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Pathways Regulating VEGF Activity During the Decidualisation of Endometrial Stromal Cells

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1.1 The endometrium and the menstrual cycle

1.1.1 The human endometrium

The endometrium is the mucosal lining of the uterus and it is a unique and dynamic multi-layered tissue consisting of a functional layer and basal layer overlaying the myometrium (Johnson and Everitt, 2007). The functional layer is highly receptive and is rely on circulating steroid hormones (Johnson and Everitt, 2013). This layer undergoes cyclic changes which separates from basal layer during menstruation, shed and regenerated within two weeks if no implantation occurs. The monthly cyclic changes in the female reproductive system coordinate the development of follicles in the ovaries to release an oocyte and prepare the endometrium for implantation of an embryo. Menstruation occurs in response to coordinated interaction between hypothalamic, anterior pituitary and ovarian hormones (Johnson and Everitt, 2007). The length of the menstrual cycle is highly variable ranging from 26 to 34 days (Mihm et al., 2011) and a typical woman have more than 400 cycles in their reproductive life.

The human uterus consists of three layers: the outer layer (perimetrium) which is composed of epithelial cells covering the uterus; the middle muscular layer (myometrium) which contains a highly organised network of arteries and veins; and the inner layer (endometrium) (Figure 1.1). The lining of the endometrium is composed of a single layer of luminal epithelium overlies a network of connective tissue and a multicellular stromal compartment (Gilbert, 2010). Uterine glands form cavities into the stromal layer. The endometrial blood supply enters the arteries arising from the myometrium, which then split to form basal arteries and spiral arterioles to supply functional layer and basal layer (Rogers, 1996) (Figure 1.1). The permanent basal layer which is adjacent to myometrium provides cells to generate a new functional layer each month of reproductive life. It is only recently studies identified the epithelial progenitor cells (Chan et al., 2004; Verdi et al., 2014) and mesenchymal stem cells in the human endometrium which are thought to play a role for maintenance of endometrium during regeneration and degeneration throughout menstrual cycle (Gargett & Masuda, 2010; Masuda et al., 2010).

Figure 1.1: The structure of the human endometrium. A schematic illustrating the multi-layered structure of the endometrium, which forms the inner lining of the uterus. The functional layer is composed of endometrial epithelial and stromal cells which undergoes cyclical changes under the control of steroid hormones. The basal layer consisted stem and progenitor cells for regeneration during menstrual cycle.

1.1.2 Cyclical changes of the endometrium

The endometrium undergoes phases of proliferation, differentiation, tissue degeneration and regeneration with a normal full cycle spans 28 days under the changes in the ovarian cycle and control of ovarian steroid hormones (Gellersen and Brosens, 2014). The ovaries undergo a monthly concurrent with the endometrial cycle. Ovarian cycle consists of the follicular and luteal phases. During the follicular phase, follicular granulosa cells produce high amount of oestrogen under stimulation of follicle stimulating hormone (FSH), reaching a peak of circulating oestrogen prior to ovulation which induces a positive feedback to trigger the luteinizing hormone (LH) surge and ovulation (John and Everitt, 2007). During the luteal phase (day 14-28), the corpus luteum becomes highly vascularized and begin to secrete high amounts of progesterone. If pregnancy occurs, human chorionic gonadotrophin (hCG) secreted by the implanting blastocyst maintains the corpus luteum. Otherwise, the corpus luteum degenerates at the end of luteal phase leading to a decline in both oestrogen and progesterone level (Critchley and Saunders, 2009) (Figure 1.2).

The endometrial cycle consists of three phases: proliferative phase, the secretory phase, and menstrual phase if no pregnancy occurs (Figure 1.2). During days 1-5, the endometrium breaks down and sloughed off in the process termed menstruation. During the proliferative phase (day 5-13), oestrogen produced by the ovarian follicles lead to the growth of the endometrial functional layer and rapid epithelial and stromal cell proliferation. The secretory phase (day 15-28) begins following the onset of the production of progesterone from corpus luteum. During the mid-secretory phase (day 20-23), the endometrium becomes receptive in the mid-secretory phase (Figure 1.2). With the rising levels of progesterone, the endometrium stops proliferating and it causes endometrial stromal cell undergoes differentiation, via a process called decidualisation (see Section 1.3) (Gellersen and Brosens, 2014; Gellersen et al., 2007) (Figure 1.2). In addition, the secretory phase also accompanied by the vascular remodelling by immune cells, pre-dominantly uterine natural killer cells (uNK) and changes in the extracellular matrix (ECM) simultaneous to stromal decidualisation.

Unlike human, animals like rodents do not undergo spontaneous preparation for pregnancy and do not menstruate, they undergo an oestrous cycle which exhibits differences compared to the human menstrual cycle. Thus, it is important to use the human samples instead of animals.

Figure 1.2: Hormone levels through the menstrual cycle. The menstrual cycle is series of degenerative and regenerative endometrial changes that occurs on a monthly basis in response to the influence of ovarian hormones. The dominant follicle enlarges with the increased of oestrogen by FSH. Following ovulation (day 14), the oestrogen level decline and increase of progesterone by newly formed corpus luteum. In the absence of pregnancy, the corpus luteum breaks leading to withdrawal of ovarian hormones resulting in the begin of menstruation. The cyclical changes involve transformation of the endometrium into a state where it is most receptive (7 days following ovulation) to the embryo implantation called as the “Window of implantation”. The progesterone levels elevated during mid-secretory phase triggers decidualisation.

1.2 Decidualisation

The process of decidualisation is marked by the transformation of elongated spindle-like stromal cells into rounded, enlarged epithelial-like decidual cells (Gellersen et al., 2007). Decidualisation can be observed around day 23 in 28-day menstrual cycle in the stromal cells surrounding terminal spiral arteries of the endometrial independent of the presence of blastocyst (de Ziegler et al., 1998, Kajihara et al., 2013). Histologically, the decidual process indicates the end of the window of implantation (Figure 1.2). The differentiation of decidual cell is one of the earliest adaptations to pregnancy which includes changes in their cytoskeletal organization, extracellular matrix production, secretion of cytokines and growth factors and increased resistance to oxidative stress (Alam et al., 2007; Oliveira et al., 2000; Christian et al., 2011). The decidual reaction spreads to the basal endometrial layer through autocrine and paracrine signals to regulate invasion of trophoblast and placental formation if pregnancy occurs (Kajihara et al., 2013; Maruyama & Yoshimura, 2008; Brosens et al., 2002).

Trophoblast invasion, growth and protection of placenta from local immune responses is important in order to have successful pregnancy. Decidual cells can express various proteins like decorin, tissue factor and plasminogen activator inhibitor to form a protective envelope around the spiral arteries for maintenance of vascular stability (Lockwood et al., 2007). Furthermore, decidual cells also stand up to the arrival of macrophages and resist inflammatory signals for uNK (Dimitriadis et al., 2005; Gellersen et al., 2007; Lash et al., 2010).

1.2.1 Differentiation of hESC

Undifferentiated hESC are mesenchymal cells with an elongated spindle-shaped fibroblastic appearance (Gellersen & Brosens, 2003; Gellersen et al., 2007). Decidualised hESC are characterised by a marked enlargement of cells in size, rounded nucleus and, an increase of nucleoli, expansion of the secretory machinery consisting rough endoplasmic reticulum and the Golgi complex, and cytoplasmic accumulation of glycogen and lipid droplets (Oliver et al., 1999; Christian et al., 2002). This transformation of the endometrial fibroblasts into highly secretory cells is accompanied by profound biochemical changes. Microarray studies have reported that decidualisation involves sequential reprogramming of functionally related genes which is involved in extracellular matrix organization, metabolism, stress response, cell cycle progression, differentiation and apoptosis (Figure 1.3) (Giudice, 2004). Therefore, hESC decidualisation adopt new biochemical functions that are critical for embryo implantation and successful pregnancy. Upon decidualisation, hESC acquire a secretory phenotype releasing various cytokines, growth factors and proteins that are important for maintaining the decidual phenotype and regulating trophoblast invasion and placental formation (Giudice, 2004). PRL and IGFBP-1 are the most common decidualisation markers (Gellersen et al., 2007; Oliver et al., 1999; Tseng et al., 1992).

Decidualisation of hESC is an interesting example of mesenchymal-epithelial transition (MET). The interconversion between epithelial and mesenchymal phenotype through MET and the epithelial-mesenchymal transition (EMT) processes is essential for embryogenesis and wound healing during differentiation (Micalizzi et al., 2010; Choi & Diehl, 2009).

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Figure 1.3: Functions of decidualised hESC. Decidualisation can be induced invitro by treating with 8-Br-cAMP and MPA. Decidualised hESC acquire a secretory phenotype and become specialised epithelioid cells that can regulate various biological functions to have a successful pregnancy such as in (1) embryo invasion, (2) window of implantation, (3) embryo selection, (4) haemostasis, (5) immunomodulation and (6) oxidative stress response mechanism.

1.2.2 The importance of hESC decidualisation in embryo implantation

Pregnancy relies upon a dynamic cross-talk between the embryo and receptive endometrium which is under the control of locally secreted factors. In order for successful implantation to occur the endometrium has to undergo changes which optimize the environment to receive and support the developing blastocysts (Hannan et al., 2010; Lee and DeMayo, 2004;Dey et al., 2004). The implantation process can be divided into three main steps: apposition, attachment and penetration (Figure 1.4) (Lee et al., 2011, Norwitz et al., 2001). There are many factors that can lead to implantation failure such as uterine abnormalities, hormonal disorders, immunological factors and infections (Timeva et al., 2014). Recently, the changes of autocrine and paracrine molecules level during the menstrual cycle have been identified as critical to uterine function (Dimitriadis et al., 2010Singh et al., 2011;Okada et al., 2014).

Figure 1.4: Embryo implantation into the endometrium. The embryo enters the human uterus approximately 4 days after fertilisation as an unhatched blastocyst. Apposition begins when the human blastocyst hatches from the zona pellucida and approached to epithelial layer to form an unstable adhesion. This mechanism is followed by the firm attachment of blastocyst to the endometrium. Penetration occurs when the embryo invades through the luminal epithelium (LE) and basal lamina deeper into the stroma of uterine lining forming a vascular connection with mother. Adapted from Greening et al., 2016. The window of implantation

It is now well-established that the endometrium receptivity is a vital part of successful implantation. This period is known as the window of implantation and usually occurs at around day 20-23 of the menstrual cycle, coinciding with the mid-secretory phase (Figure 1.2) (Harper, 1992, Achache and Revel, 2006). Deficiencies of endometrium receptivity, or the failure of a blastocyst to attach to the endometrium at the right time can cause infertility and there is a strong correlation between implantation occurs beyond the normal receptivity period and early pregnancy loss (Wilcox et al., 1999). Many studies have attempted to find the useful biomarkers which signify endometrial receptivity including histological dating, immunohistochemistry, and microarray analysis, several genes have been found but the number of common genes that were identified was minimal (Haouzi et al., 2012, Sugawara et al., 2014). However, it was shown that local factors produced during receptive period including cytokines, growth factors, transcription factors serve to specify endometrial receptivity. Embryo invasion

As discussed earlier, embryo implantation involves embryo apposition and attachement to the endometrial surface, breaching of the luminal epithelium and invasion into the ESC (Loke et al., 1995). Understanding on this process is limited due to the restricted use of animal models and in vitro co-culture system because of ethical and practical reasons. The decidualised ESC secrete a number of matrix metalloproteases (MMPs) (Anacker et al., 2011), which is motile and invasive are able to promote embryo invasion (Salamonsen et al., 2007). A study reported that the expansion of trophoblast spheroids cultured with decidualised hESC is higher compared to those cultured with undifferentiated hESC, which thought to be due to the secretion of EGF and IL (Gonzalez et al., 2011). In addition, decidualised hESC also able to distinguish and select the high-quality embryo by migrates toward the high-quality embryos (Gonzalez et al., 2011). Taken together, decidual cells are not subjected to an invading blastocyst but they can actively take part in the process of encapsulation (Brosens and Gellersen, 2010; Quenby et al., 2013). Embryo selection

It has been reported that secretion of cytokines, chemokines and growth factors from the supernatants of decidualised hESC co-cultured with blastocysts are different if the blastocyst displays signs of developmental arrest and inhibition of interleukins and growth factors (Teklenburg et al., 2010). A few studies reported that decidual hESC can acts as biosensors in recognizing compromised embryos and prevent their implantation (Salker et al., 2010; Teklenburg et al., 2010). Decidualised hESC have shown to migrate more in the presence of high-quality embryos compared to low-quality embryos using the time-lapse microscopy (Weimar et al., 2012). However, the mechanism how hESC contributes to discrimination between high- and low-quality embryos still unknown. Therefore, decidualisation is not only critical for embryo implantation, but also recognition and selecting human embryos. Haemostasis

Decidualised hESC secrete various factors such as tissue factor and VEGF that promotes haemostasis and involves in the remodelling of the spiral arteries to create an environment ready for trophoblast invasion (Cloke et al., 2010). During decidualisation, an influx of innate immune cells take place to tolerate the semi-allogenic embryo and these cells also known to regulate trophoblast invasion and vascular remodelling due to their expression of angiogenic factors, endothelial cell mitogens and chemokines (Koopman et al., 2003; Ledee et al., 2008). Immunomodulation

Pregnancy is an inflammatory related process which requires complex immuno regulation to prevent the fetal antigens being recognized by cytotoxic T-cells and to protect the fetal-maternal interface (Cloke et al., 2010). Decidualised ESC also known to act in a manner which modulates the immune responses at the fetal-maternal interface. Decidual cells plays a critical role to ensure the sufficient balance of specialised macrophages and uNK to prevent the priming of maternal T cells to paternal anti-allogens. The important of uNK cells in the angiogenesis have been studied and reported that lacking uNK cells cause angiogenesis impairment and lead to conditions such a preeclampsia and fetal growth restriction (Zhang et al., 2011). Oxidative stress responses

During early pregnancy, there is vascular remodelling and as a consequence oxygen concentration fluctuate profoundly at the fetal-maternal interface (Burton et al., 1999; Brosens et al., 2009). Due to these changes and inflammatory responses, reactive oxygen species (ROS) including hydroxy radical, superoxide anion, and hydrogen peroxide are produced. ROS when not stop by antioxidative defences can brings to nucleic acids and proteins damage and subsequently cause cell death (Valko et al., 2007). Decidualised hESC acquire the ability in oxidative cell death resistance by producing ROS scavengers such as superoxide dismutase (SOD2), which is up-regulated by the FOXO1 (Takano et al., 2007). In contrast, ROS up-regulate FOXO3A in undifferentiated cells and unable to survive under oxidative stress, but the silencing of FOXO3A can prevents apoptosis (Kajihara et al., 2006). Therefore, undifferentiated cells only adopt pro-survival behaviour upon decidualisation.

Taken together, decidualisation is not only critical for embryo implantation and placental formation, but also play a crucial role in offspring protection as well as in the maternal recognition and rejection of low-quality embryos. Hence, decidualisation failure can cause in a range of reproductive disorders, including recurrent pregnancy loss, preeclampsia, fetal growth restriction and pre-term delivery.

1.3 Decidualisation cues

1.3.1 Endocrine cues

Prior to ovulation, the ESC is controlled by oestrogens and therefore mount a decidual response in the rise of progesterone levels. It is reported the morphological signs of decidualisation are not seen until 9 days following ovulation, indicating that other factors are important to initiate this process (Gellersen and Brosens, 2003). In consistent with in vitro study in primary hESC, induction of decidual markers takes 7-10 days of stimulation with progesterone analogue and can be accelerated by other factors (Telgmann et al., 1997), which indicates that progesterone is not the primary initiating force during decidualisation (Gellersen and Brosens, 2014).

Relaxin is another factor known to promote decidualisation in culture. Relaxin is increases after the LH surge, but its increase is fairly moderate (Stewart et al., 1990). Furthermore, FSH, LH and hCG have also been implicated in the decidual process (Tang et al., 1993; Han et al., 1999; Kasahara et al., 2001). However, it is now known that the expression of relaxin and prostaglandins all contribute to the elevated levels of cAMP in ESC and therefore lead to decidualisation (Gellersen and Brosens, 2014).

1.3.2 Paracrine and autocrine cues

In addition to endocrine cues, number of growth factors, cytokines, peptides and lipids are secreted following the onset of decidualisation ESC which contribute to their own decidualisation. The heparin-binding epidermal growth factor (HB-EGF) is known to be up-regulated during the mid-secretory phase and in hESC decidualisation (Lessey et al., 2002). It is reported that blockade of HB-EGF during decidualisation reduced the decidual markers expression in hESC (Chobotova et al., 2005). TGFβ1 is a secreted cytokine that inhibits the expression of decidualisation markers such as prolactin, IGFBP-1 and tissue factor (Kane et al. 2010). Activin A is a secretory, dimeric glycoprotein of the transforming growth factor family which is an early inducer of decidualisation through up-regulating the MMPs; and modulates trophoblast differentiation and adhesion (Jones et al., 2006). Activin A also takes place in inflammation by regulating pro-inflammatory IL-8 and IL-11, and angiogenesis by regulating VEGF (Menkhorst et al. 2010; Rocha et al. 2012).

Another paracrine factor involved in decidualisation is the interleukin family of cytokines, such as interleukin-11 (IL-11), which is known to be up-regulated during decidualisation (Dimitriadis et al., 2005).  The study on IL-11 receptors in knock-out mice reported that the mice is infertile due to lack of sustained decidualisation resulting in implantation failure (van Mourik et al., 2009). Other interleukins important for decidualisation include IL-1 which is important for complete decidualisation and embryo implantation (Strakova et al., 2005). IL-6, another pro-inflammatory cytokine is mostly produced by endometrial epithelial and stromal cells during implantation. The expression of IL-6 is highest during secretoty phase and deficiciency of IL-6 during decidualisation and placenta development causes recurrent miscarriage (van Mourik et al., 2009). Leukaemia inhibitor factor (LIF), a member of the IL-6 is important cytokine for endometrial epithelial cells, yet it is unclear whether LIF is involved in human endometrial stromal cells during decidualisation. However, it is believed that it involves the expression of IL-6 and IL-15, whereas, the LIF receptor is up-regulated, perhaps for later activity in the pregnancy (Shuya et al. 2011).

1.2.3 cAMP signalling

Activation of the cyclic adenosine monophosphate (cAMP) pathway is essential to start the decidualisation and progesterone is indispensable to maintain the phenotype (Gellersen & Brosens, 2003). Many evidences suggest that decidual process is initiated by increased cAMP signalling and sustained activation of protein kinase A (PKA) pathway (Gellersen & Brosens, 2003; Telgmann et al., 1997). cAMP is a second messenger activated by increased exposure of the endometrium to relaxin, prostaglandins, corticotrophin releasing hormone (CRH) and pituitary gonadotropins following ovulation via activation of ligands of G-protein coupled receptors (GPCRs) (Christian et al., 2002; Dimitriadis et al., 2005). The cAMP signalling pathway is regulated by GPCRs activate adenylate cyclase which catalyzes the conversion of adenosine triphosphate (ATP) into cAMP. cAMP pre-dominantly activates the protein kinase A (PKA) by binding to its two regulatory subunits, causing to a conformational change that leads to the release of two catalytic subunits.

There are few studies strongly support elevated cAMP levels sensitize hESC to progesterone signalling. First, it is reported that higher expression levels of cAMP in the endometrial tissues obtained from patients in secretory phase than the proliferative phase of the menstrual cycle (Tanaka et al., 1993). Second, basal adenylate cyclase activity and prostaglandin-stimulated adenylate cyclase activity is higher in secretory phase than proliferative phase (Tanaka et al., 1993). Last, in vitro studies revealed that progesterone treatment in hESC increases intracellular cAMP level (Brar et al., 1997). In addition, the study on the effect of cAMP or progesterone alone and combination of both progesterone and cAMP on the induction of decidual marker gene PRL revealed that cAMP is a potent inducer of the decidual phenotype. Progesterone is found to be a weaker stimulus as mentioned in Figure 1.5 (Tang et al., 1993; Gao et al., 1994; Brosens et al., 1999; Reem et al., 1999).

Figure 1.5: Initiation of decidual process is primarily dependent on cAMP. hESC treated with cAMP alone markedly induces decidual marker PRL but failed to maintain the decidual phenotype. Treatment with MPA alone induces PRL after 8 to 10 days of stimulation. However, treatment with combination of both cAMP and MPA has a synergistic effect on PRL production characterized by a significant induction and maintenance of the decidual phenotype. Adapted from Brosens et al., 1999.

In the nucleus of hESC, the catalytic subunit of PKA phosphorylates a number of target proteins including cAMP response element binding protein (CREB), cAMP response element modulating protein (CREM), signal transducer and activator of transcription 5 (STAT5), CCAAT-enhancer binding protein (C/EBP) and Forkhead box protein O1 (FOXO1) as shown in Figure 1.6 (Brosens & Gellersen, 2006; Gellersen et al., 2007; Gellersen & brosens, 2003). The cAMP signalling during decidualisation is highlighted by the studies which demonstrated inhibition of PKA stops the expression of decidual gene in hESC (Brar et al., 1997; Matsuoka et al., 2010). The siRNA mediated knock-down of exchange protein directly activated by cAMP (EPAC), another target of cAMP prevents decidualisation in hESC (Kusama et al., 2013). Due to the critical role of cAMP during menstrual cycle, it is not surprise that adenylate cyclase activity as well as cAMP levels are higher in the secretory phase of the cycles in vivo and is higher in the endometrium (Tanaka et al., 1993).

Figure 1.6: The cAMP pathway. Endocrine factors stimulate the cAMP-dependent PKA pathway. Activated PKA migrates to the nucleus of hESC where it phosphorylates target proteins including FOXO1, STAT5, CREM and CREB. These factors mediate cAMP response and sensitize cells to progesterone. Adapted from Grimaldi, 2012.

1.3.4 Progesterone signalling

Progesterone is a steroid hormone that plays a critical role in the female reproductive system such as gland development, ovulation, decidualisation, implantation, menstruation and maintenance of pregnancy (Gellersen et al., 2009). Progesterone acts primarily through activation of its receptor (PR). PR knock-out in mice leads to the various reproduction defects including sexual behaviour and implantation indicating the important of progesterone in regulating diverse reproduction events (Rider, 2002; Lydon et al., 1995). There are two isoforms of PR: PR-A and PR-B as a result from alternative promoter usage and translation at two distinct start-sites (Wen et al., 1994). The progesterone receptor is driven by oestrogen acting on the ER during the proliferation phase. It is therefore essential when culturing hESC to have oestrogen present not only to promote growth but to ensure progesterone receptor expression is maintained. PR-A is known to express in the stromal cells throughout the menstrual cycle, whereas PR-B is expresses in the mid-proliferative phase (Mote et al., 1999). PR-A is the pre-dominant form in hESC (Brosens et al., 1999). Binding of progesterone to PR undergoes a conformational change which leads to its phosphorylation, dissociation from multimeric chaperone complex, and the PR is free to dimerize and bind to promoter and enhancer elements of specific target genes (Patel et al., 2015; Jones et al., 2006). PR is able to control the expression of variety genes involved in decidualisation even if they lack a progesterone response element (PRE) by hijacks other transcription factors, thus being able to bind to response elements indirectly (Maruyama & Yoshimura, 2008).

1.4.5 cAMP and progesterone cross-talk

The human endometrium is exposed to progesterone for 7-10 days before decidualisation is observed and only a few of genes respond to the hormone in short term such as decidual protein induced by progesterone (Depp) and promelocytic leukemia zinc finger protein (PLZF) (Watanabe et al., 2005). Depp is known to up-regulate within 30 minutes of progesterone treatment, however its function still remains to be further investigated (Watanabe et al., 2005). PLZF is rapidly induced by progesterone and this induction is indispensable for progesterone-dependent decidualisation (Kommagani et al., 2016).

In vitro, treating stromal cells with both cAMP and progesterone enhances decidualisation. It has now become apparent that progesterone does not trigger the decidual response, it is required for maintenance and cAMP, via activation of PKA pathway is the initiating factor sensitising the stromal cells to progesterone. There are two known mechanisms causes in convergence of cAMP and progesterone pathway as shown in Figure 1.7. First, cAMP signalling activates the expression of transcription factors such as STAT3, FOXO1, and C/EBP (Christian et al., 2002; Mak et al., 2002) which have the ability to bind to the PR in which permit PR to modulate genes without the need for progesterone response element (Gellersen and Brosens, 2003). Second, the progesterone-dependent hESC decidualisation occurs simultaneously with intracellular cAMP elevation and is stop in the presence of PKA inhibitor (Brar et al., 1997).

Figure 1.7: Crosstalk of the cAMP and progesterone signaling. The activation of G-protein coupled receptors (GPCRs)by ligands such as relaxin, prostaglandin and corticotrophin releasing hormone (CRH) causes an increase in cellular cAMP levels. Consequently, there is an accumulation of transcription factors within the nuclei of stromal cells which form a complex with the activated progesterone receptor PR-A, resulting in activativation of a number of decidua-specific genes such as prolactin.

1.3.6 Lists of some hormones, cytokines and growth factors induce in ESC decidualisation

No Abbreviation Protein Name Functions References
1 E2 Oestrogen Induces progesterone receptors Lydon et al., 1995
2 ER-α Oestrogen receptor-alpha Mediates up-regulation of progesterone receptors, endometrial proliferation and differentiation Kurita et al., 2001
3 ER-β Oestrogen receptor-beta Up-regulates progesterone receptors; regulates vascular function Lecce at al., 2001
4 P4 Progesterone Inducing and maintaining complete decidualisation Hourseman et al., 1989
5 PR-A Progesterone receptor A Predominant PR decidualisation; represses transcription of PRB Cheon et al., 2002
6 CRH Corticotropin-releasing hormone Auto-immune reaction to an inflammatory component of decidualisation Kalantaridou et al., 2003
7 FSH Follicle stimulating hormone Induces cAMP to promote decidualisation Tang and Gurpide, 1993
8 hCG Human chorionic gonadotropin Prevents degeneration of corpus luteum; variety functions in and outside of embryo-endometrium Litch et al., 2001
9 LH Luteinizing Hormone Induces cAMP to initiate decidualisation Tang and Gurpide,1993
10 PTHLH Parathyroid hormone-like hormone Inhibit decidualisation and stimulates stromal cell apoptosis Sherafat-Kazemzadeh et al., 2011
11 Activin A Activin A Trophoblast invasion, and promotes decidualisation Jones et al., 2002
12 Akt AKT1 kinase Intracellular mediator of cAMP/PKA pathway Yoshino et al., 2003
13 CBR-1 Cannabinoid receptor 1 Inhibit human decidualisation and encourages apoptosis in cAMP-dependent mechanism Kessler et al., 2005
15 Decorin Decorin Binds to TGFβ to inhibit trophoblast invasion Xu et al., 2002
16 EGF Epidermal growth factor Mediates the actions of oestrogen and progesterone on the proliferation and differentiation in human endometrium Watson et al., 1996
17 FB-1 Fibulin-1 Mediates progesterone in the extracellular matrix and as plasma glycoprotein Nakamoto et al., 2005
18 SRC-1 steroid receptor coactivator-1 Involves in functional and morphological decidualisation Maruyama et al., 2004
19 IL-1β Interleukin-1 beta Pro-inflammatory cytokine, stimulate IL-8 and IL-8; Inhibit cAMP-mediated decidualisation Yoshino et al., 2003b
20 IL-8 Interleukin-8 Stimulating trophoblast secretion of progesterone to maintain the successful pregnancy in embryo-endometrium interaction Tsui et al., 2004
21 IL-11 Interleukin-11 Mediates prostaglandins 2 and relaxin to induce decidualisation Dimitriadis et al., 2005
22 IL-15 Interleukin-15 Modulates uNK and decidualisation Ashkar et al., 2003; Godbole and Modi, 2010
23 LIF Leukaemia inhibitory factor Enhances decidualisation and up-regulates IL-6 and IL-15, implantation failure and fail to induce decidualisation if lack of it Stewart et al., 1992; Shuya et al., 2011
24 MMP2/3/9 Matrix Metallopeptidase 2/3/9 Connective tissue controlling, Extracellular matrix remodelling and trophoblast invasion Curry and Osteen, 2003
25 TIMP3 Tissue inhibitor metallopeptidase 3 Inhibit MMP9 and regulates trophoblast invasion Vassilev et al., 2005
26 p300/CBP p300/CREB-binding protein Complexes formation with progesterone receptor in the secretory phase Shiozawa et al., 2003
27 KLF-9 Kruppel-like factor 9 Progesterone receptor-interacting protein, lack of it leads to subfertility Pabona et al., 2012
28 LEFTY LEFTY Inhibit the induction of decidualisation Tabibzadeh, 2011; Li et al. 2014; Tang et al., 2010
29 PGE2 Prostaglandin E2 Synergistic with E2/P4 to enhance differentiation and PRL expression Frank et al., 1994
31 Relaxin Relaxin Induces cAMP and PKA pathway to initiate decidualisation Bartscha and Ivell, 2004
32 TGFβ1 Transforming growth factor β1 Pro-apoptotic effect via FasL/Fas system; inhibits decidualisation Chatzaki et al., 2003
33 TNFα Tumor necrosis factor alpha Activin A mediator; modulates menstruation, proliferation, implantation and decidualisation Mangioni et al., 2005
34 Wnt4/5 Wingless-type MMTV integration Progesterone mediator of cAMP by up-regulates Mn-SOD anti-apoptosis Matsuoka et al., 2010
35 BMP2 Bone morphogenetic protein 2 Induces Wnt4 and mediates progesterone to induce decidualisation Li et al., 2007
36 PKA Protein kinase A Phosphorylates prolactin promoter and induces prolactin during decidualisation Tierney et al., 2003
37 Notch1 Notch1 Dual function in the window of implantation: Initially mediates a survival signal of hCG for endometrium from the blastocyst and subsequently down-regulated for decidualisation Afshar et al., 2012

Table 1.1: Lists of the part of known hormones, cytokines and growth factors induce decidualisation in ESC via progestereone and cAMP signalling.

1.4 Decidualisation markers

1.4.1 Prolactin (PRL)

PRL was shown to start producing around day 22 of the menstrual cycle in decidualisation (Maslar et al., 1986). PRL level reaches a peak during the secretory phase of menstrual cycle and is one of the establishes markers for decidualisation (Tseng et al., 1992). PRL is synthesised by decidualised ESC. Secretion of dPRL during in vitro decidualised hESC is induced by progesterone, cAMP and combination of these, with a prominent role of the PKA signalling pathway in activating transcription of PRL (Telgmann and Gellersen, 1998; Tang et al., 1993; Brosens et al., 1999). PRL secretion increases until a peak at 20-25 weeks of pregnancy and thereafter declines toward term (Wu et al., 1995). Both PRL mRNA and protein expression increases during secretory phase in endometrium by synthetic progestin medroxyprogesterone (MPA) (Reis et al., 1999). The stimulation of the anti-progestin reduces reduces mRNA expression encoding both PRL and PRL receptor in ESC in vitro (Tseng and Mazella, 1999). PRL binds on its receptor mediates intracellular signalling, the expression of PRL receptor has been detected in the mid- and late secretory endometrium in stromal cell (Jones et al., 1998), which is consistent with the role in decidualisation. PRL plays an important role in implantation and early pregnancy. Furthermore, PRL is essential in trophoblast growth and invasion, angiogenesis, cytotoxic activity of uNK cells, as well as to regulate water transport to the maternal compartment (Corbacho et al., 2002; Stefanoska et al., 2013). Prolactin deficient mice are infertile due to a failure in embryo implantation (Horseman et al., 1997). Consistent with these findings, PRL deficiencies during the window of implantation can cause unexplained infertility in women (Garzia et al., 2004). It has been reported that PRL receptor knock-out mice led to implantation failure (Bole-Feysot et al., 1998).

1.4.2 Insulin-like growth factor binding protein-1 (IGFBP-1)

IGFBP-1 is formerly known as placental protein 12 and it is first detected in the human placenta and decidua (Bell et al., 1991). IGFBP-1 is induced in secretory phase endometrium and decidua which is coincident with decidualisation (Giudice et al., 1991; Bryant-Greenwood et al., 1993). Therefore, IGFBP-1 is considered as a marker of decidualisation and its expression is used to assess the extent of in vitro decidualisation (Giudice et al., 1992). The study using luciferase reporter assays with primary hESC reported the increased IGFBP-1 activity treated with MPA, and PR-A is the dominant transactivator that binds on progesterone response element on the IGFBP-1 promoter (Gao et al., 1999; Gao et al., 2000).

There are widespread roles for IGFBP-1 in reproduction including menstrual cycle, puberty and fetal growth. However, deregulation of IGFBP-1 has been affected by pregnancy complications like intrauterine growth restriction (IUGR), pre-eclampsia and polycystic ovarian syndrome (PCOS) (Crossey et al., 2002). The high level of IGFBP-1 in early pregnancy appeared beneficial, whereas, it will cause obstetrical disorders in late pregnancy (Giudice, 2002; Fazleabas et al., 2004).

IGFBP-1 has also been found to trigger the invasion of trophoblast (Gleeson et al., 2001). IGFBP-1 interacts with the invading extravillous trophoblasts (EVT) and regulates the insulin-like growth factor (IGFs) during decidualisation (Giudice et al., 1998). The IGFs, mediator of steroid hormones is present in stromal and decidual cells (Rutanen, 2000). Briefly, oestrogen stimulated IGF-1 to facilitate endometrial growth and expressed during proliferative and early secretory phases, whereas, IGF-II is expressed by EVT during secretory phase and early pregnancy (Zhou et al., 1994). There a study on IGP-II knockout mice led to the impaired of placenta growth and caused IUGR when lack of either IGF-I or IGF-II (Crossey et al., 2002). Furthermore, in vitro decidualisation of primary hESC co-incubated with recombinant IGF-1 caused decidualisation failure (Matsumoto et al., 2008).

1.5.3 Homeobox A10 (HOXA10)

Homeobox (HOX) transcription factors play a crucial role in embryonic development and reproductive tract in the developing human and mouse embryo (Taylor et al., 1997). HOXA10 is also an important decidualisation associated transcription factor in both human (Taylor et al., 1998) and mouse (Lim et al., 1999) endometrium. HOXA10 has been reported to regulate the expression of several genes which is involved in the differentiation process such as IGFBP-1 (Kim et al., 2003), integrin-3 (Daftary et al., 2002) and MMP26 (Jiang et al., 2014) to promote embryo adhesion during the window of implantation. HoxA10 knock-out mice are sub-fertile and the subfertility phenotype is maternal-derived which lead to endometrial dysfunction (Satokata et al., 1995). In addition, HOXA10 expression has been reported to decrease in the secretory ESC of women with unexplained infertility compared to healthy fertile women (Wu et al., 2005; Matsuzaki et al., 2009). HOXA10 expression is increased in the primary hESC treated with oestrogen and progesterone during the secretory phase (Taylor et al., 1998).

1.4.4 CCAAT- enhancer binding protein  (C/EBP)

During early phase of decidualisation, increased level of C/EBPs is detected in ESC and C/EBPs are the members of basic leucine-zipper (bZIP) superfamily of transcription factors that bind DNA sequences (Lekstrom and Xanthopoulos, 1998). C/EBP has been reported to bind to an enhancer located within MER20 transposon (Pohnke et al., 1999), which subsequently enables it to bind to other transcription factors such as FOXO1 and PR-A (Christian et al., 2002). C/EBP has been highly detected in the nuclei of decidual cells during mid-secretory phase of the menstrual cycle using immunochemistry (Plante et al., 2009). There is also evidence indicate that C/EBP is implicated in IL-11 and cell cycle during decidualisation (Wang et al., 2012). C/EBP deficiencies in female mice are found to impair decidual transformation of ESC and lead to infertility (Mantena et al., 2006). The absence of C/EBP also found to decrease the expression of bone morphogenetic protein 2, a morphogen playing an important role in mouse and human ESC decidualisation (Wang et al., 2012).

1.4.5 Forkhead box O (FOXO)

FOXO are proteins belonging to subfamily of forkhead transcription factors. FOXO play a role in increase accessibility of genomic regions within chromatin which enable it in the transcription factors binding recruitment (Lamansingh et al., 2012). FOXO are important in regulation of several diverse process including cell cycle regulation, apoptosis, DNA damage and repair, responses to oxidative stress and tumour suppression.

The transcriptional activity of FOXO is regulated by multiple post-translational modifications including phosphorylation, acetylation, ubiquitination and methylation (Calnan and Brunet, 2008). In the absence of growth factor signalling, FOXO are localized between nucleus and cytoplasm, pre-dominantly nuclear localisation (Brownawell et al., 2001).  Phosphorylation of FOXO by serine/threonine protein kinase B (AKT) on three conserved residues inhibit FOXO transcriptional activity by blocking DNA binding and re-localising FOXO to the nucleus. Phosphorylation induces 14-3-3 proteins binding to the first two AKT phosphorylation sites and subsequently blocks the DNA binding (Boura et al., 2007; Brunet et al., 1999). In addition, the phosphorylation and 14-3-3 binding also blocks nuclear import causes the shifting the localisation of FOXO from the nucleus to the cytoplasm (Figure 1.8) (Obsilova et al., 2005). Multiple kinases have been identified to regulate FOXO transcription activity in both positive and negative manner including SGK, IKK, JNK and MST1 (Calnan and Brunet, 2008) which will not be discussed in this study.

In mammals, FOXO transcription factors consists of four members: FOXO1, FOXO3, FOXO4 and FOXO6. FOXO share similar binding specificity to the DNA binding consensus sequence: TTGTTTAC due to the high conservation within the DNA binding domain (Furuyama et al., 2000).

Figure 1.8: Translocation FOXO transcription factors upon growth factor signalling. In the presence of growth factor/survival signals activates PKB, which then translocate into the nucleus. Phosphorylation of FOXO by PKB results in release from DNA, and binding to 14-3-3 proteins, and this complex is subsequently transported out of the nucleus, where it remains inactive in the cytoplasm. In the absence of growth factor/survival signals, FOXO is dephosphorylated, 14-3-3 is released and FOXO is transported back into the nucleus where is transcriptionally active. FOXO1

FOXO1 is a transcription factor that is critical for the induction of decidualisation by regulating various decidualisation genes such as PRL (Lynch et al., 2009; Christian et al., 2002) and IGFBP-1 (Kim et al., 2003). FOXO1 expression increased in the human endometrium during the secretory phase by immunohistochemistry showing that only cytoplasmic epithelial staining during the early secretory phase and both cytoplasmic and nuclear epithelial and stromal staining during late secretory phase (Christian et al., 2002). cAMP and progesterone signalling are known to positively regulate FOXO1 expression upon in vitro decidualisation of hESC. Activation of FOXO1 protects the decidual cells from oxidative stress, and also in spiral artey remodelling (Brosens and Gellersen, 2006; Buzzio et al., 2006; Takano et al., 2007). In response to progesterone withdrawal, FOXO1 translocate into the nucleus of decidual ESC and promote apoptosis (Labied et al., 2006). Various genes involved in hESC decidualisation are found to be regulated by FOXO1, overexpression of FOXO1 significantly increase the expression of IGFBP-1, TIMP3 etc, while FOXO1 deletion significantly reduced their expression (Buzzio et al., 2006). In consistent with these findings, Grinius et al. also reported that siRNA-mediated knock-down of FOXO1 in decidualised hESC significantly decrease IGFBP-1 protein (Grinius et al., 2006).

FOXO3A acts to protect the cells from oxidative stress, apoptosis and plays an important role in follicular growth (Wang et al., 2014). The FoxO3a knock-out female mice are infertile due to the impairment of follicular development and oocyte growth (Christian et al., 2011). A very recent study reported that FoxO3a expression increase in decidualisation of mouse primary stromal cells, and siRNA-mediated FoxO3a knocked down damaged the apoptosis (Long et al., 2018). In constrast with the Kajihara et al. study reported that FoxO3a appears to be repressed in decidualised hESC (Kajihara et al., 2006).

In summary, increased levels of progesterone in ESC during secretory phase of menstrual cycle activated the PI3K/Akt pathway which causes localization to FOXO1 in the cytoplasm. However, progesterone stimulation also increases the levels of intracellular cAMP in hESC which activates the PKA pathway and inactivates PI3K/Akt pathway causing FOXO1 enter the nucleus and increase gene transcription (Figure 1.9).

Figure 1.9: The model of the regulation of FOXO1 transcriptional activity in a progesterone-dependent manner in ESC through PI3K/Akt pathway and PKA pathway. The rise of progesterone levels in hESC during secretory phase of menstrual cycle activated the PI3K/Akt pathway which causes localization to FOXO1 in the cytoplasm to stop its transcription activity (Right). However, progesterone stimulation also increases the levels of intracellular cAMP in hESC which activates the PKA pathway and inactivates PI3K/Akt pathway causing FOXO1 enter the nucleus and increase gene transcription (Left).

1.4.6 Lists of known transcription factors expression in hESC decidualisation

No Abbreviation Protein Name Functions References
1 FOXO1 Forkhead box O1 Induces prolactin during decidualisation, protects against reactive oxygen species, apoptosis Labied et al., 2006
2 C/EBPβ CCAAT/enhancer-binding protein β Cooperate with FKHR in the regulation of decidual specific- genes expression eg, Prolactin Christian et al., 2002
5 PLZF Promelocytic leukemia zinc finger protein Progesterone-induced transcription factor in decidualisation Komangani et al., 2016
6 HOXA10 Homeobox A10 essential in uterine receptivity during the window of implantation Gui et al., 1999
7 HOXA11 Homeobox A11 Diminished uterine glands; modulated by sex steroid direct sequential of decidualisation and leads to implantation receptivity Hsieh-Li et al., 1995; Taylor et al., 1999
8 FOXM1 Forkhead Box M1 Downstream of HOXA10 in decidualisation Gao et al., 2015
9 STAT3 signal transducer and activator of transcription 3 Downstream of FOXM1 in decidualisation Jiang et al., 2015
10 VEGF Vascular endothelial growth factor Stimulates angiogenesis and vasculogenesis Sugino et al., 2002
11 TWIST1 TWIST homolog 1 Transcription factor associated with FOXO1 and ETS1 Schroeder et al., 2011
12 ETS1 v-ets erythroblastosis virus E26 Transcription factor enhances decidual markers eg. PRL, IGFBP-1 Kessler et al., 2006
13 HAND2 Heart and neural crest derivatives expressed transcript 2 Regulated by progesterone, important in decidualisation Huyen and Bany, 2011
14 CREB cAMP response element binding protein Central transcription factor of cAMP signaling Yoshie et al., 2015

Table 1.2: Transcription factors expression in decidualisation of hESC.

1.5 Clinical perspective

Humans are a relatively sub-fertile species compared to other mammals with only 20% of average monthly fertility rate and many couples have difficulties in to conceive naturally due to infertility or miscarriage problems (Evers, 2002; Gnoth et al., 2005). Assisted reproductive techniques (ART) have been widely used. These techniques have been advanced to improve the in vitro fertilization (IVF) techniques as well as to increase the pregnancy rate. Tools that enable the selection of high-quality embryos and assess endometrial status are now available (Diedrich et al., 2007). However, despite the advancement of these techniques including frozen embryo transfer technique and blastocysts chromosomal screening, only increased approximately 20-30% of implantation rate through IVF (Scott et al, 2013; Schoolcraft and Katz-Jaffe, 2013; Shapiro et al, 2011). In fact, there are still women who fail to carry a successful pregnancy even after several cycles of ART attempts. Successful implantation requires a receptive endometrium and a viable embryo. In order to improve pregnancy rate and prevent miscarriage, many researches have been focused at gaining clearer understanding about the mechanisms involved during the dynamic process of ESC decidualisation and implantation.

1.5.1 Endometriosis

Endometriosis is a female reproductive disorder occurs when the abnormal growth of endometrial tissue in other areas of the body mainly outer surface of the ovaries, fallopian tubes, or uterus. This disorder can cause serious pelvic pain, heavy menstrual periods and infertility. Approximately 35-50% of infertile women diagnose with endometriosis (Macer and Taylor, 2012), and eutopic endometrium was found to cause infertility phenotype in women with endometriosis. However, the cause of endometriosis still unknown and no cure exists for endometriosis until now.

The eutopic endometrium of women with endometriosis has been shown to exhibit impaired decidualisation response. A study using microarray followed by validation with real-time qPCR of endometrial samples of women with or without endometriosis identified various genes involved in decidualisation and implantation aberrantly expressed in the endometrium of women with endometriosis (Kao et al., 2003). In addition, the expression of decidualisation markers is also changed in the endometrium of women with endometriosis compared to healthy women. Low expression levels of the transcripts for PRL and IGFBP-1 was observed in the isolated primary hESC from women with endometriosis compared to cells from healthy women (Aghajanova et al., 2011). Moreover, they also reported the IGFBP-1 protein secretion in the cell culture medium also significantly decrease from the women with endometriosis compared to healthy women (Aghajanova et al., 2011). All these findings have been confirmed by several studies (Aghajanova et al., 2009; Aghajanova et al., 2010; Su et al., 2015).

FOXO1 mRNA expression was reported to be significantly decreased in the eutopic endometrium of women with endometriosis compared to the healthy women in the secretory phase of the menstrual cycle (Shazand et al., 2004). Moreover, microarray analysis of endometrial samples from women with endometriosis and healthy women demonstrated a reduction in FOXO1 expression in the endometriosis women compared to the control group in the early secretory phase, and this finding also validated by real-time qPCR (Burney et al., 2007).  Defective Notch signalling of women with endometriosis has been reported to cause decidualisation impairment which explaining the down-regulation of Notch signalling downstream target, FOXO1 expression in women with endometriosis (Su et al., 2015).

Apart from FOXO1, HOXA10 expression also found to alter in the women with endometriosis. HOXA10 mRNA expression has been reported to reduce in mid-secretory hESC from women with endometriosis compared to healthy women (Matsuzaki et al., 2009). Another study using Northern blot analysis of endometrial samples between 40 women with and without endometriosis demonstrated that HOXA10 expression failed to increase during mid-secretory phase (Taylor et al., 1999). Taken together, these findings suggest that the endometrium of women with endometriosis causes the changes of decidualisation response including the expression of PRL, IGFBP-1, FOXO1 and HOXA10 which explaining the infertility associated with endometriosis.

1.5.2 Recurrent miscarriage

Decidualisation failure results in infertility (Gellersen and Brosens, 2014), whereas, impairment of decidualisation altered embryo-maternal interactions which can causes recurrent pregnancy loss, preeclampsia, and poor embryo selection (Salker et al., 2010; Macklon & Brosens, 2014). Although many anatomical, endocrine, immunological, and genetic pertubations have been implicated as causes of recurrent miscarriage, yet none are specific (Saravelos et al., 2014). Recurrent miscarriage is among the prevalent causes of reproductive failure.

A large proportion of research has investigated endometrial defects including interaction of fetal-maternal endometrial interface and how these lead to the recurrent miscarriage. Decidual cells acting as biosensors of embryo viability by recognizing, selecting and eliminating the unwanted embryo and thus involved in terminating the endometrium window of receptivity (Teklenburg et al., 2010). The studies have reported that women with recurrent miscarriages present impaired decidualisation. A study found that hESC from recurrent miscarriage women has an abnormal decidual response and failed to transit between a pro-inflammatory and anti-inflammatory decidual response (Salker et al., 2012). In addition, the women with recurrent miscarriages had lower levels of decidual marker, PRL than control women, indicates an aberrant decidual response (Teklenburg et al., 2010; Salker et al., 2010). hESC from recurrent miscarriage patients are not able to distinguish the high or low-quality embryos (Weimar et al., 2012).

1.6 Vascular endothelial growth factor A (VEGF)

1.6.1 VEGF protein overview

Originally, VEGF was identified as vascular permeability factor (VPF) in 1983, secreted by a guinea pig tumour cell line which promote and increase vessel permeability (Senger et al., 1983). In 1989, an endothelial cell specific mitogen was isolated from the conditioned medium of bovine pituitary follicular cells by Ferrara and Hanzel and named VEGF (Ferrara and Hanzel, 1989). Subsequently, Connolly et al independently isolated and sequenced the VPF from U937 cells. The cloning and sequencing of VEGF (Leung et al., 1989) and VPF (Keck et al., 1989) cDNA was reported the end of 1989, which concluded that VEGF and VPF were the same protein. As a conclusion, the VEGF is potent and play a role in the regulation of physiological and pathological growth of blood vessels (Leung et al., 1989).

VEGF is a small disulfide-linked homodimeric glycoprotein. VEGF is then renamed as VEGF-A after discovered its homologues: placenta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D and their receptors VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt-4 (Bagheri et al, 2013; Stuttfeld and Ballmer-Hofer, 2009). VEGF is also known as VEGF-A, and it is the most common and potent isoform in angiogenesis which will be the focus of interest in this study.

VEGF have been shown to play various roles which include: promoting the vascular endothelial cells growth (Ferrara and David-Smyth, 1997); acts as a survival factor in endothelial cells to prevent the apoptosis (Alon et al., 1995), as a chemoattractant for monocytes and endothelial cells (Ferrara et al., 2003); and increase vascular permeability. VEGF expression is regulated by many factors including hormones, growth factors and oxygen tension. The studies reported that VEGF mRNA expression is up-regulated in low oxygen exposure in variety of pathological conditions (Semenze, 2003; Dor et al., 2001). Hypoxia inducible factor -1 (HIF-1) is the well-known factor that regulates VEGF gene under hypoxia conditions. Apart from that, growth factors such as platelet derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF) also reported to regulate VEGF expression (Frank et al., 1995; Pertovaara et al., 1994). Ovarian hormone such as oestrogen and progesterone has also been reported to induce VEGF in endometrium both in vivo (Ferrara et al., 1998) and in vitro (Classen-Linke et al., 2000).

The important roles played by VEGF and its receptors in blood vessels development are shown in gene knockout mice. The mice lacking VEGF-A (Ferrara et al., 1996) leads to embryonic lethality due to inadequate vascular development (Vuorela et al., 2000). VEGF-A and PIGF are known to regulate early placental vascular development and important factors in implantation success (Andraweera et al., 2012).

1.6.2 Isoform of VEGF

Early structural studies reported that VEGF must be dimerized to bind to its receptor, but VEGF can stimulate VEGFR-2 independent of glycosylation modifications. VEGF-A has several isoforms that differ in the degree of extracellular matrix binding capacity (Figure 1.9). The human VEGF gene is located on chromosome 6p21.3 (Vincenti et al., 1996), which composed of eight exons and separated by seven introns (Houck et al., 1991). The main isoforms found are VEGF121, VEGF165, VEGF189 and VEGF206 (Cross et al., 2003; Ferrara, 2009). They are formed from the alternative splicing and each with a distinct number of amino acids (Figure 1.10). VEGF121 is a freely diffusible protein which has the poor extracellular matrix (ECM) binding capacity due to its lacking heparin-binding properties in exon 6 and 7 (Park et al., 1993). VEGF165 is the predominant isoforms lacks exon 6, which can be secreted and partly bound to the cell surface and ECM. However, VEGF189 is tightly bound to ECM because it contains all eight exons (Ferrara, 2009).

Figure 1.10: Exon structure of the VEGF gene. The VEGF gene consists of eight exons. VEGF206 and VEGF189 contains all eight exons and is pre-dominantly membrane bound. VEGF165, the most common isoform, and lacks exon 6, whereas, VEGF121 is a completely soluble isoform lacking exon 6 and 7. Exon 6 and 7 is the heparin binding domain that are responsible for making VEGF membrane bound or soluble.

1.6.3 VEGF receptors

VEGF binds to two closely related receptor tyrosine kinases (RTKs), VEGFR-1/Flt-1 and VEGFR-2/KDR (Ferrara, 2009). These receptors contain seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region and others receptor tyrosine kinase sequences. A third member of this RTKs family, VEGFR-3, is not a receptor for VEGF, but instead specifically binds VEGF-C and VEGF-D (Figure 1.11). VEGF and its receptors, Flt-1 and Flk-1/KDR are necessary for the embryonic vasculature development in mice (Ferrara et al., 1996; Fong et al., 1999; and Shalaby et al., 1995). Vascular endothelial growth factor receptor 2 (VEGFR-2/KDR)

VEGFR-2 is also known as kinase domain receptor (KDR) in human, and fetal liver kinase-1 (Flk-1) in mouse (Ferrara, 2009). VEGFR-2 binds to VEGF (refer to Figure 1.11) primarily on the second and third immunoglobulin-like domains (Fuh et al., 1998). The role of VEGFR-2 in angiogenesis and hematopoiesis demonstrated in the study of Flk-1 null mice led to the death in uterus due to the lack of vasculogenesis and failure in blood vessel development (Shalaby et al., 1995). Therefore, VEGFR-2 is a key mediator of the angiogenic and permeability-enhancing effect of VEGF.

VEGF-A activates VEGFR-2 inducing VEGFR-2 dimerization and phosphorylation at various tyrosine residues resulting in a chemotactic, mitogenic and pro-survival signals (Ferrara, 2009). Phosphorylation iof various downstream mediators is induced by VEGF include PI3-kinase, phospholipases Cγ, ras GTPase activating protein, Src tyrosine kinases, protein kinase C, ERK and MAPK pathway (Ferrara, 2009).

Figure 1.11: Schematic illustration of VEGF and its receptors expression patterns and ligand specificity. VEGF receptors (VEGFR-1, VEGFR-2 and VEGFR-3) contain seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region and others receptor tyrosine kinase sequences. Arrows indicate which ligands are capable of binding to each receptor. VEGF can bind to two cell surface receptors Flt-1 and KDR. VEGF binds to Flt-1 with a higher affinity than KDR. An alternatively spliced soluble form of Flt-1, sFlt-1 is an inhibitor of VEGF and PIGF activity. Neuropilin co-receptors bind specific VEGF ligand splice variants to enhance VEGFR-2 mediated signalling. Neuropilin-1/2

Neuropilins (Figure 1.10) are considered to be VEGF “co-receptor” (Gelfand et al., 2014). Unfortunately, the function of Neuropilin-1 not fully known, but in most studies believed that Neuropilin-1’s major function is to enhance VEGFR2 signalling in response to VEGF (Soker et al., 2002, Gelfand et al., 2014). The neuropilin-1 knock-out mice were embryonic lethal indicates that neuropilin-1 is important in the development of vascular system (Kawasaki et al., 1999). Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1)

VEGFR-1 also known as fms-like tyrosine kinase (Flt-1) was the first VEGF receptor in RTK (Ferrara, 2004). However, the full functional activity of this receptor still unclear. Studies indicate that the function and signalling properties of VEGFR-1 vary depending on the cell type and developmental stage at which it is acting (Ferrara, 2004). VEGFR-1 binds to the VEGF-A, PIGF and VEGF-B primarily in the second immunoglobulin-like domain (Park et al., 1994; Olofsson et al., 1998). VEGFR-1 is also produced in a soluble form called sFlt-1 (sVEGFR-1) which is a potent physiological inhibitor of VEGF activity (Kendall and Thomas, 1993).

Although the functions of VEGFR-1 are less well understood, there are many studies report VEGFR-1 acts as a VEGF ligand-trap or “decoy receptor” which negatively regulates or modulates the access of VEGF to VEGFR-2 (Park et al., 1994). A study demonstrated that a synergism exits between VEGF and PIGF in vivo, particularly during pathologies circumstances which confirmed from a PIGF gene knockout mice, led to impaired tumorigenesis and vascular leakage (Carmeliat et al., 2001). The inhibition of Flt-1 also found to inhibit the endothelial cell migration (Gille et al., 2000), while there is also study reporter that Flt-1 can mediate a weak mitogenic signal (Maru et al., 1998) indicating Flt-1 may have other roles.

VEGFR-1 knockout mice die at embryonic between day 8.5 to day 9.5 due to the increased proliferation of endothelial progenitor cells resulting in a disorganized vasculature (Fong et al., 1999). Transgenic mice which have the tyrosine kinase domains of VEGFR-1 deleted developed a normal vasculature and were viable (Hiratsuka et al., 1998). It was suggested that VEGFR-1 acts as decoy receptor modulating embryonic development by sequestering VEGF and preventing over-expression through VEGFR-2. Unique Characteristics of sFlt-1

As mentioned previously, the VEGFR-1 gene expresses one long form of approximately 8 kb mRNA and shorter truncated mRNAs encoding most of the extracellular domain form of 2.5 to 3.0 kb. sFlt-1 isoform contains first six immunoglobulin-like (Ig-like) domains with 31 amino acid-long tail derived from 5’ region of intron 13 (Figure 1.12 & Figure 1.13) supporting VEGFR-1 acts as a scavenger (Shibuya et al., 1990).

The role of sFlt-1 is further complicated by the existence of multiple splice variants of sFlt-1 (Figure 1.12 & Figure 1.13). The two most abundant splice variants, sFlt-1-i13 (Thomas et al., 2007) (also known as sFlt1_v1 (Heydarian et al., 2009)) and sFlt-1-e15a (Thomas et al., 2007) (also known as sFlt1_v2 (Heydarian et al., 2009)), are differentially expressed in human tissues (Thomas et al., 2007; Heyderian et al., 2009; Rajakumar et al., 2009; Whitehead et al., 2011; Jebbink et al., 2011;). sFlt-1-i13 was the first sFlt-1 identified (Kendal and Thomas, 1993) and is comprised of the first 13 exons of Flt-1 with an extension of exon 13 into the intronic sequence (Figure 1.12) (Heydarin et al., 2009). It shares 657 amino acids of full-length Flt-1, encoding a unique 31 amino acid C-terminus producing an 85-95 kDa protein (Figure 1.13) (Thomas et al., 2007). It is expressed in most of the endothelium tissues including brain, heart, and kidney.

In comparison, sFlt-1-e15a is specific to humans and mainly expressed in placenta (Whitehead et al., 2011; Jebbink et al., 2011). sFlt-1-e15a encodes the first 14 exons of Flt-1 followed by a 480 nucleotide stretch of intronic sequence (Figure 1.12) (Sela et al., 2008). This encodes the unique exon 15a containing the unique polyserine tail followed by the complete AluSq sequence producing a 95-135 kDa protein (Figure 1.12) (Thomas et al., 2007; Gu et al., 2008). A study reported that more than 80% of all Flt-1 transcripts are spliced to become sFlt-1-e15a in placenta, indicates that sFlt-1-e15a may be the major isoform involved in preeclampsia (Jebbink et al., 2011).

Figure 1.12: The structure of Flt-1 and sFlt-1. The full-length homologous Flt-1 receptor encodes an mRNA transcript of 30 exons which composed of seven extracellular Ig-like domains containing the ligand-binding regions, a transmembrane domain and intracellular tyrosine kinase domains. An alternative expressed soluble truncated form of Flt-1, sFlt-1, containing the N-terminal six Ig-like domains following by a unique 31 amino acid residue C-terminal sequence functions as an inhibitor of VEGF activity. sFlt-1-i13 is generated by skipped splicing and extension of exon 13, while, sFlt-1-e15a contain alternatively spliced exons derived from intronic sequences (exon 15a). sFlt-1-e15b and sFlt-1-i14 are not discussed in this study where sFlt-1-i14 generated by skipped splicing and extension of exon 14 and sFlt-1-e15b encodes the alternative spliced exon of 15b derived from intronic sequence. Adapted from Palmer et al., 2016.

Figure 1.13: Schematic representations of Flt-1 protein isoforms. The Flt-1 protein encodes seven extracellular Ig-like domains, a plasma membrance and tyrosine kinase domain. The first three extracellular Ig-like domains are essential for ligand binding and the 4-7th for receptor dimerization.  sFlt-i13 contains the first six Lg-Like domains, 657 amino acids (aa) of Flt-1 and a unique 31 aa tail. The sFlt-1-e15a diverge from Flt-1 after 706 aa and contain a unique 28 aa tail.

Oxygen-sensing jumonji domain containing protein 6 (JMJD6) was known directly affect in regulating the splicing pattern of Flt-1 to produce sFlt-1 in endothelial cells (Palmer et al., 2016; Boeckel et al., 2011). JMJD6 was able to strive its normal enzymatic functions, hydroxylates to U2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65), a component of splicing machinery under normal conditions to produce the full-length membrane bound Flt-1 transcript (Boeckel et al., 2011). However, JMJD6 was found to decrease its activity under hypoxic conditions and reduced its ability to hydroxylate U2AF65 which leads to the spicing machinery to produce shorter alternatively spliced sFlt-1 transcripts (Boeckel et al., 2011).

sFlt-1 is suspected as a biochemical barrier between fetal and maternal circulation in the placenta by stopping excess angiogenesis and abnormal vascular permeability. Over-expression of sFlt-1 in the placenta was found in a major disease in the field of reproduction. The patients with preeclampsia were found to have abnormally high levels of sFlt-1 in serum and plasma (Maynard et al., 2003). The placental trophoblasts were the major cell types producing large amounts of sFlt-1 in preeclampsia patients. The abnormal suppression of VEGF-A level by sFlt-1 causes hypertension and proteinuria in the patients. Moreover, the overexpression of sFlt-1 in pregnant rats also induces hypertension and proteinuria, showing that sFlt-1 is at least part of the cause of preeclamptic syndromes.

1.7 Angiogenesis in the endometrium

1.7.1 Angiogenesis

Vascular system is forms through two independent processes: vasculogenesis and angiogenesis. Vasculogenesis is the de novo formation of blood vessels from endothelial progenitors within the embryo. Vasculogenesis is the formation of new blood vessels in blood islands. Angiogenesis is defined as the process by which new blood vessels are formed from pre-excisting vessels. It is essential in various physiological processes, for example, embryo development, reproductive function and wound healing and pathological conditions like tumorigenesis (Ribatti, 2005). Angiogenesis is a complex and coordinated process which requiring sequential activation of receptors by numerous families of ligands in endothelial and mural cells (Ferrara, 2004; Ferrara, 2009).

1.7.2 Angiogenesis in the endometrium

Physiological angiogenesis is rare in normal tissues except during wound healing, and in the ovary and the endometrium during the female reproductive life (Fraser and Wulff, 2003). Angiogenesis is important in development and differentiation of human endometrium throughout the implantation as well as pregnancy (Gordon et al., 1995). As discussed earlier, there is a dynamic remodelling of the vascular in the endometrium during menstrual cycle. A complex process of the proliferation, differentiation, regeneration of vascular and glandular cells occurs each month in female reproductive life. In the human uterus, the endometrial arterioles arise from arteries in the myometrium pass into arterioles in the basal endometrium supplying the basal layer and functional layer (Farrer-Brown et al., 1970). Upon decidualisation, endometrial arteries also transformed to increasingly tortuous phenotype during the secretory phase (Kaiserman-Abramof and Padykula, 1989; Rogers and Gargett, 1998), and this transformation is carried on in early pregnancy until vasculature remodelling occurs in response to trophoblast invasion (Plasier, 2011; Smith, 2004). Many angiogenic factors such as VEGF, VEGF-C, PIGF, Angiopoietins, and VEGF receptors have been identified in the endometrium which may promote angiogenesis (Li et al., 2001; Smith, 2004).

1.7.3 VEGFs and angiogenesis in female reproductive

Despite the complexity of angiogenesis, VEGF is a central regulatory factor for the blood vessels grow in the variety of processes (Ferrara, 2009). VEGF is expressed in both the epithelial and stromal cells in the human endometrium (Perrot-Applanat et al., 2000). In addition, a study in mice reported that VEGF is a prime regulator of angiogenesis during decidualisation (Douglas et al., 2009). Besides that, there are also studies reported the role of VEGF in embryonic vasculogenesis and angiogenesis (Ferrara et al., 1996; Carmeliat et al., 1996). Inactivation of single VEGF allele in mice led to the embryonic lethality between day 11 and 12. The heterozygous VEGF embryos were found in a number of development abnormalities, defects in the vasculature of several organs for example: Heart, forebrain, placenta, nervous system, decreasing number of red blood cells within blood vessels in the yolk sac (Ferrara, 2009). Therefore, these findings have indicated that VEGFs are critical for vasculogenesis and hematopoiesis.

Moreover, the inactivation of conditional VEGF gene in VEGF loxP mice has shown that the dosage of VEGF is a key determinant in vascular development in developing the nervous system. The decreasing of VEGF impaired the vascular development and subsequent hypoxia resulting in the cerebral cortex degeneration and neonatal lethality (Raab et al., 2004). Meanwhile, the overexpression of VEGF gene led to severe abnormalities in heart development and embryonic lethality at day 12.5 to day 14 (Miquerol et al., 2000). These findings demonstrate that VEGF-dosage dependence is critical during development.

It is interesting that inactivation of PIGF and VEGF-B did not cause embryonic lethality. The mice with PIGF inactivation are viable and fertile, there are only results in some impairment of wound healing (Carmeliat et al., 2001). And, inactivation of VEGF-B only reduced the heart size and impairment of coronary vasculature (Bellomo et al., 2000). So far, VEGF-C is found to play an essential role in vascular development among all other members of VEGF gene family. The inactivation of VEGF-C led to the embryonic lethality due to the failure of lymphatic development (Karkkainen et al., 2004).

Furthermore, VEGF is the most prominent pro-angiogenic factor in endometriosis,one of the major factors caused infertility. VEGF has been reported to express higher in the peritoneal fluid and lesions of endometriosis patients compared to controls (McLaren et al., 1996). These findings led to the use of VEGF inhibitors as therapeutic value for endometriosis treatment (Hull et al., 2003).

1.8 Relationship of VEGF and FOXO

Given that VEGF activates PI3K/Akt signalling and lead to many downstream pathways (Abid et al., 2006). As described previously, PI3K/Akt signalling plays role in forkhead phosphorylation and translocation from nucleus to cytoplasm. Subsequently, it was found that VEGF stimulation induces phosphorylation of forkhead transcription factors through PI3K/Akt signalling, which acts as pro-survival and mitogenic phenotype (Abid et al., 2004). A study on the role of FOXO1 in endothelial cells using FoxO1-deficient mice led to the embryonic lethality on day 11 due to the incomplete vascular development of embryos and yolk sacs (Furuyama et al., 2004). This study proposed that FOXO1 is necessary in responding to VEGF in order to have a normal vascular development.

Although VEGF is known to suppress FOXO, it is surprising that the removal of FOXO led to the endothelial disruption. This can be shown in a study on the overexpression (constitutively-active FOXO1) and deletion (siRNA FOXO1) of FOXO in the presence or absence of VEGF in endothelial cells. There is a number of genes were inhibited by the deletion of FOXO1 in VEGF addition, whereas, significantly increased treated with constitutively-active FOXO1 (Abid et al., 2006). All these genes act as a straightforward model to show that FOXO1 is essential for their expression. However, in the same study, VEGF showed to induce a number of genes, this induction requires the presence of FOXO1. These genes were induced by VEGF, highly expressed by VEGF and constitutively-active FOXO1, and decreased in FOXO1 knockdown conditions (Abid et al., 2006). VEGF acts to suppress FOXO1 levels in endothelial cells, but it does not appear to directly influence the FOXO1 transcriptional targets. A very recent study reported FOXO1 directly regulates VEGF activity in wound healing in keratinocytes cells (Jeon et al., 2018). Therefore, it is important to investigate the interplay between VEGF and FOXO.

1.9 Rationale

Decidualisation is highly regulated event and is crucial for the maintenance of a successful pregnancy. It encompasses various morphological responses underpinned by extensive biochemical changes, and the signalling pathway involved in this differentiation process are complex. Research into the changes in decidualisation may lead to greater understanding of endometriosis as well as other pregnancy complications like recurrent miscarriage, preeclampsia, intrauterine growth restriction (IUGR), preterm birth and ectopic pregnancy (Venners et al., 2004; Brosens et al., 2011). As part of the complex system of decidualisation, VEGF family members are essential for maintaining the cycling endometrium under normal hormonal regulation and successful pregnancy. VEGF is a key growth factor essential for evoking the supporting vasculature during decidualisation. VEGF is dramatically up-regulated in hESC by cAMP in a manner that appears to be dependent on the FOXO1 transcription factor.

We proposed that VEGF and its receptors are under direct control of the PKA/FOXO1 pathway in hESC undergoing decidualisation and may modify the function of these cells in addition to regulate trophoblast migration and blood vessel development. Therefore, the regulation and function of these key mediators of implantation and placentation in decidualising hESC will be examined in this project. The overall main aim of the project is to identify the pathways regulating VEGF activity during the decidualisation of endometrial stromal cells and how this impact on vascular remodeling/angiogenesis and trophoblast function.

The specific aims of this project are:

  • To determine the expression of angiogenic factors in hESC decidualisation.
  • To examine the expression of the VEGF and VEGFR-1/Flt-1 during hESC decidualisation.
  • To Identify the transcription factors regulating VEGF and Flt-1 gene expression.
  • To assess the relative functional activity of hESC decidual-derived VEGF on angiogenesis in vitro.

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