an integrative view on the physiology of human early placental villi

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Progress in Biophysics and Molecular Biology 114 (2014) 33e48

Contents lists avai

Progress in Biophysics and Molecular Biology

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Review

An integrative view on the physiology of human early placental villi

Berthold Huppertz a, Debabrata Ghosh b, Jayasree Sengupta c,*

a Institute of Cell Biology, Histology and Embryology, Medical University of Graz, AustriabDepartment of Physiology, All India Institute of Medical Sciences, New Delhi, IndiacDepartment of Physiology, North DMC Medical College, Hindu Rao Hospital, Delhi 110007, India

a r t i c l e i n f o

Article history:Available online 1 December 2013

Keywords:PlacentaTrophoblastEarly pregnancy lossPre-eclampsiaIUGRTranscriptomics

* Corresponding author.E-mail addresses: berthold.huppertz@medunigraz

0079-6107/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.pbiomolbio.2013.11.007

a b s t r a c t

Theplacenta isan indispensableorgan for intrauterineprotection,developmentandgrowthof theembryoandfetus. It provides tight contact between mother and conceptus, enabling the exchange of gas, nutrients andwaste products. The human placenta is discoidal in shape, and bears a hemo-monochorial interface aswell asvillous materno-fetal interdigitations. Since Peter Medawar’s astonishment to the paradoxical nature of themotherefetus relationship in 1953, substantial knowledge in the domain of placental physiology has beengathered. In the present essay, an attempt has been made to build an integrated understanding of morpho-logical dynamics, cell biology, and functional aspects of genomic and proteomic expression of human earlyplacental villous trophoblast cells followed by a commentary on the future directions of research in this field.

� 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342. Early placental morphological dynamism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343. Cell biological aspects of early placental villi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.1. Villous trophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.2. Syncytiotrophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.3. Villous cytotrophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353.4. Villous trophoblast turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353.5. Villous stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353.6. Hofbauer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363.7. Fetal vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

4. Molecular correlates of villous physiology in early placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365. Early placental apoptosis signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386. Early placental endocrine signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

6.1. Human chorionic gonadotropin (hCG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386.2. Growth hormone/chorionic somatomammotropin (GH/CS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396.3. Insulin like growth factors (IGFs) and their binding protein-1 (IGFBP-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406.4. Placental steroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

7. Early placental transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408. Early placental immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409. Molecular correlates of early placental disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

10. Early pregnancy loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4111. Pre-eclampsia and intrauterine growth restriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4212. Future direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

.at (B. Huppertz), [email protected], [email protected] (D. Ghosh), [email protected] (J. Sengupta).

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B. Huppertz et al. / Progress in Biophysics and Molecular Biology 114 (2014) 33e4834

The professional scientist will attack wherever he can. Thepoint of attack is always a matter of tactics.

all these trees, even at term the whole villous surface (12e15 m2) isstill covered by a single syncytiotrophoblast layer containing bil-

Crick (1966)

1. Introduction

Proper placental development and function are critical for thesurvival of the fetus. Despite the fact that all eutherian placentasshare conserved characteristics there are major differences inplacental shape (for example, diffuse, cotylendonary, zonary,discoidal, bidiscoidal), type of placental interface (for example,epitheliochorial, endotheliochorial, hemochorial), and nature ofmaterno-fetal interdigitations (for example, folded, lamellar,villous, trabecular, labyrinthine). The anatomical characteristicsof the human placenta include a discoidal shape, a hemo-monochorial interface and villous materno-fetal in-terdigitations (Bernirschke et al., 2006). The objective of thepresent review is to build an integrated understanding of firsttrimester villous trophoblast physiology. To this end, we shalldiscuss morphological dynamics, cell biology, and functionalaspects of genomic/proteomic expression in trophoblast cells ofearly placenta. Finally, we shall forward a commentary on dis-ease correlates for future direction of research in first trimesterplacental biology.

2. Early placental morphological dynamism

The trophectoderm of the blastocyst is responsible for thedevelopment of the human placenta (Huppertz, 2008a,b). In thefirst weeks after implantation, the trophoblast lineage gives rise totwo main types of trophoblast with a variety of subtypes: (i) villoustrophoblast: this type includes the syncytiotrophoblast that formsthe entire epithelial lining of the villous trees, and the villouscytotrophoblast cells that represent the main germinative popu-lation giving rise to the syncytiotrophoblast via syncytial fusion. (ii)Extravillous trophoblast: this type comprises cells derived fromtrophoblast cell columns of anchoring villi that are non-proliferating and invade into maternal tissues in the placentalbed of the uterine wall (Moser et al., 2010).

During the first week after implantation it is the early syncy-tiotrophoblast that is the embryonic tissue in direct contact tomaternal tissues. Within the syncytiotrophoblast fluid filledlacunae develop into a single large space that will become the spacefor maternal blood later in pregnancy (intervillous space). Thesurface of the syncytiotrophoblast facing this space enlarges andforms villous structures. The villi remaining in direct contact tomaternal tissues develop into anchoring villi fixing the placentaand the embryo to the uterine tissues. Using the anchoring villi assource, only in week five of pregnancy (post-menstruation) extra-villous trophoblast cells develop and come into first contact withmaternal tissues.

Villous development starts with syncytial sprouts outgrowingfrom core structures. These structures are built from the syncytio-trophoblast as outer layer followed by a complete layer of villouscytotrophoblast cells. The core of these structures, separated fromthe villous trophoblast by a basement membrane, is filled withmesenchymal cells derived from the early embryo. While furtheroutgrowing from the cores, the syncytial sprouts are now filled bycytotrophoblast cells first, then also by mesenchymal cells. Thisprocess leads to the development of a villous treewith a main villusemanating from the chorionic plate and several lines of villous sidebranches. Since the syncytiotrophoblast remains the single cover of

lions of nuclei in a continuous cytoplasm without any lateralmembranes (Bernirschke et al., 2006).

During the first trimester of pregnancy, only two out of five(mesenchymal, immature intermediate, mature intermediate, stemand terminal) villous types of the human placenta develop:mesenchymal villi and immature intermediate villi. Mesenchymalvilli start to develop around week five of pregnancy. They are themost primitive villi comprising a dense and cell-rich stroma withonly very small vessels that just start to develop and grow. Withfurther growing of the villous trees, this first villous type developsinto immature intermediate villi starting at around week eight ofpregnancy. This villous type shows a unique feature not present inany other villous type. The villous stroma of immature intermediatevilli contains fluid filled channels, called stroma channels that arenot covered by any endothelial or epithelial lining. These channelsstart and end within this villous type and hence there is noconnection to any other fluid filled structure in the villous trees. Thechannels are full of embryonic macrophages (Hofbauer cells) whilein all other villi macrophages can be found in the extracellularmatrix of the villous stroma. Beside stroma channels, the villouscore of immature intermediate villi contains small vessels that arenot necessarily localized in close vicinity to the villous trophoblast(Bernirschke et al., 2006).

Looking at the morphology of villous types in early preg-nancy, these villi are not built to enable optimal transfer of nu-trients and gases from the mother to the embryo. Rather, longdistances between the two circulatory units and only a plasmaflow on the maternal side do not provide an optimal basis forexchange. Flow of maternal blood cells into the intervillous spaceof the placenta only starts at the end of the first trimester andonly then the partial oxygen pressure rises from below20 mmHg in the first trimester to about 60 mmHg in the secondtrimester (Jauniaux et al., 2000). Villous development enablingoptimal nutrient and gas exchange only starts after midgestationwith the first appearance of terminal villi at around week 25e27of pregnancy.

3. Cell biological aspects of early placental villi

3.1. Villous trophoblast

Throughout pregnancy, the metabolic demand of the fetus in-creases with its rapid growth. The placenta and especially theplacental barrier need to adapt accordingly to meet the demands ofthe fetus. One of the main morphological changes of the villoustrophoblast is the decrease in the diffusion distance betweenmaternal and fetal blood. In the first trimester this distance rangesbetween 50 and 100 mm, while it is reduced to 4 and 5 mm at term(Bernirschke et al., 2006) (Fig. 1).

3.2. Syncytiotrophoblast

The syncytiotrophoblast is the outermost cover of all placentalvilli. It is a multinucleated layer without lateral cell borders, andhence is built of a single cytoplasm with an apical and a basalplasma membrane covering all villi of a given placenta. During thefirst trimester of pregnancy, the microvilli on the apical membraneare in contact to the maternal plasma flowing through the inter-villous space. Initial development, growth and maintenance of thesyncytiotrophoblast are achieved by continuous fusion with un-derlying cytotrophoblast cells, due to the absence of any prolifer-ative capacity of the syncytiotrophoblast. Also RNA synthesis(transcription) is reduced in the syncytiotrophoblast. Most of the

Fig. 1. First trimester placental villus displaying the typical features and components of villi at this stage of gestation. (A) Overview of a villous cross-section. (B) A mesenchymal cellof the villous stroma. (C) Hemangiogenic cells (erythroblasts containing nuclei and a white blood cell) in a developing blood vessel. (D) An early blood vessel that has alreadydeveloped a lumen surrounded by endothelial cells. (E) A placental macropaghe (Hofbauer cell) in the villous stroma. (F) The villous trophoblast layer with the outer multinucleatedsyncytiotrophoblast [S] and the inner layer of mononucleated cytotrophoblast cells [C].

B. Huppertz et al. / Progress in Biophysics and Molecular Biology 114 (2014) 33e48 35

nuclei are transcriptionally silent, while only the freshly incorpo-rated nuclei maintain their transcriptional activity for a while(Ellery et al., 2009; Huppertz et al., 1999).

3.3. Villous cytotrophoblast

Within the villous trophoblast the layer of mononucleated villouscytotrophoblast cells rests on a basement membrane and is locatedunderneath the multinucleated syncytiotrophoblast layer(Bernirschke et al., 2006). The cytotrophoblast cells are a heteroge-neous stem cell population. A subset of these cells preserve theirproliferative activity until delivery and proliferate throughout gesta-tion, the second subset displays a progenitor status since they can beinduced to differentiate into extravillous trophoblast, and the thirdsubsetof cells resides invariable levelsof differentiation, preparing forsyncytial fusiondirectedby factorssuchasGlialCellMissing-1 (GCM1)(Baczyk et al., 2004, 2009). Due to the continuous proliferation ofcytotrophoblast cells, their number increases froma fewat the timeofimplantation toabout1�109 at thebeginningof thesecondtrimester.

3.4. Villous trophoblast turnover

Different to an epithelium built of mononucleated cells, thevillous trophoblast exhibits a turnover that comprises the following

stages. Proliferation of cytotrophoblast cells is followed by differen-tiation of daughter cells that have left the cell cycle. Differentiation isfast and only takes 2e3 days, then syncytial fusion of highly differ-entiated cytotrophoblast cellswith the overlying syncytiotrophoblastoccurs. Within the syncytiotrophoblast further differentiation andmaturation takes place, leading to aging and late apoptosis of nucleiand other organelles at specific sites within the syncytiotrophoblast.This material is packed into protrusions of the apical membrane,called syncytial knots, and extruded as membrane-sealed apoptoticcorpuscles (syncytial knots) into maternal blood flowing in theintervillous space (Huppertz and Kingdom, 2004; Huppertz et al.,2006a,b; Huppertz, 2010). Due to the massive growth of thetrophoblast surface during thefirst trimester, the number of syncytialknots during this stage of pregnancy is quite low, while it dramati-cally increases at the end of pregnancy.

3.5. Villous stroma

Within the stroma of the villous core fixed and moving con-nective tissue cells are present, including mesenchymal cells, fi-broblasts and higher differentiated cells such as myofibroblasts,placental macrophages (Hofbauer cells), and placental vessels withtheir endothelial and surrounding cells (pericytes, smooth musclecells).

Table 1Morphological basis of major functional correlates in human first trimester placentalvillous cytotrophoblast (CTB) and syncytotrophoblast (STB).

Function Relevant physiological information(reference)

Cell proliferation CTB pool with mitotic index ofapproximately 1.5e3.0% (Kar et al., 2007)

Asymmetric divisionto maintain pool of CTB

Mitotic spindle forms in the plane of villousbasement membrane (Kar et al., 2007)

Bipotential behavior of CTB,development of STB andextravillous trophoblast(EVT)

In vitro model of CTB culture on matrigelshowing STB regeneration and EVToutgrowth indicative of two different sub-populations of CTB (James et al., 2005)

Two-stage model of CTBcell fusion

Pre-fusion complex established betweentransition stage CTB followed by fusioncomplex bearing punctuate areas ofmembrane breakdown with involvementof lysosomes (Kar et al., 2007)

Molecular markers of fusion Expression of hCG-a, caspases 8 and 10;transcription factor glial cell missing-1(GCM1) in CTB (Huppertz et al., 1999; Blacket al., 2004; Baczyk et al., 2004)

CTB differentiation forsyncytial fusion

Intermediate or transitional CTB cellsdifferentiated in preparation. Syncytin 1expression in intermediate trophoblast inadvance of fusion and regulated by GCM1(Lin et al., 2005; Kar et al., 2007; Gausteret al., 2009)

Fusion proteins in CTBand STB

Syncytin 2 expression only in CTB cells.Human endogenous retroviruses (HERVs) inCTB and STB. Syncytin 1 in CTB and STB.Cadherins, tight junction proteins, integrinsre-located in apical syncytial microvillousmembrane (Huppertz et al., 2006a,b; Kudakaet al., 2008; Aplin et al., 2009)

Apoptosis in CTB and STB Ultrastructural characteristics of apoptosiswith expression of Bcl-2 and TUNEL highestat 5e6 weeks, decreased at 7e9 weeks.Multiparametric analysis of proteins linkedwith apoptosis: cytokeratin 18f, Fas, TNFR1,TUNEL in CTB (approximately 5e8%) and inSTB (approximately 9e20%) withultrastructural characteristics of apoptosisat 6e8 weeks (Huppertz et al., 1999;Ishihara et al., 2000; Burton et al., 2003;Kar et al., 2007)

Apoptosis machinery andCTB fusion

Transient activation of caspase 8 for a-foldrin fragmentation and remodeling ofsub-membraneous cytoskeleton in pre-fusion CTB (Gauster et al., 2009)

Barrier to pathogens, andmaternal cells, transport,synthesis of glycoprotein,peptide and steroidhormones, cytokines,growth factors

STB layer (Desforges and Sibley, 2010;Carter, 2012)


3.6. Hofbauer cells

Hofbauer cells exhibit a very typical morphology because theircytoplasm encloses a large number of vacuoles and lysosomes, andtheir surface displays a complex arrangement of lamellipodia andmicroplicae. During the first four weeks of pregnancy, the placentalmesenchyme seems to be the source to generate placental macro-phages (Demir et al., 2004), while later in pregnancy with the onsetof the feto-placental blood flow this is amplified by macrophagesderived from fetal bone marrow (Bernirschke et al., 2006), and bymitotic division of Hofbauer cells (Castellucci et al., 1987). Hofbauercells exhibit a variety of different functions such as the regulation ofvillous trophoblast differentiation, regulation of stromal cellgrowth, regulation and modulation of placental angiogenesis andvasculogenesis, and absorption of immune complexes coming fromthe mother (Bernirschke et al., 2006).

3.7. Fetal vessels

A non-fenestrated endothelium lines the placental vasculaturethroughout pregnancy. Neighboring endothelial cells are linked byjunctional complexes, which serve two functions: stabilization ofthe endothelium, and reduction of the permeability between cells(the paracellular route), which is especially effective for anionicmolecules. Larger molecules, including immunoglobulins such asIgG, need to be transported across endothelial cells by vesiculartransport (Leach et al., 1989).

During the first trimester of pregnancy, capillaries are not sur-rounded by a basement membrane (Demir et al., 1989). Pericytesare associated with placental capillaries especially during earlygestation and involved in angiogenesis (Demir et al., 1989; Zhanget al., 2002). In immature intermediate villi, arteries and arteri-oles are found with a coat of smooth muscle cells, but withoutelastic laminae. Due to the absence of neural innervations in theplacenta, flow within placental vessels needs to be regulated byautocrine and paracrine factors produced in close vicinity of thefetal blood flow within placental vessels (Myatt, 1992).

4. Molecular correlates of villous physiology in early placenta

Early placental villi display a complex pattern of homeody-namics including proliferation, differentiation, apoptosis and in-flammatory responses (see Table 1 for details). Characteristicchanges in the proportion and organization of different cell types inplacental villi include several pools of cytotrophoblast andmesenchymal cells that are transcriptionally active with the syn-cytiotrophoblast nuclei being transcriptionally active only shortlyafter syncytial integration (Apps et al., 2011; Demir et al., 2007;Ellery et al., 2009; Huppertz et al., 1999).

Dizon-Townson et al. (2000) reported expression of a largenumber (w12K) of genes in normal first trimester placental villi. Arecent study reported that w9K genes with known functionsexhibit marked expression while displaying a defined temporo-spatial profile during 6 to 8 weeks of gestation in normal firsttrimester placental villi (Khan et al., 2013). Chen et al. (2002)examined the differential gene profiles of 9600 clones of knownregulatory genes and expressed sequence tags (ESTs) in firsttrimester placental villi and in decidua collected at 6e8 weeksgestation. Mikheev et al. (2008) compared global gene profiles inplacental villous samples obtained in normal first trimester (6.4e8.4 weeks) and second trimester (15.5e16.4 weeks), and in termplacentae. Transcriptional changes associated with in vitro transi-tion of EGF-R (þ) villous to HLA-G (þ) extravillous trophoblast cellshave been examined employing global transcription profiling ofisolated trophoblast cells collected from 8 to 12 weeks normal

placentae (Apps et al., 2011). Founds et al. (2009) provided aninteresting first report that compared global profiling of genesobtained from chorionic villus samples (CVS) collected at 10e12weeks of gestation from normal pregnancies and those from CVSsamples of women developing pre-eclampsia later in pregnancy.Table 2 provides a summary of these above findings.

Comparative analysis of global transcript profiling of genesexpressed in placental villous samples between first trimester (6e8weeks) and term conducted byMikheev et al. (2008) identified 495genes that were over-expressed (>2.0 to 333.7 fold) and 844 genesunder-expressed (<2.01 to �188.1 fold) in placental villi of firsttrimester compared with term (Mikheev et al., 2008). Functionalgene categorization revealed that early in gestation genes related tocarbohydratemetabolism, amino acids, DNA and cell cycle are over-expressed, while genes related to signal transduction are under-expressed. Khan et al. (2013) performed a critical analysis of

Table 2Summary of studies examining genomics of first trimester villous placenta

Reference Study design Observations

Dizon-Townsonet al. (2000)

Synthesis of cDNA from firsttrimester chorionic villus, 6e14 weeks, creation ofcDNA library for placentalgenes; Northern blot andISH

Around 12K genes expressedbelonging to structural andmatrix protein, endocrineproteins, enzymatic proteins &transporter proteins.

Chenet al. (2002)

High-density cDNAmicroarray withcolorimetry detectionsystem for 9600 genes; IHCfor 3 proteins. Comparisonof placental chorionic villi(CV) and decidua (D) at 6e8weeks gestation.

Seventy-five genes upregulatedin CV compared to D andcategorized under cell growthand cell cycle regulators (11),apoptotic related factors (2),hormones or cytokines (20),stress response proteins (3),modulators, effectorsintracellular signal transducers(7), cell surface antigens/celladhesion proteins (6), metabolicenzymes (6), transcriptionfactors and general DNA bindingproteins (4), cytoskeleton or ECM(6). PCNA and LIF-R proteinshighly expressed in CTB and STB,and cathepsin L in D cells.

Soleymanlouet al. (2006)

First trimester, 5e6 weeksplacenta, 12e13 weeks andterm placentae from pre-eclampsia (PE) and fromhigh altitude and moderatealtitude to study geneexpression using highthroughput functionalgenomics and qPCR inchorionic villi and inchorionic villi explantcultured with 3 and 20%oxygen in vitro organculture model

Differential expression of genesin three hypoxic conditions:in vivo and in vitro hypoxiaconditions and in pathologicalhypoxic condition of PE (term).Seven (7) HIF related genesupregulated, 12 genescommonly expressed in hypoxiccondition are altered. Aberrantglobal placental geneexpressions in PE may be due toreduced oxygenation.

Mikheevet al. (2008)

Affymetrix platform tocompare the globalgenomic expressionprofiles in placentalchorionic villus cells of 1st(45e49 days), 2nd (109e115 days) trimester andterm placentae. WB and IHCof b-catenin.

Genes related to cell cycle, DNA,amino acids and carbohydratemetabolism were significantlyoverrepresented, genes relatedto signal transduction wereunderrepresented in 1sttrimester with activation of theWnt pathway. b-cateninexpression in 1st and 2ndtrimesters and immunolocalizedin CTB and STB.

Foundset al. (2009)

Affymetrix platform tocompare global geneexpression in chorionicvillus (CVS) samples 10e12weeks from womeneventuating to PEcompared to age-matchedCVS of normal termpregnancy. RT-PCR for 3genes: LAIR2, CCK, CTAG2.

Downregulation of CFH, FN1, F11receptor, LAIR2, LRAP, MMP12,MUC15, PAEP, in PE, andassociated with immuneresponse/adhesion/complementactivation, antigen processingand blood pressure regulation,proteolysis; upregulation of:holecystokinin, S100 calciumbinding protein A8 and cancer/testis antigen 2 in PE chorionicvillus samples compared withage-matched normal. Noalterations in hypoxia andoxidative stress related genes.

Khanet al.(2010a,b)

Custom-tailored 400 genearray (Clontech) to comparegene expressions between6, 7 and 8 weeks choronicvilli. qPCR for 6 genes:COL4A4, CXCR4, ERBB2,HDAC1, HPRT1, TNFRSF1A

Baysenian prediction showedhigh degree (0.8e1.0) confidencemeasures in 15 out of 18placentae based on geneexpression profile. Seventygenes (w18%) upregulated, 53genesw14%) downregulatedbetween 6 and 8 weeks villi andinvolved cell growth andproliferation, anti-apoptosis,angiogenesis, immune and

Table 2 (continued )

Reference Study design Observations

inflammatory responses,extracellular matrix remodeling,multicellular organismaldevelopment. Oxytocin receptor,retinol binding protein-1upregulated in 6,7 and weekssamples; tenascin C and TNFR1upregulated in 6 weeks villi anddonwregulated in 7 and 8 weekssamples. Relative expression ofCXCR4 and TNFRSF1A related toinflammation and immunefunctioning was downregulatedand ERBB2 and HDAC1 related tocell proliferation wasupregulated with increase ingestational age.

Farinaet al. (2011)

Affymetrix platform tostudy global geneexpression of chorionicvillus samples (CVS) fromwomen eventuating to PEand CVS from women withnormal term pregnancy.RT-PCR for 10 genes: ADD1,BTD, CLDN6, F2, F8, HLA-DRB4, LTF, MAS1, TTN,VASH1

One hundred and eighteen genesshowed differential regulation.Downregulation of 10 genesassociated with immunefunction, inflammatory stress,complement and coagulationcascade, cell and focal adhesion,calcium signaling, actincytoskeleton, blood pressurecontrol.

Appset al. (2011)

Illumina platform to studyglobal gene expression inEGF-Rþ villus trophoblast(VT) and HLA-Gþ

extravillous trophoblast(EVT) cells isolated fromin vitro culture of placentalvilli at 8e12 weeksgestation. Flow cytometryand IHC for macrophagesand NK cells.

Transition of VT to EVT has beenexamined based on geneexpression profiles. Around 3000transcripts differentiallyregulated; STAT4 may functionas ‘master regulator’ oftranscription factors upregulatedin EVT. EVT secretes LAIR-2 thatcan regulate the functions ofLAIR-1 in decidual cells.

Khanet al. (2013)

NimbelGen platform tostudy global genomicexpression in chorionic villiof 6e8 weeks placentae.WB and IHC for CAGE-1,CD9, SLC6A2, TANK, VEGF

Around 9K genes expressed ofwhichw1Kwere over-expressedand w1K under-expressed andclustered in three clusters basedon gestation age with four co-expressional clusters of genes;167 genes showed differentialregulation and specific processesidentified from post hoc analysisassociated with: (i) immunetolerance with anti-viralimmunity, (ii) morphogenesis[involving regulation of (a)apoptosis, (b) autophagy, (c) cellcycle progression, and (d)epithelial-to-mesenchymaltransition], (iii) synthesis ofplacental proteins and steroidhormones, (iv) transporteractivity related to nutrients, ions,hormones and xenobiotics, and(v) specific neurotransmittersand olfactory receptors.


global gene expression in timed first trimester placental villoussamples at 6, 7 and 8 weeks gestation. The temporal pattern of co-expressed genes revealed a steep rise in their expression profile invillous samples between 7 and 8 weeks gestation including genesin the following categories: ‘cell adhesion molecules’, ‘Ephrin re-ceptor signalling’,‘Insulin signaling ‘, ‘Notch signaling’, ‘PPARsignaling’, ‘apoptosis and survival TNFR1signalling’ and ‘cell cycleand p53 signaling’ (Table 3). Biological significance of these genenetworks in developmental programming of normal placentationand in cases of early pregnancy loss, intrauterine growth restriction

Table 3Enriched categories of co-expressed genes in 6e8 weeks placental villi.

Co-expressedcluster number

Enriched category (gene names)

Cluster 1 Caspase activation (caspase recruitment domain familymember 12, member 14, Caspase 9, Caspase 14, Tumorprotein p53)Cell Cycle Control: G2/M DNA damage (breast cancer 1, Tumorprotein p53)Cholesterol metabolism and biosynthesis (NADPH oxidase 1,SEC14-like 2)VEGF signalling and activation (breast cancer 1, early onset andv-rel reticuloendotheliosis viral oncogene homolog A_nuclearfactor of kappa light polypeptide gene enhancer in B-cells3_p65)PDGF signalling via STATs (platelet-derived growth factorreceptor_alpha polypeptide, Platelet-derived growth factorreceptor_beta polypeptide, v-rel reticuloendotheliosis viraloncogene homolog A_nuclear factor of kappa lightpolypeptide gene enhancer in B-cells 3_p65)

Cluster 2 Cell adhesion molecules (Claudin 19; Dystroglycan 1, Integrinbeta 1, Integrin beta 3 binding protein, Syndecan 3)Ephrin receptor signalling (Ephrin-B2, G-protein signallingmodulator 1, regulator of G-protein signalling 4, sorbin andSH3 domain-containing protein 1)Insulin signalling (MAP kinase-interacting serine/threoninekinase 1, peroxisome proliferative activated, receptorgamma_coactivator 1_alpha, Sorbin and SH3 domain-containing protein 1)Notch signalling (gamma-secretase subunit APH-1B,Recombining binding protein suppressor of hairless-like,Transducin-like enhancer of split 4 E (sp1) homolog)PPAR signalling (conserved oligomeric Golgi complex subunit6, Sorbin and SH3 domain-containing protein 1

Cluster 3 Apoptosis and survival TNFR1signalling (A-kinase anchoringprotein, endoplasmic reticulum aminopeptidase associatedwith antigen processing, apoptosis inhibitor 4, phosphofurinacidic cluster sorting protein 1-like, Tumor protein p53regulated apoptosis inducing protein 1)Cell cycle and p53 signalling (ataxia telangiectasia and Rad3-related protein, chaperonin containing TCP1 subunit 6A zeta1, apoptosis inhibitor 4, PAR3-L protein, RPN10 homolog, p53-inducible ribonucleotide reductase small subunit 2 homolog,thymidylate synthetase)G-protein and alpha-i signalling cascades (A-kinase anchorprotein 13, Regulator of G-protein signaling 11)

Cluster 4 Chemokine signalling (CCL11, CCL14)Cysteine and methionine metabolism (methionineadenosyltransferase 2 subunit beta)Cytokineecytokine receptor interaction (CCL14, Frizzledhomologue 4)Tight junction (membrane-associated guanylate kinaseinverted 2)

Data are available at http://www.ncbi.nlm.nih.gov/geo (accession numberGSE37653).


(IUGR) and pre-eclampsia, in which aberrant placentation has beensuggested, remains to be defined. In the following sections, we shallhighlight a few specific aspects of placental transcriptomics:morphogenesis of placental villi, their endocrine correlates,placental transporters, and placental immunity at the feto-maternal interface (also see Table 4).

5. Early placental apoptosis signaling

The balance of the process dynamics between apoptosis andmacroautophagy isahallmarkofdevelopmental regulation, and thesetwoprocesses share commonpathways at themolecular level (Levineand Yuan, 2005; Lockshin and Zakeri, 2001; Maiuri et al., 2007). Thebalance of these processes is the key to normal development of thevillous tree, and any high dimensional error in the balance may pre-dispose villous growth to anomaly and/or insufficiency (Sharp et al.,2010). Regulation of apoptosis processes is not only important for

programmed morphogenesis, but also suggestively involved in syn-cytium formation and rendering immune tolerance. Hence, it is ofsignificant relevance to the biology of early placental villi in general(Huppertz et al., 2001; Jerzak and Bischof, 2002).

High expression levels of genes involved in apoptosis inhibition(ARRB1, CHRNB1, FYN, MTOR/FRAP1) and DNA-damage inducedapoptosis (ATR, BLM, CHRNA7) are observed in first trimesterplacental villi (Khan et al., 2013). The mTOR/FRAP-1 pathwaysinclude several machineries involved in cell growth, inhibition ofapoptosis, cell migration and invasion (Fingar et al., 2004; Zhou andHuang, 2011) and are integral to the developmental process ofplacentation and embryogenesis (Knofler, 2010; Roos et al., 2009;Wen et al., 2005). mTOR/Frap1 null mice show failed pregnancyimmediately after implantation with impaired embryogenesis andtrophoblast growth (Murakami et al., 2004). A few members of Gli-similar (GLIS) including Kruppel-like (KLF) zinc finger proteins aredownregulated in normal first trimester placental villi. Glis proteinshave been shown to interact with beta-catenin and thereby serve asa negative modulator of the canonical Wnt pathway (Kim et al.,2007). However, it remains to be explored whether the Wnt-beta-catenin signaling pathway is indirectly promoted by downregulation of Glis and whether the same module interacts withhedgehog-Glis signaling pathways in early placental villi (Gill andRosenblum, 2006; Van den Brink et al., 2004).

Transcriptionally, the hedgehog-signaling pathway in 6e8 weeksplacental villi has been identified to be one of the top-scored pro-cesses (Khan et al., 2013). Placental glial cell missing-1 (GCM1) mayprovide a transcriptional basis of placental morphogenesis (Baczyket al., 2009). Indeed, the reported high expression of GCM1 andMMP2 (matrix metalloproteinase-2) along with low expression ofTIMP4 (tissue inhibitor ofmatrixmetalloproteinase 4) in normal earlyplacental villi (Khan et al., 2013) lends strong support to this notion(Drewlo et al., 2011). GCM1 is one of the three dozens of zinc fingerproteins which we have observed to be highly expressed in earlyplacental villi (http://www.ncbi.nlm.nih.gov/geo/GSE37837). Gener-ically, zinc finger proteins are among the most abundant proteins inthe eukaryotic genome having diverse functions that include DNA/RNA recognition, RNA packaging, transcriptional activation, regula-tion of apoptosis, protein folding and assembly, and thereby aregenerally associated with developmental processes and embryogen-esis (Bilic andEllmeier, 2007;Hollemannet al.,1996;Laityet al., 2001).

6. Early placental endocrine signaling

The placenta is the site of synthesis of a large number of peptideand steroid hormones, growth factors and their receptors. Abundantcopies of transcripts for several placental protein hormones areexpressed in human early placental villi (Table 4). Furthermore, thetranscriptomic evidence in early placental villi also supports thenotion that regulation of placental protein hormones involvesdifferent signaling systems: peroxisome proliferators-activated re-ceptor gammaalongwith its coactivator1 alpha (PPARG, PPARGCIA),the activin signaling system and follistatin primarily for hCG, neu-ropeptide Y for chorionic corticotrophin (hCC), and dynorphin(PDYN) and somatostatin (SST) for chorionic somatomammotropin(hCS) (Handschuh et al., 2007; Parry and Strauss, 2005). In thefollowing sections, we shall give a stock of information for chorionicgonadotropin (CG), growth hormone/chorionic somatomammo-tropin (GH/CS), insulin like growth factors (IGFs) and their bindingprotein-1 (IGFBP-1) and placental steroid hormones.

6.1. Human chorionic gonadotropin (hCG)

Maintenance of pregnancy is directly dependent upon placentalchorionic gonadotropin (CG), which is required to maintain corpus

http://www.ncbi.nlm.nih.gov/geo/GSE37837

http://www.ncbi.nlm.nih.gov/geo

Table 4Selected groups of expressed genes in first trimester placental villi

Subcategory Specific entity (Gene symbol)

Functional categoryHormones and associated factorsHormones Activin A (INHBA)

Chorionic gonadotropin alpha polypeptide (CGA)Chorionic gonadotropin beta polypeptide 5 (CGB5)Chorionic gonadotropin beta polypeptide 8 (CGB8)Chorionic somatomammotropin homone 2 (CSH2)Chorionic somatomammotropin homone-like 1(CSHL1)Chromogranin B (secretogranin 1) (CHGB)Follistatin (FST)Pregnancy specific beta-1 glycoprotein 6 (PSG6)Relaxin (RLN1)Somatostatin (SST)

Enzymes Catechol-O-methyl-ransferase (COMT)Hydroxysteroid (11 beta) dehydrogenase (HSD1B1)Propanoyl-CoA-C-acyl transferase, SCP 2 (SP2)Sulfatase (SULF2)

Receptors Progestin and adipoQ receptor family member VIIreceptor (PAQR8)Neuropeptide Y receptor 6 (NPY6R)Retinoid X receptor (RXRG)Somatostatin receptor 5 (SSTR5)Thyroid hormone receptor associated protein 2(TRHAP2)Thyroid hormone receptor interactor 6 (TRIP6)

Neuro-transmitter receptorAdrenergic receptor Alpha 1D (ADRA1D)Cannabinoid receptor Type 1, brain type (CNR1)Cholinergic receptor Nicotinic alpha 7 (CHRNA7)

Nictonic beta 1, muscle type (CHRNB1)Dopamine receptor Type D5 (DRD5)Gamma aminobutyric

acid (GABA) receptorA receptor beta 2 (GABRB2)B receptor 1 (GABBR1)

Glutamate receptor Ionotropic AMPA 1 (GRIA1)Ionotropic AMPA 2 (GRIA2)Ionotropic N methyl D aspartate 1 (GRIN1)Ionotropic N methyl D aspartate 2B (GRIN2B)Ionotropic kainate 1 (GRIK1)

Glycine receptor Alpha 1 (GLRA1)5-Hydroxytryptamine

(serotonin) receptorType 1B (HTR1B)Type 3, family member E (HTR3E)Type 6 (HTR6)

Opioid receptor Type mu 1 (OPRM1)

Olfactory receptorFamily 1 Subfamily A member 1(OR1A1)

Subfamily M member 1 (OR1M1)Family 2 Subfamily L member 8 (OR2L8)

Subfamily W member 5 (OR2W5)Family 3 Subfamily A member 1 (OR3A1)Family 4 Subfamily A member 47 (OR4A47)

Subfamily D member 1 (OR4D1)Subfamily K member 1 (OR4K1)Subfamily Q member 3 (OR4Q3)Subfamily X member 1 (OR4X1)

Family 5 Subfamily B member 17 (OR5B17)Family 6 Subfamily A member 2 (OR6A2)Family 8 Subfamily K member 1 (OR8K1)Family 10 Subfamily S member 1 (OR10S1)Family 11 Subfamily G member 2 (OR11G2)Family 52 Subfamily D member 1 (OR52D1)

Membrane-bound transportersAquaporin Type 8 (AQP8)

Type 10 (AQP10)ATP-binding cassette

(ABC)Sub-family A member 5 (ABCA5)Sub-family A member 13 (ABCA13)Sub-family G member 1 (ABCG1)Sub-family G member 2 (ABCG2)

Ion channels Amiloride-sensitive cation channel 4, Pituitary(ACCN4)ATPase Caþþ transporting, type 2C member 1(ATP2C1)

Table 4 (continued )

Subcategory Specific entity (Gene symbol)

ATPase Naþ/Kþ transporting, beta 1 polypeptide(ATP1B1)Calcium channel oltage-dependent P/Q type, alpha 1Asubunit (CACNA1A)Calcium channel voltage-dependent alpha 1G subunit(CACNA1G)Chloride channel CLIC-like (CLCC1)Potassium voltage gated channel shaker relatedsubfamily beta member 1 (KCNAB1)Potassium voltage gated channel shaker relatedsubfamily member 7 (KCNA7)Potaasium voltage gated channel Subfamily G,member 3 (KCNG3)Potassium voltagegated channel subfamily Hmember5 (KCNH5)Potassium inwardly rectifying channel subfamily J,member 12 (KCNJ12)Potassium channel tetramerisation domain-containing 9 (KCTD9)Transient receptor cation channel subfamily C,member 5 (TRPC5)Transient receptor cation channel subfamily V,member 2 (TRPV2)

Solute carrier family(SLC)

SLC 5 (sodium/glucose co transporter) member 9(SLC5A9)SLC 6 (neurotransporter, serotonin) member 4(SLC6A4)SLC 12 (potassium-chloride transporter) member 5(SLC12A5)SLC 16 (monocarboxylic acidtransporters) member 7(SLC16A7)SLC 17 (sodium phosphate) member 2 (SLC17A2)SLC 22 (organic cation transporter) member 13(SLC22A13)SLC 22 (organic cation transporter) member 15(SLC22A15)SLC 23 (nucleobase transporter) member 2 (SLC23A2)SLC 24 (sodium/potassium/calcium exchanger)member 1 (SLC24A1)SLC 24 (sodium/potassium/calcium exchanger)member 6 (SLC24A6)SLC 25 member 34 (SLC25A34)SLC 25 member 35 (SLC25A35)SLC 26 member 6 (SLC26A6)SLC 26 member 35 (SLC26A35)SLC 27 (fatty acid transporter) member 1 (SLC27A1)SLC 29 (nucleoside transporters) member 1 (SLC29A1)SLC 35 member B3 (SLC35B3)SLC 35 member E3 (SLC35E3)SLC 36 (proton/amino acid symporter) member 2(SLC36A2)SLC organic anion transporter family member 2A1(SLCO2A1)SLC2A4 regulator (SLC2A4RG)

Data are available at http://www.ncbi.nlm.nih.gov/geo (accession numberGSE37653).


luteum function and for the synthesis of progesterone and othergestagens. CG holomolecules include CGA (hCG-a), and CGB5 andCGB8 (hCGb). Similar to earlier reports (Cocquebert et al., 2012; Rulland Laan, 2005), we also observed that out of four (CGB, CGB5,CGB7 and CGB8) coding genes for hCGb, CG5 and CG8 primarily(w80%) contribute to the total hCGb transcript pool in the placenta(http://www.ncbi.nlm.nih.gov/geo/GSE37837).

6.2. Growth hormone/chorionic somatomammotropin (GH/CS)

Of the GH/CS locus on human chromosome 17q22e24 con-taining five homologous genes, the placenta expresses four paral-ogs: GH2, CSH1, CSH2 and CSHL1. GH2 codes for placental GH(PGH), while CSH1 and CSH2 encode for placental lactogen andCSHL1 is a pseudogene (Hirt et al., 1987). GH2 expression is overtly

http://www.ncbi.nlm.nih.gov/geo/GSE37837

http://www.ncbi.nlm.nih.gov/geo


under-expressed that matches with the reported profiles of PGH inthe maternal circulation (Parry and Strauss, 2005). Also, both CSHgenes (CSH1 and CSH2) contribute to the transcript pool ofplacental lactogen, with a higher expression of CSH2 compared toCSH1 in first trimester villi. In the term placenta, however, therelative expression ratio of CSH1 to CSH2 was observed to be in theopposite direction (Chen et al., 1989; MacLeod et al., 1992; Manniket al., 2010). Our own observation that the relative abundance oftranscripts for CSHL1 was about 100% higher than that of CSH2substantiates the earlier report that CSHL1 is not a pseudogene inthe human placenta (Hu et al., 1999).

6.3. Insulin like growth factors (IGFs) and their binding protein-1(IGFBP-1)

These protein factors act in endocrine-paracrine manner topromote growth, proliferation, differentiation and migration oftrophoblast cells and decidual cells during implantation andplacentation in primates (Dhara et al., 2001; Fowden, 2003; Ghoshet al., 2011; Hill et al., 1993). Placental growth in mice is reducedafter deletion of IGFII but not in the absence of IGFI (Liu et al., 1993).Placenta-specific deletion of the paternally imprinted IGFII gene inmice provides evidence that this gene controls both the placentalsupply of, and the genetic demand for, maternal nutrients to themammalian fetus (Constância et al., 2002). The function of IGFII ismediated via its binding to IGF-IR, while high affinity binding ofIGFII to its receptor IGFIIR (M6P/IGFIIR) promotes the clearance ofIGFII. In mice, null mutation of IGF-IR results in slow prenatalgrowth (Liu et al., 1993), while null mutation of M6P/IGFIIR resultsin increased birth weight (Lau et al., 1994). It is noteworthy that thereciprocal imprinting of the IGFII and IGFIIR genes (paternallyimprinted IGFII and maternally imprinted IGFIIR) provide anexcellent example of the kinship theory of genomic imprinting thatsupports the parenteoffspring conflict hypothesis with the motherretaining overall control of resource allocation by sequestering thegrowth factor for the fitness of the offspring or the developing fetus(Wilkins and Haig, 2002).

6.4. Placental steroid hormones

The general paradigm that placental secretion of progesteronebegins at 6e8 weeks of gestation and the placental steroidogenesisoccurs without the active involvement of SF-1 (steroidogenicfactor-1) and STAR (steroidogenic acute regulatory protein) (Parryand Strauss, 2005; Petraglia et al., 2005) is substantiated by ourreport of very high expression levels of HSD1B1 (11b-HSD1) andSCP2 (sterol carrier protein) along with very low to undetectablelevels of transcripts of SF1, STAR and CYP17 (P450c17) (Khan et al.,2013). Also, the absence of CYP17 and the presence of high effi-ciency HSD1B1 allows for progesterone synthesis at a higher effi-ciency in placenta than gonads (Parry and Strauss, 2005; Petragliaet al., 2005). On the other hand, high expression levels ofCYP19A1 (aromatase) and SULF2 (sulfatase) in early placental villi(Khan et al., 2013) may yield processes for synthesis of estrogensfrom fetal DHEA sulfate (Parry and Strauss, 2005; Petraglia et al.,2005). It is plausible that relatively high transcript levels forGCMs (Glial cell missing-1 and 2) might be involved in regulatingaromatase activity in early placental villi (Yamada et al., 1999).

7. Early placental transporters

An important determinant of fetal growth is the capacity of theplacenta to deliver nutrients from the mother to the fetus and is inturn determined by the expression and function of nutrienttransporters in the placental barrier. A cluster of 22 genes was

found to be over-expressed in first trimester versus term; thesegenes were related to amine metabolism, nitrogen compoundmetabolism, amino acid metabolism and DNAmetabolism (DDAH1,ODC, FAH, SCLY, MCCC2, BCAT1, MTHFD1, SUOX,) associated withcationic amino acid (SLC7A1) and ornithine mitochondrial trans-port (SLC31A15) (Mikheev et al., 2008). Six to eight week placentalvilli exhibit a marked presence of transcripts for all the four types ofmembrane transport proteins: AQP-8 and -10 in the aquaporingroup, ABCA and ABCG in the ATP-binding cassette (ABC) trans-porters group, several types of calcium, chloride, potassium andsodium channels among ion channels, and large numbers of sub-types belonging to the solute carrier (SLC) family involved intransport of amino acids, fatty acids, glucose, ions including pro-tons, nucleobases and other organic substances (Table 4). At least15 amino acid transporters are found in the syncytial membrane forthe transport of 20 amino acids, some of them are Naþ dependentsystems whereas others are Naþ independent and differentiallydistributed in syncytiotrophoblast membranes. System A aminoacid transporters which include Naþ dependent transporters forneutral amino acids are expressed in the apical microvillous syn-cytial membrane of which SNAT1, SNAT2 and SNAT4 are found inplacenta at 6e10 weeks of gestation (Cetin, 2003; Desforges et al.,2006). A number of reports have already indicated the involve-ment of these membrane-bound transporters in the process ofmaternal-to-fetal exchange of nutrients, hormones and xenobioticsacross the placenta (Atkinson et al., 2005; Carter, 2012; Evseenkoet al., 2006; Desforges and Sibley, 2010; Ni and Mao, 2011;Prouillac and Lecoeur, 2010; Stulc, 1997; Wang et al., 2001). Inparticular, the high expression of the human placenta-specific ABC,ABCP (ABCG2) that maps to human chromosome 4q22 and ispossibly involved in multidrug resistance, appears intriguing(Allikmets et al., 1998; Lorkowski and Cullen, 2002).

8. Early placental immunity

The maternalefetal interface is one of the ‘immune privilegedsites’ where active mechanisms of cell destruction achieved byacquired immunity are muted or entirely bypassed as an adaptivefunction in the evolution of the mammalian placenta. Based on theglobal transcriptomic data analysis, we proposed that an ‘upregu-lated anti-viral response’ and ‘downregulated immune response’exhibited by first trimester villous cells functions as the first site ofdefense (Khan et al., 2013) and it works in unisonwith the immunefunctioning of decidual cells, T cells, B cells, macrophages and NKcells to achieve an ‘immune privilege status’ at the maternalefetalinterface during the course of normal pregnancy (Bulmer et al.,2010; Faridi and Agarwal, 2011; Hunt 2006; Lee et al., 2011;Tirado-González et al., 2012).

As shown in Fig. 2, enrichment and network process analysisidentified a network of three highly expressed genes in earlyplacental villi (ATF/Ap1, IL10R and OAS) associated with anti-viralimmunity and nine under-expressed genes (COL1, JAK2, JAK3, IL12,IL13, IL15, IL27, STAT3 and STAT5) associated with immune re-sponses (Khan et al., 2013). Further, normal first trimester placentalvilli exhibited a transcriptomic bias towards high levels of innatedefense against microbial invasion (Khan et al., 2013). This is clearlyevident from the high copies of transcripts for defensins, cell sur-face mucin, soluble mucin, Fc fragment of IgA, and tripartite motif(TRIM) proteins (McNab et al., 2011).

It is noteworthy that overt downregulation of genes associatedwith immune response (FN1, F11 receptor, SEMA3C, LRAP, SART3)may predispose the mother to pre-eclampsia (Founds et al., 2009).Leukocyte-associated immunoglobulin-like receptor-2 (LAIR2),homologous with inhibitory collagen receptor (LAIR-1) present onNK cells, T cells, B cells and macrophages was found to be

Fig. 2. Construction of network-pathway processes involving highly expressed (green box) three genes (ATF/AP1, IL10R and OAS) associated with anti-viral immunity and nineunder-expressed (red box) genes (COL1, JAK2, JAK3, IL12, IL13, IL15, IL27, STAT3 and STAT5) associated with immune response in enriched categories (adapted from Khan et al.,2013). It appears that integration of IL-10R, and OAS-Ap1-IRF/NF-kB plays a significant role in anti-viral function. Ap-1 and IRF/NF-kB appear to be minor and major functionalhubs, respectively. On the other hand, the overall under-expression of several ILs (IL-12, IL-13, IL-15 and IL-27) and JAKs (JAK-2 and JAK-3) is suggestive of downregulation ofimmune responses. In this process, IL-13, IL-15, JAK-2, JAK-3 and STAT-3 appear to be seeding nodes (blue filled circle) in the pathway processes.


significantly downregulated in CVS samples of women developingpre-eclampsia later in pregnancy (Founds et al., 2010). LAIR-2 isexpressed and secreted by extravillous trophoblast cells during 8e12 weeks of gestation (Apps et al., 2011). Founds et al. (2009) havereported downregulation of progestagen associated endometrialprotein (PAEP, PP14) in CVS samples of women developing pre-eclampsia later in pregnancy supporting a positive role of PP14 inimmune tolerance during pregnancy (Soni and Karande, 2010).PP14 has been reported to promote immune tolerance by reducingT effector functions and augmenting Treg differentiation(Ochanuna et al., 2010).

9. Molecular correlates of early placental disorders

It is now generally known that placental growth, differentiationand function during the first trimester is highly vulnerable, andexternal and internal insults during this phase may result in un-explained miscarriage (Ball et al., 2006), intrauterine growth

restriction (IUGR), low birth weight (Huppertz, 2008b; Pardi et al.,2002) and pre-eclampsia (Huppertz, 2008b; Jauniaux et al.,2006a,b; Kadyrov et al., 2003). In the following sections, we shallpresent a representative catalogue of molecular correlates of thoseearly placental disorders.

10. Early pregnancy loss

The commonest early pregnancy complication is spontaneousmiscarriage, which occurs in approximately 15e20% of all recordedpregnancies, a great majority occurring prior to 12 weeks ofgestation (Everett, 1997), while recurrent pregnancy loss (RPL) re-sults in miscarriage of the fetus prior to 20 weeks of gestation andaffects about 1e5% of pregnancies (Baek et al., 2007). A variety offactors have been identified to explain the patho-physiologicalbasis of RPL that include uterine anomalies, chromosomal abnor-malities, endocrine dysfunction, thrombophilia, immune disorders,lifestyle and maternal infection, polygenic disorders (Regan et al.,

Table 5Cardinal anomalies in villous placenta potentially involved in first trimester earlypregnancy loss (EPL).

Oxidative damage to trophoblast (Jauniaux et al., 2003)Impedence to oxygen and nutrient supply to trophoblast (Svensson et al., 2004)Inadequate placental antioxidant defense mechanism (Liu et al., 2006)Defective trophoblast motility and invasion (Gundogan et al., 2008)Impaired cytotrophoblat differentiation (Yurdakan et al., 2008)Reduced anti-inflammatory activity in placenta (Stortoni et al., 2010)Abnormal placental immune function (Lorenzi et al., 2012)Excessive placental inflammation (Calleja-Agius et al., 2012)


1989; Su et al., 2011a,b; Warning et al., 2011). Abnormal karyotypehas been detected in about 60% of Swedish women experiencingearly pregnancy loss (Farquharson et al., 2005) or first trimestermiscarriage (Ljunger et al., 2005). In studies of early pregnancy losswhere the well-established multifactorials known to result in RPLwere excluded, morphology and morphometry of placental villirevealed a thin trophoblastic shell with reduced trophoblast inva-sion, and incomplete plugging of maternal arterioles resulting inexcessive and early entry of maternal blood into the intervillousspace (Kurjak and Kupesic, 1997; Merce et al., 2009).

In normal pregnancy, the placenta is not considered to be trulyhemochorial until the end of the first trimester because it is sepa-rated from the maternal circulation by the plugs of endovasculartrophoblast cells within uteroplacental arteries. This results in alow intervillous partial oxygen pressure (Burton et al., 1999). Priorto 10 weeks the oxygen concentrations in the intervillous space isabout 2.5% (pO2 below 20 mmHg) and this rises to about 6e8%(pO2 of 60 mmHg) at around 14 weeks of gestation (Burton andJauniaux, 2001; Jauniaux et al., 2000). Increased oxidative stressof placental tissues appears as a strong primary defect in placen-tation in early pregnancy loss (Jauniaux et al., 2003; Jauniaux andBurton 2005) with or without chromosomal anomaly (Jauniauxet al., 2006a,b) and is associated with elevated levels of theinduced form of heat shock protein, nitrotyrosine residues andmorphological changes in mitochondria normally manifested insecond trimester trophoblast villi after the normal rise in oxygen(Jauniaux et al., 2000). Defects in placental villous remodeling andtrophoblast apoptosis are increased in cases of early pregnancy lossirrespective of chromosomal abnormality (Greenwold et al., 2003;Hempstock et al., 2003) but may arise secondarily to the increasein placental oxygen tension (Greenwold et al., 2003).

Yurdakan et al. (2008) reported a loss of E-cadherin proteinexpression in chorionic villi of early pregnancy loss. E-cadherin isknown to restrain trophoblast invasion (Zhou et al., 1997a,b) andthis calcium dependent cell-cell adhesion molecule has beenlocalized in cytotrophoblast, in non-proliferating intermediatetrophoblast and in migrating populations of extravillous tropho-blast (Floridon et al., 2000). In normal gestation, the low oxygenenvironment surrounding first trimester placental villi promotescytotrophoblast differentiation towards the invading population ofextravillous trophoblast, finally remodeling spiral arteries asendovascular trophoblast. Extravillous trophoblast cells showdownregulation of E-cadherin and induction of alpha-5 integrin topromote trophoblast migration (Arimoto-Ishida et al., 2009). Pro-tein expression profiling of E-cadherin, a binding partner of beta-catenin exhibited reduced expression with gestational age butincreased expression in trophoblast cells in IUGR/pre-eclampsiasuggestive of either exaggerated cytotrophoblast proliferation orimpaired cytotrophoblast differentiation (Brown et al., 2005).Based on our preliminary evidence and the existing literature wesuggest that increased expressions of IGFI, MAPK1, MAPK8 and E-cadherin may be indicative of a potential thrust for trophoblastproliferation (Forbes et al., 2008) with reduced differentiation to-wards an invasive phenotype (Canonici et al., 2008; Fu et al., 2010;Rahanama et al., 2006) that results in pregnancy failure.

Loss of anti-inflammatory functions with defects in thrombo-modulin expression has been identified in early pregnancy losswith associated defects in activated protein C (Stortoni et al., 2010;Svennson et al., 2004). Abnormal immune function of trophoblastcells in early pregnancy loss bearing normal chromosomal karyo-type has been suggested from increased levels of TNFa in villoustissues together with decreased secretion of IL-10 (Calleja-Agiuset al., 2012). Also, dysregulated expression of CD-45, CD-72 andCD-100 in Hofbauer cells in villous stroma and in syncytiotropho-blast (Lorenzi et al., 2012) confirm excessive local placental

inflammation. Polymorphism of IL-10, a secreted cytokine oftrophoblast cells, is an independent risk factor in early pregnancyloss (Cochery-Nouvellon et al., 2009).

Table 5 provides a brief summary of our current understandingabout the cardinal anomalies in placental physiology that result inearly pregnancy loss. Interestingly, enrichment analysis of our datafrom 8-week normal vis-à-vis pregnancy loss placental villi iden-tified several affected processes in early pregnancy loss samples,which includes oxidative stress, immunity and inflammation,nutrient transporter function and cytoskeletal organization (Fig. 3)

11. Pre-eclampsia and intrauterine growth restriction

Pre-eclampsia is a life threatening pregnancy complication thatoccurs in 5e7% of all human pregnancies with high rates ofmaternal and fetal morbidity and mortality. The clinical symptomsof pre-eclampsia include new onset of hypertension, proteinuria,and edema and can be associated with fetal/intrauterine growthrestriction (FGR/IUGR). Fetal IUGR has been traditionally includedin the diagnostic criteria of severe pre-eclampsia (Pennington et al.,2012). It has now become clear that pre-eclampsia and IUGR areindependent syndromes (Huppertz, 2008b) and that pre-eclampsiamay be independently associated with the development of IUGR(Huppertz, 2008b; Srinivas et al., 2009).

The mechanisms by which pre-eclampsia develops from aber-rant placental implantation and/or villous trophoblast develop-ment are not fully understood. It has been proposed that duringearly stages of pregnancy, the immune system plays a key role inthe alteration of trophoblast differentiation and function leading topre-eclampsia (Laresgoiti-Servitje et al., 2010). This hypothesisfinds support from two studies that explored global gene expres-sions in surplus chorionic villus (CVS) samples that were obtainedin first trimester (10e12 weeks gestation) of women who laterdeveloped pre-eclampsia compared with age-matched CVS sam-ples of normal pregnancies (Founds et al., 2009; Farina et al., 2011).Founds et al. (2009) reported a downregulation of 9 genes (CFH,FN1, F11 receptor, LAIR2, LRAP, MMP12, MUC15, PAEP, SEMA3C) thatare mainly associated with immune response/adhesion/comple-ment activation, antigen processing and blood pressure regulationand proteolysis. They also reported upregulation of three genes:cholecystokinin S100, calcium binding protein A8 and cancer/testisantigen-2. Dysregulation of cell growth, proliferation, signaltransduction and transport processes as well as of genes likeIGFBP1, OXGR1, SCARA5, SLC16A6, SLCO4A1 were also found in firsttrimester CVS samples of women who later developed pre-eclampsia. However, the above study failed to show differentialexpressions of hypoxia inducing factor -1alpha (HIF1alpha) andgenes related to oxidative stress betweenpre-eclamptic and controlsamples suggesting that ischemia-hypoxia and oxidative stress dueto reperfusion injury may not occur in pre-eclampsia. Dysregula-tion in a set of genes associated withmaternal immunity (IL8, IP-10,HLA-C, HLA-G), trophoblast invasion, angiogenesis, remodeling ofextracellular matrix (MMP-9), and hemodynamic adaptation (NKB)have also been reported in CVS samples collected at 11 weeks of

Fig. 3. Construction of network-pathway processes involving 11 highly expressed (green box with red circle) genes (EIFSA2, TMEM33, ENTPD2, JNK, TPS3AP1, WDR4, GALNTL1,GPCRs, PTPRCAP, SLC2A8, HHLA2) and six under-expressed (red box with blue circles) genes (HNF4alpha, CREBH, BAF57, ALKBH5, CHCHD5, Sec10/EXOC5) in first trimester villousplacenta of early pregnancy loss at eight weeks of gestation (unpublished data). It is of interest to note the over-expression of stress protein genes TP53AP1, JNK (MAPK8-10) isassociated with ROS-mediated endoplasmic reticulum stress, while PTPRCAP which is a transmembrane phosphoprotein specifically associated with tyrosine phosphatase PTPRC/CD45 is a key regulator of T- and B-lymphocyte activation, and HHLA2 belonging to the B7 family is essential in adaptive immune system. SLC2A8 is a facilitative glucose transporter,and WDR4tRNA is a subunit of (guanine-N (7)-)-methyltransferase. Under-expression of nuclear transcription factor HNF4a functions as the central element in modulating theabove-mentioned over-expressions of 11 genes as well as the under-expressions of BAF57 (a core subunit of the mammalian SWI/SNF chromatin remodeling complex), CREBH (acAMP responsive element-binding protein, hepatocyte specific transcription factor in the nuclear compartment), BRSM1l (a breast cancer metastasis-suppressor), Sec10/EXOC5(responsible for actin cytoskeletal remodeling and vesicle transport machinery), ALBH5 (alkylated DNA repair protein), CHCHD5 (a putative substrate of the mitochondrial Mia40-dependent import machinery), and ENTPD2 (an ecto-ATP diphosphohydrolase 2) in the cytoplasmic compartment of first trimester placental villi in early pregnancy loss at eightweeks gestation.


gestation in women exhibiting pre-eclampsia at term (Farina et al.,2011).

A large number of studies revealed that genes/proteins involvedin pro-inflammatory responses, TGFb, inhibin-A (Reddy et al.,2009), CCL8 (Struyf et al., 2009), PRDX family (Ishii et al., 2012;Shichita et al., 2012) are elevated in placentas of pre-eclampsiacompared with age-matched controls, with elevated expressionof genes and proteins of the family of heat shock proteins (Borgeset al., 2012), apolipoprotein A1 (Yin et al., 2012) and alpha-2-HS

glycoprotein (Li et al., 2011) with known anti-inflammatory func-tions. The association of pre-eclampsia with the development of aninflammatory response in the placenta is further supported by theupregulated expression of TGFb1 in global microarray studies (Toftet al., 2008; Centlow et al., 2011). Recent evidence suggests thatheme oxygenase-1 (HO-1) and its metabolite carbon monoxide(CO) may function as gatekeepers to prevent the onset of clinicalsymptoms of pre-eclampsia through negative regulation of sFlt-1and sEndoglin and thereby offer vascular protection of the


mother against oxidative stress and exacerbated inflammation(Cudmore et al., 2012).

On the other hand, IUGR is associated with reduced endovas-cular trophoblast invasion and uteroplacental artery remodeling inthe first trimester (Brosens, 1977) leading to altered perfusion ofthe intervillous space and to oxidative damage of villous tissues(Hung et al., 2001; Li et al., 2010; Makris et al., 2007). Kaufmannet al. (2003) have suggested that maladaptation and malinvasionof uteroplacental arteries, characteristic of IUGR, may result frominteractions of intrinsic and extrinsic factors in a cascade of eventsin which an abnormal biology of extravillous trophoblast (asintrinsic factors) acts in concert with impaired decidual remodel-ing, macrophage-based defense mechanisms, impaired function ofNK cells and maternal endothelial cell failure to express selectins(as extrinsic factors). IUGR, unlike early pregnancy loss, exhibits alesser defect in early trophoblast invasion since in the case of IUGRinvasion is sufficient to anchor the conceptus, but it is insufficient tofully convert the spiral arteries to low resistance vessels (Robertsonet al., 1984) resulting in increased flow velocities of maternal bloodinto the intervillous space (Burton et al., 2009). Abnormalities introphoblast-mediated spiral artery remodeling may result in fluc-tuating oxygenation of the placenta, mimicking hypoxia-reoxygenation injury in the placenta secondary to oxidative dam-age (Hung et al., 2001; Li et al., 2010; Makris et al., 2007). Sup-portive evidence from several laboratories lends further credenceto this hypothesis (see Table 6 for details).

The evolutionary conserved protein kinase, a mechanistic targetof rapamycin (mTOR), is known to integrate inputs from severalpathways, including insulin, growth factors (such as IGF-1 and IGF-

Table 6Selected candidate genes/proteins in villous placenta potentially involved in fetalintrauterine growth retardation (FGR/IUGR) and maternal pre-eclampsia (PE).

Candidate gene/protein with putative mechanism (reference)

High TNFamRNA and low superoxide dismutase, glutathione peroxidase mRNAand protein levels in PE placentas resulting in increased lipid peroxidation inPE placentas (Wang and Walsh, 1996a,b)

Increased endothelial nitric oxide synthase (NOS) in placental vessels of PE andIUGR as a likely adaptive response to increased resistance and poor perfusion(Myatt et al., 1997)

Increased mRNA expressions for IGF-1 and PlGF in IUGR placenta possiblyreflecting response to fetal growth restriction (Sheikh et al., 2001)

Decreased expression of heme oxygenase-2 in endothelial cells of PE placentacausing reduced placental blood flow (Barber et al., 2001)

Dysregulated VEGF family related proteins in PE placental cytotrophoblast(Zhou et al., 2002)

Increased E-cadherin in cytotrophoblast compared to syncytiotrophoblastsuggesting altered cytotrophoblast proliferation and/or differentiation in PE(Brown et al., 2005)

Coexistence of loss of function catalytic activity and elevated nitration of p38MAPK in PE placenta causing growth restriction generally observed in PE(Webster et al., 2006)

Decreased placental mTOR activity, which functions as a placental nutrient‘sensor’ matching fetal growth with maternal nutrient availability byregulating placental nutrient transport, resulting in IUGR (Roos et al., 2007)

mRNA expressions for IGF-II, IGFBPs-IL-6 higher in perifunicular villous tissue infetal plate in FGR possibly results from limited nutrition availability (Streetet al., 2008)

Decreased transferrin receptor 1 mRNA expression in IUGR with low TFRCprotein expression predominantly in syncytiorophoblast resulting in irondeficiency and thereby leading to low fetal birth weight in IUGR (Mandò et al.,2011)

Decreased glial cell missing-1 (GCM1) and associated dysregulated TIMP4expression contributing to the development of PE (Drewlo et al., 2011)

Imbalance of angiogenic and inflammatory cytokines and chemokines in PEplacenta contributing to the onset of PE (Schanz et al., 2011; Barrera et al.,2012)

Decreased SNAIL transcription factor which is a master regulator for epithelial-mesenchymal transition in villous progenitor trophoblast cells contributingto onset of PE (Fedorova et al., 2012)

2), and amino acids to function as a ‘sensor’ of cellular nutrient andenergy levels and redox status (Tokunaga et al., 2004). It is also aneffector of cell proliferation and growth via the regulation of pro-tein synthesis (Hay and Sonenberg, 2004). In IUGR, the activity ofkey placental amino acid transporters, systems A and L, isdecreased. Roos et al. (2007) provide evidence to suggest that themTOR pathway is decreased in IUGR placentas which results indeficiency in the activity of L-amino acid transporters but notsystem A-taurine transporters. The ‘placental phenotype’ (Sibleyet al., 2005) demonstrates a mixed phenotype in cases of IUGRthat includes an increased glucose utilization capacity butdecreased protein synthesis and growth (Hay, 2006). Insulin syn-thesis and secretion of IGF-I and PlGF are increased (Sheikh et al.,2001), while a dysregulated IGFs e IL-6 network has been foundto operate in IUGR placentas (Foltran et al., 2011; Street et al., 2008).

12. Future direction

Despite a large bulk of morphological and molecular informa-tion at our disposal, we have a serious deficiency in acquiring anintegrated understanding about physiology and pathophysiologyfor most of the physiological systems. No doubt there are severalreasons behind it and these include the proximate nature of bio-logical questions asked, the hegemony of qualitative biology insearch of ‘magic nuggets’, and lack of dimensionality and scalabilityin the strategy of exploration, to name a few, compelling us toadmit that ‘We physiologists are a notorious sloppy lot.’ (Florey,1987). However, referring specifically to placental biology, webelieve additionally the issue of evolutionary heuristic needs to beaddressed to get clue about how placental control systems may ormay not fall short of ‘optimality’ in a given situation (Bostrom andSandberg, 2009). In recent times, serious efforts are being given tounderstand human placental evolution heuristics in terms of co-evolution, convergent evolution, genetic and epigenetic adapt-ability towards structureefunction regulation and resource-demand optimization in placental biology (Carter, 2011, 2012;Crespi and Semeniuk, 2004; Frost and Moore, 2010; Monk et al.,2006; Nelissen et al., 2011). It is anticipated such endeavour shallopen up newer approaches to catch up ‘Medawarian puzzle’ aboutthe development of physiology and pathophysiology of the humanplacenta (Medawar, 1953).

Acknowledgements

The authors acknowledge the help of Ms. G. Anupa and Mr.Muzaffar Ahmed Bhat in proof reading and Mr. Akhilesh Srivastavain constructing process networks and proof reading of the article,and the grant support of the Indian Council of Medical Research [I-1760].

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