Extracellular vesicles in normal pregnancy and pregnancy-related diseases.

Abstract Extracellular vesicles (EVs) are nanosized, membranous vesicles released by almost all types of cells. Extracellular vesicles can be classified into distinct subtypes according to their sizes, origins and functions. Extracellular vesicles play important roles in intercellular communication through the transfer of a wide spectrum of bioactive molecules, contributing to the regulation of diverse physiological and pathological processes. Recently, it has been established that EVs mediate foetal‐maternal communication across gestation. Abnormal changes in EVs have been reported to be critically involved in pregnancy‐related diseases. Moreover, EVs have shown great potential to serve as biomarkers for the diagnosis of pregnancy‐related diseases. In this review, we discussed about the roles of EVs in normal pregnancy and how changes in EVs led to complicated pregnancy with an emphasis on their values in predicting and monitoring of pregnancy‐related diseases.


| INTRODUC TI ON
During the process of pregnancy, a large range of extracellular vesicles (EVs) with different sizes are extruded into the maternal circulation, including macro-vesicles (also termed as syncytial nuclear aggregates or mononuclear trophoblasts), micro-vesicles (MVs) and exosomes. [1][2][3] These distinct EVs could be distinguished by their specific features, including size, biogenesis process and biological functions. 4,5 Exosomes, with 30-150 nm in diameter and from endocytic origin, are the most studied EVs so far. Extracellular vesicles contribute to cell-to-cell communication by transporting signalling molecules including proteins and nucleic acids. The cargos in these small vesicles may reflect the physiological or pathophysiological state of the source cells. 6,7 Emerging evidence suggests that exosomes with functional cargos can be transferred between foetus and maternal bodies. 8 A variety of body fluids such as blood, urine and amniotic fluid contain EVs released from trophoblast cells, immune cells and endothelial cells, among others. [9][10][11] EVs are suggested as essential modulators of multiple processes of pregnancy, including implantation, migration and invasion of trophoblasts, and cellular adaptations to the physiological changes. [1][2][3] Changes in the concentration, composition and bioactivity of EVs have been reported to be associated with pregnancy-related diseases. 12,13 Given these important roles, EVs have great potential to be developed as non-invasive biomarkers of foetal and maternal conditions. In this review, we summarized the current knowledge about the functions of EVs in pregnancy and its complications. The contribution of macro-vesicles to normal and complicated pregnancies has been well documented in other literatures. 14,15 Therefore, we mainly focused on the roles of exosomes and MVs in normal pregnancy and its related diseases. The clinical values of EVs detection for the early diagnosis and monitoring of pregnancy-related diseases are also discussed.

| E Vs IN NORMAL PREG NAN C Y
Extracellular vesicles mediate foetal-maternal communications and participate in many important physiological activities during normal pregnancy, including embryo implantation, immunomodulation, spiral arteries remodelling, metabolism adaptations and delivery ( Figure 1).

| EVs in embryo implantation
Extracellular vesicles take part in maternal-embryo interaction within human uterine microenvironment, promoting implantation, an earliest and essential step for successful pregnancy. 16,17 Greening  have been introduced as mediators of embryo-endometrium crosstalk in the implantation process. 19 MiRNAs packaged in MVs could be detected in human uterine luminal fluid, indicating their potential role in implantation. 20,21 Vilella et al 22 provide evidence that miR-30d is present in exosomes and could be transferred from receptive endometrial epithelium to embryo trophectoderm, improving the adhesive ability of pre-implantation embryo. In addition, exosomal miR-30d could act as a transcriptomic regulator, leading to overexpression of genes involved in embryo adhesion, such as Itgb3, Itga7 and Cdh5. The uptake of exosomal miR-30d increases the rate of murine embryonic adhesion to the endometrial epithelium in vitro. They further demonstrate that maternal and/or embryonic miR-30d deficiency impairs endometrial receptivity and embryo implantation rates in vivo by using wild-type and miR-30d knockout mice. 23 Embryo itself can also generate MVs to enhance implantation by promoting the migration of trophoblast cells.
Desrochers et al 24 demonstrate that extracellular matrix proteins fibronectin and laminin α5 on the surface of MVs from embryonic stem cells can bind to integrin α5β1 and laminin receptors on trophoblast cells to trigger the activation of FAK and JNK signalling pathways.
Moreover, they confirm that the implantation efficiency is increased in surrogate mice after injecting embryonic stem cell-derived MVs into blastocysts, indicating an important role of EVs in mediating embryo implantation.

| EVs in immunomodulation
Recent studies have highlighted a critical interaction between EVs and immune cells in modulation of pregnancy during which maternal immune system tolerates the growing foetus and maintains its normal functions. 25 However, this effect is not observed in syncytin-2 (Syn-2)-silenced VCT exosomes, indicating an important function of exosomal Syn-2 in immune suppression. 29 Kovacs et al 30 suggest that trophoblastic EVs act as important players in immune tolerance, which is associated with their regulation of T reg differentiation. Extracellular vesicle-derived heat shock protein family E member 1 (HSPE1) promotes T reg differentiation from CD4 + T cells and T reg cell expansion in vitro. Placental exosome-derived miR-499 is elevated in the first trimester in cows and could inhibit NF-κB activation via Lin28B/let-7 axis, thereby repressing inflammation response and forming an immune-tolerant microenvironment in the uterus. miR-499 inhibition leads to inflammation dysregulation and increased risk of pregnancy failure in vivo. 31 Maternal immune system is essential for the uterus to prevent pathogenic infections. 32 Holder et al 33

| EVs in spiral artery remodelling and vascular function
To meet increased metabolic demands of the mother and foetus and to ensure adequate nutrients and oxygen supplies to the growing foetus, early stage of pregnancy requires sufficient spiral artery remodelling and physiological adaptations in the cardiovascular system. 35 Interestingly, the nano-vesicles extruded from first trimester human placenta cannot affect endothelium-dependent vasodilation of uterine artery whereas it affects the ability of systemic mesenteric arteries to undergo endothelium-and nitric oxide-dependent vasodilation in pregnant mice. 40 There is evidence that normal circulating piglet foetal exosomes derived from the umbilical vein are able to increase tube formation function of human umbilical vein endothelial cells (HUVECs). After coculture with normal circulating piglet foetal exosomes derived from the umbilical vein, the expression of pro-angiogenic genes VEGF and Notch1 are up-regulated while that of antiangiogenic gene TSP1 is down-regulated in HUVECs. 41 Similarly, Jia et al 42 provide evidence that both maternal and umbilical serum exosomes enhance HUVEC proliferation, migration and tube formation abilities. Furthermore, the altered expression of a subset of migration-related miRNAs, including miR-210-3p, miR-376c-3p, miR-151a-5p, miR-296-5p, miR-122-5p and miR-550a-5p, has been identified in umbilical serum exosomes.

| EVs in metabolism
Increasing evidence suggests that EVs are involved in metabolic homoeostasis. 43,44 However, little is known about the role of EVs in metabolic regulation with regard to pregnancy. Nair et al 45

| EVs in delivery
Signals of foetus maturation probably induce inflammatory responses and thus prepare the uterus for delivery. 47,48 The previous study has shown that the activated form of p38 mitogen-activated protein kinase (MAPK) is a term parturition associated marker. 49,50 Sheller et al 51 demonstrate that phosphorylated p38 MAPK is expressed in exosomes from amnion epithelial cells (AECs) and is upregulated in response to oxidative stress. Hadley et al 52 have tested if senescent foetal AEC exosomes can cause inflammatory changes F I G U R E 1 Effects of EVs in normal pregnancy. EVs mediate foetal-maternal communications in normal pregnancy. EVs contribute to embryo implantation by promoting trophoblast adhesion. Placenta can interact with immune cells via EVs to balance immune activation and suppression across the gestation. EVs can activate endothelial cell (ECs) and vascular smooth muscle cells (VSMCs) to promote angiogenesis. EVs can accelerate glucose metabolism in the placenta and skeletal muscle. Moreover, inflammation signals of maturation in EVs can prepare uterus for delivery in maternal and placental tissues. Primary AECs are grown in normal cell culture (control) or oxidative stress condition, and myometrial and decidual cells are treated with those AEC-derived exosomes.
Regardless of the source of exosomes, exosome treatments increase the secretion of IL-6, IL-8 and prostaglandin E2 (PGE2) and induce the activation of NF-κB in uterine myometrial and decidual cells. However, exosomes produced under oxidative stress condition have a more dominant pro-inflammatory effect than control exosomes. In addition, more foetal exosomes are possible to reach maternal gestational tissues at term labour. Altogether, their data imply that foetal cell exosomes may contribute to the timing of birth by increasing uterine cell inflammation. Ithier et al 53,54 suggest that foetal lung-derived C4BPA plays a role in birth timing determination by utilizing proteomics analysis of exosomes from foetal cord arterial blood. They reveal that C4BPA could bind to CD40 of placental villous trophoblast to promote p100 processing to p52. In turn, RelB/p52 heterodimers transport to the nucleus to activate noncanonical NF-κB pathway in placenta, which drives the epigenetic changes in pro-labour genes. A recent study in mouse model also suggests that non-specific inflammation builds up gradually across late gestation and reaches the peak at the day before the expected delivery. Exosomes from uterine tissues at late gestation contain pro-inflammatory cargos and could affect birth timing by promoting local inflammatory responses in foetal membranes. 55

| E Vs IN COMPLIC ATED PREG NAN C Y
Abnormal changes of EV concentration and composition are involved in pregnancy-related diseases, including pre-eclampsia (PE), gestational diabetes mellitus (GDM), preterm birth (PTB) and other adverse pregnancy outcome ( Figure 2).

| EVs in inflammation
The dysregulation of the balance between pro-and anti-inflammatory factors has been reported in complications of pregnancy. 56,57 Exosomes from extravillous trophoblast cells cultured under low oxygen tension could increase TNFα expression in HUVECs, thus inhibiting their migration. 58 In addition, EV-induced active platelets could activate NLRP3 inflammasome in trophoblast cells via ATPpurinergic signalling, leading to PE-like symptoms (ie pregnancy failure, elevated blood pressure, increased plasma sflt-1 and renal dysfunction) in mice. Notably, platelets induce placental sterile inflammation without blood clots and increase fibrin accumulation, indicating that EVs-activated platelets are linked with inflammatory reaction directly in this setting. 59 In PE patients, the level of plasma gelsolin, an anti-inflammatory factor of maternal origin, is much lower than that in healthy women at late stage of pregnancy, which may be associated with the shedding of EVs. 60 The expression of high mobility group box 1 (HMGB1), a pro-inflammatory danger signal, is increased in macro-vesicles derived from trophoblast after treating placental explants with pre-eclamptic sera. 61 Ospina-Prieto et al 62,63 show that exosomal miR-141 derived from foetal tropho-

| EVs in metabolic disorders
Metabolism dysregulation is closely related to some pregnant complications, for instance, GDM and foetal overgrowth. 83,84 Compared to AT exosomes of NGT controls, AT exosomes of GDM could increase the expression of genes associated with glycolysis and glycogenolysis in placental cells. It is possible that increased placental glycogenolysis in GDM accelerates glucose transferring to foetus, resulting in foetal overgrowth. Furthermore, ingenuity pathway analysis reveals the differential expression of mitochondrial function-related proteins in AT exosomes of GDM. 46 The work from Nair et al 45 shows that placental exosomes are capable of modulating insulin-stimulated migration and glucose uptake in primary skeletal muscle cells. Gene target and ontology analyses of differentially

| E Vs A S B I OMARK ER S FOR PREG NANC Y-REL ATED DISE A SE S
Extracellular vesicles have a tissue-specific pattern and may reflect the status of source cells, which highlights their usefulness as indicators of cellular function and as biomarkers for various diseases.
Increasing studies suggest that EV concentration and content are of great diagnostic utility for women at risk of pregnancy-related diseases (Table 1). and miR-495 are 6.4-, 3.9-and 2.1-fold higher than that in normal pregnancies, respectively, according to a study enrolling 100 PE patients. 91 In addition, miR-141, 62  in the urine of PE women and is correlated with proteinuria and diastolic blood pressure. The expression level of nephrin is reduced in pre-eclamptic renal tissue compared to that of normotensive pregnancies. The elevated urinary podocin + EVs-to-nephrin + EVs ratio is associated with renal injury in PE. 94 In addition, abnormal increased sodium reabsorption also contributes to hypertension in PE. Impaired urine sodium excretion, Na/K ratio and elevated urine plasminogen are also found in PE. 95 In urine EVs, increased expression of epithelial sodium channel (ENaC) and phosphorylation of Na-Cl2-K co-transporter 2 (NKCC2) are observed in PE,

| Pre-eclampsia
indicating the status of renal dysfunction. 96

| Gestational diabetes mellitus
The aberrant changes in placental microenvironment may affect the release and content of placenta-derived EVs. The quantity of exosomes secreted from trophoblast cells cultured under high glucose condition is approximately three times higher than that cultured under normal glucose condition. 67 Salomon et al 66  placenta-derived exosomes and circulating exosomes in GDM. 45 Gillet et al 98

| Foetal growth restriction
In a cohort study of pregnant women who give birth to small foetuses, Miranda et al 106

| CON CLUS IONS
The current studies provide us a comprehensive understanding of Moreover, human tissues usually only can be obtained after delivery and cannot accurately reflect the dynamic changes of markers during the whole pregnancy process. Therefore, more efforts should be devoted to giving an insight into the functions of EV in pregnancy and to apply EVs to the diagnosis, monitoring and treatment of pregnancy-related diseases.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.