**5. Clinical and therapeutic applications of EVs in reproductive disorders**

#### **5.1 EVs as biomarkers**

The optimal age to have a baby is a medical social problem that needs to be faced by the whole society. Based on the risk of fetal disease, male and female fertility, and many other factors, it is generally believed that the best childbearing age for men and women is between 25 and 35 years old. With the increase of age, the semen quality of men decreases, the sperm concentration and motility decrease, and the deformity rate increases; the quality of oocytes decreases in women, which limits the success of pregnancy [109]. Therefore, looking for reproductive-related markers that change with age has become a meaningful research direction. It is found that the miRNAs characteristics of follicular EVs vary with the age of women, suggesting that miRNAs of EVs are possible markers and predictors of age-related oocyte quality decline [9]. A study compared the EVs of FF in young women with that in older women and found four significantly different miRNAs: miR-99b, miR-134, and miR-190b were upregulated, and miR-21-5p was down-regulated in older women. These miRNAs regulate genes related to cell apoptosis, p53 signaling, and cytokine-cytokine-receptor interaction, so their changes may affect follicular development and oocyte maturation [110].

There are numerous EVs in SP which can easily detach and collection. A study identified that prostasomal proteins relating to sperm activity and energy production pathways have changed in non-normozoospermic men. Among them, HIST1H2B, KLK2, MIF, MPO, and MSMB are related to liquefaction of semen (in order to break through SP) and sperm-oocyte binding. LDHC, HK1, PNP, APRT, and SLC2A14 are involved in sperm energy production [111]. Others include ELSPBP1/BLVRA, GPX5, SPAM1, P34H, Aldose reductase and sorbitol dehydrogenase, PAP, PSA, TMPRSS2, pTGase, PSCA, KIF5B, ANXA2, and so on [50]. Due to these variations may have a relevant correlation with male infertility, the cargos of EVs can potentially serve as useful biomarkers of male infertility.

In ART, FF can be taken out together with the follicles, so it is an attractive biomarker to detect the quality of oocytes. The expression of miRNAs in EVs is related to fertilization status and embryo quality [112]. It has been found that the overexpression of miR-92a and miR-130b can lead to adverse results of *in vitro* fertilization (IVF), while the differential expression of miR-214, miR-454, and miR-888 is related to high quality embryos [113].

Peripheral blood is the most widely used biological sample in clinical diagnosis. The cargos in EVs in peripheral blood are good indexes for the detection of many diseases [114]. Prostasomes produced by prostate tumor cells may be involved in the spread of prostate cancer. Prostate corpuscles in peripheral plasma and their specific proteins, such as PAP, PSA, TMPRSS2, and PSCA, may be valuable biomarkers of prostate cancer [115]. Since EVs and their content in women's peripheral blood can be detected from early pregnancy, they can be used as biomarkers for the prediction or diagnosis of pregnancy complications, fetal developmental disorders, and preterm birth (PTB). About 11% of infants around the world are born prematurely every year. PTB, defined as delivery before 37 weeks of pregnancy, is the leading cause of neonatal morbidity and mortality. The exosomes in plasma can be used to predict whether pregnant women will develop PTB, so as to prepare for prenatal intervention. Several miRNAs such as hsa-miR-381, hsa-miR-154, hsa-miR-377, and hsa-miR-150-5p in circulating EVs have been predicted to be potential biomarkers of PTB. A longitudinal study analyzed the miRNAs in plasma EVs collected from multiple pregnant women throughout pregnancy and found that the number and expression profile of miRNAs of EVs in maternal plasma of PTB changed significantly compared with the term birth, especially these miRNAs associated with TGF-β, p53, and glucocorticoid receptor signaling. It is worth pointing out that these miRNAs are known to be associated with fetal membrane apoptosis [116–119]. Fetal cell-free DNA (cfDNA) carried by maternal circulating plasma and serum exocrine has been used in non-invasive prenatal diagnosis as a biomarker of pregnancy complications, such as PE and GDM [120].

MVs from vaginal microbes (e.g., Group B Streptococcus) can affect fetal placental tissue and lead to pregnancy complications [121]. Therefore, it may be possible to detect the contents of vaginal MVs to find significantly differential cargos, so as to detect pregnancy complications as soon as possible.

With the deepening of research, studies in mounting numbers have shown that the miRNAs in EVs can be used to diagnose pregnancy-related diseases, including miR-136, miR-155, miR-210, miR-486, miR-494, miR-495, miR-517-5p, miR-520a-5p, miR-525-5p, and miR-548c-5p [122].

Wrong or late diagnosis is one of the main problems of EMs. Therefore, it is of great significance to find biomarkers for early diagnosis. Extracellular nucleases are key enzymes in the process of inflammation, which participate in the occurrence and development of EMs by regulating the levels of extracellular ATP and adenosine. A study has found that the extracellular nuclease activity in the aspiration fluid of endometrial tumors is significantly higher than that of simple cysts and comes from the exosomes of these fluids, and therefore the exosomes of these lesions may become biomarkers of EMs. The proteins and miRNAs transported by endEVs may be very valuable biomarkers of human endometrial diseases [123].

The expression of DENN domain containing 1A (DENND1A) in urine-derived exosomes of PCOS patients was significantly higher than that of normal controls, suggesting that it can be used as a potential PCOS marker [124].

#### **5.2 Clinical and therapeutic applications**

As biological substances are produced by normal human bodies, the in-depth study of EVs in different physiological and disease states is helpful to identify specific proteins related to EVs functional defects, thus promoting the development of new diagnoses and treatment strategies for reproductive dysfunction. EVs have attracted much attention in the field of translational medicine because compared with other commonly used synthetic drug delivery carriers (such as liposomes), bioengineered EVs have not only have inherent targeting ability but also are low immunogenicity, easy to obtain, selective assembly, high modification flexibility, and biological barrier permeability. Considering EVs can deliver functional cargos to target cells, it is hopeful for us to use them as a drug delivery tool. For example, EVs may be able to pass through the tissue barrier (such as endothelial barrier, blood–brain barrier, endothelial barrier, and blood–brain barrier) through endocytosis. At present, the application of exosome therapy has been explored in a variety of reproductive diseases, such as the treatment of female infertility, POI, EMs, IUA, and so on. For instance, exosomes can carry specific miRNAs into receptor cells to target genes. There are also studies to target the treatment of related diseases by putting modified small interference RNA (siRNAs) into exosomes [125, 126].

EuESCs can secrete a large number of exosomes, which contain a variety of miRNAs may not only be closely related to the occurrence, development and complications of EMS, but also have different expression levels from normal endometrial cells. Therefore, exosomal miRNAs have certain application value in the early diagnosis, judgment of disease progression, and prognosis of EMs. In EMs mouse models, the delivery of miR-214 rich exosomes isolated from EuESCs can inhibit fibrosis and regulate the development of EMs [127].

Exosomes derived from endometrial epithelial cells can enhance the adhesion, implantation, and growth ability of embryo *in vivo* [128]. EVs obtained by uterine lavage carries proteins that regulate and predict embryo implantation, and its protein composition, metastatic and invasive properties, and antioxidant function are dynamically regulated throughout the menstrual cycle, so it has the potential to be used as a biomarker of embryonic development, implantation, and successful pregnancy [129].

PCOS and GDM are closely related to obesity. Adipose tissue macrophages (ATM) of obese mice secrete exosomes containing miRNAs, which can lead to impaired glucose tolerance and IR when given to thin mice. In contrast, ATM exosomes obtained from lean mice can improve glucose tolerance and insulin sensitivity when given to obese mice. These miRNAs can be transferred to insulin target cells through paracrine or endocrine regulation, and have a strong effect on cellular insulin action, insulin sensitivity, and global glucose homeostasis [88]. Therefore, the differentially expressed miRNAs of ATM in obese and thin people may be used as biomarkers of PCOS and GDM and used in their treatment.

Premature ovarian failure (POF) is defined as ovarian function lost in women before 40 years of age. The incidence of POF in women is about 1%. The occurrence of POF is closely related to the depletion of ovarian follicles, but the specific mechanism is still not clear [130]. The combination of stem cell therapy and EVs provides a glimmer of hope for the treatment of POF. In one study, exosomes collected from Bone mesenchymal stem cells (BMSCs) were injected into POF mice, and then found that follicles, FF, and corpus luteum increased in mice ovaries, while apoptosis-related genes p53 and caspase3 were down-regulated, indicating that BMSC-derived exosomes can improve the phenotype of POF. Further in *vitro* experiments showed that the expression of miR-664-5p was increased in BMSC-derived exosomes and could reverse the injury of POF granulosa cells, which was regulated by p53 [131]. Another study found that miR-144-5p in BMSC-derived exosomes inhibit POF GCs apoptosis by targeting PTEN and activating the PI3K/AKT pathway in *vitro* [132]. Furthermore, exosomes derived from human amniotic epithelial cells (hAECs), amniotic fluid stem cells (AFSCs), and placenta-derived mesenchymal stem cells (PD-MSCs) can regulate the apoptosis pathway to reduce the apoptosis of ovarian follicles through their different cargos, such as miR-10a, miR-146a, miR-1246, and antioxidant enzymes [133]. Taken together, these studies demonstrated that exosomes can mediate ovarian function, thus pointing out a new direction for the treatment of POF.
