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ResearchOBSTETRICS

First-trimestermetabolomicdetection oflate-

onsetpreeclampsia

Ray O. Bahado-Singh,

MD, MBA; RanjitAkolekar, MD;

Rupasri Mandal, PhD;

Edison Dong, BSc;

Jianguo Xia, PhD;

Michael Kruger, MS;

David S. Wishart,

PhD; Kypros

Nicolaides, MD

www.

AJOG .o

r

g

OBJECTIVE: Wesoughttoidentifyfirst-

trimestermaternalserumbio-

markersforthepredictionoflate-

onsetpreeclampsia(PE)using

metabolomicanalysis.

STUDYDESIGN: Inacase-

controlstudy,nuclearmagneticresonance

basedmetabolomicanalysiswasperformedonfi

rst-trimestermaternal serumbetween110-

136weeksofgestation.Therewere30cases

oflate-

onsetPE,ie,requiringdelivery37weeks,and59u

naffected

controls.Theconcentrationsof40metabolitesw

erecomparedbe-tweenthe2groups.Wealsocompared30early-

onsetcasestothe

RESULTS:

Atotalof14metabolitesweresignificantlyelevat

edand3sig-

nificantlyreducedinfirst-trimesterserumoflate-onsetPEpatients.Acom-plexmodelconsistingofmultiplemetabolitesandmaternaldemographicchar-acteristicshada76.6%sensitivityat100%specificityforPEdetection.Asimplifiedmodelusingfewerpredictorsyielded60%sensitivityat96.6%spec-ificity.Strongseparationoflate-vsearly-onsetPEgroupswasachieved.

CONCLUSION:

Significantdifferencesinthefirst-

trimestermetabolites

werenotedinwomenwhowentontodevelopedlate-onsetPEandbe- tweenearly-andlate-onsetPE.

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late-onsetgroup.

Keywords:metabolomics,preeclampsiaprediction

Citethisarticleas:Bahado-SinghRO,AkolekarR,MandalR,etal.First-trimestermetabolomicdetectionoflate-onsetpreeclampsia.AmJObstetGynecol2013;208:58.e1-7.

proves it is expected that thisnumber

is associated with significant fetalmor-

Metabolomics,arelat

ivelyrecent

addition totheomicsfamily

,could grow by a factor of 10.

2

Because

biditie

s.Thepathophysiologyisthought

involvesthehigh-

throughputcharacter- ization andinterpretation of the small- molecule

metabolites (1500 d) pro- duced by

cells, tissues, and organisms. To

date, 8000 human metabolites from

80 chemical classes have been

identi- fied or catalogued.1 As

technology im-

FromtheDepartmentofObstetricsand Gynecology,WayneStateUniversity,Detroit,MI(DrBahado-SinghandMrKruger);Harris

BirthrightResearchCentreforFetalMedicine,KingsCollegeHospital,London,United

Kingdom(DrsAkolekarandNicolaides);and

theDepartmentsofBiologicalSciences(Drs

MandalandWishartandMrDong)and

ComputingSciences(DrsXiaandWishart),

UniversityofAlberta,Edmonton,Alberta, Canada.

ReceivedJune18,2012;revisedNov.4,2012; acceptedNov.8,2012.Thisstudywaspartlysupportedbyagrant

fromtheFetalMedicineFoundation,Charity

Number1037116.

Theauthorsreportnoconflictofin

terest.

Reprintsnotavailablefromtheauthors.

0002-9378/$36.00

2013PublishedbyMosby,Inc.

http://dx.doi.org/10.1016/j.ajog.2012.11.0

03

SeeJournalClub,page8

7

of the wide chemical diversity of

metab-

olites,theirtightcouplingwithenviron-

mental interactions (food, drugs, gut

microbiota), and their huge phenoty-

pic-dependent concentrationvariations

6

(10

),metabolomicsoffersapowerful,

quantitative route to describe the

actual phenotype of cells, tissues, or

organisms in both normal and

diseased states. Re- cently,

significant advances have oc- curred

both in metabolite identification

techniques1,2 and computational

tech- niques3foranalyzingthelargevolumeof data

generated by metabolomic studies.

Thereiscurrentlytremendousinteresti

n the use of metabolomics for the

charac-

terizationandearlydiagnosisofcomple

x diseases.4

Preeclampsia(PE)isacommonobstet-

ricdisordercharacterizedbyhypertens

ion and proteinuria during

pregnancy. It is a cause of

significant morbidities, affectingthehealthofboththemother5 andfetus.

However, its causes and

pathophysiol-

ogylargelyremainamystery.Itnowap-

pears that PE is at least 2 fairly

distinct disorders,anearly-

onsetandalate-onset form.6,7

Theearly-onsetvarietytypically

occurs 34-35 weeks of pregnancy,

and

to be failure of trophoblast invasion

of thematernalspiralarteriole8

resultingin maintenance of highmaternal vascular resistance. This is

consistent with the high frequency

of placental underperfu- sion

reported9 in this disorder. Thelate-

onsetformisconsideredtobe

moreofamaternalconstitutionaldisor-

der10

duetounderlyingmaternalmicro-

vascular disorders such as

hypertension

orageneticpredispositioninwhichpoo

r

trophoblastinvasionisthoughttoplaya

lesssignificantrole.Late-

onsetPEissig-

nificantlymorecommonandwhileitof-

ten has a mild course can be

associated with significant clinical

morbidities.11 It is therefore

important to investigate its

pathogenesis and if possible to

develop biomarker predictors of this

disorder.

Studieshavenowconfirmedtheclinical

feasibility of first-trimester

screening for early-, late-, and

intermediate-onset vari- eties of PE

using demographic, clinical,

biomarker,anduterinearteryDoppleri

n- formation.12,13

Recently,theNationalCol-

laboratingCenterforWomensandChi

l- drens Health in the United

Kingdom issued clinical

guidelines14 for routine early

p

re

n

a

t

a

l

s

c

r

e

e

n

i

n

g

f

o

r

P

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b

a

s

e

d

o

n

58.e1 AmericanJournalofObstetrics & Gynecology

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www.AJOG.org

ObstetricsResearchTABLE1Demographicandothercharacteristics:late-onsetpreeclampsiavscontrolgroup

disorderofpregnancyandwhohadblood

collectedwithin3daysofassessmentofthe

late-onset PE case. There was no evident

source of bias in the selection of cases orLate-onset controls. Thedefinition of PE used was

Parameter preeclampsiaControl Pvalue

thatproposedbytheInternationalSociety

No.ofcases 30 59 for the Study of

Hypertension in Preg-..............................................................................................................................................................................................................................................

nancy,16 namely systolic pressure 140

Maternalage,y,mean(SD) 31.2(6.4)30.8(5.6).81

..............................................................................................................................................................................................................................................

mmHgordiastolicpressure 90mmHg

Racialorigin,n(%) .02

..................................................................................................................................................................................................................................... on

2occasions4hoursapart 20weeksWhite 14(46.7)44(74.6) of gestation,

in women who were previ-.....................................................................................................................................................................................................................................

Black 14(46.7)14(23.7) ously

normotensive. Proteinuria was de-.....................................................................................................................................................................................................................................

finedasatotalof300mgina24-hoururine

Asian 0(0) 1(1.7)

.....................................................................................................................................................................................................................................

collectionor2readingsofatleast2 pro-

Mixed 2(6.7) 0(0)

..............................................................................................................................................................................................................................................

teinuria on a midstream or catheterizedNullipara,n(%) 12(40)31(52.5).37

.............................................................................................................................................................................................................................................. urinespecimen in the absence of a 24-

Weight,kg,mean(SD) 74.9(15.7)67.7(12.2).03 hour

urine collection must also have..............................................................................................................................................................................................................................................

Crown-rumplength,mm,mean(SD)62.0(9.1)62.7(7.6).69....................................................................................................................................................................

..........................................................................

Uterinepulsatilityindex,MoM,mean(SD)1.07(0.35)0.98(0.31).22....................................................................................................................................................................

..........................................................................

MoM, multiplesofmedian.

Bahado-Singh.Late-onsetpreeclampsia,metabolomics.AmJObstetGynecol2013.

beenpresentinadditiontothehyperten-

sion. Proteinuria must also have been

present in addition to the hypertension

for the diagnosis of PE. No HELLP

syn- drome or gestational hypertension

casesmaternal demographic, historical, and ics Committee.

Briefly, women were re-

were included.

06

Nuclear magnetic resonance (NMR)

clinicalcharacteristics.Itispossiblethat,

in the future, combining clinical with

cruited at 1113

wee

ks gestation.spectrometry

was used for metabolite

biomarker predictors could further en-

hance screening accuracy. Our primary

objective was to evaluate the use of

metabolomics to identify first-trimester

biomarkers of late-onset PE. Second-

arily, we evaluated the diagnostic accu-

racy of these markers for late-onset PEprediction.Finally,weevaluatedtheca-

pability of metabolomics for distin-

guishinglate-fromearly-onsetPE.

MATERIALS AND METHODS

StudypopulationThis study is part of an ongoing

prospec- tive study being conducted by

the Fetal Medicine Foundation,

London, United Kingdom, for the first-

trimester predic-

tionofimportantfetalandobstetricdisor-

ders. Institutional review board project

#02-03-033 approval was obtained on

March14,2003.Thedetailsofpatienteval-

uation and study methods have been

ex- tensively described in a prior report

of metabolomic prediction of early-

onset PE.15 A routine population of

Britishwomenwasprospectivelyscreenedfrom

March 2003 through September 2009

andtheyallgavewrittenconsenttopartic-

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Research Obstetrics

www.AJOG.org

are significant metabolite differences be-

tweenthenormalandcontrolgroups.

PLS-DAisusedtoenhancethesepara-TABLE2Serummetaboliteconcentrationsbynuclearmagneticresonancetionbetweenthegroupsbysummarizing Late-onsetPE,mean

Controls,mean(SD)

the data into a few latent variables that

Metabolite (SD)

(concentration

(concentrationin Fold

maximize covariance between the re-

inmol/L) mol/L)

P

value

change

sponse and the predictors.18 To mini- mize the possibility that the observed..............................................................................................................................................................................

Glycerol 800.7(541.7)312(296.8) .0012.4

separation on PLS-DA is due to chance,

permutation testing was performed...............................................................................................................................................................................................................................................

1-methylhistidine70.3(40.0) 38.9(20.3) .0011.7

........................................................................................

........................................................................................

..............................................................

Thisinvolvedrepeated(2000

times)data sampling, with

different random label-

ing. A significant P valueindicates that

.......................................................................................................................................................................

.......................................................................

Acetone

22.1(11.4)

14.9(8.5)

.0031.6

the separation observed between groups

is very unlikely to be due to chance. The..............................................................................................................................................................................................................................................

Trimethylamine6.03(2.0) 7.6(3.3) .0050.88........................................................................................

........................................................................................

..............................................................

MetaboAnalyst computer

used to perform (PCA and PLS-DA)

analyses.19 Avariableimportanceinpro-

Pyruvate..............................................................

Hydroxyisovalerate_36.5(3.3)4.7(2.5)

.0081.4

jection (VIP) plot18 is a plot ranking the.....................................................................................

.........................................................................................................................................................

Acetamide

11.9(7.8)

16.1(6.4)

.

0080.7

3

metabolites based on their importance

in discriminating study from control..............................................................................................................................................................................................................................................

Glucose 4312.9(1783.0)3362.4(765.9).0081.2

.......................................................................................

.......................................................................................

................................................................

groups. Metabolites with the

Hydroxybutyrate_228.0(14.4)

21.2(7.5)

.021.3

powerful group discriminators....................................................................

...................................................................

........................................................................................................

Creatinine

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63.2(16.5)

55.1(14.7)

.0211.1

In comparing the concentrations of.....................................................................................

.........................................................................................................................................................

Creatine

metabolitesbetweengroups,outliertest- ....................................................................................................................................................................................................

ingwasperformedusingDixonQtest.20 The

Dixon Q test is used for identifica-

Citrate..........................................................

Hydroxybutyrate_349.9(46.7)

29.7(19.1)

.0381.4

tion of outliers in the dataset and re-.....................................................................................

.........................................................................................................................................................

Leucine

114.5(98.5)

87.1(61.9)

.1121.2

places that value with the one closest to.....................................................................................

.........................................................................................................................................................

Acetate

it.Replacementofoutliershelpstomeet

the assumption of normal distribution..............................................................................................................................................................................................................................................

Betaine80.6(101.5)49.1(52.5)33.3(23.6) 21.6(9.4).121.6.141.5

andequalvariancebetweengroups.Onlya

singlevalue(forvaline)wasadjustedinthis..............................................................................................................................................................................................................................................

Glutamine 253.1(131.1)218.5(66.9) .1821.2

...................

...................

...................

.........................................................................

.........................................................................

...................................

67.7(42.

6)

56.1(37.

2)

.191.2

fashion, however. Kolmogorov-Smirnov.....................................................................................

.........................................................................................................................................................

Ornithine

andShapiro-Wilktestsofnormaldistribu-

tion were performed. Metabolite concen-..............................................................................................................................................................................................................................................

Acetoacetate36.8(17.4)18.9(9.8)42.3(22.5)16.5(9.6).240.87

.271.1trations in late-onset PE vs controlswere

comparedusingthe2-tailedttest.Mann-WhitneyUtestwasusedincomparingme-..............................................................................................................................................................................................................................................

Alanine 366.8(204.8)323.8(151.2).271.1..............................................................................................................................................................................................................................................

Lactate 1213.1(564.7)1100.9(689.3).441.1........................................................................................

........................................................................................

..............................................................

tabolite concentrations between

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thatwerenotnormallydistributed.Other

independentvariablesincludingfetalCRL,

Threonine................................................

........................................................................

........................................................................

..............................................

66.2(62.5).50.94

Propyleneglycol11.1(5.0)

11.8(4.9)

.510.93

uterine artery Doppler, PI, and maternal.....................................................................................

.........................................................................................................................................................

Formate

27.0(13.8)

29.0(17.8)

.61.02

age,parity,weight,ethnicity,smoking,and.....................................................................................

.........................................................................................................................................................

Tyrosine

medical disorders were included in the...............................................................................................................................................................................

..............................

geneticcomputinganalysesalongwiththe

metabolite concentrations for PE

Proline.......................................................................................

.......................................................................................

................................................................

Serine

172.2(57.7)165.7(56.4)

148.4(103.4)158.6(92.2)

.611.04 .6350.93

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prediction.

Genetic programming is a branch of..............................................................................................................................................................................................................................................

Arginine 136.3(55.5)131.2(35.9) .651.04.......................................................................................

.......................................................................................

..........................................................

......evolutionary computing

computingisabranchofgeneticprogram-

ming.Theadvantageofgeneticcomputing

Phenyl

alanine

78.0(4

5.9)........

..................

..................

..................

..................

..................

..................

........................

........................

........................

........................

........................

..80.9(45.

4).780.96

Glycine

238.4(129.3)244.0(115.7).840.97

lies in its ability to handle nonnormally

distributed outcome measures and the

largevolumeofdatageneratedfromom-..............................................................................................................................................................................................................................................

Bahado-Singh.Late-onsetpreeclampsia,metabolomics.AmJObstetGynecol2013. (continued)58.e3

AmericanJournalofObstetrics & Gynecology JANUARY2013