Extracellular Vesicles in Reproduction: Biology, Production, and Potential Applications in Livestock Breeding.

EVs are also critical in female reproductive processes, particularly in oocyte maturation, follicular development, and embryo implantation (Machtinger et al.  2021 ). The female reproductive tract is a highly dynamic environment that undergoes cyclical changes regulated by hormonal fluctuations (Hawkins and Matzuk  2008 ). EVs mediate molecular communication within the follicular (Hasan et al.  2021 ), oviductal (Dissanayake et al.  2021 ), and uterine microenvironments (Godakumara et al.  2021 ; Piibor et al.  2024 ). Follicular fluid, which surrounds the developing oocyte within the ovarian follicle, contains EVs secreted by granulosa cells, theca cells, and the oocyte itself (Lai et al.  2015 ). These vesicles play an essential role in oocyte competence, which refers to the ability of an oocyte to undergo successful fertilisation and embryonic development (Gabryś et al.  2022 ). The cargo of follicular EVs includes growth factors, cytokines, and microRNAs that regulate follicular cell proliferation, oocyte metabolic activity, and meiotic progression (Benedetti et al.  2024 ; Uzbekova et al.  2020 ). In particular, EVs contribute to the transfer of small RNAs involved in epigenetic modifications, which may influence oocyte developmental potential (Aoki et al.  2024 ; Martinez et al.  2018 ). After ovulation, the oocyte enters the oviduct, where fertilisation occurs. The oviductal fluid provides a supportive environment for sperm capacitation, fertilisation, and early embryo development (Ferraz et al.  2019 ). EVs secreted by oviduct epithelial cells have been shown to facilitate sperm storage and survival within the oviduct by preventing premature capacitation (Alcântara‐Neto et al.  2020 ; Ferraz et al.  2019 ). These vesicles also contribute to spermatozoa selection by influencing the molecular composition of the oviductal reservoir where spermatozoa are retained before fertilisation (Lange‐Consiglio et al.  2022 ). Following fertilisation, the early embryo undergoes a series of cleavage divisions while travelling through the oviduct toward the uterus. During this pre‐implantation period, EVs in the oviductal and uterine fluids provide essential signals that regulate embryo metabolism, gene expression, and immune tolerance (Poh et al.  2022 ; Segura‐ben et al.  2025 ). The transfer of maternal RNAs and proteins via EVs has been suggested to play a role in embryo quality and implantation success (Es‐Haghi et al.  2019 ; Leal et al.  2022 ).

Alireza Fazeli: conceptualized the study, conducted the literature review, wrote the original draft of the manuscript and acquired funding. Kasun Godakumara: contributed to literature review, contributed to write the original draft of the manuscript, provided critical insight and editing of this manuscript. Suranga Kodithuwakku: contributed to literature review, contributed to write the original draft of the manuscript, provided critical insight and editing of this manuscript. Subhashini Muhandiram: contributed to literature review, contributed to write the original draft of the manuscript, provided critical insight and editing of this manuscript.

As the field of EV research continues to evolve, several exciting avenues for future exploration have emerged. One promising direction is the development of engineered EVs for targeted reproductive therapies. Researchers can design vesicles with enhanced therapeutic properties by modifying EV cargo through genetic engineering or chemical modifications. For example, EVs engineered to carry specific microRNAs or proteins involved in sperm function could be used to treat male infertility. Similarly, EVs containing pro‐angiogenic factors may be utilised to improve placental vascularisation in cases of recurrent pregnancy loss. Another important area of research is the use of EVs as drug delivery vehicles in reproductive medicine. EVs have inherent biocompatibility and the ability to cross biological barriers, making them ideal carriers for delivering drugs, hormones, or gene‐editing tools to reproductive tissues. Investigating the potential of EV‐based delivery systems for reproductive therapies could lead to innovative treatments for infertility, endometriosis, and other reproductive disorders. EVs also hold promise for advancing non‐invasive diagnostics in reproductive health. The identification of EV‐derived biomarkers for conditions such as polycystic ovary syndrome, endometriosis, and recurrent pregnancy loss could provide clinicians with novel tools for early detection and personalised treatment strategies. Liquid biopsy approaches utilising EV analysis may revolutionise reproductive medicine by enabling real‐time monitoring of fertility status and pregnancy health. In livestock reproduction, EV‐based approaches may contribute to sustainable breeding practices and genetic improvement programmes. Enhancing reproductive efficiency through EV‐mediated interventions could reduce the environmental impact of livestock production and improve food security. Further research into the role of EVs in gamete preservation and embryo transfer could optimise breeding strategies for economically important animal species.

EVs are categorised based on their size, mode of biogenesis, and functional properties. They include exosomes, which are the smallest vesicles ranging from 30 to 150 nm, microvesicles, which are larger and typically range from 150 to 1000 nm, and apoptotic bodies, which can reach up to 2000 nm in size (Welsh et al.  2024 ; Yáñez‐Mó et al.  2015 ). These vesicles are released into the extracellular environment through distinct cellular mechanisms, each contributing to their unique molecular composition and function (Dissanayake et al.  2024 ; Hagey et al.  2023 ). Exosomes originate from endosomes; they form endosomal compartments that contain intraluminal vesicles. These vesicles are released into the extracellular space through fusion of multivesicular bodies with the plasma membrane (Kalluri and LeBleu  2020 ). Microvesicles, on the other hand, are formed by outward budding of the plasma membrane, a process regulated by lipid reorganisation and cytoskeletal remodelling (Tricarico et al.  2017 ). Apoptotic bodies are generated during programmed cell death and contain remnants of cellular components, including nuclear fragments, organelles, and cytoplasmic proteins (Battistelli and Falcieri  2020 ). The cargo of EVs is selectively sorted and loaded into vesicles through complex regulatory pathways (Lee et al.  2024 ). The endosomal sorting complex required for transport, also known as ESCRT, is a major determinant of exosome biogenesis and is responsible for packaging specific biomolecules into vesicles (Frankel and Audhya  2018 ). Lipid raft‐associated pathways (De Gassart et al.  2003 ) and tetraspanin proteins also play key roles in vesicle cargo selection. The composition of EVs is highly dynamic and varies depending on the cell of origin (Hagey et al.  2023 ), physiological conditions (Hart et al.  2023 ), and environmental stimuli (Mousavi et al.  2024 ). EVs are enriched in proteins such as heat shock proteins, tetraspanins, and integrins, which are involved in cell adhesion, signalling, and stress responses. Lipid analysis of vesicle membranes has revealed a unique composition of sphingolipids, ceramides, and cholesterol, contributing to membrane stability and fusion properties (Ghadami and Dellinger  2023 ; Haraszti et al.  2016 ). The presence of microRNAs, messenger RNAs, and long non‐coding RNAs within vesicles further highlights their role in post‐transcriptional gene regulation (O'Brien et al.  2020 ). The ability of EVs to transfer functional nucleic acids between cells has profound implications for reproductive biology, as they can modulate gene expression and cellular behaviour in target tissues (Dissanayake et al.  2024 ; Es‐Haghi et al.  2019 ). Advances in omics technologies, including proteomics, lipidomics, and transcriptomics, have greatly enhanced our understanding of EVs composition (Blandin et al.  2023 ; Ghanam et al.  2022 ; Hayasaka et al.  2023 ; Lischnig et al.  2022 ). High‐throughput sequencing and mass spectrometry have allowed researchers to identify key molecular signatures associated with reproductive EVs. These insights are essential for deciphering the functional roles of vesicles in gamete development, fertilisation, and embryo implantation (Beal et al.  2023 ; Mazzarella et al.  2024 ; Piibor et al.  2023 , 2024 ). Despite significant progress, challenges remain in isolating and characterising EVs with high specificity. Current isolation methods, such as ultracentrifugation, size‐exclusion chromatography, and immunoaffinity capture, each have limitations in terms of purity, yield, and vesicle integrity (Brennan et al.  2020 ; Welsh et al.  2024 ). The development of microfluidic‐based platforms and single‐vesicle analysis techniques will be crucial for improving EV isolation and characterisation (Gao et al.  2023 ). The study of EV biogenesis has provided important insights into their regulatory mechanisms and functional relevance (Dar et al.  2021 ; Dixson et al.  2024 ). Understanding how vesicles are formed, packaged, and secreted will pave the way for developing targeted therapeutic strategies that exploit their natural signalling capabilities (Hadizadeh et al.  2022 ). Given their potential for modulating reproductive processes, EVs represent a promising avenue for advancing fertility research and clinical applications (Parvin et al.  2024 ).

The placenta is a vital organ that facilitates nutrient exchange, gas exchange, and immune modulation between the mother and fetus (Gude et al.  2004 ). EVs derived from placental trophoblasts have been identified in maternal circulation and play an essential role in pregnancy maintenance (Kupper and Huppertz  2022 ; Tong et al.  2018 ). Placental EVs regulate vascular remodelling and angiogenesis by transferring pro‐angiogenic factors to endothelial cells (Cronqvist et al.  2020 ). These vesicles contain growth factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), which stimulate the formation of new blood vessels within the maternal–fetal interface. Proper vascularisation of the placenta is critical for fetal oxygenation and nutrient delivery (Feng et al.  2022 ; Gebara et al.  2021 ). EVs also influence metabolic adaptations during pregnancy. Placenta‐derived vesicles carry metabolic enzymes and transporters involved in glucose homeostasis, lipid metabolism, and fetal nutrient uptake. These vesicles ensure optimal fetal growth by modulating maternal metabolic pathways (Renaud et al.  2023 ; Rosenfeld  2024 ). Pregnancy complications such as preeclampsia, gestational diabetes, and intrauterine growth restriction have been linked to altered EV profiles in maternal circulation (Levine et al.  2020 ; Ortega et al.  2022 ). Analysing the composition of placental EVs may provide valuable insights into pregnancy health and allow for early diagnosis of gestational disorders (Chaemsaithong et al.  2023 ).

The clinical applications of EVs in reproductive medicine are vast, with potential uses in fertility diagnostics (Muraoka et al.  2024b ; Rana et al.  2024 ), ART enhancement (Fang et al.  2023 ), and therapeutic interventions (Xue et al.  2024 ). One of the most promising applications is the use of EVs as biomarkers for assessing fertility status. Clinicians may be able to predict sperm quality (Pal et al.  2025 ), oocyte competence (da Silveira et al.  2012 ), and embryo viability (Dissanayake et al.  2021 ; Es‐Haghi et al.  2019 ) by analysing the molecular composition of EVs in seminal plasma, follicular fluid, and uterine secretions. In assisted reproductive technologies, EVs have been explored as tools for improving embryo culture conditions (Xue et al.  2024 ). Supplementing culture media with EVs derived from reproductive fluids may enhance embryo development by providing essential growth factors and protective molecules (Leal et al.  2022 ; Poh et al.  2023 ).

Despite the promising applications of EVs in reproductive medicine and livestock production, several challenges and limitations need to be addressed before their widespread implementation. One of the primary challenges in EV research is the standardisation of isolation and characterisation techniques. Various methods, including ultracentrifugation, size‐exclusion chromatography, and microfluidic‐based approaches, are used to isolate EVs from biological fluids (Welsh et al.  2024 ; Yakubovich et al.  2022 ). However, differences in isolation protocols can lead to inconsistencies in vesicle purity, yield, and functionality (Allelein et al.  2021 ; Ramirez et al.  2018 ). Developing standardised methodologies for EV isolation and characterisation is essential for ensuring reproducibility and comparability across studies. Another limitation in EV research is the heterogeneity of vesicle populations. EVs are a diverse group of particles with varying sizes, cargo compositions, and biogenesis pathways. Distinguishing between exosomes, microvesicles, and apoptotic bodies remains a challenge due to overlapping size distributions and shared molecular markers (Allelein et al.  2021 ; Wang et al.  2025 ; Willms et al.  2018 ). Advances in single‐vesicle analysis techniques (Midekessa et al.  2021 ), such as high‐resolution flow cytometry (Barranco et al.  2024 ) and super‐resolution microscopy (Bağcı et al.  2022 ), may provide more precise methods for characterising EV subtypes. The functional mechanisms of EVs in reproductive processes also remain incompletely understood. While studies have demonstrated the involvement of EVs in sperm maturation, oocyte competence, and embryo–maternal communication, the exact molecular pathways by which these vesicles exert their effects require further investigation (Dissanayake et al.  2024 ; Hasan et al.  2021 ; Hung et al.  2015 ; Muhandiram et al.  2024 ). Identifying the specific cargo molecules responsible for EV‐mediated signalling will be crucial for developing targeted therapeutic applications. In clinical settings, the scalability and cost‐effectiveness of EV‐based therapies pose additional challenges (Adlerz et al.  2020 ; Ng et al.  2022 ). Large‐scale production of EVs for therapeutic use requires optimised cell culture conditions and efficient purification methods (Busatto et al.  2018 ; Kusuma et al.  2022 ; Liaqat et al.  2024 ). Furthermore, regulatory considerations regarding the safety, stability, and delivery of EV‐based treatments need to be addressed before they can be integrated into successful reproductive medicine applications and treatments (Wang et al.  2024 ).

EVs represent a rapidly expanding field of research with significant implications for reproductive biology, clinical fertility treatments, and livestock reproduction. Their ability to mediate intercellular communication, regulate reproductive processes, and serve as biomarkers for fertility assessment highlights their potential as transformative tools in reproductive medicine. While challenges remain in standardising isolation techniques, characterising vesicle heterogeneity, and elucidating functional mechanisms, ongoing advancements in molecular biology, bioengineering, and nanotechnology are poised to address these limitations. The development of EV‐based diagnostics and therapeutics holds great promise for improving reproductive health outcomes in both humans and animals. As the scientific community continues to unravel the complexities of EV biology, the integration of vesicle‐based approaches into clinical and agricultural settings will pave the way for innovative solutions in fertility management. The coming years are likely to witness groundbreaking discoveries in EV research, leading to novel applications that enhance reproductive success and advance the fields of reproductive medicine and livestock biotechnology.

EVs have significant potential in improving reproductive outcomes in livestock species such as cattle, pigs, sheep, goats, and horses breeding programmes (Table  1 ). The global livestock industry relies heavily on assisted reproductive technologies, including artificial insemination, embryo transfer, and in vitro fertilisation, to enhance genetic traits and reproductive efficiency (Gadea et al.  2020 ; Mikkola et al.  2024 ; Verma et al.  2012 ). Despite advancements in these technologies, fertility rates remain suboptimal due to limitations in sperm cryopreservation (Donnelly et al.  2001 ; Tanga et al.  2021 ), embryo viability (Erdem et al.  2020 ; Lopera‐Vasquez et al.  2017 ), and maternal receptivity (Binelli et al.  2022 ; Paulson and Comizzoli  2021 ). Emerging research suggests that EVs can provide novel solutions for overcoming these challenges by modulating sperm function (Mahdavinezhad et al.  2022 ), embryo–maternal communication (Hu et al.  2022 ; Xue et al.  2024 ), and pregnancy maintenance (Galli et al.  2024 ). Potential applications of EVs in livestock breeding. One of the primary applications of EVs in livestock reproduction is their use as fertility biomarkers. Identifying reliable biomarkers for spermatozoa quality (Rana et al.  2024 ), oocyte competence (Uzbekova et al.  2020 ), and embryo development (Dissanayake et al.  2020 ) is crucial for selecting the most viable and functional gametes and embryos for assisted reproductive technologies. Studies have shown that EVs isolated from seminal plasma contain microRNAs and proteins that correlate with sperm motility, viability, and fertilisation capacity (Barranco et al.  2019 ; Pal et al.  2025 ). Similarly, EVs from follicular fluid have been found to carry molecular signatures associated with oocyte maturation and developmental potential (Gabryś et al.  2022 ; Hung et al.  2015 ). Veterinarians and breeders can make informed decisions regarding breeding strategies and artificial insemination protocols by analysing the EV profiles in reproductive fluids. EVs also have potential applications in sperm preservation and cryopreservation. Freezing and thawing procedures commonly used in artificial insemination can cause structural damage and reduce the viability of spermatozoa. Supplementing cryopreservation media with EVs derived from epididymal or seminal plasma has been shown to improve post‐thaw sperm motility and membrane integrity (Rodriguez‐martinez and Roca  2022 ). These vesicles provide protective effects by stabilising lipid membranes, reducing oxidative stress, and delivering key proteins involved in sperm function (Barranco et al.  2025 ). Enhancing sperm preservation techniques using EVs could lead to higher conception rates in artificial insemination programmes (Sawicki et al.  2024 ). In embryo transfer programs and in in vitro fertilisation practices, EVs can be utilised to improve embryo culture conditions and implantation success. The early embryo relies on maternal signals from the oviduct and uterus to regulate gene expression and developmental processes. Co‐culturing embryos with EVs derived from oviductal and endometrial secretions has been shown to enhance blastocyst formation rates, reduce oxidative stress, and improve embryo survival (Han et al.  2025 ; Leal et al.  2022 ; Mazzarella et al.  2024 ; Piibor et al.  2024 ). These findings suggest that EVs could be used as bioactive additives in embryo culture media to mimic the physiological environment of the reproductive tract. Another promising application of EVs in livestock reproduction is their potential use in reproductive immunomodulation. Pregnancy in mammals involves complex interactions between the maternal immune system and the developing fetus (Abu‐Raya et al.  2020 ). Inadequate immune tolerance to the embryo can lead to implantation failure or early pregnancy loss (Andreescu  2023 ). EVs secreted by the conceptus and maternal tissues help regulate immune responses by suppressing pro‐inflammatory cytokines and promoting regulatory T‐cell activity (Abeysinghe et al.  2023 ; Paktinat et al.  2021 ). Understanding how EVs contribute to maternal‐fetal immune tolerance could pave the way for developing therapeutic approaches to prevent pregnancy complications in livestock. EVs may also play a role in improving the efficiency of cloning and somatic cell nuclear transfer. Cloning techniques are often associated with low success rates due to epigenetic abnormalities and improper reprogramming of the donor nucleus (Gouveia et al.  2020 ; Srirattana et al.  2022 ). Emerging evidence suggests that EVs derived from oocytes and early embryos contain epigenetic modifiers that may enhance nuclear reprogramming (Barrera et al.  2017 ; Estill et al.  2016 ). Researchers may be able to improve the developmental competence of cloned embryos and increase the efficiency of somatic cell nuclear transfer by incorporating EVs into cloning protocols.

Extracellular Vesicles (EVs) are widely recognised as key mediators of intercellular communication and have been identified in virtually all biological fluids (Lättekivi et al.  2022 ; Rodriguez‐martinez and Roca  2022 ; Sun and Lerman  2020 ; Van Herwijnen et al.  2016 ). These vesicles, which range in size from 30 to 1000 nm, serve as carriers of bioactive molecules that regulate various physiological processes. Their presence in reproductive fluids, including seminal plasma (Reshi et al.  2021 ), follicular fluid (Hasan et al.  2021 ), oviductal fluid (Almiñana et al.  2017 ), and uterine secretions (Piibor et al.  2023 ), has sparked significant interest for studying their involvement and function in reproductive biology and physiology. Reproduction is a highly complex process that involves intricate signalling between gametes, the reproductive tract (Ghersevich et al.  2015 ; Hasan et al.  2021 ), and the developing embryo (Guzewska et al.  2023 ; Muhandiram et al.  2023 ; Segura‐Benítez et al.  2022 ). The discovery that EVs facilitate molecular crosstalk at multiple stages of reproduction has led to increasing investigations into their biological significance (Fazeli and Godakumara  2024 ). In the male reproductive system, EVs may influence spermatozoa maturation and development, capacitation (Hasan et al.  2021 ) and interactions of spermatozoa with the female reproductive tract (Reshi et al.  2023 ). In females, EVs secreted in the follicular and endometrial environment contribute to oocyte competence (Makieva, Saenz‐de‐Juano, et al. 2024 ), embryo implantation (Evans et al.  2019 ; Muhandiram et al.  2023 ), and maternal immune modulation during pregnancy (Wu et al.  2022 ). The study of EVs in reproductive medicine has expanded due to their potential as biomarkers for fertility assessment (Dissanayake et al.  2021 ; Rana et al.  2024 ) and potential applications for diagnosing reproductive disorders (Muraoka et al.  2024a ; Piibor et al.  2024 ). Their cargo composition, which includes proteins, microRNAs, and lipids, may reflect the physiological status of the reproductive system (Hart et al.  2023 ; Mousavi et al.  2024 ), making them ideal candidates for non‐invasive diagnostics. Additionally, EV‐based therapies are being explored for enhancing in vitro fertilisation outcomes (Franko and de Almeida Monteiro Melo Ferraz  2024 ), improving embryo culture conditions (Bauersachs et al.  2020 ), and even developing novel treatments for infertility (Liu et al.  2020 ; Poh et al.  2023 ). The recognition of EVs as central players in reproductive processes has prompted further research into their biogenesis, molecular function, and translational applications. However, challenges such as standardising isolation techniques, characterising heterogeneous vesicle populations, and elucidating their precise functional roles remain significant obstacles in the further development of the field. Continued advancements in molecular biology, nanotechnology, and reproductive sciences will be essential for fully understanding the function of EVs in any physiological system, including reproductive systems. In addition, such developments will allow harnessing the therapeutic and diagnostic potential of EVs in reproductive medicine.

The authors declare no conflicts of interest.

Successful pregnancy requires complex molecular communication between the developing embryo and the maternal endometrium. This process, known as embryo‐maternal crosstalk, is largely mediated by EVs, which serve as molecular messengers facilitating bidirectional signalling between the embryo and the uterine lining. Prior to implantation, the blastocyst must establish a receptive environment within the uterus. EVs secreted by trophoblast cells, the outer layer of the blastocyst, interact with maternal immune cells and endometrial epithelial cells to promote endometrial receptivity (Godakumara et al.  2021 ; Godakumara et al.  2023 ; Makieva, Giacomini, et al.  2024 ; Muhandiram et al.  2023 ; Poh et al.  2021 ). These vesicles carry signalling molecules such as cytokines, integrins, and microRNAs that regulate endometrial remodelling and vascularisation, ensuring adequate blood supply to the implantation site (Fatmous et al.  2022 ; Guzewska et al.  2023 ; Poh et al.  2023 ). EVs also play a crucial role in immune regulation during pregnancy. The maternal immune system must tolerate the presence of the semi‐allogeneic fetus while maintaining immune surveillance to prevent infections. Trophoblast‐derived EVs contribute to maternal immune tolerance by modulating the activity of immune cells, including T cells, macrophages, and natural killer cells (Favaro et al.  2021 ; Wu et al.  2024 ). These vesicles suppress inflammatory responses and promote an anti‐inflammatory environment conducive to fetal development. Dysregulation of EV‐mediated communication during implantation has been implicated in pregnancy complications such as recurrent implantation failure and early pregnancy loss (Sun et al.  2025 ; Zhang et al.  2020 ). Abnormal EV cargo, altered secretion patterns, and disrupted vesicle uptake by maternal cells may contribute to implantation failure and placental dysfunction (Makieva, Giacomini, Giacomini, et al.  2024 ; Segura‐Benítez et al.  2022 ).