Extracellular Vesicles in Human Reproduction in Health and Disease

Abstract

Extensive evidence suggests that the release of membrane-enclosed compartments, more commonly known as extracellular vesicles (EVs), is a potent newly identified mechanism of cell-to-cell communication both in normal physiology and in pathological conditions. This review presents evidence about the formation and release of different EVs, their definitive markers and cargo content in reproductive physiological processes, and their capacity to convey information between cells through the transfer of functional protein and genetic information to alter phenotype and function of recipient cells associated with reproductive biology. In the male reproductive tract, epididymosomes and prostasomes participate in regulating sperm motility activation, capacitation, and acrosome reaction. In the female reproductive tract, follicular fluid, oviduct/tube, and uterine cavity EVs are considered as vehicles to carry information during oocyte maturation, fertilization, and embryo–maternal crosstalk. EVs via their cargo might be also involved in the triggering, maintenance, and progression of reproductive- and obstetric-related pathologies such as endometriosis, polycystic ovarian syndrome, preeclampsia, gestational diabetes, and erectile dysfunction. In this review, we provide current knowledge on the present and future use of EVs not only as biomarkers, but also as therapeutic targeting agents, mainly as vectors for drug or compound delivery into target cells and tissues.

Essential Points

• Extracellular vesicles are a newly identified mechanism of cell-to-cell communication, recently discovered as a communication between the mother and the embryo

• Extracellular vesicles play an important role in normal physiology and in pathological conditions in human reproduction

• Prostasomes participate in regulating sperm motility activation

• Different extracellular vesicles and their cargo are implicated in promoting oocyte development and maturation

• Exosomes and their cargo in microRNAs play an important role in embryo implantation

• Extracellular vesicles are involved in the triggering, maintenance, and progression of reproductive and obstetric pathologies

• The participation of extracellular vesicles in human reproductive health has made them appealing players as biomarkers and to carry therapeutic agents

Intercellular communication is an essential process both for multicellular organisms and for the relationship of unicellular organisms with the environment and hosts (1). Classically, communication has been identified as indirect as endocrine, paracrine, and autocrine or direct via cell-to-cell contact, secretion, release, and uptake of chemical moieties such as hormones, growth factors, or neurotransmitters (2, 3). According to the Human Protein Atlas, nearly 39% of the human protein-coding genes are annotated to give rise to membrane (28%) and secreted (15%) forms of signaling protein variants, some producing both isoforms and posttranslational modifications that can alter function. These molecules, which constitute potential therapeutic targets, include cytokines, growth factors, and coagulation factors, among others, playing physiological and pathological roles in processes such as immune defense, blood coagulation, or matrix remodeling. Of note, >500 of these proteins are currently known as pharmacological targets, with already approved druggable targets available commercially.

A new mechanism has recently been in the spotlight for cellular communication: the release of membrane-enclosed compartments, most commonly regarded as extracellular vesicles (EVs). EVs can act to convey molecules from one cell or tissue to another. Importantly, their contents (cargo) are protected from extracellular degradation or modification. They exert their biological roles by either direct interaction with cell surface receptors or by transmission of their contents by endocytosis, phagocytosis, or fusion with the membrane of the target cells. Recipient cell specificity appears to be driven by specific receptors between the target cells and EVs (4–6). EVs have been described in different body fluids, including semen (7), saliva (8), plasma (9), breast milk (10), urine (11), and amniotic fluid (12), among others (4).

EVs can be classified in different populations based on their biogenetic pathway, composition and physical characteristics, such as size or density, giving rise to three major categories: apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) (5, 13, 14).

EV content is complex as a continually progressing field with new cargos being identified continually. Regrettably, owing to technical limitations in methods of isolation and differentiation of the different populations of EVs, mixed, heterogeneous populations are often used, making interpretation of their content and functionality difficult (15–17). This constitutes a salient notion in the field at present, that populations of EV subtypes must be considered when reviewing published literature. With homogeneous sample preparation and key developments in characterization of EVs, we now hold important insights into defining these selected communicators in far greater depth. With the implementation of high-resolution and sensitive instrumentation for characterization such as mass spectrometry and next generation deep sequencing, it has been possible to develop databases gathering information about protein, lipid, and RNA content of EVs from different sources, including ExoCarta (online source: www.exocarta.org) (18), EVpedia (online source: www.evpedia.info) (19), and Vesiclepedia (online source: www.microvesicles.org) (20).

In recent years, EVs have been shown to participate in different processes committed to the maintenance of the normal physiology of the organism such as tissue repair, maintenance of the stem cell status of progenitor cells, platelet and immune function, and nervous system homeostasis. The potential role of EVs in the pathogenesis of different diseases has also been studied, with cancer, autoimmunity, neurodegeneration, HIV-1 infection, and prion diseases being the widest studied areas (1, 6, 21). In all of these cases, EVs are unique, as they became small indicators of an organism’s homeostasis that can stably travel over the body fluids. That their content reflects cell of origin and pathophysiological states highlights their usefulness as biomarkers. Importantly, EVs are attributed with the potential to cross tissue barriers, such as the blood–brain barrier, possibly by transcytosis. This fact makes them appealing targets for therapeutics development (22). EVs can be released in response to cell activation, pH changes, hypoxia, irradiation, injury, exposure to complement proteins, and cellular stress (23–25). Interestingly, EVs are also secreted by plant cells (26, 27), as well as pathogens (28, 29), including bacteria, mycobacteria, archaea, and fungi (30, 31), suggesting an important evolutionary conserved mechanism of intercellular signaling.

In the field of reproductive biology there is growing interest in understanding the role of EVs within the male and female reproductive tracts, as they may constitute a new mechanism of communication between the reproductive tract and the immature germ cells, or between the mother and the developing embryo. Such developments offer great potential implications in the establishment of a successful pregnancy or implications with understanding associated pathological conditions (32). In the present review, we address current knowledge on the existence and functionality of EVs as cell-to-cell messengers in normal human reproductive physiology, as well as their contribution in the triggering, maintenance, and/or progression of pathological conditions in the functionality of the reproductive tract. Furthermore, we discuss their usefulness as biomarkers of altered reproductive conditions such as preeclampsia, spontaneous preterm birth (SPB), or polycystic ovaries syndrome. Finally, we summarize current knowledge on the present and future of the use of EVs as therapeutic agents, mainly as vectors for drug or compounds delivery into target cells and tissues.

Types, Isolation, and Characterization of EVs and Cargo

EV heterogeneity

EVs can be classified into selected subtypes according to different criteria, that is, cellular origin, biophysical (density and size) and biochemical (biological markers) characteristics, biological function, and biogenetic pathway. According to their biogenetic mechanism of formation and release, three main classes of EVs are defined: ABs, MVs, and EXOs (Fig. 1).

Figure 1.

Main types of EVs present in body fluids and culture media. EVs are classified in three groups according to their biogenetic pathways. EXOs are produced in the endosomal pathway by invagination of the membrane of late endosomes to form intraluminal vesicles (ILVs) enclosed in multivesicular bodies (MVBs). MVBs can then fuse with lysosomes and degrade their content, or fuse with cell plasma membrane to release ILVs, now regarded as EXOs. MVs are produced directly from the cell plasma membrane by outward budding. ABs are generated as blebs in cells undergoing programmed cell death. EE, early endosome; ExV, exocytic vesicle; LE, late endosome.

Apoptotic bodies

ABs are EVs produced by plasma membrane blebbing in cells undergoing programmed cell death. This term was coined by Kerr et al. (33) who defined them as “small, roughly spherical or ovoid cytoplasmatic fragments, some of which contain pyknotic remnants of nuclei.” Indeed, one of the events that characterizes ABs is the fragmentation and packaging of cellular organelles such as the nucleus, endoplasmic reticulum (ER), or Golgi apparatus into these vesicles (34, 35).

ABs have widely been described as 1 to 5 μm in diameter, thus overlapping with the size range of platelets (36, 37), although some groups extend this range to 50 nm (16, 38, 39). Their buoyant density in a sucrose gradient is in the range of 1.16 to 1.28 g/mL (40, 41).

This vesicle population is characterized by cytoskeletal and membrane alterations, including the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer (42). In this way, PS serves as an “eat me” signal for phagocytes to target and clear apoptotic debris (43, 44). Moreover, PS can naturally be recognized by annexin V, which is a useful marker of ABs (45). Nevertheless, care should be taken when using annexin V for this purpose, as PS flipping can also be triggered by other stimuli such as mechanical disaggregation of tissues, enzymatic treatments for detachment of cells, electroporation, chemical transfections, or retroviral infections, and PS exposure has also been described in healthy cells (46). PS flipping also induces MV formation, so these can also be recognized by annexin V detection (47, 48). Another specific feature of ABs is the oxidation of surface molecules, creating sites for recognition of specific molecules such as thrombospondin (49) or C3b complement protein (50), which are also useful as markers of ABs.

Included in newly identified potential molecular markers of ABs, VDAC1 is a protein that forms ionic channels in the mitochondrial membrane and has a role in the triggering of apoptosis. It proves to be a useful AB marker, as its biological function and subcellular localization are characteristic of this vesicular fraction (39). Calreticulin is an ER protein that could also work as an AB marker due to its subcellular localization (15), although it has also been observed in the smaller sized MV fraction (39). It is possible that, during the apoptosis process, the ER membrane is fragmented and forms vesicles smaller in size than ABs, which would contain calreticulin and would sediment at higher centrifugal forces (51, 52). Indeed, proteomic studies have related calreticulin with vesicular fractions across the full size range of MVs (53) and ABs (54).

Different functions have been attributed to ABs, although most are also features of other EVs. DNA can be horizontally transmitted between somatic cells, with possible integration of this DNA within the receptor cell where it can be functional (55). These vesicles are also a vehicle for the horizontal transfer of oncogenes, which are internalized by target cells and consequently increase their tumorigenic potential in vivo (56, 57). ABs have also been related to the immune response where they are associated with an underactivation of the immune system (58) and with antigen presentation with special regard to self-tolerance (59–61).

Microvesicles

MVs were reported for the first time by Chargaff and West (62) as being sedimented at high-speed centrifugation (31,000 × g) (not specifically at lower speeds such as 5000 × g). 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 (63, 64). Plasma membrane blebbing is triggered by different mechanisms that are accompanied by the remodeling of the membrane proteins and lipid redistribution, which modulate membrane rigidity and curvature (65). Such changes within the periphery of the plasma membrane have been associated with cargo sorting in MVs (66).

The size range of MVs has been classically established between 100 and 1000 nm (67), thus overlapping with that of bacteria (13). Some groups extend this range up to 1500 nm (68) or even 2000 nm (69–71). The buoyant density of MVs is not as clear as that of other vesicle populations: ∼1.16 g/mL in sucrose gradient (71) or 1.04 to 1.07 g/mL (72). The flotation density in iodixanol gradient is between 1.18 and 1.19 g/mL (73).

As a proposed marker for MV populations, ARF6 is a guanosine triphosphate–binding protein that is implicated in the regulation of cargo sorting and promotion of the budding and release of MVs through activation of the phospholipase D metabolic pathway (65, 74). Additionally, data coming from our current knowledge on proteomic studies suggest numerous proteins (e.g., KIF23, RACGAP1, exportin-2, chromosome segregation 1–like protein) as unique and/or enriched for MVs and potentially discriminatory markers (75). Nevertheless, care should be taken with these results, as different EV cell sources and techniques to selectively enrich may lead to differences within EV populations.

Among the functions described for MVs are pivotal roles in cancer cell invasiveness (76, 77), transformation potential (78), progression (63, 79, 80), and drug resistance (81). MVs have also been implicated in autoimmune diseases (82–84), immune system modulation and coagulation (67, 85, 86), embryo–maternal crosstalk (87), and embryo self-regulation (88).

Exosomes

The first description of EXOs in 1981 described them as a second population of vesicles that appeared in the preparations of MVs, and the term “exosome” was coined (89). Two years later, their biogenetic pathway was formally described by transmission electron microscopy (TEM), trying to follow the pathway of uptake and trafficking of transferrin molecules within reticulocytes in an anemic mice model (90). EXOs constitute a population of nano-sized EVs that arise and are trafficked through the endosomal pathway. Endosomal sorting complexes required for transport (ESCRTs) are important for the biogenesis of multivesicular bodies (MVBs, which include EXOs). MVB inward budding of the limiting membrane of late endosomes facilitates formation of intraluminal vesicles (ILVs) that remain enclosed inside the greater membrane compartment of MVBs. ESCRT-independent mechanisms, including neutral sphingomyelinase/ceramide formation and ARF6/PLD2, have been reported to also occur (73, 91). The formed MVBs can then be targeted to plasma membrane to release ILVs, now known as EXOs, or otherwise fuse with lysosomes to degrade their content (92). Members of the Rab guanosine triphosphatase (GTPase) family have been shown to modulate EXO secretion and are thought to act on different MVBs along ESCRT-dependent and -independent endocytic pathways. It is likely that ESCRT-dependent and ESCRT-independent MVB/EXO biogenesis machineries vary from tissue to tissue (or even cell type) depending on specific metabolic needs. There are several molecular mechanisms, both canonical and alternative, implicated in the formation, release, and extracellular fate of EXOs [see reviews in (5, 75)].

Most studies place EXOs in a size range of 30 to 150 nm (5, 93) or even 200 nm (94), thus establishing an overlap with viruses in terms of size (14). The buoyant density of EXOs in sucrose gradients has been set in a wide range of 1.10 to 1.21 g/mL (38, 95), and 1.10 to 1.12 g/mL in iodixanol gradients (96).

The classically associated markers of EXOs are molecules mainly implicated in the biogenesis of this population, which are incorporated during this process: tetraspanins (CD63, CD9, CD81), Alix, TSG101, and flotillin-1, among others (5, 95). Nonetheless, with the emerging interest in studying different EV populations as isolated entities, many of these classical markers have been identified as widespread between populations, although with different relative abundances. This is the case for at least CD9, CD63, HSC70, and flotillin-1. Other molecules such as TSG101 and syntenin-1 have been ratified as markers of only this vesicle population (39). PS, while being described as a broad marker of EVs, has also been reported as exposed on the surface of EXOs produced by different cell types (92, 97). Accumulating evidence from in vitro studies using cells grown in culture and ex vivo body fluids indicates the existence of more than one EXO subtype (98–105). For example, EXOs contain subpopulations, including the study of EXOs derived from apical (EpCAM-Exos) or basolateral (A33-Exos) surfaces of highly polarized cancer cells, which indicated the presence of two distinct subtypes with distinct protein (98) and RNA cargo (99, 106). The biological significance of these findings awaits further investigation.

Because of the high expectations and efforts dedicated to the study of the role of EXOs in different biological processes, both in physiological or pathological conditions, the field of EXO biology has experienced an exponential growth in recent years, with a wide range of functions identified (1, 107). EXOs are implicated in cancer physiology, participating in tumor progression and maintenance, resistance, immune modulation, and angiogenesis (108). Their function in immune regulation has also been well studied in antigen presentation modulation, immune activation, and suppression (109, 110). Importantly, knowledge of the seminal role of EXOs in reproductive biology is expanding rapidly. Such studies and the molecular markers and mechanisms identified have the potential for use as markers to discriminate between EV subtypes, as well as various applications of EXOs in clinical diagnosis.

Methods of isolation and purification of EVs

The main experimental problem when studying EVs is to achieve a homogeneous separation with appropriate yield of the EV population of interest. Different methods of isolation and purification have been developed; however, to a varying extent, all carry the bias of providing completely homogeneous EV populations of any one vesicle type (summarized in Table 1). In the field, there is a pressing need to define EV surface-exposed proteins for the purpose of generating monoclonal antibodies that would allow discrimination of EV class or subtype (i.e., stereotypical markers). Most rapid, one-step approaches for isolating EVs do not take into consideration that they are dealing with a possible mixture of vesicle classes or subtypes and coisolated contaminants such as high relative molecular mass protein oligomer and protein–RNA complexes (e.g., high-density lipoprotein/low-density lipoprotein/AGO2) complexes.

Table 1.

Classification of the Methods of Isolation of EVs Based on Their Principles

Method	Technique	Isolation Principle	General Workflow	Advantage	Limitation	References
Centrifugation	Serial differential centrifugation	Sedimentation velocity	Serial or differential centrifugation: (1) 300 × g, 10 min to remove cells → (2) 2000 × g, 10 min to remove cell debris, ABs → (3) 10,000/20,000 × g, 30 min to isolate MVs → (4) 100,000/200,000 × g, 70 min to isolate EXOs	• Broad application	• Sedimentation dependent on density, tube length, sample viscosity, concentration, and vesicle aggregation apart from size	(16, 112, 116, 138)
				• Standardization
				• Ease of use
				• Reproducibility
				• Yield
	Density gradient	Buoyant density	Generally introduced to further purify distinct types of EVs (i.e., MVs or EXOs). Various different reagents, including sucrose or iodixanol. Crude EV populations loaded either on top (float down) or at bottom (float up) of gradient. Ultracentrifugation performed under pre-established conditions	• Purification: increases EV population purity from: protein aggregates, RNA–protein complexes, separation of EV subpopulations within the same type	• Yield	(4, 112–115, 117–119,139)
				• Soft isolation approach	• Reproducibility
				• Clinically applicable medium (iodixanol)	• Trained user
				• EV homogeneisty	• Time-consuming
Size exclusion	Filtration	Size/shape	Generally interspersed within centrifugation steps: prior to centrifugation, supernatants are challenged through syringe filters of determined pore size	• Easy to use	• Yield loss within filtering membrane	(112, 120, 121)
				• Further stringency of the populations based on their canonical sized	• Risk of vesicles deformation or fragmentation.
				• Reproducibility
	Ultrafiltration	Size	Centrifugal filtration units of prefixed molecular size range that selectively retain vesicles Previous studies shown to isolate distinct subtypes of EVs using this strategy	• Easy to use	• Yield loss within filtering membrane.	(73, 122–125, 140)
				• Quick technique	• Risk of vesicles deformation or fragmentation.
				• Reproducible
	Chromatography	Size/charge	Purification of EVs based on surface charge or size	• High resolving power; improved purification of EVs from proteins and lipid particles	• Usually coupled to centrifugation to remove cell debris and recover EV containing fractions	(126–129, 142)
				• Limits EVs and protein aggregation based on buffer used	• Often issues with volume or buffer associated with elution
				• Less sensitive to the viscosity of the media
				• Respectful with EV functionalities and biological properties
				• Shorter isolation times
Immunoaffinity		Presence of specific EV surface molecules	Microbeads coupled to antibodies are incubated with EVs for specific surface markers recognition (i.e., A33, EpCAM, CD63). Afterward, beads are washed and recovered by precipitation or magnetism.	• Separation based on specific molecules further than by size	• Sometimes coupled to centrifugation and/or filtration to initially remove larger cellular debris	(96, 98, 99, 130–132)
				• Selectivity	• Select surface markers of EVs are not always known or available
				• Resolution	• Cost
				• Speed of isolation	• Yield
Polymeric precipitation		Weight increase to pellet at low centrifugal force	Incubation of polymerization kit reagents with EV solution and recovery by low-speed centrifugation	• High speed	• Possibility of coprecipitating impurities	(111, 133, 134)
				• Simple procedure	• Unable to separate EV fractions
					• Ideal only for small (60 to 180 nm) EV populations
Microfluidics		Different possible principles:	(1) EVs are passed through microfluidic system and EV-specific markers are recognized by antibodies in a device surface	• Reduced sample volume needed	• Habitually couple to centrifugation to remove undesired EV populations	(135–137, 141)
		(1) Presence of specific molecules	(2) Still not applicable for EVs	• Smaller processing times and costs, maintaining high sensitivity	• Unable to differentiate EVs populations
		(2) Physical properties such as size	(3) Combination of microfluidics and polymer filter that allow passing EVs under a certain size	• Possibility to process, quantify. and image the samples within the system itself	• Still under development
		(3) Microfluidic filtration

Serial differential centrifugation

Differential centrifugation is the most common and well-known method for the isolation of EVs. Although each group adapts the times and centrifugal speeds depending on their samples, the basic protocol as follows: (1) centrifugation at low speed for the elimination of cells (300 × g, 10 minutes), (2) centrifugation at up to 2000 × g for 10 minutes to pellet membrane debris and ABs, (3) centrifugation at 10,000 to 20,000 × g for 30 minutes to pellet MVs, and (4) a crude EXO preparation is pelleted by ultracentrifugation at 100,000 to 200,000 × g for 70 minutes. After steps 1 to 3 of centrifugation, supernatants are transferred to new tubes for the isolation of the subsequent EV type. Pellets (steps 2 to 4) containing different cell populations are washed by resuspension in phosphate-buffered saline (PBS) and recentrifugation under the same conditions. The washing step removes some impurities, but also reduces EV yield.

Apart from vesicle size, centrifugation alone cannot achieve the separation of pure populations for various reasons: sedimentation of other particles in the supernatant depending on density, distance of the particles from the bottom of the tube, and vesicle/particle aggregation (111).

To improve EV population purity, a gradient step can be added to the centrifugation protocol. This system aims to avoid as far as possible the contamination of EV pellets with large protein/protein-RNA aggregates and proteins nonspecifically bound to EVs (4). The essentials of the technique are resuspension of the pellet from the previous serial differential centrifugation in a suitable buffer (i.e., PBS), then loading on either the top or base of a prepared sucrose cushion (112, 113) or a sucrose gradient (114, 115). Following ultracentrifugation, vesicles are recovered either from the bottom of the tube (for cushions) or from a specific fraction of the gradient, depending on their buoyant density. Moreover, substitution of sucrose by a nonionic density gradient medium, called iodixanol (116), offers many advantages: better separation of viral particles from EVs; low toxicity toward biological material; is clinically applicable; and forms isosmotic solutions compatible with the size and shape of EVs in a wide range of densities (117–119).

Size exclusion methods: filtration and chromatography

Filtration for isolation of EVs is often used in combination with ultracentrifugation protocols to improve separation efficiency based on size. Filtration steps using 0.8-, 0.2-, or even 0.1-µm filters can be inserted between the centrifugation steps depending on the size of the desired population (112, 120, 121). Alternatively, ultrafiltration utilizes filtration units of different molecular mass cutoff membranes that are centrifuged at moderate centrifugal forces. They allow concentration of vesicles in the interface of the filters, from which they can be recovered by washing (122–125). All of these methods face several drawbacks. The pressure of the supernatant can cause the EVs to deform or break into smaller vesicles, and the filter membrane may decrease the yield. Gravity filtration has been proposed to cope with the problems associated with elevated pressures (120), but this can be time consuming and filters can become saturated.

Another option for EV isolation in conjunction with ultracentrifugation is based on size exclusion chromatography. In brief, the medium containing the vesicles is loaded into the chromatography device, generally a gel size exclusion column, equilibrated into the column, and eluted with PBS (126–128). The technique is usually coupled with previous low-speed centrifugation to remove larger debris and subsequent ultracentrifugation to wash and concentrate the vesicles from the different chromatography fractions (129, 142). Its advantages are enhanced separation of EVs from proteins and high-density lipoproteins, avoidance of protein and vesicle aggregate formation, reduced sensitivity to the viscosity of the vesicle media, compatibility with the biological properties and functionalities of the isolated vesicles, and preservation of the vesicular structure and conformation (126). Moreover, it offers shorter isolation times and relatively low cost. As a disadvantage, this technique offers reduced EV recovery yields in comparison with others such as ultracentrifugation or polymeric precipitation, although it is susceptible to scale-up (143, 144). Nevertheless, some studies indicate that a combination of size exclusion chromatography and ultrafiltration may produce a yield surpassing that of classical ultracentrifugation (145, 146).

Other approaches

Immunoaffinity uses microbeads coated with specific antibodies for the recognition of specific surface markers of EV populations. In brief, beads are incubated with the sample containing EVs, then beads linked to their epitopes on the EV surface are recovered by magnetism or low-speed centrifugation, depending on the nature of the beads (130, 131). The technique can follow centrifugation and/or filtration to clear large cellular products (96, 99, 132). This method differentiates EV populations based on surface markers regardless of their size. Nevertheless, care should be taken, as population-specific markers are not necessarily available, and the working surface of the beads is limiting, so the EVs may not have access when they are large or present at high concentrations (116).

“Differential centrifugation is the most common and well-known method for the isolation of EVs.”

Aiming for a quicker and simpler method to isolate EVs, a polymeric precipitation system (ExoQuick) was commercially developed. The experimental procedure is as simple as incubating the kit reagents with the EXO-containing media and recovering the resulting polymeric complex by low-speed centrifugation. A study with human ascite samples showed that ExoQuick could provide high concentrations and purity of exosomal RNA and that the high exosomal protein concentrations from the same samples compared well with other isolation methods such as ultracentrifugation, immunoaffinity isolation, and chromatography (133). Even though ultracentrifugation-based protocols are preferable for exosomal protein recovery and purity, ExoQuick obtains better results in terms of exosomal messenger RNA (mRNA) and microRNA (miRNA) yield and quality (134). The method has a series of limitations. Impurities such as lipoproteins are possibly coisolated along with EVs, and the method is unable to provide isolation of different EV subpopulations. It works ideally with small vesicles in the size range of 60 to 180 nm (111).

A new technology based on microfluidic devices has recently been developed for the isolation of EVs. It allows the reduction of sample volumes, processing times, and costs while maintaining high sensitivity. The chip technology can be based on different principles. The first developed systems relied on the recognition of EVs by specific antibodies on the surface of the device (147). The surface of the flow system was coated with anti-CD63 antibodies. When EV-containing media was pumped through the system, EXOs were restrained. The system allows scanning electron microscopy (SEM) imaging and lysis of EVs for RNA isolation directly on the chip. However, it does not provide sufficient material for protein or functional analyses. Subsequently, the system was expanded with lipophilic staining of EXOs to allow simultaneous quantitation (135). A third microfluidic scheme used physical properties as the principle for EV isolation, separating microparticles based on their size within the micrometric size range (136). Clearly this method is not applicable to EV population analysis. This technology has also been combined with porous polymers, allowing purification of vesicles in the nanometric size range: the pore size can be modulated so that only EVs under a certain size can be filtered (148). A recent study introduced the concept of using a combination of acoustics and microfluidics for a high-purity degree of EXO isolation. The platform is composed of two sequential modules that remove larger components and other EV groups (MVs and ABs, respectively), allowing the direct use of undiluted body fluid samples (tested in whole blood) or conditioned media from cell cultures in a single step. The system is based on the combination of microfluidic channel conformation and adjusted acoustic pressure, which make it possible to set the cutoff particle diameter (149).

The demands of clinical applications involving diagnostics and therapeutics such as low cost, reliability, and speed can eventually be met with modifications to existing technologies for improved scalability. Isolation of EVs from blood and urine is a challenge due to the presence of abundant and complex protein and lipoprotein networks, which undoubtedly will attenuate intrinsic EV protein/RNA signatures. Distinct clinically relevant strategies to isolate EVs are currently being investigated (75, 150, 151).

Methods for characterization of EVs

Characterization of EVs is fundamental to enable differentiation among the different subpopulations within the same biological sample, between vesicles of distinct cellular origin or even of the same origin in pathological vs physiologically normal conditions (summarized in Table 2).

Table 2.

Classification of the Methods of Characterization of EVs Based on Their Principles

Method	Technique	Principle	Main Features	Quantitative/Qualitative	References
Microscopy	TEM	Negitive staining of EVs with electron-dense molecules (heavy metals)	• Direct imaging of EV size	• Semiquantitative	(139, 152–154, 178)
			• Size distribution	• Dehydrating (fixation)
			• Can be coupled to immunogold labeling to stain specific structures	• Possibility to take measures within the imaging field
	SEM	Covering of molecules with microgold particles and electron reflection scanning	• Three-dimensional imaging of EV structures	• Semiquantitative	(156, 157, 179)
				• Possibility to take measuresments within the imaging field
	Cryo-electron microscopy	Plunge-frozen in liquid ethane/nitrogen	• Avoids fixation and contrasting steps	• Semiquantitative	(2, 73, 155)
			• Allows seeing structures closer to their native states	• Possibility to take measures within the imaging field
			• Size distribution	• Highly trained user
	AFM	Use of a cantilever with a free end that touches the surface to obtain topographical information	• Resolution at the nanometric level	• Quantitative	(137, 158–160)
			• Possibility to analyze both dry and aqueous samples	• Size-distribution profile determination
			• Can be combined with microfluidic isolation devices	• Require homogeneous EV purification
			• It does not provide direct imaging of EVs
Size distribution analysis techniques	NTA	Particles are challenged with a laser beam and forward scattered light is real-time captured by a microscope to calculate sizes based on particles using their Brownian motion.	• Size measures in the range of 50 to 1000 nm	• Qualitative: not only size populations but also EV markers can be analyzed by fluorescent labeling	(161, 180, 181)
			• Standardization is not needed but possible (interest for concentration assessments)	• Quantitative: possibility to get precise size distributions and their associated concentrations in 1-nm intervals
			• Size distribution	• Cost
			• Low sample use
			• Compatibility of fluorescence detectors
	Dynamic light scattering	Particles are challenged with a laser beam and reflected light is captured by a detector in a certain variable angle. The detector converts time-dependent fluctuations in the scattered light intensity into particle size data.	• Size measurements in the range of 1 to 6000 nm for EV concentrations from 106 to 109 particles/mL	• Mainly qualitative	(162, 182)
			• Samples can be recovered after the analysis	• Semiquantitative if standards are used
			• Limitations with polydisperse samples and those containing big EVs
	Tunable resistive pulse sensing	A transmembrane voltage is established in a porous membrane. The crossing of EVs through the pores alters the electrophoretic flow causing a resistance that can be translated into size data.	• Size measurements in the range of 70 nm to 10 µm for EV concentrations from 105 to 1012 particles/mL	• Qualitative	(163–165)
			• Single EV measures that allow multimodal EV population studies	• Quantitative
			• By modifying pore configuration, the analyzable EV size and sample volume can be regulated
	Flow cytometry	EVs are swept along by a liquid stream to align them in single file in the center of the stream until the interrogation point, where they are excited by a laser beam. Laser scattered light is gathered by detectors situated 180° (size data) and 90° (morphology or fluorescently stained structure data) to the laser beam.	• Analysis of EVs with a lower size limit of 250 to 500 nm and ability to distinguish vesicles that differ 200 nm in size	• Qualitative: not only size populations but also EV markers can be analyzed	(166–171)
			• New technological developments have reduced the limit of detection to ∼100 nm and the discrimination power to 100 to 200 nm	• Quantitative
			• Possibility to coupling to latex beads for easy marker analysis
			• No sorting capacity
			• Dependent on EV surface markers or use of EV fluorescent labels
Molecular marker characterization techniques	Western blotting/ELISA	Both techniques share the same principle: proteins are attached to support (membranes or plates, respectively) and challenge with antibodies carrying a certain label.	• Easy to perform	• Qualitative	(39, 172, 173)
			• Cheap and available	• Semiquantitative in the case of western blot and quantitative for ELISA
			• Relatively quick
	ExoScreen	ELISA sandwich-like system with modifications in the detection tandem. The method relies on that all the components of the system must stay closed (∼200 nm, within the same vesicle) for laser stimuli transfer and detection.	• Reduced time consumption	• Qualitative	(174)
			• Increased sensitivity	• Quantitative
			• EV isolation is not mandatory
			• Little sample volumes are required
	μNMR	Labeling of specific EV surface molecular markers with antibodies coupled to magnetic nanoparticles and detection by microfluidic μNMR	• Greatly higher sensitivity	• Qualitative	(175)
				• Quantitative
	Nano-plasmonic EXO assay	A gold film with nanoholes coated with specific antibodies for the recognition of exosomal proteins is light-excited, generating surface plasmons. Joining of EVs to the antibodies causes plasmon intensity changes that are proportional to the amount of joined EVs.	• Label-free	• Qualitative	(176, 177)
			• Easy to miniaturize	• Quantitative
			• Scalable for higher throughput detection
			• A magnitude order more detection sensitivity than μNMR

Abbreviations: ELISA, enzyme-linked immunosorbent assay; μNMR, micronuclear magnetic resonance spectrometry.

Microscopy: morphology and size analysis

Electron microscopy techniques are the only method available to provide the appearance of EVs related to their size. Different variants offer different data to the user. TEM was initially used by Raposo et al. (152), who described EXOs as cup-shaped vesicles. Although different protocols can be used for TEM visualization, two general schemes offer different views. EVs can be resuspended in fixative media and laid into grids for staining and visualization. Alternatively, EV pellets from centrifugation and ultracentrifugation steps can be fixed, resin embedded, and cut into ultrathin slides, which are then stained and laid in grids. The first method is simpler and less time consuming, and it offers a view of the exterior of the EVs. The second method is more informative, shows the interior of the EVs, and allows immunogold staining of specific markers that are seen as electron-dense spots (153, 154). Cryo-electron microscopy allows direct visualization of frozen EVs without previous fixation and contrast steps. The structures are seen as close as possible to their native states (not dehydrated or fixed) and demonstrate variable EV morphology (155). Indeed, such analysis showed that the classical cup shape attributed to EXOs was an artifact of fixation (2). Finally, SEM offers three-dimensional imaging of the EVs for further morphological description (156, 157).

Atomic force microscopy (AFM) is an alternative for the analysis of size distribution and quantity of EVs within a sample and is based on the scanning of the sample by a mechanical probe, which physically touches the sample, providing topographical information. AFM allows imaging at the subnanometric level. It can be adjusted to air (dry samples) or liquid modes (aqueous samples), and differences in size or number measurements are negligible between them. The possibility of measuring samples in aqueous media is advantageous, as it permits the maintenance of EV physiological properties and structure (158–160). AFM has been efficiently combined with microfluidic isolation devices to provide consecutive isolation and characterization of EVs. Mica-microfluidic chips are also of interest, as they provide a nonconductive flat surface for in situ AFM analysis (137, 160).

Size distribution analysis techniques

Nanoparticle tracking analysis (NTA), a light-scattering technique, is now widely used for the assessment of EV size distributions and concentration in the range of 50 to 1000 nm. The principle of the technique is based in the inherent Brownian motion of the particles in a solution: EVs in suspension are irradiated by a laser beam, thus emitting dispersed light. This scattered light is captured by a microscope, and NTA software tracks the movement of each particle in a time lapse. Silica nanospheres have been proposed for standardization, as their refraction index (1.46) is similar to that observed for most EVs (refraction index of ∼1.39) (161).

Dynamic light scattering is also used for the assessment of EV size distribution. Although the principle for size determination is also Brownian movement of particles in suspension, the way to attain these data varies from NTA technology. It has limitations when measuring polydisperse samples and those containing big EVs, because the bigger particles scatter more light, masking the smaller ones (162). It is also possible to calculate vesicle concentrations in the samples by direct extrapolation from the distribution representations using mathematical criteria (162).

Tunable resistive pulse sensing (or qNano by its IZON commercial name; Izon Science Ltd., New Zealand) is a novel and less expensive technique for the analysis of particle size distributions. The system is composed of a thermoplastic polyurethane membrane containing nanopores that are selected by size requirements. Currently, the system can measure individual particles in the size range of 30 nm to 10 μm and in the concentration range of 10⁵ to 10¹² particles per milliliter. Because the system analyzes the particles individually, multimodal populations can be studied. Alternatively, a configuration of only one pore type restricts measures to a narrow size range, which is particularly useful for analysis of a specific vesicle population. Combining pores of distinct size and geometry allows widening of this range and analysis of a greater volume of sample (163–165).

Flow cytometry has also been applied to the analysis of size distribution, concentration, and qualitative characteristics of the EVs within a sample. Light scatter flow cytometry allows the analysis of vesicles with a lower size limit of usually between 300 and 500 nm (166, 167), but small EVs, including EXOs, cannot be studied by this method. However, innovative new flow cytometry technology and the use of fluorescent labeling of EVs has reduced the lower limit of detection to ∼100 nm, and it is possible to discriminate between vesicles 100 to 200 nm in size (168, 169). Finally, EVs can be coupled via antibodies to their surface markers, to latex beads of greater size. In this way even nano-EVs can be analyzed, but no quantification or differentiation between vesicle populations is possible (170, 171).

Molecular marker characterization

The most effective and well-accepted approach to measure EV purity is the concentration of a specific EV surface marker antigen. Approaches including western immunoblotting, enzyme-linked immunosorbent assay (ELISA) using surface markers that can be used with adaptation for the quantitation of EVs within a sample (172, 173), and ExoScreen have been used (174).

Another approach for the characterization and quantitation of EVs is based on micronuclear magnetic resonance spectrometry (μNMR) (175) EV labeling with specific EV surface molecular marker antibodies coupled to magnetic nanoparticles enables specific detection by microfluidic μNMR. The technique offers a detection sensitivity level that greatly surpasses ELISA or flow cytometry.

Finally, transmission surface plasmon resonance can provide an alternative method for the molecular characterization and quantitation of EXOs in a system called nano-plasmonic EXO assay. This consists of a gold film patterned with a series of nanohole arrays, each of which is coated with specific monoclonal antibodies for the recognition of EXO-specific proteins. Compared with previous systems, the nano-plasmonic EXO assay is label free, easy to miniaturize and scalable for higher throughput detection, and improves detection sensitivity to a magnitude order lower than that for μNMR (176, 177).

During the past decade, recent studies and groups have used developments in proteomic profiling to characterize specific markers for highly purified EV subtypes (EXOs and MVs). Since the emergence of the interest in studying different vesicles populations as isolated entities, many of the classical markers of EXOs have been uncovered as widespread between populations, although with different relative abundances. This is the case of CD9, CD63, HSC70, EpCAM, and flotillin-1, among others (98, 100). Alternatively, some new molecular markers have been identified and ratified as markers of EXOs, including TSG101, syntenin-1, and Alix/PDCD6IP (39, 100). Numerous proteins found exclusively/enriched in MVs [e.g., KIF23, RACGAP1, chromosome segregation 1–like protein, exportin-2 (CSE1L/CAS)] warrant further study as to their potential use as discriminatory markers for MVs. Furthermore, care should be taken when analyzing PS as a marker of ABs, as it has also been reported to be exposed in the surface of EXOs produced by different cell types (92, 97) and also MVs (47, 48). An in-depth review detailing proteomic insights into EV biology and defining markers for EV subtypes and understanding their trafficking and function was provided by Greening et al. (183).

EV cargo

Membrane receptors and cargo content are the most important feature of EVs, because they define their cellular selectively, target, uptake, and functionality, respectively. EV cargo includes proteins, bioactive lipids, various RNAs (including fusion gene and splice-variant transcripts), and DNAs (described later), as well as other cell regulatory molecules (1, 4). To date, most studies have focused on their genetic (particularly RNA and miRNA) and protein content, as sensitive methods exist for their comprehensive analysis and detection.

Protein contents in EVs have been widely studied since the application of mass spectrometry–based techniques (184). EVs have been shown as to be enriched in proteins from cytoskeleton, cytosol, plasma membrane, heat-shock proteins, and proteins involved in EVs biogenesis, whereas proteins from cellular organelles are less abundant (1). From initial studies, EVs were shown to carry commonly widespread EV proteins and a specific subset of proteins, depending on the cell, the type of vesicle, and the method of isolation (5). Moreover, it has been observed that EV number, protein content, and protein concentration vary depending on the stimuli for vesiculation, even in the same subpopulation of vesicles (185).

“The most effective and well-accepted approach to measure EV purity is the concentration of a specific EV surface marker antigen.”

Cytokines have also been described to be carried by EVs (1). Interleukin (IL)-1β is among the examples of theses soluble mediators that are secreted in EVs. Indeed, secretion pathways of EVs may constitute an alternative to exocytosis for proteins that lack leader signal peptide (186). Another interesting example of cytokine cargo is IL-1α, which has been reported to be selectively carried by ABs but not by smaller-in-size vesicles (<1 μm) in endothelial cells (187), thus confirming the cargo sorting into different populations of EVs. Further examples of cytokines released into EVs are IL-18 (188), IL-32 (189), tumor necrosis factor (TNF)-α (190), and IL-6 (191), among many others. During pregnancy, EV cytokine cargo has been shown to be modified toward an increase in comparison with nonpregnancy, maybe contributing to the modulation of maternal immune response against the fetus. Levels of transforming growth factor-β1 and IL-10 were increased in EVs from pregnant women, along with an enhanced ability to induce caspase-3 activity in cytotoxic natural killer (NK) cells, thus promoting an immunosuppressive phenotype through the induction of apoptosis in these cells (192).

Lipid content of EVs has been much less studied. However, some groups have shown that EVs are enriched in certain types of lipids in comparison with their parent cells, demonstrating the sorting of these molecules. Specifically, vesicles are enriched in sphingomyelin, cholesterol, PS (193, 194), ceramide and its derivate, and, in general, saturated fatty acids (195). It is also remarkable that the lipid/protein ratios are higher in vesicles than in parent cells. In contrast, phospatidylinositols, phospatidylglycerols, phosphatidylcholine, and phosphatidylethanolamines are more present in parent cells than in vesicles (193). Recently, when using mass spectrometry, quantitative lipidomic combinations of three lipid species were shown to distinguish cancer patients from healthy controls (196).

RNAs in EVs were first described by Valadi et al. (197) in mast cells. They found that EXOs released by these cells contained mRNAs and miRNAs and were able to transfer their content to other cells, where mRNA was functional and could be translated into protein. More recent studies using high-throughput sequencing techniques have shown that EXOs contain various classes of small noncoding RNAs in addition to mRNA, that is, miRNA, small interfering RNA (siRNA), small nucleolar RNA, Y RNA, vault RNA, ribosomal RNA, transfer RNA (tRNA), long noncoding RNA, and piwi-interacting RNA (198–200). Ng et al. (171) showed that endometrial epithelial cells cultured in vitro produced EVs containing a different miRNA profile from that of parent cells, thus suggesting a sorting mechanism of these miRNAs into EXOs. This could constitute a mechanism for communication between the mother and the embryo with potential implications in embryo implantation. Indeed, bioinformatic studies on the EV miRNAs showed that some of the genes targeted by the miRNAs are involved in implantation. More recently, our investigation group deepened the knowledge of maternal–embryo crosstalk and demonstrated that EXOs containing miR-30d were actively transferred from endometrial epithelial cells to trophoblastic cells, were the miRNA was subsequently internalized (156).

A major problem concerning RNA analysis from EVs is the variability of the results depending on the methodology used for isolation of cells and obtaining data. One of the major factors affecting this variability is the possibility that the RNA present in the medium, for example, from lysed cells, could stick to the external EV walls, thus being isolated along with internal RNA. In this sense, RNase A treatment previous to EV RNA isolation should be conducted (201). Even with this procedure, it has been stated that extravesicular RNAs associated with proteins, such as miRNAs in complex with argonaute proteins, can circumvent RNase A degradation, thus leading to bias in interpretation of results. This protective role of protein complexes has been reported in extravesicular medium (202, 203) and inside EVs (204). To overcome complex protection, treatment with proteinase K has been proposed for dissociation of RNA–protein complexes (205). Nevertheless, negative impact in EV yields should be investigated, as proteases may provoke vesicle lysis.

Less has been reported regarding DNA content in EVs. Some studies have currently reported the presence of double-stranded DNA in EVs (206, 207), even distinguishing a different pattern of content among EV subpopulations (208). A previous study conducted in a similar way in tumor cells, using DNase to cleave extravesicular DNA, showed that EV DNA was more abundant in MVs from tumor cells than from normal cells and that this DNA was mainly single stranded (209). It has been shown that mitochondrial DNA can also be transported between cells inside EXOs, possibly constituting a pathway to transmit altered mitochondrial DNA and associated pathologies (210). This may serve as evidence of a trans-acting function of DNA, being able to have functional effects on the recipient cells.

Of note, both the amount and content of EV genetic cargo can be hormonally regulated in EXOs from target cells: this is of particular relevance to reproductive tissues and is further discussed later.

EV mechanism of recognition and uptake

Mechanisms of EV uptake

For EVs to act in cell–cell signaling, they must recognize their specific cellular target, bind to that cell, and undergo internalization (Fig. 2).

Figure 2.

Pathways shown to participate in EV uptake by target cells. EVs transport signals between cells and facilitate selective reprogramming. EVs have been shown to be internalized by cells through (1) phagocytosis and (2) clathrin- and (3) caveolin-mediated endocytosis. There is also evidence to support their interaction with (4) lipid rafts resulting in EV uptake. Lipid rafts are involved in both clathrin- and caveolin-mediated endocytosis. EVs may also deliver their protein, mRNA, and miRNA content by (5) fusion with the plasma membrane. EVs can be internalized by (6) macropinocytosis where membrane protrusions or blebs extend from the cell, fold backward around the EVs, and enclose them into the lumen of a macropinosome; (7) alternatively, EVs are macropinocytosed after becoming caught in membrane ruffles. On the other hand, (8) intraluminal EVs may fuse with the endosomal limiting membrane following endocytosis to deliver their protein, mRNA, and miRNA cargo and elicit a phenotypic response.

Target cell recognition.

EVs may interact with recipient cells by direct signaling through ligand/receptor molecules on their respective surfaces or by direct fusion of EV and recipient cell plasma membranes (211) through lipid raft–, clathrin-, and calveolae-dependent endocytosis, micropinocytosis, and phagocytosis (212–217).

Cell surface and integral membrane/adhesion proteins on distinct EVs are important in mediating associated cell recognition and adhesion. These include integrin pairs: for example, distinct EXO integrin repertoires, specifically integrins α₆β₄ and α₆β₁, were identified as associated with lung metastasis, whereas EXO integrin α_vβ₅ associated with liver metastasis (218). The integrin profile of each EXO subtype permits selective cellular targeting.

Differences in EXO tetraspanin complexes also appear to influence target cell interaction in vitro and in vivo, possibly by modulating the functions of associated integrin adhesion molecules (219). EXO capture by dendritic cells was reduced by 5% to 30% following coincubation with blocking antibodies specific for various integrins, adhesion molecules, or tetraspanins (212). Other membrane proteins reported as important in targeting selected EVs to recipient cells include intercellular adhesion molecule 1 and milk fat globule–epidermal growth factor VIII protein (220, 221). Furthermore, the delivery efficiency of EXOs to cells is reported to be directly related to rigidity of cargo lipids, including sphingomyelin and (N-acetylneuraminyl)galactosylglucosylceramide (23).

Recent new data indicate that proteoglycans and lectins can participate in EXO binding and internalization. Proteoglycans are cell surface proteins, whereas lectins, such as galectins 1, 3, and 5, which recognize and bind proteoglycans, are identified on EVs. Indeed, proteoglycan receptors along the plasma membranes of cells and proteoglycans on EXO surfaces have been shown to promote docking (222).

Exosome uptake and release of cargo.

EV internalization by recipient cells is reported to occur via multiple processes such as phagocytosis (197, 212, 213), clathrin-mediated endocytosis (223), macropinocytosis (217), receptor interaction (224), and direct fusion (23). However, a better understanding of the underlying mechanisms and, importantly, whether EV subtypes have distinct mechanisms of uptake at their target cell specificity is required (225–229) [reviewed in (230)].

EV uptake is readily demonstrated in cell culture using fluorescently labeled EVs (91). Uptake and cargo release occur very rapidly, within minutes to hours. However, such techniques do not absolutely prove release, as it is possible that the transfer and spread of fluorescence results from the culture conditions and lipid/membrane transfer.

Recent developments in modification of EVs have also facilitated monitoring and tracking their behavior, interaction, and transfer in vivo (231). Intracellular probes are used to fluorescently label mRNA within EVs to monitor EV-borne mRNA encoding luciferase. Developments in transgenic mice enable visualization of EV transfer to cells associated with tumor stroma (232) and immune cells (233, 234), whereas EV-mediated transfer of donor genomic DNA to recipient cells supports a mechanism for genetic influence between cells (235). Such in vivo approaches have not specifically shown whether transfer involves a direct fusion of EVs with the recipient cells, formation of gap junctions or nanotubes, or phagocytosis of live or apoptotic cell–derived EVs by the recipient cell.

Low pH is important for EXO uptake. There appears to be elevated stability and lipid/cholesterol content of exosomal membranes in an acidic environment (23).

Understanding recipient cell function and regulation by EXOs needs to focus on specific mechanisms of targeting and delivery, uptake, and transfer, including modulation of key signaling pathways in various recipient cells both in vitro and in vivo. Processes that control target cell recognition and EV uptake are not well understood.

Inhibiting EV recognition and uptake

Although several uptake mechanisms have been proposed for EVs, detailed knowledge regarding the key steps in EV target cell definition and definitive mechanisms of uptake is required (230) particularly because variability is found between cell types in vesicle internalization (236). The use of inhibitors is proving useful in elucidating cell type–specific mechanisms.

As discussed, using fluorescently labeled EVs, internalization can be readily observed in vitro within a short period of time (91, 237). Treatment with inhibitory agents such as chlorpromazine to examine clathrin-dependent uptake (214) and specific RGD inhibitory peptides (238) to target integrin-mediated EV uptake allows identification of selective processes of internalization. The efficacy of EV exchange between cells probably depends on their surface antigen repertoires because partial digestion of membrane proteins exposed on EVs with proteinase K can significantly decrease their uptake (214), and blockage of selected integrins or tetraspanins with monoclonal antibodies also has suppressive effects on EV internalization (212). Furthermore, the use of cytochalasin D, which interferes with actin polymerization and endocytosis, significantly reduces the uptake of EVs (214, 215). Similarly, the inhibition or knockout of dynamin, a GTPase responsible for formation of endosomal vesicles, significantly suppresses EV uptake (216). Further research is needed to understand the precise mechanisms that underpin distinct EV entry into selected target cells and importantly how to control this process.

EVs as Messengers in Reproductive Physiology

Normal reproductive processes are highly dynamic, with well-characterized stages. The considerable intercellular interactions involved at each stage have prompted the study of the involvement of EVs in both the male and female reproductive tracts, from preconception to birth. EVs associated with reproductive biology have been specifically identified and studied in different fluids such as prostatic and epididymal fluid (239), seminal fluid (7, 155), follicular fluid (240, 241) oviductal fluid (242, 243), cervical mucus (244), uterine fluid (156, 171), amniotic fluid (115, 245), and breast milk (246), as well as the originating tissues [reviewed in (247)] (summarized Table 3).

Table 3.

Main Functions of EVs in Reproductive Physiology Classified by Their Origin

EV Type	Main Features	Target	Function	References
Epididymosomes	First described by Pikó in 1967	SPZ	Transfer of molecules involved in sperm maturation (P25b, GliPr1L1, MIF, SPAM1, PMCA4)	(248–250, 252, 253)
	Sizes: 50 to 8000 nm or even 2 to 10 µm
			Protection from oxidative stress (BLVRA)	(251)
			Protection from lipid peroxidation (glutathione peroxidase 5)	(256)
	Two main classes: CD9-positive (affinity for live SPZ) and ELSPBP1-enriched (affinity for dead SPZ) epididymosomes		Morphology and membrane composition regulation (ADAM7)	(254, 255)
			Sperm motility (ADAM7, PMCA4)	(253–255)
			Small RNA regulation of gene expression	(257, 258, 291)
Prostasomes	First described by Ronquist et al. in 1978	SPZ	Enhancement of sperm motility (progesterone receptors, Ca2+ cascade signaling components, aminopeptidase N)	(261, 263, 264, 265, 266)
			Protection from acidic female reproductive tract environment	(262)
	Sizes: 30 to 500 nm		Protection from oxidative stress (PMCA4)	(264)
	Unusual lipid composition that provides them with increased ordered structure, rigidity, and viscosity		Prevention of premature capacitation and acrosome reaction (cholesterol)	(260, 267–269)
			Posterior induction of capacitation, SPZ hypermotility, and acrosome reaction at the moment of fertilization (cAMP, progesterone receptors, hydrolases, lipoxygenases)	(270– 274)
			Protection from the hostile female reproductive tract: immunity, oxidative stress, bacteria	(259, 260, 270)
Uterine microenvironment EVs	Wide variety of origins: serum transudates, residues from womb cell apoptosis, endometrial epithelial cells, and conceptus	Endometrium: endometrial origin	Promotion of embryo implantation (specific miRNA cargo)	(171)
	Variations throughout the menstrual cycle	Endometrium: embryo origin	Regulation of endometrial angiogenesis (specific miRNA and protein cargo) and uterine spiral arteries remodeling	(285, 286)
	—	Embryo: endometrial origin	Embryo development (enJSRV env gene RNA) and subsequent priming of the endometrium for embryo harboring	(282–284)
			Promotion of embryo implantation (miR-30d, specific protein cargo, influenced by uterine hormones—functional with trophoderm)	(91, 156)
		Embryo: embryo origin	Enhancing of trophoblast cells migratory ability and implantation efficiency (laminin, fibronectin)	(88)
		SPZ	Sperm maturation (SPAM1)	(287)
			Capacitation, acrosome reaction and motility promotion (PMCA4)	(243, 288)
Oviductal EVs	First described for their implications in SPZ final competence acquisition	SPZ	Regulation of SPZ storage and promotion of capacitation, acrosome reaction, and hypermotility (PMCA4a)	(243, 279, 280, 289)
			Regulation of molecule delivery into SPZ (integrins α5β1 and αvβ3)	(242)
		Embryo	Enhancement of embryo quality and early development	(281)
Follicular EVs	First described by da Silvera et al. in 2012	Cumulus–oocyte complex	Follicle development and oocyte growth (specific miRNA cargo, ACVR1, ID2)	(275, 277)
			Follicle maturation: proliferation of small follicles and inflammatory response of large developed follicles (specific miRNA signatures)	(276)
	miRNA cargo variation with female age and reproductive aging
			Cumulus–oocyte complex expansion and related genes upregulation	(278)

Abbreviation: SPZ, spermatozoa.

There are currently increasing data pointing to EVs as key regulators of different reproduction processes such as sperm and ovum maturation, coordination of capacitation/acrosome reaction, prevention of polyspermy, endometrial embryo crosstalk, and even communication between in vitro–cocultured embryos leading to quorum improved development (291). In these initial steps of the reproductive process (e.g., preconception) EVs are widely produced by different organs and show specific functions. Once implantation has taken place, production of EVs continues throughout pregnancy, with the placenta being the main source of EVs. During early pregnancy, EVs are released by the extravillous trophoblast (EVT). Later on, the syncytiotrophoblast (STB) is formed and establishes contact with maternal blood flow. From here on, STBs constitute the main site of EV generation, and these EVs get access to the maternal systemic vasculature, where they show important roles in immune modulation, either for the innate or the adaptive response (32). Of note, EVs are also found in amniotic fluid, where they are attributed to inflammatory and procoagulant activities (292), and in maternal breast milk. In the latter case, an important influence of EV recovery procedure has been detected on subsequent analysis (293). Among attributed roles, milk EVs have been involved in bone formation, immune modulation, and gene expression regulation, with special emphasis for long noncoding RNAs (246, 294).

EVs in the male reproductive tract: epididymis and prostate

After leaving the seminiferous tubules, spermatozoa (SPZ) are still immature cells. SPZ are stored in the epididymis where they undergo a series of morphological and biochemical modifications that provide them with motility and fertilization ability in their transit from the caput to the cauda, a process called sperm maturation (295, 296). During ejaculation, SPZ mix with seminal fluid from the seminal vesicles, the prostate, and the bulbourethral gland to form the ejaculated semen, which is ejected into the vaginal cavity. Seminal fluid composition is crucial in promoting sperm motility and genomic stability (295, 297). Moreover, it contributes to the establishment of maternal immune tolerance (298, 299). Subsequently, as SPZ travel through the female reproductive tract to the upper fallopian tube where fertilization occurs, they interact with the endometrial and tubal milieu. Finally, to achieve successful fertilization, SPZ undergo capacitation: sperm head membranes undergo a series of biochemical modifications that enable the acrosome reaction when the spermatozoon reaches the zona pellucida of the oocyte. This leads to the release of enzymes that allow SPZ to penetrate the zona pellucida and fuse with the oocyte plasma membrane (300–302). In this context, secretions from the different components of the male and female reproductive tracts have been proposed to play a sequential role in programming sperm function (303).

Epididymosomes

Epididymosomes (EVs originating from the epididymis) were first described in 1967 by Pikó (304) in the Chinese hamster as having diameters between 20 and 100 nm and being associated with the SPZ acrosomal membrane (305). More recently, it has been shown that epididymosomes are a population of roughly spherical bilayered vesicles that display heterogeneity both in size and content that varies between the different segments of the epididymis. Their sizes range from 50 to 800 nm or even to 2 to 10 μm in the first segments of the caput (239). Their lipidic composition also varies: indeed, an increase in sphingomyelin and a general decrease in the other phospholipids and in the proportion of cholesterol occurs with epididymal progression from the caput to the cauda. This is in contrast to SPZ, where the proportions remain more constant. Epididymosomes also have an increased ratio of saturated/unsaturated fatty acids from the caput to the cauda, whereas the opposite situation is found in SPZ. Taken together, these data indicate that epididymosomes tend to gain membrane rigidity whereas SPZ membranes tend to become more fluid (239).

Two main classes of epididymosomes have been identified: CD9-positive epididymosomes, which preferentially bind live SPZ, and ELSPBP1-enriched epididymosomes, which present higher affinity for dead SPZ (306). CD9-positive epididymosomes are EVs of size ranging from 20 to 150 nm (307). These were recovered by ultracentrifugation from the total epididymal fluid EVs, specifically in the epididymis cauda. CD9-positive epididymal cargo transferred to SPZ includes proteins involved in sperm maturation, namely P25b, GliPr1L1, and MIF (248–250), in contrast to ELSPBP1, which was widespread between all EVs. Moreover, CD9, in cooperation with CD26, plays a role in promoting this transfer (307).

ELSPBP1-enriched epididymosomes constitute a subpopulation of vesicles obtained from the epididymal fluid by high-speed ultracentrifugation (120,000 × g) after SPZ and debris removal at 4000 × g (308). It had been suggested that ELSPBP1 allowed distinction between dead and viable SPZ, as it was only detectable in the dead SPZ population (309). Later, the same group demonstrated that epididymosomes were the only path for the transmission of molecules including ELSPBP1 to dead SPZ (307, 308). Interestingly, ELSPBP and biliverdin reductase A (BLVRA) can associate and bind in tandem to dead SPZ, concurrently with the epididymal maturation of SPZ, a process during which these cells cease producing BLVRA. Therefore, BLVRA could act as a quencher of reactive oxygen species generated by dead and immature SPZ, protecting viable SPZ from oxidative stress. Moreover, BLVRA may be involved in hemic protein catabolism, changes also important in the SPZ maturation process (251, 306).

“Normal reproductive processes are highly dynamic, with well-characterized stages.”

Because the epididymis brings SPZ to functional maturity before they enter the vas deferens, it is not surprising that epididymosomes serve as a means for protein transfer into SPZ during their transit in the epididymal duct. Some epididymosomal proteins have proven roles in sperm maturation: these include P25b, MIF, or sperm adhesion molecule (SPAM1), among others (252, 310). SPAM1 is a hyaluronidase with roles in both fertilization and sperm maturation. It is transferred to SPZ from epididymosomes, increasing their ability for penetrating the oocyte cumulus (253). Another protein transferred to SPZ by this mechanism is ADAM7, which is important for sperm motility, morphology, and establishment of membrane correct protein composition (254, 255). Of note is the transfer of the plasma membrane ATPase 4 (PMCA4), a major Ca²⁺ efflux pump, into epididymosomes: this plays a pivotal role in SPZ maturation and motility (253). Glutathione peroxidase 5 associates with SPZ during its transit through the epididymis, protecting them from lipid peroxidation stress and, independently, is transferred to SPZ via epididymosomes (256). Finally, components of the Notch pathway are described in epididymosomes, suggesting that these vesicles transmit Notch signaling at a distance between epididymal epithelial cells, but also between the epididymis and SPZ with important implications for sperm motility (311).

Epididymosomes also convey miRNAs within the epididymal duct. As with proteins, distinct regions of the epididymis produce EVs with a specific set of miRNA whose profiles differ from those of parent cells, suggesting a sorting mechanism (290). Indeed, it has been proposed that epididymosomes may act as modulators of gene expression between sections of the epididymal duct (290). Recent analysis confirmed that they contain >350 miRNA, showing a different profile from that of parent cells and dependent on the region of the epididymis from which they originate. Finally, it was demonstrated that many of these miRNAs are transported into the SPZ (312).

An emerging concept is the transfer of traits to the offspring by epigenomic modifications. In this respect, tRNA has been attributed a new function as a modulator of genetic expression. It was initially discovered that a respiratory syncytial virus infection of lung and kidney cell lines led to the production of specific tRNA fragments (tRFs) that are able to repress the expression of specific mRNAs in the cytoplasm to favor viral replication and survival (313). Subsequently, further examples of tRFs have been described with potential implications in pathological processes, such as cell proliferation in cancer (314). Mature molecules corresponding to tRNA fragments are highly enriched in mature sperm. Interestingly, these fragments are produced by sequence-specific cleavage, giving place to fragments corresponding to the tRNA 5′ end (257). Recently, the transfer of tRFs to maturing SPZ in epididymosomes was demonstrated in mice (258), providing an explanation for the scarcity of these molecules in testicular SPZ but with an increase with SPZ maturation. A tRF (tRF-Gly-GCC) has been identified as transferred to SPZ by epididymosomes. This tRF represses MERVL, an endogenous retroelement that positively regulates a set of genes that are highly expressed in preimplantation embryos. Interestingly, male mice treated with a low-protein diet have a trend (nonsignificant) to increased tRF-Gly-GCC in mature SPZ and to downregulate tRF-Gly-GCC targets in embryos at the two-cell stage. This evidence supports that parental diet can affect the offspring epigenome; however, these preliminary data require confirmation (258).

Prostasomes

Prostasomes were first described as vesicles recovered from human prostatic fluid by centrifugation that were associated with Mg²⁺- and Ca²⁺-dependent ATPase activity (315). They are now considered a population of EVs produced by the prostate epithelial cells that interact with SPZ, epididymal, and seminal secretions at the time of ejaculation. They are EVs that range from 30 to 500 nm and are surrounded by lipoprotein bilaminar or multilaminar membranes (259, 316). It is likely that a population of prostasomes is exosomal, as they originate from structures resembling MVBs and exhibit classical EXO markers (317). The lipid composition of prostasomes is unusual and provides them with a characteristic highly ordered structure, rigidity, and viscosity due to several factors: a high cholesterol/phospholipid ratio reaching values of 2, which greatly surpasses the values for most of biological cholesterol-rich membranes; phospholipid composition domination by sphingomyelin, which accounts for almost a half of the phospholipids found in prostasomes (318); and finally prostasomes show a strongly saturated fatty acid profile in comparison with SPZ membranes (319). It has been reported that prostasome uptake decreases the fluidity of SPZ membranes by transfer of lipids directly dependent on the prostasome/SPZ ratio (316, 320). This decrement is crucial, as it stands as a regulator of the acrosome reaction, preventing a premature response (260).

Different roles have been attributed to prostasomes in sperm maturation and function, either directly or indirectly. These include protection of SPZ from the female acidic environment and immune surveillance modulation of SPZ motility, capacitation, acrosome reaction, and fertilizing ability, among others (259, 260, 316, 317).

SPZ motility is vital for a successful fertilizing ability, especially for traversing the cervical mucus and zona pellucida (321). One of the first roles attributed to prostasomes was the enhancement of SPZ motility (261) in a pH-dependent manner, suggesting that prostasomes might alleviate the negative effects of the vaginal acidic microenvironment on SPZ motility, thus showing a protective effect (262). Ca²⁺ has been well known as the major ion promoting SPZ motility and fertility, from initial studies carried out in the hamster (322). Increased SPZ Ca²⁺ levels have been linked to prostasomal delivery, directly depending on the extent of fusion/prostasome concentration and influenced by pH (323). However, it took a decade to identify a mechanism. Park et al. (263) showed that a progesterone-triggered long-term sustained Ca²⁺ stimulus is involved in SPZ motility promotion via fusion of (acidic) pH-dependent prostasomes. Specifically, prostasomes transferred progesterone receptors and different Ca²⁺ signaling cascade components to the SPZ neck region where, following progesterone stimulation, they triggered the release of Ca²⁺ from SPZ internal acidic stores to promote SPZ motility (263). Other proteins involved in intracellular Ca²⁺ homeostasis are also transported into SPZ in prostasomes, including PMCA4 (264), which along with nitric oxide synthases (NOSs) are delivered into SPZ by prostasomes. PMCA4 and NOS activity is stimulated by Ca²⁺ ions (324) and, indeed, NOS spatially interacts with PMCA4 to a degree positively related to Ca²⁺ concentration levels. This supports the theory that PMCA4 expels Ca²⁺ from SPZ in the presence of NOS to reduce nitric oxide production and thus oxidative stress, which could reduce SPZ viability, resulting in asthenozoospermia (264). Prostasomes also carry aminopeptidase N, a protein involved in modulating sperm motility, which acts through the regulation of endogenous opioid peptides, such as enkephalins, once in SPZ (265, 266).

Interestingly, EXO-like EVs found in cervical mucus have been reported to carry sialidase activity, which reaches a maximum during the ovulatory phase in healthy women. This is likely involved in modifying the properties of the highly glycoslyated mucus to favor SPZ access to the uterine cavity and tubes (244).

There are scarce data on the nucleic acid cargo of prostasomes and its implications for male reproductive physiology. Prostasomes contain various coding and regulatory RNAs, with potential modulatory functions (199). Interestingly, mRNA and miRNA do not represent most of the prostasomal RNA (317), and it has been postulated that mRNA in semen is predominantly transported inside vesicles whereas miRNA is mostly contained in the vesicle-free fraction of the semen, forming complexes with proteins (204). DNA inside prostasomes apparently represents random regions of the genome and is effectively transported into SPZ (325, 326). Nevertheless, this DNA may be a contaminant from ABs in the semen (327).

Capacitation is a cyclic adenosine monophosphate (cAMP)–regulated process, whose production is in turn promoted by bicarbonate and Ca²⁺ ions and influenced by membrane dynamic changes mainly due to cholesterol composition (259, 317). It has been proposed that prostasomes may act as inhibitors of the capacitation process and acrosomal reaction, mainly through transfer of cholesterol (267, 268). Indeed, this might represent a mechanism to avoid premature capacitation and acrosome reaction (260, 269). A switch between positive and negative regulation exerted by prostasomes may be influenced by the environment or even determined by specific prostasome subpopulations. cAMP promotes capacitation through the protein kinase A (PKA) axis by the simultaneous tyrosine phosphorylation of specific downstream proteins and plasma membrane protein and lipid remodeling. This remodeling breaks down plasma membrane asymmetry, exposing cholesterol molecules to external acceptors to trigger the capacitation process (270). In this context, coincubation of equine SPZ with prostasomes led to increased cAMP levels and tyrosine phosphorylation of PKA cascade proteins, in addition to the prostasome endogenous PKA activity described in previous reports. However, these changes were not correlated with increased capacitation and acrosome reaction rates and reverted after 3 hours of coincubation in capacitating conditions (267). Interestingly, Aalberts et al. (270) observed that at least a subpopulation of prostasomes is able to bind to live SPZ only when capacitation-inducing conditions are established, probably to promote hypermotility and acrosome reaction at the precise moment it is needed. Nonetheless, care should be taken when interpolating these results into humans, as they were obtained from a stallion model, a species that deposits its ejaculate directly in the uterus.

Following capacitation, SPZ need to undergo an acrosome reaction to enable penetration of the zona pellucida of the oocyte and fusion of plasma membranes. The zona pellucida glycoprotein ZP3 is mandatory for this process, as it facilitates sperm binding, triggering the acrosome reaction. Nevertheless, the acrosome reaction begins before the SPZ contacts the zona pellucida, probably due to the progesterone-dependent stimulus produced by cumulus cells (328). Conversely, prostasomes have been proposed as inhibitors of the acrosome reaction through the transference of cholesterol to the SPZ (268, 329) or as inducers by facilitating progesterone uptake by the SPZ (271), most likely by the transfer of progesterone receptors (263). Other studies also supporting the promotion of the acrosome reaction via prostasomes include delivery of molecules to the SPZ membrane in a pig model (272), or progesterone priming, acting via the Ca²⁺ signaling axis (330). Other acrosome reaction–promoting molecules in prostasomes include hydrolases (273) and lipoxygenases (274).

In summary, the role of prostasomes in sperm-fertilizing ability in humans is most likely the result of orchestrated actions. Initially, prostasomes would attach to SPZ after mixing during ejaculation, favored by the acidic environment of the vagina, thus transferring cholesterol to stabilize SPZ membranes and prevent premature capacitation and acrosome reaction. This would enable prostasomes to pass the barrier of cervical mucus adhered to SPZ with subsequent fusion and transfer of their content to the SPZ when the SPZ first contacts the oocyte. At this time, the progesterone secreted by the cumulus cells would activate Ca²⁺-dependent pathways that promote the capacitation process and acrosome reaction (259).

Finally, of note is the role of prostasomes in protecting SPZ from the potentially hostile female genital tract. They appear to exert roles as protectors from female immunity, antioxidants, antibacterial agents, and the process of semen liquefaction [see reviews in (259, 260, 270)].

EVs in the female reproductive tract: follicular fluid, oviduct/tube, and uterine cavity

Contemporarily with sperm maturation, coordinate oocyte development must be taking place so that both gametes can meet at the appropriate location and time inside the female reproductive tract. Developing oocytes are arrested in prophase I of meiosis in primordial follicles from the fetal period until female reproductive maturity. From this moment, cohorts of these oocytes cyclically restart growth, forming the zona pellucida, while granulosa cells proliferate to form the cumulus, which will support posterior egg fertilization. Concomitantly, meiosis is reinitiated, extruding the first polar body and arresting again at metaphase II during ovulation (331). The resumption of meiosis is stimulated by the luteinizing hormone peak, which in turn is initiated by a surge in estradiol-17β levels due to the secretion by granulosa cells from the preovulatory follicle and results in ovulation 36 hours later (332). After ovulation, the extracellular matrix of the cumulus cells serves as an adhesion dock for the fallopian tubes, through which the eggs travel as far as the ampulla where they await SPZ for fertilization (333). Following fertilization, embryo development to the blastocyst stage proceeds as the embryo passages through the fallopian tubes, reaching the uterine cavity ∼4 days after ovulation. The blastocyst undergoes final preparation for implantation into the maternal endometrium in the microenvironment of uterine fluid with implantation occurring 6 to 10 days after ovulation (334).

The process of embryo implantation can only occur during a short period of time during the luteal phase of the menstrual cycle, which has been classically regarded as the window of implantation and that typically extends from 5.5 to 9.5 days after ovulation in healthy normal cycling women (334, 335). At this point, different factors affect and limit embryo implantation, namely embryo quality, endometrial receptivity and embryo–endometrial crosstalk (336), where EVs stand as important potential mediators.

During this process, EVs carry out many different supporting actions: they assist follicle and oocyte development and maturation at the initial stages, and they further assist early embryo development and implantation as the conceptus reaches the uterus. Furthermore, female tract EVs contribute in preparing endometrial vascular net, promote embryo implantation, and prime the endometrium for harboring the embryo. Moreover, these EVs also contribute to SPZ maturity, capacitation, and acrosome reaction coordination, support SPZ storage while waiting for the oocyte, and regulate molecule delivery into SPZ during this period. All of these concepts are discussed in the following sections.

“Following capacitation, SPZ need to undergo an acrosome reaction to enable penetration of the zona pellucida.”

Follicular fluid EVs

Oocyte maturation occurs within the microenvironment of follicular fluid (337). The easy availability of this fluid during oocyte retrieval in assisted reproductive techniques makes it attractive in the search of biomarkers for oocyte quality (338). EVs (resembling EXOs and MVs) were first identified in follicular fluid by da Silveira et al. (339), who demonstrated follicular fluid EV uptake by granulosa cells, both in vivo and in vitro, and their protein and miRNA cargo. EV miRNAs were also present in the surrounding granulosa and cumulus cells, thus suggesting EVs as a vehicle for biomolecule transfer within the ovary. Of particular interest, the miRNA signature of follicular EVs varied with the age of the female, suggesting EV miRNA cargo as an indicative and possible predictor of age-related decline in oocyte quality (339). Subsequently, EV miRNAs were further evaluated and a set of four differentially expressed miRNAs based on age (young/old) was defined. However, these age-related miRNAs were studied in complete follicular fluid samples and as such cannot be confidently attributed to EVs (340).

The miRNA of bovine follicular fluid is present both as free miRNAs and in EXOs, each with different composition (275). The EXOs were taken up by granulosa cells in vitro, resulting in increased miRNA content and variations in mRNA profiles: some of the affected genes are involved in follicle development. Moreover, some of the miRNA within EXOs may also contribute to oocyte growth, as they were differentially expressed in follicles containing oocytes at different maturation stages (275). A more exhaustive characterization of the EV content of bovine follicular fluid demonstrated variation in number, protein markers, and miRNA contents depending on the developmental stage of the follicles. What is more interesting, variation in the miRNA signature suggested a switch in genetic programming concurrent with follicular maturation. As such, EV miRNAs from small follicles preferentially promoted cell proliferation pathways whereas those from large follicles related to inflammatory response pathways (276). A possible role in follicle development through the TGFB/BMP axis by follicular fluid–derived exosomes was demonstrated when granulosa cells were exposed to follicular fluid exosomes in vitro. It was proposed that these effects were triggered by the direct delivery of ACVR1 and ACRV1 regulatory miRNA within follicular EXOs to granulosa cells (277).

Cumulus–oocyte complex expansion is a critical process for ovulation. In this context, in vitro coculture experiments using bovine follicular fluid–derived EXOs 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 (278).

Oviduct/tubal EVs

Fertilization of the oocyte by SPZ occurs within the fallopian tubes/oviduct. After capacitation, SPZ must undergo an acrosome reaction and maintain hyperactivated motility to fuse with the oocyte, with both functions being regulated by high intracellular Ca²⁺ concentration levels. In this context, the major murine Ca²⁺ efflux pump PMCA4, and particularly its splicing variant PMCA4a, is predominant in oviductal fluid, compared with uterine and vaginal fluids, and is totally associated with EVs. Moreover, these PMCA4a-carrying vesicles had exosomal characteristics and were taken up by SPZ, where the efflux pump was functionally relocated to their membranes. This was the first study describing the presence of EXOs in the oviducts and introduced the relevance of PMCA4 as a tool for the maintenance of Ca²⁺ homeostasis and SPZ viability during SPZ storage, regulating capacitation and acrosome reaction timing and SPZ motility (242, 243, 279, 280). Subsequently, the same authors discovered that integrins (α₅β₁ and α_vβ₃) in oviductal EVs were transferred to SPZ and were involved in EV–SPZ fusion for cargo delivery. Although the oviductal EVs include both MVs and EXOs, the former appeared to be more efficient in fusing with SPZ (242).

Bovine oviductal EVs produced in vitro by cell lines have beneficial effects on the quality and development of in vitro–cocultured bovine embryos, suggesting a functional communication between the oviduct and embryo during the early stages of embryo development (281). However, these results must be treated with caution, as oviductal EVs produced in vitro have been observed to carry a different cargo compared with in vivo–produced EVs. This is the case, for example, for OVGP and HSPA8, oviductal proteins known to be important in the fertilization process and early pregnancy. Whereas HSPA8 was found in both in vitro and in vivo EXOs, OVGP was absent in EXOs of in vitro origin (341).

Uterine EVs

Endometrial fluid is a viscous liquid secreted by the endometrial epithelial cells from the glands into the uterine cavity. Because the endometrium is a hormonally regulated organ, the molecular composition of the fluid varies depending on the phase of the menstrual cycle (342). Uterine fluid, a biologically and clinically relevant sample source (343), also contains contributions from the oviductal fluid and a large cohort of plasma proteins along with other factors differentially exudated from the blood (344). Importantly, this uterine fluid carries information that mirrors maternal environmental exposure and possibly relays such information to the embryo, subsequently generating long-term epigenetic effects on the offspring via embryonic and placental programming.

To date, EVs have been reported throughout menstrual/estrous cycles in the endometrial fluid of different species, including humans (156, 171) and sheep (282–284, 345), and they are also released by endometrial epithelial cells in culture (156, 171).

Ng et al. (171) first described the production of EVs by human endometrial epithelial cells in primary culture and by the endometrial epithelial cell line ECC1. These EVs contained a specific subset of miRNAs, not detectable in the parent cells. Bioinformatic analysis revealed that some of the target genes of the EV miRNAs are relevant to processes involved in embryo implantation. Importantly, they also verified the presence of EVs in human uterine fluid and the associated mucus (171).

Greening et al. (91) demonstrated that the proteome of highly purified EXOs derived from human endometrial epithelial cells is regulated by steroid hormones and thus varies with the progression of the menstrual cycle. Under follicular phase hormonal conditions, when estrogen constitutes the main hormonal stimulus, the EXO proteome was enriched in proteins related to cytoskeletal reorganization and signaling cascades, coinciding with the phase of endometrial restoration. Importantly, after ovulation, when progesterone is the dominant hormone driving endometrial receptivity, the proteome altered with changes indicating enrichment in extracellular matrix reorganization and embryo implantation. As in other systems, the exosomal protein profiles were shown to be distinct from parental cells. Importantly, this study demonstrated that endometrial EXOs were transferred and internalized by human HTR-8 trophoblast cells, enhancing their adhesive capacity, partially through focal adhesion kinase (FAK) signaling (91). This was significantly higher when the EXOs were derived from cells subjected to both estrogen and progesterone to mimic the receptive phase of the menstrual cycle.

Embryonic and trophectodermal EVs

Interestingly, murine embryonic stem cells from the inner cell mass generate MVs that reach the trophectodermal layer and enhance the migration ability of trophoblast cells in culture, either as isolated cells or in the whole embryo. The presence of laminin and fibronectin in the cargo of the inner cell mass EVs enabled attachment to the integrins on the trophoblast cell surfaces and stimulated c-Jun N-terminal kinase and FAK cascades, increasing trophoblast migration. Furthermore, injection of these EVs inside the blastocoele cavity of day 3.5 blastocysts increased their implantation efficiency (88). Importantly, note that this mechanism may be particular to the mouse and to other species in which the inner cell mass is distal to the site of trophectoderm attachment to the endometrial surface: in women this is the reverse, with the extracellular matrix tightly aligned with the attaching trophectoderm.

EVs produced by ungulate trophectoderm participate in crosstalk with the maternal endometrium (283). Bidarimath et al. (285) observed that EVs from a porcine trophectodermal cell line stimulated the proliferation of endothelial cells in vitro, thus being potential regulators of maternal endometrial angiogenesis. These vesicles contained an miRNA and protein cargo likely to annotate functions in the angiogenesis process. Again, care should be taken with these data, as they were retrieved from cell lines cultured in vitro. Furthermore, the pig is a species with epitheliochorial placentation, and thus the in utero development is very different from that of the human (285). Nevertheless, study of human EVT cell (HTR-8/SVneo and Jeg3)–derived EXOs similarly showed that these vesicles promote vascular smooth muscle cells migration, which is important during human uterine spiral artery remodeling in successful pregnancies (346). Importantly, the two trophoblast cell lines (which are different stages along their differentiation pathway) produced differential migration results, raising the likelihood that cell origin as well as content and bioactivity of the exosomal cargo are of considerable importance, emphasizing the need to keep models as close to the physiological situation as possible.

EVs as vehicles for embryo–maternal crosstalk.

The first indication that the endometrium produced EVs with unique cargo was that the human endometrial epithelial cell model ECC1 (which best represents luminal epithelium) released EVs containing a different miRNA profile from that of parent cells (171). These EVs could provide a mechanism for communication between the mother and the embryo with potential implications in embryo implantation. Indeed, bioinformatic analyses on the EV miRNAs showed predominance of the genes targeted by the miRNAs as involved in implantation. Furthermore, interrogation of the proteome of ECC1 EVs, cultured under conditions to represent the proliferative (estrogen-dominant) and secretory (estrogen plus progesterone) phases of the cycle, showed that the protein cargo of EVs is hormone specific, enriched with 254 and 126 proteins, respectively (91). Importantly, 35% of the endometrial EV proteome had not been previously reported, indicating the unique cargo of endometrial EVs. These findings were validated in EVs from primary endometrial epithelial cells. Functionally, the EVs were internalized by human trophoblast cells, inducing increased adhesive capacity, that was at least partially mediated through active FAK signaling, indicating a likely role in promoting embryo implantation (109). Interestingly, among the implantation-related proteome of these endometrial EXOs were the cell surface metalloproteinases ADAM10 and matrix metalloproteinase-14 (a membrane-bound matrix metalloproteinase), for which there are abundant substrates on the trophectoderm.

“Fertilization of the oocyte by SPZ occurs within the fallopian tubes/oviduct.”

Another study showed that endometrial epithelial-derived EVs in the uterine fluid contain hsa-miR-30d during the receptive phase of the cycle. This EXO-associated hsa-miR-30d was internalized by mouse embryos via the trophectoderm, resulting in an indirect overexpression of genes encoding for certain molecules involved in the murine embryonic adhesion phenomenon—Itgb3, Itga7, and Cdh5. Functionally, in vitro treatment of murine embryos with miR-30d resulted in a notable increase in embryo adhesion, again indicating how maternal endometrial miRNAs might act as transcriptomic modifiers of the preimplantation embryo (156).

Implications of EVs in Reproductive Pathology

Given their seminal functional role and presence in various aspects of reproductive biology, a growing field of evidence is uncovering potential roles for EVs in regulating reproductive pathological conditions, including endometriosis, polycystic ovaries syndrome, erectile dysfunction (ED), early pregnancy loss, hypertension, preeclampsia (PE), and gestational diabetes (GD) mellitus (summarized Table 4). Given this importance of EVs during maternal environment and development, significant efforts are now focused on evaluating prognostic value and applicability of EVs as diagnostic and therapeutic agents (107, 370).

Table 4.

Involvement of EVs in Reproduction-Related Pathologies

Disease	EV Pathogenic Role			References
Endometriosis	Promotion of endometriotic lesions invasion and progression			(347, 348, 350)
	Enhancement of angiogenic potential			(347, 349)
Polycystic ovaries syndrome	miRNA expression regulation toward PCOS phenotype			(351)
ED	Promotion of endothelial dysfunction, vascular damage, and atherogenesis			(352, 353)
Early pregnancy loss	Induction of an excessive procoagulant activity			(354, 355, 368)
	Promotion of endothelial dysfunction			(356)
PE	Placental origin	Promotion of abnormal remodeling of uterine spiral arteries		(333)
		Enhancement of angiogenic failure and subsequent endothelial dysfunction		(358)
		Stimulation of proinflammatory and procoagulant activities		(357–360, 366)
		Generation of oxidative stress into the placenta and mother vasculature		(361, 362)
	Maternal origin	General	Transportation of PE risk factors	(363)
			Failure to ensure appropriate vascular development	(367)
		Platelet EVs	Unleashing of thrombo-inflammatory placental response	(364)
		Leukocyte EVs	Promotion of proinflammatory cytokines release by the placenta	(365)
GD mellitus	Promotion of proinflammatory cytokines production by endothelial cells			(369)

Abbreviation: PCOS, polycystic ovarian syndrome.

EVs in endometriosis

Endometriosis is an estrogen-dependent inflammatory disease that is characterized by the deposition and growth of endometrial cells outside the uterine cavity, with the pelvic peritoneum and ovaries being the most common sites for ectopic growth (371). For this reason, endometriosis is considered a benign metastasizing disease (372).

Endometriosis is characterized in part by an increase in the expression of angiogenic factors and metalloproteinases. Patients with endometriosis show higher levels of these molecules in endometriotic lesions than in eutopic endometrium, and eutopic endometrium of endometriosis patients shows higher levels than in healthy endometrial controls (347). Indeed, by inhibiting metalloproteinases it is possible to avoid the establishment of ectopic endometriotic cysts (373). In this context, EMMPRIN, a metalloproteinase inducer, is carried in EVs produced by uterine epithelial cells and stimulates the expression of metalloproteinases in stromal fibroblasts. The secretion of both EMMPRIN and metalloproteinases is positively regulated by IL-1β/α, whose secretion is increased in women under endometriosis conditions in whom there is a proinflammatory peritoneal environment. This would allow the increase of metalloproteinase production by fibroblasts to trigger endometriotic lesion invasion (348).

In terms of EV RNA cargo, EVs from endometrial stromal cells from women with endometriosis vs women without the disorder showed different profiles of exosomal miRNA content between EVs derived from eutopic and ectopic endometrium from endometriosis subjects and between eutopic endometrium from women without or with disease (349). Moreover, there was a differential miRNA signature between eutopic endometriotic and control EXOs. Among these miRNAs, miR21 is already known for a role in angiogenesis. It remains to be established whether miR-21 can promote angiogenesis following EV uptake (349).

Ectonucleotidases are enzymes involved in inflammatory processes and previously reported as expressed in the endometrium. Teixidó et al. (350) investigated ectonucleotidase activity from endometriotic cysts (endometriomas) on the ovary, one of the common sites for endometriotic lesion development. Ectonucleotidases were highly enriched in endometriomas compared with simple cysts. Interestingly, the ectonucleotidase activity was also contained by EXOs derived from endometriomas and simple cyst fluids, but it was significantly higher for EXOs from endometriomas.

Polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) is one of the most common hormonal disorders affecting women, characterized by androgen excess and insulin resistance, leading to androgenism, high risk of glucose intolerance, diabetes, and lipid abnormalities (374). Its complex phenotypic manifestation was formally described nearly a century ago as the concurrence in women of amenorrhea, hirsutism, obesity, and typical polycystic appearance of the ovaries (375).

Koiou et al. (376) observed that platelet MVs in plasma from PCOS affected women (defined by elevated circulating androgens and insulin resistance markers) were at higher levels than in healthy controls. Moreover, there was a significant positive correlation between MVs and numbers of follicles in the ovaries of these women. Subsequent confirmation of the increase in EV levels (mainly of exosomal size) in PCOS also demonstrated a direct correlation with insulin resistance markers. Furthermore, polycystic ovary–derived EVs showed a higher content in annexin V along with 16 miRNAs that are normally expressed at low levels, being increased with PCOS (377).

Sang et al. (351) described EVs in the human follicular fluid and identified 120 miRNAs within their cargo, 11 of which were highly expressed and with target genes in pathways involved in reproduction, endocrine, and metabolic processes. Two of these miRNAs, miR-132 and miR-320, were significantly decreased in the follicular fluid EVs from PCOS patients compared with nonaffected controls (351). Of note, miR-132 and miR-320 have HMGA2 and RAB5B, respectively, as target genes: these were associated with key roles in the etiology of PCOS in a previous genome-wide association study (378).

DENND1A is a PCOS candidate locus, characterized in a number of genome-wide association studies (378, 379). DENND1A variants at two levels, both at the protein and mRNA levels, were increased in theca cells of PCOS patients compared with healthy controls. In agreement with these results, mRNA for this locus was significantly increased in EXOs extracted from the urine of PCOS-affected women in comparison with normal-cycling controls. In this sense, the exosomal miRNA profile is proposed to reflect the physiological status of the source cells, providing a potential biomarker of PCOS (380). Further studies are needed to uncover the roles of EVs in the triggering and development of PCOS.

Erectile dysfunction

ED is the most studied sexual problem worldwide and mainly affects men >40 years of age. It costs up to £7 million in the United Kingdom and $15 billion in the United States. The prevalence of this condition varies greatly throughout the world, highlighting the Middle East (45.1%), United States (37.7%), and especially mainland China (varying from 17.1% to 92.3%), according to a retrospective study carried out on men of different ages (381).

Microparticles have been proposed as involved in endothelial dysfunction and atherogenesis, with special regard to ED. Initially, microparticles defined as membrane vesicles, apoptotic or not, <1.5 µm were recovered from plasma after platelet depletion at 900 × g and measured by flow cytometry using specific markers (352). These circulating endothelial-derived microparticles were increased in type 2 diabetic men with ED, compared with controls, and a positive correlation between microparticle counts and ED severity, determined by the International Index of Erectile Function, was shown. However, diabetes risk factors did not influence microparticle levels, and thus these were postulated to be independently linked to ED severity. Finally, microparticles were proposed as possible links between endothelial dysfunction and ED (352). Retrospectively, a molecular signature identified in microparticles enabled discrimination between diabetes and ED. The marker CD31 in microparticles was mainly related to diabetes, whereas CD62E was directly linked to ED, without diabetes. The CD31/CD62 ratio could be used to evaluate endothelial function, with a high ratio being related to endothelial activation and a low ratio associated with apoptosis. In the study, diabetic men with ED showed lower ratios, possibly indicating a cooperative effect of the two disorders. Finally, levels of CD31⁺ microparticles were directly correlated with ED aggressiveness (382).

La Vignera et al. (353) increased the centrifugal force to achieve a better clearance of platelets from serum (13,000 × g). They confirmed an increase in endothelial-derived microparticle levels in ED patients with arterial etiology, in comparison with patients with ED of psychogenic origin. Because a positive correlation was observed with typical ED metabolic parameters, they proposed endothelial dysfunction as the cause underlying ED and reasserted microparticles as predictors of the condition. Furthermore, their levels were directly related to the aggressiveness of arterial ED (383): a combination of disorders leading to greater vascular damage was associated with more severe ED and endothelial dysfunction, and correlated with increasing levels of endothelial microparticles (384).

ED is associated with increased endothelial apoptosis, and both can be, in part, reverted by treatment with a type 5 phosphodiesterase inhibitor such as tadalafil (385). Treatment benefits were maintained for 4 weeks after the cessation of a 1-year treatment in almost half of the analyzed cases (386). Subsequently, the effect of tadalafil treatment and discontinuation on the production of apoptotic endothelial-derived microparticles was examined. ED patients had increased levels of apoptotic microparticles compared with controls before the start of the treatment. Ninety days of tadalafil administration improved International Index of Erectile Function score and endothelial parameters and reduced apoptotic microparticle levels, although not to control levels. These improvements reverted by 6 months after treatment discontinuation (387). Interestingly, complementation of tadalafil treatment with an antioxidant maintained the tadalafil effects at least until 6 months after treatment cessation, prolonging the duration of the antiapoptotic effect within the endothelium (388). This is in accord with other studies implicating oxidative stress in endothelial dysfunction (389, 390). Patients with greater severity and duration of ED, associated with the concurrence of high cardiovascular risk profiles, were nonresponders to sildenafil, another type 5 phosphodiesterase inhibitor.

Androgen deficiency has also been proposed to contribute to the development of cardiovascular disease and endothelial function impairment (391). Six months of androgen replacement therapy (testosterone) improved endothelial and ED features and decreased endothelial derived microparticle levels in patients of ED and late onset hypogonadism (a new vascular risk factor) (392). Indeed, late-onset hypogonadism worsened metabolic parameters and increased the already high endothelial microparticle levels in ED patients (393).

Pregnancy complications

EVs from a variety of sources (epididymis, prostate, cervical mucus, ovarian follicle, embryo, and endometrium) have potential roles in both the establishment and development of a successful pregnancy. However, from the sixth week of gestation (394), placenta-derived EVs mainly of STB origin represent the main source of vesicles with potential implication in feto–maternal communication (32, 87). Their concentrations in maternal plasma increase gradually as pregnancy progresses (286). Their release and bioactivity are favored by both low oxygen tensions (395) and high d-glucose concentrations (396). Changes in concentration, composition, and bioactivity of placental and nonplacental EVs have been reported in pregnancy disorders (397). Notably, the secretion of EVs is increased in the two main EV-related pregnancy complications, that is, GD (398) and PE (399).

EVs in early pregnancy loss

Early pregnancy loss is a common complication that affects ∼15% of the gestations and shows recurrence rates of 2% to 3%. Importantly, up to 50% of these cases are usually of idiopathic etiology (400). Interestingly, the levels of plasma endothelial microparticles are decreased in pregnancy loss, especially in cases with recurrent miscarriage, compared with controls (401). However, these results should be viewed with caution, as in healthy pregnancy (their controls) there is also an increase in EV levels, mainly due to the contribution of placenta-derived EVs (394).

In pregnancy, the hemostatic balance shifts toward upregulated procoagulant activity, with increased clotting factors and fibrinogen, and concurrently decreased anticoagulant factors and fibrinolytic activity (402). An excessive procoagulant response leading to thrombosis of the uteroplacental vasculature and subsequent hypoxia has been proposed as a factor accounting for an important part of the fetal loss cases (403). In this regard, blood microparticles with procoagulant activity are increased in miscarriage cases, in parallel with the enhanced coagulation-promoting activity. These microparticles may play a role in this outcome by favoring the thrombotic phenomena (354, 355). Furthermore, pregnancy loss–affected women present with lower levels of platelet microparticles and higher levels of endothelial microparticles than do controls; although this could not be directly related to the hypercoagulation phenotype, it was suggested to reflect endothelial dysfunction (356). In contrast, plasma platelet-derived microparticles were increased in women with recurrent miscarriage compared with controls (376). However, these results may be biased by the small size of the study population (404), and the controls may be inappropriate due to the contribution of the placenta to the total EV content.

EVs in gestational vascular complications

Gestational vascular complications, which include hypertension and PE, are prevalent causes of maternal and fetal morbidity and mortality. Hypertension may appear as a consequence of abnormal placentation into the maternal uterus, and it may lead to the development of impaired liver function, progressive renal insufficiency, pulmonary edema, and the new onset of cerebral or visual disturbances that might end in the hemolysis, elevated liver enzymes, and low platelet count syndrome and/or eclampsia (405). PE is a complex disorder causing preterm birth, intrauterine growth restriction, and maternal death (406). In general, different studies point toward an increase in endothelial microparticle shedding within gestational vascular complication conditions, thus suggesting vascular injury (407).

Preeclampsia

PE is a pregnancy-related syndrome affecting between 2% and 8% of pregnancies and is characterized by a variety of systemic symptoms. It is detected by new-onset hypertension and proteinuria after the 20th week of gestation. Its etiology is not well known, but the pathogenesis of PE is conceptualized in a two-stage model with the placental defect precipitating an abnormal vascular maternal response that manifests as the signs of this pathological condition. Early PE appears before 34 weeks of gestation and involves the fetus, showing reduced placental perfusion, possibly due to abnormal trophoblast invasion and/or uterine spiral artery remodeling. Late PE appears after 34 weeks, and the maternal manifestations appear; a series of inflammatory, metabolic, and thrombotic responses compromise vascular function up to the point of producing systemic organ damage (408).

Several published studies have attempted to elucidate the relevance of EVs of both maternal and placental STB origin in the pathophysiology of PE. Changes in EV concentration and cargo affect PE development via proinflammatory and procoagulatory activity enhancement. In this section, we summarize current knowledge of EVs in relationship to PE.

Placenta-derived EVs.

The placenta plays a critical role and is undoubtedly the source of PE development. PE can develop even in the absence of a fetus, provided that trophoblast tissues are established, forming the characteristic mass known as a hydatidiform mole, a tissue abnormality formed by the distension of some or all of the chorionic villi (409).

STB-derived EXOs and MVs (STBMs) are increased in PE compared with normal pregnancies (357), perhaps in part owing to the hypoxia that results from abnormal placentation (346). This increase occurs specifically in early-onset PE cases but not in late-onset PE or normotensive intrauterine growth restriction (410, 411). Importantly, early-onset PE is established in the first trimester when trophoblast invasion and vascular remodeling occur (346), emphasizing the importance of STBMs in these processes. Furthermore, variations in protein (399, 412, 413), lipid (414), and miRNA (399) cargo of STBMs may explain the specific roles of STBMs in PE, including immune response, coagulation, oxidative stress, and apoptosis.

One of the main characteristics of PE is abnormal remodeling of the uterine spiral arteries, which in normal pregnancies ensures enough maternal blood flow to support fetal growth and development. Thus, a role for EVT-derived EVs has been proposed in PE development. Variations in concentration, cargo, and bioactivity of EVT-derived EVs as indicated above may result from a proinflammatory environment, inducing these changes, impairing their physiological roles in vascular/smooth muscle tissue remodeling, and thus stimulating the emergence of PE (346, 415). In PE, increased amounts of proinflammatory cytokines (IL-18, IL-12, TNF-α) are released by monocytes and lymphocytes. PE-increased STBMs can bind monocytes to promote the production of more inflammatory cytokines, perpetuating the proinflammatory environment and hence stimulation of EV alterations and endothelial cell damage (357). Furthermore, villous cytotrophoblast-derived EXOs carry syncytin-1 and syncytin-2, which are involved in EXO fusion with the target cells. Importantly, syncytin-2 content was reduced in EXOs derived from serum of PE patients (416).

Antiangiogenic factors, such as sFlt1 and sEng, appear to participate in PE through a series of mechanisms that lead to the imbalance of angiogenic factors and finally to the generation of endothelial dysfunction and the maternal syndrome of PE. Increasing levels of sFLT and sEng can predict PE and directly correlate with the aggressiveness of this syndrome (417). PAI-1 and, to a lesser extent, PAI-2, which is predominant in placenta, are important inhibitors of fibrinolysis. Their overactivation results in the establishment of fibrin deposits that occlude placental vasculature and spiral arteries, leading to hypertension and endothelial damage causing PE. Moreover, increasing levels of PAI-1 in plasma directly correlate with PE severity (418). Eng and PAI-2 are highly expressed and localized to the surface of STBM MVs and EXOs, and thus can readily influence the development of PE (358). Additionally, STBMs from PE patients possess increased tissue factor activity compared with normotensive patients (359), and this could increase fibrin deposition. Coagulation may be enhanced by STBM action directly by direct association with platelets leading to activation: such activity is increased in PE-derived STBMs and correlates with PE-associated thrombotic risk. Moreover, treatment with aspirin, which is usually prescribed for PE women to reduce platelet aggregation, also inhibits STBM-induced platelet aggregation (360).

Cell-free hemoglobin (HbF) is released by the placenta, and increased hemoglobin expression as well as HbF accumulation in the vascular lumen of PE placentas have been reported (419). Indeed, HbF has been proposed as an important factor marking the transition between the first and second stages of PE. HbF causes placental damage similar to that observed in PE by inducing oxidative stress, which affects the blood–placenta barrier integrity (420). Blood–placenta barrier disruption may lead to the release of placental factors, including HbF, which leak into the maternal circulation, contributing to the maternal manifestations of PE. Moreover, levels of HbF correlate with PE severity symptoms (421). Placental HbF can provoke differential alterations in STBM miRNA cargo between EV populations: three miRNAs were specifically downregulated in MV populations under HbF influence. STBMs may also transport HbF itself, although these data may be an artifact of the external HbF perfusion (361). Furthermore, STBMs from PE pregnancies exacerbated the production of superoxide radicals by neutrophils in a dose-dependent manner, also correlating with PE severity. In this way, STBMs display multiple mechanisms to cause vascular damage and dysfunction in women with PE (362).

“Early pregnancy loss is a common complication that affects ∼15% of the gestations and shows recurrence rates of 2% to 3%.”

Maternally derived EVs.

Even before pregnancy, maternal risk factors for PE are obesity, diabetes mellitus, hypertension, and systemic lupus erythematous. Pro-PE EVs have altered concentrations and modified molecular contents that may alter the functioning of maternal tissues prior to pregnancy. In particular, changes in endothelial cells and leukocyte- and platelet-derived EVs are associated with the risk of PE. All share the common feature of a general increase in endothelial and platelet-derived EV levels [see review in (363)].

Once pregnancy is established, maternal EVs of different cellular origin interact with embryonic tissues with potential implications in PE pathogenesis. Platelets have crucial roles in PE development, and several studies report decreased platelet-derived EV levels in pregnancy compared with nonpregnancy, with a further decrease in PE (363). EVs of maternal endothelial and platelet origin appear to unleash a thrombo-inflammatory response in the placenta. EVs cause activated platelet aggregation and inflammasome activation within the placental vascular and trophoblastic cells, triggering a PE-like phenotype. Furthermore, inhibition of inflammasome or platelet activation components within the placenta abrogated the PE-like phenotype (364).

In contrast to platelets, leukocytes and certain derived EVs populations are increased in PE in comparison with normotensive pregnancies, mainly those EVs of granulocyte and monocyte origin (422). Interestingly, low levels of NK cell–derived EVs are observed in PE, linking with PE-associated maternal immune tolerance disorders (NK cell death activity dysfunction) (423). Of interest, Holder et al. (365) showed that human placenta is able to internalize EXOs from macrophages via endocytosis. Importantly, macrophage EXO uptake induced the release of proinflammatory cytokines by the placenta. Previously, the same group had reported that EXOs from PE placenta can activate peripheral blood mononuclear cells, inducing a proinflammatory response to a greater extent than EVs from normal placenta, and related to their cytokine content, mainly IL-1β. Moreover, PE-derived EVs stimulated an enhanced response of peripheral blood mononuclear cells to external pathogen-associated molecular patterns such as lipopolysaccharide (365). Such outcomes may be triggered by direct stimulation by EVs of Toll-like receptor, the signal subsequently internalized via nuclear factor κB (366). Taken together, these studies indicate a potential positive feedback loop by which an inflammatory response is overstimulated under PE conditions via EVs. Endothelial-derived EV levels correlate with the increment of the antiangiogenic factor sFLt1 and the sFLt1/P1GF ratio. This combined evidence suggests that apoptosis of endothelia occurs along with inhibition of angiogenesis and correlates with PE-characteristic endothelial damage, which persists between <1 week (424) and 72 hours postpartum (425).

Regarding obesity, a link between EXO release and the progression of PE is emerging. A recent study has observed that the levels of EXOs in maternal blood are correlated with maternal body mass index (BMI). A positive correlation of BMI with EXO levels was established, leading to the decrease of placenta-derived EXO proportions throughout gestation. These increased EXO levels contributed to a further exacerbated release of IL-6, IL-8, and TFN-α from endothelial cells, thus leading to worsened systemic inflammation in a BMI-dependent manner (426).

Finally, it has been observed that serum MVs from healthy pregnant women can reduce caspase activity and stimulate migration and tube formation in endothelial cells, whereas this is abrogated when the MVs are derived from patients with gestational vascular complications such as PE and hypertension. Furthermore, similar opposing actions on early-stage trophoblast of these vesicles was observed (367).

EVs in gestational diabetes

GD is defined as a carbohydrate intolerance of variable severity that appears or is first recognized during pregnancy. Along with PE, GD represents the most common metabolic complication of pregnancy, affecting between 1% and 15% of all pregnancies and increasing concurrently with obesity rates. It is characterized by pancreatic β cell–insufficient insulin production, usually due to pregnancy and characteristic insulin resistance, and is associated with maternal and fetal morbidity. Moreover, women with GD have increased risks of developing type II diabetes in the future (427, 428).

To date, little is known about the contribution of EVs in this pathophysiology. Salomon et al. (369) showed increased serum placenta-derived EXOs in GD pregnancies compared with control pregnancies, regardless of gestational age. In vitro, GD EXOs increased the release of proinflammatory cytokines from endothelial cells contributing to the enhanced proinflammatory state in pregnancy under GD conditions.

Clinical and Therapeutic Applications of EVs

The involvement of EVs in a wide variety of pathophysiological processes has made them appealing players as biomarkers and to carry therapeutic agents. This may also be the case when considering reproductive disorders.

EVs as biomarkers

EVs have been proposed as potential biomarkers of disorders of reproductive organs. The placenta releases EVs from the sixth week of pregnancy with steady increase as pregnancy proceeds, peaking at term (360). Importantly, their release is modulated by a number of factors that arise from the placenta; hence, EVs may provide mirrors of placental/fetal health and evolution (397). Because maternal blood is the primary source of placental EXOs, it will contain both maternal and placenta-specific EV populations and thus placental alkaline phosphatase (PLAP) has been proposed as a marker for the placental EVs, because it is restricted to placental cell lineages (394).

Alterations in both the levels and cargo of placenta-derived EXOs during pregnancy are associated with different pregnancy complications. A proteomic signature of 62 proteins in microparticles was developed from plasma samples of women at 10 to 12 weeks of gestation (363). This signature was able to predict and differentiate SPBs from normal term births. Functional enrichment analyses showed processes related with preterm birth, such as inflammation, fibrinolysis, immune modulation, the coagulation cascade, or steroid metabolism. Currently, the only tool for evaluation of risk of spontaneous preterm birth is measurement of cervical length by ultrasound (364). A retrospective study on plasma samples of women at early gestational age (prior to 18 weeks) demonstrated potential for EXOs in the diagnosis of PE and SPB with higher (but not significant) levels of EXOs in both pathological conditions vs normal pregnancies. More interestingly, a specific exosomal miRNA signature could differentiate between the three conditions, being more similar between normal pregnancy and SPB compared with that of PE. When these miRNA profiles were compared with those from the EVT HTR-8/SVneo cell line cultured under normal and low-oxygen tension (LOT) conditions there was a strong correlation between the SPB and LOT conditions, with a common variation in >45% of the SPB condition miRNA profile. Placental–exosomal miRNA cargo was related to cell migration potential and inflammatory cytokine production. Particularly, LOT EXOs decreased endothelial cell migration potential and increased their TNF-α production, which could negatively impact spiral artery remodeling during placentation. Thus, under circumstances that favor a proinflammatory environment or a reduction of oxygen tension such as advanced gestational age, placental EVs may be negatively altered, impacting spiral artery remodeling and resulting in development of pathologies such as PE or SPB (422). In this sense, placental EVs may be potential early biomarkers of PE/SPB or as targets for directed therapy. Finally, both total and placenta-derived EVs are increased in women delivering low birth weight babies compared with those with normal birth weight deliveries (429).

EVs have been further investigated as biomarkers of PE. Recent publications debate the usefulness of the content of EVs for their predictive value in the diagnosis of PE. As an example, Tan et al. (430) analyzed three candidate biomarkers, TIMP-1, PAI-1, and P1GF, for their predictive ability in a large cohort of low-risk PE women from EVs isolated from bank plasma samples. They concluded that measurement of TIMP-1 and PAI-1 reinforced the value of the classical P1GF for PE prediction. Indeed, TIMP-1 and PAI-1 were analyzed in specific subgroups of EVs that can be retrieved thanks to their affinity to cholera toxin B and annexin V, both of which had been described previously in the search for PE biomarkers. In this study, EVs were purified from plasma of women at ∼32 weeks of pregnancy, using immunoadsorption to the surface proteins, GM1 ganglioside (binds to cholera toxin B chain), and PS (binds to annexin V). Using these two populations of EVs (one from each marker), a specific protein signature was identified in women with PE compared with healthy pregnant controls. Importantly, note that such biomarker discovery is highly dependent on the selected conditions, providing a possible limitation. Indeed, in this study, large cellular debris was not removed from samples prior to the immunoadsorption step, providing a major potential source of error (431). In another study, different subtypes of MVs were evaluated in plasma, compared with cord blood from normal women and those with PE. Microparticles were more abundant and had altered coagulation-related factors in cord blood in PE compared with no PE (432). Recently, Salomon et al. (433) investigated whether EXOs and their miRNA cargo might provide early biomarkers of PE. More than 300 miRNAs were identified in total and placenta-derived EXOs in maternal plasma across gestation with hsa-miR-486-1-5p and hsa-miR-486-2-5 being identified as candidates for further study. Functional analysis showed that these miRNAs are involved in migration, placental development, and angiogenesis. Because PLAP is a marker of serum placenta-derived EXOs, which trend upwards with gestational age, exosomal content of PLAP has been proposed as a potential biomarker of PE in saliva and gingival cervical fluid (434). Finally, reduced EV-associated endothelial nitric oxide synthase expression and activity, a common feature of PE, was elevated in EVs from PE placentas (defined by PLAP) in both serum and placental perfusates, compared with healthy controls (435). Importantly, considering the above information, note that current biomarkers of pregnancy complications, such as PE or gestational diabetes mellitus, allow us to diagnose these states only once the pathologies are established and when the clinical management is limited. In this sense, to advance the field, efforts should focus on discovery of new biomarkers during early gestation.

“EVs have also been proposed as biomarkers of peripartum cardiomyopathy (PPCM).”

EVs have also been proposed as biomarkers of peripartum cardiomyopathy (PPCM). PPCM is an idiopathic form of cardiomyopathy characterized by left ventricular systolic dysfunction (the ejaculation fraction is reduced normally <45%) and subsequent heart failure. It usually appears around the end of pregnancy and in the next few months and it is currently only diagnosed by exclusion of other heart failure causes (436), making a search for new biomarkers of considerable importance. Initially, Walenta et al. (437) reported increased levels of blood-derived activated endothelial microparticles in PPCM when compared with healthy postpartum, pregnant, and nonpregnant control but also with patients of ischemic cardiomyopathy and stable coronary arterial disease. These microparticles in PPCM were mainly platelet derived and monocyte microparticles. Treatment with bromocriptine, a therapy proven to work in animal models and human patients, significantly reduced endothelial and platelet-derived microparticles in PPCM compared with patients treated with standard undirected heart failure therapy. Thus, specific microparticle profiles may provide biomarkers that can distinguish PPCM from normal pregnancy, vascular diseases, and heart failure of different origin. miR146a has also been identified as a possible EXO-associated biomarker for PPCM. The 16-kDa N-terminal prolactin fragment, the primary known trigger of PPCM, stimulates the packaging of miR-146a into EXOs from human umbilical vein endothelial cells, which then are able to reach cardiomyocytes and trigger PPCM. Thus, miR-146a may provide a biomarker and therapeutic target for PPCM (438).

Placental EVs may provide indicators of infectious diseases during pregnancy. Both total and placental-derived EVs are increased in plasma from pregnant women with HIV infection compared with noninfected controls. In contrast, there were no changes in the level of plasma EVs due to malaria infection, neither for placental malaria nor for its peripheral variant. Nonetheless, miR-517c was found to be increased in microparticles from plasma of women with active placental malaria compared with noninfected controls (429).

Clinical and therapeutic aspects of EVs in reproductive biology

Intercellular transfer of genetic and protein material mediated by EVs could facilitate new diagnostic and therapeutic tools in the field of reproductive biology. As discussed, 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 (75). Specifically, bioengineered EVs are being successfully deployed to deliver potent drugs and the capacity for select cellular reprogramming (6, 41). Recently, members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles presented a framework for challenges associated with development of EV-based therapeutics at the preclinical and clinical levels (439). This discussion addresses development of best practice models and current outlook for EV therapies.

Engineered or modified EVs can be designed for cell-specific targeting and delivery (440, 441). A seminal study has demonstrated that the selective cellular uptake of EVs surpasses that of more traditional carriers such as liposomes or nanoparticles, taking advantage of the natural characteristics of EVs to deliver molecules to target cells (442). Such insights provide future possibilities for clinical applications of EVs based on their ability to circumvent the limitations of various drug delivery systems of mucosal and blood–brain barrier traversal. The physicochemical configuration of EVs can also be modified to enable extended clearance compared with synthetic nanoparticles and spatiotemporal localization (ligand and cell type–specific targeting) and controlled release (238, 443–445). With respect to modifying EV cargo, a recent, comprehensive study compared various passive and active drug-loading methods, including electroporation, saponin treatment, extrusion, and dialysis, and used porphyrins of various hydrophobicities as model drugs (446). A comprehensive overview of EV cargo loading strategies, including electroporation, sonication, direct transfection, and cellular engineering, is provided in the literature (447, 448).

The potential functional roles of EVs in human embryo development have only recently been demonstrated. Embryos may generate their own microenvironment by secreting soluble factors and membrane vesicles, which constitute a secretome with selected autocrine and paracrine signaling (91, 449–453). In reproductive biology, nanoparticles have been used experimentally to load sperm with exogenous genetic material that is subsequently transferred to the oocyte during fertilization (454, 455). EVs have been identified in uterine fluid during the estrous/menstrual cycles, including humans, sheep, and mice (75, 156, 171, 282, 456). Indeed, EVs derived from the maternal endometrium contain multiple subtypes, including mixtures of EVs, EXOs and packaged different proteins, miRNAs and endogenous retrovirus mRNA (91, 156, 282–284, 287, 348). In the broader context of trophectodermal preparation for implantation, EVs have been shown to mediate communication between the inner cell mass and the trophectoderm (88). EV-encapsulated cargo is protected from degradation and is highly stable in biological fluids. Such unique properties may greatly facilitate the translation of EVs and their selected bioactive cargo and surface ligands into clinical applications. The study of EVs in reproduction has the potential for expanding our current understanding of the normal physiology of reproduction and pathological conditions such as implantation failure (452). Recent studies have provided key insights into the functional capacity of maternal EVs and how the protein cargo is directly modulated by uterine hormones during implantation to subsequently modulate trophoblast adhesive capacity (91). This study further validated selected components in primary human endometrial cells under hormonal control.

Recent studies have observed the ability of EVs to undergo cell-selective fusion (457) and tissue-specific tropism (228, 238, 458, 459), as well as their capacity to transverse the blood–brain barrier (460) and penetrate dense structural tissue (461). Importantly, based on their surface composition, EVs may be directed to specific tissues and organs (219, 238, 458, 459). Imaging of EVs in selected targeted organs has indeed demonstrated that the interactions of EVs with target cells are highly dynamic (232, 462). Such unique properties of circulating EVs make them promising applications for the delivery of therapeutic cargo. Several studies support the utility of EVs as a novel path for drug delivery and as new drug targets. Alvarez-Erviti et al. (462), in an in vivo study, demonstrated that systemically injected neuron-targeted EXOs loaded with BACE1 siRNAs were able to significantly reduce BACE mRNA and protein, specifically in neurons (463). Furthermore, EXOs loaded with artificial siRNA against MAPK efficiently knocked down MAPK1 upon their delivery into monocytes and lymphocytes in vitro (464). Similarly, EXOs from induced pluripotent stem cells have been shown to deliver siRNA to attenuate expression of intercellular adhesion molecule 1 and neutrophil adhesion in pulmonary microvascular endothelial cells (465). Exosomes have further been applied for drug delivery to target a small-molecule, anti-inflammatory drug to selected organs and immune cells (466). These studies have demonstrated the capacity for EV-mediated targeted and delivery capacity and importantly the ability for EXOs to deliver and modulate multiple pathways simultaneously in the targeted cells. All of these studies are examples showing how EV cargo can be manipulated in a way that may be useful for target-based drug development for successful in vivo drug delivery.

Recent reviews have discussed the rationale to aim for selective silencing of EVs that promote unwanted functional effects. However, this is still an emerging concept in the field. Some of the strategies for specific silencing of EV subtypes (cell specific) are likely to require careful and detailed mechanistic studies. There are inhererent difficulties in avoiding the blocking of all EV types indiscriminately, which may interfere with and perturb physiological intercellular communication. Some examples of systems for abrogating EV formation and targeting/recipient cell uptake [reviews include (222, 230, 448, 467)]: (1) inhibition of EXO formation, including treatment with dimethyl amiloride; (2) inhibition of the endolysosomal compartment functions, including proton pump inhibitors, (3) blocking of EXO release (for example, silencing GTPase Rab11/27A/35 using siRNA or targeting ESCRT proteins and/or GTPases involved in trafficking of EXOs); and (4) prevention of fusion or uptake of EXOs by target cells, which can be done using a variety of reagents that block phosphatidyl serine such as diannexin, heparin to inhibit endocytosis (heparan sulfate proteoglycans), cytochalasin D to inhibit endocytosis and micropinocytosis, chlorpromazine to inhibit clathrin-dependent endocytosis, EIPA and LY294002 to block micropinocytosis, annexin V to inhibit phagocytosis and macropinocytosis, methyl-β-cyclodextrin, simvastatin, and filipin III to target lipid raft–mediated endocytosis, nystatin to target caveolae-mediated endocytosis, dynasore to inhibit clathrin-independent endocytosis (calveolae), and nystatin to perturb lipid raft–mediated endocytosis.

Future studies are required toward investigating EVs from primary tissues and biofluids and incorporating state-of-the-art quantitative analyses, including quantitative proteomics (183, 468) and sequencing technology that could be exploited to study protein and gene regulation during pregnancy. These would enable identification and monitoring of functional or low-abundant EV cargo, as well as cellular drivers of implantation and signaling, that hitherto have been unreported or functionally masked. Unlike small-molecule pharmaceutical compounds, there are no defined parameters or assays for current safety testing of EV-based therapeutics (469). Understanding biodistribution patterns and circulating timeframe of locally and systemically administered EVs is important to assessing safety, in addition to techniques that enable reproducible monitoring and safety testing of selected EV marker cargo. Targeted studies using EVs (modified or engineered) will hold the potential to develop novel nanodiagnostics and nanotherapeutics to increase the success of pregnancy rates during assisted reproductive technologies or in vitro fertilization. Recent work on targetable biodegradable delivery platforms for transporting biological cargo into gametes and embryos [reviewed in (470)] emphasizes the need to understand how EVs enter cells. We anticipate that future investigations into the use of EVs for the intentional targeted delivery of molecular compounds will provide new horizons for reproductive science and clinical assisted reproductive technologies, ultimately leading to improvements in pregnancy success.

“Recent reviews have discussed the rationale to aim for selective silencing of EVs that promote unwanted functional effects.”

Concluding Remarks

Considering the body of evidence treated in the present review, there is no doubt that the field of EVs and its implication in reproduction is rapidly evolving and promises a further understanding of the processes that lead to a successful pregnancy, as well as markers of correct or compromised reproductive function. Nonetheless, there is still a difficult path to negotiate. First, there is an unavoidable need to firmly define standard methods for EVs isolation, because these define the fractions considered as different EV populations and, as such, may lead to ambiguous results that cannot be compared among studies. New challenges associated with standardization of methods for isolation, quantification, and analysis of EVs from complex tissues such as blood, as well as the stability of EVs within such biofluid samples, need to be overcome before the EV field can provide reliable tools for diagnosis and therapy.

It is also necessary to define the extent to which EVs are important participants in the reproductive events that lead to the delivery of healthy normal newborns, as this knowledge will lead to new therapies and clinical tests to ensure good pregnancy outcomes. Sample availability is maybe one of the main limiting factors that hinders such progress. In this sense, much more is known about epidydimal and prostatic EV regulation of sperm compared with embryo maternal crosstalk through EVs. Nevertheless, EV communication may provide a cornerstone to enable better understanding of the conception and implantation processes. This is important as it paves the way to deal with those patients in which the present assisted reproductive techniques fail.

Finally, data regarding the involvement of EVs in the triggering, maintenance, and progression of reproductive and obstetric-related disorders is still in its infancy and further key investigations utilizing homogeneous and human-specific material are needed. The use of EVs as disease biomarkers provides the opportunity for diagnostic potential with reduced invasiveness, as they can be retrieved from body fluid instead of tissue biopsies. This is vital for embryo diagnoses, where the possibility of getting STBMs from mother blood flow appears as an interesting alternative to invasive amniocentesis and chorionic villi sampling, further offering the possibility of an earlier diagnosis. Regarding EVs used as therapeutic agents, many different variants could be exploited. EVs could be used as vectors to deliver drugs and biological compounds in a targeted manner. Nevertheless, they could potentially be used as therapeutic targets if they are produced by affected cells and present disease-promoting characteristics. This may be achieved by inhibiting EV biosynthesis, by capturing them once produced, or by blocking their uptake by target cells, and this may be applicable in diseases such as PE. Furthermore, they could be used as natural therapeutic agents when experimental strategies rely on their natural features. Understanding cell type specificity and the long-term effects of EV remodeling, the potential of EVs to impart transgenerational consequences on the offspring’s health, ranging from metabolism to sex determination, and potential epigenetic changes affecting the mother’s fertility and altering the offspring’s fertility are key factors to be addressed as the field moves forward. EVs derived from the immune cells including dendritic cells within the reproductive tissues also need examination, since such cells, once stimulated, may trigger detrimental immune responses. Advances in research on noncoding RNAs contained in EVs must also be considered (471). Understanding all these molecular signaling networks, utilizing advances in quantitative proteomics and sequencing technology, and mediated by EVs that coordinate strategies for successful implantation, may lead to approaches to improve the outcomes of natural pregnancy and pregnancy achieved using reproductive technologies.

Abbreviations

Abbreviations
 
• AB

  apoptotic body

 
• AFM

  atomic force microscopy

 
• BLVRA

  biliverdin reductase A

 
• BMI

  body mass index

 
• cAMP

  cyclic adenosine monophosphate

 
• ED

  erectile dysfunction

 
• ELISA

  enzyme-linked immunosorbent assay

 
• ER

  endoplasmic reticulum

 
• ESCRT

  endosomal sorting complex required for transport

 
• EV

  extracellular vesicle

 
• EVT

  extravillous trophoblast

 
• EXO

  exosome

 
• FAK

  focal adhesion kinase

 
• GD

  gestational diabetes

 
• GTPase

  guanosine triphosphatase

 
• HbF

  cell-free hemoglobin

 
• IL

  interleukin

 
• ILV

  intraluminal vesicle

 
• LOT

  low-oxygen tension

 
• miRNA

  microRNA

 
• mRNA

  messenger RNA

 
• MV

  microvesicle

 
• MVB

  multivesicular body

 
• NK

  natural killer

 
• NOS

  nitric oxide synthase

 
• NTA

  nanoparticle tracking analysis

 
• PBS

  phosphate-buffered saline

 
• PCOS

  polycystic ovarian syndrome

 
• PE

  preeclampsia

 
• PKA

  protein kinase A

 
• PLAP

  placental alkaline phosphatase

 
• PPCM

  peripartum cardiomyopathy

 
• PS

  phosphatidylserine

 
• SEM

  scanning electron microscopy

 
• siRNA

  small interfering RNA

 
• SPAM1

  sperm adhesion molecule 1

 
• SPB

  spontaneous preterm birth

 
• SPZ

  spermatozoa

 
• STB

  syncytiotrophoblast

 
• STBM

  syncytiotrophoblast-derived exosome and microvesicle

 
• TEM

  transmission electron microscopy

 
• TNF

  tumor necrosis factor

 
• tRF

  transfer RNA fragment

 
• tRNA

  transfer RNA

 
• μNMR

  micronuclear magnetic resonance spectrometry.

Acknowledgments

We thank Fernando Ibañez from Contextos Culturales for help in the final edition of the figures for this review.

Financial Support: This work was supported by MINECO/FEDER Grant SAF2015-67154-R to C.S; La Trobe Institute for Molecular Science Fellowship, La Trobe University Leadership Research Focus grant, and La Trobe University Start-up Fund to D.W.G; MECD Grant FPU15/02248 to D.B; Atracció de Talent Program ultraviolet-INV-PREDOC14-178329 to N.B; National Health and Medical Research Council of Australia Project Grant 1002028 and the Victorian Government’s infrastructure support funding to the Hudson Institute to L.A.S.; and by Miguel Servet Program Type I of ISCIII (CP13/00038) and FIS project (PI14/00545) to F.V.

Disclosure Summary: The authors have nothing to disclose.

Author notes

These authors contributed equally to this work.