at the maternal-fetal interface. Mechanistically, this therapeutic approach modulated both T-cell function and macrophage polarization, ultimately decreasing embryo resorption rates by 42% compared to control groups. These findings collectively establish the therapeutic potential of stem cell-derived EVs in ameliorating immune-mediated pregnancy complications through precise immune modulation ( 121 ). Another investigation showed that villi can modulate IFN-γ production by decidual natural killer cells via the EV-mediated delivery of miR-29a-3p. This finding suggests a novel therapeutic strategy involving engineered villus-derived EVs mixed with HA-Gel, which shows promise for treating unexplained RPL in both murine models and potential clinical applications ( 23 ). Collectively, the immune regulatory role of EVs offers a novel perspective for understanding pathogenesis of RPL. By exploring the specific mechanisms of EV-mediated immune regulation at the maternal-fetal interface, we may identify new targets and strategies for the early diagnosis and treatment of RPL ( 122 ). Nonetheless, the prospect of utilizing EV therapy for managing RPL appears encouraging, especially when combined with current therapeutic strategies.

Infertility has become a pressing global health concern, with modern lifestyles and environmental pollution contributing to its rapid rise ( 1 ). Intercellular communication is essential for maintaining physiological homeostasis in multicellular organisms. Disruptions in intercellular communication are increasingly recognized as a key factor in infertility ( 2 ). In addition to juxtacrine signaling through tight junctions such as gap junctions for cell-to-cell communication, cells secrete a variety of molecules, including hormones, peptides, cytokines, and growth factors, into the extracellular environment to facilitate endocrine, paracrine, and autocrine signaling ( 3 , 4 ). Recently, a novel mechanism of intercellular communication has been identified, involving the secretion and internalization of extracellular vehicles (EVs) ( 5 ). EVs are divided into microvesicles, apoptotic vesicles, and exosomes based on their nature and function ( 6 , 7 ). Exosomes are a separate subpopulation of EVs with diameters ranging from 30 to 150 nm and densities from 1.13 to 1.19 g/mL. They serve as vehicles for informational molecules involved in communication between cells, facilitating the transport of functional proteins and genetic information. This transport can alter the phenotype and function of recipient cells, leading to alterations in cellular fate and physiological activities ( 8 ). EVs are produced by the double invagination of the plasma membrane and the inward budding of the luminal membrane. These structures develop within the intralumenal vesicles of multivesicular bodies (MVBs), which extend inward from the luminal membrane. The formation of these structures involves mechanisms that are both endosomal sorting complexes required for transport (ESCRT) -dependent and ESCRT-independent, linking with autophagosomes and lysosomes for biomolecule degradation or plasma membrane interaction for release, thus engaging in the endocytic and transport functions of cellular materials ( 6 ) ( Figure 1 ). Across physiological and pathological states, nearly all cell types, including epithelial cells, macrophages, mast cells, neurons, and mesenchymal cells, are capable of secreting EVs ( 9 ). Electron microscopy has revealed that EVs are flattened, or spherical vesicles encased in a lipid bilayer membrane, displaying a distinctive cup-like morphology ( 10 ). These EVs are widely present in several biological fluids, such as blood, urine, saliva, amniotic fluid, cerebrospinal fluid, follicular fluid (FF), and semen ( 11 , 12 ). They contain a consistent set of marker proteins, specifically the tetraspannin proteins CD9, CD63, CD81, and CD82, which are currently recognized as the hallmark of EV ( 13 ). Additionally, EVs are abundant in proteins that are involved in multivesicular bodies biogenesis (such as Alix and TSG101), as well as in membrane transport and fusion (including Annexins, Flotillins, and GTPases), and heat shock proteins (for instance, Hsp60, Hsp70, and Hsp90). They also harbor significant components of the major histocompatibility complex (MHC I and MHC II) proteins, as well as a variety of lipids, including sphingomyelin, sphingosine, cholesterol, ceramide, and glycans ( 14 – 16 ). Additionally, EVs may encompass various types of cell surface proteins, intracellular proteins, nucleic acids, amino acids, and various metabolites ( 5 , 17 ) ( Figure 2 ). The formation of EVs begins with endocytosis, which has two pathways: returning the cargo to the plasma membrane as “recycling endosomes” or transforming into “late endosomes,” or MVBs. MVBs will either merge with the lysosome or the plasma membrane, releasing their cargo outside the cell. Several RAB proteins, including Rab 27a and Rab 27b, as well as protein complexes, help transport MVBs to the plasma membrane and release EVs. In contrast, microvesicles are formed by the plasma membrane’s outward budding and scission, whereas apoptotic cellular membranes’ outward bubbling results in the production of apoptotic bodies. EVs and target cells interact in three ways (1): membrane proteins on EVs bind directly to receptors on target cells, activating an intracellular signaling cascade; (2) EVs transport their contents to target cells by fusing with the cell membrane; and (3) EVs are engulfed by endocytosis, releasing signaling molecules. Created with BioRender.com . Structure and composition of EV. EV is a lipid bilayer structure that contains lipids, proteins and nucleic acids. Sphingomyelin, phosphatidylserine, cholesterol and ceramides are highly distributed on the membrane. In addition, EVs also contain a variety of proteins such as major histocompatibility complex I and II (MHC I and MHC II), proteins from the MVB machinery (ALIX, TSG101), heat shock proteins (HSP70, HSP90, HSP60), tetraspanins (CD9, CD63, CD81), receptors (FasL, TNF, TfR), adhesion molecules (Interins, Selectins, Cadherins) and cytosolic proteins, RNA and DNA. Created with BioRender.com . EVs engage in biological activities primarily through three mechanisms. Firstly, they fuse directly with the membrane of the target cell, thereby activating downstream signal pathways; secondly, EVs are internalized by the target cell through receptor-mediated endocytosis, releasing biomolecules into the cytoplasm and subsequently activating the cell; thirdly, upon recognizing specific receptors on the target cell surface, EVs initiate signal transduction pathways in effector cells ( 18 ). Overall, the interaction between EVs and target cells facilitates intercellular communication, immune modulation, cellular differentiation, and pathological processes related to related to reproductive diseases, including polycystic ovary syndrome (PCOS), premature ovarian failure (POF), endometriosis ( 9 ) ( Figure 1B ). For the reproductive system, EVs play a multifaceted role, including gamete maturation, fertilization, embryonic development, and implantation ( 19 ). Moreover, they are associated with reproductive disorders such as PCOS ( 20 ), endometriosis ( 21 ), male infertility ( 22 ), and RPL ( 23 ). The quantity and composition of EVs are considered to be innovative biomarkers for the diagnosis and prediction of reproductive diseases ( 9 ). The aim of this review is to provide a summary of the research progress of EVs in reproductive biology, to enhance our understanding of the intercellular communication mechanisms of EVs in the reproductive system.

In conclusion, EVs play a role in numerous biological functions in reproductive systems, such as gamete maturation, fertilization, embryo development, and the progression of reproductive diseases. These small vehicles carry bioactive compounds such as proteins, lipids, and nucleic acids from one cell to another, functioning as crucial regulator of cell communication. Their potential as indicators and therapeutic targets is highlighted by their involvement in these vital reproductive processes. EVs aid in the interchange of vital components that improve sperm and oocyte quality during gamete maturation, increasing the chance of successful fertilization ( Figure 6 ). EVs regulate gene expression and cellular signaling during embryogenesis, fostering proper embryonic development and differentiation. Their dysregulation is associated with reproductive disorders, highlighting the importance of understanding their mechanisms. For ART, the use of EVs is especially promising. Clinicians can utilize EVs to develop innovative diagnostic tools and therapeutic strategies to combat infertility and enhance ART outcomes. EV-based interventions could enhance the quality of gametes and embryos, reduce the risk of implantation failure, and minimize the incidence of pregnancy complications. This figure elucidates the crucial roles of EVs in Assisted Reproductive Technology (ART). EVs enhance the quality of sperm and oocytes by regulating the microenvironment of the reproductive tract and delivering signaling molecules and bioactive substances. During fertilization, they facilitate the recognition and binding between sperm and oocyte by transferring specific proteins and molecules, thereby increasing the success rate of fertilization. In the embryo development stage, EVs are vital in regulating gene expression and cell differentiation through intercellular communication, ensuring proper embryonic growth. Finally, during embryo transfer, EVs support the preparation of the uterine endometrium and enhance embryo-uterus interactions, which improves the implantation potential of the embryo. Collectively, these processes demonstrate the significant impact of EVs on the success of ART. Created with BioRender.com . Future research should focus on elucidating the specific molecular pathways and cargo of EVs involved in reproductive processes, while also exploring their potential applications in personalized medicine. Integrating EV into clinical practice has the potential to revolutionize the reproductive medicine, providing new hope to couples facing infertility diseases. In sum, research into EVs within reproductive biology and pathology deepens our comprehension of human reproduction. With ongoing advancements, the significance of EVs is anticipated to escalate, paving the way for innovative strategies in reproductive health.