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The fetal origins of adult disease, the evidence and mechanisms

Veenendaal, M.V.E.

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Marjolein Veenendaal

UITNODIGING

voor het bijwonen van de openbare verdediging van het proefschrift

The fetal origins of adult disease, the

evidence and mechanisms

door Marjolein Veenendaal

Op donderdag 25 oktober 2012om 14.00 uur in de Agnietenkapel,

Oudezijds Voorburgwal 231te Amsterdam

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Marjolein VeenendaalJohannes van der Waalsstraat 48 hs

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The fetal origins of adult disease, the evidence and mechanism

s Marjolein Veenendaal



UITNODIGING











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UITNODIGING











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The fetal origins of adult disease, the evidence

and mechanisms


Lay-out: LegatronElectronicPublishing,RotterdamPrinting: IpskampDrukkersBV,Enschede

ISBN/EAN:978-94-6191-437-8

2012©M.V.E.Veenendaal

No part of this thesismay be reproduced, stored in a retrieval systemor transmitted in anyformorbyanymeans,withoutwrittenpermissionoftheauthoror,whenappropriate,ofthepublishersofthepublications.


ACADEMISCHPROEFSCHRIFT

terverkrijgingvandegraadvandoctor

aandeUniversiteitvanAmsterdam

opgezagvandeRectorMagnificus

prof.dr.D.C.vandenBoom

tenoverstaanvaneendoorhetcollege

voorpromotiesingesteldecommissie,

inhetopenbaarteverdedigen

indeAgnietenkapel

opdonderdag25oktober2012,

te14:00uur

door

Marjolein Vera Elisabeth Veenendaal

geborenteErmelo

ProMoTiEcoMMissiE

Promotores Prof.dr.P.M.M.Bossuyt Prof.dr.J.A.M.vanderPost

Co-promotores Dr.T.J.Roseboom Dr.R.C.Painter

Overigeleden Prof.dr.R.J.B.J.Gemke Dr.J.A.Joles Prof.dr.B.W.J.Mol Prof.dr.R.J.P.M.Scholten Prof.dr.R.P.M.Steegers-Theunissen Dr.C.S.P.M.Uiterwaal

FaculteitderGeneeskunde

HetonderzoekdataanditproefschrifttengrondslagligtismogelijkgemaaktdooreensubsidievandeNederlandseHartstichting(NHS-2007B083).Het verschijnen van dit proefschrift werd mede mogelijk gemaakt door de steun van deNederlandseHartstichting.

conTEnTs

chapter 1 Introduction 7

chapter 2 Isthefetaloriginshypothesisofdiabetessupportedbyanimalresearch? 15 Asystematicreviewandmeta-analysisoftheevidence

Submitted

chapter 3 Thefetaloriginsofhypertension:asystematicreviewandmeta-analysisof 31 theevidencefromanimalexperimentsofmaternalundernutrition

Journal of Hypertension, accepted for publication

chapter 4 Gripstrengthatage58afterprenatalexposuretotheDutchfamine 55The Journal of Aging Research and Clinical Practice, 2012 vol 1

chapter 5 Prenatalfamineexposure,healthinlaterlifeandpromotermethylation 65 offourcandidategenes

Journal of Developmental Origins of Health and Disease, accepted for publication

chapter 6 AssociationsbetweenDNAmethylationofaglucocorticoidreceptor 81 promoterandacutestressresponsesinalargehealthyadultpopulation arelargelyexplainedbylifestyleandeducationialdifferences

Psychoneuroendocrinology. 2012;37(6):782-8

chapter 7 Transgenerationaleffectsofprenatalexposuretothe1944-45Dutchfamine 95Submitted

chapter 8 Consequencesofhyperemesisgravidarumforoffspring: 105 asystematicreviewandmeta-analysis

BJOG 2011;118(11):1302-1313

chapter 9 Summaryanddiscussion 125

Nederlandsesamenvatting 135Dankwoord 139CurriculumVitae 143

1Introduction

8

Chapter 1

Manystudieshavedemonstratedthatprenatalundernutritionisassociatedwiththedevelopmentofanumberofage-relateddiseasesinlaterlife.Inthelate1980’sDavidBarkerwasthefirsttodescribe an association between fetal development and adult disease. Using birthweight asaproxyfor fetaldevelopment,hefoundthatbirthweightwas inverselyassociatedwithadultsystolic blood pressure1. Moreover, in a cohort of men born in Hertfordshire between 1911and1930, he reported that thosebornwith the lowestbirthweightshad thehighest riskofdeathfromischemicheartdisease2.Withthisobservation,thefetaloriginshypothesiswasborn,suggestingthatundernutritionearlyindevelopment,andparticularlyduringintrauterinelife,canleadtopermanentchangesinphysiologyandmetabolism,whichresultinincreaseddiseaseriskinadulthood.Afterinitialscepsisandmuchdebatethefetaloriginsofadultdiseasehypothesisbecamewidelyacceptedandsupportedbymanysimilarfindingsinpopulationsworldwide. Toexperimentally test the fetaloriginshypothesis animalexperimentsarenecessary. Thefield of animal research regarding the fetal origins hypothesis has expanded rapidly over theyears,usingdifferentspeciesandexposures.Initiallytheseweredescriptive,providingevidenceforthecausalrelationshipbetweenearlylifeexposuresandmetabolicriskfactorsinlaterlife.Inthemorerecentyearsthefocushaschangedtounravellingtheunderlyingmechanisms.Thishasledtoanabundanceofstudieswithnotalwaysagreeingresults.Tostudythefetaloriginshypothesis in humans,we can study people that have been exposed to theDutch famine inutero.

ThE DuTch faMinE

TheDutchfaminewasafivemonthperiodattheendofWorldWarIIduringwhichtheurbanwesternpartoftheNetherlandswasstruckbyaseverefamine.AfterthesouthoftheNetherlandshadbeenliberatedbytheAlliedforcesinSeptember1944,theDutchgovernmentinexilecalledfora railwaystrike toaid the liberationof theprovinces stilloccupiedby theGerman forces.Despite the railway strike, theAllieswerenot able topass the riverRhine.As a reprisal, theGermanadministrationputanembargoonallfoodtransports.Foodstocksranoutinamatterofweeks.Rationsdroppedto400to800caloriesperday,lessthanaquarterofthepre-faminelevels.After liberation,thefoodsituationquickly improvedandrationsroseto2000calories3.Thefaminewasnodoubtahumanitariandisaster,butturnedouttobeauniqueopportunitytostudytheconsequencesofprenatalundernutritiononhealthinlaterlife.Thefactthatthefaminelasted5monthsandstruckapopulationthatwaswellfedbeforethefamineincombinationwiththefactthatfoodsuppliesimprovedquicklyafterliberation,allowedustostudytheeffectsofprenatalundernutritiononspecificpartsofgestation. TheDutchfaminebirthcohortisacohortof2414babies,allbornastermsingletonsintheWilhelminaGasthuisinAmsterdamwhosebirthrecordshavebeenkept.Thiscohortgaveusthe

9

Introduction

1opportunitytostudytheeffectsofmaternalundernutritionduringgestationoftheoffspring’shealth. Two previous rounds of data collection at age 50 and 58 have shown that maternalundernutritionduringgestationhaslastingnegativeconsequencesfortheoffspring’shealth.Theeffectsdependonthetimingduringgestationandtheorgansandtissuesdevelopingatthattime.Exposuretofamineduringanypartofgestationwasassociatedwithraisedglucoselevelsatadultage4,5,possiblyduetoaninsulinsecretiondefect6.Peopleexposedtofamineinearlygestationhadalteredbloodcoagulation7andamoreatherogeniclipidprofile8.Exposuretofamineinearlygestationwas also found to be associatedwith increased blood pressure response to stress9 andanincreasein,andearlieronsetofcoronaryarterydisease10,11.Womenwhowereexposedtofamineprenatallyhadmorechildren,moretwinsandstartedreproducingatanearlieragecomparedtounexposedwomen.Thesewomenalso lessoftenremainedchildless12.Astrikingfindingwasthefact thattheeffects foundwere independentof thesizeof thebabyatbirth,whichmay imply that adaptations that enable the fetus to continue to grow inunfavourablecircumstancesmayhaveadversehealthconsequencesinlaterlife. Inthelatestroundofdatacollection,thefirstevidenceoftransgenerationaleffectsoffamineexposurebecameclear.Grandmaternalexposuretofamineduringgestationdidnotaffectbirthweight or prevalence of cardiovascular or metabolic disease12. But grandoffspring was moreadiposeatbirth,andchildrenofwomenthathadbeenexposedtofamineinuterohadpoorerhealth12. This first indication of transgenerational effects of famine exposure is in line withevidence fromanimalexperimentswhereadverseeventsduringgestationnotonlyaffect theoffspringofthatpregnancy,butalsohaseffectsonthenextgeneration.Theeffectoffeedingratsa lowproteindietduringpregnancy forseveralgenerationstookthreegenerationsofnormalfeeding for fetal growth and development to return to normal13. The underlyingmechanismthatisthoughttoserveasamemoryofearlylifeexposuresandleadingto(transgenerational)changesingeneexpressionandpotentiallydiseaseinlaterlifeisepigenetics.

EPigEnETics

Epigenetics refers to processes that induce heritable changes in gene expression potentialwithoutalteringthegenesequence14.OneofthemajorepigeneticmechanismsismethylationofCpGnucleotides.MethylationofCpG’swithingenepromotersisassociatedwithtranscriptionalinactivation,incontrast,unmethylatedpromotersarepotentionallytranscriptionallyactive15.Inadditiontogenesilencingbypromotermethylation,differentialmethylationofindividualCpG’scaninducesubtlechangesintranscriptionalactivity. For example, feeding pregnant rats a protein restricted diet induced hypomethylation ofthe peroxisomal proliferator-activated receptor α (PPAR α) and glucocorticoid receptor (GR)promoters and increased the expressionof PPARαandGR in the livers of juvenile and adult

10

Chapter 1

offspring16,17. Thefirst evidenceof epigeneticprogramming afterprenatal famineexposure inhumanscamefromtheDutchfaminefamiliesstudy18.Menandwomenwhohadbeenexposedto famine in early gestation had hypomethylation of the differentially methylated region ofinsulin-like growth factor-2 gene compared to unexposed same-sex siblings18. Further studiesfrom this group suggested that the effects of prenatal famine exposure on methylation aresex- and timing specific19. Thus, both animal and human studies suggest that changes in theintrauterineenvironmentcanleadtoalteredgeneexpressionviaalterationsinDNAmethylation,possiblyresultinginanincreasedsusceptibilitytochronicdiseaseinadulthood20. The finding that the developing fetus is sensitive to its environmentmay be relevant tocurrentpregnancies. Thenutritional experienceof fetusexposed to famine inearlygestationmayresemblethatoffetuswhosemotherssufferfromhyperemesisgravidarum,asevereformofnauseaandvomitinginearlypregnancy.TheresultsfromtheDutchfaminestudyhaveshownthat the adverse effects of prenatal undernutritionwere present despite the absence of anyeffectonthesizeofthebabyatbirth.Thereforetheassumptionthatlongtermconsequencesofhyperemesisgravidarummaybelimitedbecauseofthenormalbirthweightofthebabyatbirthnolongerholds.

aiM anD ouTlinE of This ThEsis

Theworkpresentedinthisthesisexploresdifferentaspectsofthefetaloriginshypothesis. Humanstudiesontheassociationbetweenbirthweightandhealth in later lifehavebeensystematicallyreviewed21-24andgenerallysupportthefetaloriginshypothesis.Theyshowthatbirth weight is inversely related to systolic blood pressure22, type 2 diabetes risk23, ischemicheartdisease21 andmortality24. Theevidence for thishypothesis fromanimal studieshasnotbeenreviewed.There isa largebodyofevidencefromanimalstudiesexploringtheeffectsofundernutritionduringgestationonthehealthoftheoffspring.Inthesestudies,differentspeciesanddietaryregimensareused.Wesystematicallyreviewedanimalexperimentsconcerningthefetaloriginshypothesisconsideringtheeffectsonglucose-andinsulinmetabolism(chapter 2)andonbloodpressure(chapter 3). Inchapter 4theeffectsofprenatalexposuretotheDutchfamineonhandgripstrengtharereported.chapter 5 describeswhetherprenatal exposure to faminealtersmethylation levelsof promoter regions of 4 candidate genes involved in cardiovascular andmetabolic disease,andwhether there is an association betweenmethylation levels of these genes and lifestyleanddiseasemarkers.Theassociationbetweenmethylationof thepromoter regionof theGRreceptorandstressisdescribedinchapter 6.Whethertheadverseeffectsofprenatalexposuretofamineareconfinedtotheoffspringorarepassedontothenextgenerationisthesubjectofchapter 7.Maternalundernutritionduringpregnancyisstillpresentintheformofhyperemesis

11

Introduction

1gravidarum.chapter 8 is a systematic review of the literature on the effects of hyperemesisgravidarumonthechildren.chapter 9isasummaryofthisthesisanddiscussestheimplicationsofthefindingsreportedhereforfurtherresearch.

12

Chapter 1

rEfErEncE lisT

1. BarkerDJP,BullAR,OsmondC,SimmondsSJ.Fetalandplacentalsizeandriskofhypertensioninadultlife.BMJ.1990;301:259-262.

2. Barker DJP,Winter PD, Osmond C,Margetts B.Weight in infancy and death from ischaemic heartdisease.Lancet.1989;9:577-580.

3. Burger GCE, Sandstead HR, Drummond JC. Malnutrition and Starvation in Western Netherlands,September1944toJuly1945.PartIandII.TheHague:GeneralStatePrintingOffice;1948.

4. deRooijSR,PainterRC,RoseboomTJ,PhillipsDI,OsmondC,BarkerDJ,etal.Glucosetoleranceatage58andthedeclineofglucosetoleranceincomparisonwithage50inpeopleprenatallyexposedtotheDutchfamine.Diabetologia.2006;49:637-643.

5. RavelliAC,vanderMeulenJH,MichelsRP,OsmondC,BarkerDJ,HalesCN,etal.Glucosetoleranceinadultsafterprenatalexposuretofamine.Lancet.1998;351:173-177.

6. deRooijSR,PainterRC,PhillipsDI,OsmondC,MichelsRP,GodslandIF,etal.Impairedinsulinsecretionafterprenatalexposuretothedutchfamine.Diabetes Care.2006;29:1897-1901.

7. RoseboomTJ,vanderMeulenJHP,RavelliACJ,OsmondC,BarkerDJP,BlekerOP.PlasmafibrinogenandfactorVIIconcentrationsinadultsafterprenatalexposuretofamine.British Journal of Haematology. 2000;111:112-117.

8. RoseboomTJ,vanderMeulenJHP,OsmondC,BarkerDJP,RavelliACJ,BlekerOP.PlasmalipidprofilesinadultsafterprenatalexposuretotheDutchfamine.AJCN.2000;72:1101-1106.

9. PainterR.C.,deRooijSR,BossuytPM,PhillipsDI,OsmondC,BarkerDJ,etal.BloodpressureresponsetopsychologicalstressorsinadultsafterprenatalexposuretotheDutchfamine.Journal of Hypertension. 2006;24:1771-1778.

10. PainterRC,deRooij SR,RoseboomTJ,BossuytPMM,SimmersTA,OsmondC, et al. EarlyonsetofcoronaryarterydiseaseafterprenatalexposuretotheDutchfamine.AJCN.2006;84:322-327.

11. RoseboomTJ,vanderMeulenJHP,OsmondC,BarkerDJP,RavelliACJ,TankaJS,etal.CoronaryheartdiseaseafterprenatalexposuretotheDutchfamine,1944-45.Heart.2000;84:595-598.

12. PainterRC,WestendorpRG,deRooijSR,OsmondC,BarkerDJ,RoseboomTJ.Increasedreproductivesuccessofwomenafterprenatalundernutrition.Hum Reprod.2008;23:2591-2595.

13. Stewart RJ, Sheppard H, Preece R,Waterlow JC. The effect of rehabilitation at different stages ofdevelopmentofratsmarginallymalnourishedfortentotwelvegenerations.Br J Nutr.1980;43:403-412.

14. BirdA.DNAmethylationpatternsandepigeneticmemory.Genes Dev.2002;16:6-21.15. Burdge GC, Lillycrop KA. Nutrition, epigenetics, and developmental plasticity: implications for

understandinghumandisease.Annu Rev Nutr.2010;30:315-339.16. BurdgeGC,Slater-JefferiesJ,TorrensC,PhillipsES,HansonMA,LillycropKA.Dietaryproteinrestriction

ofpregnantrats intheF0generationinducesalteredmethylationofhepaticgenepromoters intheadultmaleoffspringintheF1andF2generations.Br J Nutr.2007;97:435-439.

17. LillycropKA,PhillipsES,JacksonAA,HansonMA,BurdgeGC.Dietaryproteinrestrictionofpregnantratsinducesandfolicacidsupplementationpreventsepigeneticmodificationofhepaticgeneexpressionintheoffspring.J Nutr.2005;135:1382-1386.

18. HeijmansBT,TobiEW,SteinAD,PutterH,BlauwGJ,SusserES,etal.Persistentepigeneticdifferencesassociatedwithprenatalexposuretofamineinhumans.Proc Natl Acad Sci U S A.2008;105:17046-17049.

19. TobiEW,LumeyLH,TalensRP,KremerD,PutterH,SteinAD,etal.DNAmethylationdifferencesafterexposuretoprenatalfaminearecommonandtiming-andsex-specific.Hum Mol Genet.2009;18:4046-4053.

20. WaterlandRA,JirtleRL.Earlynutrition,epigeneticchangesattransposonsandimprintedgenes,andenhancedsusceptibilitytoadultchronicdiseases.Nutrition.2004;20:63-68.

13

Introduction

121. HuxleyR,OwenCG,WhincupPH,CookDG,Rich-EdwardsJ,SmithGD,etal.Isbirthweightariskfactor

forischemicheartdiseaseinlaterlife?Am J Clin Nutr.2007;85:1244-1250.22. HuxleyRR,ShiellAW,LawCM.Theroleofsizeatbirthandpostnatalcatch-upgrowthindetermining

systolicbloodpressure:asystematicreviewoftheliterature.J Hypertens.2000;18:815-831.23. WhincupPH,KayeSJ,OwenCG,HuxleyR,CookDG,AnazawaS,etal.Birthweightandriskoftype2

diabetes:asystematicreview.JAMA.2008;300:2886-2897.24. Risnes KR, Vatten LJ, Baker JL, Jameson K, Sovio U, Kajantie E, et al. Birthweight andmortality in

adulthood:asystematicreviewandmeta-analysis.Int J Epidemiol.2011;40:647-661.

2is the fetal origins hypothesis of diabetes

supported by animal research? A systematic review and meta-analysis

of the evidence

MarjoleinVEVeenendaalShakilaThangaratinam

DerickYatesRebeccaCPainterSusanneRdeRooij

JorisAMvanderPostPatrickMMBossuytGeorgeRSaadeBenWillemJMolKhalidSKhan

TessaJRoseboom

Submitted

16

Chapter 2

absTracT

The fetal programming hypothesis states that fetal undernutrition during pregnancy resultsin permanent changes in the offspring’smetabolism. A large number of animal studies haveevaluatedtheeffectoffetalundernutritiononlatersusceptibilitytotype2diabeteswithvaryingresults.aim:We systematically reviewed the existing animal literature examining effects of prenatalundernutritiononglucoseandinsulinmetabolism.Methods: An electronic searchwasperformed inMedline and Embase to identify all articlesthatreportedstudiesinvestigatingtheeffectoffetalundernutritiononplasmainsulin,plasmaglucoseandbetacellmassinanimalmodels.Summaryestimatesoftheeffectofundernutritiononmeanglucoseconcentration,insulinlevel,andbetacellmasswereobtainedthroughmeta-analysis.results:Thesearchresultedin1827articles,ofwhich117werepotentiallyeligible,basedontitleandabstract,and49mettheselectioncriteriaandwereincludedinthereview.Prenatalproteinrestriction increasedplasmaglucoseconcentrations(0.42mmol/l (95%CI0.07to0.77)).Bothgeneralundernutritionandprotein restrictionreducedplasma insulinconcentrations (generalundernutrition:-0.03nmol/l(95%CI-0.04to-0.01),proteinrestricted:-0.04nmol/l(95%CI-0.08to0.00))andbetacellmass(generalundernutrition:-1.24mg(95%CI-1.88to-0.60),proteinrestriction:-0.99mg(95%CI-1.67to-0.31)).Inallcases,heterogeneitywassignificant.Conclusions: Despitesignificantheterogeneity,evidencefromexperimentsindifferentspeciessuggeststhatprenatalundernutrition–bothgeneralorproteinrestriction–resultsinincreasedglucoseandreducedinsulinconcentrationsaswellasbetacellmassinlaterlife.

17

Fetal origins of diabetes in animals

2

inTroDucTion

In the early 1990s, a cohort study of 64-year-old men in Hertfordshire revealed an inverseassociationbetweenbirthweightandglucoseconcentrationsand insulin resistance1.Subjectswiththelowestbirthweightswere6timesmorelikelytodeveloptype2diabetesorimpairedglucosetolerancethanthosewithhighestbirthweights.Thesefindingsledtothe‘fetaloriginshypothesis’, stating that fetal adaptations to reduced nutrient supply predispose to impairedglucosetoleranceandtype2diabetesinadultlife2.Since,morethan40studiesinpopulationsacrosstheworldhaveinvestigatedtheassociationbetweensizeatbirthandlaterriskoftype2diabetes3.Asystematicreviewofhumanstudiesonbirthweightandtype2diabetesconfirmedaninverserelationshipbetweenbirthweightandtype2diabetes3. Birthweight,however,isonlyaproxyforpoormaternalnutritionduringgestation.Animalmodelsallowustoexperimentallystudytheeffectsofmaternalundernutritionduringgestationon glucose and insulinmetabolism.While the number of animal studies is increasing,manydifferentmodels are used, ranging from large species as sheep to small rodentmodels. Theinterventionstudiesincludeavarietyofdifferentdietaryregimens,varyingfromundernutritionduringonlypartofgestation,toundernutritionduringtheentirepre-andearlypostnatallife.Theconclusionsofthesestudieshavebeendiverging,withsomeofferingsupportforthehypothesis,whileothersdonot.Theseinconsistenciesmightbeduetothedifferencesindietaryregimensorstrainsorspeciesofanimalsused.Thereforewesystematicallyreviewedtheliteratureonfetalundernutrition and glucose and insulinmetabolism in animal studies and usedmeta-analysistoobtain summaryestimatesof theeffectsofmaternalnutritionduringgestationonplasmaglucose,insulinandbetacellmass.

METhoDs

search strategyWeperformedasearchintheelectronicdatabasesMedline(1951-January2011)andEmbase(1980-January 2011) to identify all articles that reported on fetal undernutrition and plasmainsulin,plasmaglucoseandbetacellmassasdiabetes-relatedoutcomesinexperimentalanimalstudies.Thesearchterms‘undernourished’, ‘(fetal)malnutrition’, ‘famine’, ‘starvation’, ‘caloricrestriction’, ‘protein restriction’, ‘low protein diet’, ‘low calorie diet’, ‘pregnancy’, ‘diabetes’,‘glucosemetabolism’,‘glucose’,‘insulinmetabolism’,‘insulin’and‘betacellmass’wereused.OnlyarticleswritteninEnglishwereincluded.Afterscreeningoftitlesandabstracts,tworeviewersindependentlyexaminedfulltextarticlesandextracteddataonstudycharacteristics,qualityandresults.Referencelistsofreviewsandrelevantpaperswerehandsearched.

18

Chapter 2

Study selectionWeincludedstudiesthatprovideddatadescribingoutcomesinexperimentalanimalmodelsofprenatalundernutrition that reportedonplasmaglucose,plasma insulinorbeta cellmassasmeasuresofoutcome.Prenatalundernutrition included lowproteinmalnutritionandgeneralcaloric malnutrition. Studies had to report outcomes in comparison to control animals thatwereborntoamotherthatwasnormally fedthroughoutpregnancy.Eligibilitywasevaluatedindependentlybytworeaders.Disagreementswereresolvedinconsensusdiscussions.

Data extractionTworeviewers independentlyextracted informationonstudydesign,exposureperiod,animalspecies and type of undernutrition. To assess methodological quality, data on allocationconcealment,randomization,blindingandsamplesizecalculationwereextracted.Whenmorethan two experimental groups were formed, we focused on the experimental group withmalnutritionasearlyinpregnancyaspossibleandpreferablylimitedtopregnancyalone.Whenoutcomeinoffspringwasmeasuredatmultipletimepoints,wechosetheoldestageatwhichthemeasurementsweretaken.Whenmultiplegroupsweremeasuredatdifferentages,bothagegroupswere included. If resultswereonlydisplayedgraphically,outcomewasreadaspreciseas possible. Studies that reported results asmean and standard deviation or standard error,andnumberofanimalspergroupwereusedformeta-analysis.Dataonplasmaglucose,plasmainsulinandbetacellmasswereconvertedtommol/l,nmol/landmg,respectively.

Statistical analysisDatawereanalyzedusingReviewManagerVersion5.0.Toexaminepotentialpublicationbiasweconstructedfunnelplots.WeexaminedthepossibleheterogeneityinresultsacrossstudiesbycalculatingtheI²statistic. Summaryestimatesoftheeffectsofundernutritionwereobtainedusingarandomeffectsmodelformeta-analysis,whichaccountsforbothwithin-andbetween-studyvariability.Separateestimateswereobtainedformodeltype(proteinorgeneralmalnutrition)andoutcomemeasure(plasmaglucose, plasma insulin andbeta cellmass). The summaryeffectswereexpressedasmeandifferencewith95%confidenceintervals(CI).Whensignificantstatisticalheterogeneitywasdetected,thesourcesofheterogeneitywereexploredandsubgroupanalyseswereperformedfordifferentspecies,animalsex,differentexperimentalregimentsorinanimalsofdifferentagesattimeofmeasurement.Toevaluatetherobustnessofourresultsagainstinfluentialstudies,aleaving-one-outsensitivityanalysiswasperformed.

19


2

rEsulTs

The search resulted in 1827 articles, of which 117 were considered potentially eligible afterscreeningtitlesandabstracts(MVandST).Afterreadingfulltextarticles(MVandeitherDY,RPorST),49primarystudiesmettheinclusioncriteriaandweresuitablefordataextraction(Figure1).Twenty-sixstudiesreportedonproteinrestrictedundernutrition,oneusingamousemodel4, andtwenty-fiveusingaratmodel5-29.Twenty-fourreportedongeneral(caloric)undernutrition,onestudyusingguineapigs30,twoonamousemodel31,32,fiveusingasheepmodel33-37and16studiesonrats7,8,13,38-51.

figure 1Literaturesearchresultsforpublicationsreportingonprenatalundernutritionwithregardtoglucoseandinsulinmetabolism.

1827 poten�ally eligible studies iden�fied(database searches and references lists)

117 full text ar�cles reviewed

1710 studies were excluded based on theinclusion criteria and �tle and abstract review

68 studies excluded for not having the required exposure / not repor�ng the outcome of

interest / not repor�ng data in a form fit for meta-analysis / non-animal

49 studies were included in the meta-analysis

Methodological aspectsOnlyonestudyreportedblindingoftheinvestigator52.Randomizationwasreportedintwenty-fourstudies,eitherrandomizationtothedietaryregimenorrandomlyselectingthepupsthatwere studied from the litters14,15,17-24,26,28,29,31,32,34-36,39,42,44,48,50,53,54.Noneof the studies reportedasamplesizecalculationormethodsforconcealmentofallocation.Funnelplotsofallsixoutcomesshowedsymmetricalscatteringofthestudyresultsaroundthesummaryestimate.Therewasnoevidenceofasmallstudyeffectorpublicationbias.

20

Chapter 2

Plasma glucose after prenatal low protein dietTwenty-two primary animal studies provided data for meta-analysis (464 undernourishedanimals, 464 controls). Twenty-one studies were performed using rats7-16,18-21,23-29, one using a mouse model4. Using the random effects model we found a higher mean plasma glucoselevel inprenatallyundernourishedanimalscomparedtothecontrolgroup:ameandifferenceof 0.42 mmol/l (95% CI 0.07 to 0.77) (Figure 2). The results showed statistically significantheterogeneity(I²89%).Theheterogeneitypersistedevenafterseparatelypoolingfastingvalues,stratifyingforthesexoftheoffspring,or limitingtheanalysistoWistarratsonly.Offspringoflow protein undernourished adults that were older than 6 weeks of age had a 0.54mmol/lhigher plasma glucose level (95%CI 0.16 to 0.92) compared to control offspring. But glucoseconcentrationsmeasuredatday0werelowerinundernourishedoffspringcomparedtocontrolswithameandifferenceof-0.62mmol/l(95%CI-1.34to0.11).Inbothcases,heterogeneitywassubstantial,withanI²of89%and69%respectively.

figure 2 Forest plot of mean differences and 95% CIs in plasma glucose concentrations (mmol/l)afterprenatallowproteinundernutritioninallanimalstudies.Study-specificmeandifferenceswerecombinedbyusingarandom-effectsmodel.SD,standarddeviation.UN,undernourished.

21


2

Plasma glucose after prenatal general malnutritionTwentystudiesprovideddataonplasmaglucoseinoffspringafterprenatalcaloricmalnutrition.Twelvestudieshadbeenperformedinrats7,8,13,38,39,41,43-47,49,51,oneinmice31,oneinguineapigs30, and5usingasheepmodel33-37.Intotal,301undernourishedanimalsweredescribed,comparedto339controls.Themeanplasmaglucoselevelwas0.05mmol/lhigher(95%CI-0.14to0.24)inundernourishedanimalscomparedtocontrols(Figure3).Themeta-analysisshowedstatisticallysignificantheterogeneity(I²84%).Subgroupanalysisofrodentmodelsonly,stratifyingforspecies,fastingvaluesorsex,didnotremoveheterogeneity.Undernourishedanimalsmeasuredatday0hadasignificantlylowerplasmaglucoselevel,-0.49(95%CI-0.87to-0.11)mmol/l(I²78%)asopposedtorodentsolderthan6weeks,whichhadahigherplasmaglucoselevel:0.25(95%CI0.04to0.46)mmol/l (I²79%).Meta-analysisof theeffectsonsheeponly (71undernourishedanimals,79controls)showednosignificantdifference inglucoseconcentrations,withameandifferenceof0.03mmol/l(95%CI-0.31to0.26)(I²43%).

figure 3Forestplotofmeandifferencesand95%CIsinplasmaglucoseconcentrations(mmol/l)afterprenatalgeneralundernutritioninallanimalstudies.Study-specificmeandifferenceswerecombinedbyusingarandom-effectsmodel.SD,standarddeviation.UN,undernourished.

22

Chapter 2

Plasma insulin after prenatal low proteinData formeta-analysiswere available fromnineteen experimental studies.One study used apigmodel5,oneusedamousemodel4,andtheremaining17studieswereperformed inaratmodel7,8,10,11,13-19,21,24-26,28,29.Themeta-analysis,usingdata from377 lowproteinundernourishedanimalsand382controls,showedalowermeanplasmainsulinlevelinundernourishedoffspringcompared to control offspring, with amean difference of 0.04 nmol/l (95%CI -0.08 to 0.00)(I²95%) (Figure4). Theheterogeneitypersistedafter separatelypoolinganimalsaccording tospecies,sexorageorseparatelyanalyzingfastingvalues.

figure 4Forestplotofmeandifferencesand95%CIsinplasmainsulinconcentrations(nmol/l)afterprenatal low protein undernutrition in all animal studies. Study-specific mean differences werecombinedbyusingarandom-effectsmodel.SD,standarddeviation.UN,undernourished.

Plasma insulin after prenatal general malnutritionIn themeta-analysiswe could includedata from21 studies, obtained in330undernourishedanimals and 358 controls. Fourteen experiments were conducted in rats7,8,12,13,38,39,43-49,51, 4 insheep33,35-37,2inmice31,32andoneinguineapigs30.Themeanplasmainsulinlevelwas0.03nmol/llower(95%CI-0.04to-0.01)intheundernourishedgroupcomparedtocontrolanimals,I²86%(Figure5).Theheterogeneityremainedafterstratificationbyfastingvalues,sex,rodentspeciesorage.

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2

figure 5Forestplotofmeandifferencesand95%CIsinplasmainsulinconcentrations(nmol/l)afterprenatalgeneralundernutritioninallanimalstudies.Study-specificmeandifferenceswerecombinedbyusingarandom-effectsmodel.SD,standarddeviation.UN,undernourished.

In rats at day 0, there was no significant effect of prenatal undernourishment on plasmainsulin,withameandifferenceof0.23nmol/l (95%CI -0.67 to0.21) (I² 91%).However, adultundernourishedratshadalowerplasmainsulinlevelthancontrols,withameandifferenceof0.04nmol/l(95%CI-0.07to-0.01)(I²91%).Thefoursheepstudies(66undernourishedanimals,74controls)didnotshowanydifferenceinthemeanfastingplasmainsulinlevel(0.00nmol/l;95%CI-0.01to0.01,I²4%)33,35-37.

Beta cell mass after prenatal low proteinFiveratstudiesreportedbetacellmassofoffspring(94undernourished,92controlanimals)6-8,13,22.Thebetacellmasswaslowerintheundernourishedoffspringcomparedtocontroloffspring,withameandifferenceof-1.24mg(95%CI-1.88to-0.60)(Figure6).Therewasstatisticallysignificantheterogeneity,I²97%.

24

Chapter 2

figure 6 Forestplotofmeandifferencesand95%CIsinbetacellmass(mg)afterprenatallowproteinundernutrition in all animal studies. Study-specific mean differences were combined by using arandom-effectsmodel.SD,standarddeviation.UN,undernourished.

Beta cell mass after prenatal general malnutritionThe9studiesonrats(91undernourishedand91controlanimals)7,8,13,38,40,42-44,49showedareductionin beta cellmass of 0.44mg (95%CI -0.75 to -0.13) in undernourished animals compared tocontrols.Theresultsshowedstatisticallysignificantheterogeneity(I²94%)(Figure7).

figure 7 Forestplotofmeandifferencesand95%CIs inbetacellmass (mg)afterprenatal generalundernutrition in all animal studies. Study-specific mean differences were combined by using arandom-effectsmodel.SD,standarddeviation.UN,undernourished.

Sensitivity analysisInaseriesofsensitivityanalysis,weevaluatedtherobustnessofourfindingsbyrepeatingtheanalysesanumberoftimes,eachtime leavingone studyoutof themeta-analysis. If a studyappearstobeanoutlier,withresultsverydifferentfromtherestofthestudies,thenitsinfluencewillbecomeapparent,astheresultwithoutthestudywouldbeverymuchdifferentfromthe

25


2

resultofthemeta-analysisofallthestudies.Allsensitivityanalyses,foreachofthesixoutcomemeasuresevaluated,confirmedthestabilityofouranalysis.Noinfluentialindividualstudycouldbeidentified.

Discussion

Althoughheterogeneityinallmeta-analyseswassignificant,theresultssuggestthatbothgeneralandlowproteinundernutritionduringgestationresultsinincreasedglucoseandreducedinsulinconcentrationsandbeta cellmass in theoffspring. Thesefindings generally support the fetaloriginshypothesis. Themostmarkedeffectofprenatalundernutrition-bothgeneralandlowprotein-wasfoundonbetacellmass.Undernourishedoffspringhada significantdecrease inbetacellmass, theeffectwas stronger in the lowprotein group. Prenatal lowprotein diet also had a significanteffectonplasmaglucoseconcentrations,whichwerehigherinundernourishedoffspring. Theeffectofprenatalgeneralmalnutritiondependedonthetimeatwhichglucosemetabolismwasstudied.Whendatafromnewbornrodentoffspringwerepooledseparately,theseoffspringhadasignificantlylowerplasmaglucoselevel,asopposedtoadultoffspringwhichhadhigherglucoseconcentrations. In the lowproteinmodels thesameeffectofagewasseen,althoughthe effect was not significant in newborn offspring. This shows that prenatal undernutritionleads to lower glucose concentrations directly after birth, while after normal postnatal diet,glucoseconcentrationsrisemorethanincontrolanimals.Biologically,thisphenomenonmaybesimilartothehypoglycaemiathatisoftenobservedamonginfantswhoaresmallforgestationalage55. Higher glucose concentrations at later age are consistent with the findings of glucoseintoleranceinpeopleprenatallyexposedtotheDutchfamine56,57.Bothlowproteinandgeneralundernutrition models showed a slight decrease of plasma insulin concentrations, which isconsistentwithreducedinsulinproductionthroughdecreasedbetacellmass. Meta-analysesofanimalstudiesareknowntoshowsignificantheterogeneity57,58.Inlinewiththis,wefoundseverestatisticalheterogeneityinourmeta-analyses,andwehavetobecautiouswheninterpretingthemeandifferences.Manydifferentanimalmodelshavebeenusedtostudytheeffectsofprenatalundernutrition.Wefinditdefendabletopoolresultsofallanimalmodelstogether, since consistency of the resultswould indicate that the same effectsmay apply todifferentspeciesincludinghumans. Exploringpotentialsourcesofheterogeneity,thesubsequentsubgroupanalysesconductedforanimalmodel,species,ageoftheanimalsatinvestigationandprotocol(fastedornot),onlyaccountedforasmallpartoftheheterogeneity.Heterogeneitycouldalsohavebeencausedbythefactthatsomeofthearticlesthatweincludedwerenotoriginallydesignedto investigatetheeffectofprenatalundernutritiononplasmaglucoseandinsulinlevelsorbetacellmassas

26

Chapter 2

primaryoutcome.Thiscouldbeanexplanationforthegreatvarietyingroupsizesinthestudiesweidentified. Methodologicalheterogeneitywasoneofthemajorreasonsfortheheterogeneityobserved.Themethodologicalqualityofmostreportedstudieswaspoor,withonlyonestudyreportingblindingoftheinvestigators34,andlessthanhalfoftheincludedstudiesreportingrandomizationof theanimals.Noneof the studies reporteda sample size calculation. In contrast tohumanstudies,randomization,blinding,samplesizecalculationandplannedanalysiswerenotstandard.Animalstudiesthatdidnotreportrandomizationandblindinghavebeenshowntobemorelikelyto report a difference in study groups than studies that did use thesemethods58. Quality ofanimalstudiescouldbeimprovedbystandardizedreporting. Thefindingsfromanimalresearchinthisreviewareinlinewithevidencefromhumanstudies.AprospectivecohortstudyinIndiashowedsignificantlylowercordbloodinsulinconcentrationsinbabiesbornfrommalnourishedmothers,comparedtocontrols.InthatstudymalnourishmentwasdefinedasaBMIoflessthan17kg/m²59.InsubjectsprenatallyexposedtotheLeningradsiegebetween1941and1944,therewasnodifferenceinconcentrationsoffastingand2hourplasmaglucoseduringanoralglucosetolerancetestcomparedtounexposedsubjects.Inuteroexposedsubjectsalsodidnothavedifferentplasmainsulinconcentrationsoranexcessofknowndiabetesorglucoseintolerance60. Three studies have reported on the long term effects of prenatal exposure to the Dutchfamine of 1944-4556,57,61. Glucose tolerance was decreased in subjects that were prenatallyexposedtofaminewhenmeasuredatbothage50and58years56,57.Inasubsetofparticipants,an intravenous glucose tolerance test was performed. The results showed impaired glucosetolerance inprenatallyexposedsubjects,especiallythoseexposed inmidandearlygestation.Thiseffectwassuggestedtobecausedbyaninsulinsecretiondefect61.Similarly, inadultmenand women prenatally exposed to the Chinese famine (1959-1961) there was an increasedprevalence of hyperglycemia defined as increased fasting plasma glucose, impaired glucosetoleranceorapreviousdiagnosisoftype2diabetes62. Insummary,thissystematicreviewshowsthattheresultsfromanimalexperimentssupportthe fetal origins hypothesis: prenatal undernutrition leads to a disturbed glucose and insulinmetabolismandadecreaseinbetacellmassinlaterlife.

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28. Zambrano E,Martinez-Samayoa PM, Bautista CJ, DeasM, Guillen L, Rodriguez-Gonzalez GL, et al.Sexdifferencesintransgenerationalalterationsofgrowthandmetabolisminprogeny(F2)offemaleoffspring(F1)ofratsfedalowproteindietduringpregnancyandlactation.J Physiol.2005;566:225-236.

29. ZambranoE,BautistaCJ,DeasM,Martinez-SamayoaPM,Gonzalez-ZamoranoM,LedesmaH,etal.Alowmaternalproteindietduringpregnancyandlactationhassex-andwindowofexposure-specificeffectsonoffspringgrowthandfoodintake,glucosemetabolismandserumleptinintherat.J Physiol. 2006;571:221-230.

30. Kind KL, Clifton PM, Grant PA, Owens PC, Sohlstrom A, Roberts CT, et al. Effect of maternal feedrestrictionduringpregnancyonglucosetoleranceintheadultguineapig.Am J Physiol Regul Integr Comp Physiol.2003;284:R140-R152.

31. JimenezC,HernandezV,ReamerC,FisherS,JosziA,HirshmanM,etal.(beta)-cellsecretorydysfunctioninthepathogenesisoflowbirthweight-associateddiabetes:Amurinemodel.Diabetes.2005;54:702-711.

32. OgeA,IsganaitisE,JimenezC,ReamerC,FaucetteR,BarryK,etal.Inuteroundernutritionreducesdiabetesincidenceinnon-obesediabeticmice.Diabetologia.2007;50:1099-1108.

33. FordSP,HessBW,SchwopeMM,NijlandMJ,GilbertJS,VonnahmeKA,etal.Maternalundernutritionduringearlytomid-gestationintheeweresultsinalteredgrowth,adiposity,andglucosetoleranceinmaleoffspring.J Anim Sci.2007;85:1285-1294.

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35. HustedSM,NielsenMO,TygesenMP,KianiA,BlacheD,IngvartsenKL.Programmingofintermediatemetabolism in young lambs affected by late gestationalmaternal undernourishment.Am J Physiol Endocrinol Metab.2007;293:E548-E557.

36. ToddSE,OliverMH,JaquieryAL,BloomfieldFH,HardingJE.Periconceptionalundernutritionofewesimpairsglucosetoleranceintheiradultoffspring.Pediatr Res.2009;65:409-413.

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37. SmithNA,McAuliffe FM,Quinn K, Lonergan P, EvansAC. Thenegative effects of a short period ofmaternal undernutrition at conception on the glucose-insulin system of offspring in sheep. Anim Reprod Sci.2010;121:94-100.

38. AlvarezC,MartinMA,GoyaL,BertinE,PorthaB,Pascual-LeoneAM.Contrastedimpactofmaternalratfoodrestrictiononthefetalendocrinepancreas.Endocrinology.1997;138:2267-2273.

39. BlondeauB,GarofanoA,CzernichowP,BreantB.Age-dependentinabilityoftheendocrinepancreastoadapttopregnancy:along-termconsequenceofperinatalmalnutritionintherat.Endocrinology. 1999;140:4208-4213.

40. Blondeau B, Lesage J, Czernichow P, Dupouy JP, Breant B. Glucocorticoids impair fetal beta-celldevelopmentinrats.Am J Physiol Endocrinol Metab.2001;281:E592-E599.

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44. GarofanoA,CzernichowP,BreantB.Effectofageingonbeta-cellmassandfunctioninratsmalnourishedduringtheperinatalperiod.Diabetologia.1999;42:711-718.

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46. Holemans K, Van BR, Verhaeghe J, Meurrens K, Van Assche FA. Maternal semistarvation andstreptozotocin-diabetes in rats have different effects on the in vivo glucose uptake by peripheraltissuesintheirfemaleadultoffspring.Journal of Nutrition 127(7):1371-6.1997.

47. HolemansK,GerberR,MeurrensK,DeCF,PostonL,VanAsscheFA.Maternalfoodrestrictioninthesecondhalfofpregnancyaffectsvascularfunctionbutnotbloodpressureofratfemaleoffspring.Br J Nutr.1999;81:73-79.

48. IkenasioT,BreierBH,VickersMH,FraserM.Prenatalinfluencesonsusceptibilitytodiet-inducedobesityaremediatedbyalteredneuroendocrinegeneexpression.Journal of Endocrinology.2007;193:31-37.

49. Martin MA, Alvarez C, Goya L, Portha B, Pascual-Leone AM. Insulin secretion in adult rats thathad experienced different underfeeding patterns during their development. Am J Physiol. 1997;272:E634-E640.

50. Wang X, Liang L, Du L. The effects of intrauterine undernutrition on pancreas ghrelin and insulinexpressioninneonaterats.Journal of Endocrinology.2007;194:121-129.

51. HolemansK,VerhaegheJ,DequekerJ,VanAsscheFA.Insulinsensitivityinadultfemaleratssubjectedtomalnutritionduringtheperinatalperiod.J Soc Gynecol Investig.1996;3:71-77.

52. GardnerDS,TingeyK,VanBonBW,OzanneSE,WilsonV,DandreaJ,etal.Programmingofglucose-insulin metabolism in adult sheep aftermaternal undernutrition.Am J Physiol Regul Integr Comp Physiol.2005;289:R947-R954.

53. HernandezV,PattiME.AThinPhenotypeIsProtectiveforImpairedGlucoseToleranceandRelatedtoLowBirthWeightinMice.Archives of Medical Research.2006;37:813-817.

54. LutherJ,AitkenR,MilneJ,MatsuzakiM,ReynoldsL,RedmerD,etal.Maternalandfetalgrowth,bodycomposition,endocrinology,andmetabolicstatusinundernourishedadolescentsheep.Biol Reprod. 2007;77:343-350.

55. deLeeuwR,deVriesIJ.Hypoglycemiainsmall-for-datesnewborninfants.Pediatrics.1976;58:18-22.56. deRooijSR,PainterRC,RoseboomTJ,PhillipsDI,OsmondC,BarkerDJ,etal.Glucosetoleranceatage

58andthedeclineofglucosetoleranceincomparisonwithage50inpeopleprenatallyexposedtotheDutchfamine.Diabetologia.2006;49:637-643.

57. RavelliACJ,vanderMeulenJHP,MichelsRPJ,OsmondC,BarkerDJP,HalesCN,etal.Glucosetoleranceinadultsafterprenatalexposuretofamine.Lancet.1998;351:173-177.

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58. BebartaV,LuytenD,HeardK.Emergencymedicineanimalresearch:doesuseofrandomizationandblindingaffecttheresults?Acad Emerg Med.2003;10:684-687.

59. MahajanSD,SinghS,ShahP,GuptaN,KochupillaiN.Effectofmaternalmalnutritionandanemiaontheendocrineregulationoffetalgrowth.Endocrine Research 30(2):189-203.2004.

60. StannerSA,BulmerK,AndresC,LantsevaOE,BorodinaV,PoteenVV,etal.Doesmalnutritioninuterodeterminediabetesandcoronaryheartdiseaseinadulthood?ResultsfromtheLeningradsiegestudy,acrosssectionalstudy.BMJ.1997;315:1342-1348.

61. deRooijSR,PainterRC,PhillipsDI,OsmondC,MichelsRP,GodslandIF,etal.Impairedinsulinsecretionafterprenatalexposuretothedutchfamine.Diabetes Care.2006;29:1897-1901.

62. LiY,HeY,QiL,JaddoeVW,FeskensEJ,YangX,etal.ExposuretotheChinesefamineinearlylifeandtheriskofhyperglycemiaandtype2diabetesinadulthood.Diabetes.2010;59:2400-2406.

3The fetal origins of hypertension:

a systematic review and meta-analysis of the evidence from animal experiments of

maternal undernutrition

AnnetFMvanAbeelen*MarjoleinVEVeenendaal*

RebeccaCPainterSusanneRdeRooij

ShakilaThangaratinamJorisAMvanderPostPatrickMMBossuyt

SjoerdGEliasCunoSPMUiterwaalDiederickEGrobbeeGeorgeRSaadeBenWillemJMolKhalidSKhan

TessaJRoseboom*bothauthorscontributedequallytothiswork

Journal of Hypertension, accepted for publication

32

Chapter 3

absTracT

Objective:Numerousexperimentsinanimalshavebeenperformedtoinvestigatetheeffectofprenatalundernutritiononthedevelopmentofhypertensioninlaterlife,withinconclusiveresults.Wesystematicallyreviewedanimalstudiesexaminingtheeffectsofmaternalundernutritiononsystolic,diastolic,andmeanarterialbloodpressureinoffspring.Methods:AsearchwasperformedinMedlineandEmbasetoidentifyarticlesthatreportedonmaternalundernutritionandhypertensioninexperimentalanimalstudies.Summaryestimatesof the effect of undernutrition on systolic, diastolic, andmean arterial blood pressure wereobtainedthroughmeta-analysis.results: Of the 6,151 articles identified, 194 were considered eligible after screening titlesand abstracts. After detailed evaluation, 101met the inclusion criteria andwere included inthereview.Bothmaternalgeneralandproteinundernutritionincreasedsystolicbloodpressure(generalundernutrition:14.5mmHg,95%CI10.8to18.3;proteinundernutrition:18.9mmHg,95%CI16.1to21.8)andmeanarterialpressure(generalundernutrition:5.0mmHg,95%CI1.4to8.6;proteinundernutrition:10.5mmHg,95%CI6.7to14.2).Therewassubstantialheterogeneityintheresults.Diastolicbloodpressurewasincreasedbyproteinundernutrition(9.5mmHg,95%CI2.6to16.3),whilegeneralundernutritionhadnosignificanteffect.conclusion:Theresultsofthismeta-analysisgenerallysupporttheviewthatinanimalsmaternalundernutrition‒bothgeneralandprotein-resultsinincreasedsystolicandmeanarterialbloodpressure.Diastolicbloodpressurewasonlyincreasedafterproteinundernutrition.Theresultsdependedstronglyontheappliedmeasurementtechniqueandanimalmodel.

33

Fetal origins of hypertension in animals

3

inTroDucTion

The fetaloriginshypothesisproposes thathypertensionoriginates in utero. Itpostulates thatundernutritionduringimportantperiodsofgrowthanddevelopmentduringfetallifecanresultinadaptationsinstructureandfunctionofthebody.Intheshorttermtheseadaptationsmaybebeneficialforfetalsurvival,butinthelongtermtheycanleadtocardiovascular,metabolic,andendocrinediseaseinadultlife. Hypertension is one of these long-term effects of maternal undernutrition1. Numerousstudies, indifferentpopulations,have reportedassociationsbetween small sizeatbirth, as aproxy for undernutrition during fetal development, and high blood pressure or hypertensionin later life2. Most studies found an inverse association between birth weight and bloodpressure,showingthatsmallsizeatbirthisassociatedwithraisedbloodpressureinlaterlife.Asystematicreviewofeightystudiesontheassociationbetweenbirthweightandbloodpressuredemonstratedthatakilogramincreaseinbirthweightisassociatedwitha2mmHgdecreaseinsystolicbloodpressure3. Birthweight,however,isonlyaproxyformaternalundernutritionduringgestationandtheepidemiological studies inhumansarenon-experimental, lacking theability toderivedefinitecausal conclusions. Animal studies can be used to experimentally investigate the effects ofmaternalundernutritiononbloodpressureintheoffspringinlaterlife.Avarietyofanimalspecies,includingthemouse,rat,andsheep,havebeenusedtostudythiseffect.Themodelsemployeddiffer, using various protein:lipid:carbohydrate ratios of the maternal diet during gestation,and varying timing and duration of dietarymanipulation. Some of these animal experimentsobservedsignificantlyraisedbloodpressureintheoffspringofundernourishedmothers,whileothersdidnot.Theseinconsistenciesmaybeduetodifferencesindietaryregimensorstrainsorspeciesofanimalsusedbutalsotolimitedsamplesizeandchance.Wethereforesystematicallyreviewedanimalstudiesonmaternalundernutritionduringgestationandbloodpressureintheoffspringandperformedameta-analysistoobtainprecisesummaryestimatesoftheeffectsofmaternalundernutrition.

METhoDs

search strategyWeperformedasearchintheelectronicdatabasesMedline(1951–August2011)andEmbase(1980 – August 2011) to identify articles that reported on maternal undernutrition andhypertension in offspring in experimental animal studies. The search terms ‘undernutrition’,‘malnutrition’,‘famine’,‘starvation’,‘nutritiondisorder’,‘caloricrestriction’,‘proteinrestriction’,‘lowproteindiet’,‘lowcaloriediet’,‘bloodpressure’and‘hypertension’wereused.Therewerenolanguagerestrictions.

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Study selectionWe included papers describing outcomes in experimental animal models of maternalundernutrition that reportedonsystolicand/ordiastolicbloodpressureand/ormeanarterialpressureintheoffspring.Maternalundernutritionincludedlowproteinmalnutritionandgeneralcaloricmalnutrition.Studieshadtoreportoutcomesincomparisontocontrolanimalsthatwereborn to amother thatwas normally fed throughout pregnancy. After screening of titles andabstracts, two reviewers (AFMvA andMVEV) independently examined full text articles frompotentially eligible papers. Disagreementswere resolved in consensus discussions. Referencelistsofreviewsandincludedpaperswerehandsearchedtoidentifyadditionalstudies.

Data extractionFromallincludedpapers,tworeviewers(AFMvAandMVEV)independentlyextractedinformationonstudydesign,exposureperiod,animalspeciesandtypeofundernutrition,andsamplesize.Toassessriskofbias,dataonallocationconcealment,randomization,blindingwereextracted.Whenmorethantwoexperimentalgroupswereformed,wefocusedontheexperimentalgroupwithmalnutritionasearlyinpregnancyaspossibleandpreferablylimitedtopregnancyalone.Whenoutcomeinoffspringwasmeasuredatmultipletimepoints,wechosetheoldestageatwhichmeasurementsweretaken.Studiesthatreportedonfetalbloodpressurewereexcluded.Ifresultswereonlydisplayedgraphically,outcomewasreadaspreciseaspossible.Studiesthatreportedresultsasmeanandstandarddeviationorstandarderror,andnumberofanimalspergroupwereusedformeta-analysis.

Data analysisSummary estimates of the effects ofmaternal undernutritionwere obtained using a randomeffectsmodelformeta-analysis,whichaccountsforbothwithin-andbetween-studyvariability.Separateestimateswereobtainedforsex,modeltype(proteinorgeneralundernutrition),andoutcomemeasures(systolic,diastolicbloodpressureandmeanarterialpressure).Thesummaryeffectswereexpressedasmeandifferenceswith95%confidence intervals (CI).Weevaluatedheterogeneity in results across studies by calculating the I² statistic, which describes thepercentageofthevariabilityineffectestimatesthatisduetoheterogeneityratherthansamplingvariability. When significant statistical heterogeneity was detected, further stratification wasapplied to investigatewhether heterogeneity could be explained by different animal speciesormethodtomeasurebloodpressure(tailcuffand intra-arterial).Toevaluatetherobustnessofourresultsagainst influentialstudies,a leaving-one-outsensitivityanalysiswasperformed.To examinepotential publicationbiaswe constructed funnel plots.Datawere analyzedusingReviewManagerVersion5.1.

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rEsulTs

Thesearchresultedin6,151articles,ofwhich194wereconsideredpotentiallyeligible.Intotal,101primarystudiesmettheinclusioncriteriaandwereusedfordataextractionafterreadingfulltextarticles(AFMvAandMVEV)(Figure1).Thirty-fourstudiesreportedongeneral(caloric)undernutrition;18usingaWistar ratmodel4-21and4usingaSprague-Dawleyratmodel22-25,8usingasheepmodel26-33,2usingaguineapigmodel34,35,and2usingamousemodel36,37.Sixty-sevenstudies reportedonproteinundernutrition;47usingaWistar ratmodel38-84,15usingaSprague-Dawleyratmodel85-99,1usingaspontaneouslyhypertensiveratmodel100,and4usingamousemodel101-104.Theageofthestudiedanimalsrangedfromfourweeksinratstothreeyearsinsheep.

figure 1 Literature search results for studies reporting onmaternal undernutritionwith regard tohypertension.

6,151 poten�ally eligible studies iden�fied(database searches and references lists)

5,957 studies were excluded based on the inclusion criteria and �tle and abstract review

194 full text ar�cles were reviewed

93 studies were excluded for not having the required exposure / not repor�ng the outcome of interest / not

repor�ng data in a form fit for meta-analysis / non-animal or fetal studies

101 studies were included in the meta-analysis

risk of biasForty-five studies reported randomization, either randomization to the dietaryregimen5,8-11,13,18,20,21,25,27-30,32,33,40,42,48,70,72,79,81,83,92,93,103,104,orrandomizationinselectingthepupsfromthe litters thatwere studied12,22,24,45,55,57,60,68,74,82,99,102, or both6,7,14,41,67. Eighteen studies reportedblindingoftheinvestigator27,28,30,31,40,50,51,55-57,62,65,67,81,88,91,103,104.Onlyonestudyreportedthatthey

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performed a sample size calculation67. Funnel plots of all six outcomes showed symmetricalscatteringofthestudyresultsaroundthesummaryestimate.Therewasnoevidenceofasmallstudyeffectorpublicationbias.

Systolic blood pressure after maternal general undernutritionThirty studies provided data on systolic blood pressure in offspring after maternal generalundernutrition.Twenty-twostudieshadbeenperformedinrats4-25,twoinmice36,37, one in guinea pigs35,andfiveinsheep27,28,30-32. Intotal,384undernourishedanimalsand420controlanimalsweredescribed.Meansystolicbloodpressurewas14.5mmHg(95%CI10.8to18.3)higher inundernourishedanimalscomparedtocontrols(Figure2). Therewasconsiderableheterogeneity,withanI2of92%,whichpersistedafterstratifyingforsexormeasurementmethod.Stratifyingforspeciesdidnotreduceheterogeneityinthedifferentrodentmodels.Meta-analysisinsheeponly(37undernourishedanimalsand43controls)showednodifference in systolicbloodpressurebetweenundernourishedandcontrol animals,withameandifferencesystolicbloodpressureof-1.1mmHg(95%CI-6.4to4.3,I257%).Stratifyingformeasurementmethodshowedameandifferenceinsystolicbloodpressureof19.7mmHg(95%CI15.3to24.2,I292%)instudiesusingthetailcuffmethodanda4.2mmHg(95%CI-1.2to9.6,I281%)meandifferenceinstudiesusingintra-arterialcatheters. Threestudiesreportedbothblindingoftheinvestigatorandrandomization27,28,30.Separatelyanalysingthesestudiesreducedheterogeneity(I232%)andshowednosignificantdifferenceinsystolicbloodpressure(2.1mmHg,95%CI-3.0to7.2)aftermaternalgeneralundernutrition.

Systolic blood pressure after maternal protein undernutritionFifty-four animal studies provided data on systolic blood pressure in offspring aftermaternalproteinundernutrition. Fifty studieswereperformedusing rats40-42,44-71,74,76-78,80-88,90,91,93-95,100 andfourusingamousemodel101-104.We founda significantlyhighermeansystolicbloodpressurein animals prenatally exposed to a lowprotein diet (n = 1,421) compared to control animals(n =1,427),withameandifferenceinsystolicbloodpressureof18.9mmHg(95%CI16.1to21.8)(Figure3).Theresultsshowedconsiderableheterogeneity(I291%).Heterogeneitypersistedafterstratifyingtheanalysisforsexorspecies.Stratifyingformeasurementmethodshowedameandifferenceinsystolicbloodpressureof19.8mmHg(95%CI16.8to22.8,I291%)instudiesusingthe tail cuffmethodanda5.2mmHg (95%CI -2.1 to12.6, I2 0%)meandifference in studiesusing intra-arterial catheters.Separatelyanalysingstudies thatusedradiotelemetryshowedanon significant difference in systolic blood pressure (mean difference systolic blood pressure8.8mmHg,95%CI-9.8to27.3,I281%).

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figure 2Systolicbloodpressureaftermaternalgeneralundernutritionaccordingtosexoftheanimalandmeasurementmethod

M:Males,F:Females,TC:Tailcuff,IC:Intra-arterial,RT:Radiotelemetry.

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figure 3Systolicbloodpressureaftermaternalproteinundernutritionaccordingtosexoftheanimalandmeasurementmethod

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Figure3Continued


Sixstudiesreportedbothblindingoftheinvestigatorandrandomization55,57,67,81,103,104.Separatelyanalysing these studies reduced heterogeneity (I2 60%) and showed a significant but smallerdifference in systolic blood pressure (9.1 mmHg, 95% CI 5.6 to 12.6) after maternal proteinundernutrition.

Diastolic blood pressure after maternal general undernutritionTenanimalstudiesprovideddataondiastolicbloodpressureinoffspringaftermaternalgeneralundernutrition.Fivestudieshadbeenperformedinrats12,14,16,22,24,fourinsheep27,30-32,andoneinguineapigs35.Therewasnosignificantdifferenceindiastolicbloodpressure(1.6mmHg,95%CI-2.1 to5.3higher inprenatallyundernourishedanimals (n=95)comparedtocontrolanimals(n = 108) (I2 65%) (Figure 4).Meta-analysis of the effects on sheep only (29 undernourishedanimalsand31controls)demonstratedanon-significantdifferenceindiastolicbloodpressure(-3.5mmHg,95%CI-7.8to0.9,I220%).Heterogeneitywasnotfurtherreducedbystratificationforotherspecies,sex,ormeasurementmethod.Stratifyingformeasurementmethodshowedameandifferenceindiastolicbloodpressureof0.7mmHg(95%CI-9.7to11.1,I266%)instudiesusingthetailcuffmethodanda1.8mmHg(95%CI-2.5to6.1,I268%)meandifferenceinstudiesusingintra-arterialcatheters. Two studies reported both blinding of the investigator and randomization27,30. Separatelyanalysing these studies slightly reduced heterogeneity (I2 53%) and there was no significant

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Chapter 3

difference indiastolicbloodpressure(-1.8mmHg,95%CI -10.6to6.9)aftermaternalgeneralundernutrition.

figure 4Diastolicbloodpressureaftermaternalgeneralundernutritionaccordingtosexoftheanimalandmeasurementmethod


Diastolic blood pressure after maternal protein undernutritionDataformeta-analysiswereavailablefromfouranimalstudies.Threestudieswereperformedin a rat model45,48,54 and one in a mouse mode101. In total, 48 animals had been prenatallyexposedtoalowproteindietand40animalswerecontrolfed.Meandiastolicbloodpressurewassignificantlyhigherinanimalsprenatallyexposedtoalowproteindietcomparedtocontrolanimals (mean difference diastolic blood pressure 9.5mmHg, 95%CI 2.6 to 16.3) (Figure 5).There was no heterogeneity (I2 0%). Stratifying for measurement method showed a meandifferenceindiastolicbloodpressureof11.0mmHg(95%CI-12.6to34.6)inthestudyusingthetailcuffmethodandan11.0mmHg(95%CI1.2to20.8)meandifferenceinthestudyusinganintra-arterial catheter. Separately analysing studies that used radiotelemetry showed smaller,

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nonsignificantdifferencesindiastolicbloodpressure(meandifferencediastolicbloodpressure7.0mmHg,95%CI-7.7to21.6,I248%). Therewerenostudiesthatreportedbothblindingandrandomization.

figure 5Diastolicbloodpressureaftermaternalproteinundernutritionaccordingtosexoftheanimalandmeasurementmethod


Mean arterial pressure after maternal general undernutritionElevenanimalstudiesprovideddataonmeanarterialpressureinoffspringaftermaternalgeneralundernutrition. Two studieswereperformedusing rats14,18, seven studies using sheep26,27,29-33, and two studies using guinea pigs34,35. Mean arterial pressure was significantly higher inundernourished(n=105)comparedtocontrolanimals(n =114)(5.0mmHg,95%CI1.4to8.6,I271%)(Figure6). Meta-analysis of the effects on sheep only (71 undernourished and 69 control animals)showedlessheterogeneitybutdemonstratednosignificantdifferenceinmeanarterialpressure(meandifferencemeanarterialpressure1.4mmHg,95%CI-3.1to5.9,I250%).Heterogeneitywasnotlowerafterstratificationforsexormeasurementmethod.Stratifyingformeasurementmethodshowedameandifferenceinmeanarterialpressureof13.0mmHg(95%CI7.4to18.6)inthestudyusingthetailcuffmethodanda4.2mmHg(95%CI0.6to7.8,I267%)meandifferenceinstudiesusingintra-arterialcatheters. Threestudiesreportedbothblindingoftheinvestigatorandrandomization27,30,34.Separatelyanalysing these studies did not change heterogeneity (I2 66%) and there was no significantdifference inmean arterial pressure (2.9mmHg, 95% CI -4.3 to 10.2) aftermaternal generalundernutrition.

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figure 6 Meanarterialpressureaftermaternalgeneralundernutritionaccordingtosexoftheanimalandmeasurementmethod


Mean arterial pressure after maternal protein undernutritionSeventeen rat studies reportedmeanarterialpressure inoffspringafterprenatalexposure toa low protein diet38,39,43,45,48,72-75,79,87,89,92,96-99. Mean arterial pressure was significantly higher inundernourished (n =208) compared to control rats (n =201) (meandifferencemeanarterialpressure10.5mmHg,95%CI6.7to14.2,I285%)(Figure7).Heterogeneitycouldnotbeexplainedbystratifyingforthedifferentratspeciesorsex.Stratifyingformeasurementmethodshowedameandifferenceinmeanarterialpressureof17.5mmHg(95%CI11.3to23.8,I20%)instudiesusing the tail cuffmethod and a 10.7mmHg (95%CI 6.6 to 14.7, I2 85%)meandifference instudies using intra-arterial catheters. Separately analysing studies that used radiotelemetryshowed smaller, non significant differences inmean arterial pressure (meandifferencemeanarterialpressure-0.9mmHg,95%CI-5.9to4.2,I20%). Therewerenostudiesthatreportedbothblindingandrandomization.

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figure 7Meanarterialpressureaftermaternalproteinundernutritionaccordingtosexoftheanimalandmeasurementmethod


Sensitivity analysisInaseriesofsensitivityanalyses,weevaluatedtherobustnessofourfindingsbyrepeatingtheanalysesanumberoftimes,eachtime leavingone studyoutof themeta-analysis. If a studyappearstobeanoutlier,withresultsverydifferentfromtherestofthestudies,thenitsinfluencewillbecomeapparent,astheresultwithoutthestudywouldbeverydifferentfromtheresultofthemeta-analysisoftheremainingstudies. Theobservedheterogeneityinthemeta-analysisfordiastolicbloodpressureaftermaternalgeneralundernutritionwas largelyduetothestudyofOzakietal14.Onremovalofthisstudy,the I2 decreased from 62% to 27%. The overall summary estimate, however, did not changesubstantially (-0.22mmHg, 95% CI -2.45 to 2.00).We could not identify differences in study

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design or characteristics in the study of Ozaki et al14 compared to the other studies whichcouldexplaintheinfluenceontheheterogeneity.Allothersensitivityanalyses,foreachoftheotherfiveoutcomemeasuresevaluated,confirmedthestabilityofouranalyses.Noinfluentialindividualstudycouldbeidentified.

Discussion

Theresultsgenerallysupportthefetaloriginshypothesis.Wedemonstratedthatbothgeneralandproteinundernutritionduringgestationresultedinsignificantlyincreasedsystolicandmeanarterial blood pressure in the offspring, while diastolic blood pressure was only significantlyincreasedaftermaternalproteinundernutrition.Thelargesteffectofmaternalundernutrition–bothgeneralandprotein–wasfoundonsystolicbloodpressure,whichwassignificantlyincreasedinprenatallyundernourishedoffspring.Thiseffectwasstrongerintheproteinundernourishedanimals. This systematic review confirms the substantial variability in results from animalstudieswhich,inmostcases,cannotbeattributedtochancevariabilityonly:heterogeneitywasconsiderableinallmeta-analyses. Someofthestudiesincludedinthismeta-analysisusedonlymaleorfemaleanimals,whileothersusedbothordidnot specify sex.Only two studiesexplained their choice tousemaleanimalsalone80,86.Theauthorsofoneofthesepaperssuggestedthatusingmaleanimalsonlyreducesthevariability86.Furthermore,theauthorsoftheotherpaperreferredtothefindingthatmaleanimalshavebeenshowntobemorevulnerabletodevelopmentalprogrammingeffects80,97, sincemalefetusesgrowfasterthanfemalefetusesfromanearlystageofgestationandthismakesthemmorevulnerableiftheirnutritioniscompromised105,106.Maternalproteinrestrictioninratshasbeenassociatedwithfewernephronsandanincreasedbloodpressureamongmalebutnotfemale offspring97. Female gender seems to be relatively protective against the hypertensiveeffects of maternal protein restriction. However, this protection is lost with more severeproteinrestriction97.Themechanismofthisrelativeprotectionisunknown.Thismeta-analysisshows,however, thatthere isalso in femalesasignificanteffectofmaternalundernutrition–bothgeneralandprotein–onsystolicbloodpressureandofproteinundernutritiononmeanarterialpressure.Inaddition,theeffectsonbloodpressureareofsimilarsizeinmaleandfemaleoffspring. When looking at species separately, we found that sheep prenatally exposed to generalundernutritiondidnothave significantlyhigherbloodpressure. This in contrast toprenatallyundernourishedrodentoffspring,whereanincreaseinsystolicandmeanarterialpressurewasseen.Theprogrammingofanyoutcomemeasure,includingbloodpressure,maybeamplifiedintherat.Thesumweightoftheproductsofconceptionrelativetomaternalweightis25-35%inratsversus6-10%insheepand3-5%inhumans30.Thiscouldverywellexplainthefactthatwedidfindaneffectinrodentmodels,butnotinsheep.

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Inhumans,therelationbetweenmaternalundernutritionduringpregnancyandbloodpressurein theoffspring is lesswell studiedand theresultsareconflicting.TheHungerwinterFamiliesStudy,whichstudiedsubjects fromthree institutions in famine-exposedcitiesatanageof59years,foundthatpeoplewhohadbeenexposedtofaminein uterohadhigherbloodpressuresas adults107. The Dutch famine birth cohort study did not find significant effects of prenatalfamineexposureonbloodpressurebutobservedthatthoseexposedtofamineprenatallyhadahigherbloodpressureresponsetostress108.Incontrast,in uteroexposuretotheLeningradsiegebetween1941and1944ortotheChinesefamineof1959-1961wasnotassociatedwithraisedbloodpressureinlaterlife109,110. Othermeta-analysesofanimaldatashowedmarkedheterogeneity111,112.Theresultsofthepresentsystematicreviewareinlinewiththis.Stratifyingforsexorspeciesdidnotconsistentlyexplaintheheterogeneity.Thereforewechosetoprimarilyreportthepooledresultsofallstudies,sinceconsistencyoftheresultswouldindicatethattheeffectsofmaternalundernutritionarebasedonthesamemechanismsandapplytodifferentspecies.However,thesizeoftheeffectmightdifferbetweenspecies. We explored the potential sources of heterogeneity by conducting subgroup analyses foranimalmodel,animal speciesandanimal sex,but thisaccountedonly fora smallpartof theheterogeneity.Anotherpossiblesourceofheterogeneitycouldbethefactthataportionoftheincludedarticlesdidnothavebloodpressureas theprimaryoutcome.Thiscouldaccount forthevarietyinsamplesizeswefoundbetweenthestudies.Furthermore,thefactthatdifferentlaboratoriesusedifferentlycomposeddietsintheirexperimentscouldbeanotherexplanationfortheheterogeneity,reflectingtheinconsistenciesintheresults.Inastudycomparingtwolowproteindiets,onlyoneaffectedpostnatalsystolicbloodpressure65.Themaindifferencebetweenthe two dietswas the source and content of fat. That study demonstrated that exposure tolowproteinin uterodoesnotinitselfdeterminethedevelopmentofhypertensioninlaterlife.Thebalanceofothernutrientswithin thematernaldietsappears toplaya critical role65. Thestudies included in our meta-analysis used different diets for the low protein experiments,which couldexplainpartof theheterogeneity.Also, in thegeneralmalnutritionexperiments,differentregimenswereapplied.Restricteddietsvariedfrom30to70%ofnormalintake.Thesewiderangesofdietaryrestrictionhaveundoubtedlyaffectedtheoutcomeandattributedtothereported heterogeneity. Standardization of animal experimentswill improve comparability ofthesestudies. Methodological heterogeneitymust also be discussed as amajor reasonof the observedheterogeneity. The methodological quality of the included studies was poor, with only 18studies reporting on blinding of the investigators, and 46 reporting on randomization, eitherwhen allocating the diet or selecting the pups for themeasurements. In contrast to humanstudies, randomization, blinding, and sample size calculations are not standard practice inanimalexperiments. Ithasbeenshownthatanimal studies thatdonot report randomizationandblindingaremorelikelytoreportadifferenceinthestudygroupsthanstudiesthatdoreport

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onthesemethods113.Wecansupportthisinourstudy.Separatelyanalysingthemethodologicallysoundstudiesresultedinareductionordisappearanceoftheeffect.Improvementscanbemadeonthequalityofanimalstudiesbyapplyingstandardsforreportingsuchasthosethatareroutineinhumanstudies. Inrodentmodels,measurementsofbloodpressurearedonebythetailcuffmethodorbydirect intra-arterialmeasurementswhich encompass both direct indwelling catheters and 24hourradiotelemetry.Radiotelemetryisconsideredtobeadirect,minimallystressful,continuousbloodpressuremeasurement114.There isevidencesuggesting thatprenatalmalnutritiondoesnotprimarilyresultinincreasedbloodpressure,butinaheightenedstressresponseleadingtohigherbloodpressurelevels108,114.Thismaybereflectedinourfindingsofagreaterdifferencein systolic blood pressure and mean arterial pressure when measurements were performedwith the tail cuffmethod, which is considered to be a source of stress, compared to directintra-arterialmeasurements.Radiotelemetryhasonlybeenusedinthreestudies investigatingthe effects of a low protein diet. Separately analysing studies using radiotelemetry showedsmallerandnonsignificantpooledeffectestimatescomparedwiththeothermethods.Ofthetwostudiesthatreportedonsystolicanddiastolicbloodpressure,oneshowedan increase inbothsystolicanddiastolicbloodpressure101,whiletheotherreportednoeffect45.Twostudiesreportedonmeanarterialpressureafterlowproteinundernutrition,withonestudyreportingaverysmall increase45,while theother reportednoeffect39.Swaliet al. reportedan increaseinsystolicbloodpressurewhenmeasurementswereperformedbythetailcuffmethodwhileradiotelemetrydemonstratedlowermeanarterialpressureinthesameanimalsafterlowproteinundernutrition80. These resultsmay indicate that part of the increased blood pressure levelsfound after prenatal undernutritionmaybedue to an increased stress response. In our dataon systolic anddiastolic bloodpressure, althoughno longer significant, bloodpressure levelswerestillhigherafterprenatalundernutritionwhenmeasuredby radiotelemetry.This lossofsignificancemaybecausedbythesmallnumberofanimals.

conclusion

In summary, the results of this meta-analysis generally support the view that maternalundernutrition–both general andprotein – leads to an increased systolic andmeanarterialbloodpressureintheoffspringofmostanimals.Yet,studiesshowsubstantialheterogeneityintheresultswhichcouldnotbesufficientlyexplainedbyknowncharacteristicsoftheresearch.Also,theresultsdependedstronglyontheappliedmeasurementtechniqueandanimalmodel.Futureanimalstudiesshouldimprovetheirmethodologicalqualitybyapplyingrandomization,blinding,andsamplesizecalculationtechniques,topreventselection,performance,anddetectionbias.

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4Grip strength at age 58 after prenatal

exposure to the Dutch famine

MarjoleinVEVeenendaalSusanneRdeRooijRebeccaCPainterSianRobinsonCliveOsmond

AvanAihieSayerTessaJRoseboom

The Journal of Aging Research and Clinical Practice, 2012 vol 1

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Chapter 4

absTracT

background: Grip strength is a marker of current and future health. Small size at birth isassociatedwithreducedgripstrengthandpoorhealthinlaterlife.Prenatalundernutritionmayaffectadultgripstrength.WeinvestigatedtheeffectofprenatalundernutritionongripstrengthintheDutchfaminebirthcohort.Methods: We assessed grip strength in 334 men and 364 women at age 58, born as termsingletonsaround thetimeof the1944-45Dutch famine.Wecomparedgrip strengthamongmen and women who had been exposed to famine during different periods of gestation tounexposedsubjects.results: Men exposed to famine in early gestation had a 4.2 kg (95%CI 1.0 to 7.3) greatergrip strength compared to unexposedmen. After adjustment for adult height and timing ofparticipationinthestudy,theassociationwasnolongersignificant(2.9kg(95%CI-0.2to6.0)).Inwomen,prenatalexposuretofaminewasnotsignificantlyassociatedwithgripstrength.A1kilogramincrease inbirthweightwasassociatedwithan increaseof2.8kg(95%CI1.0to4.7)ingripstrengthinmenand1.5kg(95%CI0.1to2.8)inwomen,adjustmentforadultbodysizeexplainedthisrelationship.conclusions:Therewasnoevidenceforsignificantindependentassociationsbetweenprenatalfamineexposureandadultgripstrengthalthoughmenexposedinearlygestationappearedtohaveincreasedgripstrengthexplainedbytalleradultheight.Consistentwithpreviousstudies,therewasarelationshipbetweensmallsizeatbirthandlowergripstrength.

57

Grip strength at age 58 after prenatal exposure to the Dutch famine

4

inTroDucTion

Handgripstrengthcanbeusedasamarkerofoverallmusclestrength1.Decreasedgripstrengthis associatedwith the presence of chronic diseases including coronary heart disease, stroke,chronicobstructivepulmonarydiseaseanddiabetesmellitus2.Gripstrengthisknowntodeclinewithageandpoorgripstrength isassociatedwith increasedall-causemortality3.Birthweightispositivelyassociatedwithadultgripstrength4,5,andthereforegripstrengthisthoughttobeaffectedbyenvironmentalinfluencesinearlylife. TheDutchfaminewasaperiodofseverefoodshortageinthewestoftheNetherlandsthatoccurredduringthelast5-6monthsofWorldWarII.Thefamineoffersauniqueopportunitytostudytheeffectsofprenatalundernutritiononhealthinlaterlife.Amongseveraladversehealtheffects,peopleconceivedduringthefaminehadamoreatherogeniclipidprofile6andanearlieronsetandadoubledrateofcoronaryarterydisease7.Also,peopleexposedtothefamineduringgestationhadimpairedglucosetolerance8,9.Recently,wedemonstratedthatpeopleconceivedduringthefamineperformedworseonaselectiveattentiontest,acognitiveabilitythatisknowntodeclinewithage.ThiswasthefirstevidenceintheDutchfaminebirthcohortthatsuggeststhatprenatalundernutritionisassociatedwithacceleratedaging10. In the present study, we investigated grip strength inmen andwomen born around thetimeoftheDutchfamine,aged56-61years.Wehypothesizedthatpeoplewhowereprenatallyexposedtofaminewouldhavereducedgripstrengthatadultage.

METhoDs

The Dutch famine birth cohortTheDutch faminebirth cohortmemberswerebornas termsingletonsbetween1November1943 and 28 February 1947 in theWilhelminaGasthuis inAmsterdam, theNetherlands. Theselectionprocedures for this cohort havebeendescribedelsewhere9.At age58, 1423of the2414originalcohortmembers(58%)werestillalive,livingintheNetherlandsandtheiraddresswasknowntotheinvestigators.Thesepeoplewereeligibleforourstudy.Ofthegroupof1423eligiblepeople,810 (57%)agreed toparticipateatage58years.Allparticipantsgavewritteninformedconsent.

Exposure to famineExposure to famine was defined according to the official daily food-rations for the generalpopulationolderthan21years.TheofficialrationsaccuratelyreflectthevariationovertimeinthetotalamountoffoodavailableinthewestoftheNetherlands11.Weconsideredfetusestohavebeenexposedtofamineinuteroiftheaveragedailyrationsduringany13-weekperiodofgestationwerelessthan1000calories.Therefore,peoplebornbetween7January1945and8

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Chapter 4

December1945wereconsideredtobeexposedtofamine inutero.Wedefinedperiodsof16weekseachtodifferentiatebetweenthosewhowereexposedtofamineinlategestation(bornbetween7January1945and28April1945),inmidgestation(bornbetween29April1945and18August1945)andinearlygestation(bornbetween19August1945and8December1945).Peoplebornbefore7January1945(andwerethusbornbeforethefamine)andthosebornafter8December1945(andwhohadthusbeenconceivedafterthefamine)wereconsideredtobeunexposedtofamineinutero.

study parametersThemedicalbirthrecordsprovidedinformationaboutthemother,thecourseofthepregnancyandthesizeofthebabyatbirth9.BetweenSeptember2002andOctober2004atameanageof58.3(SD0.9)yearsparticipantsvisitedtheclinicwheretrainedstudynursescarriedoutallmeasurements.Asamarkerofmusclestrength,maximumgripstrengthwasmeasuredusingaJamarhandgripdynamometer(PGB,Bussum,TheNetherlands)attheclinicvisit.Gripstrengthwasmeasuredthreetimesoneachside,andthemaximumofthesemeasurementswasusedfortheanalyses.WemeasuredheightusingafixedoraportablestadiometerandweightusingSecascalesorportableTefalscales.

Statistical analysesWeusedlinearregressionanalysestocomparethecharacteristicsofindividualsexposedinlatemid or early gestationwith characteristics of thosewho had not been exposed to famine ingestation. Linear regressionwas also performed to explore the associationbetweenprenatalexposuretofamineandmaximumgripstrengthandbetweenbirthweightandmaximumgripstrength.Analyseswereadjustedforcurrentbodysizebyaddingheightintothemodel.Furtheradjustmentsweremadeforthetimeatwhichthesubjectsparticipatedduringthestudy(date)since this contributed significantly to the variance in grip strength. In accordance with theexistingliterature,menandwomenwereanalyzedseparately.Weconsidereddifferencestobestatisticallysignificantifp<0.05.Wherep-valuesaregiven,theyare2sided.WeusedSPSS17.0forallanalyses.

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4

Table 1General,birthandadultcharacteristicsaccordingtotimingofprenatalexposuretotheDutchfamine,menandwomenseparately.

Exposure to famine

born before

in late gestation

in mid gestation

in early gestation

conceived after

all (sD)

MEn

N 106 56 43 30 99 334

gestationalage(days) 283 282 286 291* 285 285(12)

birthweight(kg) 3.42 3.25* 3.27* 3.51 3.57 3.43(0.47)

weight(kg) 90.5 87.8 85.4 93.9 88.9 89.2(15)

height(m) 1.77 1.77 1.76 1.79 1.77 1.77(0.06)

maximumgripstrength(kg) 50.3 50.3 49.7 55.0* 51.5 51.0(8.4)

WoMEn

N 107 67 59 33 98 364

gestationalage(days) 285 283 285 287 285 285(11)

birthweight(kg) 3.37 3.16* 3.15* 3.41 3.41 3.31(0.46)

weight(kg) 78.2 77.0 76.1 74.3 79.2 77.6(14)

height(m) 1.65 1.64 1.64 1.64 1.64 1.64(0.06)

maximumgripstrength(kg) 29.3 30.0 29.0 30.0 28.9 29.3(6.1)

*p<0.05fordifferenceswithbornbeforeandconceivedafter

rEsulTs

Grip strength data were available for 334 men and 364 women who visited the hospital,afterexcludingparticipants (N=37)with complaintsofosteoarthritis, rheumaticdiseaseandneurological and physical complaints that influenced the grip strength measurements. Thebirthweightsofpersonsincludedinthestudy(3363g)didnotdiffersignificantlyfromthebirthweightsofthosenotincludedinthestudy(3339g;p =0.3). Table1showsthecharacteristicsoftheparticipantsaccordingtotimingofexposuretofaminein utero.One-hundredandtwenty-nine(39%)menandone-hundredandfifty-nine(44%)womenhadbeenexposedtofaminein utero.Thecontrolgroupconsistedofindividualsbornbeforethefamine and those conceived andborn after the famine. These groupswere comparablewithrespecttogestationalage,birthweight,adultweightandheightormaximumgripstrength(allp>0.05).Male and female babies exposed to famine in late ormid gestationwere lighter atbirththanthosenotexposed.Gestationalagewashighest inmenexposedto famine inearlygestation. Therewasno significantdifference in adult body size in exposedmenandwomencomparedtounexposedcontrols.

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Table2showsthedifferencesinhandgripstrengthaccordingtotimingofprenatalexposuretofaminecomparedtonon-exposedparticipants.Menexposedto famine inearlygestationhadgreaterhand grip strength thanunexposedmen (meandifference4.2 kg (95%CI 1.0 to 7.3)).Afteradjustmentforadultheightandtimingofparticipationinthestudythisassociationwasnolongersignificant(2.9kg(95%CI-0.2to6.0)).Prenatalfamineexposureinmidorlategestationwasnotassociatedwithhandgripstrengthinmen.Inwomennoassociationwasfoundbetweenprenatalexposuretofamineandhandgripstrength,neitherintheunadjustednortheadjustedanalyses.Additionof birthweightor age to the linear regressionmodels didnot change theassociations.

Table 2 Differences (and 95% confidence intervals) in hand grip strength (kg) inmen andwomen accordingtothetimingofprenatalexposuretofaminecomparedtonon-exposedparticipants(thosebornbeforeorconceivedafterthefamine).

Late gestation Mid gestation Early gestation

Men Women Men Women Men Women

Unadjusted -0.6 (-3.0to1.9)

1.0 (-0.7to2.7)

-1.2 (-3.9to1.6)

-0.1(-1.9to1.7)

4.2(1.0to7.3)*

0.9 (-1.3to3.2)

Adjustedforheight -0.6 (-3.0to1.7)

1.0 (-0.6to2.6)

-0.9(-3.5to1.7)

0.1(-1.6to1.8)

3.4(0.4to6.5)*

1.0 (-1.2to3.1)

Heightandtime -1.0 (-3.3to1.4)

0.7 (-0.9to2.3)

-1.2(-3.8to1.4)

-0.0 (-1.7to1.6)

2.9(-0.2to6.0)

1.0 (-1.2to3.1)

*p<0.05

Considering thewhole cohort irrespectiveof exposure status, both inmenandwomenbirthweightwas strongly relatedwith adult handgrip strength.An increaseof 1 kilogram inbirthweightwasassociatedwithanincreaseof2.8kginhandgripstrength(95%CI1.0to4.7)inmenand1.5(95%CI0.1to2.8)inwomen.Afteradjustmentforageandadultheight,thisassociationwasnolongersignificant.Theassociationwaslargelyexplainedbyadultheight(table3).

Table 3Differences(and95%confidenceintervals)inhandgripstrength(kg)accordingtobirthweight(kg)intheDutchfaminebirthcohort(regardlessofexposurestatus).

Men Women

Unadjusted 2.8(1.0to4.7)* 1.5(0.1to2.8)*

Adjustedforage 2.6(0.7to4.5)* 1.4(0.1to2.8)*

Adjustedforheight 1.3(-0.7to3.2) 0.1(-1.3to1.5)

Ageandheight 0.9(-1.0to2.9) 0.1(-1.4to1.4)

*p<0.05

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4

Discussion

Inthisstudy,wedidnotdemonstrateanysignificant,independentassociationsbetweenprenatalexposuretofamineandadultgripstrength.Howevermenexposedtofamineinearlygestationappeared to have increased grip strength which was an unexpected finding. As reported inpreviousstudies,wefoundapositiverelationshipbetweensizeatbirthandadultgripstrength;howeverinthiscohortofmenandwomen,theassociationwaslargelyexplainedbyadultheight. Theassociationbetweenfamineinearlygestationandincreasedgripstrengthinmenwassurprising in viewof earlier findings describing an increase in chronic disease among peopleexposedtofamineinearlygestation.Itmayrepresentachancefindinginthecontextofmultiplecomparisons,alternatively itmayreflectbias inthestudydesignorconfounding.Forexamplemenexposedtofamineinearlygestationalsotendedtohaveahighergestationalageandtobeyoungeraswellastallerandheavierinadulthood.Certainlytheassociationbetweenfamineinearlygestationandincreasedgripstrengthinmenwasnotindependentofadultsizeandtimingofparticipationinthestudy.Howeveritisalsopossiblethatthiswasatrueassociationperhapsmediatedthroughincreasedmusclesize.Thereissomeevidencefromstudiesinanimalmodelsthataperiodofreducednutritioninearlygestationcanincreasegrowthoftissuessuchasbonelengthpossiblythroughan increase in IGF-1 level12.Howevermostanimalstudiesfocusingonskeletalmusclereportthatprenatalundernutritionisassociatedwithreducedskeletalmusclefibrenumberordensityeven intheabsenceofaneffectonmuscleweight13,14.Studiesoftheeffectofearlyundernutritiononhumanmusclearesparse,howeveranothernatural‘experiment’foundthatchildrenexposedtoaperiodofundernutritioninlatechildhoodwereshorterandhadhigher appendicular leanmass/height2 after the age of 65 years but therewas no significanteffectongripstrength15. Theoverallabsenceofsignificant,independentassociationsbetweenprenatalexposuretofamineandadultgripstrengthcouldbeatruefindingbutalternativeexplanationsalsoneedtobeconsidered.Selectionbiasisacommonissueincohortstudiesandsomeinformationbiasmayhaveexistedinthisroundofdatacollectionbecausethemostfrailindividualswereseenattheendofthestudyforlogisticalreasons.Tosomeextentthiscouldbeaddressedbyadjustingthefindingsfortimeseen.Howeverallowingfortheageoftheparticipantswasmoreproblematicastheexposurevariableandagewerecloselylinked.Howeverinformationbiaswithregardtothemeasurementofgripstrengthwaslikelytobeminimalbecauseweusedastandardisedgripstrengthmeasurementprotocol16.Theageofourcohortwascomparabletocohortsreportingtherelationshipbetweenbirthweightandadultgripstrength(HertfordshireCohortStudy,59-73years17andaBritishCohortStudy,53years5,18).Lackofpower isanotherpossibleexplanationforour lackofassociations.Althoughalmost700peopleparticipatedinthestudy,therewereonly30menexposedtofamineinearlygestation.Thesemenalsohadgreateradultheightandweight, although the differencewas not significant. Our study size is smaller than the other

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cohorts reportingon the associationbetweenbirthweight and adult grip strength4,5,18-20.Ourfindingsthereforeneedreplicationinfuturestudies. Regardlessofprenatalfamineexposurewealsofound,asmanyotherstudieshavedone,thatbirthweightwaspositivelyassociatedwithadultgripstrength, theassociationbeingstrongerformenthanforwomen4,5,18-21.Themagnitudeofourfindingswascomparabletothefindingsoftheseearlierstudies.Attenuationoftherelationshipbetweenbirthweightandgripstrengthafteradjustment foradult sizehasbeendescribedpreviously4,5andwasdemonstrated inourstudywith loss of the significant association after adjustment for height. One explanation isthatadjustingforadultsizeusingheight,alsocorrectsformusclesizewhichpotentiallyliesonthecausalpathwaybetweenlowbirthweightandreducedadultgripstrength.Thenumberofhumanskeletalmusclefibresisfixedbybirthandthereispreliminaryevidencethat lowbirthweightisassociatedwithreducedmyofibrescoreinlaterlife22. In summary, there was no evidence for significant independent associations betweenprenatalexposuretofamineandadultgripstrengthinthiscohortalthoughmenexposedinearlygestationappearedtohaveincreasedgripstrengthexplainedbyadulttallerheight.Consistentwithpreviousstudies,therewasarelationshipbetweensmallsizeatbirthandloweradultgripstrength.

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rEfErEncE lisT

1. BasseyEJ,HarriesUJ.Normalvaluesforhandgripstrengthin920menandwomenagedover65years,andlongitudinalchangesover4yearsin620survivors.Clin Sci (Lond).1993;84:331-337.

2. RantanenT,MasakiK,FoleyD,IzmirlianG,WhiteL,GuralnikJM.Gripstrengthchangesover27yrinJapanese-Americanmen.J Appl Physiol.1998;85:2047-2053.

3. CooperR,KuhD,HardyR.Objectivelymeasuredphysicalcapability levelsandmortality:systematicreviewandmeta-analysis.BMJ.2010;341:c4467.

4. InskipHM,GodfreyKM,MartinHJ,SimmondsSJ,CooperC,SayerAA.Sizeatbirthanditsrelationtomusclestrengthinyoungadultwomen.J Intern Med.2007;262:368-374.

5. KuhD,BasseyJ,HardyR,AihieSA,WadsworthM,CooperC.Birthweight,childhoodsize,andmusclestrengthinadultlife:evidencefromabirthcohortstudy.Am J Epidemiol.2002;156:627-633.

6. RoseboomTJ,vanderMeulenJHP,OsmondC,BarkerDJP,RavelliACJ,BlekerOP.PlasmalipidprofilesinadultsafterprenatalexposuretotheDutchfamine.AJCN.2000;72:1101-1106.

7. PainterRC,deRooij SR,RoseboomTJ,BossuytPMM,SimmersTA,OsmondC, et al. EarlyonsetofcoronaryarterydiseaseafterprenatalexposuretotheDutchfamine.AJCN.2006;84:322-327.


9. RavelliACJ,vanderMeulenJHP,MichelsRPJ,OsmondC,BarkerDJP,HalesCN,etal.Glucosetoleranceinadultsafterprenatalexposuretofamine.Lancet.1998;351:173-177.

10. deRooijSR,WoutersH,Yonker JE,PainterRC,RoseboomTJ.Prenatalundernutritionandcognitivefunctioninlateadulthood.Proc Natl Acad Sci U S A.2010;107:16881-16886.

11. TrienekensG.Tussenonsvolkendehonger.1sted.Utrecht:Matrijs;1985.12. Osgerby JC,Wathes DC, HowardD, Gadd TS. The effect ofmaternal undernutrition on ovine fetal

growth.J Endocrinol.2002;173:131-141.13. CostelloPM,RowlersonA,AstamanNA,AnthonyFE,SayerAA,CooperC,etal.Peri-implantationand

lategestationmaternalundernutritiondifferentiallyaffectfetalsheepskeletalmuscledevelopment.J Physiol.2008;586:2371-2379.

14. QuigleySP,KleemannDO,KakarMA,OwensJA,NattrassGS,MaddocksS,etal.Myogenesisinsheepisalteredbymaternalfeedintakeduringtheperi-conceptionperiod.Anim Reprod Sci.2005;87:241-251.

15. WooJ,LeungJC,WongSY.Impactofchildhoodexperienceoffamineonlatelifehealth.J Nutr Health Aging.2010;14:91-95.

16. RobertsHC,DenisonHJ,MartinHJ,PatelHP,SyddallH,CooperC,etal.Areviewofthemeasurementofgripstrengthinclinicalandepidemiologicalstudies:towardsastandardisedapproach.Age Ageing. 2011;40:423-429.

17. SyddallHE,AihieSA,DennisonEM,MartinHJ,BarkerDJ,CooperC.Cohortprofile:theHertfordshirecohortstudy.Int J Epidemiol.2005;34:1234-1242.

18. KuhD,HardyR,ButterworthS,Okell L,RichardsM,WadsworthM,etal.Developmentaloriginsofmidlifephysicalperformance:evidencefromaBritishbirthcohort.Am J Epidemiol.2006;164:110-121.

19. Robinson SM, Jameson KA, Batelaan SF, Martin HJ, Syddall HE, Dennison EM, et al. Diet and itsrelationship with grip strength in community-dwelling older men and women: the Hertfordshirecohortstudy.J Am Geriatr Soc.2008;56:84-90.

20. SayerAA,CooperC,EvansJR,RaufA,WormaldRP,OsmondC,etal.Areratesofageingdeterminedinutero?Age Ageing.1998;27:579-583.

21. SayerAA, SyddallHE,GilbodyHJ,Dennison EM,Cooper C.Does sarcopenia originate in early life?FindingsfromtheHertfordshirecohortstudy.J Gerontol A Biol Sci Med Sci.2004;59:M930-M934.

22. PatelH,JamesonK,SyddallH,MartinH,StewartC,CooperC,etal.DevelopmentalInfluences,MuscleMorphology,andSarcopeniainCommunity-DwellingOlderMen.J Gerontol A Biol Sci Med Sci.2011.

5Prenatal famine exposure, health in later life and promoter methylation

of four candidate genes

MarjoleinVEVeenendaal*PaulaMCostello*KarenALillycropSusanneRdeRooij

JorisAMvanderPostPatrickMMBossuytMarkAHanson

RebeccaCPainterTessaJRoseboom

*bothauthorscontributedequallytothiswork

Journal of Developmental Origins of Health and Disease, accepted for publication

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absTracT

Poornutritionduringfetaldevelopmentcanpermanentlyaltergrowth,cardiovascularphysiologyandmetabolic function.Animal studieshave shown thatprenatal undernutrition followedbybalanced postnatal nutrition alters DNAmethylation of gene promoter regions of candidatemetaboliccontrolgenesintheliver.Theaimofthisstudywastoinvestigatewhethermethylationstatusoftheproximalpromoterregionsof four candidategenesdifferedbetween individualsexposed to theDutch famine inutero. Inaddition,wedeterminedwhethermethylation statusof thesegeneswasassociatedwithmarkersofmetabolicandcardiovasculardiseaseandadultlifestyle.Methylation status of the GR1-C, PPARγ, LPL and PI3kinase p85 proximal promoters wasinvestigated inDNA isolated fromperipheralblood samplesof75958yearold subjectsbornaroundthetimeofthe1944-45Dutchfamine.We observed no differences in methylation levels of the promoters between exposed andunexposedmen andwomen.Methylation status of PPARγwas associatedwith levels of HDLcholesterolandtriglyceridesaswellaswithexerciseandsmoking.HypomethylationoftheGRpromoterwasassociatedwithadverseadultlifestylefactors,includinghigherBMI,lessexerciseandmoresmoking.Thepreviously reported increased riskof cardiovascular andmetabolicdiseaseafterprenatalfamineexposurewasnotassociatedwithdifferencesinmethylationstatusacrossthepromoterregionsofthesecandidategenesmeasuredinperipheralblood.TheadultenvironmentseemstoaffectGRandPPARγpromotermethylation.

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Prenatal famine exposure, health in later life and promoter methylation of four candidate genes

5

inTroDucTion

Lowbirthweightisassociatedwithanincreasedriskofchronicdiseaseinlaterlife.Studiesaroundtheworldhaveshownthatpeoplewhowerebornwithlowbirthweighthaveincreasedratesoftype2diabetes,hypertensionandcardiovasculardiseaseatadultage1-3.Theseassociationsarethoughttoreflecttheprocessbywhichfetalresponsestolimitedfoodsupplyinducepermanentalterationsintheoffspring’sphysiologyandmetabolism,optimizingitschancesofsurvivalintheshortterminapoornutritionalenvironmentbutpossiblybeingdetrimentalinlaterlife,especiallyiffoodisabundant.Thefirstdirectevidenceinhumansthatsuchundernutritionduringgestationincreasestheriskofchronicnon-communicablediseasehascomefromtheDutchfaminebirthcohortstudy.Peoplewhosemothershadbeenexposedtothisfivemonthperiodofacutefoodshortageinearlypregnancyhadimpairedglucoseregulation4,5,amoreatherogeniclipidprofile6, andincreasedratesofcardiovasculardisease7,8andwomeninthiscohorthadincreasedratesofbreastcancerinadultlife9.Theeffectsizewasstriking;thoseexposedtofamineinearlygestationhadadoubledrateofcardiovasculardiseasecomparedtothosewhohadnotbeenexposedtofamineprenatally7. Oneofthemechanismsthatmayplayaroleininducingsucheffectsisepigeneticregulationofgeneexpression.Animalstudieshaveshownthatreducedfoodintakeduringpregnancyleadstochangesintheepigeneticregulationoftheexpressionoftranscriptionfactorsthatplayakeyroleinregulatingglucoseandlipidmetabolismintheoffspring.Forinstance,feedingaproteinrestricted diet to rats during pregnancy induces hypomethylation of the PPARα (peroxisomeproliferator-activated receptor alpha) and GR (glucocorticoid receptor) promoters, increasedexpressionoftheGRandPPARαandtheirtargetgenesandanincreaseinthemetabolicprocessesthattheycontrol,namelyβ-oxidationandgluconeogenesisintheliveroftheoffspring10,11. ThefirstevidencethatundernutritionduringgestationaltersepigeneticregulationinhumanshascomefromHeijmansetal.12whohaveshownthatprenatalexposuretotheDutchfamineis associatedwithdecreasedmethylation levelsof thedifferentiallymethylated regionof theimprintedinsulin-likegrowthfactor-2gene(IGF2DMR)inperipheralblood.Themeanlevelofmethylationofexposedindividualswas49%comparedto52%inunexposedsiblingcontrols12.Thefunctionalimplicationofthissmall,butstatisticallysignificantdifferenceisunclear.Analysisof15additionalcandidategenesrevealedthatmethylationofsixoftheselociwasassociatedwithprenatalexposuretofamine,ofwhichthreeweresex-specific13.Theauthorssuggestedthatthese epigenetic changes could provide amechanismbywhich the prenatal nutrition affectsriskof laterchronicdiseases.Theauthors,however,didnotreportwhethersuchvariationsinmethylation were associated with any phenotypic characteristic. Also, there is an increasingbody of evidence to suggest that adult lifestyle factors can also affect the epigenome14.Wehavepreviouslyshownthatprenatalfamineexposureaffectslifestylechoices15whichmightalsocontributetodiseaserisk.Genomic imprintingisaphenomenonwherebyageneisexpressedinaparent-originmanner,withoneactiveandonesilentallele.Manymethylationstudieshave

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beenlimitedtoimprintedgenes,butalsonon-imprintedgenescanbeenaffectedbyepigeneticprogrammingeffects. Wethereforeselectedfournon-imprintedcandidategeneswithrelevancetocardiovascularandmetabolicfunction.Peroxisomeproliferator-activatedreceptorgamma(PPARγ)is involvedininsulinandlipidmetabolism16.Glucocorticoidreceptor(GR)1-Cregulatesthestressresponse,developmental processes, immune responses and glucose metabolism. The p85α regulatorysubunitofphosphatidylinositol3kinase(PI3kinase)isinvolvedinplacentaldevelopmentandfetalgrowth17andakeycomponentintheinsulinsignalingpathway.Lipoproteinlipase(LPL)hydrolyseslipids into lipoproteins.A LPLdeficiency leads tohypertriglyceridemia18. Theexpressionof allfourof thesegeneshasbeenshowntobepersistentlyalteredbymaternaldietaryrestrictioninanimalmodelsofmaternalprogramming19-22.Weinvestigatedwhethermethylationstatusoftheirpromoterregionsdifferedbetween individualsexposedtofamineatdifferentperiodsofgestationcomparedtounexposed individuals.Secondly,we investigatedwhethermethylationstatusofthesefourgeneswasassociatedwithmarkersofmetabolicandcardiovasculardiseaseoradultlifestylefactors,andthuswhethertheycouldexplaintheincreasedratesofdiseaseinpeoplewhowereprenatallyexposedtotheDutchfamine.

METhoDs

Selection procedures TheDutch faminebirth cohort consists of 2,414menandwomenborn as term singletons inthe Wilhelmina Gasthuis in Amsterdam between 1 November 1943 and 28 February 1947.The selection procedure and subsequent loss to follow up have been described in detailelsewhere5,23.Atage58,1,423ofthe2,414originalcohortmembers(58%)werestillalive,livingin theNetherlands at a known address. The studywas approved by the localMedical EthicsCommitteeandcarriedoutinaccordancewiththeDeclarationofHelsinki.Allparticipantsgavewritteninformedconsent.

Exposure to famineExposuretofaminewasdefinedaccordingtotheofficialdailyrationsforthegeneralpopulationolder than21years. Theofficial rationsaccurately reflect thevariationovertime in the totalamountoffoodavailableinthewestoftheNetherlands24.Anindividualwasconsideredtobeprenatallyexposedtofamineiftheaveragedailyfoodrationofthemotherduringany13-weekperiod of gestation contained less than 1000 calories. Based on this definition, babies bornbetween 7 January 1945 and 8 December 1945were exposed in utero.We used periods of16weekseachtodifferentiatebetweenpeoplewhohadbeenexposedinlategestation(bornbetween7January1945and28April1945),inmidgestation(bornbetween29April1945and18August1945),andinearlygestation(bornbetween19August1945and8December1945).

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5

People born before 7 January 1945 (andwere thus born before the famine) and those bornafter8December1945(andwhohadthusbeenconceivedafterthefamine)wereconsideredunexposed.

study parametersThemedicalbirthrecordsprovidedinformationaboutthemother,thecourseofthepregnancy,and the body size of the baby at birth5. For the adultmeasurements of the participants,wemeasuredadultheightusingaportablestadiometerandweightwithaportableTefalscale.Bloodpressurewasmeasuredinduploontwooccasions(morningandafternoon)usinganautomateddevice (Omron705CP/IT;OmronHealthcareUK,WestSussex,UK)andappropriatecuffsizes.Meansystolicanddiastolicbloodpressurewascalculatedusingallavailablemeasurements. Weperformedanoralglucosetolerancetest(OGTT)afteranovernightfastwithastandardload of 75 grams. Participantswith pre-existing diabetes (defined as taking glucose-loweringmedication)wereexcluded fromtheOGTT.PlasmaglucoseconcentrationsweremeasuredbystandardizedenzymaticphotometricassayonaModularPanalyzer(Roche,Basel,Switzerland)and plasma insulin concentration by immuno-luminometric assay on Immulite 2000 analyzer(DiagnosticProductCorporation,LosAngeles,USA).Type2diabeteswasdefinedasa2hrglucoselevel of >11.0 mmol/l (in accordance with our previous studies and the 1999World HealthOrganizationrecommendations4,5,25,26)ortakinganti-diabeticmedication. AfastingbloodsamplewasdrawnforanalysisofLowDensityLipoprotein(LDL)-cholesterol,HighDensityLipoprotein(HDL)-cholesterolandtriacylglycerol.HDL-cholesterolandtriacylglycerolweremeasuredusinganenzymaticcolorimetricagent(Roche)onaP-800Modular(Roche).LDL-cholesterolwascalculatedusingtheFriedewaldformula.Standard12-leadelectrocardiograms(ECG)weremadeofallparticipants.Coronaryheartdiseasewasdefinedasthepresenceofoneormoreofthefollowing:anginapectorisaccordingtotheRose/WHOquestionnaire27;QwavesontheECG(Minnesotacodes1-1or1-2)orahistoryofcoronaryrevascularization(angioplastyorbypasssurgery). WeperformedB-modeultrasoundexaminationsofthearterialwallsofthecommon,bulband internalcarotidarterysegments.Detailsofthemeasurementsaredescribedelsewhere23.Meancarotidintimamediathickness(IMT)wasdefinedasthemeanIMTinmmoftherightandleftcommonartery,commonbulbandtheinternalcarotidfarwallsegments.Ifeithertherightorleftvaluewasmissingforanygivencarotidsegment,theremainingavailablesegmentwasusedtocalculatethemeancarotidIMT. ADutchtranslationoftheHADS(HospitalAnxietyandDepressionScale)wasadministeredtoallparticipantstomeasuresubclinicaldepressionandanxietysymptoms28.TheHADSconsistsoftwosubscales:adepressionsubscale(HADS-D,sevenitems,scorerange0-21,Cronbach’salpha0.80)andananxietysubscale(HADS-A,sevenitems,scorerange0-21,Cronbach;salpha0.82).Cronbach’salphawas0.88forall14HADSitems,indicatinggoodinternalconsistency.

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Informationonsocioeconomicstatus,medicalhistory,andlifestyle(smoking,exercise)andtheuseofmedicationwas retrieved fromastandardized interview.CurrentsocioeconomicstatuswascodedaccordingtoInternationalSocio-EconomicIndex92,whichisanumericscalebasedontheperson’sorhisorherpartner’soccupation,whicheverwashigher29.

Methylation-sensitive PCRDNA was extracted from a fasting blood sample and stored at 4°C. To ensure maximumcomparability with rodentmodels ofmaternal undernutrition and epigenetic changes in theoffspring,weassessedmethylationstatusbyapplyingmethylation-sensitivepolymerasechainreaction(PCR)11. For analysis of promoter methylation, genomic DNA (400 ng) was incubated with themethylationsensitiverestrictionendonucleasesAciIandHinfIasinstructedbythemanufacturer(NewEnglandBiolabs,Hitchin,Hertfordshire,UK).TheresultingDNAwasamplifiedusingrealtimePCR,whichwasperformedinatotalvolumeof25μlwithSYBR®GreenJumpstartReadyMix(Sigma)asdescribedbythemanufacturer.AfragmentofthehumanPPARαexon7whichdoesnotcontainAciIorHinfIcleavagesiteswasusedasaninternalcontrolgene.PrimersweredesignedtoamplifyregionscontainingHinfIand/orAciIcuttingsitesintheproximalpromotersofLPL(chr8:19,796,366-19,796,515) PI3kinase (chr5: 67,521,933-67,522,282), PPARγ (chr3:12,392,392-12,392,591)andtheCpGislandspanningtheGR1-Cpromoter(chr5:142,782,821-142,783,152)(seeLillycropetal21foranoverview).Cycleparameterswere94°Cfor2minutesthen,40cyclesof95°Cfor30s,60°C(GR1-C,PPARα)or55°C(PPARγ,LPL)or65°C(PI3kinasep85) for1minuteand72°Cfor1minute.PrimersequencesareshowninTable1.AllCtvalueswerenormalizedto the internal control andeach sample analyzed induplicate.Methylationwasexpressedaspercentagemethylationcomparedtothecontrolgene.

Table 1 Primer sequences (5’ to 3’) used in the measurement of promoter methylation levels bymethylationsensitivePCR.

Target gene Primers sequence

PPARα ForwardPrimer CGG-AGT-TTA-TGA-GGC-CAT-ATT-C

ReversePrimer AGG-GAG-ATA-TCA-CTG-TCA-TCC-AG

GR1-C ForwardPrimer ATT-TTG-CGA-GCT-CGT-GTC-TG

ReversePrimer CGC-AGC-CGA-GAT-AAA-CAA-CT

PPARγ ForwardPrimer AAC-CCT-TCT-TCA-TTC-TCT

ReversePrimer CTG-GTT-GAA-TCT-CTA-ATC-A

LPL ForwardPrimer GGG-AGG-ACT-GCA-AGT-GAC-AAA

ReversePrimer CAC-CAA-ACA-CAG-GTT-TAC-ATC-GA

PI3kinasep85 ForwardPrimer CCC-GCC-CGG-TGT-TCT-TA

ReversePrimer TTC-CTC-TGC-GTG-CCA-CAG-T

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5

Statistical methodsDistributionsofmethylationstatusofthepromoterregionsforthefourcandidategeneswerehighly skewed and we used logarithmic transformations to better approach normality. Weused linear and logistic regression analysis to compare maternal, birth and adult outcomesandmethylation levelsof thoseexposed in late,midorearlygestationand thoseunexposedto famine during gestation. Adjustments were made for maternal age, sex and parity. Ininvestigating possible associations between methylation status and markers of disease, wereport regressioncoefficientsandp-valuesof linearand logistic regressionanalysesusing logtransformedmethylationvalues.Weconsideredeffectstobestatisticallysignificantifp-valueswere≤0.05.SPSS16.0(SPSSInc,ChicagoIL)wasusedforallstatisticalanalyses.

rEsulTs

Characteristics of the study populationDNAwasavailablefrom759participants.Theirmeanagewas58years(SD1);349(46%)weremen.Atotalof319(42%)hadbeenexposedtofamineinutero,and440(58%)hadnotbeenexposedtofamineprenatally.Table2showsthatmothersexposedtofaminein lategestationwereolderand lessoftenprimiparous thanmotherswhohadnotbeenexposedto famine inpregnancy. Babies exposed to famine in late andmid-gestationhad lowerbirthweights thanunexposed babies. At age 58, there were no significant differences between exposed andunexposedgroupsinsmokinghabitsandsocio-economicstatus.RelativemethylationlevelsofthefourcandidategenesareshowninTable3.

Methylation and birth and maternal characteristicsBirthweightwaspositivelyassociatedwithGRmethylationbutnotwiththemethylationstatusoftheotherthreegenes.A1kilogramincreaseinbirthweightwasassociatedwith22%(95%CI4to43)increaseinGRmethylation(p0.02).Therewasnoassociationbetweenmethylationofthefourgenesandgender,maternalageorparity.

Methylation after exposure to famine in uteroCrude and adjustedmethylation differences between subjects exposed in late,mid- or earlygestation and those unexposed to famine are shown in Table 4. Exposure to famine duringgestationwasnotassociatedwithalteredmethylationstatusoftheGR,LPL,PPARγandPI3kinasepromoter.Thisalsoappliedafteradjustmentforsex,maternalageorparity.Theassociationsdidnotdifferbetweenmenandwomen(pforallinteractionterms>0.05).

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Table 2 Maternal,birthandadultcharacteristicsaccordingtotimingofprenatalexposuretotheDutchfamine.

Exposure to famine

born before

in late gestation

in mid gestation

in early gestation

conceived after

all n

general

Number 235 134 112 73 205 759

proportionofmen(%) 46 44 39 43 50 46 759

age(years) 59.3 58.5 58.2 58.0 57.4 58.4±1.0 758

Maternal characteristics

ageatdelivery(years) 28.9 31.1* 29.0 27.2 28.3 29.0±4.9 650

primiparous(%) 36.2 20.1* 33.0 42.5 38.0 34.0 759

birth outcomes

gestationalage(days) 284 283 286 289* 285 285±11 759

birthweight(g) 3389 3196* 3207* 3504 3453 3356±472 759

Adult characteristics

bodymassindex(kg/m²) 28.6 28.1 28.1 28.0 29.2 28.5±4.9 753

currentsmoker(%) 22 25 26 32 22 24 754

currentSES(ISEI-92) 48.5 51.6 52.2 46.8 50.3 49.9±14.2 746

Dataaremeans±SD,exceptwheregivenasnumbersandpercentages*p<0.05comparedtopeopleunexposedtofamineinutero.

Table 3RelativemethylationaccordingtotimingofprenatalexposuretotheDutchfamine.

Exposure to famine

born before

in late gestation

in mid gestation

in early gestation

conceived after

min - max n

Relative methylation

GRa 0.069 0.073 0.063 0.080 0.065 0.025–20.27 759

LPLa 0.021 0.022 0.022 0.022 0.020 0.006–0.91 757

PI3kinasea 0.016 0.019 0.011 0.010 0.020 0.000–14.62 757

PPARγa 0.031 0.030 0.027 0.028 0.029 0.000–0.74 756

a geometricmean

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5

Table 4 Methylation differences (%) and 95% CI’s compared to unexposed subjects using a linearregressionmodel.

Exposure to famine

in late gestation in mid gestation in early gestation

gr

Crude 0.20(-16.56to20.32) -6.20(-22.82to13.88) 5.65(-16.47to33.64)

adjusted* 0.60(-16.39to21.05) -5.26(-22.04to15.14) 6.82(-15.55to35.12)

lPl

Crude 10.52(-5.64to29.43) 12.08(-5.35to32.84) 9.42(-10.68to34.18)

adjusted* 11.07(-5.35to30.34) 12.08(-5.45to32.84) 9.20(-10.95to34.04)

Pi3kinase

Crude 11.96(-38.55to104.21) -30.02(-63.06to32.58) -40.84(-72.50to27.12)

adjusted* 6.18(-42.25to95.03) -32.36(-64.33to28.27) -40.84(-72.56to27.38)

PPARγ

Crude -3.82(-15.63to9.53) -9.15(-21.02to4.50) -6.48(-20.94to10.52)

adjusted* -2.37(-14.53to11.52) -8.70(-20.63to5.02) -6.76(-21.08to10.30)

*adjustedforsex,maternalageandparity

Methylation and markers of diseaseWetestedforassociationsbetweenDNAmethylationstatusandmarkersofcardiovascularandmetabolicdiseaseaswellaslifestylefactorspredisposingforcardiovasculardisease.RegressioncoefficientsofthesemarkersandlifestylefactorsaccordingtomethylationlevelsareshowninTable 5. The regression coefficients depict the size and direction of the association betweenthediseasemarkersandlifestylefactorsandthemethylationlevels.IncreasedGR-1CpromotermethylationstatuswasassociatedwithlowerscoreonHADSsubscaleanxietyanddepression,lowerselfperceivedhealthandlowerBMI,higherlevelsofphysicalactivityandnon-smoking.IncreasedmethylationstatusofPPARγpromoterwasassociatedwithlowerplasmatriglyceridelevel,higherLDLlevels,higherlevelsofphysicalactivityandnon-smoking.

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Table 5Healthoutcomesaccordingmethylationstatus.

n grβ

p-value lPlβ

p-value PPARγβ

p-value Pi3 kinase

β

p-value

health

Diabetes 756 -0.09 0.36 -0.11 0.37 -0.07 0.63 0.001 0.97

Glucose0‡* 585 0.000 0.96 -0.007 0.26 0.008 0.38 0.000 0.87

Glucose120‡* 566 -0.02 0.07 -0.004 0.80 -0.003 0.88 0.000 0.85

Insulin0‡* 585 -0.02 0.27 -0.02 0.43 -0.04 0.30 0.001 0.93

Insulin120‡* 563 -0.04 0.19 0.03 0.41 -0.07 0.17 0.002 0.88

chD 681 -0.06 0.72 0.19 0.27 -0.12 0.58 -0.008 0.87

HDL 755 0.03 0.07 0.01 0.57 0.04 0.05 0.007 0.14

LDL 752 0.01 0.76 -0.04 0.38 0.08 0.15 -0.009 0.41

Cholesterol 756 0.01 0.70 -0.03 0.53 0.06 0.30 -0.007 0.55

Triglycerides‡ 755 -0.03 0.12 -0.001 0.97 -0.08 0.01 -0.007 0.31

RRsys 667 -0.34 0.61 -1.12 0.16 -0.44 0.66 -0.47 0.39

RRdia 667 -0.06 0.88 -0.62 0.17 -0.22 0.69 -0.17 0.18

IMT 666 -0.004 0.95 0.08 0.26 -0.09 0.29 0.03 0.15

HADSA 731 -0.33 0.003 0.01 0.93 -0.17 0.32 0.05 0.19

HADSB 731 -0.27 0.01 0.01 0.92 -0.27 0.11 0.03 0.44

Perceptionofhealth 755 -0.10 0.001 0.008 0.82 -0.09 0.06 -0.006 0.52

Lifestyle

BMI 751 -0.38 0.02 0.08 0.70 -0.21 0.43 -0.03 0.57

Exercise 630 0.08 0.001 0.04 0.21 0.10 0.02 0.004 0.67

Smoking 754 -0.40 0.0001 0.03 0.80 -0.37 0.003 0.02 0.59

GR,glucocorticoidreceptor;LPL,lipoproteinlipase;PI3kinase,phosphatidylinositol3kinase;PPAR,peroxisomeproliferator-activatedreceptor;CHD,coronaryarterydisease;HDL,highdensitylipoprotein;LDL,low-densitylipoprotein;RRsys,systolicbloodpressure;RRdia,diastolicbloodpressure;IMT,intima-mediathickness;HADS,HospitalAnxietyandDepressionScale;BMI,bodymassindex.β: regression coefficients from linear or logistic regressionmodels in whichmethylation values were log transformed.Regressioncoefficientsdepictthesizeanddirectionoftheassociationbetweenhealthandlifestylefactorsandmethylationlevelsofthefourdifferentcandidategenes.Significantassociationsaredepictedinboldprint.‡valueswerelogtransformed*measurementsatbaselineand120minutes.

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Discussion

Inthepresentstudy,weexaminedtheepigeneticstateofnon-imprintedgenesinperipheralbloodofadultsexposed to famineduringdifferentperiodsofperinataldevelopment incomparisontounexposedcontrols.Wedidnotobservedifferences in methylationlevelsofthepromotersof our chosen candidate genes betweenmen andwomenwho had been exposed to famineineither late,midorearlygestationand thosewhohadnotbeenexposed.Wedidfind thatincreasedmethylationstatusofPPARγpromoterwasassociatedwithlowerplasmatriglyceridelevel,higherLDLlevels,andhigherlevelsofexerciseandnon-smoking.HypermethylationoftheGRpromoterwasassociatedwithlowerscoreonHADSsubscaleanxietyanddepression,lowerself-perceivedhealth,lowerBMI,andhigherlevelsofexerciseandnon-smoking. Thesedatacontrastwiththosewepreviouslyreported10,11inyoungadultratoffspringborntodamsexposedtoasimilarlevelofunbalancednutritionthroughoutthewholeofpregnancy.Thereareseveralpossiblereasonsforthisdifference. First,thelackofeffectsofprenatalnutritionalenvironmentonmethylationofthecandidategenesmaybedue to the variable levels of postnatal diet and lifestyle factorswhichhumansexperience. In comparison with the rat offspring which were fed a standardized diet, suchdietaryvariationsmight induceavariabledegreeofpre-andpostnatalmismatchandthusofepigenotype.Suchmaskingofprenatalepigeneticchanges,ifpresent,maybegreaterinolderindividuals, aswas the case for the adults studiedhere as compared toprevious rat studies.Support for this concept comes from studies ofmonozygotic twins30 inwhich changes in theepigenomeoccurredwithage.Ourfindingthat lifestylefactors inadultswereassociatedwiththemethylationofthepromotersofGR1-CandPPARγisinagreementwiththis. Asecondconsiderationisthatepigeneticprocessesareknowntobetissue-specific31.WhilstinthepresentstudyweonlyhadaccesstoDNAisolatedfromperipheralwhitebloodcells,studiesin rodents used target tissues such as liver11.In newborn twin pairsmethylation level of fourdifferentiallymethylatedregionsassociatedwiththe IGF2/H19 locusvariedinatissuespecificmannerinfivedifferenttissuesrepresentingthedifferentgermlayers32.HoweverotherstudiesmaysuggestthatDNAmethylationpatternsaresimilarbetweentissues31.Itmaybethatiftheenvironmentalconstraintoccurredearlyonindevelopment,genemethylationmaybealteredinthreegermlayersandanimprintofthisalteredepigeneticmarkwillbedetectableinalltissues.Methylationpatternsinbloodandperhapsinotherreadilyavailabletissuemaythereforeprovideusefulproxymarkersofmethylationinmoremetabolicallyrelevanttissues.Indeed,Heijmansetal12didfindchangesinDNAmethylationinperipheralwhitebloodcells,althoughthechangesweresmall,occurringinimprintedgenes,andtheirfunctionalsignificanceisnotknown.Morerecently, Godfrey et al has reported in two independent cohorts themethylation status of asingleCpGsite in thepromoter regionof the transcription factorRXRA inumbilical cordwasstronglyrelatedtochildhoodadiposityinbothboysandgirls33.

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In the present study we used methylation sensitive PCR to measure methylation across theentire proximal promoter region of our candidate genes. Thismethodwould not allowus todetectchangesatthelevelofindividualCpGs.Nevertheless,thisispreciselythetechniquewehavepreviouslyusedextensively inrodents10,11,21,34,35andinthisstudywedidfindassociationsbetweenthemethylationstatusofGR1-CandPPARγwitheitherpostnatalmetabolicoutcomesand/orpostnatallifestylefactors. Also,thesmallnumberofpeopleexposedtofamineinearlygestation(n=73)–thegroupinwhomwepreviouslyfoundadoubledrateofcardiovasculardiseaseinadultlife7‒mayhavelimitedourabilitytodetectdifferences.However,thenumberofsubjectsintheHungerwinterfamiliesstudywasloweranddidnotpreventthemfromfindingahighlysignificantdifferenceinmethylation levels between subjects exposed to famine in early gestation and unexposedsubjects12.Thereforewedonotconsiderourstudyunderpoweredtodetectfamineeffectsonmethylation. Inrats, thehepaticGR110promoter,whichshows70.6%homologywiththehumanGR1-Cpromoter36 is hypomethylated after prenatal low protein diet21. Hypomethylation of thepromoter is associatedwith an84%highermRNAexpression21; therefore,GR1-Cwas a likelycandidategeneinourstudy.Interestingly,inbothourcurrentstudyaswellasinthepreviouslymentioned Hungerwinter families study, differences in GRmethylation were not found afterprenatalexposuretofamine13.Unfortunately,mRNAwasnotavailablefromtheparticipantsofthisstudy. Althoughthiswasnotourprimaryresearchfocus,andthereisalwaysapossibilityoftype1 errors, we found an association between beneficial characteristics such as lower levels ofBMI, non-smoking and physical activity in adult life –which tend to cluster – and increasedmethylationstatusoftheGR1-Cpromoter.Smoking–whichwasmoststronglyassociatedwithGRmethylation‒isawell-knownlifestylefactorinfluencingepigeneticpatterns,bothinanimalmodelsandinhumans14.Forinstance,exposureofhumanlungcancercellstocigarettesmokeresulted indemethylationofthepromoteroftheprometastaticoncogenesynuclein-gamma37.InDNAfromperipheralblood,cigarettesmokinghasbeenlinkedtohypomethylationofgenesinvolved inplateletactivation38and serotonin regulation39,findings thatfitwith the idea thatpostnatalinfluencesalsoalterepigeneticpatterns.WhetherourfindingofsmokingandincreasedmethylationstatusoftheGR1-Cpromoterhasaclinicalsignificanceremainstobeinvestigated.WenowknowfromourdatathatGRmethylationisassociatedwithadultstressresponsiveness,although theseassociationswere largelyexplainedbydifferences in lifestyleandeducation40.OurcurrentanalysessuggestthatGRhypomethylation isalsoassociatedwithhigher levelsofdepressionandanegativehealthperception.Recentstudieshavereportedassociationsbetweenmethylation status at birth and body size in later childhood, suggesting a role for epigeneticfactorsasmediatorsforearlylifeprogrammingofdiseaseinlaterlife33,41.

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Basedontheresultsofthisstudy,wecanconcludethattheincreasedratesofchronicdegenerativedisease found after famine exposure are not mediated by differences in methylation statusof fourgenesof strongcandidacybasedonanimalandother studies.Thisargues for furtherstudies,inparticulartoaddresstheinteractionbetweenpre-andpostnatalnutritionalandotherenvironmentalinfluences,andtoexplorethemechanisticpathwaysunderlyingtheassociationbetweenprenatalfamineandlatermarkersofcardiovascularandmetabolicdisease.

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1. HuxleyR,OwenCG,WhincupPH,CookDG,Rich-EdwardsJ,SmithGD,etal.Isbirthweightariskfactorforischemicheartdiseaseinlaterlife?Am J Clin Nutr.2007;85:1244-1250.

2. HuxleyRR,ShiellAW,LawCM.Theroleofsizeatbirthandpostnatalcatch-upgrowthindeterminingsystolicbloodpressure:asystematicreviewoftheliterature.J Hypertens.2000;18:815-831.

3. WhincupPH,KayeSJ,OwenCG,HuxleyR,CookDG,AnazawaS,etal.Birthweightandriskoftype2diabetes:asystematicreview.JAMA.2008;300:2886-2897.



6. RoseboomTJ,vanderMeulenJH,OsmondC,BarkerDJ,RavelliAC,BlekerOP.PlasmalipidprofilesinadultsafterprenatalexposuretotheDutchfamine.Am J Clin Nutr.2000;72:1101-1106.

7. PainterRC,deRooij,SR.,BossuytPM,SimmersTA,OsmondC,BarkerDJ,etal.EarlyonsetofcoronaryarterydiseaseafterprenatalexposuretotheDutchfamine.Am J Clin Nutr.2006;84:322-327.

8. RoseboomTJ,vanderMeulenJH,OsmondC,BarkerDJ,RavelliAC,Schroeder-TankaJM,etal.CoronaryheartdiseaseafterprenatalexposuretotheDutchfamine,1944-45.Heart.2000;84:595-598.

9. PainterRC,deRooijSR,BossuytPM,OsmondC,BarkerDJ,BlekerOP,etal.Apossiblelinkbetweenprenatalexposuretofamineandbreastcancer:apreliminarystudy.Am J Hum Biol.2006;18:853-856.

10. BurdgeGC,Slater-JefferiesJ,TorrensC,PhillipsES,HansonMA,LillycropKA.Dietaryproteinrestrictionofpregnantrats intheF0generationinducesalteredmethylationofhepaticgenepromoters intheadultmaleoffspringintheF1andF2generations.Br J Nutr.2007;97:435-439.

11. LillycropKA,PhillipsES,JacksonAA,HansonMA,BurdgeGC.Dietaryproteinrestrictionofpregnantratsinducesandfolicacidsupplementationpreventsepigeneticmodificationofhepaticgeneexpressionintheoffspring.J Nutr.2005;135:1382-1386.


13. TobiEW,LumeyLH,TalensRP,KremerD,PutterH,SteinAD,etal.DNAmethylationdifferencesafterexposuretoprenatalfaminearecommonandtiming-andsex-specific.Hum Mol Genet.2009;18:4046-4053.

14. Mathers JC, Strathdee G, Relton CL. Induction of epigenetic alterations by dietary and otherenvironmentalfactors.Adv Genet.2010;71:3-39.

15. LussanaF,PainterRC,OckeMC,BullerHR,BossuytPM,RoseboomTJ.PrenatalexposuretotheDutchfamineisassociatedwithapreferenceforfattyfoodsandamoreatherogeniclipidprofile.Am J Clin Nutr.2008;88:1648-1652.

16. AuwerxJ.PPARgamma,theultimatethriftygene.Diabetologia.1999;42:1033-1049.17. ForbesK,WestwoodM.Maternalgrowthfactorregulationofhumanplacentaldevelopmentandfetal

growth.J Endocrinol.2010;207:1-16.18. MeadJR,IrvineSA,RamjiDP.Lipoproteinlipase:structure,function,regulation,androleindisease.J

Mol Med.2002;80:753-769.19. BurdgeGC,Slater-JefferiesJL,GrantRA,ChungWS,WestAL,LillycropKA,etal.Sex,butnotmaternal

proteinorfolicacidintake,determinesthefattyacidcompositionofhepaticphospholipids,butnotoftriacylglycerol,inadultrats.Prostaglandins Leukot Essent Fatty Acids.2008;78:73-79.

20. GluckmanPD,LillycropKA,VickersMH,PleasantsAB,PhillipsES,BeedleAS,etal.Metabolicplasticityduringmammaliandevelopmentisdirectionallydependentonearlynutritionalstatus.Proc Natl Acad Sci U S A.2007;104:12796-12800.

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21. LillycropKA,Slater-JefferiesJL,HansonMA,GodfreyKM,JacksonAA,BurdgeGC.Inductionofalteredepigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricteddietduringpregnancysuggeststhatreducedDNAmethyltransferase-1expressionisinvolvedinimpairedDNAmethylationandchangesinhistonemodifications.Br J Nutr.2007;97:1064-1073.

22. OzanneSE,JensenCB,TingeyKJ,StorgaardH,MadsbadS,VaagAA.Lowbirthweightisassociatedwithspecificchangesinmuscleinsulin-signallingproteinexpression.Diabetologia.2005;48:547-552.

23. Painter RC, de Rooij, SR., Hutten BA, Bossuyt PM, deGE,Osmond C, et al. Reduced intimamediathicknessinadultsafterprenatalexposuretotheDutchfamine.Atherosclerosis.2007;193:421-427.

24. TrienekensG.Tussenonsvolkendehonger.1sted.Utrecht:Matrijs;1985.25. deRooijSR,PainterRC,PhillipsDI,OsmondC,MichelsRP,GodslandIF,etal.Impairedinsulinsecretion

afterprenatalexposuretotheDutchfamine.Diabetes Care.2006;29:1897-1901.26. World Health Organization. Definition, Diagnosis and Classification of Diabetes Mellitus and its

Complications. Report of aWHO Consultation. Part1,Diagnosis and Classification. Geneva:WorldHealthOrganization;1999.

27. RoseGA.Thediagnosisofischaemicheartpainandintermittentclaudicationinfieldsurveys.Bulletin of the World Health Organisation.1962;27:645-658.

28. ZigmondAS,SnaithRP.Thehospitalanxietyanddepressionscale.Acta Psychiatr Scand.1983;67:361-370.

29. BakkerB,SiebenI.Matenvoorprestige,sociaal-economischestatusensocialeklassevoordestandaardberoepenclassificatie1992.Sociale Wetenschappen.1997;40:1-22.

30. FragaMF,BallestarE,PazMF,RoperoS,SetienF,BallestarML,etal.Epigeneticdifferencesariseduringthelifetimeofmonozygotictwins.Proc Natl Acad Sci U S A.2005;102:10604-10609.

31. ByunHM,SiegmundKD,PanF,WeisenbergerDJ,KanelG,LairdPW,etal.Epigeneticprofilingofsomatictissues from human autopsy specimens identifies tissue- and individual-specific DNA methylationpatterns.Hum Mol Genet.2009;18:4808-4817.

32. OllikainenM,SmithKR,JooEJ,NgHK,AndronikosR,NovakovicB,etal.DNAmethylationanalysisofmultipletissuesfromnewborntwinsrevealsbothgeneticandintrauterinecomponentstovariationinthehumanneonatalepigenome.Hum Mol Genet.2010;19:4176-4188.

33. GodfreyKM,SheppardA,GluckmanPD, LillycropKA,BurdgeGC,McLeanC,etal. EpigeneticGenePromoterMethylation at Birth Is AssociatedWith Child’s Later Adiposity.Diabetes. 2011;60:1528-1534.

34. Burdge GC, Lillycrop KA, Phillips ES, Slater-Jefferies JL, Jackson AA, Hanson MA. Folic acidsupplementationduringthejuvenile-pubertalperiodinratsmodifiesthephenotypeandepigenotypeinducedbyprenatalnutrition.J Nutr.2009;139:1054-1060.

35. Lillycrop KA, Phillips ES, Torrens C, Hanson MA, Jackson AA, Burdge GC. Feeding pregnant rats aprotein-restricted diet persistently alters themethylation of specific cytosines in the hepatic PPARalphapromoteroftheoffspring.Br J Nutr.2008;100:278-282.

36. Turner JD,Pelascini LP,Macedo JA,MullerCP.Highly individualmethylationpatternsof alternativeglucocorticoidreceptorpromoterssuggestindividualizedepigeneticregulatorymechanisms.Nucleic Acids Res.2008;36:7207-7218.

37. LiuH,ZhouY,BoggsSE,BelinskySA,LiuJ.Cigarettesmoke inducesdemethylationofprometastaticoncogene synuclein-gamma in lung cancer cells by downregulation of DNMT3B. Oncogene. 2007;26:5900-5910.

38. Breitling LP, Yang R, Korn B, Burwinkel B, Brenner H. Tobacco-smoking-related differential DNAmethylation:27Kdiscoveryandreplication.Am J Hum Genet.2011;88:450-457.

39. LaunayJM,DelPM,ChironiG,CallebertJ,Peoc’hK,MegnienJL,etal.Smokinginduceslong-lastingeffectsthroughamonoamine-oxidaseepigeneticregulation.PLoS One.2009;4:e7959.

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40. deRooijSR,CostelloPM,VeenendaalMV,LillycropKA,GluckmanPD,HansonMA,etal.Associationsbetween DNA methylation of a glucocorticoid receptor promoter and acute stress responses ina large healthy adult population are largely explained by lifestyle and educational differences.Psychoneuroendocrinology.2011.

41. ReltonCL,GroomA,StPB,SayersAE,SwanDC,EmbletonND,etal.DNAmethylationpatternsincordbloodDNAandbodysizeinchildhood.PLoS One.2012;7:e31821.

6Associations between DNA methylation

of a glucocorticoid receptor promoter and acute stress responses in a large healthy adult population are largely explained by

lifestyle and educationial differences

SusanneRdeRooijPaulaMCostello

MarjoleinVEVeenendaalKarenALillycropGrahamCBurdgePeterDGluckmanMarkAHanson

RebeccaCPainterTessaJRoseboom

Psychoneuroendocrinology. 2012;37(6):782-8

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absTracT

background: Glucocorticoids are the key regulators of the biological stress response and actby binding to glucocorticoid receptors (GR). Expression ofGR is altered byDNAmethylation.Methylation patterns in GR promoters have been shown to be highly variable betweenindividuals,butlittleisknownaboutthefunctionalconsequencesofthisvariationfortheacutestressresponse.ThepresentstudyinvestigatedassociationsbetweenmethylationstatusoftheGR1-Cpromoterandcortisol,cardiovascularandperceivedstressresponsestoapsychosocialstressprotocolinalargehealthyadultpopulation.Methods:A totalof725overallhealthymenandwomen,aged55-60years,participated inastandardized psychosocial stress protocol consisting of three different stressors. At differentstagesduringthestressprotocol,salivarycortisol levels,continuousbloodpressureandheartrate(HR)levelsaswellasperceivedstressweremeasured.Stressreactivitywascalculatedastheincreasebetweenbasalandpeakmeasurements.MethylationstatusoftheGR1-CpromoterwasassessedinDNAisolatedfromperipheralbloodsamplesusingamethylationsensitivePCRassayfor675ofthe725participants.results:Adecrease inmethylationoftheGR1-Cpromoterwasassociatedwithadecrease instressreactivityasindicatedbylowercortisolandlowerHRreactivity.A1%decreaseinGR1-Cmethylationcorrespondedwithacortisoldecreaseby0.14%(95%CI:0.03to0.25,p=0.02)andanHRdecreaseby0.10bpm(0.03to0.16,p =0.003).Adjustingforsex,lifestyleandeducationlargely abolished these associations. A decrease inmethylation of the GR 1-C promoterwasalso associatedwith an increase in stress perception as indicated by higher perceived stress(0.03points[0.00to0.06,p =0.05]),lowerperceivedperformance(-0.03points[-0.05to-0.01],p=0.02),and lowerperceivedcontrol (-0.03points [-0.05 to0.00],p=0.04).Afteradjustingfor sex and educational level the associations were no longer statistically significant. GR 1-Cmethylationstatuswasnotassociatedwithbloodpressureresponsestothestressprotocol.Discussion:Althougheffectsweresmall,variationinmethylationstatusintheGR1-Cpromoterwas associated with physical and perceived acute stress responses. Interestingly, theseassociationscouldlargelybeexplainedbydifferencesinlifestyleandeducation.

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GR promoter methylation and stress response

6

inTroDucTion

Thebiologicalstressresponseislargelyregulatedbysteroidhormonescalledglucocorticoids,ofwhichcortisolisthemostimportantinhumans.Glucocorticoidsactbybindingtoglucocorticoidreceptors (GR) andmineralocorticoid receptors (MR), which are expressedwidely across thebody. The GR, encoded by the NR3C1 gene located on chromosome 5q31, is expressed inalmosteverycellinthebody.Besidesregulatingthestressresponseitregulatesdevelopmentalprocesses, immune responses and metabolism. Responsiveness to glucocorticoids is to alargeextentdeterminedbytheexpression levelof theGR,which iscontrolledbyavarietyofmechanisms,includingDNAmethylation1.InDNAmethylation,amethylgroupisaddedtothe5’positionofcytosineinCpGdinucleotides.MethylationofCpGrichclusters,termedCpGislands,whichoftenspanthepromoterregionsofgenes,isassociatedwithtranscriptionalrepression,whereashypomethylationofCpGsisassociatedwithtranscriptionalactivity2. Methylation patterns in GR promoters have been shown to be highly variable betweenindividuals3 but little is known about the functional consequences of this variation for thebiological response to psychosocial stress. Oberlander et al. showed that in a group of 82neonates,increasedmethylationofGRpromoterregion1-Fwasassociatedwithincreasedcortisolresponses to a stress protocol, consisting of a visual information processing task, performedat threemonths of age4. These study results suggest that individual variation inmethylationpatternsoftheGRleadstodifferencesinresponsestostressfulsituations.Inthepresentstudy,wehypothesizedthatindividualvariationinmethylationstatusoftheGR1-Cpromoter(1-Cbeingapromoterbroadlyexpressedinmanytissuesincludingthebrain)atanadultageisassociatedwithdifferencesinphysicalandperceivedstressresponsestoapsychosocialstressprotocol5.Totestthishypothesis,weuseddatafromtheDutchFamineBirthCohortStudyinwhichbothstressresponsesaswellasGR1-Cpromotermethylationstatuswereassessed.Resultsfromthisstudyso far showed that prenatal exposure toundernutritionwas associatedwith increasedbloodpressureresponsestostress6,butnoassociationswithcortisolresponses7orGR1-Cpromotermethylationstatuswerefound(unpublisheddata).

METhoDs

Participants and selectionParticipantswereselectedfromtheDutchFamineBirthCohort.Forthiscohort,weincludedallsingletonbabiesbornaliveintheWilhelminaGasthuis(ateachinghospital inAmsterdam,theNetherlands)between1November1943and28February1947.Weexcludedthosewhosebirthrecordswere not available (1%) or thosewhowere born prematurely (8.9%, gestational agebelow259days)8.Inall,2414menandwomenwereincluded,ofwhomthepopulationregistryofAmsterdamtraced2155(89%).Ofthese,160babieshadnotbeenregisteredinAmsterdamat

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birth,328peoplehaddied,213peoplehademigrated,157peoplerefusedpermissiontorecordtheiraddress,125peoplewerenottraceabletoacurrentaddress,andeightpeoplerequestedtheir address to be removed from the study’s database. In 2002,we invited all 1423 eligiblecohortmembers toparticipate in a large studywhich includedapsychosocial stressprotocoland blood withdrawal. The study was approved by the local Medical Ethics Committee andcarriedoutinaccordancewiththeDeclarationofHelsinki.Allprocedureswerecarriedoutwiththe adequate understanding andwritten consent of the subjects. The consent form includedinformation stating that part of the blood sample takenwould be used for genetic analysesassociatedwithchronicdiseasesandthatforthispurposeDNAwouldbeanonymouslystoredinthehospital.

general study parametersAresearchnurseperformedastandardizedinterviewinwhichinformationwasobtainedaboutsocio-economicstatus(SES),educationallevel,medicalhistory,lifestyleanduseofmedication.Weaskedtheparticipantstoratehisorherlevelofphysicalactivity(1=veryactive,2=active,3=littleactive).Educationallevelwasmeasuredona10-pointscale(1=primaryeducationnotcompleted,10=universitycompleted).WedefinedcurrentSESaccordingtoISEI(InternationalSocio-Economic Index)-92, which is based on the participant’s or their partner’s occupation,whicheverstatusishigher9.ValuesintheISEI-92scalerangedfrom16(lowstatus)to87.

MethylationDNAmaterial was extracted from a fasting blood sample. Methylation status of the GR 1-Cpromoterwasassessedusingmethylation-sensitivepolymerasechainreaction(PCR).Foranalysisof GR 1-C promotermethylation, genomic DNA (400ng)was incubatedwith themethylationsensitive restriction endonucleases AciI and HinfI as instructed by the manufacturer (NewEnglandBiolabs,Hitchin,Hertfordshire,UK). The resultingDNAwasamplifiedusing realtimePCR,whichwasperformed in a total volumeof25μlwith SYBR®Green JumpstartReadyMix(Sigma)asdescribedbythemanufacturer.TocontrolfortheamountofDNAineachreaction,primersspecificforthehumanPPARαexon7wereused,thisregiondoesnotcontainanAciIorHinfIcleavagesite,asaninternalcontrolgene(Table1).PrimerswerealsodesignedtoamplifytheCpGislandspanningtheGR1-Cpromoter5,10. Cycleparameterswere94°Cfor2min,then40cyclesof95°Cfor30s,60°Cfor1minand72°Cfor1min.Allcyclethresholdvalueswerenormalisedtothe internalcontrolandeachsampleanalysed in duplicate and values expressed relative to the control gene. Single bands of thecorrectsizewereverifiedbygelelectrophoresis.

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Table 1Primersequencesusedinmethylation-sensitivePCR.

gene Forward primer reverse primer

GR1-Cpromoter ATTTTGCGAGCTCGTGTCTG CGCAGCCGAGATAAACAACT

PPARαexon7 CGGAGTTTATGAGGCCATATTC AGGGAGATATCACTGTCATCCAG

Psychological stress protocol Thestressprotocolwasperformedintheafternoon(between1200hand1400h),aboutanhourafterparticipantshadeatena light lunch.Theprotocolstartedwitha20-minbaselineperiod,followedbythree5-minpsychologicalstresstests(Strooptest,mirror-tracingtestandspeechtest).TheStrooptestandthemirror-tracingtestwereeachfollowedbya6-minrecoveryperiod.Thespeechtestwasfollowedbya30-minrecoveryperiod.TheStrooptestwasacomputerizedcolour-wordconflictchallenge.Afterashortintroduction,participantswereallowedtopractiseuntiltheygraspedthemeaningofthetest.Amistakeorexceedingtheresponsetimelimitof5secwasautomaticallyfollowedbyashortbeep.Inthemirror-tracingtestastarhadtobetracedthatcouldonlybeseeninmirrorimage(LafayetteInstrumentsCorp,Lafayette,IN,USA).Everydivergence from the line of the star induced a short beep. The participantswere allowed topractiseonecircuitoftracing.Participantswereinstructedtogiveprioritytoaccuracyoverspeedandweretoldthatmostpeoplecouldperformfivecircuitsofthestarwithoutdivergencefromtheline.Priortothespeechtest,participantslistenedtoanaudiotapedpre-recordedscenarioinwhichtheyweretoldtoimagineasituationinwhichtheywerefalselyaccusedofpickpocketing.Participantswereinstructedtogivea3-minresponsetotheaccusationsandweregiven2mintopreparetheresponse.Theresponsewasrecordedonvideo.Participantsweretoldthatthenumberofrepetitions,eloquenceandpersuasivenessoftheirperformancewouldbemarkedbyateamofcommunication-expertsandpsychologists. Continuousbloodpressure(BP)andheartrate(HR)recordingsweremadeusingaFinometeroraPortapresModel-2(FinapresMedicalSystems,Amsterdam,theNetherlands).Wedesignatedsixperiodsof5mineachasmeasuringperiods.Theperiodsweredefinedasfollows:baseline(15minintothebaselineperiod),Stroop,mirror-tracing,speechtest(includingpreparationtime),recovery1(5minaftercompletingthespeechtest),andrecovery2(25minaftercompletingthespeechtest).Wecalculatedmeansystolicbloodpressure(SBP),diastolicbloodpressure(DBP)and HR for eachmeasuring period. Saliva samples were collected using Salivettes (Sarstedt,Rommelsdorf, Germany) at seven time points during the protocol: at 5 and 20 min in thebaselineperiod;at6minaftercompletionoftheStrooptest;at6minaftercompletionofthemirror-drawingtest;andat10,20and30minaftercompletionofthespeechtest.SalivawasextractedbycentrifugingtheSalivettesandwasstoredat-80°Cuntilanalysis.Salivarycortisolconcentrationsweremeasuredusingatime-resolvedimmunofluorescentassay(DELFIA)11.This

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assayhadalowerdetectionlimitof0.4nmol/l,aninter-assayvarianceof9-11%andanintra-assayvarianceoflessthan10%. Perceivedstressquestionnaireshadtobefilledoutaftereachofthethreestresstests.Thequestionnairesconsistedofsixquestions(Howrelaxeddidyoufeelduringperformanceofthestresstask?;Howstresseddidyoufeelduringperformanceofthestresstask?;Howdifficultdidyoufindthetask?;Didyoufeelcommittedtothetask?;Howwelldidyouperform?;Howmuchdidyoufeelincontrol).Theanswershadtobegivenona7-pointscalewithscoresrangingfrom1(notatall)to7(verymuch).

Statistical analysesBaselinecortisolwascalculatedasthemeanofthefirstandthesecondcortisolconcentrationmeasuredduringthebaselineperiod.ThehighestaverageSBP,DBP,andHRvalueofthe5minmeasuring periods and the highest of the seven cortisol valueswere designated as the peakresponseduringthestressprotocol.Theincreasefrombaselinetothispeakvaluewasdesignatedas stress reactivity.We calculated a total perceived stress score by adding the scores on thequestionnairesperformedaftereachstresstask. Weappliedlinearregressionanalysistoanalyseassociationsbetweenpotentialconfoundersand methylation status of the GR 1-C promoter as well as to analyse associations betweenmethylationstatusandstressoutcomes.Toinvestigatewhetherpotentialassociationsbetweenmethylation status of the GR 1-C promoter and stress outcomeswere influenced by generaland lifestyle variableswe adjusted the regressionmodels for sex, educational level, smokingbehaviourandphysicalactivitylevelafterlookingattheassociationinaunivariateway.Westartedwithacrudemodelandadditionallyadjustedforpotentialconfoundingvariables.MethylationstatusoftheGR1-Cpromoterwashighlyskewedtotherightwithanumberoflargeoutliers.Because the outliers did seem clinically relevant, we included them in the analyses.We logtransformedmethylationstatustoapproachanormaldistribution.Wedidthesameforcortisolconcentrations,whichwerealsohighlyskewedtotheright.Wereporteffectsizesresultingfromtheseanalysesasunitchangeper1%changeinmethylationstatusincaseofnormallydistributedvariables (SBP,DBP,HRand theperceived stress variables) andaspercentage changeper1%change inmethylation status in case of cortisol. For table 3,we split themethylation valuesintoquartilesandreportstressoutcomesaccordingtothesequartiles.Wealsoreportp-valuesof linear regression analyses using themethylation quartiles as determinant.We considereddifferences tobestatisticallysignificant ifp-valueswere≤0.05.WeusedSPSS16.0 (SPSS Inc,Chicago,IL)toperformthestatisticalanalyses.

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rEsulTs

Characteristics of the study populationAtotalof725ofthe1423invitedcohortmemberscompletedthestressprotocol.WewereabletodeterminethemethylationstatusoftheGR1-Cpromoter in675ofthese725participants.Ofthe675includedindividuals,47%weremen(Table2).Themeanageofthestudypopulationwas58years(SD1year).

Table 2General,lifestyleandmedicationusecharacteristicsinthestudypopulation.

n

GR1-Cmethylation(range) a 0.064(0.003–20.266) 675

general

Sex(%male) 47 675

Age(years) 58(1) 675

Educationb, c 4(2) 649

SES 50(14) 667

lifestyle

Currentsmoking(%) 24 674

Alcoholconsumption(glassesp/week)b 6(14) 664

Activitylevel 2.2(0.6) 630

Medication use

Useofsystemiccorticosteroidtherapy(%) 8 675

Useofantihypertensivemedication(%) 24 675

Useofantidepressantsoranxiolytics(%) 12 675

Useoforalcontraceptives(%) 2 674

UseofHRT(%) 4 675

Data are given asmeans (SD) andpercentages, exceptwhere given as a medianor bmedian (interquartile range);HRT=hormonereplacementtherapy;cEducationallevelmeasuredona10-pointscale(1=primaryeducationnotcompleted,10=universitycompleted).

Methylation of GR 1-C promoterTable 2 shows thatmethylation status of theGR 1-C promoter ranged from 0.003 to 20.266withamedianvalueof0.064(IQR0.087).Menandwomen’smethylationstatusdidnotdiffersignificantly(p =0.39)andagewasnotsignificantlyassociatedwithmethylationstatus(p =0.33).Education was significantly positively associated with GR 1-C methylation status (p = 0.03),whereas SESwas not (p = 0.51). Current smokingwas negatively associated (p < 0.001) and

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physicalactivitylevelwaspositivelyassociated(p<0.001)withmethylationstatus.Consumptionof alcohol was not associated with methylation status (p = 0.98), nor was use of systemiccorticosteroids(p =0.80),antihypertensivemedication(p =0.50),antidepressantoranxiolytics(p=0.58),oralcontraceptives(p =0.89)orhormonereplacementtherapy(p=0.37).

Stress reactivitySBP,DBP,HR and cortisol values increased in response to all threepsychological stress tests.MeanSBP,DBPandHRresponsestothestressprotocolpeakedduringthespeechtask.Stressreactivitywas37%(48mmHg[95%CI:46to49])fortheSBPresponse,32%(21mmHg[21to22])fortheDBPresponseand16%(12bpm[11to12])fortheHRresponse.Themeancortisolresponsepeakedduringthefirstrecoveryperiodafterthespeechtest.Stressreactivitywas42%(geometricmean1.7nmol/l[1.5to1.8]).

Methylation and stress outcomesTable3showsthatmethylationoftheGR1-Cpromoterwassignificantlypositivelyassociatedwithphysicalstressreactivity.With1%decreaseinGR1-Cmethylation,cortisollevelsdecreasedby0.14%(95%CI:0.03to0.25,p=0.02)andHRdecreasedby0.10bpm(0.03to0.16,p =0.003).However,whenadjustingforsex,educationallevel,smokingbehaviourandphysicalactivity,theassociationsdiminishedenormouslyandwerebyfarnolongerstatisticallysignificant.Cortisolchangeafteradjustmentwas-0.08%(-0.19to0.04,p=0.19)andHRchangeafteradjustmentwas-0.04bpm(-0.11to0.02,p =0.21).Thiswasmainlyduetotheadjustmentforsmokingandactivitylevel.SBP(p=0.57)andDBP(p=0.87)reactivitywerenotassociatedwithmethylationstatusoftheGR1-Cpromoter. GR1-Cmethylationwassignificantlyassociatedwith threeof thesix selfperceivedstressvariables. It was negatively associated with perceived stress (0.03 points increase per 1%decrease inmethylation status [95%CI: 0.00 to0.06,p =0.05) andpositivelywithperceivedperformance(-0.03pointsdecrease[-0.05to-0.01],p=0.02)andcontrol(-0.03pointsdecrease[-0.05to0.00],p=0.04).Adjustingforsexandeducationalleveldiminishedthestrengthoftheassociationswithallperceivedvariables,whichwerenolongerstatisticallysignificant:perceivedstress(0.02points[0.00to0.05,p =0.10),perceivedperformance(-0.02points[-0.04to0.00],p=0.09),andcontrol(-0.02points[-0.04to0.01],p=0.16).Adjustmentforeducationallevelcontributedmoretotheabolishmentoftheassociationsthanadjustmentforsex.

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Table 3 StresstestoutcomesaccordingtoquartilesofGR1-Cmethylationstatus.

n all Q1 Q2 Q3 Q4 p-value

baseline

Cortisol(nmol/l)a 609 3.8(1.8) 3.7 3.6 3.7 4.0 .14

SBP(mmHg) 665 128.1(21.1) 128.3 127.9 127.3 129.1 .80

DBP(mmHg) 665 66.2(11.8) 66.6 66.5 65.9 65.6 .38

HR(bpm) 664 74.1(10.5) 73.3 73.9 74.4 74.9 .14

Stress reactivity

Cortisol(nmol/l)a 433 1.7(3.7) 1.5 1.6 1.6 2.2 .04

SBP(mmHg) 665 47.8(20.8) 48.5 46.8 46.6 49.3 .76

DBP(mmHg) 665 21.4(9.1) 21.6 20.9 21.4 21.6 .89

HR(bpm) 664 11.6(9.3) 10.7 10.4 11.5 13.9 .001

Perceived stress

Relaxedness 625 11.5(3.8) 11.4 11.3 11.4 11.8 .40

Stressfulness 626 11.1(4.0) 11.5 11.1 10.9 10.7 .08

Difficulty 625 14.4(3.5) 14.8 14.3 14.7 14.0 .09

Commitment 625 14.7(4.1) 14.8 14.7 14.9 14.5 .60

Performance 623 9.2(3.4) 8.7 9.4 9.1 9.6 .04

Control 625 10.1(3.6) 9.5 10.3 10.0 10.4 .06

Dataaregivenasmeans(SD),exceptwheregivenasa geometricmean(geometricSD);p-valuesfordifferencebetweenthequartiles.

Discussion We found that lower level of methylation of the GR 1-C promoter in peripheral blood wasassociatedwith lowerphysicalstressreactivity intermsof lowercortisolandHRresponsestoapsychosocialstressprotocolandwithhigherlevelsofperceivedstressvariables.Interestingly,these associations largely disappeared after adjusting for sex, educational level, smokingbehaviourandphysicalactivity,wherethelattertwohadthelargesteffect.ThissuggeststhatvariationinGR1-Cmethylationonlyhasasmallfunctionaleffectonthestressresponseandthattheremainderoftheeffectcanbeexplainedbydifferencesinlifestyleandeducationallevel. LittleisknownaboutthefunctionaleffectsofvariationinmethylationstatusinGR-promoterson the biological stress response. However, existing data does seem to support the presentfindingsofapositiveassociationbetweenmethylationstatusandstressreactivity.Thepreviouslymentioned study byOberlander et al. showed in neonates that increasedmethylation of GRpromoter1-Fwasassociatedwithincreasedcortisolresponsestoastressprotocol4.Inaddition,Weaver et al. showed in ratswho received little care of theirmothers early in life that high

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methylationstatusoftheexon17GRpromoter(thehomologofthe1-Fregioninhumans)wasassociatedwith lower GR expression and higher corticosterone stress reactivity12, whichwassuggestedtobeaconsequenceofreducedglucocorticoidnegativefeedbacksensitivity13. The observation of an association between educational level and an adverse lifestyle(smokingandlowphysicalactivity)andmethylationpatternoftheGR-1Cpromoterisinitselfaninterestingone.Epigeneticpatternshavebeenshowntoberatherstable,butthereisincreasingevidence that changes over time do take place14. Several environmental factors have beensuggestedtoaffectthisprocess,includingcircumstancesduringembryonicdevelopmentaswellasfactors inadult lifesuchasdiet,smokingandexposuretoenvironmentalpollution15.Otherfactors than thoseobserved in thepresent studymayplaya role in theobservedassociationbetween environmental factors and GR-1C methylation. For example, educational level andphysicalactivitymaybothbestronglyassociatedwithdiet,ofwhichseveralcomponentssuchasfolateandmethioninehavebeenshowntobeassociatedwithDNAmethylation15. While we found that an adverse lifestyle was associated with lower methylation of theGR 1-C promoter, at the same time lower physical stress responses were observed in thosewith lowGR-1Cmethylation.Theseassociationsmay seemcontradictory,butfit inagrowingbody of evidence that blunted stress reactivitymay be associated with adverse health. In aprevious reporton thepresentcohort,wehaveshownthat lowercortisolandcardiovascularstressresponsesareassociatedwith increasedsymptomsofdepressionandanxietyandpoorsubjective health perception16,17. Others have shown similar results18-20. Several biologicalexplanationscanbeputforward.Bluntedreactivitycouldbeduetoafailingstressresponseasaconsequenceofprolongedstress.Anotherexplanationmaybethatadiminishedstressresponsecouldhaveadverse immunological consequences.Undercertaincircumstances, thebiologicalstress responseupregulates the immunesystem,which isbeneficial forhealth21.Adecreasedstressresponsemaybelessabletodoso.Besidesthis,cortisolisneededtoeventuallysuppresstheinflammatoryresponse.Alackofcortisolmaythusleadtoaprolongedinflammatorystatewithnegativeeffectsonhealth. Lower methylation status of the GR 1-C promoter was also associated with increasedperceptionofstress:thosewithlowerlevelsofmethylationfeltmorestressedduringthestressprotocol,felttheyhadperformedlesswellonthestresstasksandfeltlessincontrol.Again,thisseemsatoddswiththedecreasedlevelsofcortisolandHRreactionstotheprotocol.However,weshowedthesameintheabovereferencedstudieswheresymptomsofdepressionandanxietyandpoorself-perceivedhealthwerealsoassociatedwithincreasedstressperceptionanddecreasedphysicalresponses16-17.Thisdiscrepancybetweenexperiencedstressandphysiologicalstressisnot unique. Several studies have found only small or no associations between self-perceivedstress/emotionsandbiologicalstress intheformofcortisolandcardiovascularresponses22-24.Differentexplanationscanbeputforward.Thephysicalstressresponsemaybedysfunctional.Individualsmaynotbeawareoftheirownemotionsorindividualsmaynotbesincereinreportingtheirthoughtsandemotions.

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Anumberoflimitationstothepresentstudyhavetobepointedout.WedidnotassessGR1-Cexpressionlevels,whichclearlywouldhaveaddedrelevantinformationtothestudy.WeassessedmethylationstatusoftheGR1-Cpromoter inperipheralbloodanddonotknowwhetherthisiscomparabletomethylationstatusindifferentpartsofthebodyimplicatedinthefunctionalregulation of the stress response.Wehave applied amethylation-sensitive PCR technique toassessDNAmethylationoftheGR1-Cpromoter,thismeasurestheaveragemethylationrangeacrossthepromoterregionanalysed.Itisunknownwhetherthesameresultswouldhavebeenachievedwhenwewouldhaveappliedthenowadayscommonlyusedbisulfitepyrosequencingtechnique,whichcanmeasurethemethylationstatusofindividualCpGs.Wethereforesuggestthat thepresentresultsshouldbereplicated inastudy inwhichGR1-Cmethylationstatus isassessedbythemoresensitivepyrosequencingmethod.Finally,effectsofGR1-Cmethylationstatusonstressresponseswerestatisticallysignificant,butsmall.Onlyabout1%ofthevariationinthestressreactivitydatawasexplainedbymethylationstatus.However,givenallthelimitationsreferredtoaboveandthefactthatweonlymeasuredmethylationstatusinoneexonofeighttranslatedexonsknown,itmaybeseenasremarkablethatwefoundsuchassociationsatall. Major assets of our study include a population based design and a large, well-describedstudy population, enabling us to investigate the potential confounding/mediation by a set ofdifferentvariables includingbasalcharacteristics, lifestylevariablesanduseofmedication.Anadditionalstrengthistheuseofapsychologicalstressprotocolwithdifferenttypesofstressors:cognitiveandsocialstressors.Unlikeaphysiologicalstresstest,apsychologicalstressprotocolis able toactivate stress responsesabove thehypothalamic levelwhere the limbic structuresprocesscognitiveandaffectiveinformation25. Inconclusion,wefoundevidenceforassociationsbetweenmethylationstatusoftheGR1-Cpromoterandbiologicalandperceivedstressresponses.Interestingly,theseassociationscouldlargelybeexplainedbylifestyleandeducation.

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rEfErEncE lisT

1. Turner JD, Alt SR, Cao L, Vernocchi S, Trifonova S, Battello N, et al. Transcriptional control of theglucocorticoidreceptor:CpGislands,epigeneticsandmore.Biochem Pharmacol.2010;80:1860-1868.

2. RazinA.CpGmethylation,chromatinstructureandgenesilencing-athree-wayconnection.EMBO J.1998;17:4905-4908.

3. TurnerJD,PelasciniLP,MacedoJA,Muller,C.P.Highly individualmethylationpatternsofalternativeglucocorticoidreceptorpromoterssuggestindividualizedepigeneticregulatorymechanisms.Nucleic Acids Res.2008;36:7207-7218.

4. OberlanderTF,WeinbergJ,PapsdorfM,GrunauR,MisriS,DevlinAM.Prenatalexposuretomaternaldepression,neonatalmethylationofhumanglucocorticoidreceptorgene(NR3C1)andinfantcortisolstressresponses.Epigenetics.2008;3:97-106.

5. Turner JD,MullerCP.Structureof theglucocorticoidreceptor (NR3C1)gene5’untranslatedregion:identification,andtissuedistributionofmultiplenewhumanexon1.J Mol Endocrinol.2005;35:283-292.

6. PainterRC,deRooijSR,BossuytPM,PhillipsDI,OsmondC,BarkerDJ,etal.BloodpressureresponsetopsychologicalstressorsinadultsafterprenatalexposuretotheDutchfamine.Journal of Hypertension.2006;24:1771-1778.

7. deRooijSR,PainterRC,PhillipsDIW,OsmondC,TanckMWT,BossuytPMMetal.CortisolresponsestopsychologicalstressinadultsafterprenatalexposuretotheDutchfamine.Psychoneuroendocrinology.2006;31:1257-1265.


9. Bakker B, Sieben I. Maten voor prestige, sociaal-economische status en sociale klasse voor destandaardberoepenclassificatie1992.Sociale Wetenschappen.1997;40:1-22.

10. LillycropKA,Slater-JefferiesJL,HansonMA,GodfreyKM,JacksonAA,BurdgeGC.Inductionofalteredepigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricteddietduringpregnancysuggeststhatreducedDNAmethyltransferase-1expressionisinvolvedinimpairedDNAmethylationandchangesinhistonemodifications.Br J Nutr.2007;97:1064-1073.

11. WoodPJ,KilpatrickK,BarnardG.Newdirectsalivarycortisolandcortisoneassaysusingthe‘’DELFIA”system.J Endocrinology.1997;155:Supp:P71.

12. WeaverIC,CervoniN,ChampagneFA,D’AlessioAC,SharmaS,SecklJR,etal.Epigeneticprogrammingbymaternalbehavior.Nat Neurosci.2004;7:847-854.

13. LiuD,Diorio J, TannenbaumB, Caldji C, FrancisD, FreedmanA, Sharma S, PearsonD, Plotsky PM,MeaneyMJ.Maternalcare,hippocampalglucocorticoidreceptors,andhypothalamic-pituitary-adrenalresponsestostress.Science.1997;277:1659-1662.

14. WhitelawNC,WhitelawE.2006.Howlifetimesshapeepigenotypewithinandacrossgenerations.Hum Mol Genet.2006;15SpecNo2:R131-R137.

15. AguileraO,FernandezAF,MunozA,FragaMF.Epigeneticsandenvironment:acomplexrelationship.J Appl Physiol.2010;109:243-251.

16. deRooijSR,ScheneAH,PhillipsDI,RoseboomTJ.Depressionandanxiety:Associationswithbiologicaland perceived stress reactivity to a psychological stress protocol in a middle-aged population.Psychoneuroendocrinology.2010;35:866-877.

17. de Rooij SR, Roseboom TJ. Further evidence for an association between self-reported healthand cardiovascular as well as cortisol reactions to acute psychological stress. Psychophysiology. 2010;47:1172-1175.

18. Fries E, Hesse J, Hellhammer J, Hellhammer DH. A new view on hypocortisolism.Psychoneuroendocrinology.2005;30:1010-1016.

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19. Phillips AC, Der G, Carroll D. Self-reported health and cardiovascular reactions to psychologicalstressinalargecommunitysample:Cross-sectionalandprospectiveassociations.Psychophysiology.2009;46:1020-1027.

20. PhillipsAC.Bluntedcardiovascularreactivityrelatestodepression,obesity,andself-reportedhealth.Biol Psychol.2011;86:95-97.

21. Calcagni E, Elenkov I. Stress system activity, innate and T helper cytokines, and susceptibility toimmune-relateddiseases.Ann NY Acad Sci.2006;1069:62-76.

22. Allen PI, Batty KA,Dodd CA,Herbert J, Hugh CJ,MooreGF, et al. Dissociation between emotionalandendocrineresponsesprecedinganacademicexaminationinmalemedicalstudents.J Endocrinol.1985;107:163-170.

23. FeldmanPJ,CohenS,LeporeSJ,MatthewsKA,KamarckTW,MarslandAL.Negativeemotionsandacutephysiologicalresponsestostress.Ann Behav Med.1999;21:216-222.

24. Schwerdtfeger A. Predicting autonomic reactivity to public speaking: don’t get fixed on self-reportdata!Int. J. Psychophysiol.2004;52:217-224.

25. Dickerson SS, Kemeny ME. Acute stressors and cortisol responses: a theoretical integration andsynthesisoflaboratoryresearch.Psychol Bull.2004;130:355-391.

7Transgenerational effects of

prenatal exposure to the 1944-45 Dutch famine

MarjoleinVEVeenendaalRebeccaCPainterSusanneRdeRooijPatrickMMBossuytJorisAMvanderPostPeterDGluckmanMarkAHanson

TessaJRoseboom

Submitted

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absTracT

Introduction:Wepreviouslyshowedthatmaternalundernutritionduringgestationisassociatedwithincreasedmetabolicandcardiovasculardiseaseintheoffspring.Also,wefoundincreasedneonatal adiposity among offspring of women who themselves had been undernourishedprenatally. In thepresentstudywe investigatedwhether these transgenerationaleffectshaveledtoalteredbodycompositionandpoorerhealthinadulthoodinthegrandchildren.subjects: Theadult offspring (F2) of a cohort ofmenandwomen (F1) born around thetimeof the1944-45Dutch famine.Weapproached theF2 through theirparents.ParticipatingF2s(n=360,meanage37yrs)completedanonlinequestionnaire.results: AdultoffspringofprenatallyexposedfathershadhigherweightandBMIthanoffspringofprenatallyunexposedfathers(+4.9kg,p=0.03;+1.6kg/m²,p=0.006).Nosucheffectwasfoundforoffspringofprenatallyexposedmothers.WeobservednodifferencesinadulthealthbetweentheF2generationgroups.conclusions: Offspringofprenatallyundernourishedfathers,butnotmothers,wereheavierandmoreobesethanoffspringoffathersandmotherswhohadnotbeenundernourishedprenatally.We found no evidence of transgenerational effects of grandmaternal undernutrition duringgestationonhealthofthisrelativelyyounggroup,buttheincreasedadiposity intheoffspringofprenatallyundernourishedfathersmayleadtoincreasedchronicdiseaseratesinthefuture.

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inTroDucTion

Humanandanimalstudieshaveshownthatthefetalenvironmentcanaffectthehealthofanindividualthroughoutthelife-course.Thisisthoughttoreflectprogramming,theprocessbywhichthesamegenotypecangive rise to severalphenotypesdependingon theearlyenvironment.Theseprogrammingeffectsmayevenbetransmittedacrossgenerations.Feedingratsaproteinrestricted diet during gestation not only resulted in higher blood pressure and endothelialdysfunctionintheoffspring,butalsointhegrand-offspring1.LowproteindietduringpregnancyledtoinsulinresistanceintheadultmaleandfemaleF2offspring2-4.ThereisevidencesuggestingthattheglucosemetabolismoftheF3generationisalsoaffectedbyF0undernutrition5.Inmice,maternalgeneralundernutritionduringgestationledtoreducedbirthweight,impairedglucosetoleranceandobesityintheF1andF2generationinagenderspecificmanner6. Studies reporting on transgenerational effects of prenatal undernutrition in humans arescarce.AhistoricalstudyofthreegenerationsinOverkalix,Sweden,reportedthatlimitedfoodsupplyofthegrandparentsinfluencedgrandchildren’slatermortalityanddiseaseriskinasex-specific manner, partly operating exclusively through the paternal line7. We have previouslyreportedthatindividualsexposedtotheDutchfamineof1944-45inuterohaveincreasedratesofcardiovasculardisease,type2diabetesandbreastcancer.ThefirstindicationsofapotentialtransgenerationaleffectofprenatalfamineexposuredescribedthatwomenprenatallyexposedtotheDutchfaminehadslightlysmallerbabiesthanunexposedwomen8,butlaterreportsfromthesamegroupdescribedthatfirstbornbabiesofwomenexposedtofamineinearlygestationwereheavieratbirth9. Wehavepreviouslyreportedthatprenatalfamineexposureaffectsadulthealthlaterinlifeandthattheeffectsofprenatalfamineexposuremaynotbelimitedtotheimmediatelysucceedinggeneration.Wefoundincreasedneonataladiposityandpoorerhealthamongoffspringofwomenwhothemselveshadbeenexposedto famineprenatally10.This study,however,wasbasedonparents’recalloftheiroffspring’ssizeatbirthandlaterhealth,whichmayhaveledtoalevelofinaccuracy.Inthestudyreportedherewecontactedtheoffspringdirectlytomeasuretheirbodycompositionandinvestigatetheirhealth.

METhoDs

Participants and selectionTheDutch FamineBirthCohort consistsof 2414menandwomenbornas term singletons intheWilhelmina Gasthuis, a local hospital in Amsterdam, between 1 November 1943 and 28February1947.Theselectionproceduresandlosstofollowupuntil2002havebeendescribedindetailelsewhere11;12.CohortmemberswereeligibleforparticipationinthisstudyiftheylivedintheNetherlandsonSeptember1st,2008andiftheiraddresswasknowntous.From2002on,

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31personshaddied,6hademigrated,11hadanunknownaddressand8hadrequestedtheiraddresstoberemovedfromourdatabase.Atotalof1371eligible individualswere invitedtoparticipate.ThestudywasapprovedbythelocalMedicalEthicsCommitteeandcarriedout inaccordancewiththeDeclarationofHelsinki.Allparticipantsgavewritteninformedconsent.

Exposure to famineTheofficialdailyfoodrationsforthegeneralpopulationof21yearsandolderwereusedtodefineexposuretofamine13.Apersonwasconsideredtobeprenatallyexposedtofamineiftheaveragedailyfood-rationofthemotherduringany13-weekperiodofgestationcontainedlessthan1000calories.Basedonthisdefinition,babiesbornbetween7January1945and8December1945hadbeenexposedinutero.Peoplebornbefore7January1945andconceivedandbornafter8December1945wereconsideredasunexposedtofamineinuteroandactedascontrolgroups.

GenerationsWestudiedtwogenerations.F1werethemenandwomenfromtheDutchfaminebirthcohort,bornbetweenNovember1943andFebruary1947.F2weretheoffspringofF1menorwomen.

Data collectionInformationaboutthemother,thecourseofthepregnancyandthesizeofthebabyatbirth(F1)wasextractedfrommedicalbirthrecords12.Previously,atage58,F1participantsvisitedtheclinicorwereseenathomewhereF1weightwasmeasuredwithSecascalesorTefalportablescalesandheightusingafixedorportablestadiometer.Weaskedthemaboutthebirthweight,birthlengthandgestationalageatdeliveryoftheirchildren(F2)10. In the current study, all F2 participants were asked to give information concerningtheirmedical history, lifestyle and children. Datawere collected bymeans of a standardizedquestionnairewhichwas filled out at home by the participants either on a paper or using aweb-based form. The questionnaire included questions on height, weight, smoking, alcoholconsumptionandexercisebehavior.Weobtained informationaboutsymptomsorahistoryofcardiovascular,pulmonary,psychiatricandmetabolicdiseaseandmedicationuse.ThequestionsconcerningcardiovasculardiseaseincludedtheRosequestionnaire14.Questionswerecombinedtoachievecategoriesrelatingtocardiovasculardisease,pulmonarydisease,hayfever,eczema,cholesterol,diabetesandhypertension.Foreachcondition,questionswerephrasedas“hasadoctoreverdiagnosed(condition)”or“hasadoctoreverprescribedmedicationfor(condition)”.

Statistical methodsWe used linear regression for continuous variables and logistic regression for dichotomousvariablestocompare(grand)maternal,birthandadultoutcomesofthoseexposedandthoseunexposedtofamineduringgestationandalsotocompareoffspringofthesegroups.Totakeintoaccountthecorrelationofcharacteristicsbetweensiblings,weusedmixedmodelstoanalyzethe

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associationbetweenF1famineexposureduringdifferentstagesofgestationandF2birthandhealthcharacteristics.Todealwithmissingvalues,weusedmultiple imputation.Weadjustedforpossibleconfoundingfactors,suchasF2age,sex,birthweightandF1BMIandweight.WereporttheF2characteristicsstratifiedaccordingtoF1sex.WeusedSPSS19.0(Chicago,IL,USA)forallanalyses.

rEsulTs

Intheeligiblepopulationof1371cohortmembers(F1),483F2swerewillingtoparticipateinthecurrentstudy.Ofthese,360(74.5%)completedthequestionnaire.Themeanageatparticipationwas 37 years (range 18 to 47 years). In total, 135males and 225 females participated. BirthweightorgestationalagedidnotdifferbetweenF1participantsandnon-participants(p>0.8). F2ofF1exposedmenwere2.1yearsyoungerthanoffspringfromunexposedF1men(Table1).BirthcharacteristicswerenotdifferentbetweenF2offspringoftheexposedandunexposedF1men.F2ofF1exposedwomenhadahigherponderalindexatbirthcomparedtoF2ofunexposedF1women(Table1).ExposedF1menandwomenwerecomparabletounexposedF1menandwomenwithregardstoanthropometricmeasuresatage58.

Table 1CharacteristicsofF2participantsaccordingtoF1gender.

f1 male f1 female

Exposed unexposed All +/- SD Exposed unexposed All +/- SD

N 52 99 151 106 103 209

Sex(%male) 42 35* 38 46 28* 37

Age(years) 33.9 36.0* 35.3+/-4.7 36.7 38.3* 37.5±4.6

Birthweight(grams) 3351 3342 3345+/-826 3374 3245 3313±707

Birthlength(cm) 50.5 49.4 49.8+/-6.4 50.2 50.5 50.3±3.4

Ponderalindex(kg/m³) 27.6 26.3 26.7+/-6.2 26.7* 24.7 25.8±4.8

Adultlength(m) 1.76 1.74 1.75+/-0.1 1.76 1.73 1.75+/-0.1

Adultweight(kg) 78.8‡ 73.5 75.3+/-14.4 79.1 78.9 79.0+/-18.0

BMI(kg/m²)® 25.2‡ 23.8 24.3+/-3.8 25.0 25.7 25.3+/-5.3

*p<0.05‡p<0.05aftercorrectionforageandsexofF2®geometricmeanandSD

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F2 adult body composition BMIOffspring of exposed F1 fathers had a higher BMI than offspring of unexposed F1 fathers(+1.6kg/m²,95%CI0.5to2.6)afteradjustingforF2sexandage(p0.006).AddingF2birthweightandF1BMItothemodeldidnotchangetheassociation(+1.5kg/m²,95%CI0.4to2.5,p0.005).TherewasnoeffectofmaternalF1exposuretofamineontheBMIoftheoffspring(unadjusted-0.69kg/m²,95%CI-3.5to2.1,p0.36;adjusted-0.60kg/m²,95%CI-1.7to0.5,p0.30).

Weight OffspringofexposedF1fatherswereheavierthanoffspringofunexposedF1fathers(+4.9kg95%CI0.8to9.1,p0.03)afteradjustmentforF2sexandage.ThiseffectremainedwhenadjustmentsweremadeforF2birthweightandF1weight(+4.5kg,95%CI0.9to8.1,p0.01).OffspringofF1famineexposedmotherswerenotheavierthanF2ofunexposedmothers(unadjusted-1.7kg,95%CI-5.5to2.0,p0.36;adjusted-0.7kg,95%CI-4.5to3.1,p0.72).

F2 self-reported healthWedidnotfinddifferencesintheprevalenceofcardiovascularandpulmonarydisease,hayfever,eczema,elevatedcholesterol,diabetesorhypertensionbetweenoffspringofmenandwomenexposed to famine during gestation and offspring of unexposedmen andwomen (all p>0.1)(Table2).

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Table 2PrevalenceofF2self-reporteddisease*accordingtoF1gender

f2 of f1 men f1 exposed f1 unexposed all

N 52 99 151

Cardiovascular% 5.9 2.0 3.4

Pulmonary% 9.8 8.2 8.7

Hayfever% 19.6 30.6 26.8

Eczema% 25.5 24.5 24.8

Cholesterol% 0.0 2.0 1.3

Diabetes% 0.0 2.0 1.3

Hypertension% 3.9 2.0 2.7

F2 of F1 women exposed unexposed all

N 106 103 209

Cardiovascular% 1.9 4.0 3.0

Pulmonary% 4.9 5.0 4.9

Hayfever% 20.6 18.0 19.3

Eczema% 19.4 24.0 21.7

Cholesterol% 0.0 0.0 0.0

Diabetes% 1.0 0.0 0.5

Hypertension% 3.9 5.0 4.4

*definedasansweringconfirmativetoquestionsphrasedas“hasadoctoreverdiagnosed”or“hasadoctoreverprescribedmedicationfor”thedifferentconditions.

Discussion

In this studywe found that offspring of fathers that had been exposed to famine prenatallywereheavierandhadahigherBMIthanoffspringofunexposedfathers.Thiseffectremainedafteradjustment forbirthweightandpaternalweightandBMI.Wecouldnotdemonstrateatransgenerationaleffectonhealthamongoffspringofprenatallyexposedmenorwomen. Thesefindingsfitwithinthegrowingbodyofevidencethattransgenerationalnon-genomicalinheritancespecificallytakesplaceinthepaternalline.Forinstance,embryonicexposuretotheendocrinedisruptor vinclozolin increaseda varietyof adult onsetdiseases in the subsequentgenerationsspecificallythroughthepaternalline15;16.Dietaryexposureshavealsobeenshowntoproducetransgenerationaleffectsthroughthemaleline;inSprague-Dawleyrats,highfatdietconsumptionoffathersinducedimpairedglucosetoleranceandinsulinsecretionintheirfemaleoffspring17. Inmice,paternal lowproteindiet inducedalteredexpressionofgenes involved inlipidandcholesterolmetabolisminliveroftheoffspring,comparedtooffspringofcontrolfed

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malemice. These expression differenceswere thought to be the result of alterations of theepigenome18. ThereisevidencethatprenatalexposuretotheDutchfamineinducedepigeneticalterationsin the F1 generation that persist throughout life19;20. It is unknownwhether these epigeneticalterationsaretransmittedtothenextgeneration,andwhethertheyaresex-specificsothattheycould explain the transgenerational effects thatwe foundonly in thepaternal line. Althoughepigeneticsisaverylikelymechanismexplainingtransgenerationaleffectsinmodelorganisms,inhumansothermechanismsmayalsoplayarole. Thetransgenerationaleffectswefoundmayalsohavebeentransmittedthroughenvironmentalfactors such as food preferences and physical activity. Although the transgenerational effectsreportedherewerealsoseenafteradjustmentforpaternalBMIandweight,parentalobesityisknowntobeassociatedwiththelevelofoverweightandobesityinchildren21anditsignificantlyalterstheriskofobesityinadulthood22.Parentsareresponsibleforthequalityandavailabilityofthefoodinthehome,andtheirfoodhabitswillbeadoptedbytheirchildren23.Childrenfromobeseoroverweight familieshavebeenshown tohaveahigherpreference for fatty foods,alowerlikingofvegetablesandlowerphysicalactivitythanchildrenfromleanfamilies24.WehavereportedpreviouslythatpeoplewhowereconceivedduringtheDutchfamineweretwiceaslikelytoconsumeahigh-fatdietandtohaveatendencytobelessphysicallyactive25.Transgenerationalpropagationofunhealthy lifestylepatternsmayhavecontributedtothe increasedweightandBMIofF2ofexposedF1men. Previously,wefoundthatparticipantswhowerethemselvesexposedtofamineprenatally,rated their offspring’s health more often as poor than did unexposed participants10.We setout this study to investigate the self-reported health of the F2 offspring, but we could notdemonstrateanyeffectsofF1famineexposureinuteroonthehealthoftheF2generation.AnumberofmethodologicalissuesmayexplainthefactthatwedidnotfindaneffectonF2health.Thestudiedgroupconsistedofonly360people,whichlimitsthepowertodetectaneffect.ThestatisticalpowertodetecteffectswasfurtherreducedsincethemeanageoftheF2participantswas37years,whichisrelativelyyoungwhenstudyingtheprevalenceofchronicdisease.Also,ouranalysesarebasedonself-reportedhealthquestionnaireswhichareafairlycrudemeasureofhealth. In conclusion,we did not find a transgenerational effect of prenatal famine exposure ongrand-offspring’shealth inthisstudy,butwefoundincreasedweightandBMIamongF2of inuterofamineexposedmen.TheseresultswarrantfurtherfollowupofthehealthoftheF2astheyage,sincetheirincreasedadipositymaypredisposethemtoincreasediseaserisklateron.Also,theysuggestthattransmissionthroughthepaternallinemayoccur,futurestudiesinthiscohortwill investigatemechanisms thatmay elucidate the biological processes that underliethesefindings.

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rEfErEncE lisT

1. TorrensC,PostonL,HansonMA.TransmissionofraisedbloodpressureandendothelialdysfunctiontotheF2generationinducedbymaternalproteinrestrictionintheF0,intheabsenceofdietarychallengeintheF1generation.Br J Nutr.2008;100:760-766.

2. Benyshek DC, Johnston CS, Martin JF. Post-natal diet determines insulin resistance in fetallymalnourished, low birthweight rats (F1) but diet does not modify the insulin resistance of theiroffspring(F2).Life Sci. 2004;74:3033-3041.

3. Pinheiro AR, Salvucci ID, AguilaMB,Mandarim-de-Lacerda CA. Protein restriction during gestationand/orlactationcausesadversetransgenerationaleffectsonbiometryandglucosemetabolisminF1andF2progeniesofrats.Clin Sci (Lond).2008;114:381-392.

4. Zambrano E, Martinez-Samayoa PM, Bautista CJ, Deas M, Guillen L, Rodriguez-Gonzalez GL et al.Sexdifferencesintransgenerationalalterationsofgrowthandmetabolisminprogeny(F2)offemaleoffspring(F1)ofratsfedalowproteindietduringpregnancyandlactation.J Physiol.2005;566:225-236.

5. Benyshek DC, Johnston CS, Martin JF. Glucose metabolism is altered in the adequately-nourishedgrand-offspring(F3generation)ofratsmalnourishedduringgestationandperinatallife.Diabetologia. 2006;49:1117-1119.

6. Jimenez-Chillaron JC, IsganaitisE,CharalambousM,Gesta S, Pentinat-Pelegrin T, FaucetteRRet al.Intergenerationaltransmissionofglucoseintoleranceandobesitybyinuteroundernutritioninmice.Diabetes.2009;58:460-468.

7. PembreyME,BygrenLO,KaatiG,EdvinssonS,NorthstoneK,SjostromMetal.Sex-specific,male-linetransgenerationalresponsesinhumans.Eur J Hum Genet.2006;14:159-166.

8. LumeyLH.DecreasedbirthweightsininfantsaftermaternalinuteroexposuretotheDutchfamineof1944-1945.Paediatr Perinat Epidemiol.1992;6:240-253.

9. LumeyLH,SteinAD,RavelliAC.Timingofprenatalstarvationinwomenandbirthweightintheirfirstandsecondbornoffspring: theDutchFamineBirthCohortstudy.Eur J Obstet Gynecol Reprod Biol. 1995;61:23-30.

10. Painter RC,OsmondC,GluckmanP,HansonM, PhillipsDI, RoseboomTJ. Transgenerational effectsof prenatal exposure to the Dutch famine on neonatal adiposity and health in later life. BJOG.2008;115:1243-1249.

11. PainterRC,RoseboomTJ,BossuytPM,OsmondC,BarkerDJ,BlekerOP.Adultmortalityatage57afterprenatalexposuretotheDutchfamine.Eur J Epidemiol. 2005;20:673-676.

12. RavelliACJ,vanderMeulenJHP,MichelsRPJ,OsmondC,BarkerDJP,HalesCNetal.Glucosetoleranceinadultsafterprenatalexposuretofamine.Lancet. 1998;351:173-177.

13. BurgerGCE,SansteadHR,DrummondJ.StarvationinwesternHolland:1945.Lancet.1945;282-283.14. RoseGA.Thediagnosisofischaemicheartpainandintermittentclaudicationinfieldsurveys.Bulletin

of the World Health Organisation. 1962;27:645-658.15. Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine

disruptorsandmalefertility.Science.2005;308:1466-1469.16. Anway MD, Leathers C, Skinner MK. Endocrine disruptor vinclozolin induced epigenetic

transgenerationaladult-onsetdisease.Endocrinology.2006;147:5515-5523.17. NgSF,LinRC,LaybuttDR,BarresR,OwensJA,MorrisMJ.Chronichigh-fatdiet in fathersprograms

beta-celldysfunctioninfemaleratoffspring.Nature.2010;467:963-966.18. CaroneBR, Fauquier L,HabibN, Shea JM,HartCE, Li Ret al. Paternally induced transgenerational

environmentalreprogrammingofmetabolicgeneexpressioninmammals.Cell.2010;143:1084-1096.19. HeijmansBT,TobiEW,SteinAD,PutterH,BlauwGJ,SusserESetal.Persistentepigeneticdifferences

associatedwithprenatalexposuretofamineinhumans.Proc Natl Acad Sci U S A.2008;105:17046-17049.

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20. TobiEW,LumeyLH,TalensRP,KremerD,PutterH,SteinADetal.DNAmethylationdifferencesafterexposuretoprenatalfaminearecommonandtiming-andsex-specific.Hum Mol Genet.2009;18:4046-4053.

21. Lazzeri G, Pammolli A, Pilato V, GiacchiMV. Relationship between 8/9-yr-old school children BMI,parents’BMIandeducationallevel:acrosssectionalsurvey.NutrJ. 2011;10:76.

22. WhitakerRC,WrightJA,PepeMS,SeidelKD,DietzWH.Predictingobesity inyoungadulthoodfromchildhoodandparentalobesity.N Engl J Med.1997;337:869-873.

23. Klesges RC, Stein RJ, Eck LH, Isbell TR, Klesges LM. Parental influence on food selection in youngchildrenanditsrelationshipstochildhoodobesity.Am J Clin Nutr. 1991;5:859-864.

24. WardleJ,GuthrieC,SandersonS,BirchL,PlominR.Foodandactivitypreferencesinchildrenofleanandobeseparents.Int J Obes Relat Metab Disord. 2001;25:971-977.

25. LussanaF,PainterRC,OckeMC,BullerHR,BossuytPM,RoseboomTJ.PrenatalexposuretotheDutchfamineisassociatedwithapreferenceforfattyfoodsandamoreatherogeniclipidprofile.Am J Clin Nutr.2008;88:1648-1652.

8consequences of hyperemesis

gravidarum for offspring: a systematic review and meta-analysis

MarjoleinVEVeenendaalAnnetFMvanAbeelen

RebeccaCPainterJorisAMvanderPostTessaJRoseboom

BJOG 2011;118(11):1302-1313

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absTracT

background: There is evidence that hyperemesis gravidarum (HG) is associated with apredominanceoffemalefetuses,lowerbirthweightandshortergestationalageatbirth.Astheadverseeffectsofprematurityand lowbirthweightondisease risk in later lifehavebecomeincreasinglyclear,therepercussionsofHGmightnotbelimitedtoadverseperinataloutcomes.Objectives: TosummarisetheevidenceonshortandlongtermoutcomesofpregnancieswithHG.search strategy: A literature searchwas conducted in the electronic databasesMedline andEmbase.Selection criteria: Studieswere included that reported on the fetal, neonatal and long termoutcomeofpregnanciescomplicatedbyHG.Data collection and analysis: Twoauthors independentlyselectedstudiesandextracteddata.Meta-analysiswasperformedusingReviewManagersoftware.Main results: Women with HG during pregnancy were more likely to have a female child(OR1.27,95%CI1.21to1.34).Theywerealsomorelikelytohavebabieswithlowbirthweight(LBW,<2500kg)(OR1.42,95%CI1.27to1.58),smallforgestationalage(SGA)(OR1.28,95%CI1.02to1.60)andtodeliverpremature(OR1.32,95%CI1.04to1.68).TherewasnoassociationwithApgarscores,congenitalanomaliesorperinataldeath.OnestudydescribedanassociationbetweenHGandtesticularcancerintheoffspring.conclusions: HG is associatedwith a higher female/male ratio of the offspring and a higherincidenceof LBW,SGAandprematurity. Little is knownabout the long termhealtheffectsofbabiesborntomotherswhosepregnancieswerecomplicatedbyHG.

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Consequences of hyperemesis gravidarum for offspring: a systematic review and meta-analysis

8

inTroDucTion

Many women suffer from nausea and vomiting in early pregnancy, the incidence has beenestimated to be up to 50 to 70 per cent of pregnancies1. Hyperemesis gravidarum (HG) is asevereformofnauseaandvomitingduringpregnancy.HGisassociatedwithfluid,electrolyteandacid-base imbalance,nutritiondeficiencyandweight lossoftensevereenoughtorequirehospitaladmission.Symptomstypicallystartat4-8weeksofpregnancyandcontinuetoweek14-16ofpregnancy2,3.HGoccursin0.3to2%ofpregnancies4,andisassociatedwithsignificantmaternalmorbidity5. It isunclearwhetherHGhasshortor longtermeffectsontheoffspring.Most frequently,studiesonHGreporttheeffectsasperinataloutcome,includingbirthweightandgestationalage.ThereportedeffectsofHGonbirthweightandgestationalageareconflicting,withsomestudiesreportingshortergestationalageandlowerbirthweightassociatedwithHGpregnancies4,andothers reportingnoeffects6.A largebodyofevidence fromepidemiologicandanimal studiessuggests thatundernutritionduringpregnancy increases the riskofdisease in later life7-9.HGmaythusbehypothesisedtohavelongtermhealtheffectsduetosuboptimalfetalnutrition. AsthelongtermeffectsofHGareunclearandtheshorttermconsequencesthatarereportedareinconsistent,theaimofthisreviewistosummarisetheavailableevidenceregardingthefetaloutcomeandthelongtermeffectsofpregnanciescomplicatedbyHG.

METhoDs

search strategyWeperformedanelectronicsearchtoidentifyallstudiesthatreportontheshortandlongtermoutcomes of HG.We searched Central,Medline (1953‒ January 2011) and Embase (1980‒ January 2011) using a search strategy with keywords: ‘hyperemesis gravidarum’, ‘perniciousvomitingofpregnancy’,‘neonate’,‘infant’,‘newborn’and‘longtermeffects’.Referencelistsofreviewarticlesandprimarystudieswerecheckedtoidentifycitedarticlesnotcapturedbytheelectronicsearch.

Study selectionStudieswereselectedinatwo-stageprocess.First,tworeviewers(MVandTR)scrutinizedtitlesand abstracts. Studies were selected if they reported on neonatal outcome of pregnanciescomplicatedbyHGandwerewritteninEnglish.Fullmanuscriptswereobtainedofallselectedstudies.Second, in-andexclusiondecisionsweremadeafter independentexaminationof thefullmanuscriptof selectedreferencesby tworeviewers (MVandAA). Inclusioncriteriawere:(1)patientsdiagnosedwithand/oradmittedforHG,(2)reportedneonataloutcomesincludinglowbirthweight (LBW), small forgestationalage (SGA), sexof theoffspring,perinataldeath,

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prematurity,congenitalanomaliesandlongtermhealthoutcomes.Bothstudiesthatreportedonsingleandmultiplepregnancieswereincluded.Studiesthatdidnotreportacontrolgroupwereexcluded.Anydisagreementswereresolvedbyconsensus. Twoindependentreviewers(MVandAA)extractedthedata.Meta-analyseswereconducted(ReviewManager(RevMan)version5.0,TheCochraneCollaboration,2008.).Alloutcomeswereevaluatedwitharandomeffects-model.Dichotomousoutcomeswerepooledasanoddsratio(OR)with95%CI.Differenceswere statisticallydifferent if95%CIdidnotencompass1.Datawereplotted inforestplots.The I² testwasusedtoassessstatisticalheterogeneity.Subgroupanalyseswereperformedforcasecontrolandcohortstudiesseparately.MOOSEguidelineswerefollowed10.

rEsulTs

Thesearchresultedin205studies.Afterscreeningtitlesandabstracts,163studieswereexcludedbecausetheydidnotreportonneonataloutcomeofpregnanciescomplicatedbyHG.Another2wereexcludedduetothelanguagecriterion.40paperswereretrievedforcompleteassessment(Figure1).24articlesreportedonshortandlongtermoutcomesasdescribedabove4,6,11-32(Table1).Qualityofreportingwaslimited;acleardefinitionofthereferencegroupwasnotalwaysgiven.Moststudiesreportedsingletonpregnanciesonly. Insomestudies, itwasnotstatedwhethertwinpregnancieswereincluded.Therewere11cohortstudies,12casecontrolstudiesandonecross sectional study. Four studies were conducted prospectively, all others retrospectively.Meta-analysiswasconductedforstudiesthatprovideddichotomousdata.

sex of childThirteen studies reported on gender of the offspring11-13,16,18,21,24,26-29,31,32. All studies confirmedthehigherfemale/maleratioinpregnanciescomplicatedbyHG,overall,55%oftheoffspringintheHGpregnancieswasfemale,comparedto49%inthecontrolgroup,pooledoddsratio1.27(95%CI1.21to1.34),heterogeneitywasmoderate(I²=54%(p=0.01)(Figure2A).

Low birth weightFivestudiesreportedondeliveringalowbirthweightbaby(<2500grams)4,6,18,26,31.Havingalowbirthweightbabywithabirthweightoflessthan2500gramsoccurredin6.4%ofHGpregnancies,comparedto5.0%incontrolpregnancies(OR1.42,95%CI1.27to1.58).Therewasnosignificantheterogeneityinresultsacrossthedifferentstudies(I²=0%(p=0.42))(Figure2B).

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8

sgaThepercentageofinfantsbeingsmallforgestationalage(SGA)wasreportedin4studies4,18,31,32.WomenwithHGduring pregnancyweremore likely to have a baby born SGA, this occurredin17.9%ofHGpregnancies,comparedto12.7% incontrolpregnancies (OR1.2895%CI1.02to 1.60). There was significant heterogeneity across the different studies for SGA (I² = 87%(p<0.0001))(Figure2C).

Preterm deliveryEightstudiesreportedonpretermdelivery6,18,19,24,26,29,31,32.Prematuredeliveryoccurred in7.4%ofHGpregnanciescomparedto5.8%incontrolpregnancies.HavingHGduringpregnancywasassociatedwithanincreasedriskofdeliveringbefore37weeksgestation(OR1.32,95%CI1.04to1.68).Therewassignificantheterogeneityintheresults(I²=74%(p=0.0003))(Figure2D).

figure 1Studyselectionprocess

205 poten�ally eligible studies iden�fied(database searches and references lists)

40 full text ar�cles reviewed

163 studies were excluded based on �tle and abstract review

2 studies excluded based on language criterion

16 studies excluded for not having the required exposure / not repor�ng the outcome of interest / no control group

24 studies were included in the systema�c review

110

Chapter 8

Tabl

e 1Ch

aracteris

ticsofstud

iesinclud

edin

thereview

ofsho

rtand

long

term

outcomesofp

regn

anciescom

plicated

byhy

peremesisgravida

rum.

stud

y st

udy

desi

gnDe

finiti

onAd

mitt

ed /

dia

gnos

edn

out

com

e

askl

ing etal.1

1 Birthcoho

rtre

trospe

ctive

ICD9

Ad

mitted

5.92

6HG

/1.027

.213

all

−sexofchild

baili

t4Birthcoho

rtretrospectiv

eICD9

Admitted

2466

HG/5

20.739

all

−BW

−gestati

onalage

−SG

A

−pe

rinataldeath

bash

iri eta

l.12

Casecon

trolre

trospe

ctive

Fairw

eathercriteria

2Ad

mitted

16

4HG

/20

9control

−BW

−gestati

onalage

−sexofchild

−pe

rinataldeath

−cong

enita

lano

malies

bass

o etal.1

3Ca

secon

trolre

trospe

ctive

Discha

rgediagno

ses

Admitted

6084

HG/7

6.80

4control

−sexofchild

chin

eta

l.14

Casecon

trol

Fairw

eathercriteria

2Ad

mitted

46se

vereHG/8

802control

−BW

−gestati

onalage

czei

zel eta

l.15

Casecon

trolnon

synd

romiccleft

pa

tientsretrospectiv

eICD9

ns

−HG

del M

ar M

eler

o etal.1

6Co

hort

retrospe

ctive

ICD8

Ad

mitted

&diagn

osed

4.12

6HG

/12

7.64

7all

−sexofchild

Depu

e etal.1

7Ca

secon

trolp

rospectiv

eDiagno

siscriteria

varie

dpe

rcen

ter

Diagno

sed

419HG

/83

6contro

−BW

−pe

rinataldeath

−CN

Smalform

ation

s

Dodd

s eta

l.18

Coho

rtre

trospe

ctive

Fairw

eathercriteria

2Ad

mitted

1.27

0HG

/15

4.82

1control

−BW

−SG

A

−prem

aturity

−Ap

gar

−sexofchild

−pe

rinataldeath

111


8

Table1

Conti

nued

stud

y st

udy

desi

gnDe

finiti

onAd

mitt

ed /

dia

gnos

edn

out

com

e

halla

k etal.1

9Ca

secon

trolre

trospe

ctive

Fairw

eathercriteria

2Ad

mitted

138HG

/12

.335

con

trol

−BW

−prem

aturity

−Ap

gar

−cong

enita

lano

malies

hend

erso

netal.20

Casecon

trolte

sticularc

ancerp

atien

ts

Excessivena

usea

ns

−HG

hsu etal.2

1Ca

secon

trolre

trospe

ctive

SevereHG

Admitted

66HG/2

0.79

8control

−sexofchild

Kalle

n22Co

hortre

trospe

ctive

ICD8

Diagno

sed

3068

HGcases

−BW

−sexofchild

Kaul

eta

l.23

Crosss

ectio

nal

Clinicaldiagn

osis

Diagno

sed

12HG/3

4control

−BW

Paau

w eta

l.24

Coho

rtprospectiv

eFairw

eathercriteria

2Ad

mitted

45HG/3

06con

trol

−BW

−gestati

onalage

−Ap

gar

−prem

aturity

−sexofchild

robe

rts25

Coho

rt

Clinicaldiagn

osis

ns42

HG/3

36all

−Neu

rologicaland

de

velopm

entalscores

rose

boom

eta

l.32

Birthcoho

rtre

trospe

ctive

Clinicaldiagn

osis

Diagno

sed

2190

HG/1

1970

28con

trol

−BW

−SG

A

−prem

aturity

−Ap

gar

−sexofchild

−pe

rinataldeath

Schi

ff etal.2

6Ca

secon

trolre

trospe

ctive

ICD9

Ad

mitted

2110

HG/9

783control

−BW

−prem

aturity

−sexofchild

sore

nsen

eta

l.27

Coho

rtre

trospe

ctive

ICD8&10

Admitted

650HG

/47

.931

all

−sexofchild

112

Chapter 8

Tabl

e 1

Conti

nued

stud

y st

udy

desi

gnDe

finiti

onAd

mitt

ed /

dia

gnos

edn

out

com

e

Tan etal.2

8Ca

secon

trolre

trospe

ctive

Clinicaldiagn

osis

Admitted

166HG

/47

61con

trol

−sexofchild

Tan etal.6

Casecon

trolre

trospe

ctive

Clinicaldiagn

osis

Admitted

166HG

/49

8control

−BW

−gestati

onalage

−prem

aturity

Apg

ar

Tsan

g etal.2

9Ca

secon

trolre

trospe

ctive

Clinicaldiagn

osis

Admitted

&diagn

osed

193HG

/13

.053

all

−prem

aturity

−Ap

gar

−sexofchild

−pe

rinataldeath

−cong

enita

lano

malies

Vilm

ing etal.3

0Ca

secon

trolre

trospe

ctive

ICD9

Ad

mitted

120HG

/11

5control

−BW

−sexofchild

−gestati

onalage

Zhan

g etal.3

1 Co

hortre

trospe

ctive

Clinicaldiagn

osis

severevom

iting

ns20

1cases/

166

6controls

−BW

−prem

aturity

−SG

A

−sexofchild

ns:n

otspe

cifie

d

113


8

figure 2 Perinatal outcome: Sex of offspring (a), Low birth weight (<2500 grams) (b), Small forgestationalage(c),Preterm(<37weeks)(D),Perinataldeath(E),Apgarscore<7(f)andCongenitalanomalies(g).

A Sex of offspring

B Low birth weight

C Small for gestational age

114

Chapter 8

D Preterm delivery

E Perinatal death

f apgar score <7

115


8

g congenital anomalies

Perinatal deathsSevenstudiesreportedonperinataldeaths4,6,12,17,18,29,32.Perinataldeathsoccurredin0.7%ofHGpregnanciesandin0.6%ofcontrolpregnancies(OR0.92,95%CI0.61to1.41).Heterogeneitywasmoderate(I²=42%(p=0.11))(Figure2E).

apgarApgarscores<7at5minuteswerereportedin4studies6,18,19,32.Nodifferencewasfoundintheincidenceof5minuteApgar scores<7, thisoccurred in1.9%ofHGpregnanciesand2.3%ofcontrolpregnancies(OR1.01,95%CI0.78to1.32).Heterogeneitywasnotsignificant(I²=7%(p=0.36))(Figure2F).

congenital anomaliesSix studies compared thenumberof congenitalanomalies in childrenborn toHGmothers tocontrol offspring12,15,17,19,22,29. Three studies provided data suitable for pooling. There was nodifferenceinthenumberofcongenitalanomaliesinHGpregnancies(3.1%)comparedtocontrols(3.2%)(OR1.17,95%CI0.68to2.03).Therewasnosignificantheterogeneityinresultsacrossthedifferentstudies(I²=0%(p=0.87))(Figure2G). Three other studies reported on specific congenital anomalies. A cohort study reportedseveral congenital malformations associated with HG including undescended testicles, hipdysplasiaandDownsyndrome22.AcasecontrolstudyofchildrenbornwithanoralcleftshowedanassociationbetweenearlyonsetHGandareducedriskofgivingbirthtoachildwithanoralcleft15.Anothercasecontrolstudyreportedan increase incentralnervoussystemandrelatedskeletalmalformationsinoffspringofHGmothers(OR4.0)17.

Long term effectsNostudieswerefoundthatwereconductedspecificallywiththepurposetofollowupthechildrenbornafterapregnancycomplicatedbyHGinthelongterm.Onestudyreportedtheneurologicaldevelopmentofchildrenatoneyearofageandtheobstetrichistoryoftheirmothers.TherewasnoobserveddifferenceinoutcomeassociatedwithHG25.

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Chapter 8

TheonlylongtermeffectwasanassociationofHGandtesticularcancer.Inacasecontrolstudyofmenunderage40withtesticularcancer,eightpatientmothersreportedexcessivenauseaasacomplicationoftheindexpregnancy,comparedtotwocontrolmothers.Theriskwasgreatestfornausearequiringhospitaltreatment20.

subgroup analysesFigure3showsthesummaryestimates forthedifferentstudytypes,aswellas thecombinedsummary estimates. For the outcome SGA and congenital anomalies this analysis was notpossiblebecausethesestudieswereallofthesametype.Thesubgroupanalysesshowthattheobservedheterogeneitywasnotconsistentlycausedbyonetypeofstudy.

figure 3Perinataloutcome,subanalysesbasedonstudydesignandoverallsummaryestimates:Sexofoffspring(a),Lowbirthweight(<2500grams)(b),Preterm(<37weeks)(c),Perinataldeath(D)andApgarscore<7(E)

a sex of child

figure 3Perinataloutcome,subanalysesbasedonstudydesignandoverallsummaryestimates:Sexofoffspring(a),Lowbirthweight(<2500grams)(b),Preterm(<37weeks)(c),Perinataldeath(D)andApgarscore<7(E)

a sex of child

117


8

B Low birth weight

c Preterm delivery

118

Chapter 8

D Perinatal death

E apgar score <7

119


8

Discussion

This systematic review evaluates the outcome of pregnancies complicated by HG. HG duringpregnancy is associated with a higher female/male ratio of the offspring and with a higherincidenceofachildsmallforgestationalage,withbirthweightlessthan2500gramsandwithpretermdelivery.Thesearchretrievedonlytwostudiesreportinglongtermeffects.NeurologicaldevelopmentattheageofoneyearwasnotaffectedbyHGduringpregnancy25.TherewasanassociationofHGandthedevelopmentoftesticularcancerinlaterlife20.Therewasnostatisticallysignificant difference in Apgar scores, congenital anomalies or perinatal death between HGpregnanciescomparedtocontrolpregnancies.However,heterogeneitywassignificant. The mechanism underlying the higher prevalence of female offspring after pregnanciescomplicatedbyHG is thought to behormonal. Serumhuman chorionic gonadotrophin (hCG)isoftenstatedasthemost likelycauseofHG.Thehighest incidencesofHGoccuratthetimehCG peaks33. Furthermore, HG has an increased incidence inmultiple gestations34 andmolarpregnancies35,bothconditionsassociatedwithelevatedhCGlevels.ThefactthathCGishigherinwomenbearingafemalechild36mightexplaintheassociationofafemalefetusandHG. Althoughmeta-analysis showed an increased risk of low birthweight babies, SGA babiesandprematurity,theeffectsaremodest.ThemostclinicallyrelevanteffectisprobablyforSGAbabies,17.9%inHGpregnanciescomparedto12.7%incontrolpregnancies.Theincreasedriskof lowbirthweight couldpossibly bedue toprematurity, but this cannotbe concludedwithcertaintysincedifferentstudieswereincludedinthe3differentmeta-analysesbecausenotallstudiesprovideddataforalloutcomes. Three studies investigated whether the effects of HG were mediated through maternalweightgainduringpregnancy18,24,30.Allfoundanassociationoflowerweightgainorweightlossduring pregnancy and adverse neonatal outcomes, such as lower birthweight. In one study,no difference in SGA was reported when comparing HG pregnancies to controls, but whencomparingHGwomenwithlessthan7kgweightgainduringpregnancywithHGwomenwith≥7kgweightgain,theriskofaSGAinfantwasincreased(OR1.5,95%CI1.0-2.2)18.ThisstudyalsocomparedApgarscoresofbabiesfromwomenwithHGwith7ormorekilogramsweightgaintothoseofbabiesfromwomenwith<7kgweightgain.Babiesofwomenwith<7kgweightgainhadanincreasedriskofhavinga5minuteApgarscorelessthan7(OR5.0,95%CI2.6to9.6)comparedtobabiesfromwomenwithoutHG18.TheauthorsconcludedthatHGitselfisnotariskfactorforadverseoutcomes,buttheseoutcomesaretheconsequenceofthelowweightgainassociatedwithHG.Thesefindingsareincorrespondencewithfindingsfromaretrospectivecohortstudyinvestigatingweightgainduringpregnancy.Inthisstudylowmaternalweightgainduringpregnancy,definedas<0.27kg/week,wasassociatedwith lowbirthweight,pretermlabour andpretermdelivery37.Accordingly, a population-based cohort study investigating theeffectsof lowweightgain inwomenwithnormalprepregnancyBMIfoundan increasedoddsratioforSGAinfantsinwomenthatgainedlessthan25lb(11.34kg)duringpregnancy38.These

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studiesstresstheimportanceofappropriatematernalweightgainduringpregnancyinordertoachievehealthybirthoutcomesinwomen. Asmentioned,thesearchretrievedonlytwostudiesreportingonlongtermhealtheffects20,25.ThereportedassociationofHGandtesticularcancerinlaterlifeisproposedtobeduetohormonaldisbalance.ApartfromhigherlevelsofhCG,estradioliselevatedinHGpregnanciescomparedtocontrols.Exposuretohigherlevelsofestrogenin uteromightbeacauseofundescendedtestestherebyleadingtoahigherriskoftesticularcancerinlaterlife17,20,39. Otherlongtermhealtheffects,suchascardiovascularandmetabolicdiseasehavenotbeenstudied,eventhoughthereisampleevidencefromhumanandanimalstudiesthatundernutritionduringgestation increasestheriskofdevelopingchronicdiseases7-9.Also,meta-analyseshaveshownthatbirthweightwasinverselyrelatedtosystolicbloodpressure40andtype2diabetesrisk41.Moreover, not all long term effects of prenatal environmental influences act via birthweight.StudiesofbabiesbornaftertheDutchFamineshowthatexposuretothefamineinearlygestationhasnoeffectonbirthweight,butisassociatedwithadversehealtheffects42.Therefore,followupofchildrenbornafteranHGpregnancyseemstobewarranted. The substantial heterogeneity between the studies can be due to both clinical andmethodologicalheterogeneity. Clinicalheterogeneityispartlyduetothedifficultyindefiningthecondition.Inmoststudies,Fairweather’sdefinitionforHG isused.ThisdefinesHGasaconditionof intractablevomitingduringpregnancy,leadingtofluid,electrolyteandacid-baseimbalance,nutritiondeficiencyandweightloss,oftensevereenoughtorequirehospitaladmission2.Aweaknessofthisdefinitionisthefactthatitdoesnotincludeanestimateoftheseverityofthecondition,apartfromtheneedforadmission.Differenthospitalsmayhavedifferentadmissionpolicies,andit isalsopossiblethatwhatisregardedasseverevomitinginonepopulationmaybetakenasnormalvomitinginanother.Thethresholdforhospitalizationishighlyindividual.Thisdecisioncanbeinfluencedbypsychologicalandpracticalmatters,whichcontributetothevariabilityinthediagnosis. Theincludedstudieswereconductedinavarietyofcountries,withdifferentdemographicprofiles. The incidence of HG is thought to vary across different cultures43, which will havecontributed to the clinical heterogeneity. Not every study specified the demographic profileoftheirstudypopulation.Asarecentstudyhasshown,adversepregnancyoutcomesafterHGmightbelargelyexplainedbymaternalcharacteristics,includingethnicity32. Methodologicalheterogeneitycanbeduetothefactthatmorethan50%ofincludedstudiesarecasecontrolstudies,ofwhichmostretrospective.Duetothesemattersofstudydesignthereportedeffectsmaybeoverestimated.Subgroupanalysesaccordingtothedifferentstudytypesgave no consistent explanation of the observed heterogeneity; we cannot conclude that theheterogeneitywascausedbyonetypeofstudy.Inallcases,separatelyanalysingthestudytypeswouldnothaveledtodifferentconclusions.Aconsequenceofretrospectivestudydesignisthepossibilityofrecallbias,withregardtoreportednauseaandprepregnancyweight.

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FutureresearchonHGandconsequencesfortheoffspringshouldincludestatingacleardefinitionofHGandcompletedocumentationofmaternalcharacteristicssuchasethnicity,prepregnancyweight and weight gain during pregnancy, allowing for comparison between studies andpopulations.Moreover,notonlyoutcomedirectlypostpartumshouldbedocumented,butalongtermfollowupofthesebabies,focussingoncardiovascularandmetabolicfunctioningshouldbeconducted. Inconclusion, thismeta-analysis reports thatwomenwithHGduringpregnancyaremorelikely to give birth to a girl and have a higher risk of giving birth to a LBWor SGA child andtodeliverprematurely.As little is knownabout the long termhealth consequenceson thesechildren,thereisaneedforfollowup.

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1. Jarnfelt-SamsioeA.Nauseaandvomitinginpregnancy:areview.Obstet Gynecol Surv.1987;42:422-427.

2. FairweatherDV.Nauseaandvomitinginpregnancy.Am J Obstet Gynecol.1968;102:135-175.3. NiebylJR.Clinicalpractice.Nauseaandvomitinginpregnancy.N Engl J Med.2010;363:1544-1550.4. BailitJL.Hyperemesisgravidarium:Epidemiologicfindingsfromalargecohort.Am J Obstet Gynecol.

2005;193:811-814.5. Broussard CN, Richter JE. Nausea and vomiting of pregnancy. Gastroenterol Clin North Am.

1998;27:123-151.6. TanPC,JacobR,QuekKF,OmarSZ.Pregnancyoutcomeinhyperemesisgravidarumandtheeffectof

laboratoryclinicalindicatorsofhyperemesisseverity.J Obstet Gynaecol Res.2007;33:457-464.7. HardingJ.Thenutritionalbasisofthefetaloriginsofadultdisease.Int J Epidemiol.2001;30:15-23.8. NijlandMJ, Ford SP, Nathanielsz PW. Prenatal origins of adult disease. Curr Opin Obstet Gynecol.

2008;20:132-138.9. GluckmanPD,HansonMA,CooperC,ThornburgKL.Effectofinuteroandearly-lifeconditionsonadult

healthanddisease.N Engl J Med.2008;359:61-73.10. StroupDF,BerlinJA,MortonSC,OlkinI,WilliamsonGD,RennieD,etal.Meta-analysisofobservational

studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies inEpidemiology(MOOSE)group.JAMA.2000;283:2008-2012.

11. AsklingJ,ErlandssonG,KaijserM,AkreO,EkbomA.Sicknessinpregnancyandsexofchild.Lancet. 1999;354:2053.

12. Bashiri A, Neumann L, Maymon E, Katz M. Hyperemesis gravidarum: epidemiologic features,complicationsandoutcome.Eur J Obstet Gynecol Reprod Biol.1995;63:135-138.

13. BassoO,OlsenJ.Sexratioandtwinninginwomenwithhyperemesisorpre-eclampsia.Epidemiology. 2001;12:747-749.

14. Chin RK, Lao TT. Lowbirthweight and hyperemesis gravidarum.Eur J Obstet Gynecol Reprod Biol. 1988;28:179-183.

15. CzeizelAE, SarkoziA,WyszynskiDF.Protectiveeffectofhyperemesis gravidarum fornonsyndromicoralclefts.Obstetrics and Gynecology 101(4)()(pp 737-744), 2003 Date of Publication: 01 Apr 2003. 2003;737-744.

16. delMM-M,JickH.Hyperemesisgravidarumandthesexoftheoffspring.Epidemiology.2001;12:123-124.

17. DepueRH,Bernstein L,RossRK.Hyperemesis gravidarum in relation toestradiol levels, pregnancyoutcome,andothermaternalfactors:Aseroepidemiologicstudy.American Journal of Obstetrics and Gynecology 156(5)()(pp 1137-1141), 1987 Date of Publication: 1987.1987;1137-1141.

18. DoddsL,FellDB,JosephKS,AllenVM,ButlerB.Outcomesofpregnanciescomplicatedbyhyperemesisgravidarum.Obstetrics and Gynecology 107(2 I)()(pp 285-292), 2006 Date of Publication: Feb 2006. 2006;285-292.

19. HallakM,TsalamandrisK,DombrowskiMP,IsadaNB,PrydePG,EvansMI.Hyperemesisgravidarum.Effectsonfetaloutcome.J Reprod Med.1996;41:871-874.

20. HendersonBE,BentonB,JingJ,YuMC,PikeMC.Riskfactorsforcancerofthetestisinyoungmen.Int J Cancer.1979;23:598-602.

21. HsuCD,WitterFR.Fetalsexandseverehyperemesisgravidarum.Int J Gynaecol Obstet.1993;40:63-64.22. KallenB.Hyperemesisduringpregnancyanddeliveryoutcome:aregistrystudy.Eur J Obstet Gynecol

Reprod Biol.1987;26:291-302.23. Kaul PP, SrivastavaR, Srivastava SP, KambojM, Chand S. Relationships ofmaternal blood lead and

disorders of pregnancy to neonatal birthweight.Veterinary and Human Toxicology 44(6)()(pp 321-323), 2002 Date of Publication: Dec 2002.2002;321-323.

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24. Paauw JD, Bierling S, Cook CR, Davis AT. Hyperemesis gravidarum and fetal outcome. Journal of Parenteral and Enteral Nutrition 29(2)()(pp 93-96), 2005 Date of Publication: 2005.2005;93-96.

25. RobertsCJ.Developmentalandneurologicalsequelaeofthecommoncomplicationsofpregnancyandbirth.Br J Prev Soc Med.1970;24:33-38.

26. Schiff MA, Reed SD, Daling JR. The sex ratio of pregnancies complicated by hospitalisation forhyperemesisgravidarum.BJOG: An International Journal of Obstetrics and Gynaecology 111(1)()(pp 27-30), 2004 Date of Publication: Jan 2004.2004;27-30.

27. SorensenHT,ThulstrupAM,MortensenJT,LarsenH,PedersenL,JamesWH.Hyperemesisgravidarumandsexofchild [3] (multiple letters).Lancet 355(9201)()(pp 407), 2000 Date of Publication: 29 Jan 2000 <54>.2000;407.

28. TanPC,JacobR,QuekKF,OmarSZ.Thefetalsexratioandmetabolic,biochemical,haematologicalandclinicalindicatorsofseverityofhyperemesisgravidarum.BJOG.2006;113:733-737.

29. TsangIS,KatzVL,WellsSD.Maternalandfetaloutcomesinhyperemesisgravidarum. Int J Gynaecol Obstet.1996;55:231-235.

30. VilmingB,NesheimB-I.HyperemesisgravidaruminacontemporarypopulationinOslo.Acta Obstetricia et Gynecologica Scandinavica 79(8)()(pp 640-643), 2000 Date of Publication: 2000.2000;640-643.

31. Zhang J, Cai WW. Severe vomiting during pregnancy: antenatal correlates and fetal outcomes.Epidemiology.1991;2:454-457.

32. RoseboomTJ, Ravelli AC, vanderPost JA, PainterRC.Maternal characteristics largely explainpoorpregnancyoutcomeafterhyperemesisgravidarum.Eur J Obstet Gynecol Reprod Biol.2011.

33. KauppilaA, JouppilaP,KoivistoM,Moilanen I,YlikorkalaO.Twinpregnancy.Aclinicalstudyof335cases.Acta Obstet Gynecol Scand Suppl.1975;5-12.

34. Saxena BB, Landesman R. Diagnosis and management of pregnancy by the radioreceptorassay ofhumanchorionicgonadotropin.Am J Obstet Gynecol.1978;131:97-107.

35. Landesman R, Coutinho EM, Saxena BB. Detection of human chorionic gonadotropin in blood ofregularlybleedingwomenusingcopperintrauterinecontraceptivedevices.Fertil Steril.1976;27:1062-1066.

36. DanzerH,BrausteinGD,RasorJ,ForsytheA,WadeME.Maternalserumhumanchorionicgonadotropinconcentrationsandfetalsexprediction.Fertil Steril.1980;34:336-340.

37. EhrenbergHM,DierkerL,MilluzziC,MercerBM.Lowmaternalweight,failuretothriveinpregnancy,andadversepregnancyoutcomes.Am J Obstet Gynecol.2003;189:1726-1730.

38. DeVaderSR,NeeleyHL,MylesTD,LeetTL.Evaluationofgestationalweightgainguidelinesforwomenwithnormalprepregnancybodymassindex.Obstet Gynecol.2007;110:745-751.

39. Bernstein L, Depue RH, Ross RK, Judd HL, PikeMC, Henderson BE. Highermaternal levels of freeestradiol in first compared to second pregnancy: early gestational differences. J Natl Cancer Inst. 1986;76:1035-1039.

40. HuxleyR,ShiellA,LawC.Theroleofsizeatbirthandpostnatalcatch-upgrowthindeterminingsystolicbloodpressure:asystematicreviewoftheliterature.J Hypertens.2000;18:815-831.

41. WhincupPH,KayeSJ,OwenCG,HuxleyR,CookDG,AnazawaS,etal.Birthweightandriskoftype2diabetes:asystematicreview.JAMA.2008;300:2886-2897.

42. PainterRC,RoseboomTJ,BlekerOP.PrenatalexposuretotheDutchfamineanddiseaseinlaterlife:anoverview.Reprod Toxicol.2005;20:345-352.

43. VerbergMF,GillottDJ,Al-FardanN,GrudzinskasJG.Hyperemesisgravidarum,aliteraturereview.Hum Reprod Update.2005;11:527-539.

9summary and discussion

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This thesis focuses on the fetal origins hypothesis, which states that undernutrition duringimportantperiodsofgrowthanddevelopmentcanleadtopermanentchangesinthephysiologyandmetabolismofthebody.SinceitwasformulatedbyDavidBarkerinthelate1980’s,alargebodyofevidencehasbeenassembledtosupportthehypothesis;varyingfromdirectevidencefromexperimentsinanimalmodelstoevidencefromlargeobservationalhumancohortstudies.Inrecentyears,underlyingmechanismshavebeen investigatedandtherelevanceofthefetaloriginsofhealthanddiseaseforcurrentpregnancieshasbeenfurtherexplored.Theresearchreportedinthisthesisfurtheraddstothisfieldinscience. chapters 2and3aresystematicreviewsthatsummarizetheavailableevidencefromanimalexperimentsoftheeffectsofprenatalundernutritiononbloodpressureandglucosemetabolism.chapter 2focussesontheeffectsofprenatalundernutritiononglucoseandinsulinmetabolismandbetacellmass.Themeta-analysisshowedthat-despitesignificantheterogeneityinthestudies-prenatalproteinrestrictionincreasedplasmaglucoseconcentrationsinlaterlife(0.42mmol/l(95% CI 0.07 to 0.77)). Both general undernutrition and protein restriction reduced plasmainsulinconcentrationsinlaterlife(generalundernutrition:-0.03nmol/l(95%CI-0.04to-0.01),proteinrestricted:-0.04nmol/l(95%CI-0.08to0.00))andbetacellmass(generalundernutrition:-1.24mg(95%CI-1.88to-0.60),proteinrestriction:-0.99mg(95%CI-1.67to-0.31)).chapter 3presentsasystematicreviewandmeta-analysisontheeffectsofprenatalundernutritiononbloodpressure.Weconcludedthatbothmaternalgeneralandproteinundernutritionincreasedsystolicbloodpressure in later life (generalundernutrition:14.5mmHg,95%CI10.8 to18.3;proteinundernutrition:18.9mmHg,95%CI16.1to21.8)andmeanarterialpressure(generalundernutrition:5.0mmHg,95%CI1.4to8.6;proteinundernutrition:10.5mmHg,95%CI6.7to14.2).Herealso,therewassubstantialheterogeneityintheresults.Diastolicbloodpressurewasincreasedbyproteinundernutrition(9.5mmHg,95%CI2.6to16.3),whilegeneralundernutritionhadnosignificanteffect.Bothreviewshaveshownthatevidencefromexperimentsindifferentspecies has shown that prenatal undernutrition results in increased glucose, reduced insulinconcentrations, reduced beta cell mass and generally increased blood pressure in later life,thereforegenerallysupportingthefetaloriginshypothesis. chapter 4describestheeffectofprenatalexposuretotheDutchfamineonadulthandgripstrength.PreviousstudieshaveshownthatthoseexposedtotheDutchfamineduringgestationhaveworsehealththanthoseunexposedtofamineduringgestation.Sincehandgripstrengthisamarkerofadulthealth,ourhypothesiswasthathandgripstrengthmightbeaffectedafterprenatalexposuretotheDutchfamine.Contrarytoourexpectations,wefoundthatmenexposedduringearlygestationhadgreaterhandgripstrengthcomparedtounexposedmen.Thiscouldhoweverlargelybeexplainedbyadultheight. Inchapter 5wedescribewhetherepigeneticchangesmayplayaroleintheprogrammingeffectsofprenatalexposuretofamineonadulthealthastheyhavebeenfoundintheDutchfaminebirthcohortstudy.Epigenetics,andspecificallymethylation,isthoughttobealikelymechanismthatstoresearly lifeexperiences.Forourstudy,weconsidered fourcandidategenes,allwith

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cardiovascularandmetabolic relevance,andmeasuredmethylationat theproximalpromotersites.Wefoundnoevidenceofeffectsofprenatalexposuretofamineonthemethylationstatusof thesefourgenes.Thismeansthatthephenotypiceffectswehavefoundareunlikelytobemediatedbyalteredmethylation levels in thesegenes.Wealso studiedatmethylation levelsandmarkersofadulthealthandlifestyle,andobservedthatmethylationofGRandPPARγwereassociatedwith lifestyle factors suchas smoking,BMIandexercise, suggesting thatpostnatalfactorsalsoinfluencemethylationpatterns. chapter 6 described the association between GR methylation and stress response in asubsampleof theDutch faminebirthcohort.Wefoundthatadecrease inmethylationof theGR1-Cpromoterwas associatedwith adecrease in stress activity, indicatedby lower cortisollevelsandlowerheartrateactivity.Theseassociationscouldbelargelyexplainedbydifferencesinlifestyleandeducation. Inchapter 7 weinvestigatedwhethertheoffspringofpeoplewhowereexposedtofamineprenatallywerelesshealthythantheoffspringofunexposedpeople, inotherwords,whethertheeffectsofprenatal famineexposurepassdowngenerations. In this youngpopulation,wefoundthatchildrenofprenatallyexposedmenwereheavierandhadahigherBMIthanchildrenofunexposedmen. Inwomen,we foundno transgenerationaleffects.We foundnoevidenceof transgenerationaleffectsofprenatal famineexposureonhealth in this relatively youngF2sample,buttheincreasedadiposity intheoffspringofprenatallyundernourishedfathersmayleadtoincreasedchronicdiseaseratesinthefuture. Inchapter 8wereportonastudydescribingtheconsequencesofaclinicalconditionthatresemblesthenutritionalconditionafetusexperiencedduringtheDutchfamine;hyperemesisgravidarum (HG). Studies concerning this severe form of nausea and vomiting during earlypregnancymainly focus on treatment of the pregnantwomen, but little is known about theeffectsfortheoffspring.Wepreformedasystematicreviewandmeta-analysistosummarizetheevidenceonshortandlongtermoutcomesofpregnanciescomplicatedbyHG.WefoundthatHGisassociatedwithahigherfemale/maleratiointheoffspringandahigherincidenceoflowbirthweight,beingbornsmall forgestationalageandprematurity.ApartfromacasecontrolstudydescribinganassociationbetweenHGandtesticularcancerintheoffspring,littleisknownaboutthelongtermconsequencesofHGonthehealthoftheoffspring.

Discussion

animal studiesAnimal experimentshave contributedmuch toourunderstandingofdisease.Notonly in thefieldofDevelopmentalOriginsofAdultDisease(DOHaD),butalsointhedevelopmentofmedicaltherapies.Themajorityofmedicaltherapiesinusetodayhasinitiallybeendevelopedandtestedinanimals.Inasystematicreviewevaluatinghowoftenhighlycitedanimalstudiestranslateto

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successful human research, the authors conclude that none of the highly cited studieswerenegative1. Less than half of the studies were of good methodological quality and that fewincluded randomallocationof animals or blindingof outcomeassessment,while themediancitation count of these studieswas 889. For these animal studies the results of only a thirdcouldbe replicated inhuman randomized trials and justone tenthof the interventionsweresubsequentlyapprovedforuseinpatients1. There are differences between animal studies and the situation in humans that makecomparisondifficult. For reasonsof costandefficiency,mostanimalexperimentsaredone inveryyoungandhealthyanimals,notreflectingtheconditions inmanyhumandiseases.Manyanimalstudiesarelimitedtomaleanimals,restrictingthegeneralizabilityofthestudytofemaleanimals,letalonethehumansituation.Generalizabilitymightalsobeimpairedbythefactthatanimals are keptunder extremely controlled circumstances.Also, pathophysiology in animalsandhumansisnotalwayscomparable. It appears self-evident that the quality of the design of an animal experimentwill affectitsscientificvalidity,butthishadreceivedlittleattentioninthefieldoftranslationalmedicine.Theexistingevidence shows that these issuesare crucial, just like inhuman studies2.Animalstudiesreportingoninterventionsinemergencymedicinewerethreetimesmorelikelytoreportapositiveresultifthepublicationdidnotreportrandomizationofblinding3.Asystematicreviewon treatment of acute ischemic stroke showed larger benefit of treatmentwith lower studyquality4,5.Onereviewfoundlargeoverstatementsofthereductionofinfarctvolumeinanimalstrokestudieswithoutrandomizationorblindingcomparedtorandomizedorblindedstudies5.Humanstudiessupportthesefacts;clinical trials thatdidnotreportblinding,concealmentofallocationor(double)blindingreportlargertreatmenteffectsthantrialsthatdidreportonthesemethodologicalissues6-8.Publicationbiasisanotherfactorthatplaysanimportantroleinanimalstudies. Again in the field of experimental stroke, a meta-analysis was conducted including525publications.Of these,only ten (2%)didnot report at leastone significanteffecton theoutcomemeasures9.Thissuggeststhatnegativeorneutralanimalstudiesarepublishedmuchlessfrequentlythanpositivestudies. Inthisthesis,wehavesystematicallyreviewedtheevidencefromanimalstudiesforthefetaloriginshypothesis regarding glucosemetabolismandhypertension. Experiments to study theeffectsofmaternalmalnutritionduringgestationonthehealthofthehumanoffspringwouldbeunethical,forobviousreasons.Wethereforerelyonanimalexperimentstogaininsightintheeffectofchanges inthematernaldietfortheoffspring.Weconcludethatthemethodologicalqualityaswellasthereportingofthesestudiesispoor.Inthefieldofprenatalundernutrition,differentexperimentaldietsareused,whichmakescomparisondifficult.Ithasbeendescribedthatexposuretolowproteindietsinfetallifedoesnotinitselfdeterminethedevelopmentofhypertensioninadult life.Thebalanceofthenutrientswithinthematernaldietsappearedtoplayacriticalroleintheprogrammingeffectintheoffspring10.Thereisalsoaneedforimprovingthemethodologicalqualityofanimalstudies.Reportingshouldbedoneaccordingtostandards

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similartothoseappliedinhumanstudies.Inordertoavoidpublicationbias,animalstudiescouldberegisteredcomparabletotheregistrationofclinicaltrials.Thesemeasureswillhelpreducingpublicationbiaswhileimprovingreliabilityandreproducibilityofanimalstudies.

Epigenetics Untilrecently,organismswerethoughttoconveytheirpropertiestotheiroffspringbygeneticinheritance.Themechanismsleadingtotheeffectsofmaternalundernutritionduringgestationonthehealthoftheoffspring,however,arenotsufficientlyexplainedbythismodel. The term epigenetics was first proposed by developmental biologist ConradWaddingtonin the 1940s, who used it to explain how a multicellular organism could develop from onegenome11.Later,othershavedefinedepigeneticsas“thestudyofmitoticallyand/ormeioticallyheritable changes ingene function that cannotbeexplainedbychanges inDNAsequence”12.The first molecular factor that was identified was DNA methylation in the 1970s13 followedby histonemodification in the 1990s14. In DNAmethylation,methylation of the 5’position ofcytosinesuppressesgeneexpressionbymodulatingtheaccessofthetranscriptionmachinerytothechromatinorbyrecruitingmethyl-bindingproteins15.Unlikegeneticinformation,whichisextremelystable,epigeneticmarksarereversible,respondingtoendogenousandenvironmentalsignals. Epigeneticmodificationsarealikelyexplanationforhowinfluencesintheearlyenvironmentcanleadtopermanentchangesinmetabolismtherebychanginglong-termdiseaserisks.Itisnosurprisethen,thatmanystudieshavebeenconductedtoelucidatetheroleofepigeneticsinthefieldofDOHaD. Animal experiments have shown how maternal dietary restriction resulted in alteredmethylationpatternsofgenes intheoffspring16,17.Thesemethylationpatternsappearedtobestabile,astheywerepresent inboth juvenileandadultoffspring17,andwerepasseddowntothenextgeneration18.Methylationthenseemstoactasamemoryofanevent longaftertheexposurehasceased.Thisapparentstability is insharpcontrastwithanothercharacterizationofepigenetics;reversibilitywhichisnecessaryforthedynamicepigenometointeractwiththeenvironment. DNAmethylation is the only epigenetic mechanism directly affecting the DNAmolecule.CpGdinucleotidesarethemaintargetforDNAmethylation.CpGislands,DNAsequenceswhere CpGdinucleotidesareclustered,arefoundatthe5´promoterregionsofhousekeepinggenes.A large body of evidence suggests that CpG island methylation of these promoter regionsaffectstranscription.Recently,however,alsonon-promoterCpGislandsareidentified(UMR´s,unmethylated regions). These UMR´s can become methylated and potentially modify geneexpression19.Non-CpGmethylationhasalsobeendescribed.Inthiscase,methylationdoesnotinvolve protein-binding sites, but the gene body is methylated. These findings illustrate thecomplexityofDNAmethylationandtheimportanceofinvestigatingregionsoutsideCpGislandsandpromotersinfuturestudies.Tomakemattersevenmorecomplex,DNAmethylationactsin

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concertwithhistonemodifications,mediatedbymethyl-orhistone-bindingproteinsinfluencinggeneexpression20.Futurestudiesshouldnotfocusononeepigeneticmechanism,butinvestigatecombinationsofthemechanismsleadingtoabetterunderstandingoftheepigeneticprocess. Thechangesinmethylationlevelsthatarefoundinhumansasaresultofchangesinmaternaldietappeartobemuchsmallerthanthosefoundinanimalstudies.Astudymeasuring5CpG’sin the IGF2 DMRfoundthatchildrenwhosemotherdidanddidnottake400ugramoffolicacidperdayinthepericonceptionalperiodhaswholebloodmethylationlevelsof49.5%and47.4%respectively21. Thefirst studydescribingalteredmethylation levels afterprenatal exposure totheDutchfaminefoundamethylationdifferenceof5%inpeopleexposedtofamineprenatallycomparedtotheirunexposedsiblings22.Incontrast,inanimalsdifferencesofupto200%(relativetothecontrolgroup)arebeingdescribed23.Furthermore,itisunknownwhetherthischangeinmethylation is associatedwithalterations inexpressionnorwhether it is linked todisease inhumans. Otherconsiderationsthatmustbemadeinfutureepigeneticstudiesaretheanimalmodelor tissue to use, the technique used tomeasure (either genome-wide or gene-specific), thetimingofmeasurement(perhapsmeasuringmethylationlevelslongitudinally),whichcouldhelpunderstandthesequenceofeventsandthestability(orplasticity)intime.

Implications for future research YearsofresearchinthefieldoffetaloriginsandontheDutchfaminehaveshownthatmaternalundernutritionduringfetaldevelopmenthas lastingnegativeconsequencesfortheoffspring’shealth.Extrapolatingthistocurrentpregnanciesimpliesthattheenvironmentofthedevelopingembryo is of importance. There are studies showing that undernutrition during the earliestphasesofpregnancy,evenbeforeimplantation,canpermanentlyincreasetheoffspring’sbloodpressure24. This suggests that even the veryearlypre-implantationembryoenvironmentmayhavelongtermconsequencesforthehealthoftheindividual.Anincreasingbodyofevidencesuggests that this is the case for assisted reproductive techniques (ART). Randomly assigningembryostotwodifferentcommerciallyavailableIVFculturemediashowedadifferenceinmeanbirthweightof200grams25. Long term followupof childrenbornafterassisted reproductivetechniqueshasdemonstratedthatIVFchildrenhavehighersystolicanddiastolicbloodpressureandhigher fastingglucose levels compared tocontrols conceivedspontaneously26.Totalbodyfatalsoseemstobeincreased27.GirlsbornafterICSIpregnanciesaremoreadiposeatpubertycompared to girls born after spontaneous pregnancies28. Recently, alterations in vessel wallpropertieshavebeenshownamongARTchildren29. In this thesis we discussed the outcomes of pregnancies complicated by hyperemesisgravidarum.Recently,thefirst longtermconsequencesofthisconditionhavebeendescribed.Offspringexposed tohyperemesisgravidarum inuterowas significantlymore likely tohaveapsychological and behavioral disorder compared to unexposed siblings. Depression, bipolardisorderandanxietywerethemostfrequentreporteddisorders30.Thisfindingstrengthensour

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conclusionthatchildrenbornafterpregnanciesthatarecomplicatedbyhyperemesisshouldbefolloweduptodocumentthelongtermconsequencesofthecondition. NotonlyinthefieldofARTorhyperemesisgravidarum,butmoreingeneralforallstudiescomparinginterventionsduringpregnancytoimproveneonatalhealth,thefocusshouldexpandtooutcomeslaterinlife.Followupstudiesshouldinvestigatewhetherchildrenbornafterthesepregnancies have an altered cardiometabolic risk profile due to influences during the crucialphasesinearlylife.

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1. Hackam DG, Redelmeier DA. Translation of research evidence from animals to humans. JAMA. 2006;296:1731-1732.

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Nederlandse samenvatting

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Ditproefschriftgaatoverde“fetalorigins”hypothese.Dezehypothesesteltdatondervoedingvande foetustijdensbelangrijkeperiodenvangroei enontwikkeling kan leiden totblijvendeaanpassingen inde fysiologieendestructuurvanhet lichaam.Dezeaanpassingenzoudenoplatereleeftijdkunnenleidentoteenverhoogdrisicoopziekte.DavidBarkerheeftdezehypothesegeformuleerdindejaren80,ensindsdienisveelbewijsgevondenvoordezehypothese;zowelbewijsuitdierexperimentenalsuitgroteobservationelestudiesinmensen.Indelaatstejarenisveelonderzoekgedaannaardeonderliggendemechanismenennaarderelevantievandefetaloriginshypothesevoorhuidigezwangerschappen.Hetonderzoekdatwordtbeschreven inditproefschriftdraagtbijaanditonderzoeksgebied. hoofdstuk 2 en 3 zijnsystematischereviewsenmeta-analyses(kwantitatievesamenvattingen)diehetbeschikbarebewijs samenvattenuitdierexperimentenoverdeeffectenvanprenataleondervoedingopbloeddrukenglucosemetabolisme. hoofdstuk 2 focustopdeeffectenvanprenataleondervoedingophetglucoseen insulinemetabolisme en op de hoeveelheid bèta cellen (de cellen in de alvleesklier die insulineproduceren).Demeta-analyselietziendat,ondankssignificanteheterogeniteit,prenataleeiwitondervoedingleidttoteenverhoogdeglucoseconcentratieinhetbloed(0.42mmol/l(95%BI0.07 tot 0.77)). Zowel algemene als eiwit ondervoeding verlagende insuline concentraties inhetbloed(algemeneondervoeding:-0.03nmol/l(95%BI-0.04tot-0.01),eiwitondervoeding:-0.04nmol/l (95%BI -0.08tot0.00))endehoeveelheidbètacellen (algemeneondervoeding:-1.24mg(95%BI-1.88tot-0.60),eiwitondervoeding:-0.99mg(95%BI-1.67tot-0.31)). hoofdstuk 3 iseenmeta-analyseengeefteensystematischoverzichtvandewetenschappelijkeliteratuurwat betrefthet bewijs uit dierproevenover de relatie tussenondervoeding vandemoeder tijdens de zwangerschap en bloeddruk bij de nakomelingen. Dezemeta-analyse laatzien dat zowel algemene ondervoeding als eiwit ondervoeding van demoeder leidt tot eenverhoogdesystolischebloeddruk (algemeneondervoeding:14.5mmHg,95%BI10.8 tot18.3;eiwitondervoeding:18.9mmHg,95%BI16.1tot21.8)enverhoogdegemiddeldearteriëledruk(algemeneondervoeding:5.0mmHg,95%BI1.4tot8.6;eiwitondervoeding:10.5mmHg,95%BI6.7to14.2).Diastolischebloeddrukwasalleenverhoogdnaeiwitondervoeding(9.5mmHg,95% BI 2.6 tot 16.3), terwijl algemene ondervoeding geen effect had. Ondanks significanteheterogeniteittoontdezemeta-analyseaandat,inverschillendediersoorten,ondervoedingvandemoederoverhetalgemeenleidttoteenverhoogdebloeddrukbijdenakomelingen. Inhoofdstuk 4wordtderelatietussenhandknijpkrachtenblootstellingaandeHongerwintervoordegeboorteonderzocht.Hetisbekenddathandknijpkrachteenmaatisvoorgezondheid,enaangezienhetHongerwinteronderzoekheeftlatenziendatmensendievoordegeboortezijnblootgesteldaandeHongerwintermindergezondzijn,wasonzehypothesedathandknijpkrachtwellicht verminderd zou zijn bij deze mensen. Het tegenovergestelde bleek; wij vonden datmannendie zeervroegtijdensdeontwikkelingwarenblootgesteldaanondervoedinggrotereknijpkrachthaddendanmannendienietwarenblootgesteldaanondervoedingvoordegeboorte.Derelatiewerdgrotendeelsverklaarddoorlengteopvolwassenleeftijd.

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Inhoofdstuk 5 hebbenwij onderzocht of epigenetische veranderingen een rol spelen bij degezondheidseffectendiezijngevondenbijdemensendievoordegeboortezijnblootgesteldaandeHongerwinter.Methylering,éénvandeepigenetischemechanismen,regeltdeaansturingvangenen,enbepaaltzoofeengenactiefisofniet.Hetideeisdatinvloedenvroeginhetleveneen“epigenetischeafdruk”achterlatenopgenen.Indezestudiehebbenwijviergenenonderzochtdieallenbetrokkenzijnbijhart-envaatziektenenbijmetaboleaandoeningen.WijhebbengeenbewijsgevondendatblootstellingaandeHongerwintervoordegeboorteleidttotveranderingenin demethylering van deze genen. Dit betekent dat de verhoogde gevoeligheid voor ziektendieeerderisgevondenbijdemensendieblootgesteldwarenaandeHongerwinternietwordtgemedieerd door veranderde methylering van deze genen. In deze studie hebben wij welgevondendatmethyleringwordtbeïnvloeddoorlifestylefactorenzoalsroken,BMIenbeweging.Ditsuggereertdatinvloedenlaterinhetlevenmethyleringooknogkunnenbeïnvloeden. hoofdstuk 6beschrijfthetverbandtussenmethyleringvandeglucocorticoidreceptorendestressreactiebijmensendievoorhungeboortewarenblootgesteldaandeHongerwinter.Wevondendatverminderdemethyleringvandereceptorwasgeassocieerdmeteenverminderdestress reactie; dit bleek uit lagere cortisol spiegels en lagere hartslagstijging bij stress. Degevonden associaties konden grotendeels worden verklaard door verschillen in levensstijl enopleidingsniveau. Inhoofdstuk 7 hebbenweonderzochtofdekinderenvanmensendievoorhungeboortewarenblootgesteldaandeHongerwintermindergezondwarendandekinderenvanmensendienietwarenblootgesteldaandeHongerwintervoorhungeboorte.Metanderewoorden;ofdegevolgenvanblootstellingaandeHongerwintervoordegeboortewordtovergedragenaandevolgendegeneratie.Wijhebbengevondendatkinderenvanmannendieblootgesteldwaren,eenhogerBMIhaddendankinderenvanmannendienietwarenblootgesteld.Bijkinderenvanvrouwenwerdengeeneffectengevonden.Inderelatiefjongeonderzoeksgroepkondenwegeenverschillenvindenophetgebiedvanziekteofgezondheid.Maarhetresultaatdatkinderenvanblootgesteldemannen dikker zijn zou een eerste teken kunnen zijn van een verhoogd ziekterisicoindetoekomst. hoofdstuk 8beschrijfteensystematischereviewenmeta-analysevanallebeschikbarestudiesoverhyperemesisgravidarum(HG)endeeffectenopdekinderen.HGisernstigemisselijkheidenbrakeninhetbeginvandezwangerschap.DezeaandoeningzougezienkunnenwordenalseenvormvanondervoedingvandefoetusvertoontdaarmeegelijkenismetdevoedingstoestandvandefoetusnablootstellingaandeHongerwinter.StudiesoverHGgaanvoornamelijkoverdebehandelingvandezwangere,erzijnweiniggegevensoverdegevolgenvoordekinderendienaeendergelijkezwangerschapwordengeboren.InonzestudievondenwijdatHGgeassocieerdismetlaaggeboortegewicht,tekleingewichtvoordetermijnenprematuriteit.HGkomtrelatiefvakervoorbijzwangerschappenvanmeisjesdanjongens.Behalveeenpatiënt-controlewaarineenassociatietussenzaadbalkankerenHGwerdbeschreveniserweinigbekendoverdelangetermijngevolgenvanHGvoordekinderen.

Dankwoord

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Dankwoord

Allereerst een woord van dank aan alle deelnemers aan dit onderzoek, de mensen van hetHongerwintercohort.Hetisheelbijzonderdatvelenvanhen,enhunkinderen(opnieuw)bereidwarendeeltenemen. Mijn promotoren, Prof.dr. P.M.M. Bossuyt, en Prof.dr. J.A.M. van der Post, beste Patricken Joris, hartelijk bedankt voor jullie betrokkenheid bij dit onderzoek. Van jullie deskundigcommentaarwerdendestukkenalleenmaarbeter. Dr.R.C.Painter,besteRebecca.Zonderjouwasditboekjeernooitgeweest,jijwashetdiemijnaarhetAMChaaldevanuitAlkmaar.Knaphoejemetzo’ndrukkeagendaaltijdtijdvondombetrokkentezijnbijallestukken,dankje! Dr.T.J.Roseboom,besteTessa.Ikhadmegeenbeterebegeleiderkunnenwensen!Ditprojectwasnietaltijdmakkelijk,enhetheeftveelgevergdvanonzediplomatiekekwaliteiten.Wehebbenheelwatgezuchtensomsgemopperd,maargelukkigkondenweeruiteindelijkaltijdomlachen!Hetwaseengrootplezierommetjesamentewerken. Mijn mede-Hongerwinter-onderzoekers, Susan en Annet. Susan, wat ontzettend fijn datjij terugkwam bij het onderzoek. Behalve dat het erg handigwas om iemand in de buurt tehebbendieallesoverhetonderzoekwistwashetvooralheelerggezellig.ZowelopdeKEBBals op congres-reisjes enookbuitenhetwerk. Ikwens jeheel veel succesmet jouwverderewetenschappelijkecarrière. Annet,destartwasmisschienwatstroef,maaralsnelkondenwijhetprimavinden.Onderzoekdoenwordteenstuk leukerals jehetsamendoet,en jijwasde idealeonderzoekspartner(ofmoetikzeggen‘mattie’?).Wehebbensamenheelwatleukereisjesgemaaktwaarikmetveelplezieropterugkijk,hetweekendinSouthamptonsamenmetFleurenIngewassuperleuk! Inge, Esther, Mariska, Teodora, Lotty en alle andere KEBB-collega’s, bedankt voor degezelligheid,openbuitenhetwerk. Gré,spilvandeKEBB,dankjevoorjepraktischehulp,zekerdezelaatstemaanden. Gynaecologen, collega-assistenten, verpleegkundigen, poli-medewerkers en alle anderenvan de afdeling Gynaecologie en Verloskunde in het ZaansMedisch Centrum, vanaf dag éénvoeldeikmeopmijnplekinZaandam,dankdaarvoor! Mijnparanimfen,CarianneenFleur.Carianne, toevalligkwamenwijallebei terecht inHetPaarsKot18jaargeleden.OndankshetfeitdatikAntwerpennaeenaantaljarenverlietbleefonzevriendschap.Ikbewonderjouwcarrière,enbenblijdatjemijnparanimfbent. Fleur,jijschreefhetookal,vanafdagéénopdeKEBBonafscheidelijk,datistochwelheelbijzonder!MijnKEBB-tijdheeftmebehalveditproefschriftookeengoedevriendinopgeleverd(ookalbenjijwatmeer‘roze’enikwatmeer‘grijs’..),endaarbenikblijom!Fijndatjijnaastmestaatbijmijnverdediging. ManonenLiesbeth,altijdgezelligalsweelkaarzien!Erzijnalheelwatetentjes,uitjes inAmsterdamendaarbuitenenvakantiesgeweest,ennuditproefschriftafiszullenernogvelenvolgen.Manon,numijncongres-reisjesvoorbijzijnzullenwezelfvakantiebestemmingenmoetenverzinnen.

141

Dankwoord

Alleanderevriendenenvriendinnen,dankvoorjullievriendschap! Lievemama,jijenpapahebbenonsaltijdgesteundinallekeuzesdiewijmaakten.Zonderdievrijheidwasditboekjeernietgeweest.Hetisjammerdatpapaditnietmeeheeftkunnenmaken.AnnemarieenHans,SaskiaenJurjen,hetalaltijdgezelligalswijbijelkaarzijn,bedanktvoorjulliebelangstelling.AnneenHans,ikbenbenieuwdnaarmijnkleineneefje!

curriculum Vitae

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CurriculumVitae

MarjoleinVeenendaalwerdop21januari1976geboreninErmeloalsoudsteineengezinvandriedochters.ZijbrachthaarjeugddoorinLeiderdorp,McLean(VS)enApeldoorn. Na de middelbare school werd zij uitgeloot en begon zij in Antwerpen aan de studieGeneeskunde. Na drie jaar hield zij het daar voor gezien,werd opnieuw uitgeloot en starttein 1998 met de studie Medische Biologie aan de Universiteit van Amsterdam. Deze studiewerd in2002cum laudeafgesloten. In2001werdzijuiteindelijk toch ingelootvoordestudieGeneeskundeaandeUniversiteitvanAmsterdam.In2006behaaldezijhaarartsexamenengingwerkenalsANIOSinhetMedischCentrumAlkmaar.NaeenjaarbegonzijopdeafdelingKlinischeEpidemiologie,BiostatistiekenBioinformaticainhetAMCaanhetpromotieonderzoekdatheeftgeleidtotditproefschrift. Op1januari2012begonzijaandeopleidingtotgynaecolooginhetZaansMedischCentrum.

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