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The role of extracellular vesicles in animal reproduction and diseases

Abstract

Extracellular vesicles (EVs) are nanosized membrane-enclosed compartments that serve as messengers in cell-to-cell communication, both in normal physiology and in pathological conditions. EVs can transfer functional proteins and genetic information to alter the phenotype and function of recipient cells, which undergo different changes that positively affect their structural and functional integrity. Biological fluids are enriched with several subpopulations of EVs, including exosomes, microvesicles (MVs), and apoptotic bodies carrying several cargoes, such as lipids, proteins, and nucleic acids. EVs associated with the reproductive system are actively involved in the regulation of different physiological events, including gamete maturation, fertilization, and embryo and fetal development. EVs can influence follicle development, oocyte maturation, embryo production, and endometrial-conceptus communication. EVs loaded with cargoes are used to diagnose various diseases, including pregnancy disorders; however, these are dependent on the type of cell of origin and pathological characteristics. EV-derived microRNAs (miRNAs) and proteins in the placenta regulate inflammatory responses and trophoblast invasion through intercellular delivery in the placental microenvironment. This review presents evidence regarding the types of extracellular vesicles, and general aspects of isolation, purification, and characterization of EVs, particularly from various types of embryos. Further, we discuss EVs as mediators and messengers in reproductive biology, the effects of EVs on placentation and pregnancy disorders, the role of EVs in animal reproduction, in the male reproductive system, and mother and embryo cross-communication. In addition, we emphasize the role of microRNAs in embryo implantation and the role of EVs in reproductive and therapeutic medicine. Finally, we discuss the future perspectives of EVs in reproductive biology.

Introduction

Extracellular vesicles (EVs) are heterogeneous and nanosized membranous vesicles secreted by a wide range of cells throughout the body. They are found in various body fluids, such as blood, urine, saliva, and breast milk. EVs are known for their ability to carry significant phenotype-altering cargo, such as transcription factors and microRNAs [1]. Based on their biogenesis and size, EVs are classified as exosomes (50~150 nm), microvesicles (100~1000 nm), or apoptotic bodies (500~4000 nm) [2, 3] (Fig. 1). Generally, EVs play a significant role in cellular dumping or the release of waste materials. EVs deliver various cargoes, including mRNAs, microRNAs (miRNAs), lipids, proteins, and nucleic acids, for long-distance communication between cells [3, 4]. Pathological cells, such as cancer cells, secrete specific EVs with different compositions that can be used as diagnostic markers for certain diseases and can also be used for monitoring disease progression [5,6,7]. Extracellular vesicle secretion has been observed in various reproductive cells, such as follicular cells [8], oviductal cells [9], embryos produced in vitro [10], and endometrial cells [11]. EVs regulate various reproductive physiological functions, including ovarian follicle development, oocyte maturation and fertilization, early embryo development, and endometrial–conceptus crosstalk [8, 9, 12,13,14]. Exosomes are derived from the inward pushing of the plasma membrane, which is typically 30–150 nm [15]. The endolysosomal system comprises a complicated and dynamic membranous network that begins from the early to late sorting of endosomes, formation of multivesicular bodies (MVBs), and fusion with the plasma membrane for secretion [3]. MVB formation is carried out by machinery that may either be endosomal sorting complexes required for transport (ESCRT)-dependent or ESCRT-independent. Exosomes and MVs are produced and secreted during normal cellular activity; in contrast, apoptotic bodies are larger in size (500–4000 nm), contain cell organelles within them, and are released during apoptosis, which is one of the major mechanisms of cellular death [16]. Studies have reported that EVs from bovine follicular fluid from small follicles (3–5 mm in diameter) and large follicles (>9 mm in diameter) induce cumulus expansion during in vitro maturation [13].

Fig. 1
figure 1

Biogenesis of extracellular vesicles in male and female reproductive systems. EVs are composed of functional proteins, mRNA, and microRNA. In particular, the protein content of EVs depends on the cell type from which they are secreted. Biogenesis of extracellular vesicle (EV) subtypes such as exosomes, MV, syncytial nuclear aggregates and apoptotic bodies. EVs are intraluminal vesicles which are released when a multivesicular body fuses with the cell membrane through exocytosis. MVs are formed by outward shedding of the cell membrane into extracellular space. Apoptotic bodies are generated when cells undergo apoptosis. The macromolecular components of EVs may play a significant role in cellular functions and pathological states during ovarian and uterus cycling, implantation of female as well as male reproduction

Due to their unique composition, cells of origin, and pathological characteristics, EVs are used to diagnose various diseases. EVs contain microRNAs (miRNAs) and proteins, which regulate inflammatory responses and trophoblast invasion through intercellular delivery in the placental microenvironment [17]. Maternal circulating EVs play a significant role in the formation of pro-inflammatory environments and endothelial cell dysfunction in the placenta [18]. EVs secreted from the embryo are involved in both the dialogue with the maternal endometrium [19], and in self-paracrine regulation [20]. Pig and human models show that EVs can be secreted from the trophectoderm and stimulate the proliferation of endothelial cells in vitro, thus becoming potential regulators of maternal endometrial angiogenesis [19, 21]. Embryo implantation is a crucial step in pregnancy, and failure of embryo implantation is a major limiting factor in early pregnancy and assisted reproduction. Implantation governs various physiological parameters, including embryo viability, endometrial receptivity, and embryo-maternal interactions. Embryo implantation is regulated by various types of biomolecules, particularly microRNAs , which function as transcriptional regulators of gene expression. miRNAs not only act in the cells, but can also be released by cells into the extracellular environment through multiple packaging forms, facilitating intercellular communication and providing indicative information associated with physiological and pathological conditions [22].

The function of EVs in human reproduction depends on the load of EVs and their ability to interact with receptor cells to deliver various types of cargo. EVs specifically bind to target cells, depending on the EV content and the specific receptors of the target cells or tissues. Because of their stability, versatility, and their ability to target recipient cells with specificity and transfer genetic and protein material through biological barriers [23], EVs are considered novel diagnostic and therapeutic tools in reproductive biology. Their therapeutic potential depends on their cargo composition. The release of EVs from the placenta is regulated by a number of factors that arise from the placenta. Changes in EV content and functions might be used as diagnostic biomarkers in female fertility studies [24]. This review discusses the different types of EVs, general aspects of isolation, purification, and characterization of EVs, particularly from various types of embryos. Further, we discuss EVs as mediators and messengers in reproductive biology, the effects of EVs on placentation and pregnancy disorders, the role of EVs in animal reproduction, male reproductive system, and mother and embryo cross-communication. In addition, we emphasize the role of microRNAs in embryo implantation and the role of EVs in reproductive and therapeutic medicine. Finally, we discuss the future perspectives of EVs in reproductive biology.

Types of EVs

Exosomes

EVs are classified as exosomes, microvesicles, and apoptotic bodies, based on various parameters, such as cellular origin, biophysical and biochemical characteristics, biological function, and biogenetic pathway. Exosomes are nano-sized particles that are trafficked through the endosomal pathway. Endosomal sorting complexes required for transport (ESCRTs) are important for the biogenesis of multivesicular bodies. Exosomes are derived from the inward budding of the limiting membrane of late endosomes, facilitating the formation of intraluminal vesicles (ILVs). Exosome formation is governed by two different mechanisms, ESCRT-dependent and independent; mechanisms including neutral sphingomyelinase/ceramide formation and involvement of ARF6/PLD2 have also been reported to occur [25, 26]. ILVs released from MVBs into the plasma membrane are called exosomes. On the other hand, the fusion of ILVs with lysosomes is mainly for the degradation of their contents [27]. Exosome secretion is regulated by various factors, such as members of the Rab guanosine triphosphatase (GTPase) RAB27A, RAB27B, RAB11, and RAB35. The machinery involved in the biogenesis of MVB and exosomes varies between tissues and cell types, which is governed by specific metabolic needs [23, 28]. Almost all secreted exosomes are between 30 nm and 150 nm; in some cases, it can be up to 200 nm, which is similar to the size of viruses [28,29,30]. Exosomes are typically characterized by the expression of surface markers, such as CD9, CD63, CD81, Alix, TSG101, and flotillin, as well as other markers [31]. Exosomes have been reported in various cells and parts of the body including within the zona pellucida [32, 33]. The human blastocyst cavity contains exosomes that are CD63+ and CD81+ [34]. Previous studies have reported that small EVs are located in various reproductive cells, including follicular fluid [8], oviductal fluid [9], secreted by embryos in culture media [35,36,37] and in endometrium flushing [11]. EVs from oviductal fluid facilitate oocyte and embryo quality [14]. Preimplantation embryos secrete exosomes from CD9+ cells through exocytosis or endocytosis [38, 39].

Microvesicles

MVs are a population of EVs that are formed and released directly from the cell plasma membrane by outward budding and fission from viable cells [40, 41] and are regulated by multiple mechanistic approaches. MVs are derived from budding events nucleated by the protein ARRDC1, which is recruited to the plasma membrane along with elements of the ESCRT pathway, generating 50 nm vesicles [42]. Another protein, Bin-1 (ampiphysin), facilitates the formation of curvature when recruited to the membrane. The formation and release of MVs are triggered by the remodeling of membrane proteins and lipid redistribution, which modulate membrane rigidity and curvature [43, 44]. ARF6 is a guanosine triphosphate–binding protein, a marker of MVs, and is implicated in the regulation of cargo sorting and promotion of the budding and release of MVs through the activation of the phospholipase D metabolic pathway [44, 45]. MVs play significant roles and various functions, including cancer cell invasiveness, transformation potential, disease progression and drug resistance, regulation of autoimmune diseases, immune system modulation and coagulation, embryo–maternal crosstalk, and embryo self-regulation [46,47,48,49].

Apoptotic bodies

Apoptotic bodies are produced as a result of cell death, alteration of several morphological changes, including membrane blebbing, membrane protrusion formation [50]. Apoptotic cell-derived extracellular vesicles, otherwise called ABs, are a group of subcellular membrane-bound extracellular vesicles generated during the decomposition of dying cells. ABs can be generated by many types of cells, such as stem cells, immunocytes, precursor cells, osteoblasts, and endothelial cells [51]. The production of ABs occurs in a dose-and time-dependent manner and is regulated by various factors, such as Rho-associated protein kinase (ROCK1) [52,53,54] and myosin-light chain kinase (MLCK) [55]. Inhibition of ROCK1, MLCK, and Caspases prevents the production of ABs. ABs are produced by nuclear shrinkage and plasma membrane blebbing in cells undergoing programmed cell death and MLCK contributes to the packaging of nuclear material into ABs [56]. Actomysin also plays a role in AB production by increasing cell contraction, hydrostatic pressure, and the formation of blebs [57]. The membrane of ABs reflects the main changes occurring on the cell surface of apoptotic cells. Apoptotic microvesicles ranging from 0.1 to 1 μm in diameter and small exosome-like EVs are released during apoptotic conditions [58, 59]. ABs are formed by the fragmentation and packaging of cellular organelles, such as the nucleus, endoplasmic reticulum (ER), or Golgi apparatus into these vesicles [16, 60], which range from 1 to 5 μm. The ER membrane is fragmented and forms vesicles smaller than ABs that sediment at higher centrifugal forces. ABs are divided into two types based on the type of cargo: DNA-carrying ABs, and cytoplasm-carrying ABs [61]. ABs are typically characterized by cytoskeletal and membrane alterations, including the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer [62]. VDAC1 is an apoptotic marker that forms ionic channels in the mitochondrial membrane and plays a role in triggering apoptosis; it is specifically localized in the vesicular fraction [63]. Another AB marker is calreticulin, an ER protein which is located in the subcellular localization. ABs are associated with the immune system [64,65,66]; they express chemokines and adhesion molecules, such as CX3CL1/fractalkine and ICAM3, and MHC class II molecules that can facilitate antigen presentation to CD4+ T cells and activation of immunological memory [67]. ABs are being developed as an essential tool in cell-to-cell communication between damaged and healthy cells. ABs may stimulate the proliferation of resident stem/progenitor cells, improve tissue regeneration, and replace damaged cells [68, 69]. ABs originating from different cell types have been shown to promote various functions. In the hepatic stellate, ABs can promote differentiation and cell survival [70]. ABs containing DNA from endothelial cells induce the proliferation and differentiation of human endothelial progenitor cells in vitro [71]. ABs containing microRNAs of cardiomyocytes enhance the proliferation and differentiation of resident SCs in vitro [72]. Administration of ABs carrying miR-126 inhibits atherosclerosis and induces CXCL12-dependent vascular protection [73]. ABs from cardiomyocytes enhance the proliferation and differentiation of resident stem cells (SCs) by transporting specific miRNAs [72], while ABs containing miR-221 and miR-222 derived from macrophages promote the proliferation of epithelial cells [74].

Isolation, purification and characterization of EVs

Isolation and purification of EVs

Isolation, purification, and characterization of EVs are essential for the application of EVs in a variety of fields. In particular, homogeneous separation and high yield are important in clinical applications. In this study, we provide a brief account of various isolation and purification methods. Differential centrifugation is the most commonly used method for the isolation of EVs [75]. The first step is low speed centrifugation at 300 × g for 10 min, which is required to eliminate cells. The second centrifugation at 2000 × g is for pelleting membrane debris and dead cells. The third centrifugation at 10,000 to 20,000 × g for 30 min is performed to pellet microvesicles. After these three steps, supernatants are collected and a fourth centrifugation step is carried out at 100,000 to 200,000 × g for 70 min to isolate exosomes. In this step, pellets are collected and washed with phosphate-buffered saline (PBS), and centrifuged again under the same conditions to remove impurities. Although the centrifugation process provides EVs, ultracentrifugation cannot remove contaminating lipoproteins from biological samples, such as blood. Hence, gradient centrifugation and/or other chromatography techniques are essential to remove impurities [76,77,78,79]. To improve the population purity of EVs, gradient step centrifugation is indispensable. In this step, the pellet is resuspended in PBS, loaded into a sucrose cushion or gradient, and ultracentrifugation is carried out. The vesicles are recovered either from the bottom of the tube or from a specific fraction of the gradient, depending on their buoyant density [75, 76, 80,81,82,83]. Ultracentrifugation is used to improve purification of EVs. Otherwise, ultrafiltration is utilized, where filtration membranes of different molecular mass cutoffs are centrifuged at moderate centrifugal forces. This simple and rapid method allows the concentration of vesicles at the interface of the filters. However, the filtration method has some disadvantages, such as a decreased yield, and the use of pressure can cause the EVs to deform or break into smaller vesicles.

Size-based exclusion chromatography is an efficient chromatography technique that separates particles based on their size, which can be used to separate and purify EVs from proteins in complex biological samples. However, when used to purify EVs from plasma or serum, this technique cannot efficiently separate EVs from lipoproteins of similar size [84]. To purify EVs from lipoprotein, density gradient ultracentrifugation followed by size exclusion chromatography is needed [79]. Other types of chromatography may also be used to purify EVs. Affinity purification and ion exchange are used for the purification of EVs from biological samples [85]. Immuno-affinity columns selectively purify EVs using capture agents, including heparin, tetraspanins, and epithelial cell adhesion molecule (EpCAM) [86,87,88]. Anion exchange chromatography is a simple, efficient, scalable, and dependable method for the isolation of EVs from cell culture supernatants [89, 90]. Negatively charged EVs bind to positively charged columns, and EVs are eluted from the column using increasing concentrations of salt. The precipitation method is a rapid, feasible, and cost-effective method that allows EVs to be pelleted by low-speed centrifugation using polyethylene glycol [91] or Exoquick [92]. However, the purity of EVs from the precipitation method is not absolute; it may contain EVs with other proteins and lipoproteins. In addition, EVs purified from precipitation affect the viability and biological activity of recipient cells and EVs [93, 94]. Recently, isolation of EVs has attracted microfluidic chip technology, which is useful for the capture and analysis of EVs from small volumes of clinical samples and shows promise for liquid biopsy diagnosis of disease [95]. Microfluidic devices have been engineered for immuno-capture using tumor-specific antigens, such as human epidermal growth factor receptor (HER2) and prostate-specific antigen (PSA) [96].

Characterization of EVs

The characterization of EVs is an essential step in clinical applications. The first and leading technique is microscopy, which is used for morphology and size analysis. In particular, electron microscopy techniques are the only method available to visualize the appearance of EVs, which are generally cup-shaped [97]. Atomic force microscopy (AFM) is an alternative method for analyzing the size distribution and quantity of EVs within a sample. The use of aqueous media is advantageous because it permits the maintenance of the physiological properties and structure of EVs [98, 99]. The combination of AFM and microfluidic techniques allows for the consecutive isolation and characterization of EVs.

The size distribution of EVs can be measured by nanoparticle tracking analysis (NTA), a light scattering technique which is now widely used for the assessment of EV size distributions and concentrations in the range of 50 to 1000 nm [100]. This technique is based on the inherent Brownian motion of particles in a solution. Dynamic light scattering (DLS), which uses the same principle, can be also used to assess the EV size distribution. A tunable resistive pulse is a novel and less expensive technique for the analysis of particle size distributions within the range of 30 nm to 10 μm. The system is composed of a thermoplastic polyurethane membrane containing nanopores that are selected based on size requirements. On the other hand, flow cytometry is used to measure the size distribution, concentration, and qualitative characteristics of EVs within a sample. Light scatter flow cytometry can measure within the range of 300 nm to 500 nm; however, exosomes cannot be measured because the size of exosomes is between 30 nm and 150 nm. Innovations in flow cytometry uses fluorescent labeling of EVs, which reduces the lower limit of detection to ~100 nm. Finally, antibodies coupled with surface markers of EVs can be used to measure nanosized EVs [101,102,103,104].

Isolation, purification and identification of EVs from reproductive cells/embryos

EVs play a significant role in the male and female reproductive tracts, making connections between the reproductive tract and immature germ cells, or between the mother and the developing embryo. As such, the uses of EVs have potential implications for the establishment of a successful pregnancy or understanding associated pathological conditions [105]. Hence, we focused specifically on the isolation, purification, and identification of exosomes from embryos.

The isolation and purification of exosomes from somatic cell-cloned embryos were described previously [76, 106, 107]. Embryos were cultured for 3 d on defined medium, and then the medium was subjected to differential centrifugation to remove various debris at 4 °C (300 × g, 10 min to remove cells; 2000 × g, 10 min to remove dead cells; and 10,000 × g for 30 min to remove cell debris, macroparticles, and apoptotic bodies). The supernatants were then ultracentrifuged at 100,000 × g for 70 min in 14 mm × 95 mm ultra-clear centrifuge tubes (Beckman). The pellets from a single sample were pooled, resuspended in PBS, and centrifuged again at 100,000 × g for 70 min. Each pellet was resuspended in 30 μL of the defined medium to supplement the renewed culture medium. Exosomes were identified as previously described [106].

The isolation of EVs from bovine embryos was described previously [108]. Bovine embryos were cultured on conditioned media and then sequential centrifugation was carried out to remove larger particles, which were then filtered using 0.2 μm syringe filters and used for sample dilution and EV isolation. The culture medium was then subjected to double centrifugation. Initially, the diluted samples were centrifuged at 400 × g for 10 min at 4 °C to remove dead cells and debris, and the collected supernatants were further centrifuged at 2000 × g for 10 min to remove apoptotic bodies. Finally, EV isolation was performed using qEVsingle size exclusion columns. Fractions were collected and pooled as EVs were eluted in these fractions. The size and concentration of EVs in the pooled fractions were determined using a nanoparticle tracking analyzer.

Burkova et al. [109] reported on the methods for isolation, purification, and identification of exosomes from the placenta. The human placenta is a highly specialized organ that connects mother and fetus organisms, and it protects, nourishes, and regulates the growth of the embryo. Placenta extract preparations were obtained from total placentas. Exosomes were isolated from the placenta using various methods. Supernatants were subjected to sequential centrifugation twice at 10,000 × g for 40 min at 4 °C and once for 16,500 × g for 20 min, and the supernatant was filtered through a 0.22-μm filter. The filtered supernatant was ultracentrifuged at 100,000 × g for 2 h. After the first centrifugation, the pellet was resuspended in 8 mL of TBS. The resuspended pellet was ultracentrifuged twice at 100,000 × g for 2 h. The precipitate was resuspended, filtered through a 0.1-μm filter, and purified further using gel filtration on Sepharose 4B columns. Exosomes derived from placentas underwent various purification steps. Transmission electron microscopy revealed the aggregation of exosomes, microparticles, and amorphous protein [109]. The isolated exosomes contained the typical surface markers CD81, CD63, and tetraspanins.

EVs have been isolated and characterized from human blastocoel fluid (BF), as described previously [34]. BF samples were collected from human embryos on the fifth day of development from patients undergoing IVF cycles. Exosomes were isolated from BF, and morphological and molecular characterizations were performed using various analytical techniques, such as scanning electron microscopy (SEM) and nanoparticle tracking analysis (NTA). SEM observation revealed vesicles of spherical shape with an average diameter of 75 ± 3 nm and full width at half maximum (FWHM) of 38 ± 8 nm, compatible with exosome size [34].

Simon et al. [33] reported the isolation, identification, and characterization of EVs from mouse embryos. Embryos from 10 animals were used for the identification and phenotypic characterization of EVs using electron microscopy and immunogold. Embryos were collected from conditioned media at day E4.5 and centrifuged at low speed (300 × g, 10 min) to remove larger debris. The resulting supernatant was centrifuged at 2000 × g for 10 min to recover apoptotic bodies, as previously described [110]. It was subsequently ultracentrifuged at 185,000 × g for 70 min in a P50A3 Hitachi rotor (Hitachi, Tokyo, Japan) to collect non-apoptotic EVs (naEVs) that included MVs and exosomes in the same fraction. TEM images revealed the presence of MVBs in the cytoplasm of murine oocytes. The presence of MVBs was also observed in the blastomeres at different embryonic developmental stages (E2.5 and E3.5), migrating from the cytoplasm to the plasma membrane where their content was secreted outwards through the zona pellucida, and larger vesicles were observed in the intercellular space. At the blastocyst stage (day E4.5), the secretion of vesicular structures was observed both in the extracellular medium through the zona pellucida, as well as in the blastocoel cavity.

EVs as mediator and messengers in reproductive biology

Exosomes play a significant role in the transmission of specific cargo molecules in the reproductive tract to modulate transcription and translational activity, granulosa cell proliferation and differentiation, cumulus expansion, gametogenesis, normal follicular growth, oocyte maturation, fertilization rate, embryo development, blastocyst formation and implantation, pregnancy outcomes, and fertility (Fig. 2). Human reproductive systems are highly dynamic and have well-characterized stages. EVs are involved in the intercellular communication at each stage of the reproductive system in both the male and female reproductive tracts. EVs are associated with reproductive biology and have been identified in different fluids, such as prostatic and epididymal fluid, seminal fluid, follicular fluid, oviductal fluid, cervical mucus, uterine fluid, amniotic fluid, and breast milk, as well as the originating tissues [9, 111,112,113,114,115,116,117,118]. EVs are key regulators of different reproductive processes, such as sperm and ovum maturation, coordination of capacitation/acrosome reaction, prevention of polyspermy, endometrial embryo crosstalk, and embryo development [119]. EVs are released by extravillous trophoblasts (EVTs). The syncytiotrophoblast (STB) is considered to be the main site of EV generation, and these EVs play significant roles in immune modulation, either for innate or adaptive responses [120]. EVs derived from the amniotic fluid are responsible for inflammatory and procoagulant activities [121]. EV-derived breast milk is involved in bone formation, immune modulation, and gene expression regulation, especially for long non-coding RNAs [122, 123]. In vitro and in vivo studies suggest that embryo-derived EVs act as modulators of embryo-to-embryo communication in polytocous species [39, 106]. EVs mediate communication between the inner cell mass and trophectoderm. EVs secreted by bovine embryos can be taken by zona-intact bovine embryos, increase blastocyst rates at d 7 and 8, and improve embryo quality, with significantly decreased apoptotic cells [124]. Conceptus-derived EVs are found in the cytoplasm of luminal epithelial cells and some glandular epithelial cells. These EVs can target the uterine epithelium and serve as a novel form of cell-to-cell communication during the establishment of pregnancy [19]. EVs derived from cervical mucus have sialidase activity, which is involved in modifying highly glycosylated mucus to favor spermatozoa access to the uterine cavity and tubes [113]. Bovine follicular fluid–derived exosomes and cumulus–oocyte complexes from mice and cattle revealed that follicular EVs are taken up by cumulus cells, promoting both cumulus expansion and related expansion of genes [13]. Several studies have shown that exosomes are released from various parts of the female reproductive tract, including the uterus, oviduct epithelium, endometrium, preimplantation embryos, and placental trophoblastic cells [9, 12, 125, 126]. Exosomes play an important role in intercellular communications, which is essential for preconception and post-conception, and also serve as a marker for pregnancy and pregnancy-associated pathologies in humans [127, 128]. Prattichizzo et al. [129] reported that aged-cell-derived exosomes are more proinflammatory than younger cell-derived ones. A mouse study suggested that serum-derived exosomes from young mice were able to mitigate inflammation in both the central and peripheral nervous systems of old mice, which reduces morbidity and mortality caused by age-related diseases [130]. Exosomes contain miRNAs derived from senescent cells that initiate senescence and aging in the surrounding cells. Exosomes contain intracellular miRNAs, such as the let-7, miR-34a, and miR-17-92 cluster, which are involved in regulating and developing mammalian cells [131,132,133,134]. Exosomes play a significant role in the removal of waste and biomolecules, which are essential for the maintenance of intracellular proteins, RNA homeostasis, and cellular fitness [135]. During pregnancy, exosomes are derived from various cells and tissues, including placental trophoblasts, embryos, endothelial cells, immune cells, and platelets, which mediate the necessary communication between maternal and fetal circulation during pregnancy [120, 136]. Exosomes derived from the placenta promote endothelial and vascular cell migration, which is an essential step for the establishment of fetal-maternal circulation and remodeling of uterine spiral arteries [137]. Exosomes serve as signaling molecules and are involved in the activation of signaling pathways involved in the regulation of folliculogenesis, oocyte maturation, ovulation, meiotic resumption, embryo development, and fertilization rate [8, 115, 138]. Bioinformatic analysis revealed that 14 and 5 miRNAs were found in follicular fluid of young versus old mares, respectively. Levels of miR-513a-3P, miR-181A, and miR-375 were significantly higher in exosomes, and all these miRNAs suppressed the TGFβ pathway [139]. Exosomes play a significant role in coordinating between the embryo and uterine endometrium, which is required for successful implantation [140, 141]. Extracellular vesicles released from endometrial epithelium contribute to the transfer of miRNA and connective molecules to blastocysts and endometrium, which play a significant role in implantation and fertility outcomes [19, 141]. Exosomes derived from ovine uterine stimulated trophectoderm cells to proliferate and secrete interferon tau via TLR-mediated cell signaling [12]. Placenta-derived microvesicles in the first trimester of pregnancy indicate the role of EVs in maternal-embryo crosstalk during pregnancy [142]. Exosomes released from the fetal membrane during pregnancy potentially transmit signals originating from the fetus to the maternal uterus, as well as the cervix [143].

Fig. 2
figure 2

Multifunctional roles of EVs in male and female reproduction organs. Male and female reproductive organ-derived EVs may be involved in sperm and oocyte maturation, sperm-oocyte fusion and also increase embryo viability and pregnancy efficacy

EVs are stable, versatile, cell-derived nanovesicles with target-homing specificity and the ability to transfer through in vivo biological barriers, and they hold promise for the development of new approaches in drug delivery [23]. EVs are capable of intercellular genetic transfer and can facilitate new diagnostic and therapeutic tools in the field of reproductive biology. EVs serve as potential biomarkers for disorders of reproductive organs. The release of placental EVs is modulated by a number of factors that arise from the placenta, and maternal blood is the source of EVs [144]. Placental–exosomal miRNA cargo is related to cell migration potential and inflammatory cytokine production. Low-oxygen tension exosomes decreased endothelial cell migration potential and increased TNF-α production [145]. Placental EVs play a significant role in infectious diseases during pregnancy. Both total and placental-derived EVs are increased in the plasma of pregnant women with HIV infection compared with non-infected controls [145].

Effects of extracellular vesicles on placentation and pregnancy disorders

In humans, a successful pregnancy depends on normal placental formation, normal implantation, and development of the placenta, which are responsible for fetal growth and development during pregnancy. Soluble factors are involved in normal placental development through intercellular interactions in various types of cells, including trophoblasts, endothelial cells, immune cells, mesenchymal stem cells (MSCs), and adipocytes [17]. Exosomes are released from decreased insulin sensitivity and glucose uptake in skeletal muscles, contributing to the pathophysiology of gestational diabetes mellitus (GDM) [146]. Secretory levels of exosomes are significantly higher in GDM than those in normal glucose conditions [147]. Exosomes released from other types of cells can affect placental function and are involved in regulating the physiological and pathological mechanisms of pregnancy. For example, exosomes released from adipocytes mediate placental metabolic status and contribute to GDM [147] and are also involved in mediating maternal metabolic changes in pregnancy between different organs and the placenta [147]. Concentrations of circulating placental-derived EVs increase during abnormal placentation in preeclampsia (PE) [148]. During gestational age, the level of exosomes is increased and is involved in regulating the maternal immune response during pregnancy [149,150,151,152,153]. EVs can regulate the expression and production of different cytokines during pregnancy [146, 154]. Placental EVs are believed to play a role in modulating pro-inflammatory and anti-inflammatory states by modulating cytokine release [151]. Placental EVs inhibit maternal immunity and promote fetal survival through the expression of specific immunoregulatory molecules [149,150,151,152,153]. Placental-derived EVs contain syncytin-1, which suppresses the production of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN- γ), which are inflammatory regulators in early pregnancy [155]. Placental EVs induce the release of proinflammatory cytokines from endothelial cells, including TNF-α, macrophage inflammatory protein (MIP)-1α, interleukin (IL)-1α, -6, -8, and -1β, and activate macrophages to release proinflammatory IL-1β. The activation of phagocytic cells regulates the maternal immune response to maintain a normal pregnancy and protects against infection [156, 157]. Circulating EVs induce the formation of pro-inflammatory environments and endothelial cell dysfunction in the placenta, and contribute to the formation of pro-inflammatory environments and endothelial cell dysfunction in the placenta [18]. Circulating EVs also facilitate the prediction of the physiological and pathological conditions of the cell of origin. EVs derived from trophoblasts increase the migration of monocytes through the production of IL1B, IL6, SERPINE1, and colony stimulating factor 2 (CSF2) [157]. EVs derived from PE patients inhibit the proliferation of macrophages and the expression of inflammatory cytokines, such as IL-12 and TNF [158]. Maternal plasma-derived EVs contain miR-548c-5p, which causes inflammatory responses and PB in pregnant mice [159]. A mouse study revealed that EVs derived from injured placenta induce PE-like symptoms, such as hypertension and proteinuria, by inducing endothelial injury, vasoconstriction, and hypercoagulation [160]. The secretary level of exosomes are significantly are higher in compared to normal glucose condition [161].

Role of extracellular vesicles in normal pregnancy and pregnancy-related diseases

EVs are playing an important role in intercellular communication through the transfer of a wide spectrum of bioactive molecules, contributing to the regulation of diverse physiological and pathological processes, and mediating fetal–maternal communication across gestation. EVs play a significant role in maternal-embryo interaction within the human uterine microenvironment, promoting implantation, the earliest and essential step for successful pregnancy. Studies have suggested that exosomes can be transferred between the fetus and maternal bodies [159]. EVs potentially regulate multiple processes of pregnancy, such as implantation, migration, and invasion of trophoblasts, and cellular adaptations to physiological changes (Fig. 3) [120, 162, 163]. Alterations in EVs are critically involved in pregnancy-related diseases. Moreover, EVs have shown great potential as biomarkers for the diagnosis of pregnancy-related diseases. The concentration, composition, and bioactivity of EVs can regulate pregnancy-related diseases [144, 164]. EVs are playing significant role in maternal-embryo interaction within human uterine microenvironment, promoting implantation, an earliest and essential step for successful pregnancy [161, 165]. Exosomes from endometrial epithelial cells (ECs) were treated with estrogen, or estrogen, and progesterone (EP); EP-treated ECs have exosomes that contain proteins associated with embryo implantation and extracellular matrix remodeling [26]. MicroRNAs (miRNAs) play a potential role as mediators of embryo-endometrium crosstalk in the implantation process [166, 167]. The interaction between EVs and immune cells modulates pregnancy, tolerates the growing fetus, and maintains its normal functions [168, 169]. EV-derived heat shock protein family E member 1 (HSPE1) promotes Treg differentiation from CD4+ T cells and Treg cell expansion [170]. Exosomes derived from macrophage-derived exosomes increase the release of pro-inflammatory cytokines, such as IL-6, IL-8, and IL-10, potentially facilitating protective placental immune responses during pregnancy [171]. Exosomes are derived from primary human placental trophoblasts containing chromosome 19 miRNA cluster (C19MC) miRNAs that attenuate viral replication in recipient non-placental cells by upregulating autophagy [172]. EVs are involved in metabolic homeostasis and are associated with metabolic regulation during pregnancy. Placental-derived exosomes are able to increase insulin-induced glucose uptake in the skeletal muscle of diabetic patients, suggesting that placental exosomes may engage in changes in insulin sensitivity in normal pregnancies [146]. Exosomes from adipose tissue (AT) of pregnant women with normal glucose tolerance affect the expression of glucose metabolism-related genes in placental cells [173]. Exosomes from extravillous trophoblast cells cultured under low oxygen tension increased TNFα expression in HUVECs [174]. Exosomal miR-141 derived from fetal trophoblasts induces T cell proliferation, indicating that placental EVs regulate maternal immune cells and cause immune disorders during pregnancy [175,176,177]. Exosomes from GDM pregnancies increase the release of pro-inflammatory cytokines from ECs, including GM-CSF, IL-6, and IL-8 [161]. Preterm labor (PTB)-enriched exosomes are associated with inflammatory molecules that affect the labor process [178].

Fig. 3.
figure 3

Typical structure of EVs, properties, and functional attribution of EVs on female reproductive system. EVs are involved in various physiological functions including embryo-uterus fusion, modulation of implantation, immunomodulation, regulation of male and female hormone regulation, immunotolerance of embryo

Role of extracellular vesicles in mother and embryo cross-communication

EVs play a significant role in paracrine communication between the mother and embryo [105] and are involved in synaptic plasticity, deliver neurotransmitter receptors, and modulate tissue regeneration [179]. EVs participate in regulating immune responses, particularly triggering the adaptive immune response and suppressing inflammation [180]. A previous study reported that EVs play important roles from preconception, gamete maturation to implantation and throughout pregnancy [141]. Human uterine fluid-derived EVs contain a specific subset of miRNAs that are not detectable in maternal cells by the human endometrial epithelial cell line ECC1 [11]. Similarly, the uterine fluid of pregnant sheep contains EVs positive for CD63 and HSP70, as well as small RNAs and miRNAs [181]. Exosomes derived from human endometrial epithelial cells are subject to steroid hormonal regulation by estrogen and progesterone and vary with the menstrual cycle [26]. Internalization of miR30d by mouse embryos via the trophectoderm increased the overexpression of adhesion-related genes, Itgb3, Itga7, and Cdh5, and also increased embryo adhesion; conversely, miR-30d deficiency results in reduced implantation rates and impaired fetal growth [182]. Heterogeneous nuclear ribonucleoprotein C1 (hnRNPC1) is involved in cell-to-cell communication, and previous studies suggest that maternal endometrial miRNAs act as transcriptomic modifiers of the preimplantation embryo [183, 184]. Exosomes released from human endometrial epithelium transferring molecular cargoes promote implantation to the blastocyst and endometrium [11]. A bovine model study demonstrated that embryo-derived EVs improved the growth and viability of cloned bovine embryos and increased implantation rates and full-term calving rates [185]. Mouse studies suggest that microinjections of 3-5 days old blastocysts with embryonic stem (ES) cell-derived EVs before transfer into surrogate mothers significantly increased the likelihood of implantation. In addition, ES cell-derived EVs improve the capability of TE cells within the blastocyst to migrate into the uterus and promote blastocyst implantation [20]. EVs from day 17 of pregnancy induce apoptosis of immune cells and primary endometrial epithelial cells (EECs) through increased expression of apoptosis-related genes, including BAX, CASP3, and TNFA, which are required for conceptus implantation, during which a portion of the endometrial epithelium disappears [186].

Role of microRNAs in embryo implantation

miRNAs are small non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. They regulate genes expression in blood plasma and serum, as well as other body fluids, and are involved in intercellular communication [187,188,189]. miRNAs are secreted by all types of cells, and the concentration of extracellular miRNAs is associated with physiological and pathological conditions of the body [190, 191]. Embryo implantation is a crucial step in the establishment of pregnancy in mammals and has a profound effect in reproductive efficiency. The process of implantation is under the strict regulation of ovarian hormones, estrogen, and progesterone [192]. Several molecules, such as cytokines, chemokines, growth factors, lipids, and receptors also participate in the regulation of implantation through autocrine, paracrine, and juxtacrine pathways [192]. Several studies have reported that miRNAs are involved in the regulation of oogenesis, fertilization, implantation, and placentation. Dysregulation of miRNAs causes reproductive disorders, such as polycystic ovarian syndrome and endometriosis [193, 194]. miRNAs are associated with various types of proteins such as the AGO family, nucleophosmin 1, bound to lipoproteins and apoptotic bodies [73, 195, 196]. These enzymes are involved in miRNA biosynthesis pathways, such as DICER; AGO2 leads to embryonic death around gastrulation, suggesting an important role of miRNAs in early embryonic development [197,198,199]. Regulation of the expression of Dnmt3a/b by miR-29b causes disruption of DNA methylation, which leads to early embryonic developmental blockade in mice [200]. Inhibition of this miRNA significantly reduces morula and blastocyst formation [201]. Liu et al. reported that 45 miRNAs were differentially expressed between dormant and activated mouse embryos; particularly, let-7a levels were highly expressed in dormant embryos and inhibited the expression of Dicer and prevented embryo implantation [202, 203]. A porcine embryo study suggested that there was a lower expression of miR-24 in the blastocyst stage than in in vitro fertilized (IVF) embryos [204]. Another study suggested that high level expression of miR-24 inhibited the development of embryos to the blastocyst stage [36]. Differential expression of miRNAs was observed between IVF bovine blastocysts and degenerate embryos, and relatively higher levels of miR-181a2, miR-196a2, miR-302c, and miR-25 were found in degenerate embryos [35]. Variable expression patterns of miRNAs were observed in human endometrial fluid secreted by the endometrial glands at different stages of the menstrual cycle [182]. Placenta-specific miRNAs, such as miR-515-3p, miR-517a, miR-517c, miR-518b, miR-526b, and miR-323-3p, are widely expressed in the blood plasma of pregnant women [205, 206]. A cow model study demonstrated that circulating EV-derived miRNA is not only able to identify pregnancy, but can also distinguish between successful implantation and embryonic mortality at the early stage of pregnancy [207]. EVs derived from bovine follicular fluid contain miRNAs that reflect the stage of the estrus cycle and can modulate cumulus cell transcription during in vitro maturation [208]. Murine oviductal tract EVs (oEVs) contain miR-34c-5p, which is transferred to the sperm heads, promoting the first cleavage in the zygote and controlling embryonic development [209]. EVs secreted by donor oviductal cells increase birth rates after embryo transfer in mice due to decreased apoptosis and improved cellular differentiation in embryos [210].

Role of EVs in animal reproduction

Cell communication is a crucial process for several molecular processes involved in female reproduction. EVs have been identified as one of the key players in regulating temporal sequences, spatial interaction, and cell-cell signaling in all events in sexual reproduction [211]. EVs have been observed in seminal fluids to modulate sperm capacitation in humans [212] and pigs [213] and also influence female physiology by modulating immune-related gene expression in the porcine endometrium [214]. EVs from avian uterine fluid may play an essential role in preserving sperm function [215]. EV-mediated molecules are produced by somatic cells and germ cells present in follicular fluids (FF). EVs derived from FF have been used in various animal models, such as horses [8], humans [216] and cows [217]. Follicular fluid comprises a heterogeneous EV population secreted by granulosa, cumulus, and somatic follicular cells with functions related to the control of steroidogenesis [8, 216, 217]. EVs play a significant role in reproductive processes as intercellular communicators and are found in follicular fluid [217], oviductal fluid [9] and secreted by embryos in culture media [35,36,37]. Intercellular communication within the microenvironment of the ovary is essential for oocyte and follicle development. EVs are secreted by follicular cells and are found in follicular fluid, which transmit information between cells [217]. Sohel et al. [125] reported that the majority of miRNAs from follicular fluid were in the exosome fraction, and exosome uptake by follicular cells was associated with an increase in miRNA levels in these cells [125]. Exosomes derived from follicular fluid regulate TGF-β signaling pathways; they have been shown to regulate ACVR1 and ID2 in granulosa cells in vitro by transferring mRNA, protein, and miRNAs in follicular development of granulosa cells [218]. EVs are a component of the oviductal fluid that favors oocyte and embryo quality [14]. Proteomic analysis revealed that EVs secreted proteins, such as oviductal glycoprotein (OVGP), heat shock protein A8 (HSPA8), and myosin 9 (MYH9) by bovine oviduct epithelial cells (BOECs), which are involved in fertilization, early pregnancy development, and zona pellucida maturation [219, 220]. Embryos treated with EVs from BOEC culture media induced an increased number of total cells and better survival rate after vitrification compared to embryos cultured without EVs [14]. Oviductal fluid-derived EVs from the isthmus resulted in the greatest bovine embryo survival rate after vitrification. AQP3 (Aquaporin 3) was upregulated in embryos supplemented with EVs from the isthmus compared to embryos supplemented with FCS only [221]. Supplementation of exosomes secreted by somatic cell nuclear transfer (SCNT) embryos in the culture medium of SCNT embryos increased blastocyst rate, total cell numbers, ratio of ICM/TE, and transcript levels of OCT-4 in comparison to SCNT embryos without supplementation [185]. Exosomes present in the culture medium are essential for embryo development, and changes during embryo development caused by culture medium replacement may be repaired by exosome supplementation [185]. Progesterone treatment increased the number of EVs in the uterine lumen compared to that in the P4 receptor antagonist group [222]. EVs derived from porcine trophectoderm induce aortic endothelial cell proliferation, which may stimulate angiogenesis [223]. Furthermore, porcine trophectoderm and aortic endothelial cell EVs have miRNAs predicted to modulate angiogenesis and placental development pathways, suggesting that these EVs may play an important role in the communication between the conceptus and maternal endometrium, influencing the establishment of pregnancy [223]. Small extracellular vesicles (sEVs) released from endometrial epithelial cells (EECs) activate signaling pathways in trophoblasts, thus promoting migration and invasion, which affect implantation rates. These sEVs serve as novel intercellular communication mechanisms during embryo implantation [224].

The success of pregnancy depends on the molecular dialogue between the embryo and the female reproductive tract that starts at the oviduct and continues until the placenta is formed. Cytokines and growth factors, such as interleukin-1β (IL-1β), heparin-binding epidermal growth factor (HB-EGF), integrins, and leukemia inhibitory factor (LIF), act synergistically in embryo-maternal crosstalk. For example, the expression of epithelial cell adhesion proteins increases endometrial receptivity by IL-1 [225] and stimulates angiogenesis to promote embryonic growth [226]. HB-EGF receptors on the surface of the embryo and endometrium facilitate implantation and promote the development of blastocyst [227, 228].

Roles of extracellular vesicles in the male reproductive system

EVs are important regulators of the biological function of sperm and seminal fluid in normal and pathological reproduction [105]. EVs of the male reproductive tract are a product of a diverse population of cells, and are conserved, abundant, and carry a complex payload of regulatory elements that support sperm function, which is essential for effective functions of female reproductive tract biology after mating (Fig. 4) [229, 230]. Epididymosomes are a heterogeneous population of EVs that are produced by epithelial cells lining the epididymis and have an average size between 50 and 250 nm [231, 232]. They play a significant role in mediating post-testicular sperm maturation and storage across mammalian species. They can tether and transiently fuse with sperm to facilitate protein transfer, and are also involved in sperm maturation and successful fertilization [233,234,235]. Epididymosomes contain sncRNA cargo and are directly implicated in the transfer of microRNAs (miRNAs) and transfer RNA-derived RNA fragments (tRFs) to epididymal sperm [236,237,238,239]. Epididymosomes of mice, humans, and bulls contain an abundance of antioxidant enzymes, which play an important role in the elimination of defective sperm in humans, rats, and cattle [240,241,242]. Epididymosomes also play a significant role in sperm maturation and storage, and cargoes of epididymosomes have been shown to influence the female reproductive tract [243, 244]. Seminal fluid EVs (SFEVs) isolated from vasectomized men lack epididymosomes and show reduced capacity to support motility, capacitation, and initiation of the acrosome reaction, compared to the SFEV pool of intact men [245]. The interactions between epididymosomes and sperm are potentially involved in the female reproductive tract, which assists sperm in attaining full functional maturity [245].

Fig. 4
figure 4

Role of EVs in normal pregnancy and pregnancy related diseases. EVs mediate fetal-maternal communications in normal pregnancy. EVs contribute to embryo implantation by promoting trophoblast adhesion. Placenta can interact with immune cells via EVs to balance immune activation and suppression across the gestation. EVs are involved in angiogenesis, immunomodulation, glucose metabolism, embryonic development and fetal circulation. Particularly, secreted exosomes play a critical role in cell-cell communication mediators in pathological scenarios. As a result, these can induce gestational diabetes

SFEV is not only involved in modulating sperm function, but also has an impact on the immune environment within the female reproductive tract [230]. The interaction between seminal fluid EVs and the female reproductive tract of epithelial cells initiates an inflammatory response, similar to the responses observed following exposure to seminal fluid [214, 246,247,248,249]. EVs of the reproductive tract are potentially involved as fundamental regulators of reproductive success by regulating male gamete function and influencing the female reproductive tract during pregnancy. However, alterations in EV composition not only regulate impaired fertility, but could also influence fetal development and impart long-term consequences for offspring health.

Human seminal plasma is a complex fluid produced by secretions from several glands of the male genital tract and male gametes consist of rich amount of EVs [250]. EVs are synthesized by the prostate, as well as by the epididymis, and even by the testis. EVs are classified based on testicular, prostatic or epididymal localization. EVs from seminal plasma involved in various aspects of male fertility, improving sperm function by regulating the timing of sperm capacitation, inducing acrosome reaction, stimulating sperm motility enabling them to reach the ovocyte [251]. Myelinosomes are secreted by Sertoli cells and secretory organelles loaded with specific cargoes and capable of leaving the cell in their entirety, in the form of extracellular vesicles [252]. Prostasomes are nanosized exosomes secreted by the acinar lumen of prostate epithelial cells. These prostasomes contain proteins, lipids and nucleic acids. Prostasomes contains rich level of cholesterol and sphingomyelin, with a particularly high cholesterol/phospholipid ratio [253]. Prostasomes play significant role in controlling capacitation and the acrosome reaction and also involved in preventing premature capacitation of spermatozoa and premature acrosome reaction. The prostasomal membrane is enriched in cholesterol, which contributes to its stability in the acidic vaginal environment [212]. Bovine model studies demonstrated that epididymosomes contain several proteins that are involved in the acquisition of sperm mobility, fertilisation capacity and protection against oxidative stress. The content of these vesicles also depends on the region of the [234, 254] epididymis. EVs are secreted by testis which contains sperm RNA is an important epigenetic player in the early development of the embryo and the health of the offspring [255]. Altogether, these findings suggest that the heterogeneous population of EVs present in seminal plasma is known to influence sperm functions [141, 256].

Role of EVs in reproductive and therapeutic medicine

EVs play an important role in both physiological and pathological processes as biomarkers of fertility, reproductive cancer, embryo quality, placenta quality, and early abortion [211]. The circulating level of EVs depends on the physiological level of tissues, serum or other biological fluids, animal model, time, and type of disease. In particular, EVs regulate a variety of physiological processes. Cargoes present in EVs are protected from degradation and can be used as biomarkers for non-invasive cancer diagnosis in various types of reproductive cancers [188, 257, 258]. Physiological and pathological conditions influence EV concentration, cargo, and function. Several miRNAs have been established as biomarkers in the ovarian follicle, which create a suitable microenvironment for the growth, maturation, and fertilization of oocytes [259, 260]. For example, expression of miRNA-375 in granulosa cells and oocytes facilitates follicular growth proliferation, spread, and apoptosis of cumulus cells, whereas overexpression of miR-375 inhibits the ability to proliferate, increases the apoptosis rate of cumulus cells in cows, and suppresses estradiol production and follicular development in porcine granulosa cells [261,262,263,264]. Placenta-derived EVs can induce differentiation due to the presence of placenta-specific proteins (e.g., PLAP4) and miRNAs (e.g., chromosome 19 miRNA cluster) that are exclusively expressed in the placenta and serve as biomarkers of maternal-fetal health and evolution and for diagnosis of preeclampsia [265,266,267]. EVs show immense therapeutic potential in various diseases, including reproductive cancers, such as ovarian cancer. For example, amniotic derived EVs have been used to treat endometritis in mare to attain successful pregnancy [268]. Zhang et al. [269] demonstrated that transplantation of menstrual blood-derived stromal cells (MenSCs) derived sEVs safely and effectively promoted the regeneration of endometrial glands and blood vessels, and improved fertility in IUA rats. Furthermore, treatment with MenSCs and MenSCs-sEVs increased BMP7 levels and activated the SMAD1/5/8 and ERK1/2 pathways in vivo, thereby alleviating endometrial fibrosis by inhibiting TGFβ1/SMAD3 signaling.

EVs are serving as signaling molecules to target various types of reproductive cells

EVs are serving as intercellular signaling molecules are considered imperative for the regulation and accomplishment of different physiological events including cellular proliferation and differentiation, gametogenesis, fertilization, and embryonic development [141]. The success of pregnancy greatly depends on gametogenesis, fertilization, and an adequate uterine environment for embryonic development [270]. These highly complex processes greatly rely on the crosstalk between the gametes and the different segments of the reproductive tract. EVs are regulating diverse signaling pathways in targeting the cells [271]. EVs secreted by the male reproductive tract including epididymosomes and prostasomes are significant role in the maturation process of sperm [272, 273]. EVs derived from the uterine fluids of murine exhibited the expression of certain sperm essential proteins including spermadhesionmolecule 1 (SPAM1) and plasma membrane calcium pump (PMCA4) [116, 274]. Oviductosomes contains cargoes including proteins aV integrin, CD9 tetraspanin, heat shock proteins, lactadherin oviductal specific glycoprotein (OVGP), lipids, SPAM1, RNAs, and miRNAs are involved in acrosome reaction, increases sperm viability and motility, reduces the incidence of polyspermy through zona hardening, induces the phosphorylation of sperm-associated proteins during capacitation, and modulates fertilization, fertilization and early embryo development [275,276,277,278,279,280,281]. Various type of factors including insulin, transforming growth factor-beta (TGFB) and wingless/Int (WNT) signaling members [282, 283] growth factors [284] and hormones [285] are involved in both the folliculogenesis and initiation of different signaling pathways. Tetraspanins including CD9 and CD81 involved mediating the oocyte-sperm fusion process and exclusively CD81 may facilitate the transfer of CD9 from the oocytes to the sperm plasma membrane [286]. Oviduct-derived EVs are involved in embryonic development through mediating the embryo–maternal interactions during early embryonic development, leading to improved embryo quality and successful pregnancy [287]. Endometrial-epithelial derived EVs not only facilitate endometrium-embryo crosstalk but also help in the implantation of the embryo. EVs are derived from trophoblasts carry molecules such as miRNA and significant proteins are involved in the normal placental function and angiogenesis within the trophectoderm. Further, the trophectoderm derived EVs penetrate and stimulate the proliferation of maternal endothelial cells [157]. High concentrations of p38 MAPK in EVs influencing parturition and are involved in in inflammatory responses, cell proliferation, apoptosis, and stress induced signaling [288]. All these findings suggest that all the functional molecules carried by the EVs potentially modulate different reproductive events such as gametes maturation, fertilization, and blockage of polyspermy, development, and implantation of the embryo, fetal development, and parturition.

Conclusion and future perspectives

Extracellular vesicles (EVs) are a heterogeneous population of cellular couriers and membraned structures secreted by cells that contain various biomolecules, such as proteins, lipids, RNAs, and DNAs, which can serve as long distance messengers and play a significant role in cellular communication and cell function. EVs differ in size and function. EVs have various subsets, including exosomes, microvesicles, and apoptotic bodies. These vesicles play a significant role as key regulators of human reproduction. EVs are located in various body fluids that are critical for reproduction, such as follicular fluid, endometrial fluid, semen, and Fallopian tubal fluid [105, 141]. EVs can carry significant phenotype-altering cargo, such as transcription factors and microRNAs. EVs can serve as excellent carriers for drug delivery because of their ability to transfer their contents to target cells. Several studies have documented that EVs carrying functional molecules control different reproductive events, such as gamete maturation, fertilization, blockage of polyspermy, embryo development and implantation, fetal development, and parturition. The concentration of EVs determines the physiological or pathological state of different reproductive events and may be used as markers for pregnancy term, fetal growth, placental function, and diagnosis of different pathological conditions. EV-mediated cellular communication facilitates the enhancement of diagnostics and therapeutics for fertility-related issues, pregnancy-associated abnormalities, and pregnancy loss. EV communication may provide a foundation for a better understanding of the conception and implantation processes. EVs are released from the placenta into the maternal circulation and have a wide range of functions to regulate immunologic responses to pregnancy and to establish maternal vascular function (Fig. 5). Placenta-derived EVs contain various miRNAs and proteins that play a role in the maintenance of pregnancy in the trophoblast and placental microenvironment. EVs may be utilized as disease biomarkers and drug delivery systems, which provide the opportunity for diagnostic potential with reduced invasiveness in a targeted manner. EVs play important roles in regulating cellular functions and contributing to pregnancy-related diseases. The potential diagnostic value of EVs in pregnancy depends on the concentration and content of circulating EVs. EVs support male gamete function and interact with female reproductive tract cells, are eventually involved in pregnancy, and are potentially involved as regulators of male reproductive success.

Fig. 5
figure 5

Effects of EVs in pregnancy-related diseases. Concentration and composition of EVs are released from male and female reproductive systems are involved in pregnancy-related diseases. EVs mediate dysregulation of the balance between pro- and anti-inflammatory responses in immune cells and placenta. EVs are play significant role in induction of endometrial receptivity, uterus regulation, increases pro-inflammatory cytokines, increases glucose metabolism and regulation of vascular cells

To improve the physical and molecular characterization, action, and functions of EVs, there is a need to develop more reliable isolation methods and more sensitive technologies. Although data have demonstrated the potential role of EVs in reproductive physiology and pathology, further investigations are required in this area. Furthermore, it is necessary to understand the molecular mechanism by which EVs regulate key events in pregnancy, which may help elucidate how maternal-fetal communication is established in both normal and pathologic conditions. Detailed studies are required to understand the physiological activity of EVs during early pregnancy, which could open a new avenue to overcome abnormal placentation and pregnancy disorders as well as to characterize differences in the cargo of EVs between pregnant and non-pregnant or embryonic-mortality animals, which ultimately improves fertility rates in agriculturally important animals. In order to improve fertility, it is important to increase our knowledge regarding the biology of EVs in reproductive tissues, which can create a better environment to produce embryos in vitro and consequently generate healthier pregnancies in animals and humans. Although there is considerable evidence that EVs serve as natural therapeutic agents that are able to maintain reproductive success, the progress of reproductive and obstetric-related disorders is still in its infancy, and further investigations that utilize homogeneous and human-specific material are needed. Hence, comprehensive studies on the molecular mechanisms and functional roles of EVs in both male and female reproductive systems are required to decipher the relationship between EVs from various tissues and the entire reproductive process. More studies are required to provide insight into the functions of EVs in pregnancy and to apply EVs to the diagnosis, monitoring, and treatment of pregnancy-related diseases. These studies would provide significant knowledge regarding reproductive mechanisms and contribute to the development of new therapeutic strategies to treat various reproduction-related diseases. It is necessary to address the knowledge gaps in male gamete quality and the composition of seminal plasma on pregnancy outcomes and offspring health.

Availability of data and materials

Not applicable.

Abbreviations

AFM:

Atomic force microscopy

Alix:

ALG-2-interacting protein X

AQP3:

Aquaporin 3

AT:

Adipose tissue

BAX:

BCL2 associated X protein

BF:

Blastocoel fluid

BMP7:

Bone morphogenetic protein 7

BOECs:

Bovine oviduct epithelial cells

C19MC:

Chromosome 19 miRNA cluster

CASP3:

Caspase 3

CD4:

Cluster of differentiation 4

CD63:

Cluster of differentiation 63

CD81:

Cluster of differentiation 81

CD9:

Cluster of differentiation 9

Cdh5:

Cadherin 5

CX3CL1/fractalkine:

C-X3-C motif chemokine ligand 1

CXCL12:

C-X-C motif chemokine ligand 12

Dnmt3a:

DNA methyltransferase 3 alpha

Dnmt3b:

DNA methyltransferase 3 beta

EECs:

Endometrial epithelial cells

EP:

Estrogen and progesterone

EpCAM:

Epithelial cell adhesion molecule

ESCRTs:

Endosomal sorting complexes required for transport

ER:

Endoplasmic reticulum

ERK:

Extracellular signal-regulated kinase

EVs:

Extracellular vesicles

EVTs:

Extravillous trophoblasts

FWHM:

Full width at half maximum

GDM:

Gestational diabetes mellitus

GM-CSF:

Granulocyte-macrophage colony-stimulating factor

GTPase:

Rab guanosine triphosphatase

HER2:

Human epidermal growth factor receptor

HnRNPC1:

Heterogeneous nuclear ribonucleoprotein C1

HB-EGF:

Heparin-binding epidermal growth factor

HSP70:

70 kilodalton heat shock proteins

HSPA8:

Heat shock protein A8

HSPE1:

EV-derived heat shock protein family E member 1

ICAM3:

Intercellular adhesion molecule 3

ICM:

Inner cell mass

IFN-γ:

Interferon gamma

IL:

Interleukin

ILVs:

Intraluminal vesicles degradation of their contents

Itgb3:

Integrin subunit beta 3

Itga7:

Integrin subunit alpha 7

LIF:

Leukemia inhibitory factor

MAPK:

Mitogen-activated protein kinase

miRNAs:

MicroRNAs

MLCK:

Myosin-light chain kinase

MVB:

Microvesciles

NTA:

Nanoparticle tracking analysis

oEVs:

Oviductal tract EVs

OVGP:

Oviductal glycoprotein

PBS:

Phosphate-buffered saline

PE:

Preeclampsia

PLAP4:

Placenta-specific proteins

PSA:

Prostate-specific antigen

PS:

Phosphatidylserine

ROCK1:

Rho-associated protein kinase

SERPINE1:

Serpin family E member 1

TSG101:

Tumor susceptibility 101

VDAC1:

Voltage dependent anion channel 1

MenSCs:

Menstrual blood-derived stromal cells

MHC:

Major histocompality

MIP-1α:

Macrophage inflammatory protein

MYH9:

Myosin 9

PTB:

Preterm labor

SC:

Stem cells

SCNT:

Somatic cell nuclear transfer

SEM:

Scanning electron microscopy

SFEVs:

Seminal fluid EVs

SMAD:

Mothers against decapentaplegic homolog

STB:

Syncytiotrophoblast

TNFA:

TNF alpha

TNF- α :

Tumor necrosis factor alpha

References

  1. Anand S, Samuel M, Kumar S, Mathivanan S. Ticket to a bubble ride: Cargo sorting into exosomes and extracellular vesicles. Biochim Biophys Acta Proteins Proteom. 2019;1867(12):140203. https://doi.org/10.1016/j.bbapap.2019.02.005.

    Article  CAS  PubMed  Google Scholar 

  2. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–83. https://doi.org/10.1083/jcb.201211138.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367(6478):eaau6977. https://doi.org/10.1126/science.aau6977.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750. https://doi.org/10.1080/20013078.2018.1535750.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Iaccino E, Mimmi S, Dattilo V, Marino F, Candeloro P, Di Loria A, et al. Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes. Mol Cancer. 2017;16(1):159. https://doi.org/10.1186/s12943-017-0730-8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Manna I, Iaccino E, Dattilo V, Barone S, Vecchio E, Mimmi S, et al. Exosome-associated miRNA profile as a prognostic tool for therapy response monitoring in multiple sclerosis patients. Faseb J. 2018;32(8):4241–6. https://doi.org/10.1096/fj.201701533R.

    Article  CAS  PubMed  Google Scholar 

  7. Wong PF, Tong KL, Jamal J, Khor ES, Lai SL, Mustafa MR. Senescent HUVECs-secreted exosomes trigger endothelial barrier dysfunction in young endothelial cells. Excli J. 2019;18:764–76. https://doi.org/10.17179/excli2019-1505.

    Article  PubMed  PubMed Central  Google Scholar 

  8. da Silveira JC, Veeramachaneni DN, Winger QA, Carnevale EM, Bouma GJ. Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle. Biol Reprod. 2012;86(3):71. https://doi.org/10.1095/biolreprod.111.093252.

    Article  CAS  PubMed  Google Scholar 

  9. Al-Dossary AA, Strehler EE, Martin-Deleon PA. Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm. PLoS One. 2013;8(11):e80181. https://doi.org/10.1371/journal.pone.0080181.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mellisho EA, Velásquez AE, Nuñez MJ, Cabezas JG, Cueto JA, Fader C, et al. Identification and characteristics of extracellular vesicles from bovine blastocysts produced in vitro. PLoS One. 2017;12(5):e0178306. https://doi.org/10.1371/journal.pone.0178306.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ng YH, Rome S, Jalabert A, Forterre A, Singh H, Hincks CL, et al. Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation. PLoS One. 2013;8(3):e58502. https://doi.org/10.1371/journal.pone.0058502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ruiz-González I, Xu J, Wang X, Burghardt RC, Dunlap KA, Bazer FW. Exosomes, endogenous retroviruses and toll-like receptors: pregnancy recognition in ewes. Reproduction. 2015;149(3):281–91. https://doi.org/10.1530/rep-14-0538.

    Article  PubMed  Google Scholar 

  13. Hung WT, Hong X, Christenson LK, McGinnis LK. Extracellular vesicles from bovine follicular fluid support cumulus expansion. Biol Reprod. 2015;93(5):117. https://doi.org/10.1095/biolreprod.115.132977.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lopera-Vásquez R, Hamdi M, Fernandez-Fuertes B, Maillo V, Beltrán-Breña P, Calle A, et al. Extracellular vesicles from BOEC in in vitro embryo development and quality. PLoS One. 2016;11(2):e0148083. https://doi.org/10.1371/journal.pone.0148083.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Doyle LM, Wang MZ. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. 2019;8(7). https://doi.org/10.3390/cells8070727.

  16. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495–516. https://doi.org/10.1080/01926230701320337.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yang C, Song G, Lim W. Effects of extracellular vesicles on placentation and pregnancy disorders. Reproduction. 2019;158(5):R189–r96. https://doi.org/10.1530/rep-19-0147.

    Article  CAS  PubMed  Google Scholar 

  18. Elfeky O, Longo S, Lai A, Rice GE, Salomon C. Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. Placenta. 2017;50:60–9. https://doi.org/10.1016/j.placenta.2016.12.020.

    Article  PubMed  Google Scholar 

  19. Burns GW, Brooks KE, Spencer TE. Extracellular vesicles originate from the conceptus and uterus during early pregnancy in sheep. Biol Reprod. 2016;94(3):56. https://doi.org/10.1095/biolreprod.115.134973.

    Article  CAS  PubMed  Google Scholar 

  20. Desrochers LM, Antonyak MA, Cerione RA. Extracellular vesicles: Satellites of information transfer in cancer and stem cell biology. Dev Cell. 2016;37(4):301–9. https://doi.org/10.1016/j.devcel.2016.04.019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bidarimath M, Tayade C. Pregnancy and spontaneous fetal loss: A pig perspective. Mol Reprod Dev. 2017;84(9):856–69. https://doi.org/10.1002/mrd.22847.

    Article  CAS  PubMed  Google Scholar 

  22. Jiang NX, Li XL. The complicated effects of extracellular vesicles and their cargos on embryo implantation. Front Endocrinol (Lausanne). 2021;12:681266. https://doi.org/10.3389/fendo.2021.681266.

    Article  Google Scholar 

  23. Xu R, Greening DW, Zhu HJ, Takahashi N, Simpson RJ. Extracellular vesicle isolation and characterization: toward clinical application. J Clin Invest. 2016;126(4):1152–62. https://doi.org/10.1172/jci81129.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Mobarak H, Rahbarghazi R, Lolicato F, Heidarpour M, Pashazadeh F, Nouri M, et al. Evaluation of the association between exosomal levels and female reproductive system and fertility outcome during aging: a systematic review protocol. Syst Rev. 2019;8(1):293. https://doi.org/10.1186/s13643-019-1228-9.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Xu R, Greening DW, Rai A, Ji H, Simpson RJ. Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct. Methods. 2015;87:11–25. https://doi.org/10.1016/j.ymeth.2015.04.008.

    Article  CAS  PubMed  Google Scholar 

  26. Greening DW, Nguyen HP, Elgass K, Simpson RJ, Salamonsen LA. Human endometrial exosomes contain hormone-specific cargo modulating trophoblast adhesive capacity: Insights into endometrial-embryo interactions. Biol Reprod. 2016;94(2):38. https://doi.org/10.1095/biolreprod.115.134890.

    Article  CAS  PubMed  Google Scholar 

  27. Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2(8):569–79. https://doi.org/10.1038/nri855.

    Article  CAS  PubMed  Google Scholar 

  28. Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30:255–89. https://doi.org/10.1146/annurev-cellbio-101512-122326.

    Article  CAS  PubMed  Google Scholar 

  29. Colombo M, Moita C, van Niel G, Kowal J, Vigneron J, Benaroch P, et al. Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J Cell Sci. 2013;126(Pt 24):5553–65. https://doi.org/10.1242/jcs.128868.

    Article  CAS  PubMed  Google Scholar 

  30. Lane RE, Korbie D, Anderson W, Vaidyanathan R, Trau M. Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing. Sci Rep. 2015;5:7639. https://doi.org/10.1038/srep07639.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteomics. 2010;73(10):1907–20. https://doi.org/10.1016/j.jprot.2010.06.006.

    Article  CAS  PubMed  Google Scholar 

  32. Vyas P, Balakier H, Librach CL. Ultrastructural identification of CD9 positive extracellular vesicles released from human embryos and transported through the zona pellucida. Syst Biol Reprod Med. 2019;65(4):273–80. https://doi.org/10.1080/19396368.2019.1619858.

    Article  PubMed  Google Scholar 

  33. Simon B, Bolumar D, Amadoz A, Jimenez-Almazán J, Valbuena D, Vilella F, et al. Identification and characterization of extracellular vesicles and its DNA cargo secreted during murine embryo development. Genes (Basel). 2020;11(2):203. https://doi.org/10.3390/genes11020203.

    Article  CAS  PubMed Central  Google Scholar 

  34. Battaglia R, Palini S, Vento ME, La Ferlita A, Lo Faro MJ, Caroppo E, et al. Identification of extracellular vesicles and characterization of miRNA expression profiles in human blastocoel fluid. Sci Rep. 2019;9(1):84. https://doi.org/10.1038/s41598-018-36452-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kropp J, Salih SM, Khatib H. Expression of microRNAs in bovine and human pre-implantation embryo culture media. Front Genet. 2014;5:91. https://doi.org/10.3389/fgene.2014.00091.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kropp J, Khatib H. Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development. J Dairy Sci. 2015;98(9):6552–63. https://doi.org/10.3168/jds.2015-9510.

    Article  CAS  PubMed  Google Scholar 

  37. Kropp J, Khatib H. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development. Front Genet. 2015;6:273. https://doi.org/10.3389/fgene.2015.00273.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Bobrie A, Colombo M, Raposo G, Théry C. Exosome secretion: molecular mechanisms and roles in immune responses. Traffic. 2011;12(12):1659–68. https://doi.org/10.1111/j.1600-0854.2011.01225.x.

    Article  CAS  PubMed  Google Scholar 

  39. Saadeldin IM, Oh HJ, Lee BC. Embryonic-maternal cross-talk via exosomes: potential implications. Stem Cells Cloning. 2015;8:103–7. https://doi.org/10.2147/sccaa.S84991.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 2009;19(2):43–51. https://doi.org/10.1016/j.tcb.2008.11.003.

    Article  CAS  PubMed  Google Scholar 

  41. Muralidharan-Chari V, Clancy JW, Sedgwick A, D'Souza-Schorey C. Microvesicles: mediators of extracellular communication during cancer progression. J Cell Sci. 2010;123(Pt 10):1603–11. https://doi.org/10.1242/jcs.064386.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nabhan JF, Hu R, Oh RS, Cohen SN, Lu Q. Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein. Proc Natl Acad Sci U S A. 2012;109(11):4146–51. https://doi.org/10.1073/pnas.1200448109.

    Article  PubMed  PubMed Central  Google Scholar 

  43. D'Souza-Schorey C, Clancy JW. Tumor-derived microvesicles: shedding light on novel microenvironment modulators and prospective cancer biomarkers. Genes Dev. 2012;26(12):1287–99. https://doi.org/10.1101/gad.192351.112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tricarico C, Clancy J, D'Souza-Schorey C. Biology and biogenesis of shed microvesicles. Small GTPases. 2017;8(4):220–32. https://doi.org/10.1080/21541248.2016.1215283.

    Article  CAS  PubMed  Google Scholar 

  45. Muralidharan-Chari V, Clancy J, Plou C, Romao M, Chavrier P, Raposo G, et al. ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles. Curr Biol. 2009;19(22):1875–85. https://doi.org/10.1016/j.cub.2009.09.059.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Xiong Z, Oriss TB, Cavaretta JP, Rosengart MR, Lee JS. Red cell microparticle enumeration: validation of a flow cytometric approach. Vox Sang. 2012;103(1):42–8. https://doi.org/10.1111/j.1423-0410.2011.01577.x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Jorfi S, Inal JM. The role of microvesicles in cancer progression and drug resistance. Biochem Soc Trans. 2013;41(1):293–8. https://doi.org/10.1042/bst20120273.

    Article  CAS  PubMed  Google Scholar 

  48. Clancy JW, Sedgwick A, Rosse C, Muralidharan-Chari V, Raposo G, Method M, et al. Regulated delivery of molecular cargo to invasive tumour-derived microvesicles. Nat Commun. 2015;6:6919. https://doi.org/10.1038/ncomms7919.

    Article  CAS  PubMed  Google Scholar 

  49. Menck K, Scharf C, Bleckmann A, Dyck L, Rost U, Wenzel D, et al. Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN. J Mol Cell Biol. 2015;7(2):143–53. https://doi.org/10.1093/jmcb/mju047.

    Article  CAS  PubMed  Google Scholar 

  50. Atkin-Smith GK, Tixeira R, Paone S, Mathivanan S, Collins C, Liem M, et al. A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure. Nat Commun. 2015;6:7439. https://doi.org/10.1038/ncomms8439.

    Article  PubMed  Google Scholar 

  51. Jiang L, Paone S, Caruso S, Atkin-Smith GK, Phan TK, Hulett MD, et al. Determining the contents and cell origins of apoptotic bodies by flow cytometry. Sci Rep. 2017;7(1):14444. https://doi.org/10.1038/s41598-017-14305-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol. 2001;3(4):339–45. https://doi.org/10.1038/35070009.

    Article  CAS  PubMed  Google Scholar 

  53. Gregory CD, Dransfield I. Apoptotic tumor cell-derived extracellular vesicles as important regulators of the onco-regenerative niche. Front Immunol. 2018;9:1111. https://doi.org/10.3389/fimmu.2018.01111.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Aoki K, Satoi S, Harada S, Uchida S, Iwasa Y, Ikenouchi J. Coordinated changes in cell membrane and cytoplasm during maturation of apoptotic bleb. Mol Biol Cell. 2020;31(8):833–44. https://doi.org/10.1091/mbc.E19-12-0691.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Mills JC, Stone NL, Erhardt J, Pittman RN. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol. 1998;140(3):627–36. https://doi.org/10.1083/jcb.140.3.627.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zirngibl M, Fürnrohr BG, Janko C, Munoz LE, Voll RE, Gregory CD, et al. Loading of nuclear autoantigens prototypically recognized by systemic lupus erythematosus sera into late apoptotic vesicles requires intact microtubules and myosin light chain kinase activity. Clin Exp Immunol. 2015;179(1):39–49. https://doi.org/10.1111/cei.12342.

    Article  CAS  PubMed  Google Scholar 

  57. Orlando KA, Stone NL, Pittman RN. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells. Exp Cell Res. 2006;312(1):5–15. https://doi.org/10.1016/j.yexcr.2005.09.012.

    Article  CAS  PubMed  Google Scholar 

  58. Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, et al. The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. Sci Transl Med. 2015;7(318):318ra200. https://doi.org/10.1126/scitranslmed.aac9816.

    Article  CAS  PubMed  Google Scholar 

  59. Karpman D, Ståhl AL, Arvidsson I. Extracellular vesicles in renal disease. Nat Rev Nephrol. 2017;13(9):545–62. https://doi.org/10.1038/nrneph.2017.98.

    Article  CAS  PubMed  Google Scholar 

  60. Taylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol. 2008;9(3):231–41. https://doi.org/10.1038/nrm2312.

    Article  CAS  PubMed  Google Scholar 

  61. Hauser P, Wang S, Didenko VV. Apoptotic bodies: Selective detection in extracellular vesicles. Methods Mol Biol. 2017;1554:193–200. https://doi.org/10.1007/978-1-4939-6759-9_12.

    Article  CAS  PubMed  Google Scholar 

  62. van Engeland M, Kuijpers HJ, Ramaekers FC, Reutelingsperger CP, Schutte B. Plasma membrane alterations and cytoskeletal changes in apoptosis. Exp Cell Res. 1997;235(2):421–30. https://doi.org/10.1006/excr.1997.3738.

    Article  PubMed  Google Scholar 

  63. Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, et al. Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J Extracell Vesicles. 2014;3:25011. https://doi.org/10.3402/jev.v3.25011.

    Article  PubMed  Google Scholar 

  64. Lavoie C, Lanoix J, Kan FW, Paiement J. Cell-free assembly of rough and smooth endoplasmic reticulum. J Cell Sci. 1996;109(Pt 6):1415–25. https://doi.org/10.1242/jcs.109.6.1415.

    Article  CAS  PubMed  Google Scholar 

  65. Savill J, Dransfield I, Gregory C, Haslett C. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol. 2002;2(12):965–75. https://doi.org/10.1038/nri957.

    Article  CAS  PubMed  Google Scholar 

  66. Abas L, Luschnig C. Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. Anal Biochem. 2010;401(2):217–27. https://doi.org/10.1016/j.ab.2010.02.030.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Caruso S, Poon IKH. Apoptotic cell-derived extracellular vesicles: More than just debris. Front Immunol. 2018;9:1486. https://doi.org/10.3389/fimmu.2018.01486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Collino F, Bruno S, Incarnato D, Dettori D, Neri F, Provero P, et al. AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs. J Am Soc Nephrol. 2015;26(10):2349–60. https://doi.org/10.1681/asn.2014070710.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Brock CK, Wallin ST, Ruiz OE, Samms KM, Mandal A, Sumner EA, et al. Stem cell proliferation is induced by apoptotic bodies from dying cells during epithelial tissue maintenance. Nat Commun. 2019;10(1):1044. https://doi.org/10.1038/s41467-019-09010-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Jiang JX, Mikami K, Venugopal S, Li Y, Török NJ. Apoptotic body engulfment by hepatic stellate cells promotes their survival by the JAK/STAT and Akt/NF-kappaB-dependent pathways. J Hepatol. 2009;51(1):139–48. https://doi.org/10.1016/j.jhep.2009.03.024.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Hristov M, Erl W, Linder S, Weber PC. Apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro. Blood. 2004;104(9):2761–6. https://doi.org/10.1182/blood-2003-10-3614.

    Article  CAS  PubMed  Google Scholar 

  72. Tyukavin AI, Belostotskaya GB, Golovanova TA, Galagudza MM, Zakharov EA, Burkova NV, et al. Stimulation of proliferation and differentiation of rat resident myocardial cells with apoptotic bodies of cardiomyocytes. Bull Exp Biol Med. 2015;159(1):138–41. https://doi.org/10.1007/s10517-015-2909-6.

    Article  CAS  PubMed  Google Scholar 

  73. Zernecke A, Bidzhekov K, Noels H, Shagdarsuren E, Gan L, Denecke B, et al. Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal. 2009;2(100):ra81. https://doi.org/10.1126/scisignal.2000610.

    Article  PubMed  Google Scholar 

  74. Zhu Z, Zhang D, Lee H, Menon AA, Wu J, Hu K, et al. Macrophage-derived apoptotic bodies promote the proliferation of the recipient cells via shuttling microRNA-221/222. J Leukoc Biol. 2017;101(6):1349–59. https://doi.org/10.1189/jlb.3A1116-483R.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Gurunathan S, David D, Gerst JE. Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast. Embo j. 2002;21(4):602–14. https://doi.org/10.1093/emboj/21.4.602.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006;30(1):3.22.1-3..9. https://doi.org/10.1002/0471143030.cb0322s30.

  77. Yuana Y, Levels J, Grootemaat A, Sturk A, Nieuwland R. Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation. J Extracell Vesicles. 2014;3:1. https://doi.org/10.3402/jev.v3.23262.

    Article  Google Scholar 

  78. Sódar BW, Kittel Á, Pálóczi K, Vukman KV, Osteikoetxea X, Szabó-Taylor K, et al. Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci Rep. 2016;6:24316. https://doi.org/10.1038/srep24316.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Karimi N, Cvjetkovic A, Jang SC, Crescitelli R, Hosseinpour Feizi MA, Nieuwland R, et al. Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins. Cell Mol Life Sci. 2018;75(15):2873–86. https://doi.org/10.1007/s00018-018-2773-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Lamparski HG, Metha-Damani A, Yao JY, Patel S, Hsu DH, Ruegg C, et al. Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods. 2002;270(2):211–26. https://doi.org/10.1016/s0022-1759(02)00330-7.

    Article  CAS  PubMed  Google Scholar 

  81. Cantin R, Diou J, Bélanger D, Tremblay AM, Gilbert C. Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol Methods. 2008;338(1-2):21–30. https://doi.org/10.1016/j.jim.2008.07.007.

    Article  CAS  PubMed  Google Scholar 

  82. Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P. Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med. 2011;9:86. https://doi.org/10.1186/1479-5876-9-86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Greening DW, Xu R, Ji H, Tauro BJ, Simpson RJ. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. Methods Mol Biol. 2015;1295:179–209. https://doi.org/10.1007/978-1-4939-2550-6_15.

    Article  CAS  PubMed  Google Scholar 

  84. Böing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R. Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles. 2014;3:1. https://doi.org/10.3402/jev.v3.23430.

    Article  Google Scholar 

  85. Stranska R, Gysbrechts L, Wouters J, Vermeersch P, Bloch K, Dierickx D, et al. Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med. 2018;16(1):1. https://doi.org/10.1186/s12967-017-1374-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods. 2012;56(2):293–304. https://doi.org/10.1016/j.ymeth.2012.01.002.

    Article  CAS  PubMed  Google Scholar 

  87. Balaj L, Atai NA, Chen W, Mu D, Tannous BA, Breakefield XO, et al. Heparin affinity purification of extracellular vesicles. Sci Rep. 2015;5:10266. https://doi.org/10.1038/srep10266.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Oksvold MP, Neurauter A, Pedersen KW. Magnetic bead-based isolation of exosomes. Methods Mol Biol. 2015;1218:465–81. https://doi.org/10.1007/978-1-4939-1538-5_27.

    Article  CAS  PubMed  Google Scholar 

  89. Kosanović M, Milutinović B, Goč S, Mitić N, Janković M. Ion-exchange chromatography purification of extracellular vesicles. Biotechniques. 2017;63(2):65–71. https://doi.org/10.2144/000114575.

    Article  CAS  PubMed  Google Scholar 

  90. Heath N, Grant L, De Oliveira TM, Rowlinson R, Osteikoetxea X, Dekker N, et al. Rapid isolation and enrichment of extracellular vesicle preparations using anion exchange chromatography. Sci Rep. 2018;8(1):5730. https://doi.org/10.1038/s41598-018-24163-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Ludwig AK, De Miroschedji K, Doeppner TR, Börger V, Ruesing J, Rebmann V, et al. Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales. J Extracell Vesicles. 2018;7(1):1528109. https://doi.org/10.1080/20013078.2018.1528109.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Ding M, Wang C, Lu X, Zhang C, Zhou Z, Chen X, et al. Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling. Anal Bioanal Chem. 2018;410(16):3805–14. https://doi.org/10.1007/s00216-018-1052-4.

    Article  CAS  PubMed  Google Scholar 

  93. Paolini L, Zendrini A, Di Noto G, Busatto S, Lottini E, Radeghieri A, et al. Residual matrix from different separation techniques impacts exosome biological activity. Sci Rep. 2016;6:23550. https://doi.org/10.1038/srep23550.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Macías M, Rebmann V, Mateos B, Varo N, Perez-Gracia JL, Alegre E, et al. Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis. Clin Chem Lab Med. 2019;57(10):1539–45. https://doi.org/10.1515/cclm-2018-1297.

    Article  CAS  PubMed  Google Scholar 

  95. Salmond N, Williams KC. Isolation and characterization of extracellular vesicles for clinical applications in cancer – time for standardization? Nanoscale Adv. 2021;3(7):1830-52. https://doi.org/10.1039/D0NA00676A.

  96. Vaidyanathan R, Naghibosadat M, Rauf S, Korbie D, Carrascosa LG, Shiddiky MJ, et al. Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing. Anal Chem. 2014;86(22):11125–32. https://doi.org/10.1021/ac502082b.

    Article  CAS  PubMed  Google Scholar 

  97. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183(3):1161–72. https://doi.org/10.1084/jem.183.3.1161.

    Article  CAS  PubMed  Google Scholar 

  98. Yuana Y, Oosterkamp TH, Bahatyrova S, Ashcroft B, Garcia Rodriguez P, Bertina RM, et al. Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles. J Thromb Haemost. 2010;8(2):315–23. https://doi.org/10.1111/j.1538-7836.2009.03654.x.

    Article  CAS  PubMed  Google Scholar 

  99. Hardij J, Cecchet F, Berquand A, Gheldof D, Chatelain C, Mullier F, et al. Characterisation of tissue factor-bearing extracellular vesicles with AFM: comparison of air-tapping-mode AFM and liquid Peak Force AFM. J Extracell Vesicles. 2013;2:1. https://doi.org/10.3402/jev.v2i0.21045.

    Article  CAS  Google Scholar 

  100. Filipe V, Hawe A, Jiskoot W. Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res. 2010;27(5):796–810. https://doi.org/10.1007/s11095-010-0073-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen TG, Nieuwland R. Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost. 2010;8(12):2596–607. https://doi.org/10.1111/j.1538-7836.2010.04074.x.

    Article  PubMed  Google Scholar 

  102. Momen-Heravi F, Balaj L, Alian S, Tigges J, Toxavidis V, Ericsson M, et al. Alternative methods for characterization of extracellular vesicles. Front Physiol. 2012;3:354. https://doi.org/10.3389/fphys.2012.00354.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. van der Vlist EJ, Nolte-'t Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protoc. 2012;7(7):1311–26. https://doi.org/10.1038/nprot.2012.065.

    Article  CAS  PubMed  Google Scholar 

  104. Nolte-'t Hoen EN, Buermans HP, Waasdorp M, Stoorvogel W, Wauben MH, 't Hoen PA. Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions. Nucleic Acids Res. 2012;40(18):9272–85. https://doi.org/10.1093/nar/gks658.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Simon AL, Kiehl M, Fischer E, Proctor JG, Bush MR, Givens C, et al. Pregnancy outcomes from more than 1,800 in vitro fertilization cycles with the use of 24-chromosome single-nucleotide polymorphism-based preimplantation genetic testing for aneuploidy. Fertil Steril. 2018;110(1):113–21. https://doi.org/10.1016/j.fertnstert.2018.03.026.

    Article  PubMed  Google Scholar 

  106. Saadeldin IM, Kim SJ, Choi YB, Lee BC. Improvement of cloned embryos development by co-culturing with parthenotes: a possible role of exosomes/microvesicles for embryos paracrine communication. Cell Reprogram. 2014;16(3):223–34. https://doi.org/10.1089/cell.2014.0003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Saadeldin IM, Abdelfattah-Hassan A, Swelum AA. Feeder cell type affects the growth of in vitro cultured bovine trophoblast cells. Biomed Res Int. 2017;2017:1061589. https://doi.org/10.1155/2017/1061589.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Dissanayake K, Nõmm M, Lättekivi F, Ressaissi Y, Godakumara K, Lavrits A, et al. Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers. Theriogenology. 2020;149:104–16. https://doi.org/10.1016/j.theriogenology.2020.03.008.

    Article  CAS  PubMed  Google Scholar 

  109. Burkova EE, Grigor'eva AE, Bulgakov DV, Dmitrenok PS, Vlassov VV, Ryabchikova EI, et al. Extra purified exosomes from human placenta contain an unpredictable small number of different major proteins. Int J Mol Sci. 2019;20(10). https://doi.org/10.3390/ijms20102434.

  110. Szatanek R, Baran J, Siedlar M, Baj-Krzyworzeka M. Isolation of extracellular vesicles: Determining the correct approach (Review). Int J Mol Med. 2015;36(1):11–7. https://doi.org/10.3892/ijmm.2015.2194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Rejraji H, Sion B, Prensier G, Carreras M, Motta C, Frenoux JM, et al. Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation. Biol Reprod. 2006;74(6):1104–13. https://doi.org/10.1095/biolreprod.105.049304.

    Article  CAS  PubMed  Google Scholar 

  112. Uszyński W, Uszyński M, Zekanowska E. Thrombin activatable fibrinolysis inhibitor (TAFI) in human amniotic fluid. A preliminary study. Thromb Res. 2007;119(2):241–5. https://doi.org/10.1016/j.thromres.2006.01.012.

    Article  CAS  PubMed  Google Scholar 

  113. Flori F, Secciani F, Capone A, Paccagnini E, Caruso S, Ricci MG, et al. Menstrual cycle-related sialidase activity of the female cervical mucus is associated with exosome-like vesicles. Fertil Steril. 2007;88(4 Suppl):1212–9. https://doi.org/10.1016/j.fertnstert.2007.01.209.

    Article  CAS  PubMed  Google Scholar 

  114. Uszyński W, Zekanowska E, Uszyński M, Zyliński A, Kuczyński J. New observations on procoagulant properties of amniotic fluid: microparticles (MPs) and tissue factor-bearing MPs (MPs-TF), comparison with maternal blood plasma. Thromb Res. 2013;132(6):757–60. https://doi.org/10.1016/j.thromres.2013.10.001.

    Article  CAS  PubMed  Google Scholar 

  115. Santonocito M, Vento M, Guglielmino MR, Battaglia R, Wahlgren J, Ragusa M, et al. Molecular characterization of exosomes and their microRNA cargo in human follicular fluid: bioinformatic analysis reveals that exosomal microRNAs control pathways involved in follicular maturation. Fertil Steril. 2014;102(6):1751–61.e1. https://doi.org/10.1016/j.fertnstert.2014.08.005.

    Article  CAS  PubMed  Google Scholar 

  116. Al-Dossary AA, Bathala P, Caplan JL, Martin-DeLeon PA. Oviductosome-sperm membrane interaction in cargo delivery: Detection of fusion and underlying molecular players using three-dimensional super-resolution structured illumination microscopy (SR-SIM). J Biol Chem. 2015;290(29):17710–23. https://doi.org/10.1074/jbc.M114.633156.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Foster BP, Balassa T, Benen TD, Dominovic M, Elmadjian GK, Florova V, et al. Extracellular vesicles in blood, milk and body fluids of the female and male urogenital tract and with special regard to reproduction. Crit Rev Clin Lab Sci. 2016;53(6):379–95. https://doi.org/10.1080/10408363.2016.1190682.

    Article  CAS  PubMed  Google Scholar 

  118. Franz C, Böing AN, Montag M, Strowitzki T, Markert UR, Mastenbroek S, et al. Extracellular vesicles in human follicular fluid do not promote coagulation. Reprod Biomed Online. 2016;33(5):652–5. https://doi.org/10.1016/j.rbmo.2016.08.005.

    Article  CAS  PubMed  Google Scholar 

  119. Machtinger R, Rodosthenous RS, Adir M, Mansour A, Racowsky C, Baccarelli AA, et al. Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: an exploratory study. J Assist Reprod Genet. 2017;34(4):525–33. https://doi.org/10.1007/s10815-017-0876-8.

    Article  PubMed  PubMed Central  Google Scholar 

  120. Tannetta D, Dragovic R, Alyahyaei Z, Southcombe J. Extracellular vesicles and reproduction-promotion of successful pregnancy. Cell Mol Immunol. 2014;11(6):548–63. https://doi.org/10.1038/cmi.2014.42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Hell L, Wisgrill L, Ay C, Spittler A, Schwameis M, Jilma B, et al. Procoagulant extracellular vesicles in amniotic fluid. Transl Res. 2017;184:12–20.e1. https://doi.org/10.1016/j.trsl.2017.01.003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Karlsson O, Rodosthenous RS, Jara C, Brennan KJ, Wright RO, Baccarelli AA, et al. Detection of long non-coding RNAs in human breastmilk extracellular vesicles: Implications for early child development. Epigenetics. 2016;11(10):721–9. https://doi.org/10.1080/15592294.2016.1216285.

    Article  PubMed  PubMed Central  Google Scholar 

  123. Wang X. Isolation of extracellular vesicles from breast milk. Methods Mol Biol. 1660;2017:351–3. https://doi.org/10.1007/978-1-4939-7253-1_28.

    Article  CAS  Google Scholar 

  124. Pavani KC, Hendrix A, Van Den Broeck W, Couck L, Szymanska K, Lin X, et al. Isolation and characterization of functionally active extracellular vesicles from culture medium conditioned by bovine embryos in vitro. Int J Mol Sci. 2018;20(1):38. https://doi.org/10.3390/ijms20010038.

    Article  CAS  PubMed Central  Google Scholar 

  125. Sohel MM, Hoelker M, Noferesti SS, Salilew-Wondim D, Tholen E, Looft C, et al. Exosomal and non-exosomal transport of extra-cellular microRNAs in follicular fluid: Implications for bovine oocyte developmental competence. PLoS One. 2013;8(11):e78505. https://doi.org/10.1371/journal.pone.0078505.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Almiñana C, Caballero I, Heath PR, Maleki-Dizaji S, Parrilla I, Cuello C, et al. The battle of the sexes starts in the oviduct: modulation of oviductal transcriptome by X and Y-bearing spermatozoa. BMC Genomics. 2014;15(1):293. https://doi.org/10.1186/1471-2164-15-293.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Tsochandaridis M, Nasca L, Toga C, Levy-Mozziconacci A. Circulating microRNAs as clinical biomarkers in the predictions of pregnancy complications. Biomed Res Int. 2015;2015:294954. https://doi.org/10.1155/2015/294954.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Koh YQ, Peiris HN, Vaswani K, Reed S, Rice GE, Salomon C, et al. Characterization of exosomal release in bovine endometrial intercaruncular stromal cells. Reprod Biol Endocrinol. 2016;14(1):78. https://doi.org/10.1186/s12958-016-0207-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Prattichizzo F, Micolucci L, Cricca M, De Carolis S, Mensà E, Ceriello A, et al. Exosome-based immunomodulation during aging: A nano-perspective on inflamm-aging. Mech Ageing Dev. 2017;168:44–53. https://doi.org/10.1016/j.mad.2017.02.008.

    Article  CAS  PubMed  Google Scholar 

  130. Wang W, Wang L, Ruan L, Oh J, Dong X, Zhuge Q, et al. Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance. Faseb J. 2018;32(11):fj201800059R. https://doi.org/10.1096/fj.201800059R.

    Article  Google Scholar 

  131. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–9. https://doi.org/10.1038/ncb1596.

    Article  CAS  PubMed  Google Scholar 

  132. Hackl M, Brunner S, Fortschegger K, Schreiner C, Micutkova L, Mück C, et al. miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging. Aging Cell. 2010;9(2):291–6. https://doi.org/10.1111/j.1474-9726.2010.00549.x.

    Article  CAS  PubMed  Google Scholar 

  133. Li X, Khanna A, Li N, Wang E. Circulatory miR34a as an RNAbased, noninvasive biomarker for brain aging. Aging (Albany NY). 2011;3(10):985–1002. https://doi.org/10.18632/aging.100371.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Peng A, Rotman Z, Deng PY, Klyachko VA. Differential motion dynamics of synaptic vesicles undergoing spontaneous and activity-evoked endocytosis. Neuron. 2012;73(6):1108–15. https://doi.org/10.1016/j.neuron.2012.01.023.

    Article  CAS  PubMed  Google Scholar 

  135. Baixauli F, López-Otín C, Mittelbrunn M. Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness. Front Immunol. 2014;5:403. https://doi.org/10.3389/fimmu.2014.00403.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Adam S, Elfeky O, Kinhal V, Dutta S, Lai A, Jayabalan N, et al. Review: Fetal-maternal communication via extracellular vesicles - Implications for complications of pregnancies. Placenta. 2017;54:83–8. https://doi.org/10.1016/j.placenta.2016.12.001.

    Article  CAS  PubMed  Google Scholar 

  137. Salomon C, Yee SW, Mitchell MD, Rice GE. The possible role of extravillous trophoblast-derived exosomes on the uterine spiral arterial remodeling under both normal and pathological conditions. Biomed Res Int. 2014;2014:693157. https://doi.org/10.1155/2014/693157.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Zhang M, Ouyang H, Xia G. The signal pathway of gonadotrophins-induced mammalian oocyte meiotic resumption. Mol Hum Reprod. 2009;15(7):399–409. https://doi.org/10.1093/molehr/gap031.

    Article  CAS  PubMed  Google Scholar 

  139. da Silveira JC, de Andrade GM, Nogueira MF, Meirelles FV, Perecin F. Involvement of miRNAs and cell-secreted vesicles in mammalian ovarian antral follicle development. Reprod Sci. 2015;22(12):1474–83. https://doi.org/10.1177/1933719115574344.

    Article  CAS  PubMed  Google Scholar 

  140. Nakamura K, Kusama K, Bai R, Sakurai T, Isuzugawa K, Godkin JD, et al. Induction of IFNT-stimulated genes by conceptus-derived exosomes during the attachment period. PLoS One. 2016;11(6):e0158278. https://doi.org/10.1371/journal.pone.0158278.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Machtinger R, Laurent LC, Baccarelli AA. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum Reprod Update. 2016;22(2):182–93. https://doi.org/10.1093/humupd/dmv055.

    Article  CAS  PubMed  Google Scholar 

  142. Tang L, He G, Liu X, Xu W. Progress in the understanding of the etiology and predictability of fetal growth restriction. Reproduction. 2017;153(6):R227–r40. https://doi.org/10.1530/rep-16-0287.

    Article  CAS  PubMed  Google Scholar 

  143. Menon R, Richardson LS. Preterm prelabor rupture of the membranes: A disease of the fetal membranes. Semin Perinatol. 2017;41(7):409–19. https://doi.org/10.1053/j.semperi.2017.07.012.

    Article  PubMed  PubMed Central  Google Scholar 

  144. Mitchell MD, Peiris HN, Kobayashi M, Koh YQ, Duncombe G, Illanes SE, et al. Placental exosomes in normal and complicated pregnancy. Am J Obstet Gynecol. 2015;213(4 Suppl):S173–81. https://doi.org/10.1016/j.ajog.2015.07.001.

    Article  CAS  PubMed  Google Scholar 

  145. Moro T, Tinsley G, Bianco A, Marcolin G, Pacelli QF, Battaglia G, et al. Effects of eight weeks of time-restricted feeding (16/8) on basal metabolism, maximal strength, body composition, inflammation, and cardiovascular risk factors in resistance-trained males. J Transl Med. 2016;14(1):290. https://doi.org/10.1186/s12967-016-1044-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  146. Nair S, Jayabalan N, Guanzon D, Palma C, Scholz-Romero K, Elfeky O, et al. Human placental exosomes in gestational diabetes mellitus carry a specific set of miRNAs associated with skeletal muscle insulin sensitivity. Clin Sci (Lond). 2018;132(22):2451–67. https://doi.org/10.1042/cs20180487.

    Article  CAS  PubMed  Google Scholar 

  147. Jayabalan N, Lai A, Nair S, Guanzon D, Scholz-Romero K, Palma C, et al. Quantitative proteomics by SWATH-MS suggest an association between circulating exosomes and maternal metabolic changes in gestational diabetes mellitus. Proteomics. 2019;19(1-2):e1800164. https://doi.org/10.1002/pmic.201800164.

    Article  CAS  PubMed  Google Scholar 

  148. Salomon C, Guanzon D, Scholz-Romero K, Longo S, Correa P, Illanes SE, et al. Placental exosomes as early biomarker of preeclampsia: Potential role of exosomal microRNAs across gestation. J Clin Endocrinol Metab. 2017;102(9):3182–94. https://doi.org/10.1210/jc.2017-00672.

    Article  PubMed  Google Scholar 

  149. Germain SJ, Sacks GP, Sooranna SR, Sargent IL, Redman CW. Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles. J Immunol. 2007;178(9):5949–56. https://doi.org/10.4049/jimmunol.178.9.5949.

    Article  CAS  PubMed  Google Scholar 

  150. Hedlund M, Stenqvist AC, Nagaeva O, Kjellberg L, Wulff M, Baranov V, et al. Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function. J Immunol. 2009;183(1):340–51. https://doi.org/10.4049/jimmunol.0803477.

    Article  CAS  PubMed  Google Scholar 

  151. Kshirsagar SK, Alam SM, Jasti S, Hodes H, Nauser T, Gilliam M, et al. Immunomodulatory molecules are released from the first trimester and term placenta via exosomes. Placenta. 2012;33(12):982–90. https://doi.org/10.1016/j.placenta.2012.10.005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Stenqvist AC, Nagaeva O, Baranov V, Mincheva-Nilsson L. Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus. J Immunol. 2013;191(11):5515–23. https://doi.org/10.4049/jimmunol.1301885.

    Article  CAS  PubMed  Google Scholar 

  153. Varga Z, Yuana Y, Grootemaat AE, van der Pol E, Gollwitzer C, Krumrey M, et al. Towards traceable size determination of extracellular vesicles. J Extracell Vesicles. 2014;3:1. https://doi.org/10.3402/jev.v3.23298.

    Article  CAS  Google Scholar 

  154. Southcombe J, Tannetta D, Redman C, Sargent I. The immunomodulatory role of syncytiotrophoblast microvesicles. PLoS One. 2011;6(5):e20245. https://doi.org/10.1371/journal.pone.0020245.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Holder BS, Tower CL, Jones CJ, Aplin JD, Abrahams VM. Heightened pro-inflammatory effect of preeclamptic placental microvesicles on peripheral blood immune cells in humans. Biol Reprod. 2012;86(4):103. https://doi.org/10.1095/biolreprod.111.097014.

    Article  CAS  PubMed  Google Scholar 

  156. Atay S, Gercel-Taylor C, Taylor DD. Human trophoblast-derived exosomal fibronectin induces pro-inflammatory IL-1β production by macrophages. Am J Reprod Immunol. 2011;66(4):259–69. https://doi.org/10.1111/j.1600-0897.2011.00995.x.

    Article  CAS  PubMed  Google Scholar 

  157. Atay S, Gercel-Taylor C, Suttles J, Mor G, Taylor DD. Trophoblast-derived exosomes mediate monocyte recruitment and differentiation. Am J Reprod Immunol. 2011;65(1):65–77. https://doi.org/10.1111/j.1600-0897.2010.00880.x.

    Article  CAS  PubMed  Google Scholar 

  158. Wang Z, Wang P, Wang Z, Qin Z, Xiu X, Xu D, et al. MiRNA-548c-5p downregulates inflammatory response in preeclampsia via targeting PTPRO. J Cell Physiol. 2019;234(7):11149–55. https://doi.org/10.1002/jcp.27758.

    Article  CAS  PubMed  Google Scholar 

  159. Sheller-Miller S, Trivedi J, Yellon SM, Menon R. Exosomes cause preterm birth in mice: evidence for paracrine signaling in pregnancy. Sci Rep. 2019;9(1):608. https://doi.org/10.1038/s41598-018-37002-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Han C, Han L, Huang P, Chen Y, Wang Y, Xue F. Syncytiotrophoblast-derived extracellular vesicles in pathophysiology of preeclampsia. Front Physiol. 2019;10:1236. https://doi.org/10.3389/fphys.2019.01236.

    Article  PubMed  PubMed Central  Google Scholar 

  161. Salomon C, Scholz-Romero K, Sarker S, Sweeney E, Kobayashi M, Correa P, et al. Gestational diabetes mellitus is associated with changes in the concentration and bioactivity of placenta-derived exosomes in maternal circulation across gestation. Diabetes. 2016;65(3):598–609. https://doi.org/10.2337/db15-0966.

    Article  CAS  PubMed  Google Scholar 

  162. Burnett LA, Nowak RA. Exosomes mediate embryo and maternal interactions at implantation and during pregnancy. Front Biosci (Schol Ed). 2016;8:79–96. https://doi.org/10.2741/s448.

    Article  Google Scholar 

  163. Chiarello DI, Salsoso R, Toledo F, Mate A, Vázquez CM, Sobrevia L. Foetoplacental communication via extracellular vesicles in normal pregnancy and preeclampsia. Mol Aspects Med. 2018;60:69–80. https://doi.org/10.1016/j.mam.2017.12.002.

    Article  PubMed  Google Scholar 

  164. Salomon C, Rice GE. Role of exosomes in placental homeostasis and pregnancy disorders. Prog Mol Biol Transl Sci. 2017;145:163–79 https://doi.org/10.1016/bs.pmbts.2016.12.006.

    Article  CAS  Google Scholar 

  165. Kurian NK, Modi D. Extracellular vesicle mediated embryo-endometrial cross talk during implantation and in pregnancy. J Assist Reprod Genet. 2019;36(2):189–98. https://doi.org/10.1007/s10815-018-1343-x.

    Article  PubMed  Google Scholar 

  166. Krawczynski K, Bauersachs S, Reliszko ZP, Graf A, Kaczmarek MM. Expression of microRNAs and isomiRs in the porcine endometrium: implications for gene regulation at the maternal-conceptus interface. BMC Genomics. 2015;16:906. https://doi.org/10.1186/s12864-015-2172-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Liu W, Niu Z, Li Q, Pang RTK, Chiu PCN, Yeung WS-B. MicroRNA and embryo implantation. Am J Reprod Immunol. 2016;75(3):263–71. https://doi.org/10.1111/aji.12470.

    Article  CAS  PubMed  Google Scholar 

  168. Mincheva-Nilsson L, Baranov V. Placenta-derived exosomes and syncytiotrophoblast microparticles and their role in human reproduction: immune modulation for pregnancy success. Am J Reprod Immunol. 2014;72(5):440–57. https://doi.org/10.1111/aji.12311.

    Article  CAS  PubMed  Google Scholar 

  169. Nair S, Salomon C. Extracellular vesicles and their immunomodulatory functions in pregnancy. Semin Immunopathol. 2018;40(5):425–37. https://doi.org/10.1007/s00281-018-0680-2.

    Article  CAS  PubMed  Google Scholar 

  170. Zhao ST, Wang CZ. Regulatory T cells and asthma. J Zhejiang Univ Sci B. 2018;19(9):663–73. https://doi.org/10.1631/jzus.B1700346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  171. Holder B, Jones T, Sancho Shimizu V, Rice TF, Donaldson B, Bouqueau M, et al. Macrophage exosomes induce placental inflammatory cytokines: A novel mode of maternal-placental messaging. Traffic. 2016;17(2):168–78. https://doi.org/10.1111/tra.12352.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Delorme-Axford E, Donker RB, Mouillet JF, Chu T, Bayer A, Ouyang Y, et al. Human placental trophoblasts confer viral resistance to recipient cells. Proc Natl Acad Sci U S A. 2013;110(29):12048–53. https://doi.org/10.1073/pnas.1304718110.

    Article  PubMed  PubMed Central  Google Scholar 

  173. Jayabalan N, Lai A, Ormazabal V, Adam S, Guanzon D, Palma C, et al. Adipose tissue exosomal proteomic profile reveals a role on placenta glucose metabolism in gestational diabetes mellitus. J Clin Endocrinol Metab. 2019;104(5):1735–52. https://doi.org/10.1210/jc.2018-01599.

    Article  PubMed  Google Scholar 

  174. Truong G, Guanzon D, Kinhal V, Elfeky O, Lai A, Longo S, et al. Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - Liquid biopsies for monitoring complications of pregnancy. PLoS One. 2017;12(3):e0174514. https://doi.org/10.1371/journal.pone.0174514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  175. Ospina-Prieto S, Chaiwangyen W, Herrmann J, Groten T, Schleussner E, Markert UR, et al. MicroRNA-141 is upregulated in preeclamptic placentae and regulates trophoblast invasion and intercellular communication. Transl Res. 2016;172:61–72. https://doi.org/10.1016/j.trsl.2016.02.012.

    Article  CAS  PubMed  Google Scholar 

  176. Shao J, Zhao M, Tong M, Wei J, Wise MR, Stone P, et al. Increased levels of HMGB1 in trophoblastic debris may contribute to preeclampsia. Reproduction. 2016;152(6):775–84. https://doi.org/10.1530/rep-16-0083.

    Article  CAS  PubMed  Google Scholar 

  177. Salazar Garcia MD, Mobley Y, Henson J, Davies M, Skariah A, Dambaeva S, et al. Early pregnancy immune biomarkers in peripheral blood may predict preeclampsia. J Reprod Immunol. 2018;125:25–31. https://doi.org/10.1016/j.jri.2017.10.048.

    Article  CAS  PubMed  Google Scholar 

  178. Menon R, Dixon CL, Sheller-Miller S, Fortunato SJ, Saade GR, Palma C, et al. Quantitative proteomics by SWATH-MS of maternal plasma exosomes determine pathways associated with term and preterm birth. Endocrinology. 2019;160(3):639–50. https://doi.org/10.1210/en.2018-00820.

    Article  PubMed  PubMed Central  Google Scholar 

  179. van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213–28. https://doi.org/10.1038/nrm.2017.125.

    Article  CAS  PubMed  Google Scholar 

  180. Chaput N, Théry C. Exosomes: immune properties and potential clinical implementations. Semin Immunopathol. 2011;33(5):419–40. https://doi.org/10.1007/s00281-010-0233-9.

    Article  CAS  PubMed  Google Scholar 

  181. Burns G, Brooks K, Wildung M, Navakanitworakul R, Christenson LK, Spencer TE. Extracellular vesicles in luminal fluid of the ovine uterus. PLoS One. 2014;9(3):e90913. https://doi.org/10.1371/journal.pone.0090913.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  182. Vilella F, Moreno-Moya JM, Balaguer N, Grasso A, Herrero M, Martínez S, et al. Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome. Development. 2015;142(18):3210–21. https://doi.org/10.1242/dev.124289.

    Article  CAS  PubMed  Google Scholar 

  183. Balaguer N, Moreno I, Herrero M, González M, Simón C, Vilella F. Heterogeneous nuclear ribonucleoprotein C1 may control miR-30d levels in endometrial exosomes affecting early embryo implantation. Mol Hum Reprod. 2018;24(8):411–25. https://doi.org/10.1093/molehr/gay026.

    Article  CAS  PubMed  Google Scholar 

  184. Balaguer N, Moreno I, Herrero M, Gonzáléz-Monfort M, Vilella F, Simón C. MicroRNA-30d deficiency during preconception affects endometrial receptivity by decreasing implantation rates and impairing fetal growth. Am J Obstet Gynecol. 2019;221(1):46.e1–e16. https://doi.org/10.1016/j.ajog.2019.02.047.

    Article  CAS  Google Scholar 

  185. Qu P, Qing S, Liu R, Qin H, Wang W, Qiao F, et al. Effects of embryo-derived exosomes on the development of bovine cloned embryos. PLoS One. 2017;12(3):e0174535. https://doi.org/10.1371/journal.pone.0174535.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  186. Kusama K, Nakamura K, Bai R, Nagaoka K, Sakurai T, Imakawa K. Intrauterine exosomes are required for bovine conceptus implantation. Biochem Biophys Res Commun. 2018;495(1):1370–5. https://doi.org/10.1016/j.bbrc.2017.11.176.

    Article  CAS  PubMed  Google Scholar 

  187. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–97. https://doi.org/10.1016/s0092-8674(04)00045-5.

    Article  CAS  PubMed  Google Scholar 

  188. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008;105(30):10513–8. https://doi.org/10.1073/pnas.0804549105.

    Article  PubMed  PubMed Central  Google Scholar 

  189. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 2014;15(8):509–24. https://doi.org/10.1038/nrm3838.

    Article  CAS  PubMed  Google Scholar 

  190. Kosaka N, Iguchi H, Ochiya T. Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer Sci. 2010;101(10):2087–92. https://doi.org/10.1111/j.1349-7006.2010.01650.x.

    Article  CAS  PubMed  Google Scholar 

  191. Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, et al. The microRNA spectrum in 12 body fluids. Clin Chem. 2010;56(11):1733–41. https://doi.org/10.1373/clinchem.2010.147405.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  192. Zhang S, Lin H, Kong S, Wang S, Wang H, Wang H, et al. Physiological and molecular determinants of embryo implantation. Mol Aspects Med. 2013;34(5):939–80. https://doi.org/10.1016/j.mam.2012.12.011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  193. Teague EM, Print CG, Hull ML. The role of microRNAs in endometriosis and associated reproductive conditions. Hum Reprod Update. 2010;16(2):142–65. https://doi.org/10.1093/humupd/dmp034.

    Article  CAS  PubMed  Google Scholar 

  194. Tesfaye D, Gebremedhn S, Salilew-Wondim D, Hailay T, Hoelker M, Grosse-Brinkhaus C, et al. MicroRNAs: tiny molecules with a significant role in mammalian follicular and oocyte development. Reproduction. 2018;155(3):R121–r35. https://doi.org/10.1530/rep-17-0428.

    Article  CAS  PubMed  Google Scholar 

  195. Wang K, Zhang S, Weber J, Baxter D, Galas DJ. Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res. 2010;38(20):7248–59. https://doi.org/10.1093/nar/gkq601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  196. Tabet F, Vickers KC, Cuesta Torres LF, Wiese CB, Shoucri BM, Lambert G, et al. HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells. Nat Commun. 2014;5:3292. https://doi.org/10.1038/ncomms4292.

    Article  CAS  PubMed  Google Scholar 

  197. Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, Li MZ, et al. Dicer is essential for mouse development. Nat Genet. 2003;35(3):215–7. https://doi.org/10.1038/ng1253.

    Article  CAS  PubMed  Google Scholar 

  198. Alisch RS, Jin P, Epstein M, Caspary T, Warren ST. Argonaute2 is essential for mammalian gastrulation and proper mesoderm formation. PLoS Genet. 2007;3(12):e227. https://doi.org/10.1371/journal.pgen.0030227.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  199. Pernaute B, Spruce T, Rodriguez TA, Manzanares M. MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo. Cell Cycle. 2011;10(4):584–91. https://doi.org/10.4161/cc.10.4.14728.

    Article  CAS  PubMed  Google Scholar 

  200. Zhang J, Wang Y, Liu X, Jiang S, Zhao C, Shen R, et al. Expression and potential role of microRNA-29b in mouse early embryo development. Cell Physiol Biochem. 2015;35(3):1178–87. https://doi.org/10.1159/000373942.

    Article  CAS  PubMed  Google Scholar