**2.2 EVs derived from the oviduct**

Oviduct is the place where occur oocyte transportation, fertilization, and initial embryo development. Oviductal extracellular vesicles (oEVs), as the main components of oviduct fluid, can orchestrate gamete/embryo-oviduct and embryo-maternal interactions, hence supporting oocyte mature (in canine), embryonic development, and motility, capacitation, and fertilizing ability of sperm [14, 15]. The contents of oEVs (mRNAs, miRNAs, and proteins) are dynamic at different stages of the estrous cycle, suggesting that cargos of oEVs are under hormonal control [16]. The first oEVs in the oviductal fluid was described in mouse, and then they were identified in other mammals including human. In humans, oEVs (oviductosomes) arising from the apocrine pathway carry and deliver plasma membrane Ca2 + -ATPase (PMCA) which are fertility-modulating proteins. PMCA delivered to sperm can prevent premature capacitation and maintain Ca2 + levels homeostasis; lack of PMCA leads to sperm motility loss and male infertility in mice [17]. Bovine oEVs can pass through the zona pellucida and then be internalized by embryonic cells to increase blastocyst rate and improve the quality of embryo. The possible mechanism may involve: (1) The proteins of bovine oEVs involved in cell communication, cell metabolism, localization, and reproduction [18]; (2) The miRNAs of bovine oEVs contribute to a successful pregnancy through inducing changes of embryonic transcriptome [15]; (3) Exosomes derived from bovine oviduct epithelial cells (BOECs) improve the mitochondrial health of bovine embryo because they re-establish the tricarboxylic acid cycle (TCA-cycle) flux [19].

#### **2.3 EVs derived from the endometrium**

Successful pregnancy not only needs intercommunication in ovary and oviduct, but also the crosstalk between endometrium and blastocyst especially the trophoblasts. Indeed, several studies have found that the secretions in endometrium affect the embryo. The first reported EVs of the female reproductive system are endometrium EVs (endEVs) in mouse uterine cavity [20]. EndEVs were found in endometrial fluid during the menstrual/estrous cycle, and were also released from endometrial epithelial cells cultured *in vitro* [21]. Several studies reported that the EVs in the endometrium have autocrine/paracrine effects on regulation of receptivity and implantation of uterine [22].

In EndEVs of primary endometrial epithelial cells (ECCs), 35% of the proteins had not been reported before, indicating that the contents of EndEVs are unique [23]. EndEVs influence blastocyst implantation through their hormone-specific protein cargos to modulate trophoblast capacity of adhesion, migration, invasion, and so on. Among these protein cargos, metalloproteinases (MMP-14 and ADAM10) regulated by the endometrial receptivity-related hormone are essential to trophoblast invasion and pregnancy outcome because they regulate (activate and degrade) other factors in endEVs. *In vitro*, when EndEVs are internalized by trophoblast cells, they can activate Focal Adhesion Kinase (FAK) signaling, thereby enhance the adhesive capacity of trophoblast cells [24–26]. The proteins of bovine endEVs in ECCs change during the peri-implantation period: endEVs enhance the expression of cell apoptosis genes in the preimplantation stage, but cell adhesion genes in the post-implantation stage [27].

MiRNAs of EndEVs act as modifiers for implantation. EndEVs contain specific miRNAs cargo which targets predictably genes involved in blastocyst implantation and crucial signaling pathways such as the VEGF, the Jak–STAT, and the Toll-like receptor in primary ECC and ECC1 (an endometrial epithelial cell line) [21]. During the window of implantation (the period allows blastocyte invasion), maternal miRNAs are differently expressed in human EndEVs. One of the miRNAs, miR-30d upregulates Itgb3, Itga7, and Cdh5, which are involved in embryo adhesion in murine; treating miR-30d *in vitro* to murine embryo can increase embryo adhesion [28].

Sperm adhesion molecule 1(SPAM1)/PH-20 is a hyaluronidase which enhances sperm fertility. EVs in murine uterine luminal fluid can deliver SPAM1 to sperm membranes to increase sperm penetration through cumulus cell layers around oocyte, and adhesive to zona pellucida, same as SPAM1 transferred from epididymosome to SPZ play a significant role in sperm maturation and motility [2, 20]. While as mentioned earlier, in oEVs SPAM1 interaction with sperm to inhibit premature acrosomal reaction, showing that the EVs with the same cargo in different organs have multiple functions [17].

### **2.4 EVs derived from the placenta and embryo**

Pregnancy is a unique immunomodulatory state in which the maternal immune system temporarily tolerates the paternal antigen so as to protect the fetus from allogeneic rejection, at the same time maintaining immune surveillance to protect the mother from external pathogens. EVs have been identified in trophoblastic cells, placenta, and maternal circulation [22]. Maternal immune response is effected by the EVs from endometrium, embryo, and trophoblast cells in early pregnancy, while by the EVs from the human placenta in late pregnancy [29].

#### *Roles of Extracellular Vesicles in Human Reproduction DOI: http://dx.doi.org/10.5772/intechopen.101046*

Placenta is an important organ and performs a variety of functions to support pregnancy. Placental-derived EVs are important media for intercellular communication, considered to be potential mediators in regulating maternal immune response to achieve a successful pregnancy and maternal and infant health outcomes [30]. These EVs inhibit the immune response to the developing fetus and establish and maintain a systemic inflammatory response against infectious invaders [31]. The underlying mechanisms include: (1) Compared with non-pregnancy, during pregnancy, the cytokines such as transforming growth factor-β 1 and IL-10 increase, and the ability to induce caspase-3 activity in cytotoxic natural killer (NK) cells enhance in peripheral blood EVs, which promoting immunosuppressive phenotype by inducing apoptosis to help regulate the maternal immune response to the fetus [32]. (2) In amnion (the innermost layer of placenta), miR-21 helps in embryo growth; histone 3 (H3), heat shock protein 70 (HSP70), activated form of prosenescenceP-p38, MAPK participate in stress response and are related to pregnancy outcome [33].

In addition to the placenta, embryos can also secrete EVs (embryo extracellular vesicle, eEVs) to participate in the regulation of implantation and other pregnancy processes. Embryos transferred to uterine are known as blastocyst, which consists of a fluid-filled cavity, inner cell mass (ICM), and trophectoderm/trophoblast (T) cells. Mouse embryonic stem cells from the ICM can produce MVs which reach the trophoblast ectoderm, thereupon enhancing the migration ability of trophoblast cells, both as isolated cells and in the whole embryo. Laminin and fibronectin exist in the eEVs of ICM orchestrate ICM to attach to the integrin on the surface of trophoblast cells and stimulates the cascade of c-Jun N-terminal kinase and FAK, increasing the migration of trophoblast cells. Injecting these eEVs into the blastocyst cavity of day 3.5 blastocysts can improve successful implantation rate [34]. EEVs-derived PIBF alters the maternal immune system by increasing IL-3, IL-4, and IL-10 to achieve a Th2-dominant cytokine balance. The aberrant expressions of PIBF may lead to pregnancy failure [35]. In eEVs of trophoblast also carry a variety of factors regulating maternal immunity to protect the fetus, including: tissue factors (TFs), soluble vascular endothelial growth factor receptor1 (sFlt-1), immunosuppressive factors, and so on [33].
