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tainedfromthematernitycomputerizedrecordsorthegeneralmedicalpractitio-nersofthewomen,wereaddedintothedatabaseassoonastheybecameavailable.Venousbloodwasobtainedfromwomenwhogavewritteninformedsenttoprovidesamplesforresearchintoearlypredictionofpregnancycomplica-tions,whichwasapprovedbytheNationalResearchEthicsServiceoftheNationalHealthService.BloodwascollectedinEDTABDVacutainertubes(BectonDickinsonUKLtd,Oxfordshire,UnitedKingdom)andprocessedwithin15min-utesofcollection.Thetubeswerecentri-fugedat2000for10minutestoseparateplasmafrompackedcellsandbuffycoatandsubsequentlyat16,000for10min-utestofurtherseparatecelldebris.Plasmasamplesweredividedinto0.5-mLaliquotsinseparateEppendorftubes(JenconsSci-entiÞcLtd,VWRInternational,WillardWay,Bedfordshire,UK)labeledwithauniquepatientidentiÞerandstoredat80¡Cuntilsubsequentanalysis.Wesearchedourdatabaseandselectedallcaseswithatleast2mLofavailablestoredplasma,collectedfromOctober2010throughJanuary2011.Thepopula-tionconsistedof2230singletonpreg-nancies.Weexcluded74pregnancieswheretherewasnofetalkaryotypeandtheoutcomewasmiscarriage,stillbirth,termination(n28),ornofollow-up46).Wealsoexcluded7pregnan-cieswhereCVSdemonstratedthatthefetalkaryotypewasabnormalbutotherthantrisomy21ortrisomy18;2casesoftriploidy;and1caseeachofTurnersyn-i(8)(p10),46,XY,del(13)(q33),46,XX,dup(10)(q26.1q24.1),andmosaic45,X/47,XXX.Therewere2149casesafterexcludingfortheabovereasons.LaboratoryanalysisPlasmasamples(4tubesof0.5mLperpatient)fromtheselectedcasesweresentovernightondryicefromLondon,UnitedKingdom,tothelaboratoryofAriosaDiagnosticsIncinSanJose,CA.TheinformationprovidedtoAriosaDi-agnosticsIncforeachcasewas:patientuniqueidentiÞer,maternalage,gesta-tionalage,dateofbloodcollection,andfetalsex,butnotfetalkaryotypeorbirthPriortoevaluationforfetaltrisomy,AriosaDiagnosticsIncdetermined29caseshadinadequatesamplevolume,1casehadtubelabelsthatdidnotmatchthesamplemanifest,and70caseshadissuesofsamplemixingduringtheman-ualpoolingprocessofindividualEppen-dorftubesbylaboratorypersonnel.Therefore,theeligiblestudypopulationconsistedof2049cases.Plasmasamplesfromthestudypopula-tionwereanalyzedusingachromosome-selectiveassay(HarmonyPrenatalTest,AriosaDiagnosticsInc).Resultswereprovidedontheriskoftrisomy21andtri-somy18oneachcasetoK.H.N.whothendeterminedthecorrelationbetweentheas-sayresultswiththefetalkaryotypeorbirthoutcome.Theriskscoreswererepresentedasapercentagewithrangescappedat99%andStatisticalanalysesDescriptivedatawerepresentedinme-dianandinterquartilerangeforcontinu-ousvariablesandinnumbersandpercent-agesforcategoricalvariables.ComparisonbetweentheoutcomegroupswasbyFisherexacttestforcategoricalvari-ablesandMann-Whitneytestforcontinuousvariables.InallcasesweusedBonferronicorrectionwithad-valueofThestatisticalsoftwarepackageSPSS20.0(IBMCorp,Armonk,NY)wasusedfordataanalyses.StudypopulationThetotalstudypopulationincluded2049pregnancies.In86(4.3%)cases,thefetalkaryotypewasdeterminedbyCVSoram-niocentesisandwasnormalin75cases,tri-somy21in8,andtrisomy18in3.There-maining1963pregnanciesresultedinthelivebirthofphenotypicallynormalneo-natesassumedtobeeuploidandtherefore,thetotalnumberofprovenorassumedeu-ploidpregnancieswas2038.MaternalcharacteristicsandresultsfromconventionalÞrst-trimesterscreen-ingwithserummarkersandultrasoundofthestudypopulationarepresentedinthe.Intrisomy21,comparedtotheeu-pregnancies,themedianmaternalage,deltaNT,andserumfreeweresigniÞcantlyhigher,andtherewasahigherprevalenceofabsentNB,TR,andreverseda-waveinDV.Intrisomy18,deltaNTandserumfree-hCGwerehigherandPAPP-Aandfetalcrown-rumplengthwerelowerandsimilartotrisomy21,therewasahigherprevalenceofabsentNB,TR,andreverseda-waveinDV.Theexpectednumberofcasesoftri-somy21andtrisomy18inourstudypopulation,onthebasisofthematernalagedistributionandtheage-relatedriskforthesetrisomiesat11-13weekswere7.89and3.21,respectively,whichweresimilartotheobservednumbersof8and3,respectively.RiskscoreresultsfromNIPTResultsfromchromosome-selectivese-quencingwereavailablefor1949ofthe2049cases(95.1%).In46cases(2.2%),thefetalfractionwasbelowtheminimalrequirementof4%andin54cases(2.6%)therewasassayfailure.Oneofthetrisomy18casesfailedtogenerateanas-sayresult.Riskscoresfortrisomies21and18wereprovidedfor1949samples,including8casesoftrisomy21,2oftri-somy18,and1939euploid.Inall8casesoftrisomy21,theriskscorefortrisomy21was99%andtheriskscorefortrisomy18was).Forthe2casesoftrisomy18,theriskscorefortrisomy18wasandtheriskscorefortrisomy21wasInthe1939provenorassumedeuploidpregnancies,theriskscoresfortrisomies21and18were0.01%in1936(99.85%)1%in1937(99.9%).In2euploidpregnanciestheriskscorefortrisomy18was9.8%and11.7%,respectively.Apply-ingariskscorecutoffof1%todelineatehigh-riskfromlow-riskpatients(basedonAriosaDiagnosticsInccutofffortheHar-monyPrenatalTest),theoveralltrisomyDRwas100%(10of10cases)withacom-binedFPRof0.1%.Inthispopulationof1939euploidand10aneuploidpregnancies,theperfor-manceofscreeningbythecombinedtest(NT,free-hCG,andPAPP-A),attheriskcutoffof1:150recommendedbytheUnitedKingdomNationalScreeningCommittee,wasdetectionofallcasesoftrisomies21and18atFPRof4.5%(87/www.AJOG.orgReportsofMajorImpactNOVEMBER2012AmericanJournalofObstetrics 1939).Inscreeningbythecombinedtestandtheadditionalultrasoundmarkers(NB,TR,andDV),attheriskcutoffof1:150,allcasesoftrisomies21and18weredetectedatFPRof3.0%(59/1939).PrincipalÞndingsofthisstudyThestudypopulationwasderivedfromwomenundergoingÞrst-trimesterscreen-ingforaneuploidiesaspartoftheirroutineantenatalcareinaninner-cityhospital.Theobservednumberoftri-somieswasasexpectedonthebasisofthematernalagedistributionofthestudypop-ulation,whichwassimilartothenationalaverageinEngland,UnitedKingdom.ThiscohortstudyofpregnantwomenundergoingroutinescreeninghasshownthatNIPTwithachromosome-selectivesequencingapproachishighlyaccurateforfetalaneuploidydetectionwithverylowFPR.Theestimatedtrisomyriskscorewas99%inallcasesoftrisomy21andtri-somy18and1%in99.9%oftheeuploidLimitationsofthestudyInthisstudy,whichwasbasedononly1sampleof2mLofstoredplasmapercase,Characteristicsofstudypopulation Trisomy21Trisomy18Medianmaternalage,y(IQR)31.8(27.7–35.4)31.8(27.7–35.4)39.6(33.3–41.7)39.4(23.4–42.3)..............................................................................................................................................................................................................................................................Medianmaternalweight,kg(IQR)65.2(58.5–76.0)65.2(58.5–76.0)64.5(60.5–88.5)64.0(56.0–126.0)..............................................................................................................................................................................................................................................................Medianmaternalheight,cm(IQR)164(160–169)164(160–169)168(164–170)165(158–165)..............................................................................................................................................................................................................................................................Racialorigin,n(%)..............................................................................................................................................................................................................................................................1431(69.8)1423(69.8)6(75.0)2(66.7)..............................................................................................................................................................................................................................................................422(20.6)419(20.6)2(25.0)1(33.3)..............................................................................................................................................................................................................................................................SouthAsian82(4.0)82(4.0)..............................................................................................................................................................................................................................................................EastAsian57(2.8)57(2.8)..............................................................................................................................................................................................................................................................57(2.8)57(2.8)..............................................................................................................................................................................................................................................................Cigarettesmoker,n(%)131(6.4)131(6.4)..............................................................................................................................................................................................................................................................Methodofconception,n(%)..............................................................................................................................................................................................................................................................2007(98.0)1996(97.9)8(100.0)3(100.0)..............................................................................................................................................................................................................................................................Ovulationdrugs19(0.9)19(0.9)..............................................................................................................................................................................................................................................................Invitrofertilization23(1.1)23(1.1)..............................................................................................................................................................................................................................................................Preexistingdiabetesmellitus,n(%)..............................................................................................................................................................................................................................................................Type110(0.5)10(0.5)..............................................................................................................................................................................................................................................................Type29(0.4)9(0.4)..............................................................................................................................................................................................................................................................Medianfetalcrown-rumplength,mm(IQR)62.4(57.3–67.3)62.4(57.3–67.3)64.1(59.5–70.4)47.7(45.5–59.2)..............................................................................................................................................................................................................................................................Fetalgender,n(%)..............................................................................................................................................................................................................................................................1069(52.2)1063(52.2)4(50.0)2(67.7)..............................................................................................................................................................................................................................................................980(47.8)975(47.8)4(50.0)1(33.3)..............................................................................................................................................................................................................................................................MedianPAPP-AMoM(IQR)1.048(0.722–1.491)1.052(0.724–1.494)0.700(0.274–1.325)0.195(0.152–0.300)..............................................................................................................................................................................................................................................................Medianfree-hCGMoM(IQR)1.089(0.729–1.641)1.089(0.730–1.631)3.809(1.852–8.602)0.339(0.253–0.573)..............................................................................................................................................................................................................................................................Mediandeltanuchaltranslucency(IQR)0.112(0.083to0.349)0.110(0.084to0.347)1.366(0.797–3.590)6.341(5.872–8.721)..............................................................................................................................................................................................................................................................Absentnasalbone,n(%)122(6.0)115(5.6)4(50.0)3(100.0)..............................................................................................................................................................................................................................................................Tricuspidregurgitation,n(%)41(2.0)34(1.7)4(50.0)3(100.0)..............................................................................................................................................................................................................................................................Reverseda-waveinductusvenosus,n(%)59(2.9)51(2.5)5(62.5)3(100.0)..............................................................................................................................................................................................................................................................Medianfetalfraction,%(IQR)10.0(7.8–13.0)10.0(7.8–13.0)12.5(9.2–21.3)9.3(5.6–13.0)..............................................................................................................................................................................................................................................................Medianestimatedriskfortrisomy21(range)1:8469(1:2–1:23527)1:8547(1:2–1:23527)1:2(1:2–1:3)1:6(1:4–1:13)..............................................................................................................................................................................................................................................................Medianestimatedriskfortrisomy18(range)1:14894(1:2–1:47472)1:14980(1:3–1:47472)1:177(1:2–1:1562)..............................................................................................................................................................................................................................................................humanchorionicgonadotrophin;interquartilerange;multipleofthemedian;pregnancy-associatedplasmaprotein.ComparisonsbetweenaneuploidandeuploidpregnanciesarebyMann-WhitneytestforcontinuousvariablesandbyorFisherexacttestforcategoricalvariables,withBonferronicorrectionwithvalueofNicolaides.Noninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedpopulation.AmJObstetGynecol2012.ReportsofMajorImpactwww.AJOG.orgAmericanJournalofObstetricsNOVEMBER2012 theassayfailureratewas2.6%andtherewereanadditional2.2%casesforwhichnoriskscorewasprovidedbecausethefetalfractionwas4%.Whenthefrac-tionisbelowthisminimalrequirementthesmalldifferencesincirculatingcfDNAbe-tweentrisomicanddisomicpregnanciesmaynotbedetectable.Theseresultsofassayfailureandlowfetalfractionaresimilartothe2.8%and1.8%,respec-tively,reportedinapreviouscohortTheproblemoflowfetalfrac-tionmaybeimpossibletoovercomebycurrentlyavailableNIPTtechniques.Thefetalfractionincreaseswithplacen-talmassisinverselyrelatedtomaternalandthemaincauseoflowfrac-tionisobesity.FurtherstudyisthereforewarrantedtoinvestigatetheoptimalmethodofaneuploidyscreeningandtheroleofNIPTinobesewomen.Alimitationofthestudywasthatwedidnotperformkaryotypinginallcasesandtheassumptionofeuploidywasbasedonthelackofphenotypicfeaturesofaneu-ploidyintheneonates.Thiswasaninevi-tableconsequenceofthenatureofthestudythatwasbasedonapopulationun-dergoingroutinescreeningforaneup-loidies,ratherthanahigh-riskpopula-tionundergoinginvasivetesting.TheprimarysourceofcfDNAinthematernalcirculationisthoughttobetheOnepossibleexplanationforthe2false-positivecasesinwhichtheriskscorefortrisomy18wasabout10%,ratherthan0.01%asinallotheras-sumedeuploidcases,isplacenta-con-Þnedmosaicism.ComparisonoftheÞndingswithpreviousstudiesintheliteratureNumerousstudiesinhigh-riskpregnan-ciesreportedthatNIPTbycfDNAanal-ysisofmaternalplasmacandetectofpregnancieswithfetaltrisomy21atFPRof1%,buttheperformanceofscreeningfortrisomies18and13hasbeenlessrobust.Inapreviouscase-controlstudyweusedchromo-selectivesequencingtoexaminecfDNAinplasmaobtainedbeforeCVSfrom300euploid,50trisomy-21,and50trisomy-18pregnanciesat11-13weeksanddetectedallcasesoftrisomy21and98%oftrisomy18withFPRof0%forTheÞndingsofthisstudy,inapopu-lationofpregnanciesundergoingrou-tineaneuploidyscreening,demonstratethattheaccuracyofNIPTobtainedfromtheinvestigationofpregnanciesathighriskforaneuploidiesisapplicabletothegeneralpopulationwheretheprevalenceoffetaltrisomy21ismuchlower.ThissupportsthesuggestionthattheabilitytodetectaneuploidywithcfDNAisde-pendentuponassayprecisionandfetalDNApercentageinthesampleratherthantheprevalenceofthediseaseinthestudypopulation.ImplicationsforpracticeThesensitivityandspeciÞcityofNIPTisnot100%andthereforeitshouldnotbeconsideredadiagnostictesttoreplacein-vasivetestinginhigh-riskpregnancies.ItisanewscreeningtestthatidentiÞesahigh-riskgrouprequiringfurtherinves-tigationbyinvasivetesting.Theperfor-manceofthisnewscreeningmethodforbothtrisomy21andtrisomy18isfarsu-periortothatofcurrentlyavailablescreeningmethodswithasubstantialin-creaseinDRanddecreaseinFPR.Onthebasisofexistingdata,NIPTcanpotentiallybeusedinuniversalscreen-ingfortrisomies21and18inallsingle-tonpregnanciesandthemainlimitingfactorforsuchwidespreadapplicationofthetestatpresentistheassociatedcost.Anotherlimitingfactorintheapplica-tionofNIPTinuniversalscreeningre-latestothedelayof1-2weeksbetweensamplingandobtainingresults.ThisRiskscoresfortrisomy21and18inpregnancieswithtrisomy21,trisomy18,andeuploidfetuses Nicolaides.Noninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedpopulation.AmJObstetGynecol2012. www.AJOG.orgReportsofMajorImpactNOVEMBER2012AmericanJournalofObstetrics problemcanbeovercomebytakingthebloodsample1-2weeksbeforethescheduledÞrst-trimesterultrasoundex-amination,whichisideallyperformedat12weeks.Atthis12-weekvisit,ontheresultsofNIPTandtheultra-soundÞndings,theparentscanbecounseledconcerningtheoptionofin-vasivetesting.InthosecaseswhereNIPThasfailedtoprovidearesulttheparentswouldstillhavetheoptionofÞrst-trimesterscreeningbycombinedtestingwithfetalNTandserumfree-hCGandPAPP-AandtheadvantageofÞrst-ratherthansecond-trimesterterminationofpregnancyshouldthefetusbefoundtobeaffectedbyamajorInthelast40yearsprenatalscreeningforaneuploidieshasfocusedontrisomy21.AbeneÞcialconsequenceofsuchscreeninghasbeenthedetectionofmanyadditionalclinicallysigniÞcantaneup-loidiesinthescreen-positivegroupunder-goinginvasivetestingandfullkaryotyping.AdditionallytheincreasingapplicationofmoleculartechniquesintheanalysisofobtainedbyinvasivetestingidentiÞesmanyclinicallysigniÞcantmi-crodeletion/duplicationsyndromes.Con-sequently,apotentialcriticismofreplac-ingthetraditionalmethodsofscreeningfortrisomy21byNIPTisthatmanyclin-icallysigniÞcantcytogeneticabnormali-tiesthatarecurrentlydetectablewouldbemissed.However,thebiomarkerpro-Þleformanyoftherareaneuploidiesandmicrodeletion/duplicationsyndromesisnotclearlydeÞnedanditisuncertainwhethertheirincidenceinthescreen-positivegroupfortrisomy21ishigherthaninthescreen-negativegroup.Inthecaseofsomeaneuploidies,suchastrisomies18and13,triploidy,andTurnersyndrome,theirincidenceinthescreen-positivegroupfortrisomy21ismuchhigherthaninthescreen-negativegroupbecausetheyhaveasimilarpat-ternintheexpressionofbiophysicalandbiochemicalmarkers.Thepotentiallossofmissingsomeofthesedefectsbyre-placingtraditionalmethodsofscreeningbyNIPTcanbeovercomebyÞrstly,ex-pandingtheconditionsthatcanbede-tectedbyNIPTandsecondly,retainingatleastsomeoftheelementsofcurrentmethodsofscreeningasanmethodofselectingpatientsforinvasivetesting.SpeciÞcally,invasivetestingshouldbeofferedtofetuseswithincreasedNTthicknessandstructuraldefects,includingexomphalos,holoprosencephalymegacys-tis,anddiaphragmatichernia,evenincaseswithanegativeNIPTresultfortrisomies21and18.Consequently,theintro-ductionofNIPTasanewhigh-perfor-mancetestforuniversalscreeningshouldnotbeconsideredasamethodofreplacingthe11-to13-weekscan.Thelatterisnotonlyusefulinscreeningforaneuploidiesbutitisadiagnostictestformanymajorfetaldefectsandincombinationwithbio-chemicalandotherbiophysicalmarkerscanprovideeffectiveearlyscreeningforpregnancycomplications,includingpre-eclampsiaandpretermbirth.Theperformanceofscreeningfortri-somy21andtrisomy18byNIPTusingchromosome-selectivesequencinginaroutinepopulationisaseffectiveaspre-viouslyreportedinhigh-riskgroupswith99%andFPR NicolaidesKH.Nuchaltranslucencyandotherrst-trimestersonographicmarkersofchromosomalabnormalities.AmJObstetGy-necol2004;191:45-67.NicolaidesKH.Screeningforfetalaneup-loidiesat11to13weeks.PrenatDiagn2011;ChittyLS,HillM,WhiteH,WrightD,MorrisS.Noninvasiveprenataltestingforaneuploidy–readyforprimetime?AmJObstetGynecolChiuRW,AkolekarR,ZhengYWL,etal.Non-invasiveprenatalassessmentoftrisomy21bymultiplexedmaternalplasmaDNAse-quencing:large-scalevaliditystudy.BMJ2011;ChenEZ,ChiuRWK,SunH,etal.Noninva-siveprenataldiagnosisoffetaltrisomy18andtrisomy13bymaternalplasmaDNAsequenc-ing.PLoSOne2011;6:e21791.EhrichM,DeciuC,ZwiefelhoferT,etal.Non-invasivedetectionoffetaltrisomy21byse-quencingofDNAinmaternalblood:astudyinaclinicalsetting.AmJObstetGynecol2011;204:PalomakiGE,KlozaEM,Lambert-MesserlianGM,etal.DNAsequencingofmaternalplasmatodetectDownsyndrome:aninternationalclinicalvalidationstudy.GenetMed2011;13:913–20.SehnertAJ,RheesB,ComstockD,etal.Op-timaldetectionoffetalchromosomalabnormal-itiesbymassivelyparallelDNAsequencingofcell-freefetalDNAfrommaternalblood.ClinChem2011;57:1042-9.BianchiDW,PlattLK,GoldbergJD,etal.Genome-widefetalaneuploidydetectionbymaternalplasmaDNAsequencing.ObstetGy-necol2012;119:1-12.PalomakiGE,DeciuC,KlozaEM,etal.DNAsequencingofmaternalplasmareliablyidenti-estrisomy18andtrisomy13aswellasDownsyndrome:aninternationalcollaborativestudy.GenetMed2012;14:296-305.SparksAB,StrubleCA,WangeET,etal.Non-invasiveprenataldetectionandselectiveanalysisofcell-freeDNAobtainedfromma-ternalblood:evaluationfortrisomy21andtrisomy18.AmJObstetGynecol2012;AshoorG,SyngelakiA,WagnerM,BirdirC,NicolaidesKH.Chromosome-selectivese-quencingofmaternalplasmacell-freeDNAforrst-trimesterdetectionoftrisomy21andtrisomy18.AmJObstetGynecol2012;NortonME,BrarH,WeissJ,etal.Non-invasivechromosomalevaluation(NICE)study:resultsofamulticenterprospectivecohortstudyfordetectionoffetaltrisomy21andtri-somy18.AmJObstetGynecol2012;207:KaganKO,WrightD,ValenciaC,MaizN,NicolaidesKH.Screeningfortrisomies21,18and13bymaternalage,fetalnuchaltranslu-cency,fetalheartrate,freebeta-hCGandpreg-nancy-associatedplasmaprotein-A.HumRe-prod2008;23:1968-75.SparksA,WangE,StrubleC,etal.Selec-tiveanalysisofcell-freeDNAinmaternalbloodforevaluationoffetaltrisomy.PrenatDiagnSnijdersRJM,SebireNJ,CuckleH,Nico-laidesKH.Maternalageandgestationalage-specicrisksforchromosomaldefects.FetalDiagnTher1995;10:356-67.SnijdersRJM,SundbergK,HolzgreveW,HenryG,NicolaidesKH.Maternalageandges-tation-specicriskfortrisomy21.UltrasoundObstetGynecol1999;13:167-70.OfceforNationalStatistics.BirthsanddeathsinEnglandandWales,2010.AvailableAccessedJuly23,2012.AshoorG,PoonL,SyngelakiA,MosimannB,NicolaidesKH.Fetalfractioninmaternalplasmacell-freeDNAat11-13weeks’gesta-tion:effectofmaternalandfetalfactors.FetalDiagnTher2012;31:237-43.AlberryM,MaddocksD,JonesM,etal.FreefetalDNAinmaternalplasmainanem-bryonicpregnancies:conrmationthattheor-iginisthetrophoblast.PrenatDiagn2007;WirtzA,GloningKP,MurkenJ.Trisomy18inchorionicvillussampling:problemsandconse-quences.PrenatDiagn1991;11:563-7.SnijdersRJ,NobleP,SebireN,SoukaA,ReportsofMajorImpactwww.AJOG.orgAmericanJournalofObstetricsNOVEMBER2012 NicolaidesKH.UKmulticenterprojectonassessmentofriskoftrisomy21bymaternalageandfetalnuchal-translucencythicknessat10–14weeksofgestation:FetalMedicineFoundationFirstTrimesterScreeningGroup.Lancet1998;352:343-6.KaganKO,AvgidouK,MolinaFS,Gajew-skaK,NicolaidesKH.Relationbetweenin-creasedfetalnuchaltranslucencythicknessandchromosomaldefects.ObstetGynecolChittyLS,KaganKO,MolinaFS,WatersJJ,NicolaidesKH.Fetalnuchaltranslucencyscanandearlyprenataldiagnosisofchromosomalabnormalitiesbyrapidaneuploidyscreening:observationalstudy.BMJ2006;332:452-5.PergamentE,AlamilloC,SakK,FiddlerM.Geneticassessmentfollowingincreasednuchaltranslucencyandnormalkaryotype.PrenatDiagn2011;31:307-10.LeungTY,VogelI,LauTK,etal.Identicationofsubmicroscopicchromosomalaberrationsinfetuseswithincreasednuchaltranslucencyandapparentlynormalkaryotype.UltrasoundObstetGynecol2011;38:314-9.NicolaidesKH.Turningthepyramidofpre-natalcare.FetalDiagnTher2011;29:183-96.www.AJOG.orgReportsofMajorImpactNOVEMBER2012AmericanJournalofObstetrics NoninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedÞrst-trimesterpopulationKyprosH.Nicolaides,MD;ArgyroSyngelaki,RM;GhaliaAshoor,MD;CahitBirdir,MD;GiseleTouzet,MD WesoughttoassessperformanceofnoninvasiveprenataltestingforfetaltrisomyinaroutinelyscreenedÞrst-trimesterpregnancySTUDYDESIGN:Thiswasacohortstudyof2049pregnantwomenun-dergoingroutinescreeningforaneuploidiesat11-13weeksÕgestation.Plasmacell-freeDNAanalysisusingchromosome-selectivesequenc-ingwasused.Laboratorytestingonasingleplasmasampleof2mLwascarriedoutblindlyandresultswereprovidedasriskscore(%)fortrisomies21and18.Trisomyriskscoresweregivenfor95.1%(1949of2049)ofcasesincludingall8withtrisomy21and2ofthe3withtrisomy18.Thetrisomyriskscorewas99%inthe8casesoftrisomy21and2oftrisomy18and1%in99.9%(1937of1939)ofeuploidcases.Noninvasiveprenataltestingusingchromosome-selec-tivesequencinginaroutinelyscreenedpopulationidentiÞedtrisomies21and18withafalse-positiverateof0.1%.Keywords: Citethisarticleas:NicolaidesKH,SyngelakiA,AshoorG,etal.NoninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedÞrst-trimesterpopulation.AmJObstetGynecol2012;207:374.e1-6. nthelast40years,screeninganddiagnosisoffetalaneuploidieshasshiftedfromsecond-trimesteramnio-centesisforadvancedmaternalage,withadetectionrate(DR)oftrisomy21of30%atfalse-positiverate(FPR)of5%,toÞrst-trimesterchorionicvilloussam-pling(CVS)inthehigh-riskgroupiden-tiÞedbyscreeningwithfetalnuchaltranslucency(NT)andserumbiochem-istry,withDRofabout90%atFPRofInvasivetestingwithCVSor amniocentesisisdiagnostic,butassoci-atedwiththeriskofmiscarriage.Recently,noninvasiveprenataltesting(NIPT)byanalysisofcell-freeDNA(cfDNA)inmaternalbloodhasshownpromiseforhighlyaccuratedetectionofcommonfetalautosomaltrisomies.AnalysisofcfDNAhasbeenvalidatedinclinicalstudiesutilizingnext-generationDNAsequencingtechno-Clinicalstudieshaveprimar-ilyincludedwomenidentiÞedbypriorscreening,withmaternalageandchemicaland/orsonographictestingintheÞrstorsecondtrimesterofpregnancy,tobeathighriskforaneuploidies.IthasthereforebeenuncertainiftheresultsofNIPTinsuchhigh-riskpregnanciesareapplicabletothegeneralpregnancyTheobjectiveofthisstudyistoassesstheperformanceofscreeningbyNIPTfortrisomies21and18usingachromo-sequencingmethodofcfDNAinmaternalplasmaobtainedfromapopulationundergoingroutinescreeningat11-13weeksÕgestation.ATERIALSAND FromHarrisBirthrightResearchCentreforFetalMedicine,King’sCollegeHospital,King’sCollege(allauthors),andtheDepartmentofFetalMedicine,UniversityCollegeLondonHospital,UniversityCollegeLondon(DrNicolaidesandMsSyngelaki),UniversityofLondon,London,England,UK.ReceivedJuly27,2012;revisedAug.9,2012;acceptedAug.22,2012.ThestudywassupportedbyagrantfromtheFetalMedicineFoundation(UnitedKingdomcharitynumber1037116).ThecostofcollectionandanalysisofthesampleswascoveredbyAriosaDiagnosticsInc,SanJose,Theauthorsreportnoconictofinterest.Reprintsnotavailablefromtheauthors.©2012PublishedbyMosby,Inc. ForEditors’Commentary,see ReportsofMajorImpactwww.AmericanJournalofObstetricsNOVEMBER2012 tainedfromthematernitycomputerizedrecordsorthegeneralmedicalpractitio-nersofthewomen,wereaddedintothedatabaseassoonastheybecameavailable.Venousbloodwasobtainedfromwomenwhogavewritteninformedsenttoprovidesamplesforresearchintoearlypredictionofpregnancycomplica-tions,whichwasapprovedbytheNationalResearchEthicsServiceoftheNationalHealthService.BloodwascollectedinEDTABDVacutainertubes(BectonDickinsonUKLtd,Oxfordshire,UnitedKingdom)andprocessedwithin15min-utesofcollection.Thetubeswerecentri-fugedat2000for10minutestoseparateplasmafrompackedcellsandbuffycoatandsubsequentlyat16,000for10min-utestofurtherseparatecelldebris.Plasmasamplesweredividedinto0.5-mLaliquotsinseparateEppendorftubes(JenconsSci-entiÞcLtd,VWRInternational,WillardWay,Bedfordshire,UK)labeledwithauniquepatientidentiÞerandstoredat80¡Cuntilsubsequentanalysis.Wesearchedourdatabaseandselectedallcaseswithatleast2mLofavailablestoredplasma,collectedfromOctober2010throughJanuary2011.Thepopula-tionconsistedof2230singletonpreg-nancies.Weexcluded74pregnancieswheretherewasnofetalkaryotypeandtheoutcomewasmiscarriage,stillbirth,termination(n28),ornofollow-up46).Wealsoexcluded7pregnan-cieswhereCVSdemonstratedthatthefetalkaryotypewasabnormalbutotherthantrisomy21ortrisomy18;2casesoftriploidy;and1caseeachofTurnersyn-i(8)(p10),46,XY,del(13)(q33),46,XX,dup(10)(q26.1q24.1),andmosaic45,X/47,XXX.Therewere2149casesafterexcludingfortheabovereasons.LaboratoryanalysisPlasmasamples(4tubesof0.5mLperpatient)fromtheselectedcasesweresentovernightondryicefromLondon,UnitedKingdom,tothelaboratoryofAriosaDiagnosticsIncinSanJose,CA.TheinformationprovidedtoAriosaDi-agnosticsIncforeachcasewas:patientuniqueidentiÞer,maternalage,gesta-tionalage,dateofbloodcollection,andfetalsex,butnotfetalkaryotypeorbirthPriortoevaluationforfetaltrisomy,AriosaDiagnosticsIncdetermined29caseshadinadequatesamplevolume,1casehadtubelabelsthatdidnotmatchthesamplemanifest,and70caseshadissuesofsamplemixingduringtheman-ualpoolingprocessofindividualEppen-dorftubesbylaboratorypersonnel.Therefore,theeligiblestudypopulationconsistedof2049cases.Plasmasamplesfromthestudypopula-tionwereanalyzedusingachromosome-selectiveassay(HarmonyPrenatalTest,AriosaDiagnosticsInc).Resultswereprovidedontheriskoftrisomy21andtri-somy18oneachcasetoK.H.N.whothendeterminedthecorrelationbetweentheas-sayresultswiththefetalkaryotypeorbirthoutcome.Theriskscoreswererepresentedasapercentagewithrangescappedat99%andStatisticalanalysesDescriptivedatawerepresentedinme-dianandinterquartilerangeforcontinu-ousvariablesandinnumbersandpercent-agesforcategoricalvariables.ComparisonbetweentheoutcomegroupswasbyFisherexacttestforcategoricalvari-ablesandMann-Whitneytestforcontinuousvariables.InallcasesweusedBonferronicorrectionwithad-valueofThestatisticalsoftwarepackageSPSS20.0(IBMCorp,Armonk,NY)wasusedfordataanalyses.StudypopulationThetotalstudypopulationincluded2049pregnancies.In86(4.3%)cases,thefetalkaryotypewasdeterminedbyCVSoram-niocentesisandwasnormalin75cases,tri-somy21in8,andtrisomy18in3.There-maining1963pregnanciesresultedinthelivebirthofphenotypicallynormalneo-natesassumedtobeeuploidandtherefore,thetotalnumberofprovenorassumedeu-ploidpregnancieswas2038.MaternalcharacteristicsandresultsfromconventionalÞrst-trimesterscreen-ingwithserummarkersandultrasoundofthestudypopulationarepresentedinthe.Intrisomy21,comparedtotheeu-pregnancies,themedianmaternalage,deltaNT,andserumfreeweresigniÞcantlyhigher,andtherewasahigherprevalenceofabsentNB,TR,andreverseda-waveinDV.Intrisomy18,deltaNTandserumfree-hCGwerehigherandPAPP-Aandfetalcrown-rumplengthwerelowerandsimilartotrisomy21,therewasahigherprevalenceofabsentNB,TR,andreverseda-waveinDV.Theexpectednumberofcasesoftri-somy21andtrisomy18inourstudypopulation,onthebasisofthematernalagedistributionandtheage-relatedriskforthesetrisomiesat11-13weekswere7.89and3.21,respectively,whichweresimilartotheobservednumbersof8and3,respectively.RiskscoreresultsfromNIPTResultsfromchromosome-selectivese-quencingwereavailablefor1949ofthe2049cases(95.1%).In46cases(2.2%),thefetalfractionwasbelowtheminimalrequirementof4%andin54cases(2.6%)therewasassayfailure.Oneofthetrisomy18casesfailedtogenerateanas-sayresult.Riskscoresfortrisomies21and18wereprovidedfor1949samples,including8casesoftrisomy21,2oftri-somy18,and1939euploid.Inall8casesoftrisomy21,theriskscorefortrisomy21was99%andtheriskscorefortrisomy18was).Forthe2casesoftrisomy18,theriskscorefortrisomy18wasandtheriskscorefortrisomy21wasInthe1939provenorassumedeuploidpregnancies,theriskscoresfortrisomies21and18were0.01%in1936(99.85%)1%in1937(99.9%).In2euploidpregnanciestheriskscorefortrisomy18was9.8%and11.7%,respectively.Apply-ingariskscorecutoffof1%todelineatehigh-riskfromlow-riskpatients(basedonAriosaDiagnosticsInccutofffortheHar-monyPrenatalTest),theoveralltrisomyDRwas100%(10of10cases)withacom-binedFPRof0.1%.Inthispopulationof1939euploidand10aneuploidpregnancies,theperfor-manceofscreeningbythecombinedtest(NT,free-hCG,andPAPP-A),attheriskcutoffof1:150recommendedbytheUnitedKingdomNationalScreeningCommittee,wasdetectionofallcasesoftrisomies21and18atFPRof4.5%(87/www.AJOG.orgReportsofMajorImpactNOVEMBER2012AmericanJournalofObstetrics problemcanbeovercomebytakingthebloodsample1-2weeksbeforethescheduledÞrst-trimesterultrasoundex-amination,whichisideallyperformedat12weeks.Atthis12-weekvisit,ontheresultsofNIPTandtheultra-soundÞndings,theparentscanbecounseledconcerningtheoptionofin-vasivetesting.InthosecaseswhereNIPThasfailedtoprovidearesulttheparentswouldstillhavetheoptionofÞrst-trimesterscreeningbycombinedtestingwithfetalNTandserumfree-hCGandPAPP-AandtheadvantageofÞrst-ratherthansecond-trimesterterminationofpregnancyshouldthefetusbefoundtobeaffectedbyamajorInthelast40yearsprenatalscreeningforaneuploidieshasfocusedontrisomy21.AbeneÞcialconsequenceofsuchscreeninghasbeenthedetectionofmanyadditionalclinicallysigniÞcantaneup-loidiesinthescreen-positivegroupunder-goinginvasivetestingandfullkaryotyping.AdditionallytheincreasingapplicationofmoleculartechniquesintheanalysisofobtainedbyinvasivetestingidentiÞesmanyclinicallysigniÞcantmi-crodeletion/duplicationsyndromes.Con-sequently,apotentialcriticismofreplac-ingthetraditionalmethodsofscreeningfortrisomy21byNIPTisthatmanyclin-icallysigniÞcantcytogeneticabnormali-tiesthatarecurrentlydetectablewouldbemissed.However,thebiomarkerpro-Þleformanyoftherareaneuploidiesandmicrodeletion/duplicationsyndromesisnotclearlydeÞnedanditisuncertainwhethertheirincidenceinthescreen-positivegroupfortrisomy21ishigherthaninthescreen-negativegroup.Inthecaseofsomeaneuploidies,suchastrisomies18and13,triploidy,andTurnersyndrome,theirincidenceinthescreen-positivegroupfortrisomy21ismuchhigherthaninthescreen-negativegroupbecausetheyhaveasimilarpat-ternintheexpressionofbiophysicalandbiochemicalmarkers.Thepotentiallossofmissingsomeofthesedefectsbyre-placingtraditionalmethodsofscreeningbyNIPTcanbeovercomebyÞrstly,ex-pandingtheconditionsthatcanbede-tectedbyNIPTandsecondly,retainingatleastsomeoftheelementsofcurrentmethodsofscreeningasanmethodofselectingpatientsforinvasivetesting.SpeciÞcally,invasivetestingshouldbeofferedtofetuseswithincreasedNTthicknessandstructuraldefects,includingexomphalos,holoprosencephalymegacys-tis,anddiaphragmatichernia,evenincaseswithanegativeNIPTresultfortrisomies21and18.Consequently,theintro-ductionofNIPTasanewhigh-perfor-mancetestforuniversalscreeningshouldnotbeconsideredasamethodofreplacingthe11-to13-weekscan.Thelatterisnotonlyusefulinscreeningforaneuploidiesbutitisadiagnostictestformanymajorfetaldefectsandincombinationwithbio-chemicalandotherbiophysicalmarkerscanprovideeffectiveearlyscreeningforpregnancycomplications,includingpre-eclampsiaandpretermbirth.Theperformanceofscreeningfortri-somy21andtrisomy18byNIPTusingchromosome-selectivesequencinginaroutinepopulationisaseffectiveaspre-viouslyreportedinhigh-riskgroupswith99%andFPR NicolaidesKH.Nuchaltranslucencyandotherrst-trimestersonographicmarkersofchromosomalabnormalities.AmJObstetGy-necol2004;191:45-67.NicolaidesKH.Screeningforfetalaneup-loidiesat11to13weeks.PrenatDiagn2011;ChittyLS,HillM,WhiteH,WrightD,MorrisS.Noninvasiveprenataltestingforaneuploidy–readyforprimetime?AmJObstetGynecolChiuRW,AkolekarR,ZhengYWL,etal.Non-invasiveprenatalassessmentoftrisomy21bymultiplexedmaternalplasmaDNAse-quencing:large-scalevaliditystudy.BMJ2011;ChenEZ,ChiuRWK,SunH,etal.Noninva-siveprenataldiagnosisoffetaltrisomy18andtrisomy13bymaternalplasmaDNAsequenc-ing.PLoSOne2011;6:e21791.EhrichM,DeciuC,ZwiefelhoferT,etal.Non-invasivedetectionoffetaltrisomy21byse-quencingofDNAinmaternalblood:astudyinaclinicalsetting.AmJObstetGynecol2011;204:PalomakiGE,KlozaEM,Lambert-MesserlianGM,etal.DNAsequencingofmaternalplasmatodetectDownsyndrome:aninternationalclinicalvalidationstudy.GenetMed2011;13:913–20.SehnertAJ,RheesB,ComstockD,etal.Op-timaldetectionoffetalchromosomalabnormal-itiesbymassivelyparallelDNAsequencingofcell-freefetalDNAfrommaternalblood.ClinChem2011;57:1042-9.BianchiDW,PlattLK,GoldbergJD,etal.Genome-widefetalaneuploidydetectionbymaternalplasmaDNAsequencing.ObstetGy-necol2012;119:1-12.PalomakiGE,DeciuC,KlozaEM,etal.DNAsequencingofmaternalplasmareliablyidenti-estrisomy18andtrisomy13aswellasDownsyndrome:aninternationalcollaborativestudy.GenetMed2012;14:296-305.SparksAB,StrubleCA,WangeET,etal.Non-invasiveprenataldetectionandselectiveanalysisofcell-freeDNAobtainedfromma-ternalblood:evaluationfortrisomy21andtrisomy18.AmJObstetGynecol2012;AshoorG,SyngelakiA,WagnerM,BirdirC,NicolaidesKH.Chromosome-selectivese-quencingofmaternalplasmacell-freeDNAforrst-trimesterdetectionoftrisomy21andtrisomy18.AmJObstetGynecol2012;NortonME,BrarH,WeissJ,etal.Non-invasivechromosomalevaluation(NICE)study:resultsofamulticenterprospectivecohortstudyfordetectionoffetaltrisomy21andtri-somy18.AmJObstetGynecol2012;207:KaganKO,WrightD,ValenciaC,MaizN,NicolaidesKH.Screeningfortrisomies21,18and13bymaternalage,fetalnuchaltranslu-cency,fetalheartrate,freebeta-hCGandpreg-nancy-associatedplasmaprotein-A.HumRe-prod2008;23:1968-75.SparksA,WangE,StrubleC,etal.Selec-tiveanalysisofcell-freeDNAinmaternalbloodforevaluationoffetaltrisomy.PrenatDiagnSnijdersRJM,SebireNJ,CuckleH,Nico-laidesKH.Maternalageandgestationalage-specicrisksforchromosomaldefects.FetalDiagnTher1995;10:356-67.SnijdersRJM,SundbergK,HolzgreveW,HenryG,NicolaidesKH.Maternalageandges-tation-specicriskfortrisomy21.UltrasoundObstetGynecol1999;13:167-70.OfceforNationalStatistics.BirthsanddeathsinEnglandandWales,2010.AvailableAccessedJuly23,2012.AshoorG,PoonL,SyngelakiA,MosimannB,NicolaidesKH.Fetalfractioninmaternalplasmacell-freeDNAat11-13weeks’gesta-tion:effectofmaternalandfetalfactors.FetalDiagnTher2012;31:237-43.AlberryM,MaddocksD,JonesM,etal.FreefetalDNAinmaternalplasmainanem-bryonicpregnancies:conrmationthattheor-iginisthetrophoblast.PrenatDiagn2007;WirtzA,GloningKP,MurkenJ.Trisomy18inchorionicvillussampling:problemsandconse-quences.PrenatDiagn1991;11:563-7.SnijdersRJ,NobleP,SebireN,SoukaA,ReportsofMajorImpactwww.AJOG.orgAmericanJournalofObstetricsNOVEMBER2012 NicolaidesKH.UKmulticenterprojectonassessmentofriskoftrisomy21bymaternalageandfetalnuchal-translucencythicknessat10–14weeksofgestation:FetalMedicineFoundationFirstTrimesterScreeningGroup.Lancet1998;352:343-6.KaganKO,AvgidouK,MolinaFS,Gajew-skaK,NicolaidesKH.Relationbetweenin-creasedfetalnuchaltranslucencythicknessandchromosomaldefects.ObstetGynecolChittyLS,KaganKO,MolinaFS,WatersJJ,NicolaidesKH.Fetalnuchaltranslucencyscanandearlyprenataldiagnosisofchromosomalabnormalitiesbyrapidaneuploidyscreening:observationalstudy.BMJ2006;332:452-5.PergamentE,AlamilloC,SakK,FiddlerM.Geneticassessmentfollowingincreasednuchaltranslucencyandnormalkaryotype.PrenatDiagn2011;31:307-10.LeungTY,VogelI,LauTK,etal.Identicationofsubmicroscopicchromosomalaberrationsinfetuseswithincreasednuchaltranslucencyandapparentlynormalkaryotype.UltrasoundObstetGynecol2011;38:314-9.NicolaidesKH.Turningthepyramidofpre-natalcare.FetalDiagnTher2011;29:183-96.www.AJOG.orgReportsofMajorImpactNOVEMBER2012AmericanJournalofObstetrics theassayfailureratewas2.6%andtherewereanadditional2.2%casesforwhichnoriskscorewasprovidedbecausethefetalfractionwas4%.Whenthefrac-tionisbelowthisminimalrequirementthesmalldifferencesincirculatingcfDNAbe-tweentrisomicanddisomicpregnanciesmaynotbedetectable.Theseresultsofassayfailureandlowfetalfractionaresimilartothe2.8%and1.8%,respec-tively,reportedinapreviouscohortTheproblemoflowfetalfrac-tionmaybeimpossibletoovercomebycurrentlyavailableNIPTtechniques.Thefetalfractionincreaseswithplacen-talmassisinverselyrelatedtomaternalandthemaincauseoflowfrac-tionisobesity.FurtherstudyisthereforewarrantedtoinvestigatetheoptimalmethodofaneuploidyscreeningandtheroleofNIPTinobesewomen.Alimitationofthestudywasthatwedidnotperformkaryotypinginallcasesandtheassumptionofeuploidywasbasedonthelackofphenotypicfeaturesofaneu-ploidyintheneonates.Thiswasaninevi-tableconsequenceofthenatureofthestudythatwasbasedonapopulationun-dergoingroutinescreeningforaneup-loidies,ratherthanahigh-riskpopula-tionundergoinginvasivetesting.TheprimarysourceofcfDNAinthematernalcirculationisthoughttobetheOnepossibleexplanationforthe2false-positivecasesinwhichtheriskscorefortrisomy18wasabout10%,ratherthan0.01%asinallotheras-sumedeuploidcases,isplacenta-con-Þnedmosaicism.ComparisonoftheÞndingswithpreviousstudiesintheliteratureNumerousstudiesinhigh-riskpregnan-ciesreportedthatNIPTbycfDNAanal-ysisofmaternalplasmacandetectofpregnancieswithfetaltrisomy21atFPRof1%,buttheperformanceofscreeningfortrisomies18and13hasbeenlessrobust.Inapreviouscase-controlstudyweusedchromo-selectivesequencingtoexaminecfDNAinplasmaobtainedbeforeCVSfrom300euploid,50trisomy-21,and50trisomy-18pregnanciesat11-13weeksanddetectedallcasesoftrisomy21and98%oftrisomy18withFPRof0%forTheÞndingsofthisstudy,inapopu-lationofpregnanciesundergoingrou-tineaneuploidyscreening,demonstratethattheaccuracyofNIPTobtainedfromtheinvestigationofpregnanciesathighriskforaneuploidiesisapplicabletothegeneralpopulationwheretheprevalenceoffetaltrisomy21ismuchlower.ThissupportsthesuggestionthattheabilitytodetectaneuploidywithcfDNAisde-pendentuponassayprecisionandfetalDNApercentageinthesampleratherthantheprevalenceofthediseaseinthestudypopulation.ImplicationsforpracticeThesensitivityandspeciÞcityofNIPTisnot100%andthereforeitshouldnotbeconsideredadiagnostictesttoreplacein-vasivetestinginhigh-riskpregnancies.ItisanewscreeningtestthatidentiÞesahigh-riskgrouprequiringfurtherinves-tigationbyinvasivetesting.Theperfor-manceofthisnewscreeningmethodforbothtrisomy21andtrisomy18isfarsu-periortothatofcurrentlyavailablescreeningmethodswithasubstantialin-creaseinDRanddecreaseinFPR.Onthebasisofexistingdata,NIPTcanpotentiallybeusedinuniversalscreen-ingfortrisomies21and18inallsingle-tonpregnanciesandthemainlimitingfactorforsuchwidespreadapplicationofthetestatpresentistheassociatedcost.Anotherlimitingfactorintheapplica-tionofNIPTinuniversalscreeningre-latestothedelayof1-2weeksbetweensamplingandobtainingresults.ThisRiskscoresfortrisomy21and18inpregnancieswithtrisomy21,trisomy18,andeuploidfetuses Nicolaides.Noninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedpopulation.AmJObstetGynecol2012. www.AJOG.orgReportsofMajorImpactNOVEMBER2012AmericanJournalofObstetrics 1939).Inscreeningbythecombinedtestandtheadditionalultrasoundmarkers(NB,TR,andDV),attheriskcutoffof1:150,allcasesoftrisomies21and18weredetectedatFPRof3.0%(59/1939).PrincipalÞndingsofthisstudyThestudypopulationwasderivedfromwomenundergoingÞrst-trimesterscreen-ingforaneuploidiesaspartoftheirroutineantenatalcareinaninner-cityhospital.Theobservednumberoftri-somieswasasexpectedonthebasisofthematernalagedistributionofthestudypop-ulation,whichwassimilartothenationalaverageinEngland,UnitedKingdom.ThiscohortstudyofpregnantwomenundergoingroutinescreeninghasshownthatNIPTwithachromosome-selectivesequencingapproachishighlyaccurateforfetalaneuploidydetectionwithverylowFPR.Theestimatedtrisomyriskscorewas99%inallcasesoftrisomy21andtri-somy18and1%in99.9%oftheeuploidLimitationsofthestudyInthisstudy,whichwasbasedononly1sampleof2mLofstoredplasmapercase,Characteristicsofstudypopulation Trisomy21Trisomy18Medianmaternalage,y(IQR)31.8(27.7–35.4)31.8(27.7–35.4)39.6(33.3–41.7)39.4(23.4–42.3)..............................................................................................................................................................................................................................................................Medianmaternalweight,kg(IQR)65.2(58.5–76.0)65.2(58.5–76.0)64.5(60.5–88.5)64.0(56.0–126.0)..............................................................................................................................................................................................................................................................Medianmaternalheight,cm(IQR)164(160–169)164(160–169)168(164–170)165(158–165)..............................................................................................................................................................................................................................................................Racialorigin,n(%)..............................................................................................................................................................................................................................................................1431(69.8)1423(69.8)6(75.0)2(66.7)..............................................................................................................................................................................................................................................................422(20.6)419(20.6)2(25.0)1(33.3)..............................................................................................................................................................................................................................................................SouthAsian82(4.0)82(4.0)..............................................................................................................................................................................................................................................................EastAsian57(2.8)57(2.8)..............................................................................................................................................................................................................................................................57(2.8)57(2.8)..............................................................................................................................................................................................................................................................Cigarettesmoker,n(%)131(6.4)131(6.4)..............................................................................................................................................................................................................................................................Methodofconception,n(%)..............................................................................................................................................................................................................................................................2007(98.0)1996(97.9)8(100.0)3(100.0)..............................................................................................................................................................................................................................................................Ovulationdrugs19(0.9)19(0.9)..............................................................................................................................................................................................................................................................Invitrofertilization23(1.1)23(1.1)..............................................................................................................................................................................................................................................................Preexistingdiabetesmellitus,n(%)..............................................................................................................................................................................................................................................................Type110(0.5)10(0.5)..............................................................................................................................................................................................................................................................Type29(0.4)9(0.4)..............................................................................................................................................................................................................................................................Medianfetalcrown-rumplength,mm(IQR)62.4(57.3–67.3)62.4(57.3–67.3)64.1(59.5–70.4)47.7(45.5–59.2)..............................................................................................................................................................................................................................................................Fetalgender,n(%)..............................................................................................................................................................................................................................................................1069(52.2)1063(52.2)4(50.0)2(67.7)..............................................................................................................................................................................................................................................................980(47.8)975(47.8)4(50.0)1(33.3)..............................................................................................................................................................................................................................................................MedianPAPP-AMoM(IQR)1.048(0.722–1.491)1.052(0.724–1.494)0.700(0.274–1.325)0.195(0.152–0.300)..............................................................................................................................................................................................................................................................Medianfree-hCGMoM(IQR)1.089(0.729–1.641)1.089(0.730–1.631)3.809(1.852–8.602)0.339(0.253–0.573)..............................................................................................................................................................................................................................................................Mediandeltanuchaltranslucency(IQR)0.112(0.083to0.349)0.110(0.084to0.347)1.366(0.797–3.590)6.341(5.872–8.721)..............................................................................................................................................................................................................................................................Absentnasalbone,n(%)122(6.0)115(5.6)4(50.0)3(100.0)..............................................................................................................................................................................................................................................................Tricuspidregurgitation,n(%)41(2.0)34(1.7)4(50.0)3(100.0)..............................................................................................................................................................................................................................................................Reverseda-waveinductusvenosus,n(%)59(2.9)51(2.5)5(62.5)3(100.0)..............................................................................................................................................................................................................................................................Medianfetalfraction,%(IQR)10.0(7.8–13.0)10.0(7.8–13.0)12.5(9.2–21.3)9.3(5.6–13.0)..............................................................................................................................................................................................................................................................Medianestimatedriskfortrisomy21(range)1:8469(1:2–1:23527)1:8547(1:2–1:23527)1:2(1:2–1:3)1:6(1:4–1:13)..............................................................................................................................................................................................................................................................Medianestimatedriskfortrisomy18(range)1:14894(1:2–1:47472)1:14980(1:3–1:47472)1:177(1:2–1:1562)..............................................................................................................................................................................................................................................................humanchorionicgonadotrophin;interquartilerange;multipleofthemedian;pregnancy-associatedplasmaprotein.ComparisonsbetweenaneuploidandeuploidpregnanciesarebyMann-WhitneytestforcontinuousvariablesandbyorFisherexacttestforcategoricalvariables,withBonferronicorrectionwithvalueofNicolaides.Noninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedpopulation.AmJObstetGynecol2012.ReportsofMajorImpactwww.AJOG.orgAmericanJournalofObstetricsNOVEMBER2012 NoninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedÞrst-trimesterpopulationKyprosH.Nicolaides,MD;ArgyroSyngelaki,RM;GhaliaAshoor,MD;CahitBirdir,MD;GiseleTouzet,MD WesoughttoassessperformanceofnoninvasiveprenataltestingforfetaltrisomyinaroutinelyscreenedÞrst-trimesterpregnancySTUDYDESIGN:Thiswasacohortstudyof2049pregnantwomenun-dergoingroutinescreeningforaneuploidiesat11-13weeksÕgestation.Plasmacell-freeDNAanalysisusingchromosome-selectivesequenc-ingwasused.Laboratorytestingonasingleplasmasampleof2mLwascarriedoutblindlyandresultswereprovidedasriskscore(%)fortrisomies21and18.Trisomyriskscoresweregivenfor95.1%(1949of2049)ofcasesincludingall8withtrisomy21and2ofthe3withtrisomy18.Thetrisomyriskscorewas99%inthe8casesoftrisomy21and2oftrisomy18and1%in99.9%(1937of1939)ofeuploidcases.Noninvasiveprenataltestingusingchromosome-selec-tivesequencinginaroutinelyscreenedpopulationidentiÞedtrisomies21and18withafalse-positiverateof0.1%.Keywords: Citethisarticleas:NicolaidesKH,SyngelakiA,AshoorG,etal.NoninvasiveprenataltestingforfetaltrisomiesinaroutinelyscreenedÞrst-trimesterpopulation.AmJObstetGynecol2012;207:374.e1-6. nthelast40years,screeninganddiagnosisoffetalaneuploidieshasshiftedfromsecond-trimesteramnio-centesisforadvancedmaternalage,withadetectionrate(DR)oftrisomy21of30%atfalse-positiverate(FPR)of5%,toÞrst-trimesterchorionicvilloussam-pling(CVS)inthehigh-riskgroupiden-tiÞedbyscreeningwithfetalnuchaltranslucency(NT)andserumbiochem-istry,withDRofabout90%atFPRofInvasivetestingwithCVSor amniocentesisisdiagnostic,butassoci-atedwiththeriskofmiscarriage.Recently,noninvasiveprenataltesting(NIPT)byanalysisofcell-freeDNA(cfDNA)inmaternalbloodhasshownpromiseforhighlyaccuratedetectionofcommonfetalautosomaltrisomies.AnalysisofcfDNAhasbeenvalidatedinclinicalstudiesutilizingnext-generationDNAsequencingtechno-Clinicalstudieshaveprimar-ilyincludedwomenidentiÞedbypriorscreening,withmaternalageandchemicaland/orsonographictestingintheÞrstorsecondtrimesterofpregnancy,tobeathighriskforaneuploidies.IthasthereforebeenuncertainiftheresultsofNIPTinsuchhigh-riskpregnanciesareapplicabletothegeneralpregnancyTheobjectiveofthisstudyistoassesstheperformanceofscreeningbyNIPTfortrisomies21and18usingachromo-sequencingmethodofcfDNAinmaternalplasmaobtainedfromapopulationundergoingroutinescreeningat11-13weeksÕgestation.ATERIALSAND FromHarrisBirthrightResearchCentreforFetalMedicine,King’sCollegeHospital,King’sCollege(allauthors),andtheDepartmentofFetalMedicine,UniversityCollegeLondonHospital,UniversityCollegeLondon(DrNicolaidesandMsSyngelaki),UniversityofLondon,London,England,UK.ReceivedJuly27,2012;revisedAug.9,2012;acceptedAug.22,2012.ThestudywassupportedbyagrantfromtheFetalMedicineFoundation(UnitedKingdomcharitynumber1037116).ThecostofcollectionandanalysisofthesampleswascoveredbyAriosaDiagnosticsInc,SanJose,Theauthorsreportnoconictofinterest.Reprintsnotavailablefromtheauthors.©2012PublishedbyMosby,Inc. ForEditors’Commentary,see ReportsofMajorImpactwww.AmericanJournalofObstetricsNOVEMBER2012