Molecules and Prostaglandins Related to Embryo Tolerance

It is generally understood that the entry of semen into the female reproductive tract provokes molecular and cellular changes facilitating conception and pregnancy. We show a broader picture of the participation of prostaglandins in the fertilization, implantation and maintenance of the embryo. A large number of cells and molecules are related to signaling networks, which regulate tolerance to implantation and maintenance of the embryo and fetus. In this work, many of those cells and molecules are analyzed. We focus on platelets, polymorphonuclear leukocytes, and group 2 innate lymphoid cells involved in embryo tolerance in order to have a wider view of how prostaglandins participate. The combination of platelets and neutrophil extracellular traps (Nets), uterine innate lymphoid cells (uILC), Treg cells, NK cells, and sex hormones have an important function in immunological tolerance. In both animals and humans, the functions of these cells can be regulated by prostaglandins and soluble factors in seminal plasma to achieve an immunological balance, which maintains fetal-maternal tolerance. Prostaglandins, such as PGI2 and PGE2, play an important role in the suppression of the previously mentioned cells. PGI2 inhibits platelet aggregation, in addition to IL-5 and IL-13 expression in ILC2, and PGE2 inhibits some neutrophil functions, such as chemotaxis and migration processes, leukotriene B4 (LTB4) biosynthesis, ROS production, and the formation of extracellular traps, which could help prevent trophoblast injury and fetal loss. The implications are related to fertility in female when seminal fluid is deposited in the vagina or uterus.


INTRODUCTION
Prostaglandins (PGs) belong to a subclass of eicosanoids known as prostanoids, these are comprised of C20 atoms, including a cyclopentane ring. PGs are hormone-like chemical messengers which act as autacoids (1) through prostanoid receptors (G protein-coupled receptors) and their variants or isoforms such as E 1-4 , DP 1-2 , FP, TP, and IP (1,2). The main precursor of eicosanoids is arachidonic acid (AA), this is released by the action of phospholipases A2 (PLA2) and C (PLC) (3), AA is then converted into different metabolites through the COX, LOX, and CYP450 pathways (4). The importance of prostaglandins becomes evident when ovulation and fertilization are affected, e.g., as cyclooxygenase (COX) is inhibited by aspirin or indomethacin (5).
PGs have a significant role in maternal immune tolerance and the conception process. We consider prostaglandins in seminal fluid as key in modulating responses in different types of cells participating in fetal-maternal tolerance.
The balance of the immune response in maintaining fetalmaternal tolerance is due to a complex network of soluble molecules and cells, such as macrophages, and dendric, decidual, and NK cells. In Table 1, cells and biological processes are summarized. Moreover, many molecules are released by these cells and have a fundamental role in the tolerance process. Table  2 summarizes the most important of these.
The accumulated evidence indicates that when sexual intercourse occurs and seminal fluid is deposited in the female reproductive tract, the prostaglandins in the seminal fluid, i.e., PGE2, PGE1, PGE3, and PGF2 (109), initiate a signaling cascade toward the woman's innate immune cells. The cells mentioned in Table 1, such as platelets, polymorphonuclear leukocytes, and Group 2 innate lymphoid cells participate in the physiological mechanisms in embryo tolerance and implantation, allowing successful fertilization.

PREIMPLANTATION, IMPLANTATION, AND DECIDUALIZATION
Implantation begins by apposition and adhesion of the embryo to the luminal epithelium of the endometrium. Following its invasion toward the stromal bed, the union of the embryo to the luminal epithelium transforms the underlying stromal fibroblasts into secretory cells of the epithelioid type, or decidualization (110). Through different molecules such as IL-1b, steroid hormones, insulin-like growth-factor-binding protein-1 (IGFBP-1) and prostaglandin-endoperoxide synthase-2 (PTGS-2), the decidualized cells regulate this stage with the invasion of embryos, and the formation of the placenta (110).
Prostaglandins participate in each stage of the interaction of the embryo with the endometrium, for example in preimplantation, implantation (apposition, adhesion/attachment, invasion/ penetration) and decidualization; as well as affecting many other cells and molecules. PGs have a complex role in each of these stages, e.g., the essential role of prostaglandin E2 (PGE2) in the oocyte is to enhance the cumulus expansion in ovulation for sperm penetration, to regulate extracellular matrices disassembly (111), and also, importantly, to participate during transport and embryo implantation (112).

PROSTAGLANDIN SIGNALING BY SEMINAL FLUID AND FERTILIZATION
Preceding evidence shows that sperm induces immunosuppression against hapten-modified self and alloantigens, including cytotoxic Tcell in mice responses (113). Also, seminal plasma contains high concentrations of prostaglandins, key molecules in the regulation of sexual intercourse signaling (114). The female immune response tolerates seminal plasma and supplies cytokines and prostaglandins, which are synthesized in the male accessory glands. In addition, it causes molecular and cellular changes in the endometrium. This facilitates the development and implantation of the embryo when prostaglandins, cytokines and hormones bind to receptors in target cells in the cervix and uterus (115).
The prostaglandins present in seminal fluid have a role in immune modulation. They regulate the pathways that may exacerbate inflammation in the female reproductive tract during physiological processes such as ovulation, implantation, and parturition (116), e.g., ejaculation or the spermatozoa induce an inflammatory response in the endometrium in the preimplantation period after mating, in which IL-1 (alpha and beta), and TNF-alpha participate (117).
Seminal plasma derived from the male accessory sex glands performs a fundamental function in fertilization in animals. The components of seminal plasma participate in the transport and survival of viable sperm and the elimination of non-viable sperm from the uterus (118). In the quail species, the cloacal gland produces prostaglandin F2a (PGF2a), which contributes to successful fertilization and acts as a natural mechanism for the protection of sperm from rejection or death by the female reproductive tract (119). Seminal fluid factors exert significant effects on the female reproductive tract, as shown by Shahnazi et al. (120). Also, in the uterine tissues of mice that were paired with mice without seminal vesicles, implantation rates, enzyme cytosolic PGE synthase (cPGES), microsomal PGE synthase (mPGES) and receptors EP2 and EP4 involved in the signaling pathway of PGE2, were all significantly low (120). In addition, 19-hydroxy PGE and 19-hydroxy PGF are regulators of sperm motility, and its effects may be mediated by the content of ATP in sperm (121). Prostaglandins such as PGE-1 are potent stimulators of adenylate cyclase in various Widen maternal blood vessels and promote fetal growth. Interact with resident myeloid cells and participate in the induction of regulatory T cells IL-24, Angiopoietin 1 and 2 (Ang 1, Ang 2), vascular endothelial growth factor C (VEGF-C), TGF-b1, SDF-1, pleiotrophin, osteoglycin, IL-8, protein-10.
Suppression of their activity has been observed in humans and mice by PGE2.

Decidual stromal cells (DSCs)
Differentiation and development of dNK during decidualization. Induce the downregulation of activating NK receptors and inhibit NK cell proliferation, cytotoxicity, and IFN-g production.

IL-24, TGF-b
The DSC-induced inhibition is primarily mediate by PGE2.
Mayoral Andrade et al.

Molecules Related to Embryo Tolerance
Frontiers in Immunology | www.frontiersin.org November 2020 | Volume 11 | Article 555414 cellular systems (122). An increase in adenylate cyclase activity and subsequent entry into cAMP levels may also be involved. PGs stimulate the fertilization capacity of human sperm by facilitating the transport of calcium through their plasma membrane (123). The amplification of effects by microparticles from epididymal fluid (epididymosomes) and prostasomes could lead to the activation of many genes and the expression of related molecules, as reported in humans and mice, some species of cows, pigs and sheep (123,124). More specifically, signaling may affect the enzymes of the cyclooxygenase pathway and other molecules related to the metabolism of arachidonic acid, e.g., Cytochrome P450 in blastocyst implantation (125), and prostaglandin D2 in the maintenance of pregnancy through Th1/Th2 and T-cytotoxic (Tc) 2 cells balance (126,127).
The change induced by seminal plasma in a porcine uterus makes conception and pregnancy possible (128), it also reduces embryonic mortality in pigs and other livestock (129). In addition, seminal plasma possesses potent immunosuppressive activity caused by immune-deviating soluble factors, inducing tolerance, with molecules, such as Transforming growth factor-b (TGFB) and prostaglandin E (PGE).

EFFECTS OF PROSTAGLANDINS AND RELATED MOLECULES ON INNATE IMMUNITY AND FEMALE REPRODUCTIVE TRACT CELLS
Cells of the innate immune response are modulated by prostaglandins (130), among them, are the following: 1. M1 macrophages (Mø1) which produce proinflammatory cytokines (TNFa, IL-6, IL-12, IL-23, and IL-1b), M2 macrophages(Mø2) which produce IL-10 and TGFb (transforming growth factor b) and have anti-inflammatory and immune down-regulating properties. Both are regulated by prostaglandins in pregnancy (9) ( Table 1). 2. Dendritic cells (DCs) have several subclasses, e.g., CD103 + , myeloid, plasmacytoid, the latter are related to the production of high IFNa levels. In infertile patients with endometriosis, CD4 + , CD25 + , and CD103 + dendritic cells are increased in peritoneal fluid (131), dendritic cells CD103 + have a relevant role in implantation (132); in addition, CD103 + dendritic cells are regulated by prostaglandin D2 in different disorders (133). (134). In patients with preeclampsia (PE), in the presence of vascular endothelial growth factor (VEGF), these cells increase levels of prostacyclin (135). In the pathogenesis of PE, VEGF (VEGF-A) participates in the proliferation, migration and angiogenesis of endothelial cells, and works through the receptors VEGFR-1 (or Flt-1) and VEGFR-2. In PE this increases the release of FMS-like tyrosine kinase-1 (sFlt-1) and blocks free VEGF to protect the fetus from toxicity (136). 4. Neutrophils (PMN) are regulated by cytokines and prostaglandins (137). The aspirin (ASA) is used for prevention of preeclampsia in high-risk patients (138,139). ASA triggers    Platelet-activating factor is an acetylated Glycerophospholipid, releasing histamine from platelets, which increase vascular permeability.

Endothelial cells have innate and immune tolerogenic function
PAF is related to processes of ovulation, implantation and parturition, and is regulated by ovarian steroid hormones. PAF is associated with sperm motility, acrosome reaction, and fertilization. Harper, 1989. (50) Tieman, 2008. (51) Roudebush, 2001. (52) Cytokine mediators of implantation and decidualization IL-6 IL-6 is a cytokine with functions in immunity, metabolism and tissue regeneration. It is produced in the endometrial epithelium and stromal cells during implantation.
Variation in the expression of pro-inflammatory cytokines such as IL-6, CSF-1, macrophage colony-stimulating factor (CSF-1), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin 1-alpha, interleukin 1-beta, and tumor necrosis factor-alpha (TNF alpha) has been reported in the uterus immediately after mating in mice. Changes in the bioavailability of IL-6 are important for pregnancy. The increase of IL6 is related to unexplained infertility, recurrent miscarriage, preeclampsia and preterm delivery and inhibition of the generation of CD4 + regulatory T cells in pregnancy tolerance. Local IL-6 insufficiency could also contribute to recurrent spontaneous abortion. IL6 activate cathepsin S (CTSS) in dendritic cells, in decidualized endometrial stromal cells, this process is regulated by cystatins CST7 and CST3.  It is a member of the interleukin-6 family of cytokines. Upregulation of poFUT1, promotes trophoblast cell migration, invasion and differentiation at the fetal-maternal interface through activating the Janus kinase/signal transducers and fetal transcription (JAK/STAT) and a mitogen-activated protein kinase (MAPK) signaling pathway.

Lipoxins
These are derived from arachidonic acid, an w-6 fatty acid. They exert their anti-inflammatory effects through binding to high-affinity G protein-coupled lipoxin receptors.
Lipoxins, calcitonin, leukaemia inhibitory factor, and homeobox A10 are essential in implantation. Lipoxin A4 is regulated by human chorionic gonadotrophin (hCG) during early pregnancy and it has anti-inflammatory activity in human endometrium and decidua tissue.  transcellular biosynthesis of eicosanoids by acetylation of PGHS-2. Eicosanoids correspond to 15R-epimers of lipoxins (ATL) and are potent inhibitors of leukotriene B4-mediated neutrophils (140). Considering that preeclampsia is associated with increased proinflammatory, antiangiogenic and PMNendothelial cell adhesion, Gil-Villa et al. (141) shows that PMN adhesion in patients with preeclampsia is reduced by Aspirintriggered lipoxin (ATL) when aspirin is used. 5. Natural killer and innate lymphoid cells (ILC). According to the cytokine profile and transcription factor, ILCs are divided into two groups, cytotoxic and "helper"-ILC (17). The cytotoxic ILC group is represented by Natural Killer (NK). The "helper"-ILC in humans has three subclasses, ILC1 with two subsets, producing IFNg; ILC2 produces IL-5, IL-13, and IL-4; and ILC3 releases IL-17 and IL-22. The NK cells in a decidua (dNK) microenvironment are around 50% to 70% of the total of lymphoid cells in decidual tissue. They have CD56 bright CD16 − KIR + CD9 +, and activate the NK receptor phenotype, participate with cytokines, which mediate new vessel formation, aid in the renovation of existing tissues and placentation through the release of VEGF, stromal-derived factor-1 (SDF-1) and IFN-g-inducing protein 10 (9). In stromal tissue, the decidual stromal cells (DSCs) participate in the induction of maternal tolerance, physically concur and have a regulatory mechanism in dNK, and CD14 + myelomonocytic cells, and induce regulatory Treg. Also, DSCs inhibit dendritic cells through prostaglandin E2 (PGE2) and Indoleamine 2,3dioxygenase (IDO), this inhibition favors the maintenance of the pregnancy (18).
In the normal eutopic endometrium, the Mø2 together with the Tregs predominate, providing an anti-inflammatory environment for the implantation of the embryo, while in endometriosis, they can cause infertility. The Mø1 provide a pro-inflammatory environment which affects embryo implantation, the dendritic cells (DC) do not increase in endometrial tissue, also the Treg is dysregulated. Therefore, DC does not eliminate the cellular debris which could migrate to the peritoneal cavity and grow in ectopic sites, developing as endometriosis. On the other hand, Treg and NK have abnormal behavior, the first favors a pro-inflammatory state and the second is less cytotoxic which impacts embryo implantation (142). COX2 and PGE2 are related to the pathogenesis of endometriosis. A high level of COX-2 due to various factors such as estrogens, hypoxia and environmental pollutants could suppress apoptosis and increase cell proliferation through PGE2 and its receptors EP2, and EP4 in endometriosis (143). In addition, experimental studies with intralesional injections of ASA, in rabbits with peritoneal endometriosis, eliminate endometriotic lesions (144).

PROSTAGLANDINS IN IMPLANTATION AND MAINTENANCE OF GESTATION
The generation of prostaglandins and expression of receptors in a mouse uterus has demonstrated their importance during implantation and decidualization (145). In mice, PGE2 levels increase from the 2-cell embryo stage to the blastocyst, Alpha-fetoprotein (AFP) It is released by trophoblasts during pregnancy. Acts as a fetal transport protein. Influences fetal-maternal immunologic relationships during the first trimester and helps to protect the foetus against attacks by the maternal immune system. Suppresses the production of TNFa and IL-1b. Controls the production of HLA-G and the Ia antigen, it stimulates the growth of trophoblasts containing paternal H2 antigens. Inhibits macrophage expression of Ia antigens. AFP is capable of driving B cells into apoptosis to avoid maternal B cells in order to reach the foetus. It is a fifteen amino acid linear peptide secreted by embryos two-cell, four-cell and six-cell stages in mice, in humans and bovines, respectively PIF promotes immunological tolerance due to increasing the expression of HLA-G, -C, -E, and -F slightly. It also potentiates the effect of the endogenous steroid and promotes the secretion of Th1/Th2 cytokines.

Hakam et al. (105)
Zare et al. (106) demonstrating the importance of PGE2 in early development (112). PGE2 also plays a significant role in peri-implantation in a mouse uterus through the expression of EP2 and EP4 receptors, which increase cAMP levels during the implantation and decidualization processes. EP4 induces the activation of VEGF (growth factor vascular endothelial), increasing vascular permeability of the endometrium (146), implantation and decidualization, together with PGF2 (132). Inadequate production of prostaglandins in mice, and possibly in humans, may explain some cases of infertility (147). Low concentrations of PGE2, PGF and PGI2 cause failure in ovulation, fertilization, implantation, and decidualization (133). In mice, prostacyclin (PGI2) is the primary prostaglandin at the implantation site. It participates in implantation and decidualization through the peroxisome proliferator-activated receptor (PPAR-d) and the RXRa signaling pathway in the uterus (148).
As an example, PGF2a is used in fertilization procedures, in addition to GnRH, to pre-synchronize ovulation before applying for a resynchronization program in cows in dairy herds with acceptable pregnancy outcomes (149).

PROSTAGLANDINS IN MATERNAL IMMUNE TOLERANCE
When intercourse occurs, endothelial cells release IL-8, IL-1, INF-a, and TNF-a to recruit immune cells (150). Neutrophils are mobilized in the oviduct in female mammals in response to the presence of sperm (151). This process may also induce a state of unresponsiveness by the presence of anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, and TGF-b (152) Figure 1.
In order to prevent a compromised systemic maternal immune response, local immune regulation in the fetalmaternal interface is very important. This is achieved by several mechanisms. One of these is local immunoregulation at the fetal-maternal interface, e.g., Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) release factors such as indoleamine 2,3 dioxygenase (IDO), TGF-b, prostaglandin E2 (PGE2), and others inducing immunomodulatory effects (153).
Prostaglandin E (PGE), specifically, induces T-helper type 3 (Th3) and T-regulatory 1 cells (Tr1), as shown by Lewis´rat and mouse test (154,155). PGE2 secretion by human deciduous cells in the first trimester of pregnancy blocks the activation of maternal leukocytes in the decidua and inhibits IL-2 production and its receptor (156).
Other cells assisting in the decidualization of endometrial stromal cells (ESCs) and pregnancy maintenance are decidual natural killer (dNK) cells (157) and CD14 + cells for Treg induction and immunosuppression (158). Also, Treg and Breg may contribute to the regulation of type 1 and 2-like T helper anti-fetal immune mechanisms during human pregnancy (159) ( Table 1).

PLATELETS
It is evident that platelets may be important in tolerance mechanisms. Platelet activity is inhibited post-coitus, and this inhibition depends on prostaglandins (160). Seminal fluid has factors that favor clot formation, similar to peripheral blood, such as Factor VIII: Ag, FVIII: C and Von Willebrand factor (vWF), in addition to other factors (161). vWF (162), fibronectin (163), and vitronectin (164) are proteins that favor platelet adhesion (165). This implies that inhibition of platelet aggregation by PGI2 could be a compensatory mechanism for pro-adhesive molecules.
Using a mouse model, Etulain et al. (166) found that platelets act through P-selectin glycoprotein ligand-1 (PSGL-1), and directly affect neutrophil extracellular traps (NETosis). Platelet P-selectin is crucial for neutrophil recruitment (167). Furthermore, NETs cause the recruitment and activation of platelets and induce procoagulant activity due to the expression of histones H3 and H4, toll-like receptor 2 (TLR2) and TLR4 platelets. NETs present a surface for the activation of coagulation factor XII (168) in order to promote thrombosis as a mechanism of rejection (169).
Platelets cause a decrease in the formation of extracellular traps when preincubated with PGI2, followed by stimulation with lipopolysaccharide (LPS), arachidonic acid, and a synthetic diacylated lipopeptide (Pam3SCK4). This highlights the physiological role of PGI2 in platelet modulation (170). Prostaglandins may also inhibit the function of neutrophils by increasing levels of cyclic adenosine monophosphate (cAMP) (171).
The interaction of PMN-platelets releases products of arachidonic acid serving as precursors of neutrophil eicosanoids (172). In polymorphonuclear neutrophils (PMN), PGE2 modulates their response through the expression of EP2 and EP4 receptors (173).
The above mentioned may explain why platelet aggregation is inhibited post-intercourse and has a possible reduction in the formation of NETs to protect the embryo. It is possible that the release of extracellular traps may contribute to trophoblast lesions.
Many other cells mentioned above participate through high complexity fetal-maternal interface interaction to induce a tolerance stage, which protects the embryo (175).

POLYMORPHONUCLEAR CELLS
In mammalian species, PMNs are implicated in endometrial remodeling as being receptive to oocyte implantation. Human neutrophils exposed to progesterone and estriol hormones promote the establishment of maternal tolerance through the induction of CD4+ T cells (176).
In humans, during coitus, sperm is deposited into the female reproductive tract (FRT). Neutrophils are then recruited for the elimination of excess sperm through phagocytosis (177).
However, bovine seminal plasma is shown to reduce the ability of PMNs to phagocytize bull sperm. Furthermore, equine seminal plasma is reported to contain factors that reduce the binding of neutrophils to sperm, avoiding the formation of NETs (178). In humans, when granulocytes are exposed to the seminal plasma, the respiratory burst is inhibited (179). These mechanisms allow more of the healthy motile sperm to reach the oviduct, which makes it clear that seminal plasma contains factors that modulate the response of PMN.
In addition, PGE2 can exert anti-inflammatory action on neutrophils and other innate immune cells such as macrophages, natural killer cells, dendritic cells, and monocytes (180,181). Also, it inhibits the production of IFN-a in plasmacytoid dendritic cells and the production of IL-12 in myeloid dendritic cells.
Finally, polymorphonuclear leukocytes contribute to preterm labor by activating prostaglandin production from human fetal membranes (182).

GROUP 2 INNATE LYMPHOID CELLS
Specific ILC2s (Group 2 innate lymphoid cells) and uterine innate lymphoid cells (uILCs, uILC1, uILC2, and uILC3) (183) in the uterus are regulated by PGD2, PGE2, PGI2, and sex hormones, in particular, oestrogen (151,184). Together, these may play a role in the balance between immunity and tolerance at the beginning of placenta formation and could be related to pregnancy loss, as shown in mice (185). Some studies show that ILC2 is the most abundant subset in the human fetal-maternal interface during premature and full-term pregnancies, in which its presence is regulated by sex hormones (e.g., oestrogen) (186). PGI2 decreases the proliferation of ILC2 and significantly inhibits the expression of IL-5 and IL-13 induced by IL-33 (187).
The production of PGE2 could also suppress the function of neutrophils and uILCs, a particular cell, similar to ILC2, through its EP2 and EP4 receptors in both healthy humans and mouse models (188,189). PGE2 inhibits the expression of GATA-3, as well as the production of type 2 cytokines (IL-5 and IL-13) (144). These effects are mediated by the action of the EP2 and EP4 prostanoid receptors, which are specifically expressed in ILC2 (151,190). In addition, Group 1ILCs, uNK cells, and uILC3s significantly increase in abortion in mice. They also have a lower proportion of uILC2s (183).

DISCUSSION
Of the hundreds of molecules released with cells in the preimplantation, implantation, and decidualization processes; prostaglandins are integrated into each of these stages by seminal fluid, even until parturition. In particular, some of these molecules are found to be related to infertility and abortions, such as PGE2, PGF, and PGI2, which, in turn, are related to ovulation, fertilization, implantation, and decidualization (133). Increased levels of IL6 are also related to unexplained infertility, recurrent miscarriage, and pre-eclampsia among other disorders (9), e.g., in humans, cases of placental insufficiency, manifesting as intrauterine fetal growth restriction, are observed where the level of melatonin, a molecule with pleiotropic effects that regulates inflammatory processes (191), is decreased (192). Melatonin inhibits prostaglandin synthesis and is a potent inducer of uterine contractility (54,193), in addition, there is evidence that in fish, melatonin is produced in the granulosa cells and is a critical factor for ovulation (194). Likewise, in women, it increases progesterone and regulates the corpus luteum (195). Also in a recent clinical trial, melatonin is shown to improve intrafollicular oxidative balance and gives a slight increase in the rate of human live births (196). Another example is Polish landrace gilts treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) (PMSG/hCG-induced). Treatment with exogenous progesterone increases pregnancy success through the expression of genes responsible for vascular function and PGE2 synthesis (197). Therefore, the administration of inhibitors of prostaglandin synthesis, e.g., PGE2, must be carefully considered due to the multiple mechanisms of female fertility in which they participate (111).
Also, the mechanism of control over the rate of gene transcription or transcriptional regulation is altered in genes involved in chronic endometritis and the inflammatory response (IL-11, CCL4), growth factors (IGFBP1), and apoptotic proteins (BCL2, BAX, CASP8) in infertile patients (198).
Another mechanism of transcriptional regulation is that of Uterine Vascular Endothelial Growth Factor (UVEGF), in which PGE2 regulates vascular development through receptors EP2 and EP4.