research article differential expression of placental...

Research ArticleDifferential Expression of Placental Glucocorticoid Receptorsand Growth Arrest-Specific Transcript 5 in Term and PretermPregnancies Evidence for Involvement of Maternal Stress

D Mparmpakas1 E Zachariades1 G Sotiriadis1 A Goumenou12 A J Harvey3

Y Gidron14 and E Karteris1

1 Biosciences Centre for Cell and Chromosome Biology Brunel University Uxbridge UB8 3PH UK2Department of Obstetrics and Gynecology University Hospital University of Crete Medical School Heraklion 71003 Crete Greece3 Biosciences Brunel Institute for Cancer Genetics and Pharmacogenomics Brunel University Uxbridge UB8 3PH UK4Free University of Brussels (VUB) 1090 Jette Belgium

Correspondence should be addressed to E Karteris emmanouilkarterisbrunelacuk

Received 20 September 2013 Revised 18 January 2014 Accepted 12 March 2014 Published 11 May 2014

Academic Editor Curt W Burger

Copyright copy 2014 D Mparmpakas et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pregnancy-specific stress predicts birth outcomesWe hypothesized that there is amaternal stress-GR interaction that can influencefetal birth weight This study examined the relationship between mothersrsquo stress and attitude towards their pregnancies placentalglucocorticoid receptors (GRs) and growth arrest-specific transcript 5 (GAS5) expression and the status of GR polymorphism withtheir infantsrsquo birth weights GAS5 and GR120572were the predominant transcripts in both term and preterm placentas with GAS5 beingprimarily localized in the syncytiotrophoblasts In an attempt to mimic moderate and high stress environment in vitro BeWo andJEG-3 cytotrophoblast cell lines were treated with 10 nMndash1000 nM cortisol Only expression of GAS5 was significantly upregulatedby cortisol in all treatments comparedwith basal levels but none of theGRs changed expression significantly In an attempt to assessa stress versus gene interaction we studied four GR polymorphisms In the homozygous group for Tth111I polymorphism motherswith negative attitudes towards the pregnancy gave birth to infants with significantly lower birth weights compared to women withpositiveneutral attitudes None of the GR splice variants were associated with maternal stress However placental GAS5 levelswere inversely correlated with maternal stress This study points towards a potential gene-environment interaction that could be ofpredictive value for fetal weight

1 Introduction

Pregnancy is associated with major physiological and futurepsychosocial changes and adaptation to these changes iscrucial for normal fetal development and for healthy infant-mother relationshipsMaternal pregnancy-specific stressmaybe a more powerful contributor to birth outcomes thangeneral stress [1] Biologically the stress hormone cortisol actsby activating glucocorticoid receptors (GRs) To date foursplice variants of the GR gene have been reported formedby alternative splicing termed GR120572 GR120573 GR120574 and GR-P[2] Numerous studies have also reported the expression of

different GR splice variants in different cell and tissue types[3ndash6] Growth arrest-specific transcript 5 (GAS5) encodesa single strand noncoding RNA (ncRNA) and as its namesuggests can accumulate in growth-arrested cells [7] In arecent study by Kino and colleagues it was demonstratedthat GAS5 ncRNA may be a repressor for the GR by actingas a decoy ldquoglucocorticoid response element (GRE)rdquo thuscompetingwithDNAGREs for binding to theGRAs a resultGRs cannot bind to their DNA GRE and subsequently theirfunction is compromised [8]

Changes in the epigenetic regulation of the fetal GR pro-moter have been associated with exposure to prenatal

Hindawi Publishing CorporationObstetrics and Gynecology InternationalVolume 2014 Article ID 239278 9 pageshttpdxdoiorg1011552014239278

2 Obstetrics and Gynecology International

maternal stress [9] reflecting a possible effect of maternalstress on the expression and function of the GR in thefetus A GR BclI polymorphism has been associated withincreased glucocorticoid sensitivity and was overrepresentedin pregnant women with pathological fetomaternal immuneadaptation Indeed a number of polymorphisms have beendescribed in the gene coding for the GR although it isstill unclear whether the variability in the glucocorticoidresponses observed is due to the polymorphisms or toother factors [10] This sensitivity could be one mechanismlinking maternal stress to fetal development Only few ofthese polymorphisms are functionally relevant and theseare the Tth111I the ER2223EK the N363S themdashalreadymentionedmdashBclI and the GR-9120573 Studies have shown thatat least three polymorphisms are associated with alteredglucocorticoid sensitivity and also with changes in bodycomposition and metabolic parameters [10 11] which couldaffect fetal development as well

This study examined the relationship between mothersrsquostress levels and attitude towards their pregnancies placentalGR and GAS5 expression and the status of GR polymor-phismwith their infantsrsquo birthweightsWe also examined thematernal attitude versus gene interaction in relation to fetalbirth weight Moreover we tested the effect of cortisol on GRsplicing and GAS5 expression of BeWo and JEG-3 placentalcells in an attempt to resemble a low moderate and highstress milieu in vitro

2 Methodology

21 Subjects The study population consisted of pregnantwomen attending the Department of Obstetrics and Gynae-cology University Hospital University of Crete The par-ticipants were in the third trimester of their pregnancyAll participants gave informed consent to participate in thestudy and ethical approval was granted by the local ethicscommittee of the hospital Questionnaires regarding mater-nal stressmaternal attitudes as well as anthropometric datawere collected as previously described [12 13] Briefly mater-nal stressmaternal attitudes towards their pregnancy wereassessed in the 3rd trimester using a questionnaire approachA research nurse or a gynecologist conducted a face-to-face interview with each woman lasting approximately 20ndash30 minutes Maternal attitudes were related to self-reportedstress status ranging from low and medium (low stressresponse) to high and very high (high stress response) Theexact cause of maternal stress was not identified Maternalattitudes were divided into two groups negative attitudes(48) and positive (357)neutral attitudes (163) Imme-diately after delivery the weight of the newbornwas recorded

22 Placental Tissue Placental tissues were obtained fromwomen delivering at term (gt37 weeks of gestation) andpreterm (lt37 weeks of gestation) (total 119899 = 23) Of the 13term placentas 12 were in labor (mode of delivery 10 cae-sarean sections [CS] 2 vaginal deliveries) and 1 nonlaborCStissue None of the term or preterm women were adminis-tered any antidepressants or synthetic glucocorticoids during

pregnancy Of the 10 term placentas 4 were nonlabor and allCS and 6 were from laboring tissues (mode of delivery 3CS 3 vaginal deliveries) The average age was 296 plusmn 4 yearsthe mean pregnancy days were 240 plusmn 42 and mean fetalweight was 2805 plusmn 95 gr After delivery thematernal and fetalsurfaces of the placenta were dissected and fetal membraneswere peeled away from the placenta Placental samples werewashed in PBS and immediately stored in RNAlater (AppliedBiosystems UK) atminus80∘C Ethical approval was granted fromthe local ethics authority

23 Cell Culture BeWo and JEG-3 cell lines were main-tained at standard culture conditions of 5 CO

2in air at

37∘C BeWo cells were cultured in Ham F12 (Sigma-AldrichUK) containing 10 heat-inactivated fetal bovine serum(FBS) and 05 penicillin streptomycin whereas JEG-3 cellswere maintained in MEME (Sigma-Aldrich UK) containing10 heat-inactivated FBS and 05 penicillin streptomycin05 L-glutamine 05 sodium pyruvate and 05 MEMnonessential amino acids Prior to cortisol treatment bothcell lines were maintained for 3 hours in phenol red-freemedia containing charcoal stripped FBS

24 RNA Isolation cDNA Synthesis and PCR Total ribonu-cleic acid was isolated using an RNA extraction kit (Sigma-Aldrich UK) according to manufacturerrsquos instructions RNAconcentration was determined by spectrophotometric analy-sis (NanoDrop Thermo Scientific UK) and agarose gel elec-trophoresis RNA (200 ng from placental tissue and 500 ngfrom cell lysates) was reverse-transcribed into cDNA using5 IU120583L RNase H reverse transcriptase (Invitrogen PaisleyUK)

25 Quantitative RT-PCR Relative expression of the genes ofinterest was assessed by quantitative PCR (Q-PCR) on anABIPrism 7900HT Sequence detection system (Applied Biosys-tems) using SYBR Green-PCR reaction mixture (Sigma-Aldrich UK) and specific primers (Table 1) as previouslydescribed [14] For the quantitative PCR the following equa-tions were used ΔCt = Ct

(gene of interest) minus Ct(house keeping gene)ΔΔCt = ΔCt

(sample) minus ΔCt(calibrator) and Relative Quantity(RQ) = 2minusΔΔCt as previously described [12]

26 Immunofluorescent Analysis Following a series of depar-affinisation and dehydration placental tissue sections wereincubated with 10 bovine serum albumin (BSA) for 1 hrThis was followed by incubation for 1 hr with antibodiesagainst GR120572 at a 1 200 dilution in 1 BSAPBS Cells werethen washed with PBS prior to an incubation with a TRITC-conjugated secondary antibody (Santa Cruz BiotechnologyUSA) for 1 hr Slides were washed with PBS and mountedin Vectashield Mounting Medium (Vector Labs) containingthe dye 46-diamido-2-phenylindole (DAPI) to counterstainnuclei Images were captured using a Plan Apo Neofluor63X NA 125 oil objective (Zeiss) on a Zeiss Axiovert 200Mmicroscope and viewed using the AxioVision software

Obstetrics and Gynecology International 3

Table 1 Sequences of primers used for quantitative RT-PCR

GR120572 Sense 51015840-CTATGCATGAAGTGGTTGAAAA-31015840 96 bpAntisense 51015840-TTTCAGCTAACATCTCGGG-31015840

GR120573 Sense 51015840-GAAGGAAACTCCAGCCAGAA-31015840 81 bpAntisense 51015840-CCACATAACATTTTCATGCATAGA-31015840

GR120574 Sense 51015840-TTCAAAAGAGCAGTGGAAGGTA-31015840 264 bpAntisense 51015840-GGTAGGGGTGAGTTGTGGTAACG-31015840

GR-P Sense 51015840-GCTGTGTTTTGCTCCTGATCTGA-31015840 194 bpAntisense 51015840-TGACATAAGGTGAAAAGGTGTTCTACC-31015840

GAS5 Sense 51015840-CAGTGTGGCTCTGGATAGCA-31015840 168 bpAntisense 51015840-TTAAGCTGGTCCAGGCAAGT-31015840

120573-Actin Sense 51015840-AAGAGAGGCATCCTCACCCT-31015840 216 bpAntisense 51015840-TACATGGCTGGGGTGTTGAA-31015840

27 Western Immunoblotting Proteins from placental lysatesand BeWo cells treated with 10ndash1000 nM cortisol were sepa-rated on an SDS-12 polyacrylamide gel and transferred toa nitrocellulose membrane The membrane was blocked inTBS containing 5driedmilk powder (wv) and 01Tween-20 overnight at 4∘C After three washes with TBS-01Tween-20 the nitrocellulosemembraneswere incubatedwithprimary antibodies against GR120572120573 (Abcam ab3580 UK) andGAPDH (Sigma-Aldrich UK) The primary antisera wereused at a 1 1000 dilution for GR120572120573 and 1 2000 for GAPDHovernight at 4∘C The membranes were washed thoroughlyfor 30min with TBS-01 Tween before incubation with thesecondary HRP-conjugated immunoglobulin (1 2000) for1 hr at room temperature and further washing for 30minwithTBS-01 Tween-20 The immunoreactive bands were ana-lyzed using Image J 134s image-analysis software (NationalInstitute of Health USA)

28 RNA Fluorescent In Situ Hybridization (FISH) The slidesof the paraffin embedded placental tissue sampleswere placedin a coplin jar and deparaffinised for 30minutes at 37∘C usingHisto-Clear (Fisher Scientific UK) Thereafter the tissuesections were rehydrated by placing the slides in ethanolsolution of different concentrations (100 90 80 7050 and 30) for 3 minutes A brief wash in 1x PBS solutionwas performed after this Pepsin (001 in 001M HCl) wasused to treat the tissue for 5 minutes at 37∘C and a brief rinseinDEPC treatedH

2O followedThen the tissue samples were

dehydrated by placing them in ethanol solutions (70 90and 100) for 5 minutes A specific Alexa 488-conjugatedGAS5 hybridization probe (GTGCTATCCAGAGCCACA-CTGCATCTGCACCCAGCACCATACCTCACAG) wasutilised as previously described [8] and followed by anovernight incubation at 37∘C in a humidified chamber Afterthis incubation three 10-minute washes were followed in2xSSC at 37∘C The slides were then briefly rinsed in DEPCtreated H

2O and mounted in Vectashield Mounting Medium

containing DAPI prior to examining the emitted fluorescentsignal under a Zeiss Axiovert 200Mmicroscope viewed usingthe AxioVision software

29 GR Polymorphisms For the BclI N363S Tth111I andER2223EKGRpolymorphismsDNAwas extracted using thePhusion Blood Direct PCR Kit (New England Biolabs UK)according to the manufacturerrsquos instructions For the BclIthe following primers were used 51015840-AAGCTTAACAAT-GGCCAT-31015840 and 51015840-TGCTGCCTTATTTGTAAATTCGT-31015840 The PCR conditions used were 40 cycles of 98∘C for 5 secannealing at 50∘C for 5 sec and elongation at 72∘C for 15 secTo confirm the presence of the BclI polymorphism 17 120583L ofthe PCR product was digested with 2120583L of 10x RestrictionEndonuclease Buffer and 15U of the restriction enzyme BclI(New England Biolabs UK) for 15 hr at 50∘C The resultingdigested fragments were separated on a 2 agarose gel todetermine the genotypes For the N363S polymorphism spe-cific PCR primers were used 51015840-AGTACCTCTGGAGGACAG AT-31015840 and 51015840-GTCCATTCTTAAGAAACAGG-31015840 underthe same PCR conditions The restriction enzyme Tsp509I(10U) was used to digest the PCR products at 65∘C for4 hours For the ER22EK23 polymorphism we have usedthe following primers 51015840-GATTCGGAGTTAACTAAAAG-31015840 and 51015840-CTACCCTTTACTGGACCCTA-31015840 followed byrestriction digest using MnI I (New England Biolabs UK)at 37∘C for 4 hours Finally the Tth111I polymorphismwas assessed using the PCR primers 51015840-GGCCACAAC-AATAACCCAGT-31015840 and 51015840-CCTATGACACGTATTTTG-TGAAAGT-31015840 The restriction endonuclease Tth111I (NewEngland Biolabs UK) was used to digest the PCR productsat 65∘C for 4 hours

210 Statistical Analysis Q-PCR data are reported as themean plusmn SEM Statistical analysis was performed by Studentrsquost-test and by ANOVA 119875 lt 005 was regarded as statisticallysignificant We also tested the relationships between con-tinuous predictors (womenrsquos age stress) and infant weightusing Pearson correlations and partial correlations (whenadjusting for age and BMI) and using t-tests for dichotomouspredictors (eg planned pregnancy) and ANOVA for testingthe relationship between attitude type and infant birthweightwhile controlling statistically for relevant confounders suchas mothersrsquo age BMI pregnancy planning and pregnancynutritional habits


3 Results

31 Expression of Placental GAS5 and GRs QuantitativeRT-PCR revealed that GAS5 and all GRs were expressedin human placentas (119899 = 23) 13 were preterm labor(lt37 gestational weeks) and 10 were term (gt37 gestationalweeks) labor with GAS5 and GR120572 being the predominanttranscripts In term labor GR120572rsquos ΔCt was 0244 comparedwith 0006 for GR120573 0006 for GR120574 0023 for GR-P and026 for GAS5 At preterm the values were 0278 00040003 0006 and 0180 for GR120572 GR120573 GR120574 GR-P and GAS5respectively (see Figure 1(a)) It is evident that the majortranscripts in the human placenta are GR120572 and GAS5 Whenrelative mRNA abundance was calculated an approximate 4-fold change in the GR120572GR-P in the preterm labor group wasnoted compared with the term group In addition a twofoldincrease in theGR120572GAS5 ratio was also noted in the pretermgroup compared to the term group (see Table 2)

In term placentas there was a significant correlationbetween GR120573 and GR120574 (119903 = 0580 119875 = 0038) and with GR-P(119903 = 0980 119875 lt 0001) In the preterm group the dynamicsof GR splicing were altered since GR120572 correlated with GR-P(119903 = 0710 119875 = 0021) and GR120573 correlated with GAS5 (119903 =0792 119875 = 0011) However no significant correlation wasnoted in the preterm group between fetal weight or maternalstress and the relative expression of GRs and GAS5 In theterm group the only significant correlation was betweenGAS5 and maternal stress (119903 = minus0711 119875 = 0021)

Due to ethical restrictions we were only able to assessthe expression of GR120572120573 in 4 term and 3 preterm placentasSince GR is present as different isoformsmultiple bands wereobserved and there was an expected interpatient variation inthe protein expression Scanning densitometry of the bandscorresponding to GR120572120573 normalized over GAPDH revealedno apparent differences in the expression of these variantsbetween the term and the preterm groups (Figure 1(b))

32 Cellular Distribution of GR120572 and GAS5 in Human Pla-centas Immunofluorescence analysis of the GR120572 proteinwas performed in human placenta tissue sections Stronghomogeneous staining mainly in the cytoplasm is detectedin the syncytiotrophoblast cells on the outermost layer ofthe placental villi with some scattered expression in cytotro-phoblast cells (Figure 2(a)) UsingRNAFISHGAS5 localizedprimarily in syncytiotrophoblasts (Figure 2(c)) This is a firsttime that GAS5 localization has been studied in humanplacentas

33 Effects of Cortisol on GRs and GAS5 In Vitro BeWoand JEG-3 cells were treated overnight with cortisol tomimicmoderate andhigh stress environments in vitro Beforedoing so the expression of GAS5 and GRs was examined inBeWo and JEG-3 cells without administering cortisol GAS5was significantly lower in expression in JEG-3 cells whencompared to BeWo under basal conditions (Figure 3(a)) Forthe GRs significantly higher expression levels were detectedonly for GR120574 and GR-P in the JEG-3 cells compared to theBeWo cells (Figure 3(b))

Table 2 Relative mRNA abundance of GR120572 over GR120573 GR120574 GR-Pand GAS5

Ratio Term (119899 = 13) Preterm (119899 = 10)GR120572GR120572 1 1GR120572GR120573 47 65GR120572GR120574 38 97GR120572GR-P 11 43GR120572GAS5 08 15

When BeWo and JEG-3 cells were treated with cortisol10 nM 100 nM or 1000 nM the expression of GAS5 wassignificantly upregulated (all 119875 lt 005) compared withtheir corresponding basal levels (Figure 4(a)) The maximalstimulation for GAS5 was at 10 nM of cortisol in JEG-3 cellsand at 1000 nM in BeWo None of the cell lines exhibiteddose dependency However cortisol treatments did not exertany significant changes in the gene expression of GR120572(Figure 4(b)) GR120573 (Figure 4(c)) GR120574 (Figure 4(d)) andGR-P in either of the cell lines (Figure 4(e)) In additionthere were no apparent changes in protein expression ofGR120572120573 in BeWo cells treated with 10ndash1000 nM of cortisolcorroborating the qPCR studies (data not shown)

34 Maternal and Infant GR Polymorphisms Maternal Atti-tudes and Fetal Birth Weight Four GR polymorphisms wereinvestigated BclI N363S Tth111I and ER2223EK (Table 3)Since the maternal attitude towards the pregnancy was asignificant predictor of fetal birth weight we reexaminedthis association as a function of the GR genepolymorphismsresembling a gene-environment interaction and statisticallycontrolling for mothersrsquo age BMI pregnancy planning andpregnancy nutritional habits to remove any potential sourcesof statistical bias that could skew our data

Statistical analysis of the maternalTht111I polymorphismhas shown an inverse correlation between negative maternalattitude and infant birth weight (119903 = minus041 119875 = 0030)only in Tht111I CC polymorphism subgroup In Tht111I CTno significant correlations were noted controlling for ageBMI pregnancy planning and consumption of fast foodduring pregnancy These data point towards an interactionbetween stress and genetics since only in the CC polymor-phic GR group did negative maternal attitude predict fetalweight reduction but not in the CT group independentof confounders Hence the effects of maternal attitudes onfetal weight depend on the motherrsquos polymorphism of GRgene None of the remaining GR polymorphism subgroupsdemonstrated any differential correlations between maternalattitudes and fetal weight (data not shown)

4 Discussion

The present study extends previous findings and providesevidence for the first time how maternal stress and GRpolymorphisms can potentially affect fetal outcome togetherAs reviewed previously prenatal maternal stress has beenshown to have long-term effects on the psychological as


045

04

035

03

025

02

015

01

005

0

GR120572 GR120573 GR120574 GR-P GAS5

RQ

val

ues

TermPreterm

(a)O

D u

nits

Term Preterm

15

10

05

00

(b)

Figure 1 (a) Expression of GR120572 GR120573 GR120574 GR-P and GAS5 in term (blue 119899 = 13) and preterm (red 119899 = 10) placentas (b) Proteinexpression of GRs correcting over GAPDH in term (119899 = 4) and preterm (119899 = 3) placentas

Syncytia layer

Cytotrophoblasts

(a) (b)

(c)

Figure 2 (a) Immunofluorescent analysis demonstrated expression of GR120572 primarily in the syncytiotrophoblastic layer of term placentas(b) Negative control confirmed specificity of immunostaining (c) RNA FISH confirmed expression of GAS5 in cytotrophoblasts cells (dottedarrows) and syncytiotrophoblasts cells (white arrows)

well as behavioral development of the offspring [15 16] Inour cohort women with negative attitudes exhibited higherlevels of stress during pregnancy compared to women withneutralpositive attitudes and gave birth to infants withlower birth weights than those with positiveneutral attitudestowards their pregnancy (500 gr difference [13])

In terms of the polymorphisms analyses only thematernal Tth111I polymorphism was suggestive of a gene-environment interaction since only in Tth111I (CC) nega-tive versus positiveneutral maternal attitudes towards thepregnancy predicted fetal weight reduction but not in theTth111I (GC) group independent of important confounders


16

14

12

1

08

06

04

02

0

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWo JEG-3

lowast

(a)

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWoJEG-3

lowast

lowast

8

7

6

5

4

3

2

1

0

GR120572 GR120573 GR120574 GR-P

(b)

Figure 3 (a) Expression of GAS5 in BeWo and JEG-3 cells lowast119875 lt 005 (b) Expression of GR120572 GR120573 GR120574 and GR-P in BeWo and JEG-3cells lowast119875 lt 005

45

40

35

30

25

20

15

10

5

0

NSCortisol treatment10nM 100nM 1000nM

lowast

lowast

lowast

lowast

lowast

lowast

Rela

tive q

uant

ity

(RQ

) val

ues

(a)

16

14

12

1

08

06

04

02

0

NSCortisol treatment

10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(b)

1618

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(c)

16

14

12

1

08

06

04

02

0

Rela

tive q

uant

ity

(RQ

) val

ues


(d)

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(e)

Figure 4 Effects of cortisol on the expression of GAS5 (a) GR120572 (b) GR120573 (c) GR120574 (d) andGR-P (e) in JEG-3 (red) and BeWo (blue) placentalcell lines NS no supplement lowast119875 lt 005


Table 3 Distribution of GR polymorphisms

Polymorphism Nucleotide change Genotype Distribution ()119899 = 81

BclI C rarr GGG 5 (62)CC 19 (235)GC 57 (704)

119899 = 81

N363S Asp rarr Lys wt 79 (975)Hmz 2 (25)

119899 = 81

ThtIIII C rarr TCC 35 (432)TT 2 (25)CT 44 (543)

119899 = 81

ER2223EK Arg rarr Lys wt 78 (963)Htz 3 (37)

These confounders included womenrsquos age and BMI and werenot explained by gestational age Additionally multiple otherimmune symptoms and demographic variables tested werealso not predictive of fetal weight This is the first time that agene-environment interaction between a GR polymorphismand maternal attitudes towards pregnancy was found inrelation to fetal weight To this date there has been somecontradicting evidence as to the role of these polymorphismsin fetal outcomes [17ndash19]

The human placenta is exposed to increased levels ofcortisol as pregnancy progresses [20] and its effects aremediated via binding and activating the GR However con-troversy surrounds the exact mechanisms by which theseresponses are regulated GR alternative splicing might alsoinfluence the subsequent activation of signalling pathwaysby glucocorticoids [20] In our study we have shown thatall known transcripts of GR splice variants are expressedin the human placenta with GR120572 being the predominanttranscript in all categories studied These data corroborate aprevious preliminary study of placental GRs [20] Moreoverwe demonstrate for the first time the expression of GAS5 inhuman placentas We decided to incorporate GAS5 in thecurrent study as it can act as a GR DNA binding decoy andas a result compromise its activity [8] Interestingly GAS5and GR120572 were the predominant transcripts in both term andpreterm placentas

Here we provide further information about the regula-tion of this transcript by cortisol using two in vitromodels asit is upregulated by cortisol in a dose-independent mannerThis finding provides further evidence of regulation of GAS5by stress and corroborates initial in vivo data in mice [21]In this study using C57BL6 male mice stress inducedGAS5 RNA levels in the hippocampus and this increase wasaccompanied by a rise of corticosterone levels [22] These invitro and in vivo observations together are highly suggestiveof a functional link between stress and this ncRNAMoreoverwhen we performed RNA FISH the GAS5 transcript waslocalized almost exclusively in the syncytiotrophoblast layer

of the human placenta and colocalizes GR120572 In view ofprevious data in HeLa cells where GAS5 translocates fromthe cytoplasm into the nucleus with GR in response todexamethasone [8] this colocalization is highly suggestive ofa potential crosstalk at placental level between GAS5 and theGR

Preterm labor is associated with high mortality andmorbidity [22] and a recent study pointed towards anassociation between maternal stress and complications ofpregnancy especially preterm birth [23] GR120573 differs fromGR120572 on the C-terminus of the receptor protein There issome controversy surrounding the exact function of GR120573but it appears to exert a dominant-negative effect on GR120572-induced transcriptional activity [8] Moreover it fails tobind glucocorticoids and activate subsequent transcriptionalevents [24] GR120574 is a ligand-dependent transcription factorwith reduced transactivating activity and its function is stillunder investigation In our clinical samples GR120574 is presentat much lower levels than the GR120572 isoform in the placentasstudied Similarly little is known about the role of GR-P atruncated isoform that lacks a large part of the ligand-bindingdomain including the domains for silencing of GR in theabsence of hormone and transcriptional activation [24]

In our study an approximate 4-fold change in theGR120572GR-P in preterm labor was detected Johnson et alhave shown that placental GR-P mRNA levels were reducedsignificantly after spontaneous labor [20] A twofold increasein the GR120572GAS5 ratio was also noted in the preterm groupcompared with the term group of women It is attractivetherefore to hypothesise that the change in the ratio of thesplicing isoforms alters the responsiveness of placental GRs tocortisol and that thismay affect gestational ageWewould liketo propose a potential model where during preterm birthGR120572 is the predominant receptor since there is a decreasein negative regulators such as GR120573 GR120574 and the ldquopseudo-GRErdquo GAS5 These ratio changes will ultimately lead to anaugmented response towards glucocorticoids with potentialdetrimental effects for the mother and the fetus It is possible


that this change of relative transcript abundance ratio mightbe involved in the correlation between negative maternalattitudes (hence highly stressed) and fetal birth weight

In terms of the polymorphic study our study providesa novel insight into the involvement of GR polymorphismsin pregnancy outcome We have identified a specific groupof mothers (ThtIIII polymorphism CC group) in whommaternal attitudes predicted fetal weight In a past studya similar gene-environment interaction was noted betweenthe ER2223EK polymorphism and the effect of childhoodadversity on depression [17]

We acknowledge that measurement of GR polymor-phisms posed certain limitations for example the use of celllines as in vitro experimentalmodels Although both cell lines(JEG-3 andBeWo) could represent a cytotrophoblasticmilieuin vitro they have differences in their fusogenic capacitiesIn addition microarray analyses demonstrated that manytranscripts were differentially expressed between JEG-3 andBeWo cells [25] It would be of interest to repeat these experi-ments in primary cell lines of syncytialised trophoblastsThisexperiment would provide a better insight into the regulationof GAS5 by cortisol However and despite these limitationsthere is a wealth of literature using those two cell lines asexperimental models to study placental function It wouldalso be of interest to expand these observations in humanmyometrium that is a key organ responsible for quiescenceand contractility responses and also assess whether changesin GR transcripts are due to labor or nonlabor To test thiswe have performed qPCR for all GRs and GAS5 in the samecohort of placentas divided this time to labor (119899 = 18) andnonlabor (119899 = 5) There was no apparent change in theexpression of any of the genes in these two categories (datanot shown)Therefore at placental level the contractile statusdoes not really affect GR transcription

We acknowledge that our sample included small numbersin certain categories of polymorphic groups Moreover thenature of maternal stress was not identified and further ana-lytical approaches are needed to provide conclusive evidencefor a gene-environment interaction Nevertheless the statis-tically significant effect and the size of differences observedbetween mothers with negative versus positive and neutralattitudes in theTht111I CC polymorphism group suggest thatthis effect may be robust Second the distribution of BclIpolymorphism seen in this sample may be unique sincethere are important geographicalethnic differences in theprevalence of these polymorphisms It should be emphasizedthat in the present study we included a fairly homogeneouscohort of Mediterranean patients from Crete Despite theselimitations this is the first study to demonstrate a gene-maternal environment synergism in relation to infant birthweight using a very brief assessment of maternal attitudes topregnancy Should these data be replicated in a much widercohort given the simplicity in assessing such attitudes andthe feasibility to identify the homozygous group of womenearly on in pregnancy these findings may have significantimplications for public health and prevention For examplebased on the polymorphic profile and our brief assessmentof mothersrsquo attitude towards the pregnancy we could screennoninvasively and identify mothers during pregnancy that

may benefit from stress-management strategies and thiscould possibly dramatically improve health outcomes for themother as well as the fetus

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] M Lobel D L Cannella J E Graham C DeVincent JSchneider and B AMeyer ldquoPregnancy-specific stress prenatalhealth behaviors and birth outcomesrdquo Health Psychology vol27 no 5 pp 604ndash615 2008

[2] W J E Tissing J P P Meijerink M L den Boer and RPieters ldquoMolecular determinants of glucocorticoid sensitivityand resistance in acute lymphoblastic leukemiardquo Leukemia vol17 no 1 pp 17ndash25 2003

[3] L Pujols JMullol J Roca-Ferrer et al ldquoExpression of glucocor-ticoid receptor 120572- and 120573-isoforms in human cells and tissuesrdquoAmerican Journal of Physiology Cell Physiology vol 283 no 4pp C1324ndashC1331 2002

[4] M Honda F Orii T Ayabe et al ldquoExpression of glucocorticoidreceptor 120573 in lymphocytes of patients with glucocorticoid-resistant ulcerative colitisrdquo Gastroenterology vol 118 no 5 pp859ndash866 2000

[5] R H Oakley J C Webster C M Jewell M Sar and J A Cid-lowski ldquoImmunocytochemical analysis of the glucocorticoidreceptor alpha isoform (GR120572) using a GR120572-specific antibodyrdquoSteroids vol 64 no 10 pp 742ndash751 1999

[6] M de Castro S Elliot T Kino et al ldquoThe non-ligand binding120573-isoform of the human glucocorticoid receptor (hGR120573) tissuelevels mechanism of action and potential physiologic rolerdquoMolecular Medicine vol 2 no 5 pp 597ndash607 1996

[7] C Schneider R M King L Philipson et al ldquoGenes specificallyexpressed at growth arrest of mammalian cellsrdquoCell vol 54 no6 pp 787ndash793 1988

[8] T Kino D E Hurt T Ichijo N Nader and G P ChrousosldquoNoncoding RNA Gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptorrdquo ScienceSignaling vol 3 no 107 p ra8 2010

[9] T F Oberlander J Weinberg M Papsdorf R Grunau S Misriand A M Devlin ldquoPrenatal exposure to maternal depressionneonatal methylation of human glucocorticoid receptor gene(NR3C1) and infant cortisol stress responsesrdquo Epigenetics vol3 no 2 pp 97ndash106 2008

[10] E F C van Rossum and S W J Lamberts ldquoPolymorphismsin the glucocorticoid receptor gene and their associations withmetabolic parameters and body compositionrdquo Recent Progressin Hormone Research vol 59 no 1 pp 333ndash357 2004

[11] L Manenschijn E L T van den Akker S W J Lambertsand E F C van Rossum ldquoClinical features associated withglucocorticoid receptor polymorphismsrdquo Annals of the NewYork Academy of Sciences vol 1179 pp 179ndash198 2009

[12] D Mparmpakas E Zachariades A Goumenou Y Gidronand E Karteris ldquoPlacental DEPTOR as a stress sensor duringpregnancyrdquo Clinical Science vol 122 no 7 pp 349ndash359 2012

[13] D Mparmpakas A Goumenou E Zachariades et al ldquoImmunesystem function stress exercise and nutrition profile can affectpregnancy outcome lessons from a Mediterranean cohortrdquo


Experimental and Therapeutic Medicine vol 5 no 2 pp 411ndash418 2013

[14] Y Koga A Matsuzaki A Suminoe H Hattori S Kanemitsuand T Hara ldquoDifferential mRNA expression of glucocorticoidreceptor 120572 and 120573 is associated with glucocorticoid sensitivity ofacute lymphoblastic leukemia in childrenrdquo Pediatric Blood andCancer vol 45 no 2 pp 121ndash127 2005

[15] E J H Mulder P G Robles de Medina A C Huizink B RH van den Bergh J K Buitelaar and G H A Visser ldquoPrenatalmaternal stress effects on pregnancy and the (unborn) childrdquoEarly Human Development vol 70 no 1-2 pp 3ndash14 2002

[16] R M Reynolds J Labad C Buss P Ghaemmaghami and KRaikkonen ldquoTransmitting biological effects of stress in uteroimplications for mother and offspringrdquo Psychoneuroendocrinol-ogy vol 38 no 9 pp 1843ndash1849 2013

[17] P M Bet B W J H Penninx Z Bochdanovits et alldquoGlucocorticoid receptor gene polymorphisms and childhoodadversity are associated with depression new evidence for agene-environment interactionrdquo American Journal of MedicalGenetics B Neuropsychiatric Genetics vol 150 no 5 pp 660ndash669 2009

[18] M J J Geelhoed EA P Steegers JWKoper et al ldquoGlucocorti-coid receptor gene polymorphisms do not affect growth in fetaland early postnatal life The Generation R Studyrdquo BMCMedicalGenetics vol 11 no 1 article 39 2010

[19] R Bertalan A Patocs B Vasarhelyi et al ldquoAssociation betweenbirth weight in preterm neonates and the BclI polymorphismof the glucocorticoid receptor generdquo Journal of Steroid Biochem-istry and Molecular Biology vol 111 no 1-2 pp 91ndash94 2008

[20] R F Johnson N Rennie V Murphy T Zakar V Clifton andR Smith ldquoExpression of glucocorticoid receptor messengerribonucleic acid transcripts in the human placenta at termrdquoJournal of Clinical Endocrinology and Metabolism vol 93 no12 pp 4887ndash4893 2008

[21] I Meier L Fellini M Jakovcevski M Schachner and FMorellini ldquoExpression of the snoRNA host gene gas5 in thehippocampus is upregulated by age and psychogenic stress andcorrelates with reduced novelty-induced behavior in C57BL6micerdquo Hippocampus vol 20 no 9 pp 1027ndash1036 2010

[22] S Beck D Wojdyla L Say et al ldquoThe worldwide incidence ofpreterm birth a systematic review of maternal mortality andmorbidityrdquo Bulletin of the World Health Organization vol 88no 1 pp 31ndash38 2010

[23] N Roy-Matton J-M Moutquin C Brown N Carrier andL Bell ldquoThe impact of perceived maternal stress and otherpsychosocial risk factors on pregnancy complicationsrdquo Journalof Obstetrics and Gynaecology Canada vol 33 no 4 pp 344ndash352 2011

[24] B Sanchez-Vega N Krett S T Rosen and V Gandhi ldquoGlu-cocorticoid receptor transcriptional isoforms and resistance inmultiple myeloma cellsrdquo Molecular Cancer Therapeutics vol 5no 12 pp 3062ndash3070 2006

[25] D W Burleigh C M Kendziorski Y J Choi et al ldquoMicroarrayanalysis of BeWo and JEG3 trophoblast cell lines identificationof differentially expressed transcriptsrdquo Placenta vol 28 no 5-6pp 383ndash389 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


maternal stress [9] reflecting a possible effect of maternalstress on the expression and function of the GR in thefetus A GR BclI polymorphism has been associated withincreased glucocorticoid sensitivity and was overrepresentedin pregnant women with pathological fetomaternal immuneadaptation Indeed a number of polymorphisms have beendescribed in the gene coding for the GR although it isstill unclear whether the variability in the glucocorticoidresponses observed is due to the polymorphisms or toother factors [10] This sensitivity could be one mechanismlinking maternal stress to fetal development Only few ofthese polymorphisms are functionally relevant and theseare the Tth111I the ER2223EK the N363S themdashalreadymentionedmdashBclI and the GR-9120573 Studies have shown thatat least three polymorphisms are associated with alteredglucocorticoid sensitivity and also with changes in bodycomposition and metabolic parameters [10 11] which couldaffect fetal development as well

This study examined the relationship between mothersrsquostress levels and attitude towards their pregnancies placentalGR and GAS5 expression and the status of GR polymor-phismwith their infantsrsquo birthweightsWe also examined thematernal attitude versus gene interaction in relation to fetalbirth weight Moreover we tested the effect of cortisol on GRsplicing and GAS5 expression of BeWo and JEG-3 placentalcells in an attempt to resemble a low moderate and highstress milieu in vitro

2 Methodology

21 Subjects The study population consisted of pregnantwomen attending the Department of Obstetrics and Gynae-cology University Hospital University of Crete The par-ticipants were in the third trimester of their pregnancyAll participants gave informed consent to participate in thestudy and ethical approval was granted by the local ethicscommittee of the hospital Questionnaires regarding mater-nal stressmaternal attitudes as well as anthropometric datawere collected as previously described [12 13] Briefly mater-nal stressmaternal attitudes towards their pregnancy wereassessed in the 3rd trimester using a questionnaire approachA research nurse or a gynecologist conducted a face-to-face interview with each woman lasting approximately 20ndash30 minutes Maternal attitudes were related to self-reportedstress status ranging from low and medium (low stressresponse) to high and very high (high stress response) Theexact cause of maternal stress was not identified Maternalattitudes were divided into two groups negative attitudes(48) and positive (357)neutral attitudes (163) Imme-diately after delivery the weight of the newbornwas recorded

22 Placental Tissue Placental tissues were obtained fromwomen delivering at term (gt37 weeks of gestation) andpreterm (lt37 weeks of gestation) (total 119899 = 23) Of the 13term placentas 12 were in labor (mode of delivery 10 cae-sarean sections [CS] 2 vaginal deliveries) and 1 nonlaborCStissue None of the term or preterm women were adminis-tered any antidepressants or synthetic glucocorticoids during

pregnancy Of the 10 term placentas 4 were nonlabor and allCS and 6 were from laboring tissues (mode of delivery 3CS 3 vaginal deliveries) The average age was 296 plusmn 4 yearsthe mean pregnancy days were 240 plusmn 42 and mean fetalweight was 2805 plusmn 95 gr After delivery thematernal and fetalsurfaces of the placenta were dissected and fetal membraneswere peeled away from the placenta Placental samples werewashed in PBS and immediately stored in RNAlater (AppliedBiosystems UK) atminus80∘C Ethical approval was granted fromthe local ethics authority

23 Cell Culture BeWo and JEG-3 cell lines were main-tained at standard culture conditions of 5 CO

2in air at

37∘C BeWo cells were cultured in Ham F12 (Sigma-AldrichUK) containing 10 heat-inactivated fetal bovine serum(FBS) and 05 penicillin streptomycin whereas JEG-3 cellswere maintained in MEME (Sigma-Aldrich UK) containing10 heat-inactivated FBS and 05 penicillin streptomycin05 L-glutamine 05 sodium pyruvate and 05 MEMnonessential amino acids Prior to cortisol treatment bothcell lines were maintained for 3 hours in phenol red-freemedia containing charcoal stripped FBS

24 RNA Isolation cDNA Synthesis and PCR Total ribonu-cleic acid was isolated using an RNA extraction kit (Sigma-Aldrich UK) according to manufacturerrsquos instructions RNAconcentration was determined by spectrophotometric analy-sis (NanoDrop Thermo Scientific UK) and agarose gel elec-trophoresis RNA (200 ng from placental tissue and 500 ngfrom cell lysates) was reverse-transcribed into cDNA using5 IU120583L RNase H reverse transcriptase (Invitrogen PaisleyUK)

25 Quantitative RT-PCR Relative expression of the genes ofinterest was assessed by quantitative PCR (Q-PCR) on anABIPrism 7900HT Sequence detection system (Applied Biosys-tems) using SYBR Green-PCR reaction mixture (Sigma-Aldrich UK) and specific primers (Table 1) as previouslydescribed [14] For the quantitative PCR the following equa-tions were used ΔCt = Ct

(gene of interest) minus Ct(house keeping gene)ΔΔCt = ΔCt

(sample) minus ΔCt(calibrator) and Relative Quantity(RQ) = 2minusΔΔCt as previously described [12]

26 Immunofluorescent Analysis Following a series of depar-affinisation and dehydration placental tissue sections wereincubated with 10 bovine serum albumin (BSA) for 1 hrThis was followed by incubation for 1 hr with antibodiesagainst GR120572 at a 1 200 dilution in 1 BSAPBS Cells werethen washed with PBS prior to an incubation with a TRITC-conjugated secondary antibody (Santa Cruz BiotechnologyUSA) for 1 hr Slides were washed with PBS and mountedin Vectashield Mounting Medium (Vector Labs) containingthe dye 46-diamido-2-phenylindole (DAPI) to counterstainnuclei Images were captured using a Plan Apo Neofluor63X NA 125 oil objective (Zeiss) on a Zeiss Axiovert 200Mmicroscope and viewed using the AxioVision software


















3 Results











4 Discussion



045

04

035

03

025

02

015

01

005

0

GR120572 GR120573 GR120574 GR-P GAS5

RQ

val

ues

TermPreterm

(a)O

D u

nits

Term Preterm

15

10

05

00

(b)


Syncytia layer

Cytotrophoblasts

(a) (b)

(c)





16

14

12

1

08

06

04

02

0

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWo JEG-3

lowast

(a)

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWoJEG-3

lowast

lowast

8

7

6

5

4

3

2

1

0

GR120572 GR120573 GR120574 GR-P

(b)


45

40

35

30

25

20

15

10

5

0


lowast

lowast

lowast

lowast

lowast

lowast

Rela

tive q

uant

ity

(RQ

) val

ues

(a)

16

14

12

1

08

06

04

02

0


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(b)

1618

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(c)

16

14

12

1

08

06

04

02

0

Rela

tive q

uant

ity

(RQ

) val

ues


(d)

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(e)






119899 = 81


119899 = 81


119899 = 81
















References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































3 Results











4 Discussion



045

04

035

03

025

02

015

01

005

0

GR120572 GR120573 GR120574 GR-P GAS5

RQ

val

ues

TermPreterm

(a)O

D u

nits

Term Preterm

15

10

05

00

(b)


Syncytia layer

Cytotrophoblasts

(a) (b)

(c)





16

14

12

1

08

06

04

02

0

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWo JEG-3

lowast

(a)

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWoJEG-3

lowast

lowast

8

7

6

5

4

3

2

1

0

GR120572 GR120573 GR120574 GR-P

(b)


45

40

35

30

25

20

15

10

5

0


lowast

lowast

lowast

lowast

lowast

lowast

Rela

tive q

uant

ity

(RQ

) val

ues

(a)

16

14

12

1

08

06

04

02

0


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(b)

1618

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(c)

16

14

12

1

08

06

04

02

0

Rela

tive q

uant

ity

(RQ

) val

ues


(d)

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(e)






119899 = 81


119899 = 81


119899 = 81
















References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















045

04

035

03

025

02

015

01

005

0

GR120572 GR120573 GR120574 GR-P GAS5

RQ

val

ues

TermPreterm

(a)O

D u

nits

Term Preterm

15

10

05

00

(b)


Syncytia layer

Cytotrophoblasts

(a) (b)

(c)





16

14

12

1

08

06

04

02

0

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWo JEG-3

lowast

(a)

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWoJEG-3

lowast

lowast

8

7

6

5

4

3

2

1

0

GR120572 GR120573 GR120574 GR-P

(b)


45

40

35

30

25

20

15

10

5

0


lowast

lowast

lowast

lowast

lowast

lowast

Rela

tive q

uant

ity

(RQ

) val

ues

(a)

16

14

12

1

08

06

04

02

0


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(b)

1618

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(c)

16

14

12

1

08

06

04

02

0

Rela

tive q

uant

ity

(RQ

) val

ues


(d)

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(e)






119899 = 81


119899 = 81


119899 = 81
















References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















16

14

12

1

08

06

04

02

0

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWo JEG-3

lowast

(a)

Relat

ive q

uant

ity (R

Q) v

alue

s

BeWoJEG-3

lowast

lowast

8

7

6

5

4

3

2

1

0

GR120572 GR120573 GR120574 GR-P

(b)


45

40

35

30

25

20

15

10

5

0


lowast

lowast

lowast

lowast

lowast

lowast

Rela

tive q

uant

ity

(RQ

) val

ues

(a)

16

14

12

1

08

06

04

02

0


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(b)

1618

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(c)

16

14

12

1

08

06

04

02

0

Rela

tive q

uant

ity

(RQ

) val

ues


(d)

14121

080604020


10nM 100nM 1000nM

Rela

tive q

uant

ity

(RQ

) val

ues

(e)






119899 = 81


119899 = 81


119899 = 81
















References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















119899 = 81


119899 = 81


119899 = 81
















References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article differential expression of placental...

Documents