Mechanisms regulating cytotrophoblast invasion in normal pregnancy and pre-eclampsia

Australian and New Zealand Journal of Obstetrics and Gynaecology 2006; 46: 266–273

266 © 2006 The AuthorJournal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists

Blackwell Publishing Asia Invited ReviewMechanisms underlying trophoblast invasion

Mechanisms regulating cytotrophoblast invasion in normal pregnancy and pre-eclampsia

Fiona LYALLMaternal and Fetal Medicine Section, Institute of Medical Genetics, University of Glasgow, Yorkhill, Glasgow, UK

AbstractThe placental bed underlies the placenta and includes the decidua basalis and underlying myometrium containingthe uterine spiral arteries. For successful human haemochorial placentation, the placental bed spiral arteries mustundergo considerable alterations. These physiological modifications are thought to be brought about by the inter-action of invasive cytotrophoblast with the spiral artery vessel wall. Failure of spiral artery transformation is thoughtto play an important role in the sequence of events that gives rise to pre-eclampsia. The mechanisms that controlhuman trophoblast invasion in normal, let alone abnormal pregnancy, are still poorly understood. Much of theinformation on the early physiological changes within the placental bed comes from studies on intact hysterectomyspecimens.1 Details of such events in late pregnancy and in pregnancies complicated by pre-eclampsia and fetalgrowth restriction are principally derived from the study of placental bed biopsies taken at Caesarean section. Themethods of sampling the placental bed have been reviewed elsewhere.2 Many investigators have relied on in vitromodels of trophoblast invasion. In vitro models can be extremely useful in dissecting out some of these processesbut may be open to artefacts. The mechanisms underlying normal and failed trophoblast invasion appear to becomplex. In this manuscript the mechanisms that control the invasion of trophoblast into the decidua and myo-metrium are reviewed. Along with this is a review of the purported mechanisms underlying failed spiral arterytransformation. Particular emphasis has been placed on topics that have been best studied.

Key words: cytotrophoblast, invasion, placental bed, pre-eclampsia, pregnancy.

Extravillous trophoblast

Extravillous trophoblast (EVT) is made up of all trophoblastfound outside the placental villi. Extravillous trophoblast is ahighly migratory, proliferative and invasive population ofcells that emerges from tips of anchoring villi. Trophoblastinvasion of the uterus involves attachment of these cells tothe extracellular matrix (ECM), degradation of the matrixand subsequent migration. It is a tightly controlled processregulated by decidual cells, the trophoblast cells themselvesand many diffusible factors within the placental bed.

Trophoblast invasion during normal pregnancy and pre-eclampsia

There are two populations of EVT: interstitial cytotropho-blast, which invades the decidual stroma and superficialmyometrium,1,3 and endovascular cytotrophoblast, whichinvades the lumen of the spiral arteries.4 This process resultsin transformation of the small muscular decidual and myo-metrial arteries into distended flaccid vessels. Myometrialarteries from women with pre-eclampsia often fail to show

evidence of physiological change.5,6 Decidual arteries also oftenshow abnormalities.7,8 The result of these defective vascularchanges is that blood flow into the intervillous space isimpaired in pre-eclampsia.

Cell adhesion molecules

Cell adhesion molecules (CAMs) expressed on the surface ofinvasive cytotrophoblast interact with the ECM in the deciduato control invasion.9,10 The adhesion of cells to other cells andto the ECM relies on the expression of CAMs and their ligands.Interstitial and intravascular invasion of maternal tissue bytrophoblast requires a ‘switching’ of cell adhesion molecule

Correspondence: Professor Fiona Lyall, Maternal and Fetal Medicine Section, Institute of Medical Genetics, University of Glasgow, Yorkhill, Glasgow G3 8SJ, UK. Email: [email protected]

DOI: 10.1111/j.1479-828X.2006.00589.xReceived 02 March 2006; accepted 11 March 2006.

Mechanisms underlying trophoblast invasion

© 2006 The Author 267Journal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists; 46: 266–273

expression. Pre-eclampsia has been linked to abnormalities inexpression of particular CAMs and/or their ligands.11 MostCAMs fall into one of four families: the immunoglobulinsuperfamily, the integrins, the selectins and the cadherins.11

The cadherins include E-, N-, P, R-, B- and VE-cadherins.The integrins bind to ECM proteins (e.g. fibronectin, laminin,collagen) and to members of the immunoglobulin familysuch as intercellular adhesion molecule (ICAM)-1, -2and -3, vascular cell adhesion molecule (VCAM-1) andplatelet endothelial CAM (PECAM-1), Selectins are CAMsthat bind to carbohydrates expressed on cells. The threemembers of this family, E, P and -selectin are involved inleucocyte extravasation.

Cytotrophoblast invasion and cell adhesion molecules

Cytotrophoblast invasion is accompanied by a reduction inproliferation as well as expression of specific proteinases.12,13

This is accompanied by altered expression of cell adhesionmolecule phenotypes and matrix-degrading enzymes.

In vivo studies suggest cytotrophoblast invasion is associated with switching of integrins and altered extracellular matrix expression

Much of the information on human trophoblast invasion hascome from studies on placental bed biopsies taken at term;these contain basal decidua and underlying myometrium withone or more uteroplacental (spiral) arteries. Other studieshave used first trimester implantation sites. Such studiessuggest that marked changes in the expression of adhesionmolecules and ECM components occur in parallel to thespatial distribution of cytotrophoblasts from the chorionicvilli through to the uterine wall. The exact timing of thesechanges is not clear.

In the placental villi, where cytotrophoblast exists as anepithelial monolayer anchored to the trophoblast basementmembrane, the laminin receptor α6/β4 is the major integrinexpressed by cytotrophoblasts.14–18 Some cytotrophoblasts weaklyexpress α3/β1. Within cell columns, where cytotrophoblastsare no longer associated with the basement membrane fibronectinand collagen IV and laminin are no longer found.17,19,20 Theα3 integrin is no longer detectable; however, α6/β4 remainsintensely expressed.17

In the distal regions of the columns, cellular fibronectin(A+B+) and collagen IV expression increases.17 Oncofetalfibronectin is also found21 Matching the increase in fibro-nectin is an increase in the α5/β1 fibronectin receptor oncytotrophoblasts22 while the laminin receptor α6/β4 typicallydecreases.14,17

Within the placental bed, cytotrophoblast expressesα1/β1 and α5/β1 integrins.17 Decidual cells express α1/β1 andα6/β1 integrins17 which interact primarily with matrix

associated with maternal cells (i.e. fibronectin A+B+, collagenIV and laminins). Collagen IV laminin, heparan sulphate,fibronectins and vitronectins are found.

In vitro studies also suggest that invasion is associated with switching of integrin repertoires

Studies performed on isolated first trimester placentacytotrophoblasts plated onto a basement membrane material,matrigel, have shown that the cells attach and then migratethrough the matrigel.18 Cytotrophoblasts within matrigelproduce ECM molecules characteristic of those produced bycells about to enter the uterine wall. The integrin pattern alsogenerally matches that seen in vivo.18 Culture studies haveshown that α1/β1–laminin/collagen interactions promoteinvasion, whereas α5/β1–fibronectin interactions restraininvasion.18,23 In vitro studies have also been performed usingvillous tissue cocultured with decidua parietalis.24 In thismodel contact with the decidua stimulates formation ofcolumns. The columns show similar alterations to those seenin vivo.

Invading cytotrophoblast cells express endothelial cell adhesion molecules

Cytotrophoblast cells that invade spiral arteries have beenreported to switch their adhesion molecule repertoire so asto mimic an endothelial phenotype.25–28 For example Zhouet al. (1997) reported that VCAM-1, PECAM-1 and E-selectin are not expressed on villous cytotrophoblasts butVCAM-1 and PECAM-1 are upregulated during invasionand are expressed on cytotrophoblast within the uterine wall,on endovascular cytotrophoblasts and on maternal endothe-lium. In contrast, three studies29–31 found that, while villousendothelial cells and endothelial cells of spiral arteries werePECAM positive, all cytotrophoblast cells in villous tissueand cytotrophoblast in the placental bed and associatedwith spiral arteries were PECAM negative. There are anumber of possibilities for these conflicting findings.29 Inearly pregnancy NCAM-positive EVT cells are abundantin all parts of the basal plate including intraluminal tro-phoblast.32 ICAM-1 is expressed on maternal endothelialcells, large granular lymphocytes, macrophages, interstitialtrophoblast around uteroplacental vessels and intravasculartrophoblast.15

Cytotrophoblast invasion, cell adhesion molecules and pre-eclampsia

Pre-eclampsia is associated with impaired trophoblast invasionand consequent failed spiral artery modification. Pre-eclampsiais reported to be associated with abnormal expression ofintegrins by invasive cytotrophoblasts. Zhou et al. (1993)

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268 © 2006 The AuthorJournal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists; 46: 266–273

reported that in pre-eclampsia, cytotrophoblasts in floatingvilli failed to down regulate α6β4 and up regulate α1β1 indistal columns and in the uterine wall.33 In pre-eclampsiacytotrophoblasts were also reported not to express the nor-mal repertoire of endothelial CAMs.25,34 Pre-eclampsia wasassociated with differences in all three αV family members,and fewer villous cytotrophoblast cells were positive for β5.Expression of αvβ6 increased and extended beyond the col-umns into the superficial deciduas and αvβ3 immunostainingwas absent. In pre-eclampsia, VCAM-1 was not expressed bydecidual cytotrophoblasts, and PECAM-1 expression wasnot present in column or decidual cytotrophoblasts. Again otherstudies differ in their findings. Divers et al. (1995) reportedno differences in integrin expression in the amniochorion andthe placental basal plate of women with pre-eclampsia andnormal pregnancies.31 Others failed to identify PECAM-1 oncytotrophoblasts in placental bed biopsies taken from normalor pre-eclampsia cases.28,30

Labarrere and Faulk (1995) reported that endovascularEVT of decidual vessels were ICAM-1 negative in normalpregnancies.35 However, in pre-eclampsia endovasculartrophoblast cells were ICAM-1 positive perhaps aresult of cytokines released from the surroundinginflammatory cells.

The nitric oxide synthase/nitric oxide pathway and trophoblast invasion

Human placental syncytiotrophoblast expresses endothelialnitric oxide synthase (eNOS) but not inducible nitric oxidesynthase (iNOS). eNOS is also expressed on villous endothe-lial cells and nitric oxide produced is an important vasodilatorwithin the placental vasculature.36 Pijnenborg et al. (1983)suggested that interstitial cytotrophoblast close to spiralarteries may produce vasoactive mediators such as nitricoxide. Nanaev et al. (1995) reported that guinea pig interstitialtrophoblast expressed eNOS and iNOS.3,37 This suggested,at least in the guinea pig, that local production of nitric oxideby EVT may be a mediator of spiral artery dilatation. Humanstudies have shown that that while syncytiotrophoblast wereeNOS positive, the cytotrophoblast cells of cell columns wereeNOS negative.38 Examination of placental bed specimensshowed positive eNOS staining on the spiral artery vesselendothelium but interstitial cytotrophoblast did not expresseNOS. None of the trophoblast cells within the vesselsexpressed eNOS. With regard to iNOS, no expression wasfound on any cytotrophoblast. These findings suggest thatcytotrophoblast-derived nitric oxide is not responsible fordilatation of the spiral arteries during human placentation.Finally, no differences in nitric oxide synthase expressionwere found on EVT in pre-eclampsia or fetal growth restric-tion. Direct comparison of the animal studies with results inhumans is difficult because human placentation differs fromthe guinea pig and cell columns do not exist in rodents.Furthermore, the nitric oxide synthase isoforms expressed inthe guinea pig differ from humans.

The carbon monoxide/hemeoxygenase pathway and trophoblast invasion

Carbon monoxide is produced by hemeoxygenase (HO), anenzyme that cleaves heme, to produce biliverdin and carbonmonoxide.39–41 Carbon monoxide, like nitric oxide, producesthe second messenger cyclic guanosine monophosphate(cGMP). The HO-1 isoform is inducible while HO-2 is con-stitutive. HO-2 is widely distributed throughout the body. Ithas been proposed that HO-2 and eNOS may have compli-mentary and coordinated physiological roles.42 Carbon mon-oxide acts as a neurotransmitter, inhibits platelet aggregationand is a vascular smooth muscle relaxant.39–42 The similaritiesbetween nitric oxide and carbon monoxide effects led toinvestigations that concluded that carbon monoxide maycontribute to spiral artery dilatation and placental blood flow.43

In the first trimester, HO-2 was primarily localised to the villoustrophoblast layer with little expression on villous endothelialcells. In contrast, trophoblast expression was reduced in thirdtrimester samples and endothelial expression was greater.Selective blockage of HO-2 in the isolated perfumed placentaincreased placental perfusion pressure. HO-2 on syncytiotro-phoblast may have similar roles to those proposed for eNOS.Nitric oxide produced by syncytiotrophoblast has at leastthree physiological targets: the intervillous space, autocrineeffects on trophoblast function and paracrine interactionswith villous core components. Nitric oxide inhibits plateletaggregation and leucocyte adhesion while modulating theimmune response. Similar effects by carbon monoxidewould benefit both the maternal blood flow through theintervillous space and the avoidance of immune recognitionof the fetoplacental allograft. The same workers also showedthat placental endothelial HO-2 expression was significantlyreduced in pre-eclampsia and fetal growth restriction whichcould contribute to reduced of blood flow.44 Within theplacental bed all EVT were positive for HO-2 in normalpregnancy and this did not alter in pre-eclampsia or fetalgrowth restriction. The expression of HO-2 by invasivecytotrophoblast opens the possibility that carbon monoxidecould be produced from these cells and could contribute tospiral artery dilatation.

Matrix metalloproteinases and their tissue inhibitors

Matrix metalloproteinases (MMPs) play a pivotol role ininvasion processes by degrading basement membranes andECM components.45 Matrix metalloproteinases are secretedinto the ECM as inactive proforms and must be cleaved tobe activated. There are four main groups of MMPs46: colla-genases (e.g. MMP-1, MMP-8, MMP-13), which degradefibrillar collagens I, II and III; gelatinases (e.g. MMP-2,MMP-9), which degrade denatured collagens (gelatin) andcollagen IV; stromelysins (e.g. MMP-3, MMP-7, MMP-10,MMP-11, MMP-12), which have a much wider range ofsubstrates including fibronectin, laminin, elastin, proteoglycansand collagens III, IV, V and IX; and membrane-type matrix



metalloelastases (e.g. MMP-14, MMP-15, MMP-16,MMP-17), which are located at cell surfaces. Their substratespecificities are unclear although they appear to activateprogelatinase-A. Natural inhibitors of MMPs can be dividedinto plasma (α-macroglobulins) and tissue inhibitors (TIMPs).46

There are four tissue inhibitors of matrix metalloproteinases(TIMPs): TIMP-1, which inhibits all activated MMPs andboth latent and activated MMP-9; TIMP-2, which blocksactivity of all active MMPs; and TIMPs-3 and 4, which areless well characterised.

Human implantation requires that trophoblast cellsbecome highly invasive but trophoblast invasion is tightlyregulated.47 The trophoblast cells must break through thematernal ECM and is thought to involve several MMPs andtissue inhibitors of MMPs. Normal invasion is achieved by abalance between secretion of MMPs by trophoblasts anddecidual cells and their inhibition by tissue inhibitors of MMPsproduced by the same cells. Abnormalities in these processescould lead to excessive invasion such as choriocarcinoma orrestricted invasion as in early pregnancy failure, pre-eclampsiaand fetal growth restriction.

Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in normal pregnancy

Studies on placental samples and cultured cells suggest thatMMP-1, MMP-2, MMP-3 MMP-9, MMP-11 and MMP-14 may all be involved in controlling trophoblast invasion.48–58

Cytokines such as interleukin-1β, leukaemia inhibitoryfactor and corticosteroids may regulate MMP release andinvasion.59,60

As for tissue inhibitors of MMPs, it appears that TIMP-3is highly expressed on invasive trophoblast cells and decidualcells.45,61–64 In vitro, both TIMP-1 and TIMP-2 completelyinhibit cytotrophoblast invasion.51 Overall the evidence abovesupports MMP-1, MMP-2, MMP-3, MMP-7, MMP-9,MMP-11 and TIMPs 1–3 involvement in normal trophoblastinvasion of decidua. There are inconsistencies in the litera-ture on decidual invasion, a lack of accurate gestational datesand an absence of data on myometrial invasion that requiresobtaining true placental bed samples.

Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in pre-eclampsia

There is much less known about pre-eclampsia. The beststudied is MMP-9. In vitro studies have shown that theinactive MMP-9 is the principle MMP secreted from cyto-trophoblast cultures prepared from cases of pre-eclampsiawhereas the active form is more abundant in uncomplicatedpregnancies.65 Purified cytotrophoblast cells from the casesof pre-eclampsia showed reduced invasive potential andfailed to modulate expression of MMP-9. In vivo data are

also limited but changes in MMP-7 and MMP-1 have beenreported in pre-eclampsia.

Transforming growth factor-ββββs and trophoblast invasion in normal pregnancy and pre-eclampsia

Transforming growth factor-βs (TGF-βs) are composedof three related proteins TGF-β1, 2 and 3. TGF-βs66 actthrough receptors designated types I, II and III. Transform-ing growth factor-βs, produced primarily by the decidua,may regulate trophoblast invasion.67 Caniggia and colleagues,in 1999, reported that placental villous tissue, expression ofTGF-β3 was low at 5–6 weeks’ gestation, peaking at 7–8weeks’ gestation, and undetectable by 9 weeks.68 In contrast,Simpson et al. (2002) examined the expression of TGF-β1,2 and 3 in placental bed biopsies and placentae from 7 to19 weeks’ gestation;69 a dramatic reduction in trophoblastTGF-β3 expression at the time of the first or secondwave of trophoblast invasion was not found and TGF-β1,TGF-β2 and TGF-β3 did not change between 7 and 19weeks of pregnancy.

Interpretation of early studies of TGF-βs in placenta isconfounded by the use of antibodies that are not specificfor individual isoforms.70–72 More recent studies, includingthe one by Simpson et al., used antibodies specific for eachisoform.69 Comparison of studies is further confounded bythe variability of techniques used for tissue preparation.Incorporation of techniques which measure bioactive formsis likely to yield more functionally relevant information.There is evidence that transforming growth factor-βs canregulate trophoblast invasion in vitro, although the data arenot consistent.68,73,74

With regard to pre-eclampsia, Caniggia et al. (1999)reported that TGF-β3 is overexpressed in the placenta andsuggested that this may be responsible for failed trophoblastinvasion. Others found no changes in expression of eitherisoform in placenta or placental bed in pre-eclampsia or fetalgrowth restriction compared with normal pregnancy.75 Thelatter data rule out a role for TGF-β3 in failed trophoblastinvasion in pre-eclampsia or fetal growth restriction.

Oxygen tension and trophoblast invasion in normal pregnancy and pre-eclampsia

Oxygen tension at the fetomaternal interface may mediatesome of the changes associated with cytotrophoblast proli-feration and invasion. There is no real blood flow into theintervillous space prior to 10 weeks’ gestation.76 Thereafter,oxygen pressure in the intervillous space increases.77

Pre-eclampsia and fetal growth restriction have beenreported to be associated with placental hypoxia, although thisis controversial.78 Oxygen concentration can alter expressionof many different proteins. In vitro, cytotrophoblasts fromfirst and second trimester placentae grown under hypoxic

F. Lyall


conditions (2% oxygen) fail to upregulate the integrin α1β1(just as in pre-eclampsia).79

First trimester villi that have been explanted onto matrigeland cultured in 20% oxygen form new invasive sites at thetips of the explants,12,13,80,81 while those cultured in 2% oxygenare maintained in a proliferative, non-invasive immaturestate.80,82–84 Cytotrophoblast cultured in low oxygen does notundergo the switch in integrin receptors normally found as theinvasive phenotype is acquired.80 Several studies have exam-ined other responses of trophoblasts to low oxygen includingincreased production of inflammatory cytokines, vascularendothelial growth factor, PAI-1 and syncytialisation.85–87

The effects of oxygen are mediated by transcriptionfactors such as hypoxia inducible factor 1α (HIF1α), anuclear protein that activates gene transcription in responseto low concentrations of oxygen.88 Caniggia et al. (2000) andRajakumar and Conrad (2000) have reported that TGFβ3expression is linked to HIF-1, although some differences infindings were reported between both groups.83,89 HIF-1α andHIF-2α proteins are both overexpressed in pre-eclampsia.90

The TGFβ3 gene has the potential to be directly regulatedby HIF.91 Janatpour et al., however, reported that expressionof HIF-1α was dramatically upregulated in standard (20%)oxygen conditions, whereas hypoxic (2% oxygen) conditionsdiminished expression of HIF-1α. Further studies arerequired.92 Recent data by Burton’s group suggest that anischemia-reperfusion-type phenomenon may be important inmediating trophoblast damage in pre-eclampsia.93

Human leucocyte antigen-G

Human leucocyte antigen (HLA)-G is a class 1B majorhistocompatibility antigen expressed by EVT and may pro-tect cells from natural killer cell lysis.94,95 Cytotrophoblastcells that invade the decidua express HLA-G. In culture,purified cytotrophoblasts upregulate HLA-G as they becomeinvasive.96 Expression of HLA-G protein by EVT is reportedto be reduced in pre-eclampsia.97–99 Thus, there may be a linkbetween HLA-G expression and failed trophoblast inpre-eclampsia.100 Lim et al. (1997) have shown that culturedvillous cytotrophoblasts obtained from normal pregnanciesupregulate HLA-G as they differentiate in vitro, whereascytotrophoblasts prepared from cases complicated by pre-eclampsia did not express HLA-G. Expression of HLA-Gmay also allow trophoblasts to evade cell damage byinterleukin-2.101 The combinations of maternal killerinhibitory receptors and fetal HLA-C genes influence therisk of pre-eclampsia and reproductive success.102

Apoptosis

Apoptosis is a physiological and pathological process thatregulates the number of cells in tissues.103 Little is knownabout apoptotic events in the placental bed of women withpre-eclampsia. One study showed that normal controlsamples showed almost no apoptosis but between 15 and

50% of cytotrophoblasts within the uterine wall of womenwith pre-eclampsia were positive.104 The same cells did notexpress Bcl-2, a survival factor normally expressed by thesecells. The authors suggested that programmed cell death inthese cells may account for some of the symptoms of pre-eclampsia. In contrast, Kadyrov et al. (2003) found, in theirstudy of placental bed biopsies, that the number of apoptoticEVT was reduced in pre-eclampsia compared with normalpregnancy.105

Conclusions

Trophoblast invasion is tightly controlled by a plethora offactors expressed within the decidua and on the trophoblaststhemselves. Abnormalities in any one of these mechanismshave the potential to lead impaired cytotrophoblast invasion.The precise mechanisms are still poorly understood andrequire further investigation. Understanding these patholog-ical mechanisms may help in strategies for the developmentof therapeutic targets.

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Mechanisms regulating cytotrophoblast invasion in normal pregnancy and pre-eclampsia

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