REVIEW ARTICLE: Governing the Invasive Trophoblast: Current Aspects on Intra- and Extracellular Regulation

Authors


  • All authors have contributed to similar amounts to this review article.

Udo R. Markert, Placenta-Labor, Abteilung für Geburtshilfe, Friedrich-Schiller-Universität, D-07740 Jena, Germany.
E-mail: markert@med.uni-jena.de

Abstract

Citation Fitzgerald JS, Germeyer A, Huppertz B, Jeschke U, Knöfler M, Moser G, Scholz C, Sonderegger S, Toth B, Markert UR. Governing the invasive trophoblast: current aspects on intra- and extracellular regulation. Am J Reprod Immunol 2010

This review summarizes several aspects especially of regulating factors governing trophoblast invasion. Those include the composition of the extracellular matrix containing a variety of matrix metalloproeinases and their inhibitors, but also intracellular signals. Furthermore, a newly described trophoblast subtype, the endoglandular trophoblast, is presented. Its presence may provide a possible mechanism for opening and connecting uterine glands into the intervillous space. Amongst others, two intracellular signalling pathways are crucial for regulation of trophoblast functions and development: Wnt- and signal transducer and activator of transcription (STAT)3 signalling. Wnt signalling promotes implantation, placentation and trophoblast differentiation. Several Wnt-dependent cascades and regulatory mechanisms display different functions in trophoblast cells. The STAT3 signalling system is fundamental for induction and regulation of invasiveness in physiological trophoblastic cells, but also in tumours. The role of galectins (Gal) in trophoblast regulation and placenta development comes increasingly into focus. The Gal- 1–4, 7–10 and 12–14 have been detected in humans. Detailed information is only available for Gal-1, -2, -3, -4, -9 and -12 in endometrium and decidua. Gal-1, -3 and -13 (-14) have been detected and studied in trophoblast cells.

Introduction

Trophoblast cells are a very heterogenous cell type that display manifold basic and fundamental functions during the development of the placenta. Trophoblast cells cannot only be divided into villous and extravillous cells or into cells responsible for exchange of metabolic products and anchoring cells. Trophoblast cells invade the decidua where they possess a variety of capacities: communication with maternal immune cells, hormone and cytokine production, substitution of endothelial cells of maternal arterioles, angiogenesis, fusion to giant cells and recent studies insinuate substitution of glandular epithelium. The regulation of these features depends upon plentiful extra- and intracellular signals, a few prominent of which have been briefly described in this review.

Extracellular matrix (ECM)

The maternal ECM within the endometrium consists of several compounds such as fibronectin, fibrin, vitronectin, and laminin.1 During the implantation process, trophoblast cells express mediators facilitating attachment and degradation of the ECM to be able to invade the endometrium and reach the maternal blood supply.2 Along with other well-known mediators such as hormones, growth factors and cytokines, several integrins [e.g. integrin alpha(IIb) beta(3)] facilitate the anchoring of the trophoblast to fibrin.3 While integrin alpha(5) beta(1) improves the attachment to fibronectin of maternal origin, other integrins, such as integrin alpha(4) beta(4), are downregulated during the implantation process.4 However, integrin expression is also present on endometrial cells, particularly integrin alpha(v) beta(3) and alpha(4) beta(1).5

The expression of proteinases such as matrix metalloproteinases (MMPs) remains crucial for the invasion potential of trophoblast cells and contributes to the degradation of the ECM, while tissue inhibitors of metalloproteinases (TIMPs) act as counterparts and limit the invasion process.6

During the ‘window of implantation’, expression of MMPs and other invasion factors is tightly regulated by a bidirectional foeto-maternal cross-talk involving chemokines, cytokines and growth factors expressed by endometrial cells that contribute to a paracrine change in the gene expression profile of the trophoblast.7

CX3CL1 and CCL14, two cytokines produced by endometrial decidual cells, are found to regulate trophoblast gene expression, including MMP-12, integrin beta(5) and osteopontin, leading to an increased trophoblast adhesion to fibronectin.8 Furthermore, MMP-12 is known to degrade many components found at the implantation site, such as type IV collagen, laminin, fibronectin, vitronectin, myelin basic protein and chondroitin sulphate. Additionally, MMPs are expressed on trophoblast cells in a time-dependent manner, with a dominant expression of MMP-2 [one of the key enzymes (gelatinase) for trophoblast invasion] during the early phase of implantation followed by MMP-9 expression that is essential for the invasiveness of the trophoblast. Both MMP genes are shown to be stimulated by epidermal growth factor and forskolin in vitro.9 Furthermore, interleukins (IL), such as IL-1beta and IL-6, stimulate the autocrine production of MMP-9 within trophoblasts,10,11 while IL-10 inhibits MMP production.12

Leukaemia inhibitory factor (LIF), another cytokine of endometrial origin, increases trophoblast invasion by downregulating integrin beta(4) and by enhancing the potential of trophoblast cells to adhere to ECM components (such as fibronectin, vitronectin and laminin) in vitro.13 This cytokine is also able to regulate TIMP expression, but not MMP expression, and therefore may be capable of controlling trophoblast invasion in human pregnancy.13

Finally, the ECM itself regulates the expression of different MMPs within the trophoblast.14

Role of galectins in trophoblast invasion

Galectin (Gal) is a general name proposed in 1994 for a family of animal lectins.15 Gals are defined as lectins having both galactose-binding ability and amino-acid sequences that characterize Gals.16 Gals are members of the mammalian β-galactoside-binding proteins and recognize Galβ1-4GlcNAc sequences of several cell surface oligosaccharides (Fig. 1a).17–19 Gal-1 binds preferentially to the Thomsen–Friedenreich (TF) antigen (Galβ1-3GalNAc) on placental cells (Fig. 1b).20–22 At the moment, 15 different Gals are under investigation. There is evidence that Gal 1–4, 7–10 and 12–14 are present in humans, whereas Gal-5, -6 or -11 cannot be detected. Gal-5 is only present in rats and Gal-6 only in mice. Gal-11 has been used as an alternative name for two non-human Gal-like proteins. Gal-15 is expressed in the uteri of sheep, cow and goat.

Figure 1.

 (a) Structure of N-acetyl-lactose. (b) Structure of the Thomsen–Friedenreich antigen.

Gal-1 regulates cell apoptosis, cell differentiation and hormone production. It binds TF, CD45, CD95, CD3 and CD4 and inhibits CD45 protein phosphatase activity.20,22–25 On trophoblast cells Gal-1 induces phosphorylation of vascular endothelial growth factor receptor 3 and inhibits phosphorylation of rearranged during transfection and Janus Kinase (JAK)2.26 Experiments in mice showed that Gal-1 prevents foetal loss and restored foetal tolerance through multiple mechanisms, including the induction of tolerogenic dendritic cells, which promoted the expansion of IL-10-secreting regulatory T cells in vivo.27 Gal-1 is a pivotal regulator of the fetomaternal tolerance that has potential therapeutic implications in threatened pregnancies.28 Gal-1 may also promote NK cell licensing towards pregnancy protection on the level of progenitor cells.29

Gal-2 binds to beta1 integrins on T cells (or a closely associated glycoprotein). The carbohydrate-dependent binding of Gal-2 induces apoptosis in activated T cells.30–33

Gal-3 consists of carbohydrate recognition and collagen-like domains. It is a mediator/modulator of cell to ECM adhesive interactions. Cells, particularly epithelial cells that lack Gal-3 expression, interact poorly with their extracellular matrices. Gal-3 has also been identified in the human placenta, and its abundance was found to be inversely correlated with trophoblast invasiveness during the course of gestation.34 Up-regulation of Gal-3 in both pre-eclamptic and Hemolytic anemia Elevated Liver enzymes and Low Platelet count extravillous trophoblast (EVT) may compensate for the reduced trophoblast invasion observed in both types of pathologic pregnancies.35–39

Gal-4 binds to glycosphingolipids.40,41 Furthermore, Gal-4 promotes adhesion of human colon adenocarcinoma cells. Sulphated glycosphingolipids (SB1a) and carcino embryonic antigen on the cell surface of these cells could be biologically important ligands for Gal-4.40

Gal-7 is associated with the differentiation and development of epithelia. It is also associated with epithelial cell migration. In addition, Gal-7 is designated as the product of the p53-induced gene 1 and is a regulator of apoptosis through c-Jun N-terminal kinases activation and mitochondrial cytochrome c release. It also acts as a transcriptional regulator via its interaction with Smad3.42–45

Gal-8 has a di- or multi-valent structure. This is essential for the induction of cell adhesion and exhibits broad specificity for leukocytes. The Gal-8-induced cell adhesion is accompanied by stress fibre formation, which suggests that intracellular signalling is required. The phosphorylation of proline-rich tyrosine kinase and extracellular signal-regulated kinase (ERK)1/2, indicators of integrin-mediated signalling, was up-regulated on treatment with Gal-8. A primary target of Gal-8 could be the sugar chains on members of the integrin family, which are abundantly expressed on the surface of leukocytes.46–50

Gal-9 is able to induce decreased levels of pro-inflammatory cytokines such as IL-17, IL-12 and IFNγ. Gal-9 decreases numbers of CD4(+) TIM-3(+) T cells in peripheral blood. Gal-9-deficient mice showed increased numbers of CD4(+) TIM-3(+) T cells and decreased numbers of Treg cells. Gal-9 induces differentiation of naive T cells to Treg cells.51–56

Gal-10 is constitutively expressed in human CD25(+) Treg cells, while it is nearly absent in resting and activated CD4(+) T cells. Gal-10 was found to be essential for the functional properties of CD25(+) Treg cells.57,58

Gal-12 is preferentially expressed in peripheral blood leukocytes and adipocytes. Previously, it was shown that Gal-12 is induced by cell cycle block at the G(1) phase and causes G(1) arrest when overexpressed. Gal-12 is required for signal transduction and induction of adipogenic factors essential for adipocyte differentiation.59,60

Gal-13 is also known as Placental Protein 13 (PP13) and was cloned from human term placenta. It shows weak lysophospholipase activity. N-acetyl-lactosamine, mannose and N-acetyl-glucosamine residues had the strongest binding affinity to Gal-13, which also lead to agglutination of erythrocytes. Gal-13 is a homodimer of 16-kDa subunits linked together by disulphide bonds. Strong Gal-13 labelling of the syncytiotrophoblasts confirmed its galectin-like externalization to the cell surface. PP13/galectin 13 may have special haemostatic and immunobiological functions in the placenta. In addition, first trimester PP-13 levels may be useful in predicting pre-eclampsia, and the accuracy of the method increases when coupled with second trimester Doppler measurement.57,61,62

Gal-14 is known as PP13-like (Charcot–Leyden crystal protein 2). It is highly expressed in the human placenta and contains one galectin domain. Less is known about the function of this protein.63–66

The role of Wnt signalling in trophoblast invasion

Wnt (Wingless)-dependent signalling cascades play fundamental roles during embryonic development, tissue homeostasis and progression of cancer.67,68 They affect numerous key cellular processes such as primary axis formation, maintenance of intestinal stem cells or axon guidance by controlling proliferation, differentiation and cell motility. The Wnt ligands are glycoproteins acting through different signalling pathways of which the canonical Wnt β-catenin-dependent pathway has been extensively analysed.69

In canonical Wnt signalling, Wnt ligands interact with a heterodimeric receptor consisting of lipoprotein receptor-related protein-5 or -6 (LRP-5/6) and a seven transmembrane Frizzled (Fzd) protein. To date, 19 different Wnt ligands and 10 different Fzd receptors have been described. Formation of the Wnt/LRP-5/6/Fzd complex activates Dishevelled, a critical signalling molecule of both canonical and non-canonical Wnt pathways, and induces recruitment of Axin, a negative regulator of β-catenin stability, to the cytosolic portion of LRP-5/6. In the absence of a Wnt signal Axin together with adenomatous polyposis coli (APC), casein kinase Iα and glycogen synthase kinase 3β (GSK3β) form the so-called β-catenin destruction complex promoting phosphorylation and continuous degradation of β-catenin through a proteasomal pathway. However, receptor binding of Wnt inhibits β-catenin degradation through a series of events resulting in cytosolic accumulation of the protein and translocation to the nucleus. Within the nucleus, β-catenin acts as a transcriptional co-activator of particular DNA-binding proteins, the T-cell-specific factor (TCF)/lymphoid enhancer-binding factor 1 (LEF1) transcription factor family.70 Upon binding, β-catenin displaces transcriptional inhibitors, such as Groucho proteins, from TCFs and recruits co-activators such as histone acetylases and the Legless family docking protein BCL9. TCF/β-catenin complexes finally promote transcription of genes controlling cell proliferation and migration/invasion, such as cyclin D,71 c-myc 72 and MMPs.73 Mutations in the critical Wnt components APC, Axin and β-catenin are frequently found in tumours mainly provoking abnormal stabilization and nuclear abundance of β-catenin.74 Beside the canonical pathway, Wnts may signal through a variety of other downstream effectors, commonly known as non-canonical Wnt pathways. In the Wnt-planar cell polarity pathway, non-canonical Wnts such as Wnt-5a or Wnt-11 provoke cell polarity and movement, through Rho GTPases such as RhoA and Rac,75 whereas the Wnt-Ca2+ pathway was shown to activate protein kinase C and calcium–calmodulin-dependent kinase II.76 Ligands such as Wnt-3A may also activate other β-catenin-independent signalling cascades such as ERK or protein kinases B (AKT).77,78

Accumulating evidence suggests that Wnt signalling is also critically involved in implantation and placental development. In mice, various Wnts and Fzds were detected at the time of implantation,79 and blastocyst attachment was shown to induce endometrial Wnt-4 expression.80 Accordingly, blocking of the canonical Wnt pathway with the soluble inhibitors secreted frizzled-related protein 2 (sFRP-2)81 or Dickkopf-1 (Dkk1),82 which blocks Wnt signalling upon binding to LRP-5/6, impaired frequency of mouse implantation.

In humans, endometrial cells were shown to produce Wnt ligands and Dkk1 in a menstrual cycle–dependent manner.83 Also, Dkk1 had been identified as a progesterone-regulated gene.84 In addition, incubation of decidualized stromal cells with trophoblast supernatants resulted in the induction of Wnt signalling genes.85 Hence, similar to mice, Wnt signalling could be an essential part of the human decidual response to the implanting blastocyst.

With respect to placenta formation, gene knock-out (ko) of the ligands Wnt-2, Wnt-7b and of TCF-1/LEF-1 affected critical steps of murine extraembryonic development such as chorion-allantoic fusion or placental vascularization.86–88 Moreover, Wnt signalling may play a critical role in trophoblast development because Wnt-3A was shown to promote trophectoderm formation in embryonic stem cells by inducing the critical transcription factor Cdx2 in a LEF-1-dependent manner.89

However, Wnt signalling may not be only required for early trophoblast lineage determination but also control function of differentiated trophoblast subtypes, such as the invasive, EVT. Recent studies identified nuclear β-catenin in a subset of EVT in vivo as well as in vitro after outgrowth from chorionic villous explant cultures.90 Interestingly, TCF-3 and TCF-4 were also mainly detected in EVT, the latter being almost exclusively expressed in non-proliferating p57/KIP2-positive trophoblasts. Hence, TCF-4 could be an essential transcription factor maintaining the differentiated EVT phenotype. Upon stimulation with the recombinant Wnt ligand Wnt-3A, elevated migration and invasion of primary trophoblasts and the EVT cell line SGHPL-5 could be observed. Similarly, Wnt-3A increased trophoblast outgrowth from villous explant cultures and provoked phosphorylation of AKT in primary EVT and SGHPL-5 cells.91 Dkk1 did not inhibit AKT phosphorylation, suggesting that a classical LRP-5/6-Fzd receptor is not involved in activation of the particular kinase. In other cells, AKT can induce canonical Wnt signalling by phosphorylation/inactivation of GSK-3β and subsequent accumulation of β-catenin.92 However, this cross-talk may not exist in trophoblasts because chemical inhibition of AKT neither increased nuclear abundance of β-catenin nor luciferase activity of a canonical Wnt reporter. However, stimulation of both pathways (non-canonical and AKT) provoked Wnt-dependent secretion of MMP-2, which could be one of the critical Wnt targets promoting trophoblast invasion.91 Dkk-1 treatment of primary trophoblasts and SGHPL-5 not only abolished Wnt-3A-dependent cell motility but also reduced basal migration and invasion, suggesting expression of endogenous Wnt ligands that may act in a autocrine manner. Indeed, 14 of 19 Wnt ligands and 8 of 10 Fzd receptors could be detected in human placenta.93 Interestingly, gestation-dependent as well as trophoblast subtype-specific expression of Wnts and Fzds were noticed. For example, Wnt-1, Wnt-7A, Wnt-10A and Wnt-10B were strongly expressed in first trimester trophoblasts but largely absent from term trophoblasts, indicating that they could mainly play a role in early pregnancy. Wnt-1 and Wnt-2B were abundantly expressed in EVT suggesting that these Wnts could be the prime regulators of Wnt-dependent trophoblast invasiveness. In addition to Wnts, however, other ligands and receptors may contribute to β-catenin/TCF-dependent signalling. For example, gene silencing of protease-activated receptor-1, PAR1, provoked β-catenin destabilization and reduced trophoblast motility.94

The role of Wnt signalling in trophoblast invasion is further emphasized by the fact that promoters of some Wnt inhibitors such as sFRP-2 were shown to be methylated in first trimester trophoblasts potentially leading to their reduced expression and activation of Wnt signalling.95 Interestingly, these genes are hypermethylated in choriocarcinomas, which may correlate with further gene silencing and Wnt-dependent progression of trophoblast towards a malign phenotype. Similar to tumour cells, elevated nuclear expression of β-catenin was observed in invasive trophoblasts of complete hydatidiform mole placentae suggesting that aberrant Wnt signalling could also play a role in the particular gestational disease.90 Moreover, Wnts may also modulate other trophoblast processes such as phospholipid uptake and transport. StarD7, a member of the StAR1 lipid transfer proteins, was identified as direct target gene of TCF/βcatenin in trophoblasts.96

In conclusion, Wnt signalling has been identified as an essential signalling pathway promoting implantation, placentation and trophoblast differentiation. Despite the facts that several Wnt-dependent cascades and regulatory mechanisms have been elucidated, much remains to be learned about putative trophoblast subtype-specific roles of Wnts, their respective receptors interactions and critical downstream targets.

Signal transducer and activator of transcription (STAT)3 signalling

Signal pathways are indicated to convey crucial processes involved in conception, development and progression of pregnancy, such as trophoblast invasion and differentiation. One signalling pathway that draws interest because of its role in reproduction is the JAK–STAT signalling pathway.

The JAK–STAT pathway initially attracted a great deal of attention as a rapidly induced form of signal transduction. It was quickly demonstrated, though, that this pathway is responsible for far more than rapid induction of immunoregulatory signals, but rather, it was also vital for a number of responses, including cell growth, differentiation processes, longevity and migration.97 Especially the STAT3 protein has been described as a candidate with oncogenic potential, because either its expression or activation profile is constitutively higher in a number of malignancies, including those pertaining to the reproductive system or the STAT3-dependent transcription regulatory network is involved in metastatic progression (reviewed in98,99).

In short, cytokine-specific receptors aggregate upon mediator binding leading to the juxtaposition of receptor-associated tyrosine kinases termed Janus kinases or JAKs (named after the two-headed Roman mythological god, Janus, because of its ‘two-headed’ structure). Thus, JAKs can, through cross-phosphorylation, reciprocally activate each other as well as specific cytoplasmic domains of their respective cytokine receptor. Intracellular STATs are now recruited to these receptor domains, with the goal of bringing STATs within vicinity of JAKs so they may also in turn be phosphorylated and activated by the latter. Following activation, STATs disassociate from the receptor ligand and proceed to form homo- and heterodimers that translocate into the cell’s nucleus. Here, STATs influence the transcription of target proteins by binding and manipulating the promoter regions of these proteins. One of the transcribed proteins is the suppressors of cytokine signalling (SOCS) protein, a regulator that is specifically and non-specifically able to negatively modulate the duration of the cytokine signalling response by binding to phosphotyrosine residues as seen for instance on JAKs.100–102

Indeed, STAT3 is a main mediator of several cytokine and growth factor signals that are also relevant for reproduction: IL-6, IL-11, HGF, LIF (cytokines that mediate through gp130) or IL-10.103–105

The fact that Lif (ko) mice are incapable of implantation, although fertile, invites the hypothesis that STAT3 is involved in the process of implantation, possibly through mediation of invasion.106,107 An interesting observation is that STAT3 is expressed in the murine and human pre-implantation stage embryo at as early as the morula stage. Shortly after fertilization, STAT3 expression in the human and murine embryo is polarized, meaning that there are spacial and temporal differences in protein expression among the blastomeres such that by the eight-cell stage, unique cellular domains consisting of STAT3 rich- and poor cell populations are generated. In the morula, it appears that the ‘inner’ cells contain virtually no STAT3, while the ‘outer’ cells contain both rich and poor STAT3 expressing trophoblast. In the blastocyst stage (contrary to the aforementioned, these experiments were undertaken only in mice), STAT3 immunofluorescence was restricted to the trophoblast, but with pronounced differences in intensity, suggesting that expression might be limited to certain trophoblast populations.108

However, targeted gene disruption of the STAT3 protein was detrimental to murine pregnancy shortly after implantation. Stat3 ko embryos degenerated and died in the early post-implantation period on E7.5, while wild-type embryos expressed the protein in the extraembryonic tissues, especially in extraembryonic columnar visceral endoderm (destined to form the visceral wall of the yolk sac), but also including the parietal endoderm, the ectoplacental cone and throughout the extraembryonic ectoderm, suggesting a vital role for STAT3 in early development.109,110 The extraembryonic ectoderm is known to differentiate into the ectoplacental cone from which spongiotrophoblast and giant trophoblast cells originate (reviewed in111). Culture of spongiotrophoblast cells leads to the eventual formation of trophoblast giant cells,112 and trophoblast giant cells are considered the murine correlate to invasive human EVT cells.113 This simultaneously indicates that STAT3 is not exclusively responsible for trophoblast invasion during implantation; however, without STAT3 signalling, the maintenance of pregnancy beyond initial implantation is impossible. Whether this might be because, at least in part, of inadequate post-implantation trophoblast invasion remains speculative.

Chan et al.114 revealed via immunohistochemistry of first trimester placentae that especially serine-activated STAT3 protein is detectable in both cytoplasm and nuclei of syncytiotrophoblast, cytotrophoblast and villous intermediate trophoblast, while this expression profile disappears at term. Serine phosphorylation in trophoblastic cells is mainly mediated via mammalian target of rapamycin.115

Interestingly, expression appears more intensive in the cytotrophoblast and intermediate trophoblast subsets. These are considered the trophoblast responsible for invasion in the placenta, and thus important for placentation and, later, adequate sustenance of the foetus.116,117 The fact that this expression profile is augmented in samples derived from mole pregnancy and gestational trophoblastic disease underscores the idea that STAT3 activation is necessary for the capability of the trophoblast to invade the vicinity.

In comparative experiments between carcinoma cell lines and physiological primary cells from first trimester and term placentae, constitutive activity of STAT3 as seen through elevated DNA binding of STAT3 dimers could be correlated with invasiveness of trophoblast and its derivates.118 Furthermore, it appears that LIF transmits an up-regulation of STAT3 expression, activation and DNA-binding activity, which results ultimately in a protease profile alteration that dose-dependently boosts trophoblastic (human choriocarcinoma) invasion and proliferation.119 Here, cDNA macroarrays found that the expression of TIMP-1 was down-regulated, with TIMP-1 being a protein that binds and thus inactivates MMP-9, or MMP-9, a protein critical for cytotrophoblast invasion.120,121 Knocking down STAT3 expression in Jeg-3 choriocarcinoma cells, first trimester and term placentae trophoblast through short interfering RNA (siRNA) resulted in a dramatic reduction in invasive potential regardless of LIF supplementation.105

LIF is also able to promote the adhesion of first trimester EVT adhesion to an ECM, a process generally accepted as a prerequisite for the differentiation of trophoblast into the invasive phenotype.13 There is some controversy whether LIF has the same effect on invasiveness of physiological trophoblast cells as it has on choriocarcinoma cell lines. LIF has been reported to decrease gelatinase activity in first trimester trophoblast cells,122,123 and the aforementioned work by Tapia et al.13 demonstrated that LIF supplementation of EVT resulted in a higher secretion of TIMP-1 and TIMP-2 as detectable through ELISA. Taken together, these data suggest a largely inhibitory effect on EVT invasion. Whether this discrepancy is because of the differences in cell physiology (primary trophoblast versus choriocarcinoma cell line) or because of differences in utilized concentrations or cytokine stimulation periods has not yet been resolved. It is known, however, that LIF suppresses its own effects by means of negative feedback regulation of the JAK–STAT pathway through SOCS3.124 In this context, both too low and too high levels of LIF in uterine flushings have been suggested to have negative predictive value in implantation success.125,126

In human trophoblast cells, peripheral blood mononuclear cells (PBMC) induced phosphorylation of STAT1 and STAT3,127 and in Jeg-3 choriocarcinoma cells, ablation of Stat3 by siRNA leads to an alteration of gene expression profiles including that of SOCS3, whose gene expression decreased by almost twofold especially after JEG-3 cells were confronted with conditioned medium of activated PBMC.128

In the murine model, deletion of the Socs3 gene leads to embryonically lethal placental insufficiency and a phenotypical placenta that exposes poorly formed spongiotrophoblast and labyrinthine layers, as well as an increase in trophoblast giant cell differentiation similar to placentae of wild-type mice overexposed to LIF (reviewed in129,130). Interestingly, decreased SOCS3 expression is observed especially in the villous tissue of placentae from women with pre-eclampsia,131 and in loose analogy to the Socs3 ko placenta, pre-eclampsia placentae are characterized by shallow invasion of the decidua by the trophoblast, yet with an excess of proliferative immature trophoblast.132 These findings might appear counterintuitive upon initial inspection, because low SOCS3 expression would in turn portend high STAT3 activation, and thus sufficient trophoblast invasion. However, the finding that the expression of the LIF-receptor is abnormally persistent on EVT cells from placental bed biopsies stemming from patients suffering from a combination of early onset pre-eclampsia and intrauterine growth retardation133 only further denotes the importance of LIF and SOCS3 fine-tuning. In conclusion, it seems that the LIF–JAK–STAT–SOCS signalling pathway is involved in many functions of the trophoblast and their derivate cells as can be recognized in murine models, in vitro assays and descriptional studies. The functions that are influenced encompass trophoblast properties such as differentiation, adhesion, migration/invasion, and these characteristics underlie a strictly regulated fine-tuning mechanism (serine versus tyrosine phosphorylation for activation, negative feedback, etc.) that invites further investigation.

Endoglandular trophoblast invasion

About 2 weeks after fertilization, the subset of EVTs comes into first contact with maternal tissues and starts to invade into the decidual stroma. During this process of migration and invasion, EVTs pass capillaries and glands of the uterine interstitium, finally reaching the myometrium and/or spiral arteries. So far, trophoblast invasion has been described to serve (1) attachment of the placenta to the uterus and (2) transformation of spiral arteries into large open conduits.134 Spiral arteries are first invaded by intravasation of interstitial EVTs,135 then blocked by cells that have passed the media of the vessels and have become endovascular trophoblasts. These plugs of cells remain until the end of the first trimester, and only with the beginning of the second trimester, maternal blood flow through the intervillous space of the placenta is established. Hence, only then is haemotrophic nutrition of the embryo ascertained.136 This has led to the question as to how the embryo gains enough nutrition prior to the onset of maternal blood flow through the placenta, i.e. during the first trimester of pregnancy.

The non-pregnant endometrium, as well as the pregnant decidua, contains huge numbers of uterine glands that open towards the uterine cavity. The glandular secretion products reach the uterine cavity as well as the fallopian tubes and play an essential role in feeding and maintaining the very early embryo prior to implantation.137 The secretion products that contain a mixture of proteins, lipids and carbohydrates do not only play a role in feeding the early embryo but additionally seem to have immunosuppressive features and to be involved in controlling implantation.138

Only recently, it was assumed that glandular secretion products may play a role in nutrition of the human embryo even after implantation. The presence of such secretion products has been demonstrated in the intervillous space of the first trimester placenta.139 These authors have shown that secretory components of the uterine glands are part of the clear fluid found in the intervillous space of a first trimester placenta.139 However, they could not show how the glandular secretion products reach the intervillous space of the placenta. The glandular openings are towards the uterine cavity rather than the intervillous space, and thus so far, it can only be speculated that invasive EVTs might use the uterine glands as targets, similar to the uterine spiral arteries.

Using double immunohistochemical staining for cytokeratin 7 and human leukocyte antigen-G, EVTs were detected in the vicinity of uterine glands and even in very close contact to glandular epithelial cells. In all samples from gestational week 6–11 endoglandular trophoblasts have been perceived, i.e. trophoblasts replacing glandular epithelial cells or present in the lumen of uterine glands.140

Using novel co-culture confrontation model systems with decidual tissues as matrices for trophoblast invasion from villous explants, trophoblast invasion into the decidual interstitium and towards uterine glands has been analysed. Also in these in vitro systems, EVTs invaded the decidual tissues towards the glands and could be detected within the glands.140 These data may provide first evidence for trophoblast invasion into the lining of the glandular epithelium, thus opening the way for glandular secretion products to reach the intervillous space – similar to the pathway of arterial remodelling.

According to the aforementioned data, a new subset of EVT, the ‘endoglandular trophoblast’, can be suggested. Replacement of glandular epithelial cells by endoglandular trophoblasts may provide a possible mechanism for opening and connecting uterine glands towards the intervillous space. This would enable histiotrophic nutrition of the embryo during the first trimester of pregnancy prior to onset of the maternal blood flow. Thus, according to the definition found in Figure 9.3 of Benirschke et al.,141 the different phenotypes of EVT need to be extended to interstitial trophoblast, endovascular trophoblast and endoglandular trophoblast.

Conclusive remarks

The literature summarized in this review demonstrates that Wnt and STAT3 signalling are main intracellular governors of trophoblast invasiveness and further functions. The large variety of Gals has a strong potential to influence these systems, but most of the current knowledge has been obtained from other cell types than trophoblast. The ECM is simultaneously a multivalent key controller and target of trophoblast invasion. Newest findings suggest that a part of invasive trophoblast cells form a new subtype, the endoglandular trophoblast.

Ancillary