**4. Implications of EVs in reproductive pathology**

#### **4.1 Polycystic ovarian syndrome**

Polycystic ovary syndrome (PCOS) is one of the most common reproductive endocrine diseases in women of childbearing age (global prevalence rate: 4–21%). The clinical features of PCOS are: hyperandrogenemia, polycystic ovary (PCO) morphology, and oligo-ovulation/anovulation [71]. PCOS as a disease of follicular abnormal, there is a close relationship between EVs in follicles and the pathogenesis of PCOS [72].

The amount of EVs (mainly exosomes) in plasma of patients with PCOS are significantly increased and positively correlated with the number of follicles [73]. The expression of non-coding RNAs altered in exosomes of FF in PCOS, including miRNAs, piRNAs, and tRNAs [74]. Further study found that there is differential expression of miRNAs in plasma exosomes of women with PCOS, which was related to the menstrual cycle, antral follicle count (AFC, means the number of antral follicle) and hormone levels [75]. LncRNAs and circRNAs are also differentially expressed in follicular exocrine bodies of patients with PCOS. They may regulate ovarian steroid production, aldosterone synthesis, and secretion, and involved in Jak–STAT signal pathway, hippo signal pathway, and MAPK signal pathway. A highthroughput lncRNAs sequencing study found that there are 1253 upregulated and 613 down-regulated lncRNAs in FF exosomes of patients with PCOS infertility compared with patients with non-PCOS infertility, and nine lncRNAs with significant changes may play an important role in the pathogenesis of PCOS (lncRNA-LINC00173, lncRNA-H19, lncRNA-HDAC6, lncRNA-POP4, lncRNA-PTEN, lncRNAAKT3, lncRNA-DICER1, lncRNA-NF1, and lncRNA-MUM1). High-throughput sequencing of circRNAs also found the expression of 167 circRNAs in FF exosomes of PCOS patients are significantly upregulated and 245 circRNAs are significantly down-regulated compared with the control group, suggesting that these abnormally expressed circRNAs may play some roles in the study of pathophysiological mechanism of PCOS [76–78]. Similarly, proteins in exosomes of FF also changed. A proteomic study of exosomes from FF in patients with PCOS and healthy controls found that exosomes rich in S100-A9 can activate NF-κB signaling pathway in GCs and may play key roles in the progression of PCOS [79].

### **4.2 Endometriosis**

Endometriosis (EMs) is an estrogen-dependent inflammatory disease, characterized by the deposition and growth of endometrial stromal cells (ESCs) outside the uterine cavity, resulting in the appearance of endometrial tissue with growth activity *Roles of Extracellular Vesicles in Human Reproduction DOI: http://dx.doi.org/10.5772/intechopen.101046*

outside the uterine body. Pelvic peritoneum and ovary are the most common sites of ectopic endometrial stromal cells (EuESCs) growth. The main clinical manifestations are pelvic pain, pelvic adhesion, infertility and so on. EMs affects millions of women around the world, and the cause remains to be further determined [80].

The number of EVs in cervical and vaginal samples of rhesus monkeys with EMs decreased, indicating that the synthesis pathway of EVs in EMs has changed [81]. EMs are closely related to the formation of blood vessels. The biologically functional exosomes released by ESCs are transported to other parts through blood countercurrent in the endometrial and peritoneal microenvironment, and mediate intracellular signal transduction to ESCs itself or neighboring cells through the intercellular space in an autocrine or paracrine manner, thus regulating angiogenesis. *In vitro*, exosomes mediate the promotion of angiogenesis by EuESCs in the development of EMs [82]. Previous studies have shown that miRNAs can be extracted from the exosomes of ESCs, and these miRNAs potentially regulate the angiogenesis of ESCs. The expression of miRNA-21 related to angiogenesis in the exosomes of women with EMs is significantly higher than that of women without Ems [83]. Our research group identified the differential expression patterns of exosomal miRNAs in patients with EMs and found that 49 miRNAs expressed differentially in EuESC exosomes compared with normal endometrial stromal cells (NESCs) exosomes. Many exosomal miRNAs may be involved in regulating endometrial receptivity in women with EMs-related infertility through their predicted target genes: homeobox A10 (HOXA10) and leukemia inhibitory factor (LIF) which are essential for normal implantation. Our finding provides a new sight on how EVs participate in the occurrence and development of EMs [84].

#### **4.3 Pregnancy complications**

Common pregnancy complications include gestational hypertension, diabetes, and preeclampsia. Serious pregnancy complications may endanger the lives of fetuses and mothers [85]. The concentration and biological activity of EVs changed in a variety of pregnancy complications, such as preeclampsia (PE), gestational diabetes mellitus (GDM), preterm delivery (PTB), intrauterine growth restriction (IUGR), recurrent abortion and unexplained abortion, suggesting that EVs are closely related to pregnancy complications [86, 87].

Maternal obesity is a risk factor for GDM and several other pregnancy complications. Adipose tissue hypertrophy or metabolic stress can change the cargos in EVs (mainly miRNAs), leading to systemic inflammation and insulin resistance (IR) in obese patients with gestational diabetes. These altered EVs may also change the physiological function of placenta and remove the regulation of placental nutrition signal pathway, resulting in obesity-related pregnancy complications [88–90].

There is a class of syncytial nuclear aggregates (SNAs) in placental-derived EVs the level of which increases with the progression of pregnancy and is associated with pregnancy complications such as PE [91]. Numerous placental EVs were detected in the serum of pregnant women with PE. These EVs have pro-inflammatory, antiangiogenic, and procoagulant activities, which may lead to activation of the blood coagulation system, systemic inflammation, and vascular endothelial dysfunction [92]. In addition, the abundance of syncytin-2 in serum-derived EVs attenuates in women with PE. This may lead to immunosuppressive reduction and pathological inflammation in pregnancy complications [93].

## **4.4 Male infertility**

Infertility has becoming a global health problem, the incidence rate is as high as 15%, of which about 50% of infertility cases are caused by male reproductive disorders [94]. Just as oocytes need normal FF microenvironment to provide nutrition and support, sperm also need SP to provide a safe environment in order to survive and transport in the female reproductive tract. As mentioned earlier, there are a train of semen proteins in SP, most of which are transported by EVs (prostasomes and epididymosomes). Since only EVs proteins from normal sperm can regulate the movement of SPZ and trigger SPZ capacitation, the changes of proteomic characteristics in semen EVs may indicate male reproductive tract dysfunction; at the same time, they can be used as a biomarker of male infertility. In male infertility, the proteins transported by reproduction-related EVs are differentially expressed in azoospermia, asthenospermia, oligozoospermia, teratospermia, or other male infertility compared with normal sperm, and the prostatic proteins related to sperm energy production and sperm activity are under expressed in abnormal sperm, indicating that SP proteome map may be a potential indicator of sperm dysfunction [36].

Azoospermia may be one of the causes of male infertility. There are two types of azoospermia: non-obstructive (NOA) and obstructive azoospermia (OA) caused by seminal tract obstruction [95]. A study found that deficiency of EVs contributes to lower ejection volume, and changes of various nutritional components in semen [96].

At present, a sea of EV cargos has been identified to be differentially expressed in male infertility patients, which can be used as diagnostic markers and treatment of infertility as detailed in the next section.

### **4.5 Ovarian and cervical cancer**

Gynecological malignant tumors such as ovarian cancer (OC) and cervical cancer (CC) are two of the three major tumors of the female reproductive system, the former with the highest mortality rate and the latter with the second incidence and the third fatality rate [97, 98]. The difficulty of early diagnosis, high metastasis rate, and strong drug resistance are still the main obstacles in gynecological cancer diagnosis and treatment. As a member of tumor microenvironment, exosomes not only play important roles in tumor occurrence and development, drug resistance and immunosuppression, but also can be used as a new tumor marker and clinical target molecule in clinical work.

The latest study has proved that there is a difference in the expression of exosomes in the blood of patients with OC and normal people, and the expression in the body fluid of patients with OC is related to the stage of the tumor [99]. A total of 1017 co-expressed proteins were screened from the exosomes secreted by two kinds of OC cells, among which tubulin beta 3 class III (TUBB3), epithelial cell adhesion molecule (EpCAM), claudin 3 (CLDN3), proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR), and fatty acid synthase (FASN) were highly enriched in tumor-related signal pathways. Claudin-4 positive exosomes can be used in the diagnosis of OC, with a specificity of 98% and a sensitivity of 51%, while CA125 has a specificity of 98% and a sensitivity of 71%. Although claudin-4 is not a better diagnostic marker than CA125, this study confirms that exosome-related proteins can be used in the diagnosis of OC. These results suggested that the related proteins in exosomes may become markers for the diagnosis of OC [100, 101]. In addition, the level of exosomal proteins extracted from the serum of patients with OC were

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

higher than that of patients with benign ovarian disease and healthy women, and that of patients with advanced OC was higher than that of patients with early stage. The expression of tumor-specific antigen MAGE3/6 and transforming growth factor β1 (TGF- β1) in patients with OC was significantly higher than that in patients with benign ovarian disease and healthy women [102]. CD24 is a marker of poor prognosis in OC and other types of cancer. CD24 positive exosomes were screened from ascites of patients with OC, which is a good marker for early diagnosis of OC [103].

There are a large number of exosomes in cervicovaginal lavage specimens of women with CC, which carry miRNAs playing important roles in CC. It was found that many differentially expressed miRNAs, such as miR-483-5p, miR-1246, miR-1275, microRNA-21, microRNA-146a and miR-222-3p, were up-regulated in vaginal lavage and cell culture of CC, while some miRNAs, such as let-7d-5p, miR-92a-3p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-99-5p, miR-100 5p and miR-320a, were down-regulated. It is suggested that the contents of miRNAs in exosomes may be related to the occurrence of CC and is expected to become a new diagnostic marker of CC [104]. There was a significant difference in the expression of exosomemediated let-7d-3p and miR-30d-5p between cervical tumors and adjacent normal tissues, suggesting that these two plasma exosomes let-7d-3p and miR-30d-5p can be used as valuable biomarkers for non-invasive screening of CC [105]. The exosomes of cervical squamous cell carcinoma (CSCC) transfers miR-221-3p from cancer cells to vascular endothelial cells and promote angiogenesis by down-regulating thrombospondin-2 (THBS2) [106]. In addition, exosomal miR-221-3p secreted by CC cells promotes the invasion, migration, and angiogenesis of CC microvascular endothelial cells (MVECs) by down-regulating the expression of MAPK10 [107]. The expression of activating transcription factor 1 (ATF1) and RAS genes were significantly upregulated in primary and recurrent CC mouse models, and ATF1 and RAS could also be detected in blood exosomes of mouse models. These results suggest that exosomemediated ATF1 and RAS may become potential diagnostic markers for CC, which provides a new idea for individual detection and treatment of CC [108].
