The impact of folic acid supplementation on gestational and long term health: critical temporal windows, benefits and risks

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Porto

Biomedical

Journal

h tt p://w w w . p o r t o b i o m e d i c a l j o u r n a l . c o m /

Review

articles

The

impact

of

folic

acid

supplementation

on

gestational

and

long

term

health:

Critical

temporal

windows,

beneﬁts

and

risks

Carla

Silva

a

_,

_Elisa

_Keating

b,c

_,

_Elisabete

_Pinto

d,e,∗ a_Faculty_of_Medicine_of_University_of_Porto,_Porto,_Portugal

b_Department_of_Biomedicine_–_Biochemistry_Unit,_Faculty_of_Medicine_of_University_of_Porto,_Porto,_Portugal c_CINTESIS_–_Center_for_Health_Technology_and_Services_Research,_Porto,_Portugal

d_CBQF_–_Centre_of_{Biotechnology}_and_Fine_Chemistry_–_School_of_{Biotechnology,}_Portuguese_Catholic_University,_Porto,_Portugal e_Institute_of_Public_Health_of_the_University_of_Porto,_Porto,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19February2017

Receivedinrevisedform10May2017 Accepted31May2017

Availableonline12July2017 Keywords: Folicacid Pregnancy Supplementation Offspring Periconceptional

a

b

s

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Maternalfolicacid(FA)supplementationisoneofthemostpopularnutritionalinterventionsduring pregnancyforitsprotectiveeffectagainstneuraltubedefects(NTDs).

Thepurposesofthisrevieware:(a)togatherthecurrentevidenceregardingsupplementationof maternaldietwithFAand(b)toproblematizetheavailableliteratureintermsofdosages,criticaltemporal windows,anditspotentialbeneﬁtsandrisks.

Theexpression(pregnancyORfetusORoffspringORmother)AND(“folicacid”AND supplemen-tation)wassearchedonPubMeddatabase,ﬁlteringforarticlespublishedfrom2005to2014.Publications referringtoFAsupplementationduringthepericonceptionalperiodorpregnancyinwhichtherewasa conclusionabouttheeffectsofisolatedFAsupplementationonpregnantwoman,pregnancyoroffspring wereincluded.Oftheinitial1182papers,109fulﬁlledtheinclusioncriteria.

ThemajorityofthepublicationsreportedFAsupplementationoutcomesonoffspring’shealth,with emphasisinNTDs,allergy/respiratoryproblems,cancerandbehaviourproblems.Someinconsistencyis observedontheimpactofFAsupplementationondifferentoutcomes,exceptforNTDs.Itisalsovisiblean increasedconcernabouttheimpactofexcessivesupplementation,eitherintermsofdosesorexposure’s duration.

Inconclusion,thereisagrowinginterestinFAsupplementationissues.TheprotectiveeffectofFA supplementationoverNTDshasbeenconﬁrmed,beingthepericonceptionalperiodacriticalwindow, anditisfrequentlysuggestedthatallergy/respiratoryoutcomesarisefrom(excessive)FA supplemen-tationparticularlylaterinpregnancy.Furtherresearchoncriticaldosesandtimeofexposureshouldbe conducted.

Introduction

Folateisatermthatreferstoagroupofwater-solublevitamins

ofthecomplexBwhicharenaturallyfoundinfoodssuchasleafy

greenvegetables,citrus fruitsandliver.Folicacid(FA),the

syn-theticandcompletelyoxidizedformoffolate,isusedinvitamin

supplementsandinfortiﬁedcerealproducts.

Within the cells, folates act as cofactors in reactions which

are determinant in cell division and cell maintenance, as well

∗ Correspondingauthor.

E-mailaddresses:carla.rib.silva@gmail.com(C.Silva),keating@med.up.pt

(E.Keating),epinto@porto.ucp.pt(E.Pinto).

as in the regulation of gene expression through epigenetic

mechanisms.1_Indeed_folates_are_a_source_of_{s-adenosylmethionine,}

the main cellular methyl donor that modulates genome-wide

methylation thus regulating the expression of genes. This is

believed to be the process by which folates affect foetal

pro-gramming. In addition, folates have a determinant role in the

re-methylationofplasmahomocysteinetomethionine,2_for_which

bloodfolatelevelscorrelatenegativelywithbloodhomocysteine

levels.

MaternalFAsupplementationisoneofthemostpopular

nutri-tionalinterventionsduringpregnancyandithasbeenextensively

studiedforitseffectonneuraltubeformationandfoetalgrowth3

asfolatedeﬁciencyatconceptionandduringpregnancymaylead

toabnormaldevelopment.4

http://dx.doi.org/10.1016/j.pbj.2017.05.006

2444-8664/©2017PBJ-AssociaçãoPortoBiomedical/PortoBiomedicalSociety.PublishedbyElsevierEspa ña,S.L.U.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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1182 papers

174 papers

Abstract excluded

(n=

1008

)

109 papers included

paper

Full

excluded (n=65)

Studies not focused on the effects of isolated FA supplementation (n=539) Review articles (n=226)

Studies testing the capacity of FA reverting environmental, chemically or genetically-induced deleterious effects (n=119)

Letters or editorials (n=55) Clinical recommendations (n=38) Case reports (n=19)

Study set-up descriptions (n=6) Withdrawn studies (n=3) Duplicated studies (n=2) Study with human cells (n=1)

Studies that were not focused on the effects of isolated FA supplementation (n=52)

Studies testing the capacity of FA reverting environmental, chemically or genetically-induced deleterious effects (n=10)

Review (n=1)

Study set-up descriptions (n=1) Study with human cells (n=1)

Fig.1. Flowchartofprocessofsystematicliteraturesearch.

Particularly since the 1990s, when evidence emerged about

the protective effect of FAsupplementation against the

recur-renceandoccurrenceofneuraltubedefects(NTDs),5,6 _there_has

beenincreasingconcernabouttheroleofpericonceptionaldietary

supplementationandfortiﬁcationstrategies,asNTDsrepresentsan

importantcauseofmorbidityandmortality.7

Manycountriesworldwidepracticemandatoryfortiﬁcationof

cerealgrainproductswithFA,asapublichealthpolicy,and

sev-eralEUcountriesmaintaina voluntaryfortiﬁcation ofdifferent

foodstuffs.8 _In _addition, _the _WHO _preconizes _the

supplemen-tationwithFA,inthepericonceptionalperiodandpregnancy,with

0.4mg FA/day (together with 30–60mg iron), increasingup to

5mg/dayforwomenathighriskofbirthdefects.9

Notwithstand-ing,theWHOalsopointedoutthattheprotectionagainstNTDs

onlyhappenswhenthesupplementationoccursuntilthefourth

gestationalweek,while supplementation in othertime periods

is likely to be beneﬁcial in relation to other issues of

mater-naland foetalhealth,suchas foetalgrowthand pretermbirth.

However, the evidence regarding these other outcomes is not

consistent.

Itisknownthatat0.4mg/day,alltheoxidizedformofFAis

con-vertedintobiologicallyactivemetabolitesduringabsorption,and

so,itsconsumptionisgenerallyconsideredsafe.Intakesabovethe

recommendedlevelsarelikelytoleadtotheappearanceof

unme-tabolizedFAin foetaland maternalcirculation,which hasbeen

demonstratedincountrieswithfoodfortiﬁcation.10_Although_the

impactofthisisnotcompletelyunderstood,itissuggestedthat

unmetabolizedFAinmaternalandfoetalbloodmayactasmethyl

donorfortheregulationofgeneexpression,withconsequencesin

foetalprogramming.11

InPortugal, althoughtherecommendationsfollow theWHO

guidelines,12 _and _there _are _{multivitamin-mineral} _supplements

containing0.4mgFAorsimilar(whicharesoldwithoutamedical

prescription), theavailable reimbursed pills containingisolated

FAhavea totaldoseof5mg. Thisdosecorresponds to

approx-imately 12.5 times the WHO recommended dose for low risk

pregnancy.Furthermore,thelackofsystematizedhealthpractices

inmanycountries,beingPortugalpartoftheproblem,maylead

toexcessiveFAsupplementationresultingfromthesimultaneous

(3)

containing multivitamin-mineral supplements, sometimes,

throughoutallpregnancy.13_In_addition,_there_is_also_a_big_portion

of non-planned pregnancies all over the world14 _and _several

studiessuggestthatthecomplianceofwomentopericonceptional

FAsupplementationvarieswidely.15

Bothfolaterestrictionandexcessivesupplementationleadto

epigenetic marks, raising a potential concern about its role in

inducingunforeseenchangesinthemethylomaandthusinthe

phe-notypeofthemotherandtheoffspring1_at_a_short_or_a_long-term.

Accordingly,aremarkablenumberofstudieshavereportedthe

out-comesofmaternalFAsupplementationontheoffspring’shealth,

addressinginteractionssuchasinchildren’sasthma,cancer,insulin

resistance,1_autism16_and_{behaviour/inattention}_problems.17

Allthesefactscontributetoarenewedinterestinthe

impli-cationsofmaternalnutrition, beforeandduringpregnancy,and

itsinﬂuencesinpreandpost-nataldevelopment,raisingquestions

relatedtothemagnitude,timing,typeanddurationofexposureto

FA.18

Thisreviewhastwomainpurposes:ﬁrst,togatherevidence

publishedworldwideregardingsupplementationofmaternaldiet

withFAduringpregnancyandthemainoutcomesofthis

supple-mentation either in pregnant women or in the offspring, in a

short-to-longterm;second,withthepulledinformation,to

prob-lematize this supplementation in terms of FA dosages, critical

temporalwindows,aswellasitspotentiallybeneﬁtsandrisks,to

themothersandoffspring,regardingthecurrentstateoftheart.

Methods

To conduct this systematic review, the authors searched

PubMed database and included the MeSH terms “pregnancy”,

“fetus”,“offspring”,“mother”,“folicacid”and“supplementation”

combinedinthefollowingexpression:(pregnancyORfetusOR

off-springORmother)AND(“folicacid”ANDsupplementation).The

searchwasperformedinDecember2014withthefollowing

ﬁl-ters:allarticlesavailablepublishedfrom2005to2014,writtenin

English,Portuguese,SpanishorFrench.Thesearchretrievedatotal

of1182publications.

Basedonourobjectives,thethreeauthorsestablishedaﬁrstlist

ofexclusioncriteria.Duringtheprocessofabstractreading,some

criteriawereaddedtothisﬁrstlist.Abstractswerereviewedbytwo

oftheauthorsseparately–eachofthemevaluated650abstracts,

withanoverlapofapproximately100abstracts,inorderto

cali-bratetheagreementintheabstracts’revision.Thethirdresearcher

participatedinthemeetingwheretheconcordancebetweenthe

othertwowerediscussedandparticipatedinthedecisionof

dis-cordantclassiﬁcations,aswellasinthedeﬁnitionofnewexclusion

criteriathroughoutalltheprocess.Theagreementbetweenboth

researcherswasveryhigh.Wheneverdoubtswereraisedtheywere

sharedbythethreeresearchersandthethirdelementwascritical

fortheﬁnaldecision.

The inclusion criteria was: all original papers referring to

isolated FA supplementationduring periconceptional period or

pregnancyinwhichtherewasaconclusionabouttheeffectsofFA

supplementationonthepregnantwoman,pregnancyoroffspring.

Whenevertheabstractwasnotavailableor ifitdidnotclearly

revealedanykindofassociationbetweentheFAsupplementation

andtheoutcomes,theauthorsaccessedthefulltextarticletodecide

aboutinclusionorexclusion.

The exclusion criteria (and the number of excluded articles

afterabstractreading)were:reviewarticles(n=226),casereports

(n=19),letterstotheeditororeditorials(n=55),clinical

recom-mendations(n=38),studyset-updescriptions(n=6),withdrawn

studies(n=3),duplicatedstudies(n=2),studieswithhumancells

(n=1),studiesthatwerenotfocusedontheeffectsofisolatedFA

supplementation(n=539),studiestestingthecapacityofFA

revert-ingenvironmental,chemicallyorgenetically-induceddeleterious

effects(n=119).

So,theﬁrstabstracts’assessmentallowedtheexclusionofatotal

of1008ofthe1182abstractsretrieved.Atthispoint,theremaining

174full-textarticleswerereadandanalyzedandthisresultedin

theexclusionof65furtherpublications:reviews(n=1),study

set-updescriptions (n=1),studieswithhumancells (n=1), studies

testingthecapacityofFArevertingenvironmental,chemicallyor

genetically-induceddeleteriouseffects(n=10),studiesthatwere

notfocusedontheeffectsofisolatedFAsupplementation(n=52).

Afterwards,fromtheinitial1182retrieved,1073wereexcluded

and109publicationscompletelyfulﬁlledtheinclusioncriteriaof

thisreview(seeFig.1).

Inordertosystematizetheﬁndings,threetableswerecreated

containingthemain features ofeach paper: author(s)and year

ofpublication,country, studiedpopulation, samplesize,typeof

study,intervention(timinganddosages ofFAsupplementation)

andresultsandmainconclusions.Theﬁrsttabledescribesincluded

animal studies (Table 1), the second one, the included human

studiesreportingeffectsofFAsupplementationovertheoffspring

(Table2)andthelastonetheincludedhumanstudiesreporting

effectsofFAsupplementationoverthemotherandoverpregnancy

outcomes(Table3).

Results

Ofthe109includedpapers,themajorityofthem(n=92)were

performedinhumansanda totalof17 correspondedtoanimal

studies.Consideringthetypeofstudy,ofthe109,mostofthem

(n=85,78%)wereobservationalinnature(whethercase–control

studies(n=38),cohortstudies(n=40),orcross-sectionalstudies

(n=7))andonly24(22%)wereinterventiontrials,agreat

propor-tionofwhichconductedwithlaboratoryanimals(n=17;70.8%).

In termsof chronology,signiﬁcantly morepublications were

foundinthelastfiveyears(n=84;77%)comparingtothefirstfive

yearsofresearch(2005–2009).

Whenthepaperswereidentiﬁedbycountry,thereisavast

dis-persionaroundtheworld.However,themostrepresentedones

were the USA (n=20), The Netherlands (n=11), China (n=11),

Canada(n=11)andUK(n=9).Thepapersselectedweredivided

in2 maincategories:animal trialsandhumanstudies.Inorder

tofurthersystematizetheanalysis,humanstudiesweredivided

onthosefocusedonhealthoutcomesoftheoffspringandthose

onhealth outcomesofthemotherorpregnancyResultsofeach

categoryareshownseparately.

Animaltrials

Intheanimaltrialscategory(n=17,Table1),allthe

publica-tionswere focused onhealth outcomesin the offspring, and a

preponderanceofthoserelatedtotheroleofFAsupplementation

towardsgeneexpression(n=4),genemethylation(n=2)andits

consequencesinembryogenesis(n=4)wasfound.Otheroutcomes

werecarcinogenesis(n=4),includingmammarytumoursinthe

off-spring(n=2),colorectalcancer(n=1)andmedulloblastoma(n=1);

metabolicsyndrome(n=1);behaviouralchanges(n=1)andseizure

(n=1).

Genemethylationandgeneexpression

Sixofthe17animaltrialsfocusedtheirattentionongene

expres-sionormethylation andglobally folatesupplementationduring

pregnancyisshowntoregulategeneexpression.19–21_Many_of_the

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Table1

Descriptionoftheincludedanimalstudies(n=17).

Author,year reference

Country StudiedPopulation Sample size

Typeofstudy Intervention(doseandtiming ofFAsupplementation)

Results/mainconclusions Baruaetal.,201434 _USA _Pregnant_rats ₄₀ _Animal_trial _0.4_mg_FA/kg_diet_vs.₄_mg

FA/kgdietstartingoneweek beforemating

SupplementationwithhighdoseFA duringpregnancymodulatesthe expressionofgenesinvolvedin development.

Huangetal., 201433

China Pregnantratsand offspring

30 Animaltrial 2mg/kg;5mg/kg;40mg/kg; beforeandthroughout pregnancy

Supplementationofpregnantmice’s dietwithhighdoseFAexacerbatesthe glucoseintoleranceandinsulin resistanceinducedbyhighfatfeeding adultmaleoffspring.

Beenetal.,201331 _USA _Pregnant_rats ₁₂₆ _Animal_trial _0.3_mg/kg;₂_mg/kg;₈_mg/kg;

onemonthbeforepregnancy andduringpregnancy

Incidenceofmedulloblastomawas lowerinoffspringborntolowFA (0.3mg/kgdiet)supplementeddams whencomparedtocontrols(offspring borntorecommendedFA,2mg/kg diet).LowFAintakeduring preconceptionandgestationmay decreasemedulloblastomaincidence inmicegeneticallypredisposedto tumourdevelopment.

Domoslawska etal.,201328

Poland Pregnantbitches(Pugs andChihuahuas)

37 AnimalTrial 0.6mg/kg/day;5mg/dayfor Pugsbitches;2.5mg/dayfor Chihuahuasbitchessincethe onsetofheatandduring pregnancy

Thepercentageofpuppieswithcleft lip/cleftpalateingroupswithhigher dosagesofFAdecreasesinpredisposed PugsandChihuahuapuppies. Girottoetal.,

201335

Canada Pregnantrats Not reported

Animaltrial 4mg/dayvs.0mg/day;before andduringpregnancy

HighdosesofFA,whencomparedto none,administeredtoratsbeforeand duringgestationdecreasetheir offspringseizurethreshold.

Liuetal.,201319 _China _Newborn_piglets ₈ _Animal_trial _1.3_mg/kg/day;₃₀_mg/kg/day _Maternal_FA_{supplementation}_changes

theexpressionofhepaticproteinsthat areinvolvedinmetabolicregulation, oxidativeresponses,and

cancer-relatedprocesses. Mikaeletal.,

201326

Canada Pregnantmice 52 AnimalTrial 2mg/kg/day;20mg/kg/day;6 weekspreconceptionand duringpregnancy

SupplementationwithhighdosesofFA fromthepreconceptionthroughout pregnancymayadverselyaffect embryonicmousedevelopment. Sieetal.,201322 _Canada _Pregnant_rats ₁₅₀ _Animal_trial ₂_or₅_mg_folic_acid/kg_diet₃

weekspriortomatingand throughoutpregnancyand lactation

MaternalandpostweaningFA supplementationsigniﬁcantlyaffects globalandgene-speciﬁcDNA methylationintheratoffspring. Royetal.,201224 _India _Pregnant_rats ₄₇ _Animal_trial ₂_mg/kg/day_or₈_mg/kg/day_in

dietswithorwithoutadequate levelsofB12

SupplementationwithhighFAinan adequateB12statusdoesnot signiﬁcantlychangetheoutcomes. Nevertheless,highmaternalFA supplementationonaB12deﬁcient dietincreasesoxidativestressinboth motherandpups.

Swayneetal., 201225

Canada Rats(maleandfemale and3generations)

78 AnimalTrial 0mg/kg/day;2mg/kg/day; 6mg/kg/day

ChronicexposuretoFAthroughout generationsatdosesclosetothose achievedthroughfortiﬁcationdoesnot instigateorprotectagainst

chromosomedamage. Sieetal.,201132 _Canada _Rat_offspring ₂₂₀ _Animal_trial ₂_mg_FA/kg_diet_vs₅_mg_FA/kg

diet;starting3weeksbefore mating,throughoutpregnancy andlactationand

post-weaning

SupplementationwithFAinadose equivalenttotheaveragetotalfolate intakeinNorthAmericaafter fortiﬁcationpolicyimplementationis protectiveagainstthedevelopmentof colorectalcancerinratoffspring. Chmurzynskaand

Malinowska, 201121

Poland Pregnantratsand offspring

20 Animaltrial 2mg/kg/day;5mg/kg/day FAsupplementationduringpregnancy decreasestheexpressionofenzymesof themethioninehomocysteinepathway (phosphatidylethanolamine

N-methyltransferase,cystathionine ␤-synthase,andbetaine-homocysteine methyltransferase)butitdoesnot affectserumlevelsofhomocysteine. Lyetal.,201129 _Canada _Pregnant_rats _Different

ninthe different phases

Animaltrial 2mg/kg/day;5mg/kg/day;3 weeksbeforeconception, duringpregnancyandlactation

FAsupplementationduringpregnancy increasestherisk,acceleratestherate ofappearanceandincreasesthe multiplicityofmammary

adenocarcinomasintheoffspringand reducesglobalDNAmethylation.

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Table1(Continued)

Author,year reference

Country StudiedPopulation Sample size

Typeofstudy Intervention(doseandtiming ofFAsupplementation)

Results/mainconclusions Kulkarnietal.,

201123

India Pregnantrats 47 Animaltrial 2mg/kg/dayor8mg/kg/dayin dietswithorwithoutadequate levelsofB12

SupplementationwithhighFAinan adequateB12statusdoesnot signiﬁcantlychangetheoutcomes. NeverthelesstheratioofFAand vitaminB12mayplayanimportant roleindeterminingglobalDNA methylation.

Sableetal.,201120 _India _Pregnant_rats ₈₀ _Animal_trial ₂_mg/kg/day_or₈_mg/kg/day_in

dietswithorwithoutadequate levelsofB12

SupplementationwithFAinan adequateB12statusdoesnotaffectthe levelsofneurotrophins.

Caldwelletal., 201027

USA Pregnantmice 10 Animaltrial 3differentfolatedietsforfour weeksbeforemating:0mg/kg folate,2mg/kgfolate,and 8mg/kgfolate

Maternalhighfolatesupplementation (ordeﬁciency)duringpregnancy drasticallyaltersgeneexpressionin offspring’sheart,leadingtosimilar phenotypicoutcomesintheembryos. Sieetal.,200930 _Canada _Pregnant_rats_and

offspring

20 Animaltrial 2mgofFA/kgdiet;5mgFA/kg diet;3weekspriortomating, throughoutpregnancyand lactation

PerigestationalFAsupplementation reducesthenumberofterminalend budsareliablepredictorofmammary tumourriskatadulthoodinrodents. USA–UnitedStatesofAmerica;FA–folicacid.

ongenemethylation.Namely,Sieetal.demonstratedthat

mater-nalFAsupplementationmodulatesglobalandgene-speciﬁcDNA

methylationintheratoffspring22_and,_for_instance,_Kulkarni_et_al.

showedthattherewasareductioninDNAmethylationlevelsof

pla-centaltissuesoccurringafterexposureofpregnantdamstoexcess

offolates.23_In_what_concerns_critical_doses_of_{supplementation,}_it

seemsthatexcessivesupplementation(8mg/kginsteadofthe

rec-ommendeddoseof2mg/kg)hasadeterminantrolebothingene

expressionandgenemethylation.24_However,_another_study

con-cludedthatchronicexposuretoFAthroughoutgenerationsatdoses

closetothoseachievedthroughfortiﬁcationdoesnotinstigateor

protectagainstchromosomedamage.25

Embryogenesis

Ofthefourtrialsstudyingembryogenesisoutcomes,one

sug-geststhathighlevelsofFAsupplementationmayadverselyaffect

foetalmouse embryonic development26 _and _that _the_excessive

maternalFAsupplementationon adeﬁcient B12diet, although

notaffectingneurodevelopment,mayincreaseoxidativestressin

mothersandpups.25

Inaddition,folateintakeduringpregnancyissuggestedtohave

adualeffectoncardiogenesis,dependingonthepresenceof

envi-ronmentaltoxins.27

In all thesecases, there is a common trend suggesting that

highdosesofFAsupplementation,andalsolongerexposures,may

indeedinduceharmfuloutcomesintheoffspring.Theexceptionis

theprotectiveeffectofhighdosesofFAregardingorofacialclefts

intwospeciﬁcpredisposedbreedsofdogs.28

Tumours,behaviouralchanges,metabolicsyndromeandseizure

Regardingmammarytumours,trialsdescribingopposite

out-comeswerefound:exposuretohighlevelsofFA(5mg/kgdiet)

duringpregnancyandlactationmayincreasetheriskofmammary

tumoursinratoffspring29_and_the_same_dose_of_FA_{supplementation}

issuggestedtolowertheriskofthissameoutcome.30_Although_the

dosesandtheexperimentalmodelsusedwerethesame,theﬁrst

paperinvestigatestheeffectofFAsupplementationon

chemically-inducedmammarytumours,whilethesecondpaperexploresthe

effectofthissupplementationonspontaneousmammarytumours.

ItislikelythatahighmaternalFAenvironmentmaybeprotective

againstthedevelopmentofspontaneouspreneoplasticmammary

lesions,29_but,_on_the_other_hand,_it_may_increase_the_{susceptibility}

tochemically-inducedbreastcarcinogenesisintheoffspring.

Inthecaseofmedulloblastoma,theonlytrialconcludedthat

lowmaternalpericonceptionalFAlevels,whencomparedtothe

recommendedlevels,maydecreasetheincidenceofthesetumours

inmicegeneticallypredisposedtotumourdevelopment.31

Stillregardingcancer,Sieetal.32_suggest_that_maternal,_but_not

post-weaning,supplementationwithFAinadoseequivalenttothe

averagetotalfolateintakeinNorthAmericaafterfortiﬁcationpolicy

implementationisprotectiveagainstthedevelopmentof

colorec-talcancerinratoffspring.Theyalsosuggestthatthisprotective

effectmayresultfromanincreaseinglobalDNAmethylationand

adecreaseinepithelialproliferationinthecolorectum.

Inwhatconcernsmetabolicsyndrome,onestudyshowedthat

highFAsupplementationduringpregnancymayhaveanadverse

effectbyexacerbatingthedetrimentaleffectof highfatdiet on

glucoseintoleranceandinsulinresistanceinmaleoffspring.33

Inaddition,itwassuggestedthatunregulatedhighFA

supple-mentation during pregnancy may lead to alterations in brain

developmentresultinginchangesinbehaviour.34_And_ﬁnally,₄_mg

FA/daybeforeandduringgestation,seemstodecreaseby42%the

offspring’sseizurethreshold.35

Humanstudies:healthoutcomesoftheoffspring

Almost 2/3(n=74) of theincluded papers were focused on

studying,inhumans,theroleofmaternaldietaryFA

supplemen-tationoverthefoetalandchild’shealth(Table2).

Ofthe74 papers,arecurrentoutcomestudiedwasembryo’s

defects(n=29),including:NTDs(n=8),orofacialclefts(n=7),heart

defects (n=5), bladderexstrophy/epispadias/hypospadias (n=4)

andothercongenitalabnormalities(n=5).

Sevenoftheincludedpapersfocusedonoffspring’stumours

(braintumours(n=4)andleukaemia(n=3))and4oftheincluded

papersconcludedaboutoffspring’sasthma(n=4),lowtract

infec-tions(n=2)orallergy/atopy(n=4).

Alsowecanobservea majorconcernregardinginfantfoetal

growth(n=5),behaviourproblems(n=3)orautisticdisorder(n=2)

andneurodevelopment/memory (n=5).Otherstudiedoutcomes

wereoffspring’sboneturnover/development(n=2),DNA

methyla-tion(n=3),semencharacteristics(n=1),bodycomposition(n=1),

blood pressure (n=1), metabolic syndrome (n=1) and, at last,

schizophrenia/psychoticsymptoms(n=1)andpsychomotor

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Table2

Includedhumanstudiesdescribinghealthoutcomesoftheoffspring(n=75).

Reference Country Studiedpopulation Samplesize Typeofstudy Intervention(doseand timingofFA supplementation)

Results/mainconclusions

Yangetal.,201575 _China _Pregnant_women

andoffspring

150cases; 212controls

Case-control FAsupplementationduring pregnancy(with quantiﬁcation)

Supplementationofwomenwitha highdoseofFAduringpregnancyis associatedwithincreasedriskofinfant asthma.Supplementationofwomen withlowdoseFAduringpregnancyis associatedwithdecreasedriskof infantasthma.

Ajroucheetal., 201470

France Casesofchildhood leukaemia

Case-control FAsupplementationduring periconception(with timing)

Slightinverseassociationisobserved betweenFAsupplementationstarting inthe3monthspreconceptionandthe riskofchildhoodleukaemia. Csaky-Szunyogh

etal.,201451

Hungary Casesofcongenital heartdefects

Case-control FAsupplementationduring pregnancy(yesorno)

HighdosesFAareprotectiveagainst left-ventricularoutﬂow-tract, particularlycoarctationoftheaorta. Greenopetal.,

201466

Australia Casesofchildhood braintumours

Case-control FAsupplementationduring pregnancy(withtiming)

Reducedriskofbraintumoursin childrenofmothersthattookFA corroboratingfolate’simportant contributiontogenomicintegrityand DNAmethylation.

Mashudaetal., 201463

Tanzania Infantswith congenital anomalies

445 Cross-sectional

FAsupplementationduring periconception(yesorno)

NouseofFAinthepericonceptional periodissigniﬁcantlyassociatedwith congenitalanomalies.

Valera-Granetal., 201487

Spain Pregnantwomen andoffspring

2213 Cohort FAsupplementationduring pregnancy(with quantiﬁcation)

Poorerpsychomotordevelopment observedinchildrenbornfrom motherswhoweresupplementedwith highdoseofFA(>5mg/day)during pregnancywhencomparedtochildren borntomothersusingthe

recommendeddoseofFA (0.4–1mg/day)duringpregnancy. Veerankietal.,

201473

USA Pregnantwomen andoffspring

167,333 Cohort FAsupplementationduring pregnancy(withtiming; majority:1mg/day)

ChildrenofwomenwhohadFA supplementationonlyintheﬁrst trimesterofpregnancyhavehigher probabilitytohaveadiagnosisof bronchiolitisandgreaterseverityof bronchiolitis,whencomparedto childrenofwomenthatdidnothave FAsupplementationduringpregnancy. Agopianetal.,

201339

USA Casesofneural tubedefects

Case-control FAsupplementationinthe periconceptionandﬁrst trimesterofpregnancy

LackofFAsupplementation(after fortiﬁcationestablishment)isnota majordeterminantofspinabiﬁdaor anencephaly.

Boekeetal.,201385 _USA _Pregnant_women

andoffspring

895 Cohort FAsupplementationearly inpregnancy(with quantiﬁcation)

SupplementationwithFAearlyin pregnancyisnotassociatedwithchild memoryat7years. Esmaeilietal., 201341 Iran Casesof lipomyelomeningo-cele (LipoMMC) 35cases;70 controls

Case-control FAsupplementationduring periconceptionandﬁrst trimesterofpregnancy

PericonceptionalFAsupplementation issigniﬁcantlylowerincaseswhen comparedtocontrols.FA

periconceptionalsupplementationis anindependentprotectivefactor againstLipoMMC.

Haggartyetal., 2013101

UK Pregnantwomen 913 Cohort FAsupplementationduring pregnancy(with quantiﬁcation)

FAuseafterthe12thweekof pregnancyaffectsDNAmethylationin theoffspring,particularlyinrepeat elementsandinIGF2gene. Hollisetal.,201362 _USA _Cases_of_trisomy₂₁ ₉₀₇_cases;

983controls

Case-control FAsupplementationinthe preconception

FAsupplementationinthe

preconceptionisassociatedspeciﬁcally withmeiosisIIerrorsinoldermothers. LiXetal.,201355 _China _Cases_of_congenital

heartdefects

Case-control FAsupplementationduring periconceptionand pregnancy(withtiming)

SupplementationwithFAisassociated withreducedriskofcongenitalheart defects.TheearlierFA

supplementationbeginsbefore pregnancyandthelonger

supplementationlasts,thelowerthe riskofCHDsis. Rozendaaletal., 201350 The Nether-lands

Casesofclefts 367cases; 2945controls

Case-control FAsupplementationduring pregnancy(withtiming andquantiﬁcation)

FAsupplementuseduring0–12weeks postconceptionisassociatedwith increasedriskoflip/alveoluscleft. Surenetal.,201391 _Norway _Pregnant_women

andoffspring

85,176 Cohort 0.4mg/dayinthemonth beforepregnancyand duringtheﬁrsttrimester

FAsupplementationinthe preconceptionandintheﬁrst trimesterofpregnancyisassociated withadecreasedriskofautistic disorderintheoffspring.

(7)

Table2(Continued)

vandenHiletal., 2013104 The Nether-lands Pregnantwomen andoffspring

2863 Cohort FAsupplementation(no use,usewhenpregnancy wasknown,usingduring thepericonception)

FAsupplementationduringpregnancy isnotassociatedwithchildhood systolicordiastolicbloodpressure. Vereczkeyetal., 201352 Hungary Casesof atrioventricular canaldefects 77cases; 38,151 controls

Case-control FAsupplementationduring pregnancy(withtiming)

HighdosesofFAearlyinpregnancyare associatedwithareducedrateof atrioventricularcanaldefects. Wehbyetal.,

201348

Brazil Womenintending togetpregnant

273 Humantrial 0.4mg/dayor4mg/day beforepregnancyand duringtheﬁrsttrimester

HighdoseofFAsupplementationin thepericonceptionalperioddoesnot changerecurrenceratesoforalcleft. HighdoseofFAalsodoesnotimpair foetalgrowth.

Wangetal.,201340 _China _Cases_of_neural

tubedefects

Case-control FAsupplementationinthe preconceptionandduring theﬁrsttrimester(with timing)

FAsupplementationduringthe periconceptionalperioddecreasesthe riskofneuraltubedefects.

Bekkersetal., 201281 The Nether-lands Pregnantwomen andoffspring 3786 Cohort FAsupplementation between30and36 gestationalweeks(with quantiﬁcation)

PrenatalFAsupplementationisnot associatedwithadverserespiratoryor allergicoutcomesinchildren(1–8 yearsofage),apartfromaslight increasedriskofwheezeat1yearold. Chandleretal., 201237 USA Casesof anencephaliaand spinabiﬁda 954cases; 6268controls

Case-control FAsupplementationinthe 3monthsprevious conceptionandduringall pregnancy(with quantiﬁcation)

FAintake,aswellasother micronutrients,includingthiamin, betaine,riboﬂavin,vitaminB6,vitamin

C,vitaminE,niacin,iron,retinol,and vitaminA,maybeprotectiveagainst anencephaly.However,folateintake doesnotaffecttheriskforspinabiﬁda. Chatzietal.,201283 _Greece _Pregnant_women

andoffspring

553 Cohort FAsupplementationearly inpregnancy(with quantiﬁcation)

Dailysupplementationwithhighdose FA(5mg/day)inearlypregnancyis associatedwithimprovedvocabulary development,communicationalskills andverbalcomprehensionat18 monthsofage.HigherdosesofFAdo notshowanyassociationwith additionalimprovementsinthe neurodevelopmentscales. Dunstanetal.,

201278

Australia Pregnantwomen andoffspring

628 Cohort FolateintakeandFA supplementationinthe thirdtrimesterof pregnancy(with quantiﬁcation)

FAsupplementationishigherin childrenwithsubsequenteczema,but itwasnotassociatedwithother allergicoutcomes.

Fornsetal.,201288 _Spain _Adolescents ₃₉₃ _Cohort _FA_{supplementation}_during

pregnancy

Supplementationofthematernaldiet withFAmayinﬂuenceinattentiveand hyperactive/impulsive symptomatologyduring pre-adolescence. Kiefte-deJong etal.,201277 The Nether-lands Pregnantwomen andoffspring

8742 Cohort FAsupplementationduring periconceptionofafter,but beforethe10thgestational week(0.4-0.5mg/day)

PericonceptionalFAsupplementation andsupplementationwithintheﬁrst 10weeksofpregnancyarenot associatedwithhigherprevalence wheezing,shortnessofbreathoratopic dermatitisduringchildhood.High folateandvitaminB-12concentrations duringpregnancyisassociatedwith higherprevalenceofatopicdermatitis intheoffspring.

Martinussenetal., 201276

1499 Cohort FAsupplementationduring pregnancy(with quantiﬁcation)

FAsupplementationinpregnancyis notassociatedwiththeriskofasthma inchildrenat6yearsofage. Milneetal.,201267 _Australia _Cases_of_childhood

braintumours

Case-control FAsupplementationinthe monthpreviouspregnancy andineachtrimester

SupplementationwithFAbeforeand likelyduringpregnancymaybe protectiveagainstchildhoodbrain tumours. Paranjothyetal., 201261 UK Casesoffoetus withgastroschisis 124cases; 217controls

Case-control FAsupplementationinthe ﬁrsttrimesterofpregnancy

SupplementationwithFAfora minimumof6weeksduringtheﬁrst trimesterofpregnancyisprotective againstfoetalgastroschisis. Schmidtetal.,

201290

USA Casesofautism spectrumdisorders

Case-control FAsupplementation3 monthsbeforepregnancy andineachmonthof pregnancy(with quantiﬁcation)

PericonceptionalFAmaybeprotective againstautismspectrumdisorder particularlyformothersandchildren withinefﬁcientfolatemetabolism.

(8)

Table2(Continued)

Wangetal.,201295 _China _Pregnant_women

andoffspring

1388 Humantrial FAsupplements;Iron-FA supplements;multivitamin supplementsduring pregnancy

Resultsdonotsupportagreater advantageoftheeffectofmaternal multi-micronutrientsupplementation onchildgrowthoveriron-FAorFA onlyduringtheﬁrst30months. Ahrensetal.,

201142

USA Casesofspina biﬁda

Case-control Dietaryfolateintake, includingfortiﬁed foods+FA

supplementationinthe2 monthsbeforeand2 monthsafterconception

OverasettingofFAfortiﬁcation,FA supplementationinaregularbasis(≥4 daysperweek)aroundtheconception momentorinitiatedinearlypregnancy isnotassociatedwithspinabiﬁda. Beanetal.,201153 _Georgia _Cases_of_Down

syndrome

1011 Case-control FAsupplementationbefore conception,andduring pregnancy(withtiming)

LackofmaternalFAsupplementation isassociatedwithseptaldefectsin infantswithDownsyndrome. Binkleyetal.,

201180

Canada Casesofchildren withpeanutallergy

Case-control FAsupplementationinthe periconception

FAsupplementationbeforeorafter conceptionisnotassociatedwiththe riskpeanutallergyinchildhood. Campoyetal.,

201186

Spain Pregnantwomen andoffspring

161 Humantrial After20weeksof pregnancy:0.4mgof 5-MTHF;0.4mgof 5-MTHF+DHA;onlyDHA

FAand/orDHAsupplementationare notsigniﬁcantlyassociatedwitha bettercognitivefunctionofchildren. DeMarcoetal.,

201136

Italy Casesofneural tubedefects

Case-control FAsupplementationinthe 3monthsprevious conceptionand3months afterconception

Lackoffolatepericonceptional supplementationincreasestheriskof spinabiﬁda.

Hossein-nezhad etal.,201197

Japan Pregnantwomen andoffspring

113 Cohort FAsupplementationduring pregnancy(withtiming andquantiﬁcation; 1mg/day)

DailysupplementationwithFAduring thewholepregnancyhasapositive impactontheboneturnovermarkers inmothersandtheirnewborns,when comparedwithFAsupplementation onlyuntiltheendofsecondtrimester. Hoyoetal.,201199 _USA _Pregnant_women ₄₃₈

Cross-sectional

FAsupplementationinthe preconceptionandduring pregnancy(with quantiﬁcation)

MethylationlevelsattheH19DMR decrease(inamorepronounced fashioninmaleinfants)with increasingmaternalFAintake. Jacobsenetal.,

2011102

Denmark Singletonsonsof mothersenrolled inacohort

347 Cohort FAsupplementationduring pregnancy

Semencharacteristicsaresimilar amongsonsofmotherssupplemented withFAduringpregnancywhen comparedwithsonsofmothersnot supplementedwithFA(orwith unknownFAsupplementation). HoweverFAsupplementedgrouphas higherlevelsofFSHandLH. Jiaetal.,201144 _China _Cases_of

non-syndromic cleftlip with/withoutcleft palate 537cases; 221controls

Case-control FAsupplementationearly inpregnancy

MaternalFAsupplementationduring earlypregnancyhasaprotectiveeffect againstcleftlipandcleftpalate.

Magdelijnsetal., 201179 The Nether-lands Pregnantwomen andoffspring

2834 Cohort FAsupplementationinthe preconceptionandduring pregnancy

FAsupplementationofmaternaldietis notassociatedwithincreasedriskof atopicoutcomesincludingwheezing andasthmaintheoffspring. Mohammadzadeh etal.,201156 Iran Casesof hypospadias 25cases; 6124controls

Case-control FAsupplementationinthe 3monthsbefore conceptionandduringﬁrst trimesterofpregnancy

IronandFAsupplementationmayhave preventiveeffectinhypospadias.

Pastor-Valeroetal., 201192

Spain Pregnantwomen 786 Cohort FAsupplementationduring pregnancy:non-users, moderateusers (0.2-0.9mg/day)andhigh users(2.5-10.5mg/day)

Periconceptionalsupplementation withFAindosesover1mg/dis associatedwithdecreasedbirthlength andmayincreasetheriskoflowbirth weight.

Reutteretal., 201158

Germany Familiesofcases bladder exstrophy-epispadias complex 441 Cross-sectional FAsupplementsinthe periconceptionorinthe ﬁrst10weeksofgestation FAsupplementationinthe periconceptionalperiodseemsto preventagainstthedevelopmentof theseverephenotypeofbladder exstrophy-epispadiascomplex (associationsarenotstatistically signiﬁcant).

Rothetal.,201184 _Norway _Pregnant_women

andoffspring

38,954 Cohort FAsupplementation1 monthbeforeconception andduringpregnancy, untilthe17thgestational week(withquantiﬁcation)

FAsupplementationinearlypregnancy (eitherFAaloneorincombinationwith othersupplements)isassociatedwith areducedriskofseverelanguagedelay inchildrenat3yearsofage.

(9)

Table2(Continued)

Wuetal.,201147 _China _Cases_of

nonsyndromic orofacialclefts

Case-control FAsupplementationduring theﬁrsttrimesterof pregnancy

Maternalfolicacidsupplementation duringtheﬁrsttrimesterofpregnancy showsaprotectiveeffectonthe aetiologyofnonsyndromicorofacial clefts. Carmichaeletal., 2010a43 USA Casesof craniosynostosis 815cases; 6789controls

Case-control FAsupplementationduring pregnancy

FAsupplementationduringpregnancy isnotassociatedwithcraniosynostosis occurrence. Carmichaeletal., 2010b122 USA Casesof anencephalyand spinabiﬁda 330cases; 625controls

FAsupplementationduringpregnancy isonlymodestlyassociatedwitha reducedriskofNTDs.

Glaseretal., 2010106

UK Pregnantwomen andoffspring

5344 Cohort FAsupplementationduring pregnancy+dietaryfolate intake

FAsupplementationduringpregnancy doesnotcontributetoaetiological pathwaysthataresharedbetween schizophreniaandnon-clinical psychoticsymptomsinadolescents. Gongetal.,201038 _China _Cases_of_neural

tubedefects(<2 years)

Case-control FAsupplementationinthe preconception

FAsupplementationinthe

preconceptionpreventsagainstNTDs. Maetal.,201060 _USA _Cases_of_microtia ₄₂₀_cases;

6789controls

Case-control FAsupplementationone monthbeforeconception and/orduringpregnancy (withtiming)

PericonceptionalFAsupplementation reducestheriskformicrotia,only amongnon-obesewomen. Milneetal.,201071 _Australia _Cases_of_acute

lymphoblastic leukaemia

Case-control FAsupplementationinthe preconceptionandduring pregnancy(withtiming andquantiﬁcation)

Maternalfolatesupplementation duringthepreconception,theﬁrst trimesterortheﬁnal6monthsof pregnancyisnotassociatedwiththe riskofchildhoodacutelymphoblastic leukaemia.

Nilsenetal.,201094 _Norway _Pregnant_women

andoffspring

2934 Cohort FAsupplementationduring pregnancy(with quantiﬁcation)+dietary folateintake

Noevidencethatlowdietaryfolate intakeorlowplasmafolate concentrationduringthesecond trimesterisassociatedwithinfant birthsize.

Ortega-Garcia etal.,201069

Spain Incidentcasesof nervoussystem tumours,aged<15 years

67cases;155 controls

Case-control FAsupplementationduring pregnancy(>=0.4mg/day)

FAsupplementationinpregnancyis protectiveagainstnervoussystem tumour(NST),especiallycentralNST. Rozaetal.,201017 _The

Nether-lands

Pregnantwomen andoffspring

4214 Cohort FAsupplementationinthe preconceptionandduring theﬁrsttrimesterof pregnancy(0.4mg/dayif FAsupplementsassingle; nodosagespeciﬁedinFAin multivitamins)

InadequateFAsupplementationduring earlypregnancymaybeassociated withahigherriskofbehavioural problemsintheoffspring.

Schlotzetal., 201089 UK Pregnantwomen andoffspring 100 Cohort FA supplementation+dietary folateintakeduring pregnancy(withtiming andquantiﬁcation)

Lowmaternalfolatestatus(redblood cellfolateanddietaryintake)is associatedwithhighhyperactivity scoresintheoffspring.

Stalbergetal., 201068

Sweden Casesofbrain tumours,aged<15 years

Case-control FAsupplementationinthe preconceptionandduring pregnancy

PrenatalFAsupplementationshowsa tendencytoprotectionagainstbrain tumourintheoffspring.

vanBeynumetal., 201054 The Nether-lands Casesofcongenital heartdefects 611cases; 5744controls

Case-control FAsupplementationduring periconception

(0.4mg/day)

PericonceptionalFAsupplementation isrelatedto20%reductioninthe prevalenceofanycongenitalheart defect. Timmermansetal., 200993 The Nether-lands Pregnantwomen andoffspring

6353 Cohort FAsupplementationinthe periconception

(0.4-0.5mg)

PericonceptionalFAsupplementation isassociatedwithincreasedfoetal growthresultinginhigherplacental andbirthweight,anddecreasedrisks oflowbirthweightandSGA. Fryeretal.,2009100 _UK _Pregnant_women

(atdelivery)

24 Cross-sectional

FAsupplementationduring pregnancy,considering dailydosageortotal amountduringpregnancy

Folateindicesduringpregnancyare inverselyassociatedwithfoetalLINE-1 methylation.

Habergetal., 200974

Norway Pregnantwomen andoffspring

32,077 Cohort FAsupplementationduring pregnancy(withtiming)

FAsupplementation(intheﬁrst trimesterofpregnancyandlater)is associatedwithincreasedriskof wheezeandlowerrespiratorytract infectionsintheoffspringupto18 monthsofage.

(10)

Table2(Continued)

Julvezetal.,200982 _Spain _Pregnant_women

andoffspring

420 Cohort FAsupplementationduring theﬁrsttrimesterof pregnancy

FAsupplementationduringpregnancy ispositivelyassociatedwith neurodevelopment(verbal,motor, verbal-executivefunction,social competenceandattention)ofthe offspringat4yearsofage. Lewisetal.,

2009103

UK Pregnantwomen andoffspring

5783 Cohort FAsupplementationduring pregnancy+dietaryfolate intake

FAsupplementationat18thor32nd weeksofpregnancyisnotassociated withchangesinbodycompositionof theoffspringinchildhood(9yearsof age). Ormondetal., 200957 UK Casesof hypospadias 471cases; 490controls

Folatesupplementationduringtheﬁrst trimesterofpregnancyassociatedwith areducedriskofhypospadias. Stewartetal.,

2009105

Nepal Pregnantwomen andoffspring

4926women and3524 live-born infants

Humantrial Womengivendaily supplementsfromthetime ofenrolmentuntil3mo postpartum:1)vitaminA aloneasthecontrol;2)FA (400mcg);3)FAwithiron (60mcg);4)FAwithiron andzinc(30mg);or5)a multiplemicronutrient supplementcontainingFA

FAsupplementationduringpregnancy presentsabeneﬁcialeffectonboth offspringkidneyfunctionandriskof metabolicsyndrome.

Whitrowetal., 200911

Australia Pregnantwomen andoffspring

557 Cohort FAsupplementationduring preconceptionandduring pregnancy(withtiming andquantiﬁcation)

FAsupplementationlateinpregnancy increasestheriskofasthmainchildat 3.5yearsandpersistentasthmaat3.5 yearsand5.5years.

Gambhiretal., 200859

Germany Familiesofcases bladder exstrophy-epispadias complex 214 Cross-sectional FAsupplementationinthe preconceptionandduring theﬁrsttrimesterof pregnancy

Paradoxically,mothersofchildren withcloacalexstrophyCE,themost severedefectofexstrophy-epispadias complex,aremorecomplianttoFA supplementationstartingbeforethe 10thweekofpregnancythanmothers ofthecombinedgroupofisolated epispadias/classicexstrophyofthe bladder(E/CBE),theleastsevere manifestations.Thismaybeduetothe factthatthepreventionoftherelated NTDsandomphalocelearemost responsivetoFAsupplementation startedinthepreconception. Littleetal.,200849 _Canada _Cases_of_orofacial

clefts

Case-control FAsupplementation3 monthsbeforeand3 monthsafter

conception+dietaryfolate intake

Totalfolateintakeordietaryfolate intakeduringpregnancyarenot associatedwithcleft-lippalateorcleft palate. Michelsetal., 200864 The Nether-lands Casesofposition plagiocephaly 18cases; 7625controls Nested case-control FAsupplementationduring periconception(with quantiﬁcation,basedon thetypeofFA supplements) PericonceptionalFAsupplementation inhighdosagemaycausemore positionplagiocephaly.

vanEijsdenetal., 200896 The Nether-lands Pregnantwomen andoffspring

3153 Cohort FAsupplementationduring pregnancy(withtiming)

Folatedepletionapparently contributestotheexcessriskoffoetal growthrestrictionthatisassociated withshortinterpregnancyintervals. Wehbyand

Murray,20084

6774 Cohort FAsupplementationinthe threemonthspreviousto beawareofpregnancyand threemonthsafter

FAsupplementationinthespeciﬁed timeframeinpregnancymayimprove developmentofoffspringat3yearsof age.

Billeetal.,200745 _Denmark _Cases_of_acute

lymphoblastic leukaemia

Case-control FAsupplementationduring theﬁrsttrimesterof pregnancy

Supplementationwith0.4mg/dayin theﬁrst12weeksofpregnancyhada protectiveeffectfororalclefts. Dockertyetal.,

200772

Canada Casesofacute lymphoblastic leukaemia

97cases;303 controls

Case-control FAsupplementationduring pregnancy(withor withoutother micronutrients)

Noassociationwasfoundbetween reportedFAintakeduringpregnancy andchildhoodacutelymphoblastic leukaemia.

Wilcoxetal., 200746

Norway Casesofcleftlip 573cases; 763controls

Case-control FAsupplementationearly inpregnancy(with quantiﬁcation:noFA;0.1 to0.39mg/day; ≥0.4mg/day)

FAsupplementationduringearly pregnancywasassociatedwith33% decreasedriskofisolatedcleftlip(with orwithoutcleftpalate)butnot associatedwithreducedriskofcleft palatealone.

(11)

Table2(Continued)

Boweretal.,200665 _Australia _Cases_of_NTDs ₄₇₅_cases;

578controls

Case-control FAsupplementationinthe monthpreviousconception andduringpregnancy (withquantiﬁcation: ≤0.2mg/dayor >0.2mg/day)

Folatesupplementationwasnot preventiveagainstbirthdefectsother thanNTDs(e.g.orofacialclefts, congenitalheartdefects,urinarytract defects)

Tobiasetal.,200598 _UK _Pregnant_women

andoffspring

4588 Cohort FAintakeduringpregnancy Maternalfolateintakeduring pregnancywassigniﬁcantlyassociated withspinalbonemineralcontent. FA–folicacid;5-MTHF–5-methyltetrahydrofolate;DHA–docosahexaenoicacid;NTDs–neuraltubedefects.

NTDs

FocusingourattentiononNTDs, ofthe8papers, 6suggesta

protectiveeffectofFAsupplementationintheoccurrenceofsuch

defects.Importantlythesepapershighlighttheimportanceofthe

pre/periconceptional period.36–41 _The _remaining ₂ _articles

con-cludethat,inabackgroundofFAfortiﬁcation,FAsupplementation

oritsabsencearenotassociatedwiththeriskofNTDs.42,43_Notably,

alltheincludedstudiesfocusingonNTDswereofthecase–control

type,withalargesamplesize,exceptone41_that_included_about

onehundredparticipantsamongcasesandcontrols.So,itseems

reasonablethatpre/periconceptionalFAsupplementationindeed

playsaroleinthepreventionofNTDs.

Oralclefts

Looking towardsFA supplementationand the occurrence of

orofacialclefts, 4papershighlightedapossibleprotectiveeffect

inearlypregnancy.44–47_Other_two_publications_discussed_the

pos-sibleprotectiveeffectsofFAsupplementationonthiscongenital

defectbuttheydidnotshowanyassociationregardlesstheuse

of0.4mgor4mg/dayofFAbymothers.48,49_Just_one_publication

reportedanincreasedriskoforofacialcleftswhentherewasa

con-sistentsupplementuseduringtheaetiologicallyrelevantperiod

(weeks0–12postconception)50_and_these_results_led_their_authors

todecisively emphasize theidea that it would be advisableto

restrictFAsupplementationtotheperiodrecommendedforNTDs

untilmore information isavailable.Indeed, when theexposure

toFAisrestrictedto“early pregnancy”period (ﬁrstweeks/ﬁrst

trimesterpostconception),amongﬁvestudiesonlyone

demon-stratesanadverseeffect,whilefourofthemconsistentlyshoweda

beneﬁcialprotectiveeffectagainstorofacialclefts.Importantlyno

extraeffectswerereportedonhigherdosesofsupplementedFA

(4mg/daycomparingto0.4mg/day).

Heartdefects

Offspring’sheartdefectswerecoveredbyﬁveoftheincluded

papers,fourofthemreportingareductionintheriskofheartdefects

amongoffspringofmotherssupplementedwithFA,51–54

regard-lessofthetimeanddoseofexposure.Infact,theauthorsobserved

beneﬁcialeffectsevenforhighdoseofFA51,52₍₅_and₆_mg/day)_and

othersshowthesameeffectwithonly0.4mgFA/day.54_In_addition,

oneofthestudiesrevealedthatFAsupplementationcanberelated

toareducedriskofcongenitalheartdefects,thepreconceptional

periodasacriticalwindowforthisprotectiveeffect.55

Othergeneralcongenitaldefects

Five studies reported the effects of maternal FA

supple-mentationonbladderdefects(epispadias, hypospadias,bladder

exstrophy),mostofthem concludingonthepossibleprotective

effectofFAsupplementationinthepericonceptional period56,57

orsuggestingthatthissupplementationcouldpotentiallyalleviate

phenotypicseverityofthesedefects.58,59_Other_ﬁndings_regarding

congenitaldefectsalsorevealedaprotectiveeffectofFA

supple-mentationontheriskofmicrotia60_or_{gastroschisis,}61_while_the

lackofFAsupplementationseemstobeassociatedwithobserved

meiosisIInondisjunctionerrorsinoldermothers.62_Another_study

support the protective effect of FA against several congenital

defects.63 _In _contrast _to_the _beneﬁcial _outcomes, _it _seems_that

higherdosesofFA,althoughwithlowstatisticcorrelation,might

haveanadverseeffectbycausingmoreplagiocephalycases.64_An

Australiancase–controlstudyreportednoevidenceoffolate(either

FAsupplementsordietaryfolateintake) asanimportantfactor

in thepreventionof birth defectsotherthan NTDs.65 _With _the

obtainedresults,wecannotfundamentahardstatementontimings

anddosesbut,inthecaseofbladderdefects,aprotectiveeffectofFA

supplementationwasunanimouslyreported,whenstartedbefore

conception.Intheothercases,nostatementscanbedrawnbecause

therearefewcasesofeachreportedoutcome.

Offspring’stumours

Severalpapers(n=7)werefocusedontherelationshipbetween

FA supplementation in pregnancy and the risk of offspring’s

tumours.Regardingbrain/nervoussystemtumours,alltheselected

studiesindicateapossibleprotectiveroleofsupplementationwith

FAduringpregnancyinchildhoodbraintumours.66–69_No_critical

temporal windowsofthemaximumbeneﬁt ofthis

supplemen-tationare reportedbut,in three ofthepapers, preconceptional

periodandtheﬁrsttrimesterofpregnancyseemtobedeterminant.

On theotherhand, ﬁndingsregarding therisk of offspring’s

leukaemiaarelessexpressive,asFAsupplementationisreported

ashavinganinverseborderlineassociationifsupplementationis

startedthreemonthsbeforeconception,70_or_no_association_with

theriskofchildhoodacutelymphoblasticleukaemia.71,72

Respiratoryandallergicoutcomes

The interest on this area of knowledge is covered by the

publications(n=10)regardingchildhoodasthma,lowrespiratory

infections oratopy/allergy.Onestudysuggeststhat offspringof

mothersthatweresupplementedwithFAduringtheﬁrsttrimester

ofpregnancy,had higherrelative oddsof beingdiagnosed with

bronchiolitiscompared withthegroupofnosupplementation73

andotherstudyreportsthatFAsupplementationinpregnancyis

associatedwithslightlyincreasedriskofwheezeandlower

respi-ratory tractinfections upto 18months ofage, consistentwith

epigeneticmechanisms.74_Other_study_suggests_a_higher_general

riskofasthmaintheoffspringofsupplementedmothersand,

(12)

Table3

Includedhumanstudiesdescribinghealthoutcomesofthemotherandthepregnancy(n=17).

Reference Country Studied population Sample size Type of study Intervention (dose and timing ofFAsupplementation)

Results/main conclusions Gaskins et al., 2014118 _USA _Pregnant_women _11,072

(15,950 pregnancies)

Cohort FAsupplementationinthe preconception(with quantiﬁcation)

Therewasobservedaninverseassociation betweenprepregnancysupplementalfolateand theriskofspontaneousabortion,regardlessthe gestationalperiodofloss.Asimilarinversetrend wasobservedwiththeriskofstillbirth,butthe associationwasnotstatisticallysigniﬁcant. Kimetal.,2014107 _Iran _Mothers_in_the

puerperium

215 Cross-sectional FAsupplementationduring pregnancy

RatesofpreeclampsiaandSGAlowerinthe groupsupplementedwithFA,indicatingthat supplementationwithFAmaycontributetothe preventionofthoseoutcomes.

Sengpieletal.,2014114 _Norway _Pregnant_women _>106,000 _Cohort _FA_{supplementation}_during

periconception(withtiming andquantiﬁcation)

NoprotectiveeffectofFA(dietorsupplements) onspontaneouspretermbirthisobserved.FA supplementationstartingmorethan8weeks preconceptionisassociatedwithincreasedrisk ofspontaneouspretermbirth.

LiZetal.,2013110 _China _Pregnant_women _193,554 _Cohort _FA_{supplementation}_early_in

pregnancy(0.4mg/day)

0.4mg/dayofFAduringearlypregnancydoes notreducederiskofgestationalhypertensionor preeclampsia.

Papadopoulouetal., 2013111

Greece Pregnantwomen andoffspring

1279 Cohort FAsupplementationearlyin pregnancy(quantiﬁed)

Dailyhigh(5mg)supplementationwithFAwas protectiveagainstpretermbirthanddelivering lowbirthweightandsmallforgestationalage neonates.

Vila-Novaetal.,2013116 _Brazil _Pregnant_women ₂₆₈ _Human_trial _FA_{supplementation:}_0.4_mg/day

or4mg/daybeforeconception untiltheendofﬁrsttrimester

Similarmiscarriageratebetweenthelowdose (0.4mg/day)andthehighdose(4mg/day) groups.HighdoseFAdoesnotaffectmiscarriage riskinthestudiedpopulation.

Shawetal.,2011115 _USA _Mothers ₅₉₅₂ _{Cross-sectional} _FA_{supplementation}₃_months

previousconceptionandduring pregnancy(withquantiﬁcation)

FAsupplementationbeganatanytimeduring pregnancyrevealsa∼20%decreasedriskof pretermdeliverycomparedwithFA supplementationbeganbeforepregnancy. However,lowerFAdietaryintakesshowaslight increasedriskofpretermdelivery.TheroleofFA inthepretermdeliveryisnotclear.

Zhangetal.,2011117 _China _Cases_of_missed

abortion

Case-control FAsupplementationearlyin pregnancy

FAsupplementationisassociatedwith preventionofmissedabortion. Timmermansetal.,

2011123

The Nether-lands

Pregnantwomen 5993 Cohort FAsupplementationinthe preconceptionandduringthe ﬁrsttrimesterofpregnancy (0.4–0.5mg/day)

PericonceptionalFAsupplementationassociated withloweruteroplacentalvascularresistance andhigherbloodpressuresduringpregnancy. Theeffectsdonotseemtobeassociatedwith theriskofhypertensivepregnancydisorders. Czeizeletal.,2010112 _Hungary _Pregnant_women ₆₇₇₃ _Cohort _FA_{supplementation}_during

pregnancy(withtimingand quantiﬁcation)

HighFAsupplementationduringpregnancy (particularlyinthethirdtrimester)isrelated withlongergestationalageanddecreasedrate ofpretermbirths.

Katreetal.,2010120 _India _Pregnant_women ₁₈₄ _Cohort _FA_{supplementation}_during

IncreasingFAsupplementationisnotassociated withreductionofhomocysteineconcentration. Bukowskietal.,2009113 _USA _Pregnant_women _34,480 _Cohort _FA_{supplementation}_in_the

preconception(withtiming)

Preconceptionalfolatesupplementationis associatedwithareductionintheincidenceof earlyspontaneouspretermbirth.Durationof preconceptionalfolatesupplementationis inverselyrelatedwiththeriskofearly spontaneouspretermbirth. Zhangetal.,2009119 _USA _Pregnant_women _164,667 _Cohort

(prospective)

FAsupplementationduring pregnancy(withtimingand quantiﬁcation)

FAsupplementationduringthepericonceptional periodisassociatedwithreducedriskof anaemiainthe1sttrimester,butnotlaterin pregnancy.

Nilsenetal.,2008109 _Norway _Pregnant_women _280,127 _Cohort _FA_{supplementation}_in_the

preconceptionandduring pregnancy

FA(ormultivitamin)supplementationduring pregnancyisassociatedwithalowerriskof developingplacentalabruption(when comparedtonouseofsuchsupplements)witha strongerassociationforpretermabruption. Wenetal.,2008108 _Canada _Pregnant_women ₂₉₅₁ _Cohort _FA_{supplementation}_during

FAsupplementationinearlysecondtrimesterof pregnancyisassociatedwithincreasedserum folate,decreasedplasmahomocysteineand lowerrateofpreeclampsiawhencomparedto nouseofFA(althoughthestatisticalsigniﬁcance oftheresultsispoor).

Klingleretal.,2006124 _Germany _Pregnant_women ₅₅ _Human_trial _FA_{supplementation}_from_week

20ofpregnancyonwards: placeboor0.4mg/dayof 5-MTHF

Supplementationwith5-MTHFinlate pregnancydoesnotchangetheparameters reﬂectingplacentalproliferationorapoptosis. Charlesetal.,2005125 _UK _Pregnant_women

andoffspring

2928 Humantrial FAsupplementationbeginning underweek30ofpregnancy: placebo;0.2mg/day;5mg/day

FAsupplementationbeginningunderweek30of pregnancyhasnoteffectoverbirthweight, gestationalageatdeliveryorstillbirth/neonatal death.

(13)

theremightbeasigniﬁcantlyincreasedriskofasthma.11_Higher

risksweredocumentedwithhigherdosesofFAsupplementation

(above72mgduringthewholepregnancy,correspondingto180

daysofsupplementationwith0.4mg/day).75_Only_one_study_did

notshowanyrelationwithFAsupplementationandhigherriskof

asthmaattheageofsixyears.76

Focusingonallergicoutcomes,itissuggestedthathigh

mater-nalplasmafolateandserumvitaminB-12concentrationscanbe

associated with the development of atopic dermatitis, but not

withwheezingand shortnessof breathin childhood.77 _A

dose-dependent effect was reported in the third trimester: infants

exposedto≥0.5mgFA/dayweremorelikelytodevelopeczema

thanthosetaking<0.2mgFA/day.78_In_contrast,_one_of_the

publi-cationsfoundnoassociationbetweenFAsupplementationduring

pregnancyandatopicdiseasesintheoffspring.79_Lastly,_it_seems

thatspeciﬁcriskofchildhoodpeanutallergyisnotmodiﬁedby

maternalFAsupplementation.80

Anotherstudyalsodidnotﬁndassociationbetweenprenatal

FAsupplementationandadverserespiratoryorallergicoutcomes

inchildrenof1–8yearsofage.81

So,of the10articlesthatcover respiratoryand allergic

out-comes of the offspring, seven report an adverse effect of FA

supplementation,threeofthemhighlightingtheadverseeffectof

supplementationinlatepregnancy.Howeveradverseeffectsare

also seen for periconceptional period exposure or for reported

high FA intake. Of the 10 articles, only three report no

asso-ciation between FA supplementation during pregnancy or the

periconceptionalperiodoverthesesameoutcomes.Thus,itseems

plausiblethatFAsupplementation,particularlyinlatepregnancy,

willincrease theriskof respiratory orallergic outcomesinthe

offspring.

Behaviourproblems,neurodevelopmentandpsychomotor

disorders

Thesearealsosomeofthemostaddressedoutcomesinwhat

concernsFAsupplementation(n=10).Theincludedevidence

sug-geststhatFAsupplementsinpregnancycanbepositivelyassociated

withchildren’sneurodevelopmentattheageoffouryears,82_being

corroborated by other studies showing a possible effect of FA

supplementationonenhancedvocabularydevelopment,

commu-nication skills and verbal comprehension at 18 months of age

(butnotdoses≥5mg/day)83_as_well_as_a _reduced_risk_of_severe

languagedelayinchildrenattheageofthree.84_Nonetheless,

evi-dencealsodemonstratesnoassociationwithchildmemoryatseven

years old85 _and_no_signiﬁcant_effect_of _{supplementation}_on_the

cognitivefunctionofchildren.86_{Contradictory}_ﬁndings_about

psy-chomotordevelopmentwereestablishedassomeevidencepoints

towardsahigherriskofdelaywithintheuseofFAsupplements,

atdosesabove5mg/day,87_while_other_authors_suggest_that

pre-natalFAsupplementationmayimprovedevelopmentattheage

ofthree, reportingalso asigniﬁcantpoorer performance inthe

personal-socialdomain.4 _Facing_these_results,_no_conclusion_can

beextrapolatedas dataseem tobeindiscordance.In terms of

behaviourproblems,higherriskofhyperactivityandinattention

hasbeenattributedtoaninadequateuseofFAduringtheﬁrst

trimesterofpregnancy,17_also_estimating_that_omission_errors_seem

tobelowerinchildrenwhosemotherstookdietary

supplemen-tationwithFAduringpregnancy.88_{Additionally,}_although_in_a_small

associationandwithpossibleconfounding,lowerfolatestatusin

earlypregnancymightimpairfoetalbraindevelopmentandinduce

hyperactivityand inattention problemsin childhood.89 _Though,

stillinwhatconcernsbehaviourproblems,itseemsthatthereis

noassociationbetweenlowerfolatestatusduringpregnancyand

theappearanceofsuchkindofoutcomes.

Autistic disorders are also one of the possible outcomes

for the offspring and it seems that a lower intake (less than

0.6mg/day) increases the risk of having children with autism

spectrumdisorders,andthisriskwasstrongestformothersand

childrenwithinefﬁcientfolatemetabolism(MTHFR677C>T

vari-antgenotypes).90_Also,_a_more_recent_cohort_study_suggests_that

prenatalFAsupplementsaroundthetimeofconception(doseof

0.4mg/day)seemtobepositivelyassociatedwithlowerriskofthis

typeofdisorders.91

Foetalgrowthandotheroutcomes

A high FA dose (4mg/day) in the periconception seems

not to compromise foetalgrowth, when compared witha

rec-ommended dose of 0.4mg/day.48 _Other _study _reported _that

the periconceptional use of FA supplementation greater than

1mg/daycanbeassociatedwithdecreasedbirthweight.92

How-ever,periconceptionalFAsupplementationinrecommendeddoses

(0.4–0.5mg/day)canbeassociatedwithincreasedfoetalgrowth,

andhigherplacentalweight,resultinginhigherbirthweightand

decreased risk of low birth weightor smallfor gestational age

(SGA).93_On_the_other_hand,_low_dietary_folate_intake_or_low_plasma

folateconcentrations duringthesecond trimester ofpregnancy

seem not to affect birth size.94 _In _addition, _a _trial _comparing

maternalmulti-micronutrientwithiron-FAorFAonlydidnot

sup-portagreateradvantageoftheﬁrstonchildgrowthduringthe

ﬁrst30months.95_{Notwithstanding,}_foetal_growth_restriction

com-monlyobserved in situations of shortinterpregnancy intervals,

wasmorefrequentwhenfolatedepletionoccurred.96_Once_again,

inconsistencyregardingtheeffectsofFAsupplementationinthe

periconceptionalperiodisobserved.Regardingtheimpactofalow

folatestatus,itseemsrelevantforfoetalgrowthimpairmentonly

whenitresultsfromshortinterpregnancyintervals.

OtherobservedeffectsofFAsupplementationduringpregnancy

wereboneturnover/developmentwiththesuggestionthatdaily

FAsupplementationof1mgduringpregnancycouldhavea

posi-tiveimpactinboneturnoverbothinmothersandnewborns,thus

concludingthatbothpregnantmothersandtheirfoetusescould

beneﬁt,inthisparticularparameter,fromFAsupplementation

dur-ingthewholepregnancy.97

Another older publication refers to an association between

maternalfolateintake(cohortstudywithmeanFA

supplemen-tation of 0.257mg/day) and bone development in childhood,

observedasvolumetricbonemineraldensityofthespine

regard-lessofheightandweight.98_Both_studies_lead_to_the_expectation

thatFAsupplementationmaybebeneﬁcialintermsofbonehealth

laterinlife.

Inaddition,FAintakeduringfoetaldevelopmentwascausally

relatedtosubtleandpersistentDNAmethylation(IGF2/H19)inthe

offspringofFAsupplementedmothersandsuchresultsmaylead

toafurtherunderstandingofthefoetaloriginofadultdiseases,99

alsocorroboratedbyotherauthors.100,101_Other_observed_isolated

outcomes were, for instance, semen characteristics of the

off-spring,withnodifferencesfoundbetweenthosewhosemothers

didnottakeFAorwithunknownsupplementation,inrelationto

thesupplementedones.102_No_evidence_was_found_to_support_the

hypothesisthat maternaldietaryfolateintakeandintra-uterine

exposure to folate is related to body composition of the

off-springat theageofnine years,103 _as _well_as_no_association_on

offspring’sbloodpressurewasdocumented.104 _An_experimental

studyinNepalrevealedthatFAsupplementationduringpregnancy

appearedtohavethegreatestbeneﬁcialeffectonbothoffspring’s

kidneyfunctionandlowerriskofmetabolicsyndromeamongthe

offspring.105_Finally,_there_was_no_support_on_the_idea_that