Extracellular Vesicles Mediated Early Embryo–Maternal Interactions

Embryo–maternal crosstalk is an important event that involves many biological processes, which must occur perfectly for pregnancy success. This complex communication starts from the zygote stage within the oviduct and continues in the uterus up to the end of pregnancy. Small extracellular vesicles (EVs) are part of this communication and carry bioactive molecules such as proteins, lipids, mRNA, and miRNA. Small EVs are present in the oviductal and uterine fluid and have important functions during fertilization and early embryonic development. Embryonic cells are able to uptake oviductal and endometrium-derived small EVs. Conversely, embryo-derived EVs might modulate oviductal and uterine function. In this review, our aim is to demonstrate the role of extracellular vesicles modulating embryo–maternal interactions during early pregnancy.


Introduction
In mammals, the perfect embryo-maternal communication is necessary to allow the establishment and maintenance of pregnancy. For this, oocyte maturation occurs in the preovulatory follicle, followed by the fertilization in the oviduct, and early development in the oviduct and uterus during the luteal phase [1]. Upon ovulation, the oocyte starts its journey through the oviduct, is fertilized, and embryo development begins, followed by its first cleavages [2]. Importantly, the major embryonic gene activation (EGA) occurs during the embryo passage through the oviduct [3]. During this phase, the embryo starts to transcribe more actively, decreasing its dependency on the maternal mRNA stock. The epithelial line of the oviduct is formed by ciliated and secretory cells. These cells play roles in gamete transport, capacitation and fertilization [4,5], and on early embryonic development [6,7], mostly by oviductal secretions [8]. Small extracellular vesicles (EVs) are present in oviductal fluid content and participate in these important reproductive events [5,7].
The embryo enters the uterus 4 to 5 days after oocyte fertilization, in bovine [2,9]. During embryonic development, the embryo reaches the blastocyst stage composed of two cell lines: the inner cell mass that will originate the embryo proper and the trophectoderm cell monolayer, which ultimately will form the fetal adnexa/placenta [2,10]. Fetal appendages will establish contact with the endometrium, and thus establish the interchange interface between embryo/fetus and maternal tissues. However, in order to allow embryo development and placentation, uterine epithelial and glandular cells secrete the uterine fluid that is very important for embryo nutrition because it contains proteins, lipids, amino acids, growth factors [11], and small EVs [12][13][14].
In most domestic mammalian species, the embryo-when in the uterus-increases the secretion of biological molecules related to the maternal recognition of pregnancy (MRP). These molecules have

Embryo-Maternal Interactions Mediated by Embryotropins
Embryotropins are bioactive molecules such as proteins, lipids, and miRNAs secreted from both in vivo-or in vitro-produced mammalian embryos [32]. These molecules may act in autocrine and/or paracrine ways, modulating the embryo's development (in vitro culture) and the maternal endometrial cells, respectively [32].
In domestic ruminants, the mostly known and well-characterized embryotropin is the IFNT. IFNT is a cytokine secreted by the trophoblastic cells of the ruminant embryo and conceptus that can act in a paracrine and endocrine way. In the uterus, IFNT acts in a paracrine way, decreasing estrogen and oxytocin receptor expression in the endometrium, which is an essential step to maintaining a viable corpus luteum and producing progesterone [33]. Besides antiluteolytic function, IFNT has endocrine effects, stimulating the expression of IFN-stimulated genes (ISGs) in the endometrium [34], in the corpus luteum [35][36][37], in white blood cells [38], and in the liver [39]. The effects of IFNT secreted by the bovine embryo in the maternal organism can be detected as early as day 7 of development [40,41].
The IFNT function on the maternal recognition of pregnancy is well established for ruminants. There are parallels for such a response in other species. One example is the conserved response of interferon-stimulated gene 15 (ISG15), which is stimulated by IFNs and other cytokines. ISG15 is up-regulated in the endometrium of ruminants [34], primates [42], and mice [43] during early pregnancy. However, its function during the maternal recognition of pregnancy in non-ruminant species is not well established. Mouse knockout for Isg15 results in 50% fetal loss, which can be explained by change decidual gene expression that is functionally related to cell survival and adhesion pathways [44].
Besides IFNT, day 13 bovine embryos can also secrete prostaglandins, such as prostaglandin F2 alpha (PGF), prostaglandin E2 (PGE2), and prostaglandin I2 (PGI2) [45]. These prostaglandins act in a paracrine way in the endometrium, increasing ISGs' expression and function, which can be important for uterine receptivity as well as conceptus growth and development during early pregnancy [45]. In addition, PGE2 and PGI2 can modulate blastocyst implantation, decidualization, and endometrial vascular permeability during early pregnancy in mice and rats [46]. In large domestic species, PGE2 is secreted by the endometrium and embryo, showing an important role as the local antiluteolytic factor by oocyte cytoplasm after 72 h of maturation in bitch, demonstrating the role of EVs in improving the oocyte maturation rate [64]. Furthermore, another study using murine oviductal EVs demonstrated that EVs carrying plasma membrane Ca 2+-ATPase 4 (PMCA4) were uptaken by sperm, thus inducing sperm capacitation prior to fertilization [5].

Embryo-Maternal Interactions through Oviductal EVs
Oviduct used to be considered just a tubular connection between the ovary and the uterus where the oocyte and sperm passed through [73]. However, the oviduct is composed by ciliated and secretory cells that secrete oviductal fluid [8]. Several studies demonstrated the important biological role of the oviductal fluid during sperm capacitation [74], fertilization [4], and the outset of embryonic development [6]. Additionally, oviductal fluid contents include extracellular vesicles that have an important role during oocyte fertilization [5] and early embryonic development [7].
Recently, the functional effects of the EVs derived from the oviduct on gametes and embryos were summarized by Almiñana and Bauersachs (2019) [75]. In this review, we highlight the key findings related to EVs derived from oviductal cells and their effects in early embryonic development.
EVs from bovine oviduct epithelial cells (BOEC) were used in in vitro embryo production and demonstrated to improve embryo quality based on the increase in the number in trophectoderm and total cells and survival after vitrification [7]. In addition, space-specific EVs secreted from isthmus oviductal fluid were able to increase the survival rate and improve the development as well as the quality of in vitro produced blastocysts [67]. In vitro embryos were able to uptake EVs derived from in vivo oviduct epithelial cells, and this communication benefits the embryo blastocyst rate, survival, and quality [66]. Moreover, EVs secreted by donor oviductal cells increase birth rates after embryo transfer in mice due to decreased apoptosis and improved cellular differentiation in embryos [68]. Altogether, the se data show the importance of embryo-maternal interactions mediated by EVs derived from the oviduct during early embryonic development, leading to improved embryo quality and successful pregnancy.
An important problem that may occur during the passage of the zygote through the oviduct is ectopic pregnancy (EP), which occurs when the embryo after fertilization implants outside of the uterine cavity due to structural abnormalities in the fallopian tubes, for example [76,77]. Approximately 1.5%-2% of all the pregnancies are ectopic [78]; 97% are in the fallopian tube (oviduct) (reviewed by [79]). Currently, two diagnosis methods are used to detect EP: measurements of human chorionic gonadotropin(hCG)and progesterone in the serum [77]. The serial serum hCG measurement with intervals of 48 h is needed for the diagnostic; however, during this timecourse, tubal rupture might occur in patients, leading to possible complications in clinical status [77,80]. Therefore, only these two biomarkers are not precise and efficient to detect EP. Recently, new approaches showed that circulating miRNA miR-323-3p can be associated with serum hCG and progesterone to improve EP diagnostic [81]. Thus, the presence of miR-323-3p in serum could serve as a marker for EP. However, more studies are necessary to demonstrate if this miRNA is carried by small EVs or not, in order to become a reliable diagnostic marker.

Embryo-Maternal Interactions between Uterus and Embryo Mediated by EVs
The mammalian uterus is designed to allow sperm transport [82] as well as embryonic and fetal development [12]. In bovine, the morula stage embryo will arrive at the uterus at the uterotubal junction portion around Day 5 of embryo development [2]. Uterine fluid, termed histotroph in ruminants, is the result of glandular cells secretion inside of the uterine lumen [11,83]. This fluid is very important for the nutrition of the embryo since it contains proteins, lipids, amino acids, growth factors [11], and recently described extracellular vesicles that carry bioactive substances that are important for the early embryonic development [12][13][14]. During early embryo development, intense crosstalk starts between the embryo and the maternal uterine environment. This communication is necessary to induce the maternal recognition of pregnancy; thus, it is important to understand the role of extracellular vesicles in the embryo-maternal interface.
During maternal recognition of the pregnancy period, EVs were isolated within the uterine flushing of ewes on day 14 of the estrus cycle were fluorescently labeled with PKH67 and observed inside conceptus trophectoderm cells. This finding demonstrates that EVs are involved in paracrine communication between the endometrium and conceptus during the early pregnancy period [21].
In addition to that, sheep endometrial epithelium can secrete exosomes containing ovine endogenous jaagsiekte retroviruses (enJSRV) mRNA, which acts on trophectoderm via toll-like receptors (TLR) to induce IFNT production [69]. Trophoblast cells from the conceptus at day 15 and 17 secrete EVs containing IFNT that are able to stimulate ISGs' expression in endometrial cell culture [22,70]. Furthermore, macrophage-capping protein (CAPG) and aldo-keto reductase family 1, member B1 protein (AKR1B1) proteins are present in EVs isolated from the uterine flushing of pregnant cows on days 15 and 17 of gestation [22]. Besides that, EVs isolated from uterine flushing in the pre-implantation period increase the expression of apoptotic genes (BAX, CASP3, TNFA, and TP53 transcripts) in endometrial cells [70]. In addition to that, endometrial cells treated with EVs from the post-implantation induced and increase in vascular cell adhesion molecule 1 (VCAM) transcript, indicating the modulation of adhesion molecules [70]. Furthermore, exosomes isolated from uterine flushing obtained from pregnant cows on days 17, 20, and 22 were used to treat trophoblast CT-1 cells and did not induce changes in IFNT and CDX2 mRNA expression, suggesting that the pregnancy period may influence EVs' contents [70]. Together, this information highlights the EVs biological role during the period of maternal recognition of pregnancy, which may enhance embryo-maternal communication and consequently contribute to the maintenance of pregnancy.
Steroid hormones, such as progesterone and estradiol, can induce changes in the EVs secretion in human endometrial cells [26]. Progesterone, which is secreted by corpus luteum, is necessary to the establishment and maintenance of pregnancy and acts in the elongation and survival of the conceptus [84]. Progesterone induces myometrium relaxation and stimulates the production of mucin 1 (MUC-1), which is a protein that prevents conceptus adhesion to endometrium; thus, it can continue elongating and producing IFNT [85] as well as stimulating histotroph production by endometrial glands [11]. On day 10 to 14 of the estrous cycle in ovine, an increase in EVs secretion by endometrial luminal cells occurs, suggesting that progesterone is responsible for this event [71]. Besides that, EVs from the uterine lumen had miRNAs upregulated by progesterone that were predicted to modulate phosphoinositide 3-kinase/ Serine/threonine kinase 1 (PI3K/AKT), bone morphogenetic protein (BMP), and post-transcriptional silencing by small RNA pathways [71]. These results reinforce that progesterone is very important during the onset of pregnancy because it can modulate the endometrial function and consequently contribute to embryo development.
Extracellular vesicles are also secreted by endometrium and chorioallantoic membrane cells as well as trophectoderm and maternal endothelial cells from sows on day 20 of pregnancy [72]. MiRNAs and proteins within EVs were able to modulate the angiogenesis pathway within trophectoderm and maternal endothelial cells [72]. Moreover, EVs derived from the porcine trophectoderm are uptaken by maternal endothelial cells and stimulate the cellular proliferation of these cells [72]. Together, the se results demonstrate that EVs have an important biological role in conceptus-endometrium crosstalk during the establishment of pregnancy in porcine.
Local vascularization between the uterine horn ipsilateral and the corpus luteum is more prominent during the estrous cycle luteal phase than the contralateral phase, suggesting that the oviduct and uterine could signal to CL and adjacent tissue for future pregnancy and/or luteal vascularization maintenance [86]. This elevated vascularization on the ipsilateral horn could be involved with the early onset of pregnancy recognition. As an example, exosomal miRNAs were identified in serum samples of nonpregnant and pregnant mares on days 9, 11, or 13 postovulation [87]. These miRNAs were increased in nonpregnant mares and predicted to target the pathway of focal adhesion molecules (FAMs) in the endometrium [87], which are involved in the regulation of the extracellular matrix [88]. These data suggest that in pregnant mares, FAMs are normally abundant, which suggests that exosomal miRNAs are less necessary to modulate focal adhesion pathway mRNAs in the endometrium, allowing the embryo to move inside the uterus, which contributes to the maternal recognition of pregnancy [87].
In bovine, 27 miRNAs were highly abundant in the serum small EVs of cows with embryonic mortality compared to the pregnant group on day 17 [89]. These miRNAs modulate pathways associated with many important processes such as inflammation, cell proliferation, endometriosis, cell cycle progression, contraction, infection, late-onset preeclampsia, apoptosis, differentiation, uterine leiomyoma, ovarian endometriosis, and cell viability [89]. However, in a retrospective study examining EVs isolated from the blood plasma of pregnant cows on day 21 of gestation, a low abundance of 27 miRNAs was identified in samples from initial somatic cell nuclear transfer (SCNT) embryonic loss compared with full-term SCNT pregnancies and full-term artificial insemination pregnancies. These miRNAs modulate the pathways associated with pregnancy establishment as well as cell proliferation, differentiation, apoptosis, angiogenesis, and uterus embryonic development [90]. In addition, 29 miRNAs from serum small EVs were differently detected in the 30 days of pregnancy group compared to the normal group [91]. Different pathways involved in metabolism are modulated by these 21 up-regulated miRNAs and eight down-regulated miRNAs in pregnant cows [91].
These data suggest that there is EV-mediated communication between the uterus and peripheral circulation. This communication can be direct and modulated by biological factors secreted by the embryo, or it can be indirect, where the embryo stimulates an endometrium response. Thus, extracellular vesicles can be part of this intricate mechanism improving embryo-maternal interactions and consequently pregnancy success in mammals. Moreover, the se studies demonstrate the potential role of circulating exosomal miRNAs as biomarkers in early embryonic mortality or early pregnancy diagnosis.

Conclusions
In this review, we demonstrate the biological roles of extracellular vesicles in events occurring during the onset of pregnancy and involved in the communication between the embryo and the maternal organism in different mammalian species. EVs carry important bioactive molecules that are capable of modulating key reproductive events during the early pregnancy period. Further investigations are necessary to elucidate if EVs secreted by the oviduct and endometrium as well as embryos can arrive in peripheral circulation and modulate different pathways in maternal organisms. Thus, the progression in our understanding related to this type of communication can advance the tests to detect pregnancies, abnormal pregnancies (EP), and predict pregnancy loss, as well as push the development of new technologies to modulate early embryo-maternal interactions.