Extracellular vesicles and assisted reproductive technology Extracellular vesicles and assisted reproductive technology Jennifer Nagashima, Marcia Ferraz, Nucharin Songsasen Center for Species Survival, Smithsonian Conservation Biology Institute National Zoological Park, Front Royal, VA Abstract Research in biogenesis, composition, uptake, and functionality of extracellular vehicles (EVs) has dramatically expanded in recent decades. These lipid membrane bound particles secreted from all cells contain regulatory molecules (proteins, peptides, RNAs, DNA fragments, and lipids) that are transferred to target cells upon binding to the plasma membrane. Roles of EVs were studied in cell to cell communication, mediation of cancer development and immune response, as biomarkers for disease and potential drug delivery systems. More recently, studies were conducted to investigate roles of EVs in reproduction, ranging from gamete maturation, fertilization, and embryonic development to implantation. As such, potential use of EVs for assisted reproductive technologies (ART) has garnered substantial interest. Objectives of this review are to present current understanding of EVs in reproduction, with particular regard to recent research in understanding influence of reproductive cycles on EV production and cargo composition, and to discuss therapeutic potential of EVs in reproductive technologies, including cryopreservation and areas of research that remain to be explored for efficient application of EVs in ART. Keywords: Extracellular vesicles, gametes, cryopreservation, organs on a chip, synthetic vesicle Introduction Extracellular vehicles (EVs) are lipid membrane bound vesicles secreted by all cells.1 EVs include exosomes (30 - 100 nm in size), released via fusion of a multivesicle body with a cell’s plasma membrane; microvesicles (MV, also known as ectosomes, 100 - 1000 nm), shed via budding from plasma membrane;2 and apoptotic bodies (up to 5000 nm), formed through contraction and membrane blebbing of dying cells3 (Figure 1). EVs contain regulatory molecules, including proteins, peptides, RNA species, lipids, and DNA fragments which may be taken up by nearby cells via either fusion or endocytosis of vesicles, contributing to cell to cell communication.4 Owing to size overlap between exosomes and MV, it is difficult to separate 1 population from another and the 2 groups are often studied together as extracellular vesicles, or “EVs” in healthy cells (i.e. excluding apoptotic bodies). Multiple methods have been evaluated for EV isolation (reviewed5), including ultracentrifugation (most commonly utilized method6), density gradient centrifugation,7 size-exclusion chromatography,8 ultrafiltration,9 and sequential filtration.10 Differences in isolation method become significant in that subpopulations of EVs (i.e. more exosomes versus MV, or EV with specific surface markers) may be preferentially purified, based on isolation technique. For example, biological activity of endometrial EVs isolated via a commercially available system were compared with those purified via ultracentrifugation and ultrafiltration.11 Although ultracentrifugation had highest EV yield, EVs isolated via Total Exosome Isolation Kit were more readily taken up by trophoblast cells, indicating better preservation of biological activity. Further, regardless of purification method, copurified protein aggregates can confound the results of 2 most commonly utilized EV quantification methods, namely, colorimetric protein assays (e.g. BCA) and nanoparticle tracking analysis (NTA).12 Additionally, our ability to fully characterize EVs (both in terms of their composition and functionality) is challenged by the heterogenous nature of isolated EVs. Nevertheless, some characteristic features of EV subpopulations have been described. For example, owing to differing methods of release, proteins related to endosomal sorting complexes required for transport pathway are common to exosomes.13 Exosomes are also enriched in tetraspanins (involved in cell adhesion and signaling14) and heat shock proteins.15,16 MV contain higher concentrations of proteins associated with plasma membrane.1 Although lipid characterization has been particularly understudied in EVs field as a whole,17 lipid Clinical Theriogenology • Volume 12 Number 2 • June 2020 89 composition and high cholesterol/phospholipid ratio of EVs results in notable membrane stability, which presumably allows for greater protection of cargo and EVs in vitro longevity.18 In response to confusion associated with the heterogeneity of EVs and diversity of purification and quantification methods, in 2018, the International Society for Extracellular Vesicles published updated standards for minimal information for EV studies.19 These serve as a guide for EV isolation, classification, and nomenclature, and are important to reduce variability in research results among laboratories which are due to inconsistent appellations, or attribution of functions specifically to MV/exosomes when isolation protocols produce heterogenous populations. Improved understanding of EV sub-population compositions are also poised to hasten elucidation of the mechanisms of EV uptake into ‘recipient’ cells. It is currently understood that EVs are taken up by cells through a variety of pathways, including clathrin-mediated endocytosis, caveolin-mediated uptake, and lipid raft-mediated internalization (reviewed20). Whereas EV cargo transfer to recipient cells is documented,21 understanding molecular mechanisms of delivery is an area necessitating further study. Still, EVs are well studied for their role in cell to cell communication, including in mediating cancer development (reviewed5) and immune response (reviewed22). Release of EVs by cancer cells and immune system stimulated interest in them as a source of biomarkers for early disease detection (reviewed23). As a result of their natural in vivo stability, cargo carrying capacity, and ability to be taken up by cells, there is also growing interest in utilizing EVs for in vivo drug and therapeutic protein delivery.24,25 Although the field of reproduction-specific EVs is still relatively new, several excellent reviews published in recent years highlighted growing interest and understanding of roles of EVs in successful gamete functions, fertilization, embryo development, and pregnancy.26-30 Objectives are to outline current understanding of EVs in reproduction, including recent evaluations of reproductive stage on EV production and composition, and to discuss potential practical/therapeutic applications of EVs in assisted reproduction, with a primary focus on cryopreservation. Figure 1. Schematic of extracellular vesicle (EV) biogenesis and composition, with exosomes secreted via fusion of multivesicle bodies from early endosome fusing with cell’s plasma membrane, microvesicles secreted via budding, and apoptotic bodies released via blebbing. Collectively termed EVs, these membrane-bound vesicles carry lipid, RNA, DNA, and protein cargo from their cells of origin. Clinical Theriogenology • Volume 12 Number 2 • June 202090 Extracellular vesicles in reproduction Overview of male reproductive tract extracellular vesicles To date, studies on male reproductive tract EVs have focused on vesicles derived from epididymis (epididymosomes), and prostate gland (prostasomes). Epididymosomes are released into lumen from large blebs that form and detach from parent epithelial cells.31 Epididymosomes fuse with sperm during their transit through epididymis and deliver small RNA,32,33 and protein cargo,34,35 and alter lipid composition of sperm’s plasma membrane,36 essential for sperm maturation process (reviewed37). Specifically, epididymosomes reduce cholesterol/phospholipid ratio of bovine caput epididymal sperm,36 that results in increased membrane fluidity necessary for the sperm’s future ability to capacitate. Gene ontology analyses of bovine epididymosomes classified proteins into 6 functional groups, including adhesion molecules, chaperones, enzymes, structural proteins, signal transducers, and transporters.37 Many of these proteins are involved in sperm egg interactions, motility, plasma membrane modification, and elimination of defective gametes.38 Transfer of small RNAs (e.g. miRNA and tsRNA both small, noncoding RNA that primarily function in RNA silencing and posttranscriptional regulation39) from epididymosomes to sperm is a mechanism for transgenerational epigenetic inheritance.32,40 Coincubation of immature sperm with epididymosomes in domestic cats sustained in vitro motility.41 Delivered miRNA from epididymal EVs is linked to pregnancy success in mice, as intracytoplasmic sperm injection with sperm from caput epididymis displayed significant implantation failure following embryo transfer; a phenotype rescued by injection of cauda-specific miRNA into embryos.42 It is apparent that these vesicles have a profound influence on not only male gametes, but also pregnancy success and, potentially, offspring/subsequent generation traits via epigenetic inheritance. Prostasomes (reviewed43) are challenging to characterize, as they are mixed in with EVs from other sources in seminal fluids. Proteomic analyses of human prostasomes identified proteins in broad categories (enzymes, transport or structural integrity, GTP proteins, chaperones, signal transduction, and unannotated).44 Supplementation of sperm with prostasomes improved sperm motility, attributed to delivery of calcium signaling45 and ATP-generating46 machinery to the gamete. Human prostasome membranes are high in cholesterol, sphingomyelin and glycosphingolipids.47,48 Fusion with prostasomes enriched sperm’s plasma membranes in key lipids, including cholesterol.49 This, in turn, is apparently responsible for the prevention of premature acrosomal exocytosis in sperm exposed to prostasomes in cattle50 and humans.51,52 Prostasomes also protect sperm against female immune system, including decreasing natural killer cell activity in vitro,53 indicating prostasomes function both to maintain sperm function and support survival. Overview of female reproductive tract extracellular vesicles Extracellular vesicles derived from vaginal (vaginosomes54), uterine (uterosomes, reviewed 55,56), oviductal (oviductosomes or oEVs, reviewed27) and follicular (ffEVs, reviewed30,57,58), fluids, and secretions from in vitro cultured embryos (reviewed59) are characterized in a variety of species. Supplementation of EVs from these sources benefit gamete function and/or embryo development. For example, coincubation of vaginal EVs with murine sperm increased rates of progesterone-induced acrosome exocytosis compared to unsupplemented controls.54 This effect was attributed to transfer of tyrosine phosphorylated proteins and plasma membrane Ca2+ ATPase ([PMCA], which removes calcium from cells) from EVs to sperm, essentially priming cells for capacitation while preventing premature acrosome reaction. Endometrial mesenchymal stem cell EVs improved blastomere count and hatching rates in murine embryos.60 Coincubation with EVs also stimulated embryos to release vascular endothelial growth factor and platelet derived growth factor, which in turn may modulate endometrial receptivity to implantation.60 Our laboratory recently demonstrated that domestic cat oviductal extracellular vesicles (oEVs) bound to acrosomal region and midpiece of sperm and prevented premature acrosomal exocytosis (in vitro), while improving sperm motility and fertilizing capacity61 (Figure 2). We postulated that enrichment in proteins identified in oEVs was related to energy metabolism, calcium transport and Clinical Theriogenology • Volume 12 Number 2 • June 2020 91 membrane function, may have regulated this effect. In cattle, oEVs modulate sperm calcium concentrations62 and they also modulated sperm capacitation in cattle and mice.62,63 Porcine oEVs reduced rates of polyspermy in vitro,64 and murine oEV supplementation during in vitro fertilization and embryo culture improved live birth rates after embryo transfer.65 Hence, recently, oEVs have been utilized to improve in vitro embryo production technologies.27 Follicular fluid EVs, apparently produced by granulosa and cumulus cells, are taken up into cumulus cell layer of domestic cat oocytes (Figure 2) and, in cattle, transferred to an oocyte via transzonal projections.66 Bovine ffEVs stimulated granulosa cell proliferation67 and cumulus cell expansion,68 and improved blastocyst production.68 Bovine embryos produced in vitro in the presence of ffEVs exhibited varying levels of global DNA methylation compared to nonffEV counterparts.69 Taken together, it is clear that supplementation of EVs to in vitro embryo production systems promises to improve not only production rates, but also embryo quality and potentially live birth success. Extracellular vesicles and reproductive cycles Despite strong evidence of beneficial actions of EVs on reproduction, our understanding of how EV biogenesis and composition are modulated by reproductive cycle is limited. Nevertheless, there is evidence that changes in secreted EVs are under hormonal control.66,70-73 For example, ovariectomized sheep treated with progesterone for 14 days had higher EV count (based on NTA) and mode diameter in their uterine fluid compared to untreated cohorts, indicating biogenesis and secretion of endometrial EVs were modulated by progesterone.71 In an in vitro study with primary cell cultures of human endometrial epithelial cell lines, there were no differences in EV amounts (based on protein concentrations) from cells cultured only with estrogen (i.e. proliferative phase-mimetic) versus estrogen and progesterone (i.e. secretory phase mimetic).72 However, there were 380 differentially expressed proteins between EVs of 2 treatment groups, many of which were involved in cytoskeletal organization, cell migration, cell adhesion, and extracellular matrix organization. These studies indicated that uterine EV production and their protein compositions are under hormonal control. Figure 2. Schematic of female reproductive tract EVs and interaction with gametes/embryos, including pre-release uterosomes in a multivesicular body in ovine endometrial luminal epithelium (top left, TEM image from Burns et al 2016), oviductosomes or oEV from domestic cats (lower middle, TEM image fusing with sperm tail from Ferraz et al 2019), and feline follicular fluid (top middle, fluorescent image with EV lipids labeled in green (DiO) and EV proteins in blue (Ghost Dye) taken up by cumulus cells, manuscript under review) Clinical Theriogenology • Volume 12 Number 2 • June 202092 Recently, counts, transcriptomes, and proteomes of oEVs recovered during various stages of reproductive cycles have been characterized in several mammalian species. In murine oEV, very few differences in miRNA content were observed among reproductive stages (metestrus/diestrus versus proestrus/estrus).73 In domestic cats, we observed an increase in concentration, but not size distribution, in oEVs recovered during midluteal phase compared to early luteal and late follicular phases (manuscript under review). Conversely, protein and mRNA/small RNA content of bovine oEV isolated during postovulatory and early luteal phases were enriched in proteins related to protein translation and transport, whereas those obtained during late luteal phase contained proteins related to vesicles, cytoskeleton, metalloexopeptidase activity, and innate immune system.70 Furthermore, miRNA with high abundance in recently ovulated and late luteal phase included those involved in fatty acid biosynthesis and metabolism, cell cycle, oocyte meiosis, and multiple signaling pathways.70 These results were further evidence for potential targeted messaging to gametes and/or embryos to promote fertilization and development by reproductive tract. Also in a bovine model, miRNA from ffEV of 3 - 6 mm diameter follicles at various stages of the estrous cycle were compared.66 Follicular fluid EVs from early luteal ovaries (with evidence of recent ovulation, low follicular fluid progesterone concentration) were enriched in miRNA involved in multiple signaling pathways, including FoxO, Hippo, MAPK, and TGFβ compared to ffEVs from midluteal phase ovaries (tan corpus luteum, high progesterone).66 When early luteal phase ffEVs were supplemented to culture media during 24 hour in vitro oocyte maturation, expression of genes associated with pathways related to oocyte development, ovulation, and Notch signaling were upregulated in cumulus cells, indicating that reproductive stage specific cargo of ffEVs may have key roles in regulating gamete function. More tangentially, there is also evidence linking metabolic status as result of lactation to miRNA composition of bovine follicular fluid EVs.74 Namely, cows in negative energy balance had ffEV with downregulation of miRNA associated with metabolic processes and oocyte growth compared to metabolically normal lactating heifers, which has implications for folliculogenesis during lactational anestrus. Although in the earlier bovine ffEV study, supplementing ffEVs during IVM upregulated gene expression in the cumulus cell, no differences in oocyte maturation rates were observed between ffEVs gametes versus controls.66 It is possible that 24 hour time frame of IVM was not long enough to facilitate transfer of EV cargo into the oocyte and influence meiotic maturation outcome. In another bovine study, ffEVs were detected in cumulus cells via confocal microscopy 16 hours after incubation.60 We have similarly observed uptake of ffEV lipids and proteins only in cumulus cells of feline oocytes after 18 hours coincubation (manuscript under review). However, in ova from domestic dogs, oEV contents were observed in the oocyte after 72 hours incubation.75 It is clear, therefore, that an improved understanding of the kinetics of EV uptake into oocytes is necessary to further our understanding of their functional effects and any subsequent reproductive stage-specific differences. Potential for dramatic differences in EV composition by reproductive stage/status are not limited to female reproductive tract. Differences in protein composition of seminal plasma during breeding versus nonbreeding seasons have been characterized in sheep76,77 and goats.78,79 Notably, there is some evidence that certain protein components of breeding season (autumn/winter) seminal plasma improved motility of frozen-thawed ram sperm; however, this effect was not observed in nonbreeding season or unsupplemented sperm.80 While focus of these studies was on seminal plasma, rather than isolated EVs, it is possible that some proteins identified as interacting with sperm membranes are derived from EVs. This assertion is supported by the finding that there were no quantitative differences in abundance of most metabolic enzymes isolated from ram seminal plasma extracellular vesicles versus whole seminal plasma.81 Extracellular vesicles and assisted reproductive technologies of cryopreservation Potential of EV for assisted reproductive technologies, especially in vitro embryo production, has been elegantly reviewed recently.28 Here, we focus on new research into potential beneficial effects of EVs in cryopreservation. Recent reports of EV supplementation to gamete and/or embryo cryopreservation yielded varying results. Significantly improved survival and pregnancy success were Clinical Theriogenology • Volume 12 Number 2 • June 2020 93 observed with cryopreserved and thawed in vitro-derived bovine embryos cultured in amniotic MV (from days 5 - 7 following IVF), compared to unsupplemented controls.82 Zygotes coincubated with oEVs from either the isthmus or ampulla of the oviduct were compared to unsupplemented or fetal calf serum supplemented controls.83 Blastocysts developing from isthmic oEV-supplemented cultures displayed improved post-thaw survival rate (determined via reexpansion of blastocoel cavity) compared to control and FBS supplemented groups. Conversely, bovine embryos cultured with varying concentrations (based on protein content) of postovulatory oEVs had improved day 7 blastocyst rates, but supplementation had no effect on cryotolerance of blastocysts, as evaluated by hatching rates.84 Further, postthaw survival of bovine blastocysts sequentially cultured in oEVs and uterus-derived EVs was not significantly improved compared to controls.85 As each study utilized different EV sources (amniotic, oviductal and uterine fluid alone or in combination), isolation methods, and concentrations, as well as coculture timings and metrics of success, the full influence of reproductive EVs on bovine embryo cryopreservation is not yet clear. In our laboratory, ffEV supplementation during cryopreservation and thawing of domestic cat oocytes did not improve survival based on morphology, but did enhance ability of frozen and thawed oocytes to resume meiosis in vitro (manuscript under revision). Still, proteomic analyses of cat ffEVs identified proteins with known cryomodulating effects, including heat shock proteins (HSP), cytoskeletal elements, and oxidoreductases. For example, treatment of oocytes with cytoskeleton stabilizing agents improved cryopreservation success in mice86 and cows.87 Significant loss of chaperone proteins like HSPs and oxidoreductases have been observed following sperm cryopreservation in sheep88 and carp,89 respectively. Transfer of these and other potentially cryoprotective proteins may have supported postthaw recovery of gametes; however, much more work is necessary in this area as well. For sperm, the addition of bovine oEVs postthaw maintains viability while supporting capacitation during incubation, compared to sperm incubated under capacitating conditions in oEV absence.62 Cryopreservation significantly alters sperm membrane’s lipid compositions (reviewed90), including reducing sterols, phosphatidyl choline, and unsaturated fatty acids in caprine sperm membranes.91 As previously mentioned, EVs contain high concentrations of lipids and proteins associated with lipid-rafts, which likely supports their membrane fusion and stability in vitro,92 but also could support recovery of cryodamaged sperm membranes upon fusion. Lipid component is part of rationale behind addition of egg yolk to sperm cryopreservation media.93 More recently, a liposome-based medium (OptiXcell) was evaluated for cryopreservation of ram,94 buffalo,95 bull,96,97 deer,98 and rhino99 sperm, with promising results overall, even when compared to egg yolk, the current ‘gold-standard’. Similar to EVs, liposomes are phospholipid membrane vesicles, though they lack protein cargo. It is feasible that combining our understanding proteins present in EV membranes, particularly those likely to target gametes and promote uptake, we may be able to engineer vesicles with specific lipid and/or protein compositions to support cryosurvival while maintaining high affinity for gametes. Future extracellular vesicles production for therapeutics Applications of EVs to assisted reproduction is faced with several challenges, including the aforementioned need to improve our ability to isolate, purify and characterize EVs. However, even once we have a better understanding of EVs and optimal (and standardized) methods to purify populations of interest, EVs with consistent characteristics with low variability must be producible in large quantities. This is not feasible with in vivo sources. Beyond inherent variability, flushing both oviducts (Figure 3a) of a single individual with the aforementioned Total Exosome Isolation Kit, we isolate on average 3.1 x 1010 and 4.5 x 1010 EV/ml (in 1 ml) from domestic cats and dogs, respectively. In contrast, for our experimental purposes, we typically utilize ~ 3 x 107 EVs/ml. Though this is sufficient in a laboratory setting, high throughput production of EVs is necessary for clinical applications. In vitro extracellular vesicles production To improve our ability to consistently increase EV production, much work on EVs utilize in vitro produced vesicles from cell lines. Impact of isolation and culture conditions on EV production has been reviewed,6 yet much is still unknown, in large part because our understanding of biogenesis of these Clinical Theriogenology • Volume 12 Number 2 • June 202094 vesicles is not fully elucidated. Different cell types secreted different amounts of EVs in vitro,6 but whether this is intrinsic to cells or perhaps in response to pre-established culture methods is not certain. Presence or absence of serum in culture medium for neuroblastoma cells has been demonstrated to alter both quantity of produced EVs as well as their protein expression levels.100Alterations in lipid composition are also apparent from cell lines from same organ, as lipid profiling of prostate-derived cell line EVs identified differences in molecular lipids between cell lines with different tumorgenicity.101 Although culture conditions, once optimized, should allow for production of EVs with definable characteristics, high throughput production remains challenging.102 Owing to broad interest in EVs in regenerative medicine and cancer research, this area has already begun to be explored. EV production from prostate cancer cells is significantly improved when cells are cultured in a bioreactor compared to a conventional system,103 with EV morphology and select surface markers not different between these 2 conditions. However, metabolic analyses indicated differences in EV composition between conventional versus bioreactor derived EVs, particularly with regard to lipid contents. A 3D printer fabricated bioreactor has also been shown to enhance EV production for human endothelial cells.104 Still, as trauma and cell stress have also been demonstrated to increase EV secretion105 and mRNA/protein compositions,106 respectively. Mechanical forces on cells are known to promote exocytosis,107 therefore it is also possible that shear forces on cells grown in bioreactor systems may be stimulating mechanical, stress-driven EV production. Under these conditions, EV composition is likely altered and, potentially, less efficacious. More studies are needed to assess: 1) if dynamic culture systems consistently result in improved EV production across cell types, compared to conventional culture; and 2) how quality/composition/functionality of these EVs compare with in vivo-derived counterparts and/or those produced by conventional culture systems. In a sense, platforms for improved in vitro production of reproductive EV are already in progress. Organs on a chip technology, which has been applied to recreate in vitro microenvironment in more naturalistic conditions by mimicking tissue architecture, cell heterogeneity, and dynamic exposure to nutrients, growth factors and hormones, represent microfluidic bioreactors. Automated and controlled flow with specifically timed hormone supplementations can mimic normal pulsatile hormone pattern of reproductive cycle, and flow-through containing EVs readily collected (Figure 3b). A bovine oviduct-on-a-chip, mimicking in vivo conditions in this organ, demonstrated epigenetic patterns of chip derived embryos to be more similar to fresh controls than those produced via conventional IVF.108 An artificial uterus has similarly been produced via microfluidic chip, consisting of Figure 3. In vivo and vitro alternatives for collection of EVs, with: a) flush of fluids containing EV from domestic cat oviducts, versus b) dynamic fluidic culture system where tissues/cells of interest can be maintained in channels (inset) with culture medium delivered via syringe pump and flow through containing EV collected in tubes. Clinical Theriogenology • Volume 12 Number 2 • June 2020 95 two channels separated with a porous membrane, with cell culture (endometrial and embryos) on apical side and perfusion on basolateral.109 Higher rates of blastocyst development were obtained in chip compared to a conventional Petri dish. No microfluidic systems for vaginal cells have been reported, though a human vaginal epithelial cell model with 3D organoid culture has been described.110 A combination ovary, fallopian tube, uterus, cervix, and liver reproductive system on a chip with human and murine tissues/cells has been produced (termed EVATAR), that mimics hormonal profiles of human menstrual cycle in vitro.111 We have previously described an ovary-on-a-chip for culture of domestic dog and cat ovarian tissues,112 and substantial strides have been made in development of in vitro spermatogenesis with testis on chip in recent years.113-115 Conversely, microfluidic or bioreactor systems for accessory sex glands and/or epididymis are lacking. Still, 3D prostate organoid cultures (human and mouse sources) have been described116 for application primarily to prostate cancer research. Moreover, coculture of human cauda epididymal epithelial cells (spheroids) with caput epididymal sperm improved sperm motility and ZP-binding compared to non-cocultured controls.117 Although EV production has not been specifically compared with regards to culture system in any of described dynamic and/or 3D culture systems, we are optimistic that tools being developed to produce improved in vitro models of reproductive organs have potential to serve as controllable sources of reproductive EVs for ART as well. Synthetic or manufactured extracellular vesicles Several methods to manufacture EVs have also been reported outside of those secreted ‘naturally’ in cell culture. These have been of particular interest for their potential as nanoscale drug delivery systems, taking advantage of their inherent cargo transfer (reviewed118-121) and gamete/embryo (reviewed29) abilities. Most nanovesicles or “exosome mimetics” have been produced via disruption of cell membranes. For example, filter-based extrusion of natural killer cells were shown to maintain antitumor effects of parental/source cells, in both in vitro and in vivo mouse models.122 EV mimetics have been produced by treating cells with cytochalasin B, by which dissociated cell membranes roughly size of MV can be obtained.123,124 These ‘nanovesicles’ have similar morphology to natural EVs (based on TEM) and reportedly have similar content and functionality as parental cells.124 Membrane extrusion has also been utilized to engineer ‘hybrid’ EVs. For example, EVs secreted from 3T3 fibroblast and A549 tumor cell line were fused with synthetic lipids.125 This method allowed for a significant increase in number of controlled size vesicles compared to EVs purified from cell line secretions. Further, engineered EVs were demonstrated to be effective at drug delivery, as small interfering RNA (siRNA) loaded by electroporation into EV induced gene silencing in target cells comparable to commercially available kits. Fusion of EVs secreted from murine macrophages with engineered liposomes increased yield while maintaining targeted binding functionality of EVs.126,127 These fusion EVs also had demonstrable success as drug delivery vehicles when tested in vitro. Getting further away from in vivo derived EVs and cell sources, synthetic vesicles have been produced (reviewed92), including liposomes (previously discussed in cryopreservation section), composed of natural lipids and fully synthetic ‘polymersomes’, composed of amphiphilic block copolymers.128 Though cargo of naturally secreted EVs is not replicated in these manufactured vesicles, above studies demonstrated their ability to be loaded with specific cargos of interest. As a result, engineered EVs have potential for drug delivery or other clinical and ART applications. Conclusion Extracellular vesicle research represents a rapidly evolving field with diverse potential clinical applications. Although work on reproductive EVs is relatively new, epididymosomes, prostasomes, vaginasomes, uterosomes and oviductosomes have been characterized in numerous studies. This interest is owed in no small part to important effects observed on gamete maturation and function observed thus far. Substantially more work is needed to fully understand biogenesis, uptake, and downstream effects of varying EV, particularly within context of reproductive tract. However, if new methods for in vitro production can consistently generate high quality and quantity vesicles, there is broad clinical potential of EVs in assisted reproduction. Clinical Theriogenology • Volume 12 Number 2 • June 202096 Conflict of interest There are no conflicts of interest to declare. 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