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Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14208

Human extravillous trophoblast invasion: intrinsic and extrinsic regulation E. Menkhorst A,B,D, A. Winship A,B, M. Van Sinderen A,B and E. Dimitriadis A,B,C A

MIMR-PHI Institute of Medical Research, 27–31 Wright St, Clayton, Vic. 3168, Australia. Monash University, Clayton, Vic. 3800, Australia. C Department of Anatomy and Developmental Biology, Monash University, Clayton, Vic. 3800, Australia. D Corresponding author. Email: [email protected] B

Abstract. During the establishment of pregnancy, a human blastocyst implants into the uterine endometrium to facilitate the formation of a functional placenta. Implantation involves the blastocyst adhering to the uterine luminal epithelium before the primitive syncytiotrophoblast and subsequently specialised cells, the extravillous trophoblast (EVT), invade into the decidua in order to engraft and remodel uterine spiral arteries, creating the placental blood supply at the end of the first trimester. Defects in EVT invasion lead to abnormal placentation and thus adverse pregnancy outcomes. The local decidual environment is thought to play a key role in regulating trophoblast invasion. Here we describe the major cell types present in the decidua during the first trimester of pregnancy and review what is known about their regulation of EVT invasion. Overall, the evidence suggests that in a healthy pregnancy almost all cell types in the decidua actively promote EVT invasion and, further, that reduced EVT invasion towards the end of the first trimester is regulated, in part, by the reduced invasive capacity of EVTs shown at this time. Additional keywords: decidua, macrophage, placenta, spiral artery, T cell, uterine natural killer. Received 15 June 2014, accepted 27 July 2014, published online 28 August 2014 Introduction During the establishment of pregnancy, a human blastocyst implants into the uterine endometrium to facilitate the formation of a functional placenta. Implantation involves the blastocyst adhering to the uterine luminal epithelium before the primitive syncytiotrophoblast and subsequently specialised cells, the extravillous trophoblast (EVT), invade into the decidua in order to engraft and remodel uterine spiral arteries, creating the placental blood supply at the end of the first trimester (Burton et al. 2010). Defects in EVT invasion lead to abnormal placentation and thus adverse pregnancy outcomes (Kno¨fler 2010). Inadequate or inappropriate implantation and placentation is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications (Aplin 2010; Kno¨fler 2010). Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are associated with inadequate spiral arteriole remodelling (Cartwright et al. 2002); however, the reasons for this remain unknown. Conversely, excessive EVT invasion can lead to placenta accreta (Khong 2008), where the placental villous apposes directly onto the myometrium, with or without the presence of a decidua. The local decidual environment plays a key role in regulating trophoblast invasion (Burton et al. 2010). There have been many recent reviews highlighting molecules and signalling pathways Journal compilation Ó CSIRO 2014

important for regulating EVT invasion (Kno¨fler 2010) so, for the most part, specific molecules are not discussed here. In any case, individual cell types have been shown to express numerous factors that are shown in vitro to both promote and restrict EVT invasion, making it difficult to determine how these cells would regulate EVT invasion in vivo. Here we describe the major cell types in the decidua during the first trimester of pregnancy and review what is known about their regulation of EVT invasion. EVT invasion Placental trophoblast (cytotrophoblast) cells (Fig. 1) differentiate into two main cell types, the multinucleated syncytiotrophoblast and EVT. EVT residing at the base of the placental cell columns, which are adhered to the endometrial decidua, proliferate before subpopulations invade into the maternal decidua from as early as 5 weeks gestation (,14 days after implantation; Lunghi et al. 2007). This EVT invasion results in occlusion of endometrial spiral arteries by 8 weeks (begins as early as 5 weeks) and myometrial artery invasion by 14 weeks (Lunghi et al. 2007). There are two subpopulations of invasive EVT (Fig. 1): (1) endovascular EVTs, which initially plug, then remodel maternal spiral arterioles into low-resistance, highflow vessels; and (2) interstitial EVTs, the role of which is unclear, although they are thought to also assist in vascular www.publish.csiro.au/journals/rfd

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Placental villi Cytotrophoblast Syncytiotrophoblast

Cell column

eEVT

Spiral artery

iEVT Decidual cell

Macrophage

T-cell Decidua

uNK MGC Endothelial cell Gland

Vascular smooth muscle

Myometrium

Fig. 1. Regulation of extravillous trophoblast (EVT) invasion during the first trimester by the major cell types in the decidua. Arrows between EVTs and other decidual cell types indicate that EVT invasion is regulated by factors produced by that cell. The arrow direction indicates whether EVT invasion is enhanced (up) or restricted (down). eEVT, endovascular EVT; iEVT, invasive EVT; MGC, multinucleated giant cell; uNK, uterine natural killer cell.

remodelling, secrete hormones and modulate fetal–maternal tolerance (Pollheimer et al. 2014). Invasion is a tightly regulated multistep process controlled by locally produced and temporally regulated factors, including cell surface adhesion molecules (e.g. integrins) and secreted products, such as cytokines, chemokines, growth factors, matrix-degrading enzymes and their inhibitors (Irving and Lala 1995; Lash et al. 2005; Hannan et al. 2006; Fafet et al. 2008; Tapia et al. 2008; Paiva et al. 2009b; Jovanovic´ et al. 2010; Chau et al. 2013; Jiang et al. 2013; Stefanoska et al. 2013). EVT invasion involves dynamic cross-talk between trophoblast cells and the cells in the maternal decidua, including decidualized stromal fibroblasts, leucocytes, vascular endothelial cells, glandular epithelium and the endometrial extracellular matrix (ECM; Salamonsen et al. 2009). Decidualization Prior to implantation and in preparation for pregnancy, stromal cells of the uterine endometrium become ‘decidualized’. Decidualization describes the dramatic terminal differentiation of endometrial stromal cells into decidual cells, which involves the categorical reprogramming of endometrial stromal cells

such that different genes are expressed at different stages of the differentiation process (Popovici et al. 2000). In women, decidualization begins spontaneously in stromal cells adjacent to spiral arterioles during the mid-secretory phase of the menstrual cycle (5–10 days after the LH surge) in response to progesterone and regardless of the presence of a functional blastocyst (Paiva et al. 2009a). If implantation occurs, decidualization intensifies and continues to form the decidua of pregnancy (Dimitriadis et al. 2010). What has also become clear is that numerous locally produced factors are critical in progressing or accelerating progesterone-induced decidualization, including prostaglandin (PG) E2, relaxin, activin and interleukin (IL)-11 (Tabanelli et al. 1992; Frank et al. 1994; Dimitriadis et al. 2002; Jones et al. 2002). Both the progesterone and cAMP pathways are required for decidualization (Gellersen and Brosens 2003). Recent evidence in women indicates that decidualization is important in the formation of a functional placenta, with impaired decidualization associated with recurrent miscarriage, PE and placenta accreta (Khong and Robertson 1987; Founds et al. 2009; Salker et al. 2010). Immune cells are present in the uterus at all stages of the menstrual cycle. During the mid-secretory phase, which is associated with the onset of decidualization, there is an influx of macrophages and proliferation of uterine natural killer (uNK) cells (Salamonsen et al. 2009), such that during the first trimester, approximately 30%–40% of the cells in the decidua are leucocytes (Bulmer et al. 2010). Of these, 70% are uNK cells, approximately 20% are macrophages and variable concentrations of T lymphocytes (10%–20%), with B cells, dendritic cells, mast cells, regulatory T cells and natural killer T cells present but rare (Bulmer et al. 2010). Leucocytes populations within the decidua are highly specialised, comprising decidual-specific populations compared with peripheral leucocytes (Ka¨mmerer et al. 2003; Koopman et al. 2003), suggesting pregnancyspecific functions to optimise pregnancy outcome. Innate invasion The fact that EVTs are innately invasive is clear. The cytotrophoblast at the base of the cell columns differentiates from a proliferative, non-invasive phenotype towards the invasive EVT phenotype (Kno¨fler 2010). The invasive EVTs continue to differentiate within the decidua with large, multinucleate giant trophoblast cells found at the endometrial–myometrial border (Kemp et al. 2002). Studies comparing trophoblast invasion between tubal and endometrial pregnancies indicate that the invasive potential of the trophoblast is likely associated with their degree of differentiation, with multinucleate giant cells potentially acting to terminate invasion (Kemp et al. 2002). Regulation of this differentiation is attributed to oxygen tension and decidual-derived factors (Genbacev et al. 1997; Hannon et al. 2012). Recent studies also suggest that human leukocyte antigen G (HLAG), acquired as cytotrophoblast differentiate towards the EVT phenotype, itself promotes the invasive capability of EVT (Guo et al. 2013; Liu et al. 2013a, 2013b). The main growth factors and signalling pathways that control EVT invasion have been discussed recently in detail elsewhere

Regulation of extravillous trophoblast invasion

(Kno¨fler 2010; Kno¨fler and Pollheimer 2012) and so will not be repeated here. What is also becoming clear is that the invasive potential of differentiated EVT may be associated with the gestational age of the placenta. Both the percentage of placental villous explants that produce EVT outgrowths (James et al. 2006) and the number of EVTs invading from villous explants (Lash et al. 2006a) decline as gestation proceeds. Age-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 is found in first trimester cytotrophoblasts isolated between weeks 6 and 11 of gestation (Xu et al. 2000). MMP-2 activity decreases, whereas MMP-9 activity increases as gestation proceeds (Xu et al. 2000); however, because both MMP-2 and MMP-9 are associated with ECM degradation and EVT invasion, this does not explain the differential invasive potential associated with gestational age. This altered invasive potential associated with gestational week suggests that although EVTs are innately invasive, their invasive potential may be restricted primarily by their inability to proliferate and their reduced invasive capacity as gestation proceeds. Decidual regulation of EVT invasion In vivo, invasive EVTs are exposed to factors from many sources in the decidual environment, including the predominant cells of the decidua, decidual cells and leucocytes. This complex environment makes it very difficult to tease out the mechanisms that lead to highly regulated EVT invasion. Studies investigating EVT invasion in pregnancies with placenta creta suggest that the decidua restrains EVT invasion. Total EVT number in placenta creta is higher compared with normal implantation sites (Kim et al. 2004; Hannon et al. 2012). In support there are significantly fewer EVTs in a placenta creta implantation site with local decidua than at a placenta creta implantation site without a local decidua present (Hannon et al. 2012). In contrast, in placenta creta with no local decidua, multinucleated giant cells are absent in implantation sites of tubal pregnancies (Kemp et al. 2002) or significantly reduced in number (Hannon et al. 2012). This suggests that differentiation of EVT to giant trophoblast cells is likely regulated by factors within the decidua. It has been proposed previously that the phenotype of EVTs is critical in modulating the invasive potential of these cells (Kemp et al. 2002). It remains to be investigated what factors within the decidua regulate EVT differentiation. Despite the numerous studies investigating the regulation of EVT invasion by various cell types described below, to date only one study (Lash et al. 2010) has investigated the effect of factors released by the decidua as a whole, using conditioned media from decidual cell isolates. This is likely due to the experimental difficulties associated with investigating the effect from more than one source. Conditioned media (CM; 33% v/v) from decidual isolates (collected at both weeks 8–10 and 12–14) enhances EVT invasion (Lash et al. 2010) compared with medium only control; however, surprisingly, given EVT invasion slows at the end of the first trimester, there was no difference in the effect of gestational age on decidual CM regulation of EVT invasion. The use of medium alone as a control is somewhat problematic because it does not account for

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any non-specific effects of changes to the medium associated with cell culture; however, obtaining suitable control cell isolates is equally problematic. Overall, it is likely that regulation of EVT invasion is highly localised, such that EVT proximity to the various cell types contained within the decidua, and particularly the proportions of these cells, will regulate EVT differentiation and invasion. Decidualized stromal cell regulation of EVT invasion To date, much research into decidual regulation of EVT function, particularly invasion, has focused on the role of leucocytes; however, in contrast, the role of the decidualized stromal cells themselves, contributing .50% of the cells within the decidua, is largely understudied. The decidua is thought to regulate trophoblast invasion and placental formation by regulating the expression of locally produced factors (Xu et al. 2002; Lash et al. 2005; Spessotto et al. 2006; Singh et al. 2011; Menkhorst et al. 2012; Stefanoska et al. 2013), including chemokines (Hannan et al. 2006; Jovanovic´ et al. 2010), cytokines (Paiva et al. 2009b; Shuya et al. 2011) and integrins (Lessey et al. 1994). In vivo studies Recent evidence in women indicates that impaired decidualization is associated with recurrent miscarriage, PE and placenta accreta (Khong and Robertson 1987; Founds et al. 2009; Salker et al. 2010). These studies strongly indicate that decidualized stromal cells regulate EVT function. The critical importance of decidualization for the formation of a functional placenta has been unequivocally demonstrated by studies in genetically modified mouse where decidualization defects lead to pregnancy failure (Benson et al. 1996; Robb et al. 1998; Tsai et al. 2008). For example, the loss of or blocking decidual IL-11 impairs decidualization leading to unregulated trophoblast invasion and pregnancy failure mid-gestation (Bilinski et al. 1998; Robb et al. 1998; Menkhorst et al. 2009). Unlike in women, decidualization in mice is initiated by blastocyst implantation; thus, the systems are not analogous. In vitro studies Decidual cells secrete numerous individual factors that, in vitro, have been shown to enhance or impair EVT invasion (e.g. IL-11, leukaemia inhibitory factor (LIF), high-temperature requirement factor A3 (HTRA3), decorin, prolactin, activin, inhibin, elastin microfibril interface-located protein 1 (EMILIN1); Xu et al. 2002; Jones et al. 2006; Spessotto et al. 2006; Tapia et al. 2008; Paiva et al. 2009b; Chen et al. 2011; Singh et al. 2011; Stefanoska et al. 2013). However, to establish the overall effect of the decidual cell secretome, it is necessary to use either coculture techniques or culture EVT with decidual CM. In the seminal study performed using immortalised human EVT (HTR8SV/neo), CM (15% v/v) from isolated cells of first trimester decidual explants impaired invasion compared with culture medium alone (Graham and Lala 1991). However, the isolated cells in that study were not a pure decidual cell culture, containing as many as 30% non-decidual cells (Parhar et al. 1988), and the control medium was not ‘conditioned’ with control cells, suggesting that factors in CM, not specifically the decidual cells, may have contributed to the inhibition observed.

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Eighteen years later, a second study demonstrated that there was a concentration-dependent effect of CM (collected from decidual stromal cells isolated from first trimester decidua) on immortalised human cytotrophoblast (B6Tert) cell invasion (5% v/v increased, 10% v/v no effect and 20% v/v inhibited B6Tert cell invasion; Zhu et al. 2009). This invasive capability was correlated with MMP-2 activity in these cells (Zhu et al. 2009). However, again, the control medium was not conditioned in that study. Recent studies using predecidualized cell CM as a control suggest that decidual cell factors may, in fact, enhance EVT invasion. Godbole et al. (2011) reported enhanced invasion of choriocarcinoma cell lines (JEG-3 and ACH-3P) following treatment with CM from primary stromal cells decidualized in vitro compared with untreated cells (CM ¼ 100% v/v). A third treatment group, namely cells treated with steroid hormones for a shorter time period defined as ‘predecidualised’, were also included; however, there was no significant difference in the amount of invasion between the decidualized and predecidualized cells. Unfortunately, as that paper stands, much of the data compares the effect of cells treated with and without steroid hormones (used to decidualise stromal cells), which is likely to have a confounding effect on the data (Chen et al. 2011). In further studies, AC1M88 (fusion of JEG-3 and term trophoblasts) spheroids showed enhanced expansion when cultured on top of primary stromal cells decidualized in vitro (with cAMP, which was washed out before experiments) compared with non-decidualized cells (Gonzalez et al. 2011). A role has been suggested for the other way around also, namely that decidual cells ‘move out of the way’ of trophoblast cells (Gellersen et al. 2010). Decidualized cells are invasive in the presence of trophoblast cells (Cohen et al. 2010; Gellersen et al. 2010) and they express many factors required for ECM degradation. Overall, the role of decidual factors in the regulation of EVT invasion is still of conjecture; however, accumulating evidence suggests that the decidual cells may act to promote EVT invasion. Certainly, it seems well accepted that decidual cells are not passive in the regulation of EVT invasion. Of critical importance for future studies investigating the role of decidual cells in EVT invasion is the use of carefully designed controls, particularly for CM and decidualization stimuli (e.g. steroids, cAMP). Leucocyte regulation of EVT invasion During the first trimester of pregnancy, 30%–40% of cells in the endometrium are leucocytes; of these, up to 70% are uNK cells, up to 30% are macrophages and up to 20% are T cells (Williams et al. 2009). To our knowledge, only the role of uNK cells, macrophages and T cells in regulating EVT invasion in humans has been investigated to date. The other roles of leucocytes in the decidua are well reviewed by Bulmer et al. (2010) and therefore not reprised here. In vivo, mice deficient in uNK, T and B cells show delayed trophoblast invasion (Hofmann et al. 2014). However, as the authors note, this is associated with a delay in the timing of uterine lumen closure; thus, it is not possible yet to categorically

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state that these cell types act to promote trophoblast invasion in mice. Regulation by uNK cells of EVT invasion During the first trimester uNK cells (CD56þ) comprise up to 70% of decidual leucocytes, but their numbers fall during pregnancy to be rare at term (Bulmer et al. 2010). The uNK cells are phenotypically distinct from other natural killer (NK) cells (Koopman et al. 2003). Intriguingly, ‘peripheral’ NK cells are never observed in the uterus and, likewise, uNK cells are never observed in the rest of the body, suggesting that locally produced factors act to different peripheral NK cells into a uterine phenotype, for example transforming growth factor (TGF)-b (Keskin et al. 2007). Studies in mice demonstrate that uNK cells are likely recruited from the periphery and that T and B cells are involved (but not absolutely required) in their differentiation towards the uNK phenotype (Hiyama et al. 2011; Zavan et al. 2012). Due to their abundance, uNK cells are perhaps the most well studied of the decidual leucocytes, and many studies have examined the role of uNK cell-secreted factors in the regulation of EVT function, particularly EVT invasion. uNK cells are an important source of growth factors, cytokines and angiogenic factors (Saito et al. 1993; Lash et al. 2011), which have been shown individually in vitro to inhibit (e.g. TGF-b, interferon (IFN)-g, tumour necrosis factor (TNF)-a; Lash et al. 2005, 2006b) or promote (e.g. IL-8; De Oliveira et al. 2010) EVT invasion. Overall, it is likely that uNK cell-secreted factors act to direct EVT invasion towards uNK cells, particularly uNK cells clustered around spiral arteries undergoing remodelling (Bulmer et al. 2010). Supporting this, uNK cells act as chemoattractants for EVTs (Hanna et al. 2006) and uNK supernatants enhance EVT invasion in vitro (De Oliveira et al. 2010; Lash et al. 2010) and in vivo (Hanna et al. 2006). This action is mediated, in part, by uNK secreted IL-8: inhibition of IL-8 in uNK CM impairs invasion (Hanna et al. 2006; De Oliveira et al. 2010) and, in vitro, IL-8 stimulates HTR8/SVneo invasion (Jovanovic´ et al. 2010). uNK supernatants have a gestation week-dependent effect on EVT invasion: supernatants (33% v/v) collected from uNK cells isolated from week 8–10 decidua have no effect on invasion, whereas supernatant collected from week 12–14 decidua promotes invasion (De Oliveira et al. 2010; Lash et al. 2010). This may be due, in part, to the significantly higher IL-8 secreted by the week 12–14 uNK cells compared with week 8–10 uNK cells (De Oliveira et al. 2010); however, the mechanism behind enhanced EVT invasion remains unclear. Exogenous IL-8 at very high concentrations stimulates MMP-2 expression by week 8–10 EVTs (De Oliveira et al. 2010); however, uNK supernatants do not (Lash et al. 2010). Interestingly, it appears likely that once EVTs associate with uNK cells, the uNK cells cease signalling to recruit more EVTs. In an experiment investigating the effect of uNK cells on EVT outgrowth from villous explants, coculture with uNK cells halted EVT outgrowth (Hu et al. 2006). Correspondingly, direct contact between uNK cells and EVTs inhibits cytokine secretion, including IL-8 (Lash et al. 2011), suggesting that direct interaction between EVTs and uNK cells abrogates the chemotactic signalling by uNK cells to attract EVTs.

Regulation of extravillous trophoblast invasion

It is also important to note that uNK cells, despite being capable of being cytotoxic, are not (Croy et al. 1997). In fact, in vitro, uNK supernatants (12–14 weeks) reduce the expression of M30 (a cell death marker) in weeks 12–14 EVTs (Lash et al. 2010). Overall, the effect of uNK cells appears to be to promote EVT invasion towards uNK cells clustered around spiral arteries. Macrophage regulation of EVT invasion Macrophages comprise up to 20%–30% of the decidual leucocytes with their numbers unchanged throughout pregnancy (Bulmer et al. 2010). Unlike uNK cells, macrophages are found throughout the endometrium and myometrium in both the nonpregnant and pregnant uterus (Bulmer et al. 2010). During pregnancy, macrophages localise near spiral arteries, glands and EVTs (Bulmer et al. 2010). Reports of macrophage numbers and localisation vary due to the markers used (Bulmer et al. 2010). It appears likely that there are subpopulations of macrophages in the decidua functionalis, basalis and myometrium, however their localisation and function is not well understood. Investigations into the function of decidual macrophages have focused on their role in EVT apoptosis. The role of macrophages in regulating EVT invasion is not as well studied, although many studies suggest an inverse correlation between macrophage number and endovascular EVT invasion (Reister et al. 1999), with increased numbers of macrophages found in the placental bed of PE pregnancies (Huang et al. 2008). This correlation is possibly due to the role of peripheral blood mononuclear cell (PBMC)-derived macrophages in inducing apoptosis of HTR8/SVneo cells (Wu et al. 2012). Certainly, macrophages from PE pregnancies are positive for Fas ligand (FasLþ), which can induce apoptosis, whereas macrophages from healthy controls are not (Petsas et al. 2012). PBMCmacrophages activated by proinflammatory cytokines (TNF-a, IL-1b, corticotropin-releasing hormone (CRH)) induce apoptosis of HTR8/SVneo and AM1C88 cells (Petsas et al. 2012; Wu et al. 2012). Importantly, decidual secreted granulocyte– macrophage colony-stimulating factor and macrophage colony-stimulating factor are thought to modulate PBMCmacrophage activation (Wu et al. 2012), and macrophages closely surrounding EVTs during the first and second trimester are thought to protect EVT from uNK cytotoxic activity via secreted TGF-b (decidual-derived leucocytes used in this study; Co et al. 2013). Some studies have suggested that activated macrophages can impair EVT invasion, for example Renaud et al. (2005) who utilised PBMCs and Huang et al. (2008) who utilised the THP-1 cell line, but these studies do not control for the effect of macrophages on apoptosis. However, pre-exposure of PBMCmacrophages to IL-10 abrogated their ability to impair invasion (Renaud et al. 2007). There are a limited number of functional studies using decidual-derived macrophages. The in vivo relevance of the studies not using decidual-derived macrophages is of concern given that decidual macrophages exhibit a different phenotype from PBMC-macrophages (Gustafsson et al. 2008). Importantly, however, the factors that activate macrophages

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and result in impaired invasion (IL-1, IL-4, TNF, lipopolysaccharide) are suppressed in a healthy pregnancy and factors that impair macrophage activation (IL-10, PGE) are elevated (Hunt and Petroff 2013). Overall, it seems likely that in healthy pregnancy macrophages have little effect on EVT apoptosis or invasion, but in a pregnancy complicated by, for example, PE, macrophages are activated and have negative effects on EVT invasion. The mechanism(s) resulting in aberrant macrophage activation are unclear; however, we have shown previously that EVTs exposed to impaired decidualization express proteases such as annexin A2 and cathepsin C, which activate macrophages in other tissues and may also alter macrophage activation in decidua (Menkhorst et al. 2012). T cell regulation of EVT invasion T cells contribute approximately 10% of the total leucocyte population in the decidua during early pregnancy (Bulmer et al. 2010) and these comprise the main T cell subsets, including T regulatory (Treg) cells and NK T cells. Few studies have investigated the role of T cells in the regulation of EVT invasion. In vivo, placenta creta, which is characterised by the overinvasion of EVT, is associated with increased forkhead box P3 (FoxP3þ) Treg cells (Schwede et al. 2014), which may act to repress the maternal immune response. IL-8 is an important regulator of EVT invasion when expressed by uNK cells (Hanna et al. 2006; De Oliveira et al. 2010) and is produced by CD8þ T cells (Scaife et al. 2006; De Oliveira et al. 2010). However, despite producing IL-8 at similar levels to uNK cells, CD8þ T cells do not affect EVT invasion (De Oliveira et al. 2010), unless they are stimulated. For example, supernatant from CD8þ T cells stimulated with phytohemagglutinin-P (a mitogen that triggers T lymphocyte division) enhances EVT invasion (Scaife et al. 2006), suggesting that other factors expressed by uNK and stimulated CD8þ T cells may act in concert with IL-8 to regulate EVT invasion. The presence of gdTreg cells within the decidua is of some debate; however, studies show that these cells could comprise 30%–50% of CD3þ T cells within the decidua (MinchevaNilsson et al. 1997; Fan et al. 2011). In vitro, gdTreg cells promote EVT invasion, in part via secreted IL-10 (Fan et al. 2011). Although there are a limited number of studies investigating whether T cells play a role in regulating EVT invasion, the available evidence suggests that T cells may promote EVT invasion via the secretion of cytokines, including IL-8 and IL-10. This is in contrast with tumour invasion, where T cells are thought to limit cell invasion and/or metastasis (Kim and Cantor 2014). Endothelial and vascular smooth muscle cell chemotactic regulation of EVT invasion There have been many reviews regarding spiral artery remodelling and EVT invasion of spiral arteries (Cartwright et al. 2010; Harris 2011), thus the present review focuses on the recruitment of EVTs to the remodelling spiral artery. Remodelling spiral arteries are surrounded by uNK cells and

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macrophages that act to disrupt the vascular smooth muscle cells (VSMCs) and endothelium before the arrival of EVTs (Smith et al. 2009). It appears likely that the uNK cells at least secrete factors that are chemotactic to EVTs, thus helping EVTs ‘hone’ towards uNK cells surrounding remodelling spiral arteries (Hanna et al. 2006). Arterial endothelial cells themselves also express factors (e.g. chemokines, vascular endothelial growth factor) to stimulate EVT migration (Lash et al. 2003; Chau et al. 2013). In contrast, venous factors limit EVT interactions with veins in the decidua (Red-Horse et al. 2005) or are ‘silent’: VSMCs around veins produce fewer chemokines compared with VSMCs surrounding arteries (R. Keogh, pers. comm.). Interestingly, endothelial cells under hypoxia express fibroblast growth factor (FGF) 2, which impairs TEV-1 cell invasion (Luo et al. 2011), suggesting that EVTs may be inhibited from migration to spiral arteries above the trophoblast plugs. Glandular epithelial regulation of invasion Prior to the initiation of the functional blood supply to the placenta at the end of the first trimester, glandular secretions are thought to provide histiotrophic nutrition to the fetus (Burton et al. 2002) because there is dramatically reduced blood flow to the intervillous space during this time due to the plugging of the maternal arteries. Intriguingly, one recent study suggests EVTs invade the glandular epithelium as they do spiral arteries (Moser et al. 2010). The extent of EVT invasion shown is limited compared with that of spiral arteries; however, this discovery provides a potential mechanism by which histiotrophic nutrition to the fetus may be regulated by cells of the placenta. To date, there are no studies investigating the mechanisms by which the glandular epithelium may regulate EVT invasion. Myometrial regulation of invasion Despite EVT invasion progressing only into the junctional zone (inner third) of the myometrium in a normal pregnancy, nothing is known about myometrial regulation of EVT invasion. Certainly, the myometrial junctional zone undergoes changes associated with endometrial decidualization that are likely critical for vascular remodelling within the myometrium and may well contribute to the regulation of EVT invasion (Brosens et al. 2002). Studying EVT invasion in pathological pregnancies One significant impediment to understanding the regulation of EVT invasion is our inability to identify pregnancies during the first trimester that have impaired EVT invasion and will go on to develop PE and/or IUGR. Many laboratories use tissue collected from first trimester terminations and the ability to determine which of these placentas were from pregnancies with impaired EVT invasion would significantly enhance our ability to elucidate the critical factors that regulate EVT invasion. Uterine artery Doppler ultrasound is one method that may be suitable to identify pregnancies that are at risk of developing PE and/or IUGR before termination (Prefumo et al. 2004; Whitley et al. 2007). Although this method does not categorically predict PE

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or IUGR, pregnancies can be classified as being ‘at high risk’, allowing researchers to compare between low- and high-risk placentas. Limitations of current methodologies The most significant limitation of our current methodologies is that these are all in vitro models. The available animal models (e.g. mouse) are not ideal due to differences in decidualization and trophoblast invasion. Rodent models of placentation are very useful in answering very specific questions, however they are not as useful when attempting to elucidate overlying mechanisms. The development of nuanced, ex vivo models using primary tissue able to be manipulated in culture is required. The use of primary cells to model EVT in vivo is also to be desired. EVTs are, by definition, non-proliferative cells. All EVT-like cell lines are proliferative cells and thus do not display one of the hallmark features of EVTs. Gene array studies show that EVTs most closely resemble cytotrophoblasts and have distinct differences compared with both the choriocarcinomaderived and SV40-transformed cell lines (Bilban et al. 2010). If cell lines are used, careful justification as to why the particular cell line is used to model EVTs must be demonstrated. Primary cells are also required to investigate the role of decidual factors in the further differentiation of invasive EVTs to, for example, multinucleated giant cells and also to differentiate between intrinsic (EVT) and decidual regulation of invasion. It is therefore highly desirable that cell line data are verified using primary EVT (Hannan et al. 2010). Models for examining the effect of decidua and decidual cells on EVT invasion To date, most studies have investigated the effect of CM (from one cell type or whole decidual explants) on EVT invasion. The concentration of CM used varies widely between studies (from 15% to 100%), making it difficult to compare between studies. The controls used for CM also vary widely: some studies use culture medium alone (unconditioned, no cells), however this does not exclude the possibility that the results could be nonspecific due to cell-secreted factors rather than the specific celltype being investigated. Further, in the case of studies investigating the effect of decidualized factors on EVT invasion, decidualization treatments (e.g. cAMP, progesterone) have been shown to alter EVT function (Hohn et al. 2000; Chen et al. 2011; Gonzalez et al. 2011). Therefore, the decidualization treatments must be stringently controlled in EVT function experiments. Some studies use withdrawal of decidualization treatments for 24–48 h (wash out) before use of the CM; however, in our hands, wash out resulted in reduced secretion of prolactin (a decidualization marker), suggesting that secretion of other decidual proteins may also be reduced following wash out (pers. obs.). We submit that more carefully controlled studies are required to finally tease out the regulation of EVT invasion. Specific attention must be paid to the development of proper controls, as well as the effect that gestational week and EVT differentiation may play, given EVT invasive capacity is reduced as gestational week increases.

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Conclusion Decidualization and the associated changes in leucocyte infiltration are likely an adaptation in women to promote EVT invasion. Indeed, the evidence available suggests that in a healthy pregnancy EVT invasion is restricted mainly by time, with EVTs from older gestation placentas not as invasive as those from younger gestation placentas, and possibly by space: we speculate that the physical barrier provided by the decidua (which is not present in placenta creta) may limit the depth of the non-proliferative EVT invasion. Although the effect of individual factors on EVT function have been studied extensively, the proportional role of these factors and the overall effect of each cell type in the context of the decidual environment is not understood. Overall, there exists an exquisite balance between trophoblast over- and underinvasion. Any alteration to the cell types and factors that modulate EVT invasion can be catastrophic to the pregnancy. Extrinsic factors, such as gestational age, epigenetics and inflammatory conditions within the decidua are also likely to have an important role. Understanding the role of the decidua in regulating EVT invasion will improve our understanding of what leads to a healthy versus pathological pregnancy, ultimately resulting in the discovery of therapeutics to treat abnormal pregnancies. Funding The authors’ work reported herein was supported by the Victorian Government’s Operational Infrastructure Support Program. ED was supported by a National Health and Medical Research Council of Australia (NHMRC) Senior Research Fellowship (#550905). EM was supported by an NHMRC Early Career Fellowship (#611827). AW was supported by an Australian Postgraduate Award. References Aplin, J. D. (2010). Developmental cell biology of human villous trophoblast: current research problems. Int. J. Dev. Biol. 54, 323–329. doi:10.1387/IJDB.082759JA Benson, G. V., Lim, H., Paria, B. C., Satokata, I., Dey, S. K., and Maas, R. L. (1996). Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10 expression. Development 122, 2687–2696. Bilban, M., Tauber, S., Haslinger, P., Pollheimer, J., Saleh, L., Pehamberger, H., Wagner, O., and Kno¨fler, M. (2010). Trophoblast invasion: assessment of cellular models using gene expression signatures. Placenta 31, 989–996. doi:10.1016/J.PLACENTA.2010.08.011 Bilinski, P., Roopenian, D., and Gossler, A. (1998). Maternal IL-11Ra function is required for normal decidua and fetoplacental development in mice. Genes Dev. 12, 2234–2243. doi:10.1101/GAD.12.14.2234 Brosens, J. J., Pijnenborg, R., and Brosens, I. A. (2002). The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature. Am. J. Obstet. Gynecol. 187, 1416–1423. doi:10.1067/MOB.2002.127305 Bulmer, J. N., Williams, P. J., and Lash, G. E. (2010). Immune cells in the placental bed. Int. J. Dev. Biol. 54, 281–294. doi:10.1387/IJDB. 082763JB Burton, G. J., Watson, A. L., Hempstock, J., Skepper, J. N., and Jauniaux, E. (2002). Uterine glands provide histiotrophic nutrition for the human

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