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. Author manuscript; available in PMC: 2021 Nov 1.
Published in final edited form as: Theriogenology. 2020 Feb 19;150:106–112. doi: 10.1016/j.theriogenology.2020.01.083

Extracellular Vesicles: Novel Regulators of Conceptus-Uterine Interactions?

Eleanore V O’Neil 1, Gregory W Burns 1, Thomas E Spencer 1,*
PMCID: PMC8559595  NIHMSID: NIHMS1574752  PMID: 32164992

Abstract

This review focuses on extracellular vesicles (EV) in the uterus and their potential biological roles as mediators of conceptus-uterine interactions essential for implantation and pregnancy establishment. Growing evidence supports the idea that EV are produced by both the endometrium and conceptus during pregnancy. Exosomes and microvesicles, collectively termed EVs, mediate cell-cell communication in other tissues and organs. EV have distinct cargo, including lipids, proteins, RNAs, and DNA, that vary depending on the cell of origin and regulate processes including angiogenesis, adhesion, proliferation, cell survival, inflammation, and immune response in recipient cells. Molecular crosstalk between the endometrial epithelium and the blastocyst/conceptus, particularly the trophectoderm, regulates early pregnancy events and is a prerequisite for successful implantation. Trafficking of EV between the conceptus and endometrium may represent a key form of communication important for pregnancy establishment. Increased understanding of EV in the uterine environment and their physiological roles in endometrial-conceptus interactions is expected to provide opportunities to improve pregnancy success.

Keywords: extracellular vesicle, uterus, conceptus, pregnancy

1. Introduction

In ruminants, establishment of pregnancy requires pregnancy recognition signaling by the conceptus followed by implantation and placentation [1]. The morula-stage embryo enters the uterus by day 6 and then forms a blastocyst with an inner cell mass and a blastocoele, or central cavity, surrounded by a monolayer of trophectoderm cells. Following hatching from the zona pellucida, the blastocyst develops into an ovoid and then tubular shaped conceptus (embryo and associated extraembryonic membranes) that begins to elongate on day 12 (sheep) or day 15 (cattle) into a filamentous form that eventually occupies the entire length of the uterine horn ipsilateral to the corpus luteum [2]. Elongation of the conceptus is critical for production of pregnancy recognition signals including interferon tau (IFNT) and is a prerequisite for successful implantation. Trophectoderm cell remodeling and proliferation [3] drive the exponential increase in conceptus length during elongation [4]. Subsequent implantation is a multi-step process involving apposition, attachment and adhesion of the trophectoderm to the endometrial luminal epithelium (LE) [5].

Although blastocysts can be developed entirely in vitro, the efficiency and resulting quality is markedly lower than in vivo embryos and the blastocyst must be transferred into the uterus for it to elongate and form a filamentous type conceptus [6]. The epithelia, LE and glandular epithelia (GE), of the uterus produce embryotrophic factors in response to progesterone, IFNT and other factors that stimulate blastocyst survival, growth, conceptus elongation, and implantation [1]. Intercellular communication is an essential hallmark of conceptus-maternal interactions and can be mediated, classically, through direct cell-cell contact or transfer of secreted molecules. More recently, transfer of membrane-bound vesicles between cells has emerged as a novel method of communication [7].

Extracellular vesicles (EV) are membrane-bound nanovesicles of endosomal or plasma membrane origin present in most if not all bodily fluids [7–9]. They contain surface receptors/ligands from the originating cell and include cargo of lipids, RNAs, and proteins. Following contact or uptake by a recipient cell, EV can regulate gene expression and elicit biological effects including increased cell proliferation, migration and invasiveness. Most cell types release EV including ovine endometrial GE cells and human trophoblast cells in culture [10–16]. Notably, EV have been identified in the uterine lumen of humans, sheep, cattle, pigs and mice [16–18]. Recent evidence supports the idea that EV are a novel component of uterine histotroph and represent a new paradigm for crosstalk between the developing conceptus and uterine endometrium [9, 19].

2. Extracellular Vesicle Characteristics, Cargo and Function

EV are lipid bilayer-enclosed vesicles released by cells that can transport DNA, RNAs, lipids, and proteins between cells. The term EV can be used to describe a heterogenous mixture of exosomes and larger microvesicles (MV) of 50–150 nm and 100–1000 nm in diameter, respectively [20, 21], that are classified based on their biogenesis or release pathways [22]. Exosomes are of endocytic origin and are released from cells upon fusion of a multivesiclular endosome (MVE) with the outer plasma membrane. In contrast, MVs bud directly from the cell surface [7]. Both types of EV can elicit biological effects, such as increased cell migration and immunomodulation that are important for pregnancy establishment [12, 13, 23]. The International Society for Extracellular Vesicles has recommended using the term EV due to the difficulty of isolating and distinguishing vesicle subtypes [20].

2.1. Exosomes

Exosomes are small membrane-bound vesicles ranging from 50 to approximately 150 nm in size formed as intraluminal vesicles within MVEs as part of the endosomal network [24, 25]. Endosomal sorting complexes required for transport (ESCRT) complexes selectively sort proteins with ubiquitin and ubiquitin-like modifications, such as SUMO-dependent SUMOylation or interferon-stimulated gene 15 (ISG15)-dependent ISGylation into MVEs for release [26, 27]. There are also ESCRT-independent pathways for cargo loading and MVE formation, in which ceramide rich lipid rafts and GPI-anchored proteins cause invaginations of the cell plasma membrane forming endosomes that can eventually release exosomes [28]. The intraluminal vesicles secreted into the extracellular space by fusion of the MVE and the cell plasma membrane are termed exosomes [29]. Notably, MVE destined for secretion in polarized epithelial cells typically migrate toward the apical pole of the cell for exosome release.

2.2. Microvesicles (MV)

Microvesicles are released directly from the plasma membrane with a typical diameter of 100 to 1000 nm [30]. The mechanisms required for MV formation and release are not fully understood, but involve organization of lipid rafts, including phosphatidylserine and phosphatidlyethanolamine, contraction of the cytoskeleton, ESCRT complexes, and Rab GTPases [31–33]. Briefly, phospholipid translocases rearrange phosphatidylserine to the outer membrane inducing membrane curvature, and bud formation is completed by actin-myosin cytoskeleton contractions. MV cargo appears similar to exosomes, but differences may arise from the contrasting biogenesis [34].

2.3. Interaction of EV with target cells

Major constituents of exosome membranes include cholesterol, sphingomyelins, and phosphatidylcholines with significant enrichment of sphingomyelin, gangliosides, phosphatidylserine, and disaturated lipid populations [35, 36]. As a result of possessing a lipid bilayer enriched in these lipid populations, EV cargo is highly stable and protected from degradation in the extracellular environment due to a very rigid lipid bilayer [37, 38]. Additionally, EV may contain bioactive lipids such as prostaglandins, leukotrienes, endocannabinoids, or lysophospholipids that can signal in recipient cells [39, 40].

Both MV and exosomes may contain many ubiquitous proteins, but also unique proteins dependent on the cell type of origin. Exosomes are enriched in major histocompatibility complex class II (MHC class II) and tetraspanins (CD37, CD53, CD63, CD81, CD82), endosomal sorting complex proteins (Alix, TSG101), and chaperones, which are often used as exosome markers. Exosomes are also enriched in glycoproteins and transmembrane proteins as compared to their cell of origin, including integrins, glycoprotein Ib (GPIb), and P-selectin [22]. Finally, the proteins contained in EV do not require a signal peptide, thus EVs allow for transfer of proteins that are classically seen as intracellular, non-secreted proteins between cells.

Nucleic acids are a notable component of EVs, suggesting that EV can serve as a pathway for the transfer of genetic information from one cell to another. A variety of nucleic acids have been found in EV including DNA, mRNA, noncoding RNA, miRNA, and tRNA. Indeed, functional proteins can be derived from translated mRNA delivered via EV [41]. Similarly, EV are particularly enriched in miRNAs, which can regulate protein translation in recipient cells through RNA-induced silencing [42]. There are recent reports of EV associated tRNAs and tRNA fragments that may regulate protein translation, but universal presence of these molecules in EV requires further investigation [43, 44]. Increased cell proliferation, migration and adhesion, which are important for conceptus elongation in placental development, are commonly reported biological effects of EV in other tissues and systems that may be mediated through uptake of RNAs [45].

The effects of EV in a target cell can result from interactions at the cell surface, with or without vesicle uptake, or from delivery of EV cargo contents. Interactions with receptors on the surface of a recipient cell can alter signaling pathways without requiring EV uptake [46]. Uptake of vesicles by cells appears to be an active process, but may be secondary for EV that elicit cellular responses via membrane interactions. For example, EV that are docked at the cell surface may be internalized as a function of plasma membrane recycling [47]. Conversely, the observed effects on recipient cells may depend on delivery of the EV nucleic acids and proteins to the cytosol.

Evidence supporting EV content delivery to the cytoplasm of target cells is very strong [47]. Fusion of EV and cellular membranes is the most direct route for cargo delivery. Evidence that EV can deliver cargo by membrane fusion has been reported under acidic conditions and may be associated with the lipid composition of EVs. The most common method of uptake appears to be endocytosis, where contents would be released into the cytoplasm upon intracellular membrane fusion [7]. The mechanisms regulating vesicle endocytosis are varied and include clathrin-mediated endocytosis, phagocytosis and macropinocytosis. The diversity of internalization pathways coupled with the heterogeneity of EV may explain the difficulty of experimentally blocking EV uptake for investigation of biological functions.

2.4. EV isolation methods

Current methods used for EV isolation are based on markers, ultracentrifugation, or particle size [34]. Tetraspanins, particularly CD9 and CD63, are among the most universal markers due to their role in EV biogenesis and can be used for immunocapture [21, 31]. Immunocapture from samples pre-filtered to remove large vesicles results in purified EV with few contaminants but targets only a subpopulation of the vesicles present. A more complete understanding of signaling pathways affected by EV demands subpopulation analyses, which is dependent on a priori knowledge of the markers in a given sample.

The majority of isolation protocols utilize differential centrifugation based on the sedimentation properties of EV [20, 48]. Cellular debris, apoptotic blebs, and large MV are pelleted by sequential centrifugation at increasing speeds [49].Pelleted EV can be collected by ultracentrifugation and analyzed directly or further purified using density gradient separation [25, 50]. Ultracentrifugation through a density gradient removes contaminating proteins, but the process is time consuming and inefficient [50, 51]. Alternatively, vesicles can be precipitated from pre-filtered samples [51–53]. Precipitation is the foundation of almost all commercially available kits to isolate EV due to ease of use and low speed centrifugation. The reagents used for EV precipitation are the same as those used to isolate viral particles [54]. Consequently, EV isolated by precipitation contain viral particles, lipoprotein complexes, and protein aggregates.

Size exclusion chromatography (SEC) is an alternative to ultracentrifugation and precipitation methods with faster isolation times and less protein contamination [53, 55]. The diameter of particles is the basis of purification by SEC allowing for unbiased isolation of EV from a sample. Briefly, vesicles flow through a column more quickly than proteins that elute in later fractions. Ultrafiltration devices that retain vesicles can be utilized to concentrate starting material prior to SEC purification [55]. A comparison of methods found that SEC isolated EV contained less contaminating proteins than precipitation methods and recovered more particles than ultracentrifugation with a density gradient [51]. SEC has been used to successfully purify and characterize EV from the uterine lumen of sheep [56].

3. EV in the Female Reproductive Tract

EV are a constituent of the female reproductive tract in humans and domestic animals with potential biological roles in follicular development as well as oocyte maturation, embryo fertilization and pregnancy establishment [9, 57–60]. In pigs, embryo-derived EV influence embryo growth and viability in vitro [61]. Another study in the mouse found that the EV from the inner cell mass affected the trophectoderm by promoting cell migration [62]. EV have been isolated from mouse and bovine oviduct fluid and also in vitro cultured bovine oviductal epithelial cells [58, 63, 64]. In cats, oviductal EV fuse with the sperm cell acrosome and improve sperm motility and fertilizing capacity in vitro [65]. Additionally, evidence indicates that that oviduct EV improves blastocyst yield and quality and extends embryo survival in vitro [66]. Moreover, embryo-derived EV can be delivered into human primary endometrial epithelia and stroma cells [67], supporting the idea that crosstalk between uterus and blastocyst/conceptus can be mediated by EVs.

3.1. Uterine lumen

Recent evidence supports the idea that EVs, produced by the endometrial epithelia, are present in the uterine lumen and regulate blastocyst implantation. Both exosomes and MV are detected in uterine lumen of fertile women in the secretory phase [16, 17] as well as both sheep [56, 68–70] and cattle [71]. In women, the protein and miRNA content of endometrial epithelial cell EV change with ovarian steroid hormone treatment [16, 18, 32, 67, 70, 72, 73]. Alterations in uterine lumen EV content during cycle could provide a mechanism to synchronize development of an embryo and the uterine endometrium. For instance, miR-30d was increased in the uterine lumen during the window of implantation in women, and EV associated miR-30d was taken up by the trophectoderm of mouse blastocysts and increased trophectoderm adhesion in vitro [17]. These observations support the idea that EV support embryo attachment to the endometrial epithelium for implantation via effects on trophectoderm adhesion. Accordingly, EV in the uterine lumen could serve as potential biomarkers of uterine receptivity useful to time embryo transfer [72, 74].

3.2. Sheep uterus

Electron microscopy analysis found that the endometrial LE and GE were the primary source of EV in the uterus of cyclic sheep [56]. EV isolates from the uterine lumen of day 14 cyclic and pregnant sheep contained vesicles that ranged from 50 to 200 nm in diameter [68]. The isolated EV were positive for two common markers of exosomes (CD63 and HSP70). Proteins in the EV were similar to many expressed by the endometrial epithelia and/or conceptus trophectoderm including the enzymes cathepsin L one (CTSL1) and prostaglandin synthase two (PTGS2). A total of 195 proteins were identified in the EV from the uterine lumen with 40 and 76 unique to the day 14 cyclic and pregnant ewes, respectively. The EV contained a large number of small RNAs and miRNAs including 81 conserved mature miRNAs. Interestingly, cyclic and pregnant EV contained endogenous Jaagiekte sheep retroviruses (enJSRVs) envelope (env) and gag RNAs that could be delivered to heterologous cells in vitro. Thus, EV may deliver enJSRVs RNA to the conceptus, which is important as enJSRVs envelope regulate conceptus trophectoderm development [75, 76]. Overall, these studies support the idea that EV containing select miRNAs, RNAs and proteins are present in the uterine lumen and likely have a biological role in conceptus-endometrial interactions important for the establishment and maintenance of pregnancy.

In sheep, progesterone regulates EV production and alters cargo content [56]. Total EV number in the uterine lumen increased from day 10 to 12 in cyclic sheep, which is at the onset of conceptus elongation. Total EV number in the uterine lumen was increased over two-fold by progesterone treatment in ovariectomized sheep. A total of 768 miRNAs were detected in the endometrium and EVs by small RNA sequencing with 488 miRNAs in common, 273 unique to the endometrium, and 7 unique to EVs. A recent study demonstrated that endometrial-derived EV could target the conceptus trophectoderm in vivo [69]. Day 14 ovine conceptuses were cultured ex vivo and found to release EV into the culture medium that could target the endometrial LE and superficial GE but not stroma or myometrium of cyclic ewes. Proteomics analysis of the day 14 conceptus-derived EV identified 231 proteins that were enriched for extracellular space and several protein classes, including proteases, protease inhibitors, chaperones and chaperonins. RNA sequencing of day 14 conceptus-derived EV detected expression of 512 mRNAs. The top-expressed genes were overrepresented in ribosomal functions and components. Another study found that EV from the uterine lumen stimulated trophoblast proliferation and IFNT secretion in vitro [70].

3.3. Bovine uterus

In the bovine uterus, EV have been isolated from uterine lumen of cyclic and early pregnant cattle [71, 77]. Early pregnant ULF EV detected 596 proteins, including IFNT, and 172 differentially expressed proteins were detected in EV compared from days 17, 20 and 22 pregnancy. Treatment of primary bovine endometrial epithelial cells with exosomes from day 17 of pregnancy increased expression of apoptosis-related genes, whereas treatment with exosomes from day 20 and 22 of pregnancy increased the expression of adhesion molecules. Of note, in vitro treatment of bovine endometrial epithelial cells with EV isolated from the uterine lumen of day 15 and 17 pregnant cattle increased expression of several IFNT-stimulated genes of ISGs [77]. Collectively, studies support the ideas that EV emanate from both the conceptus trophectoderm and endometrial epithelia of the uterus, and are involved in intercellular communication between those cells during the establishment of pregnancy in ruminants. Of note, pregnancy-associated circulating miRNAs have been profiled from EVs isolated from the blood of dairy and beef cattle and are purported to be potential biomarkers of pregnancy success and loss [78, 79].

3.4. Human uterus

More than 200 miRNAs as well as other non-coding RNAs were found in EV derived from the ECC1 human endometrial epithelial cell line in vitro [16]. Those miRNAs were predicted to regulate several genes involved in embryo implantation, including cadherins required for maintaining epithelial and trophoblast layer integrity and integrins involved in attachment of the trophectoderm to the endometrial epithelium. The protein cargo of the ECC1-derived EV has also been characterized and found to contain over 1,000 proteins [18]. Further, the protein content of the EV secreted by the ECC1 are were regulated by ovarian steroid hormones. Proteins in the EV included enzymes such as ligases, oxidoreductases, transferases, lyases, isomerases, phosphatases, kinases, metalloproteinases, and hydrolases.

The effects of ECC1-derived EV on trophoblast invasion were investigated using in vitro assays employing the HTR8 trophoblast cell line [18]. Of note, EV derived from ECC1 cells, treated with either estrogen or progesterone, increased the adhesive capacity of the trophoblast cells via focal adhesion kinase signaling and increase in fibronectin. Indeed, ECC1-derived EV are taken up by human trophectoderm spheroids in vitro, increased cell adhesion and invasive capacity by horizontal transfer of adhesion proteins, and also altered the secretome of the trophectoderm spheroids [73]. These studies support the idea that EV from the endometrial epithelia are a component of the uterine luminal fluid and act in a paracrine manner on the trophectoderm of the blastocyst to aid in trophoblast adhesion to the endometrial LE for implantation.

4. EV from the placenta and in the circulation during pregnancy

Release of membranous materials from the syncytiotrophoblasts (STB) of the placenta into the maternal circulation during pregnancy is long known [80]. Not surprisingly, EV of placental origin are well characterized [81–84]. In general, STB are the primary source of placenta-derived EVs, and EV have been isolated from trophoblast cell lines, placenta cultures, and maternal circulation. Placental EV are detectable in maternal circulation as early as six weeks and identified by the presence of placental alkaline phosphatase [23, 85, 86]. The release of EV can be increased in response to low oxygen tension, pH changes, cellular damage, or stress [87–89]. Placental EV may then serve as an indicator of placental function useful for diagnosing gestational disorders even prior to the presentation of clinical symptoms [60, 90].

4.1. Circulation in pregnant women

The concentration of EV in plasma is 50 times higher in pregnant than nonpregnant women during the first trimester of pregnancy [23]. Placental-derived EV are detectable in circulation of women by the sixth week of gestation, and both placental and total EV abundance increased two-fold throughout pregnancy [23, 86]. Placental EV affect peripheral immune cells by suppressing or activating immune responses depending on the EV type [91]. STB exosomes induced apoptosis in activated immune cells [92], induced apoptosis in T lymphocytes, and suppressed the cytotoxic activity of natural killer (NK) cells [93, 94]. In contrast, MV from STB stimulated pro-inflammatory cytokine release from leukocytes and activated monocytes [95, 96]. These results support the hypothesis that placental EV subpopulations are involved in the successful progression of pregnancy and suggest that immune activating and suppressive EV may be involved in the etiology of gestational disorders such as preeclampsia [97].

4.2. Trophectoderm-derived EV confer viral immunity

The secretome of in vitro cultured human embryos contains EV that can be delivered into primary human endometrial epithelial and stromal cells in vitro [67]. Trophoblasts in the human placenta highly express chromosome 19 miRNA cluster (C19MC) miRNAs that are released in EV and detected in maternal circulation [98, 99]. The chromosome 19 miRNA cluster (C19MC) is the largest in the human genome at around 100 kb and produces 59 mature miRNAs. Primary term human trophoblasts are largely resistant to viral infection and can confer resistance to non-trophoblastic cells through EV containing C19MC miRNAs [100]. Those studies extended the functional effects of placental EV beyond the immune system and support the hypothesis that placental EV are active participants in multiple physiological adaptations of the maternal body to support pregnancy.

Conclusions

Production of EV by endometrial epithelial cells and the developing conceptus trophectoderm with unique protein and miRNA cargo suggests a biological role in mediating cell-cell interactions important for pregnancy establishment. Trophoblast cells treated with EV have higher adhesive potential and there is adequate in vitro evidence that EV are important for embryo implantation, placentation, maintenance of pregnancy and, consequently, pregnancy complications under their dysregulation. However, this evidence is complicated by a few issues. First, different studies have used various EV isolation methods, including SEC, reagent precipitation, and differential centrifugation. Different preparations lead to varying degrees of protein contamination and might alter the ratio of EV subpopulations used in an experiment, leading to inconsistent or potentially misleading results. Most of the data on EV cargo are derived from immortalized or long-term passaged cell lines and very few from primary tissue. Further, the experimental data on pro-implantation effects of endometrial epithelial cell-derived EV is largely based on in vitro trophoblast adhesion/invasion assays and needs to be supported by in vivo evidence.

The present data support roles for protein and RNA cargo of EV in embryo implantation, but the lipid cargo of endometrial EV has been largely ignored. In vivo studies with labeled EV in sheep support the idea that EV provide a source of lipids for the elongating conceptus during early pregnancy [69]. Further, the role of steroid hormones on EV sorting and cargo needs to be determined. The current idea that EV have a definitive role in implantation remains speculative. Experiments to define the biological role of exosomes and MV in pregnancy necessitate targeting EV biogenesis and uptake in the endometrium and conceptus, which has remained elusive. Knowledge of intercellular communication via EV has resulted in a more complete understanding of the physiology and pathophysiology of processes critical to the establishment of pregnancy, including angiogenesis, immunomodulation, and cell survival. Further research is warranted to establish the role of EV in conceptus-uterine interactions during pregnancy.

Fig. 1.

Fig. 1.

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Schematic illustrating working hypothesis on the function of extracellular vesicles (EV) in early pregnancy. Recent evidence supports the hypotheses that the endometrial epithelia (LE and GE) of the uterus secrete EV that have biological effects on trophectoderm survival and proliferation to promote conceptus elongation. Additionally, the trophectoderm of the elongating conceptus secretes EV that may signal to the uterine epithelia to coordinate adhesion and attachment for implantation.

Highlights.

  • Growing evidence supports the idea that EV are produced by both the endometrium and conceptus during pregnancy in women and domestic animals.

  • Extracellular vesicles (EV), including exosomes and microvesicles, mediate cell-cell communication in other tissues and organs.

  • EV have distinct cargo, including lipids, proteins, mRNAs, microRNAs, and DNA, that vary depending on the cell of origin and regulate processes including angiogenesis, adhesion, proliferation, cell survival, inflammation, and immune response in recipient cells.

  • Molecular crosstalk between the endometrial epithelium and the blastocyst/conceptus, particularly the trophectoderm, regulates early pregnancy events and is a prerequisite for successful implantation.

Acknowledgments

This research was supported, in part, by AFRI competitive grants 2015-67015-23678 and 2016-67015-24741 from the United States Department of Agriculture National Institute of Food and Agriculture (T.E.S.) and National Institutes of Health T32 HD087166 (G.W.B).

Footnotes

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