Systematic review on the extracellular vesicles in reproductive medicine and gamete union.

The landscape of EVs in the context of reproductive physiology and ART is notably diverse, encompassing distinct entities with unique biological roles. EVs including exosomes and microvesicles, are formed through distinct pathways: apical secretion and multivesicular bodies (MVBs). Among these vesicular structures, exosomes, microvesicles, and microparticles play crucial roles in governing reproductive events [ 9 , 25 ]. The design of EVs involves several key steps, including isolation, surface modification, and cargo loading. Isolation techniques such as ultracentrifugation, size-exclusion chromatography, and immunoaffinity capture ensure high purity and consistency of the vesicles. Surface modification strategies, such as conjugating targeting ligands or altering the surface charge, enhance the specificity and efficiency of delivery to target cells. Drug encapsulation within EVs can be achieved through various methods, each with specific advantages. Passive loading involves incubating the vesicles with the drug, allowing natural incorporation during vesicle formation. Electroporation uses electric pulses to create temporary pores in the vesicle membrane, facilitating drug entry. Chemical conjugation covalently bonds the drug molecules to surface proteins or lipids of the vesicles, ensuring stable attachment. Exosomes, ranging between 30 and 150 nm, are endosomal-derived vesicles enriched with bioactive molecules [ [26] , [27] , [28] ]. Implicated in long-distance cell-to-cell communication during crucial events like gametogenesis and embryo implantation, exosomes modulate gene expression profiles, establishing their central regulatory role in reproductive processes [ [29] , [30] , [31] ]. Microvesicles, larger in size (100–1,000 nm), shed directly from the plasma membrane, encapsulate cytoplasmic components [ 27 , 31 , 32 ]. Identified in seminal, follicular, and uterine luminal fluid, microvesicles act as vehicles for intercellular communication during sperm maturation, oocyte development, and embryo implantation, leveraging their larger size to influence a broader array of cellular functions [ [33] , [34] , [35] ]. Microparticles, akin to microvesicles in size, are fragments released from apoptotic or activated cells, carrying cellular components and exhibiting procoagulant properties [ [34] , [35] , [36] ]. Detected in biofluids like seminal fluid and follicular fluid, microparticles contribute to the regulation of reproductive processes [ 37 , 38 ]. In summary, the diversity in size, cellular origins, and cargo compositions of EVs, namely exosomes, microvesicles, and microparticles, collectively contributes to the rich tapestry of reproductive physiology and ART ( Fig. 2 ). Understanding the distinct roles of these vesicles provides a foundation for exploring targeted interventions that leverage the unique properties of each EV subtype to enhance fertility and improve outcomes in assisted reproductive technologies. Fig. 2 The diverse landscape of extracellular vesicles (EVs), including exosomes, microvesicles, and microparticles, intricately weaves into the fabric of reproductive physiology and assisted reproductive technology (ART). (Created by Figdraw2.0). Fig. 2 The diverse landscape of extracellular vesicles (EVs), including exosomes, microvesicles, and microparticles, intricately weaves into the fabric of reproductive physiology and assisted reproductive technology (ART). (Created by Figdraw2.0). The physiological relevance of EVs in reproductive events is a compelling area of investigation, providing profound insights into male and female fertility, natural fertilization, and ART. Exosomes, microvesicles, and microparticles emerge as crucial mediators orchestrating diverse cellular processes, contributing to the regulatory framework of reproductive physiology. In the male reproductive tract, the importance of EVs, particularly epididymosomes and prostasomes, is highlighted by their integral role in shaping sperm functionality [ 11 , 39 , 40 ]. Epididymosomes, secreted by the epididymal epithelium, modulate sperm maturation and confer essential attributes such as motility and fertilization competence [ 13 , [41] , [42] , [43] ]. Similarly, prostasomes, derived from the prostate gland, influence sperm capacitation and the acrosome reaction, underscoring their physiological significance in fine-tuning male reproductive processes [ [44] , [45] , [46] , [47] ]. In the female reproductive tract, EVs play a pivotal role in conveying critical information during key events such as oocyte maturation, fertilization, and embryo implantation [ 27 , [48] , [49] , [50] ]. EVs in follicular fluid, originating from granulosa cells, influence oocyte maturation and developmental competence ( Fig. 3 ). Oviductal and uterine EVs participate in the intricate dialogue between the embryo and maternal tissues, guiding the embryo through its developmental journey [ [51] , [52] , [53] , [54] ]. Fig. 3 In the intricate orchestration of reproductive events, the regulation of oocyte maturation emerges as a finely tuned process, with extracellular vesicles (EVs) playing a pivotal role. GnRH: gonadotropin-releasing hormone; DA: dopamine; LH: luteinizing hormone; LH-R: luteinizing hormone receptor; T, 17α-OHP: testosterone, 17α-hydroxyprogesterone; MIH: mullerian inhibiting hormone; E2: estradiol; MIH-R: mullerian inhibiting hormone receptor; EGF: epidermal growth factor; EGF-R: epidermal growth factor receptor; ACT-R: activin receptor; Smads: small mothers against decapentaplegic proteins (signaling molecules involved in the TGF-β signaling pathway); KISS-1: KISS1 gene (a regulator of puberty and reproductive function); GPR54: g-protein coupled receptor 54 (receptor for kisspeptins, which are important in regulating puberty and reproductive hormones); cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; PKC: protein kinase C; IGF-I: insulin-like growth factor I; IGF-R: insulin-like growth factor receptor; Follistatin: a protein that inhibits the activity of activins and other signaling molecules. (Created by Figdraw2.0). Fig. 3 In the intricate orchestration of reproductive events, the regulation of oocyte maturation emerges as a finely tuned process, with extracellular vesicles (EVs) playing a pivotal role. GnRH: gonadotropin-releasing hormone; DA: dopamine; LH: luteinizing hormone; LH-R: luteinizing hormone receptor; T, 17α-OHP: testosterone, 17α-hydroxyprogesterone; MIH: mullerian inhibiting hormone; E2: estradiol; MIH-R: mullerian inhibiting hormone receptor; EGF: epidermal growth factor; EGF-R: epidermal growth factor receptor; ACT-R: activin receptor; Smads: small mothers against decapentaplegic proteins (signaling molecules involved in the TGF-β signaling pathway); KISS-1: KISS1 gene (a regulator of puberty and reproductive function); GPR54: g-protein coupled receptor 54 (receptor for kisspeptins, which are important in regulating puberty and reproductive hormones); cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; PKC: protein kinase C; IGF-I: insulin-like growth factor I; IGF-R: insulin-like growth factor receptor; Follistatin: a protein that inhibits the activity of activins and other signaling molecules. (Created by Figdraw2.0). The physiological significance of these EVs lies in their ability to modulate gene expression profiles, transfer regulatory molecules, and create a conducive environment for successful fertilization [ 10 , [55] , [56] , [57] ]. Additionally, EVs impact gametogenesis and early embryonic development by carrying bioactive cargo, contributing to the regulatory network governing the fate and function of germ cells and early embryos. Understanding the physiological relevance of EVs in reproductive events opens avenues for targeted interventions in ART, enhancing the efficiency of in vitro handling of gametes, fertilization processes, and embryo culture [ [58] , [59] , [60] ].

Yutao Wang: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Honghao Sun: Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Fangdie Ye: Writing – original draft, Visualization, Resources, Methodology, Data curation, Conceptualization. Zhiwei Li: Writing – original draft, Visualization, Software, Investigation. Zhongru Fan: Writing – original draft, Methodology, Formal analysis, Data curation. Xun Fu: Writing – original draft, Visualization, Software, Investigation. Yi Lu: Writing – original draft, Resources, Methodology. Jianbin Bi: Writing – review & editing, Visualization, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Hongjun Li: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

Exosome engineering is expected to break new ground in the search for targeted drug delivery systems. With the advent of precision medicine, research has increasingly focused on customizing exosomes with homing capabilities that target specific cells within the reproductive system. Innovations may include the incorporation of targeted ligands, such as peptides or antibodies, that can selectively bind to receptors overexpressed in diseased tissues, enhancing the delivery of small molecule drugs to treat diseases such as endometriosis or uterine fibroids. In addition, the development of exosomal controlled release drugs is expected to expand therapeutic efficacy and minimize systemic side effects. Although exosome-based therapeutic approaches demonstrate significant potential in the field of reproductive medicine, there is currently a lack of in-depth research on the application of exosomes in the treatment of infertility. At present, there is relatively little literature discussing standardized treatment protocols for extracellular vesicles, particularly in terms of clinical application. Existing infertility treatment methods, such as filtration using 0.22-μm filters, ultraviolet irradiation, and chemical processing, while utilized in laboratory settings, have not been adequately validated for their impact on the integrity and functionality of exosomes, necessitating further research to determine their efficacy and safety. Exosomes are in a unique position to catalyze the shift to personalized medicine in the field of reproductive health. Utilizing a patient's own exosomes for drug delivery can significantly reduce immunogenic responses and enhance drug efficacy. Personalized exosome therapy can be tailored not only to the disease profile, but also to the specific physiological and genetic makeup of the patient, optimizing treatment options for infertility, pregnancy-related disorders, and genetic pathologies that affect reproductive health. There is no doubt that the future will see an explosion of collaborative and interdisciplinary research efforts. Bridging the gap between reproductive biologists, nanotechnologists, and clinical practitioners is critical to moving exosom-based therapies from the lab to clinical practice. In addition, combining bioinformatics and artificial intelligence can simplify the identification of exosomal biomarkers and drug candidates, accelerating the development process. The establishment of ethical guidelines and regulatory oversight is essential to ensure the safe and effective use of these advanced therapies. As collaboration expands, creating cohesive networks and a shared knowledge base is critical to continuous progress and fostering innovation that can be seamlessly integrated into the healthcare system. Together, the forward momentum of exosome research promises to have a transformative impact on reproductive health, laying the foundation for efficient, patient-centered therapies that address the complexity of individual conditions with unprecedented precision.

Exosome-mediated small molecule drug delivery represents an emerging frontier in reproductive medicine, promising targeted therapies that improve efficacy and reduce side effects. This cutting-edge strategy uses exosomes (nanoscale vesicles naturally secreted by cells) to encapsulate the therapeutic agent and transport it directly to reproductive tissue. Several case studies and preclinical research efforts have demonstrated the potential and challenges of this novel approach. A pioneering preclinical study has clarified the use of exosomes to deliver siRNA molecules to inhibit endometrial cancer cells, demonstrating the ability of exosomes to act as small molecule delivery vehicles within reproductive tissue [ 68 , 69 ]. Exosomal drug delivery for endometrial cancer typically involves local administration, such as intratumoral injection, or intravenous (IV) routes to target metastases. Exosomes usually have a slightly negative zeta potential, enhancing stability and interaction with cell membranes, though surface modifications can be made to improve targeting. Optimal particle sizes range from 30 to 150 nm, with an average around 100 nm for effective biodistribution and cellular uptake. In-cell stability is ensured by the exosomal membrane protecting encapsulated drugs from degradation, verified through techniques like fluorescence microscopy and biochemical assays. Formulation involves isolating exosomes via ultracentrifugation or chromatography, loading them with drugs through passive loading, electroporation, or chemical conjugation, and ensuring quality control through nanoparticle tracking analysis and electron microscopy. Although siRNA is not a traditional small-molecule drug, this research paves the way for similar approaches for small molecules. The exosomes used are derived from dendritic cells and take advantage of their inherent tumor environment homing ability. The results showed that tumor progression was significantly reduced and there was no systemic toxicity common to conventional chemotherapy, which heralds a new approach to treating gynecological malignancies. Another case study focuses on the treatment of polycystic ovary syndrome (PCOS), a common endocrine disorder that impairs fertility. The researchers used exosomes to deliver metformin, a small-molecule drug often used to treat polycystic ovary syndrome [ 70 ]. Metformin-loaded exosomes target ovarian tissue in rodent models, improving insulin sensitivity and restoring the ovulation cycle [ 71 ]. Exosomal delivery of metformin for PCOS involves intravenous or direct ovarian injection, with exosomes typically exhibiting a slightly negative charge to aid stability and cellular interaction. Optimally sized around 100 nm, exosomes ensure efficient uptake and stability, protecting the drug from degradation. The formulation includes isolating exosomes via ultracentrifugation or chromatography, loading metformin through passive methods or electroporation, and ensuring quality control with techniques like nanoparticle tracking analysis. This approach minimizes the gastrointestinal side effects of the drug and is a significant advance over traditional drug delivery methods. In the field of male fertility, exosom-mediated strategies have been explored to address issues such as sperm motility and motility [ 72 ]. One particular study encapsulated sildenafil, a small molecule phosphodiesterase inhibitor, in exosomes to enhance its delivery to penile tissue [ 73 ]. Sildenafil-loaded exosomes for male fertility are typically administered intravenously to ensure targeted delivery to penile tissue, enhancing erectile function with minimal systemic side effects. Exosomes generally exhibit a slightly negative surface charge, aiding stability and cellular interaction, though modifications can optimize targeting. Optimal exosome size is around 100 nm for efficient uptake and stability, with narrow size distribution preferred. In-cell stability is ensured as exosomes protect the drug from degradation, with stability assessed via fluorescence microscopy and biochemical assays. Formulation involves isolating exosomes through ultracentrifugation or chromatography, loading sildenafil via passive methods or electroporation, and ensuring quality control with techniques like nanoparticle tracking analysis. In animal models, this targeted delivery system improved erectile function without affecting systemic blood pressure, a problem often encountered with traditional oral sildenafil. However, clinical trials are still in their early stages. A pilot clinical study is currently underway investigating exosom-mediated delivery of clomiphene citrate, an ovulation stimulant. The aim of this study was to assess the efficiency of exosomal targeting of ovarian tissue and to measure the effect on ovulation rates compared to standard treatment regimens [ 70 ]. These case studies and trials highlight the versatility and potential of exosomes as reproductive health drug delivery systems. By providing a way to transport small molecule drugs directly to target tissues, exosomes can significantly enhance the therapeutic effectiveness of various reproductive diseases. While the transition from preclinical to clinical poses significant challenges, such as scaling up production and regulatory hurdles, early findings are promising. They show that reproductive medicine is moving towards a more personalized and precise approach to treatment, harnessing the natural abilities of exosomes to revolutionize fertility therapy. Innovative applications of small molecule drugs delivered by exosomes have extended to reproductive health, demonstrating potential breakthroughs in the treatment of a range of diseases that affect fertility and pregnancy outcomes. These studies highlight the versatility of exosomes as delivery carriers to encapsulate therapeutic agents and efficiently deliver them to target reproductive tissues. One notable area of research is the treatment of endometriosis, a common condition characterized by the growth of endometrial tissue outside the uterus, leading to pain and infertility. In a groundbreaking preclinical study, researchers explored the use of exosomes to deliver anti-micNA molecules designed to silence specific miRNAs associated with the pathogenesis of endometriosis [ [74] , [75] , [76] ]. Targeted delivery of these molecules resulted in a significant reduction in the size of endometrial lesions in animal models, providing a promising non-surgical treatment option that could potentially move into clinical trials. In the context of male reproductive health, exosome-mediated strategies have been explored for the delivery of small molecule drugs that target sperm motility and motility. Preliminary studies have shown that exosomes have the potential to encapsulate fertility enhancers and deliver them directly to sperm cells, thereby improving motor parameters and increasing the chances of successful fertilization [ 77 , 78 ]. The findings suggest a new way to treat male infertility that could complement existing therapies. Another promising application is the prevention of preterm birth, a leading cause of neonatal morbidity and death. Recent preclinical work has focused on the use of exosomes to deliver progesterone and other drugs that promote uterine quiescence directly to pregnancy tissue [ 79 , 80 ]. This targeted approach aims to improve the effectiveness of fetal-preserving treatment and reduce the incidence of preterm birth. Although still in its early stages, this research could pave the way for clinical trials and ultimately more effective strategies for managing high-risk pregnancies. These emerging applications of exosome-delivered small molecule drugs in reproductive health represent the frontier of medical research and have the potential to provide novel, targeted treatments for diseases that challenge fertility and pregnancy. The significance of exosome-mediated small molecule drug delivery in reproductive health lies in its potential to revolutionize the treatment of various reproductive disorders. By providing a method to transport drugs directly to target tissues, exosomes can enhance therapeutic effectiveness and minimize systemic side effects. As research progresses and these innovative applications advance from preclinical studies to clinical trials, exosome-mediated drug delivery could become a cornerstone of personalized and precision medicine in reproductive health, offering new hope to patients facing infertility and other reproductive challenges.

In conclusion, the exploration of exosomal drug delivery systems in reproductive medicine has revealed a transformative approach to the treatment of infertility and reproductive disorders. This review provides a comprehensive overview of the evolution and current state of drug delivery systems, highlighting a major shift in the utilization of extracellular vesicles (especially exosomes) due to their superior cellular communication capabilities and inherent biocompatibility. The review emphasizes the mechanisms of exosome operation, discussing their advantages over synthetic nanoparticles, particularly in terms of delivery specificity and reduced immunogenicity.

In the field of intracellular communication, exosomes are complex mediators with complex biochemical components that give them unique functions. These vesicles are characterized by a phospholipid double layer similar to the progenitor cell plasma membrane, maintaining structural integrity as they move through biological fluids [ 61 ]. Exosomes transport a diverse array of biomolecules inherited from their parent cells, including a variety of proteins, RNA species (such as mRNA and microRNA (miRNA)), and lipids. These molecules are selectively packaged during exosome biogenesis, reflecting the specific function of the source cell. The selection of these molecules is a highly selective process that reflects the specific function of the source cell. As a result, exosomes are emerging as important facilitators of intercellular communication, with the ability to transmit biological signals that can significantly alter gene expression and alter cell activity in complex biological systems. Given these unique properties, exosomes are increasingly recognized for their potential applications in therapeutic contexts. In the field of reproductive medicine, the intrinsic targeting specificity and delivery efficiency of exosomes reveal promising pathways for innovative therapies designed to modulate germ cells with previously unattainable precision [ 62 ]. Improving exosome targeting to specific germ cells in reproductive medicine involves various strategies such as surface modification by attaching target-specific ligands like peptides or antibodies, and genetic engineering to express specific surface proteins. Selecting appropriate cell types, such as reproductive epithelial cells or mesenchymal stem cells, can leverage their natural homing properties. Preconditioning donor cells under specific environmental conditions or with chemical treatments can enhance targeting capabilities. Additionally, creating fusion proteins or using nanoparticle coatings can improve stability and targeting efficiency. Exosomes act as delicate channels for cell-cell dialogue, a feature that is particularly prominent in the complex reproductive medicine environment. These extracellular vesicles use a range of surface biomolecules to precisely identify and establish complex and specific delivery mechanisms to designated recipient cells. Exosomes, due to their unique properties such as low immunogenicity and high stability, offer a promising approach for therapeutic applications, including drug delivery and as potential vaccines. However, challenges like low circulation time, targeting issues, and production limitations hinder their clinical implementation [ 63 ]. These challenges impact clinical implications significantly. Low circulation time can reduce the effectiveness of exosomal therapies, requiring higher or more frequent doses, which can increase the cost and complexity of treatment. Targeting inefficiencies can lead to off-target effects, potentially causing unintended side effects and reducing the overall therapeutic benefit. Production limitations can hinder the availability and affordability of exosome-based treatments, limiting their accessibility to patients. Once engaged, these vesicles are able to trigger signal transduction cascades or integrate with the membrane of the target cell, ultimately depositing their cargo directly into the recipient cytoplasm. This interaction can trigger significant changes in gene expression profiles or adjustments in cell function. In therapeutic applications, these vesicles can be designed to encapsulate drug compounds, facilitating their delivery to specific cellular destinations. This exosom-based carrier system proposes a less invasive approach that is expected to improve the success rate of reproductive therapy [ 64 ]. Overall, while exosomes present an innovative and promising avenue for drug delivery in reproductive medicine, addressing these challenges is essential for their successful clinical translation. Continuous research and technological advancements are crucial to overcoming these hurdles and realizing the full potential of exosomal therapies. Exosomal systems present a unique advantage over synthetic nanoparticles in drug delivery, especially in the delicate context of reproductive medicine. Biocompatibility is a paramount benefit—exosomes are endogenous to the body, significantly reducing the risk of immune rejection compared to foreign synthetic particles [ 65 ]. Additionally, their natural origin means that exosomes inherently possess the cellular machinery necessary for efficient navigation and uptake by target cells, thereby facilitating more precise drug delivery [ 66 , 67 ]. This natural propensity for targeted therapy can be leveraged to enhance the specificity of treatment, a crucial consideration where systemic side effects are a major concern. Moreover, exosomes have the capability to cross biological barriers that are often impenetrable to synthetic alternatives, potentially broadening the therapeutic window. These characteristics position exosomal systems as a potentially transformative platform in the targeted treatment of reproductive health issues, from infertility to genetic diseases affecting the reproductive tract.

The emergence of exosom-based therapies is reshaping the frontiers of reproductive medicine, particularly in the transport of proteins and genes, with the promise of tackling infertility and hereditary diseases. Exosome-delivered proteins and gene therapy have become highly targeted and effective means of treating a range of reproductive health problems. Polycystic ovary syndrome is a common endocrine disorder that has been the focus of exosome-mediated gene therapy [ 89 ]. The study examined the delivery of specific gene silencers, or promoters, encapsulated in exosomes to regulate hormonal imbalances at the ovarian level [ 90 , 91 ]. In animal models, this approach has yielded optimistic results, with a return to normal ovulation cycles and a reduction in ovarian cyst formation observed, thus demonstrating a potential pathway for clinical application. In the field of male infertility, numerous preclinical efforts have focused on delivering proteins and genes to improve sperm parameters. In laboratory settings, exosomes loaded with specific proteins have been shown to enhance sperm motility and DNA integrity [ 92 , 93 ]. In the case of hereditary infertility, the delivery of exosome-based corrected gene sequences or gene-editing tools such as CRISPR has shown the potential to restore fertility in male animal models with specific genetic mutations that cause infertility. In addition, exosome-mediated gene delivery shows promise in the treatment of uterine-induced infertility. For example, preclinical trials have successfully used exosomes to deliver growth factors and genes to promote endometrial growth and repair, which are critical for embryo implantation and pregnancy maintenance [ 94 ]. While still awaiting clinical translation, these advances mark an important step toward addressing uterine pathology through regenerative medicine. Exosomes also prevent preterm birth by providing anti-inflammatory proteins that reduce intrauterine inflammation associated with preterm birth [ 95 ]. Although clinical data are still limited, preclinical models suggest that this strategy can be effective in prolonging pregnancy, thereby reducing the risks associated with preterm birth [ 96 ]. The preclinical success demonstrates that therapeutic delivery of proteins and genes via exosomes holds great promise in reproductive health. As research progresses, these novel treatments have the potential to revolutionize the treatment of reproductive disorders, offering hope to those who face challenges in conceiving and maintaining a pregnancy.

At the forefront of reproductive medicine, the use of exosomes to deliver therapeutic agents has provided a series of important discoveries. Exosomes, as vectors for targeted drug delivery, revolutionize current treatment practices by enabling highly specific and efficient delivery of therapeutic molecules, reducing systemic side effects, and improving patient outcomes. For example, exosomes have been used to transport miRNAs and specific proteins associated with enhanced ovarian and testicular function, leading to new strategies for treating infertility [ 9 ]. Empirical evidence from in vitro experiments suggests that exosomes have the ability to deliver specific growth factors, potentially improving the efficiency of embryo culture systems and thus the success rate of in vitro fertilization (IVF) techniques [ 81 , 82 ]. In addition, the application of exosomal transfer RNA molecules has been linked to changes in sperm motility and motility, heralding an innovative approach to addressing male infertility [ 9 , 83 ]. By offering a targeted delivery mechanism, exosome-mediated drug delivery can ensure that therapeutic agents reach specific cells or tissues in the reproductive system with high precision. This targeted approach minimizes off-target effects and maximizes the therapeutic impact, which is particularly beneficial in delicate and complex reproductive environments. Furthermore, exosomes' natural biocompatibility and ability to cross biological barriers enhance their suitability for delivering a wide range of therapeutic agents, from small molecules to nucleic acids and proteins. This versatility positions exosomes as a transformative tool in reproductive medicine, potentially addressing various reproductive disorders more effectively than traditional methods. Mesenchymal stem cells derived-extracellular vesicles (MSC-EVs) in treating female reproductive disorders such as intrauterine adhesion (IUA), premature ovarian insufficiency (POI), and PCOS. MSC-EVs are highlighted for their potential therapeutic benefits, attributed to their origin from parent cells, high biological stability, and low immunogenicity. EVs function as mediators by transferring molecules like proteins, miRNAs, lipids, and cytokines to recipient cells, showing promise in repairing the endometrium, reducing fibrosis, regulating immunity and anti-inflammatory responses, and preventing apoptosis in ovarian granulosa cells [ 84 ]. The emergence of exosomal drug delivery systems heralds a transformative chapter in the field of fertility therapy. Studies have shown that exosomes have the potential to inject selected Rnas or proteins designed to regulate the reproductive environment, improve the quality of eggs and sperm, and even improve molecular level defects in gametes or embryos [ 9 ]. The utility of exosomes in fertility applications depends on their ability to encapsulate and protect therapeutic drugs throughout their journey to their target, thereby improving the prospects for successful treatment outcomes while mitigating systemic adverse reactions commonly associated with traditional fertility drugs. Multiple pregnancies from transferring multiple embryos in assisted reproductive treatments (ART) pose risks, which could be mitigated by transferring a single embryo. However, existing methods for selecting the highest quality embryo, such as morphology assessment and preimplantation genetic diagnosis (PGD), are either unreliable or invasive. Emerging research on exosomes, a subtype of extracellular vesicles, suggests their potential in non-invasive embryo selection by influencing gene expression and endometrial receptivity, offering a promising advancement in improving ART's efficiency and safety [ 85 ]. Studies have shown that exosome treatment can increase implantation rates by approximately 20%, enhance blastocyst formation rates from 40% to 60%, and improve clinical pregnancy rates by 15%. Additionally, exosome therapies have been associated with a 10% reduction in miscarriage rates and a 25% improvement in endometrial receptivity markers. In addition, exosomes provide an opportunity to develop non-invasive diagnostic modalities that can help evaluate treatment effectiveness and embryo viability in real time. These exosomes are key in processes such as angiogenesis, endothelial cell migration, and embryo implantation, and are associated with pregnancy-related conditions like gestational diabetes and preterm birth. Additionally, exosomes influence infertility in both sexes through their transport of signaling molecules, with emerging research suggesting their potential in diagnosing and treating fertility disorders [ 86 ]. These aspects of exosomal drug delivery, from its biological origins to its ability to target delivery, set the stage for a revolutionary shift in reproductive health care, where treatments are not only individualized but potentially more effective. Within the sphere of reproductive health, the function of exosomes in the development of gametes and embryos is eliciting considerable scientific interest for their aptitude in mediating intracellular communication and substance conveyance. Exosomes are replete with a wealth of molecular cues pivotal to the processes of maturation, fertilization, and initial cellular division in gametes and embryos ( Fig. 4 ). By ferrying specific proteins, mRNA, and miRNAs, exosomes can modulate gene expression, thereby influencing the developmental trajectory of these cells. The study identifies 13 miRNAs with differential expression in plasma and plasma exosomes of patients experiencing implantation failure, with specific miRNAs showing consistent directional changes in the endometrium of patients with recurrent implantation failure (RIF) and in blood samples. The findings conclude that implantation failure during the window of implantation (WOI) is linked to unique miRNA profiles in plasma and exosomes, highlighting the potential of miRNAs as biomarkers or therapeutic targets for addressing implantation issues [ 87 ]. It was found that embryos leading to failed pregnancies had, on average, more detected miRNAs compared to those resulting in pregnancies, with miR-634 showing 71% accuracy and 85% sensitivity in predicting positive pregnancy outcomes [ 88 ]. This role is of particular consequence in the context of enhancing IVF techniques and decrypting the intricacies of early embryonic growth. Progress in this field may precipitate improved methodologies for embryo assessment and selection, with the overarching goal of bolstering the efficacy of IVF interventions. Fig. 4 In the dynamic landscape of reproductive science, the exploration of extracellular vesicle (EV)-mediated delivery into gametes and embryos represents a cutting-edge avenue. MVBs: multi-vesicular bodies; IUI: intrauterine insemination; IVF: i n vitro fertilization; IVM: in vitro maturation of oocytes; ICSI: intracytoplasmic injection of sperminto oocytes. (Created Figdraw2.0). Fig. 4 In the dynamic landscape of reproductive science, the exploration of extracellular vesicle (EV)-mediated delivery into gametes and embryos represents a cutting-edge avenue. MVBs: multi-vesicular bodies; IUI: intrauterine insemination; IVF: i n vitro fertilization; IVM: in vitro maturation of oocytes; ICSI: intracytoplasmic injection of sperminto oocytes. (Created Figdraw2.0).

Extracellular vesicles (EVs) have emerged as pivotal mediators of cellular communication with profound regulatory effects in both physiological and pathological contexts [ [1] , [2] , [3] , [4] ]. Consisting of exosomes, microvesicles, and microparticles, EVs are membrane-bound vesicles transporting a diverse cargo of bioactive molecules [ [5] , [6] , [7] ]. EVs in the reproductive system originate from various cell types and biological fluids, playing crucial roles in intercellular communication. In males, epididymosomes from the epididymis and prostasomes from the prostate gland enhance sperm maturation, motility, and capacitation. In females, EVs from follicular fluid secreted by granulosa cells aid oocyte maturation; oviductal EVs facilitate fertilization and early embryonic development; and uterine luminal EVs support embryo implantation and maternal communication. In the realm of reproductive physiology and assisted reproductive technology (ART), EVs wield significant influence, shaping male and female fertility, natural fertilization, and ART outcomes [ [8] , [9] , [10] ]. In male reproductive physiology, epididymosomes and prostasomes, subsets of EVs, crucially regulate sperm motility, capacitation, and the acrosome reaction, contributing to post-testicular sperm maturation [ [11] , [12] , [13] ]. In the female reproductive tract, EVs in follicular fluid, oviductal fluid, and uterine luminal fluid serve as information carriers during oocyte maturation, fertilization, and embryo-maternal crosstalk [ 9 , 13 , 14 ]. EVs play a vital role in natural fertilization, participating in gametogenesis, fertilization, and early embryonic development [ 15 , 16 ]. Their ability to transport bioactive molecules positions them as central regulators of molecular events crucial for successful reproduction. Selecting appropriate cells for exosome-mediated drug delivery in the reproductive system involves ensuring biocompatibility, relevance to target tissues, production efficiency, cargo loading capacity, and targeting ability. Cells such as mesenchymal stem cells (MSCs), reproductive epithelial cells, and dendritic cells are ideal candidates due to their high yields, biocompatibility, and natural targeting capabilities. MSCs are favored for their regenerative properties and high exosome production, while epithelial cells from reproductive tissues offer tissue-specific targeting, making them suitable for ovarian, uterine, and fallopian tube treatment. Challenges in studying gamete structure and function, including their resistance to exogenous substances, have spurred investigations into innovative intracellular delivery methods. Nanomaterial-mediated delivery, particularly biodegradable platforms, offers a promising avenue for overcoming these challenges. This approach mimics natural molecular cargo trafficking, and within this landscape, EVs, especially exosomes, stand out. Combining the benefits of engineered nanomaterials with biodegradability, EVs hold promise as not only biomarkers but also therapeutic targeting agents, revolutionizing infertility treatment and reproductive health [ [17] , [18] , [19] ]. In male reproductive physiology, knowledge of sperm development and the role of EVs provides insights into post-testicular sperm maturation, capacitation, and the acrosome reaction, directly applicable to improving sperm performance in ART [ 20 , 21 ] ( Fig. 1 ). Fig. 1 In the realm of assisted reproductive technology (ART), intracytoplasmic sperm injection (ICSI) stands as a pivotal technique addressing male infertility. (Created by Figdraw2.0). Fig. 1 In the realm of assisted reproductive technology (ART), intracytoplasmic sperm injection (ICSI) stands as a pivotal technique addressing male infertility. (Created by Figdraw2.0). In female reproductive physiology, comprehension of oocyte maturation, fertilization, and embryo implantation is crucial for refining ART [ 22 , 23 ]. EVs in reproductive fluids are implicated in conveying information during these processes, suggesting their potential as key mediators for successful ART procedures [ 20 , 24 ]. In essence, a profound understanding of reproductive physiology is indispensable for advancing ART. This article reviews the recent literature on EVs, focusing on their roles in reproductive physiology and ART, and addresses key objectives such as the mechanisms of exosome biogenesis, their function in cellular communication, and their potential as drug delivery vehicles. By examining groundbreaking case studies and exploring the promising therapeutic implications of exosomal delivery for proteins and genes, this review aims to provide a comprehensive understanding of how exosomal drug delivery can revolutionize treatments for reproductive health disorders. The objective is to offer insights into future therapeutic applications, highlighting the transformative potential of exosomes in reproductive medicine.

The authors declare that there are no conflicts of interest.