additionalamh

RESEARCHOpenAccessAnti-Mullerian-HormoneduringpregnancyandperipartumusingthenewBeckmanCoulterAMHGenIIAssayA.
Kninger1*,B.
Schmidt2,P.
Mach1,D.
Damaske1,S.
Nieen2,R.
Kimmig1,T.
Strowitzki3andA.
Gellhaus1AbstractBackground:AMHlevelsdeterminedbytheconventionalAMHassaydeclinedduringpregnancyandpostpartum.
AnewBeckmanCoulterAMHGenIIassayremovesthepotentiallyassay-interferingcomplementwhichisactivatedinpregnancy.
TheaimofthisstudywastoevaluateifthedeclineofAMHlevelsintheserumofpregnantwomenduringthecourseofpregnancyandperipartumwasassay-dependentandthusartificial.
Methods:Inthiscross-sectionalstudyprepartalbloodsampleswerecollectedfrom62patients(medianage30.
6years[interquartilerange:25.
6-34.
5])inthethirdtrimesterofpregnancyandagain1–4daysafterdeliverybetween2011and2012.
Inanothercohortof11patients(medianage34.
1years[interquartilerange:32.
6-37.
8])bloodsamplesweretakenindifferenttrimestersofpregnancybetween1995and2001.
TheconventionalandthemodifiedAMHassaywereperformedinthesamepatientserumsamples.
WeusedtheconventionalandthemodifiedAMH-Gen-IIELISA(BeckmanCoulter,Immunotech,Webster,USA)fortheassessmentofAMHlevels.
TheWilcoxonsignedranktestwasusedfordeterminingdifferencesbetweenAMHlevelspre-andpostpartum.
ThemethodofBlandandAltmanwasappliedforanalyzingtheagreementofbothmethodsfordeterminingAMHlevels.
Results:AMHvaluesperipartumwerelowerthanthoseexpectedinfertilenon-pregnantwomenofcomparableage.
Anoverallmeandifferenceof0.
44ng/mlwasobservedbetweentheconventionalandthemodifiedassay.
Measurementswiththemodifiedassayshowedasignificantdeclineofpostpartallevelscomparedwithprepartallevelswhichisconsistentwithvaluesobtainedusingtheconventionalassay(bothpAMHlevelsdeterminedusingtheconventionalassay,AMHlevelsobtainedusingthemodifiedassaysuggestasteeperdeclineofvaluesduringthecourseofpregnancy.
Conclusion:BycomparingtheconventionalassayforAMHdeterminationwiththemodifiedassaythepresentstudyconfirmedthatAMHlevelsdeclineduringthecourseofpregnancyandearlyafterdelivery.
Keywords:Anti-Mullerian-Hormone,pregnancy,peripartal,conventionalBeckmanCoulterAMHGenIIassay,modifiedBeckmanCoulterAMHGenIIassay*Correspondence:angela.
koeninger@uk-essen.
de1DepartmentofGynecologyandObstetrics,UniversityofDuisburg-Essen,Hufelandstrasse55,45122Essen,GermanyFulllistofauthorinformationisavailableattheendofthearticle2015Kningeretal.
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Kningeretal.
ReproductiveBiologyandEndocrinology(2015)13:86DOI10.
1186/s12958-015-0082-4BackgroundAnti-MullerianHormone(AMH)isknowntobethemostprecisepredictorofovarianreserveinwomen[1,2].
Itisproducedbythegranulosacellsofsmallantralandpre-antralfolliclesandreflectsthesizeofthepoolofthesefolli-cles[3,4].
AMHdeclineswithage[5]butremainsstableduringthemenstrualcycle[6,7]allowingitsdeterminationatrandom[8].
WehaverecentlyobservedthatAMHde-creasesduringpregnancy,showingthelowestvaluesperi-partum,followedbyasignificantincreaseofAMHduringthefirstfourdaysafterdelivery[9].
WeconcludedthattheextremelyhypogonadotropicstatusinpregnancycouldberesponsibleforthedeclineofAMHduringpregnancy,followedbyarapidrecoveryafterdelivery.
Intherespectivestudy,weusedtheBeckmanCoulterGenIIAssaywhichwasavailableuntilJuly2013.
RecentstudieshavesuggestedthatstorageconditionsledtofluctuationsofAMHvaluesdeterminedbythisassay,whereasamodifiedAMHBeck-manCoulterGenIIAssay(availablesinceJuly2013)re-sultedinmorestablevaluesofAMHindependentofstoragetimeandconditions[10,11].
Additionally,theAMHlevelsdeterminedbythemodifiedassayarereportedtobehigher[11].
Accordingtothemanufacturer,thiseffectmayreflectapossibleinteractionofcomplementwiththeconventionalassayresultinginfluctuationsduetoindivid-ualcomplementactivationduringstorage.
Themodifiedassayincludesapre-mixingstepwithahighlyanionicbuf-ferwhichremovesthiscomplement.
Itisthoughtthatpregnancyisassociatedwithcomple-mentactivation[12–15]thatmayleadtoamorepro-nounceddifferenceofAMHdeterminedbytheconventionalassaycomparedtothemodifiedassayinpregnantwomenasaresultofsamplehandlingandstor-ingconditions[11].
Accordingtotheseobservations,de-creasedAMHlevelsperipartallymaysimplybeanartificialphenomenon.
InthepresentstudywehavedeterminedAMHlevelsduringpregnancyandperipartumwiththenewmodifiedAMHassayinasubgroupofourstudypopulationinwhichtheconventionalassaywasrecentlyusedtodeter-mineAMHvalues[9].
TheaimwastoexplorewhetherthelowprepartallevelsofAMHmayhavebeenassay-dependentandartificialduetoprogressivecomplementactivationinthecourseofpregnancy.
MethodsStudypopulationPrepartalbloodsampleswerecollectedfrom62patientsagedbetween18to41years(medianage30.
6years[interquartilerange(IQR):25.
6-34.
5])inthethirdtri-mesterofpregnancyasthepatientspresentedforlabourandagain1–4daysafterdelivery.
Noneofthepatientsincludedinthisstudyexhibitedahistoryofinfertility,operationsontheovaries,chemotherapyorradiationinthepast.
Allwomenwereexaminedbetween2011and2012.
Informedwrittenconsentwasobtainedfromallwomenandthestudywasapprovedbythelocalresearchethicscommittee(number11–4643).
BloodsamplesweretakenbetweenOctober10th,2011andFebruary21th,2012.
TheconventionalassaywasusedinfrozensamplesbetweenNovember9th,2011andFebruary27th,2012.
ThemodifiedassaywasperformedusingfrozenaliquotsofthesamepatientsamplesonJuly18th,2014.
Inanothercohortof11patientsagedbetween29to39years(medianage34.
1years[IQR:32.
6-37.
8])whopresentedinourclinicbetween1995and2001,wetookbloodsamplesduringvarioustrimestersofpregnancyandevaluatedAMHlevelsatseveralpointsduringthecourseofthepregnancy.
ThedeterminationofvalueswiththeconventionalassaytookplacebetweenDecember6th,2011andFebruary12th,2012.
ThedeterminationofvalueswiththemodifiedassaytookplaceonAugust8th,2014.
Allpatientsincludedinthepresentstudywerepartofthestudypopulationsusedforourrecentlypublishedstudy[9].
Aliquotsfromidenticalbloodsampleswereusedforbothstudies.
Asnoadditionalaliquotswereavail-ableforsomeparticipantsoftheoriginalstudypopulation,only62outoftheoriginal69and11outoftheoriginal15patientswereincludedinthepresentstudy.
NosubstantialdifferencesinAMHlevels(assessedusingtheconven-tionalassay)weredetectedbetweentheoriginalstudypopulationandthesubgroupincludedinthepresentstudy.
SamplingofbloodserumBloodsamples(9ml)werecollectedfromeachwomanusingS-Monovettes(SarstedtAG&Co.
),immediatelystoredat4°Candprocessedwithin4htoavoidbloodcelllysis.
Bloodfractionationwascarriedoutbycentrifu-gationfor10minat2500xg.
Subsequently,3to4mloftheupperphaseconstitutingbloodserumwereremovedfortheassessmentofAMHlevels.
Sampleswerepipet-tedinto4aliquotsandstoredat80°C.
DeterminationofAMHlevelswiththeconventionalassayAspreviouslystated[9],wefirstusedtheenzymaticallyamplifiedtwo-siteAMH-Gen-IIELISA(BeckmanCoulter,Immunotech,Webster,Texas,USA)whichwasavailableuntilJuly2013.
Undilutedserumsamplesandcontrolsweredispensedintothewellswhichwerecoatedwithanti-AMHantibody,followedbytheadditionoftheanti-AMHdetectionantibodylabelledwithbiotin.
100μlofthestreptavidin-horseradishperox-idase(HRP)wasaddedafterwashing,followedbytheadditionof100μlofsubstratesolutioncontainingTMBfor8–12min.
UsinganautomaticELISAreader(Bio-Kningeretal.
ReproductiveBiologyandEndocrinology(2015)13:86Page2of7Rad,Hercules,CA)thedegreeofenzymaticturnoverofthesubstratewasdeterminedbydualwavelengthab-sorbancemeasurementat450nmandbetween600and630nm.
Theabsorbancemeasuredwasdirectlypropor-tionaltotheAMHconcentrationinthesampleswhichwascalculatedfromthecalibrationcurve.
Theresultswereexpressedinng/ml.
Concentrationsbelow0.
08ng/mlwereconsideredundetectable.
DeterminationofAMHlevelswiththemodifiedassayTherevisedtestprocedureincludesanadditionalassaystepbeforeaddingAMHGenIICalibrators,controls,orAMHsamplestothemicroplate.
Thisadditionalstepeliminatesthecomplementinterference.
BeforeaddingasampletotheAMHGenIIELISAmi-croplate,allcalibrators,controls,andsampleswerepre-paredwiththeAMHGenIIAssayBuffer(A56021)asfollows.
Inasampletube,1partofeachcalibrator,con-trol,ortestsamplerespectivelywasthoroughlymixedwith5partsAMHGenIIAssayBuffer(forexample,60μLcalibrator,control,orsample+300μLAMHGenIIAssayBuffer).
Nodilutionfactorwasrequired.
Withinonehour,120μLofthepremixedcalibrators,controlsandsampleswereaddedtotheappropriatewellsandthetestproceededliketheconventionalAMHGenIIassay.
StatisticalanalysisAMHlevelswerefoundnottobenormallydistributedinthestudypopulations(basedonvisualinspectionandShapiro-Wilkstest;pAMHlevelspre-andpostpartumwereanalyzedusingtheWilcoxonsignedranktestwiththelevelofstatisticalsignificancesetatα=0.
05.
AgreementofmethodsfordeterminingAMHlevelswasanalyzedusingthemethodofBlandandAltman[16].
StatisticalanalysesandboxplotswereperformedusingtheRstatis-ticalpackageversion3.
0.
2[17].
Allotherplotswerecre-atedbyusingSPSSversion20[18].
ResultsAMHlevelspre-andpostpartumComparedtotheconventionalassay,measurementofAMHvalueswiththemodifiedassayresultedinhighervalueswithamedianlevelof1.
04ng/ml(IQR:0.
40–1.
87)prepartaland0.
77ng/ml(IQR:0.
31–1.
52)postpartal(Table1).
Measure-mentswiththemodifiedassayshowedasignificantdeclineofpostpartallevelscomparedtoprepartallevelswhichisconsistentwithvaluesobtainedusingtheconventionalassay(bothpAMHmeasurementsarecombined.
TheresultssuggestthatdifferencesbetweenbothassaysincreasewithrisingAMHvaluesirrespectiveofwhetherAMHwasassessedpre-orpostpartally.
ThisisalsoreflectedbythelowermeandifferenceandthesmallerlimitsofagreementobservedforpostpartalAMHlevelscomparedtoprepartal,aspostpartalAMHlevelswereonaveragelowerthanthosemeasuredprepartal(Fig.
2bandc).
AMHlevelsduringthecourseofpregnancyComparedtothelongitudinalmeasurementsofAMHlevelsdeterminedusingtheconventionalassay(Fig.
3a)graphicalanalysisofAMHlevelsdeterminedusingthemodifiedassaysuggestsasteeperdeclineofvaluesdur-ingthecourseofpregnancy(Fig.
3b).
DiscussionTheaimofthisstudywastoinvestigatewhetherdecreas-ingprepartallevelsofAMHreportedpreviouslymayhavebeenassay-dependentandthusartificial.
Asrecentlyre-portedbyus[9]AMHlevelsdeclinedwithongoinggesta-tionalageuntilthefirstdaypostpartum.
Thisdeclinewasquestionedasbeingartificial,sincetothedevelopmentofamodifiedassay,availablesinceJuly2013,eliminatedthecomplementandresultedinlessstorage-dependentAMHfluctuationsaswellasconsistentlyhigherAMHvalues.
Itwassupposedthataserologicalfactorlikecomplementwasabletointerferewiththeconventionalassayandindi-vidualcomplementactivationresultedinfluctuations,whereasnoconsistentfluctuationpatternsweredescribedwiththemodifiedassay[11].
Accordingtothemanufac-turer'sinstructions,theconsistentlyhighervaluesdeter-minedwiththemodifiedassaywereattributabletoastepduringtheassayprocedurewherebytheeffectsofcomple-mentwereeliminated.
Sincecomplementisactivatedinpregnancy,thedeclinedescribedinourrecentlypublishedstudycouldbeconsideredasartificialduetothecomple-mentbindingactionoftheassay.
Table1AMHvaluesdeterminedwiththeconventionalandthemodifiedassay(MedianandIQR)N=62AMHprepartalinng/mlAMHpostpartalinng/mlp-valueconventionalassay0.
59(0.
22–1.
13)0.
44(0.
18–0.
91)AMHvaluesdeterminedusingthecon-ventionalaswellasthemodifiedassayseemtodeclineinthecourseofpregnancyandperipartum.
PeripartalAMHvalueswerelowerthanthoseexpectedinfertilenon-pregnantwomenofcomparableage[5].
Thedeterminationattheendofthethirdtrimesterresultedinverylowvalueswithbothassays,reflectingthegestationalageassociateddecline.
TheongoingdeclineearlyafterdeliverywasFig.
1a:BoxplotsillustratingthedistributionofAMHlevelsinwomenwithAMHmeasurementsprepartumandpostpartumassessedusingtheenzymaticallyamplifiedtwo-siteAMH-Gen-IIELISA(n=62).
Wilcoxonsignedranktest:pAMHlevelsinwomenwithAMHmeasurementsprepartumandpostpartumassessedusingtheBeckmanCoulterGenIIassay(n=62).
Wilcoxonsignedranktest:pAMHvaluesinadvancedstagesofpreg-nancyaswellasduringthefirstfourdayspostpartum.
Thus,thedeclineinAMHlevelsduringpregnancyisex-pectedtobemorepronouncedusingthemodifiedassay,ashighAMHlevelsintheearlystagesofpregnancywouldbemoreaffectedbytheinducedAMHincrease.
Wehavehypothesized[5]thatthehypogonadotropicstatusmayberesponsiblefortheAMHdecline.
RecentlypublisheddatademonstratedthatinhypogonadotropicamenorrhoeaAMHlevelswerenotdecreased[19].
An-otherstudyhasexaminedtherelationshipbetweenAMH,gonadotropins,estradiolandprogesteroneinthefirstandsecondtrimesteranddidnotfoundassociations[20].
Therefore,itmaybepossiblethatadditionalfactors(e.
g.
,theFSH-antagonistfollistatinorproductsoftheplacentawhichareincreasingduringpregnancy[21])arecontributingtothepregnancy-associatedandreversibleAMH-decline.
RecentlypublishedstudiesshowedmeaningfulAMHfluctuationsdependingonstoragetimeatroomtemperaturewithanincreaseinvaluesafter8h[11].
Themanagementofourprocessingprocedureofsam-pleswasverystrictwithastoragetimeatroomtemperatureoflessthan4handstorageat80°Cim-mediatelyafterprocessing.
Noneofthesampleswasfro-zentwiceormore.
Therefore,thestrictmanagementofsamplehandlingandstorageconditionsmayhavecon-tributedtotheconsistentresultsofthetwoassays.
Thestoragetimesat80°Cforsamplesofthelongitudinalmeasuredpatientsvariedbetween10andnearly20yearsbeforeAMHwasdeterminedusingtheconventionalassayin2011/2012andusingthemodifiedassayinFig.
2a:Bland–Altmanplots(incl.
meandifferencesand95%limitsofagreement)toillustratedifferencesinAMHbetweentheconventionalandthemodifiedassay(N=62).
Scatterplotsincludinglinearregressionlinesandlinesofequalityillustratethedifferencebetweenvaluesdeterminedwithbothassayspre-andpostpartum.
b:Bland–Altmanplots(incl.
meandifferencesand95%limitsofagreement)toillustratedifferencesinprepartalAMHlevelsbetweentheconventionalandthemodifiedassay(N=62).
Scatterplotsincludinglinearregressionlinesandlinesofequalityillustratetheprepartumdifferencebetweenvaluesdeterminedwithbothassays.
c:Bland–Altmanplots(incl.
meandifferencesand95%limitsofagreement)toillustratedifferencesinpostpartalAMHlevelsbetweentheconventionalandthemodifiedassay(N=62).
ScatterplotsincludinglinearregressionlinesandlinesofequalityillustratethepostpartumdifferencebetweenvaluesdeterminedwithbothassaysKningeretal.
ReproductiveBiologyandEndocrinology(2015)13:86Page5of72014,respectively.
Thishastobementionedtogetherwiththesmallsamplesizeof11patientsaslimitationsofourlongitudinalstudy.
Storage-dependentfluctua-tionsofAMHhavenotbeeninvestigatedsofarforstorage-timesofmorethan30weeks[11].
Thus,wecan-notruleoutthatstoragetimeandconditionshadanim-pactonourresults.
Ourstudydesignwasnotsuitabletoclarifythestorage-dependentvariance,however,wedidnotobserveanyindicationofstorage-dependentdiffer-encesinthetendencyofAMHdecliningduringpregnancy.
Incontrast,thetendencyofAMHlevelsdeterminedwiththeconventionalandthemodifiedassaywascomparablewiththedeclineinpregnancyaswellaspostpartum.
Inaddition,adilutionsteptoeliminatethecomple-ment(AMHGenIIAssayBuffer(A56021))ispartofthemodifiedassaybutnotoftheconventionalassaymethod.
Toensurecomparabilityofourresultstothoseoffurtherstudieswestrictlyfollowedthemanufacturer'sinstructionsinperformingbothassayswhichincludedmethodsoftherespectivedilutionprocedure.
However,investigatingtheexactdifferencesbetweentheappliedmethodswasnottheaimofthestudy,butrathertoshowthatthedeclineofAMHlevelsinpregnancyaswellaspostpartumwaspresentirrespectiveoftheassaysappliedforAMHdeterminationandthepreanalyticalstepforcomplementremoval.
However,newcommer-ciallyavailableassaysmaybeabletofurtherimproveAMHassessmentinthefuture.
ConclusionSincecomplementisactivatedinpregnancy,anartificialdeclineofAMHduringthecourseofpregnancycouldnotberuledoutwhendeterminationwasdonewiththecon-ventionalassayasrecentlypublishedbyus.
ThisstudyusingthemodifiednewBeckmanCoulterAMHGenIIAssaywhichremovedthepotentiallyassay-interferingcomplementverifiedtheAMHdeclineduringthecourseofpregnancyandearlyafterdelivery.
Accordingtoourdata,theAMHdeclineinpregnancywasnotartificial.
Fig.
3a:LongitudinalmeasurementofAMHlevelsdeterminedwiththeconventionalassay.
b:LongitudinalmeasurementofAMHlevelsdeterminedwiththemodifiedassayKningeretal.
ReproductiveBiologyandEndocrinology(2015)13:86Page6of7CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsA.
K.
gavesubstantialcontributionstoconceptionanddesign,toacquisitionofdataandinterpretationofdata,draftingthearticleandfinalapprovaloftheversiontobepublished.
B.
S.
andS.
N.
gavesubstantialcontributionstoanalysisandinterpretationofdata,revisingthearticlecriticallyforimportantintellectualcontentandfinalapprovaloftheversiontobepublished.
P.
M.
,D.
D.
andR.
K.
gavesubstantialcontributionstoacquisitionofdata,revisingthearticlecriticallyforimportantintellectualcontentandfinalapprovaloftheversiontobepublished.
T.
S.
andA.
G.
gavesubstantialcontributionstoconceptionanddesign,draftingthearticle,revisingitcriticallyforimportantintellectualcontentandfinalapprovaloftheversiontobepublished.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsTheauthorswouldliketothankthelaboratorystaffforthesupportandcontinualhelpinsamplingandstorageofthebloodsamplesespeciallyJensRasch,UteKirsch,SieglindeArndtandGabrieleSehn.
WefurtheraregratefultoGülenYerlikayaandAlexisKauthforobtainingthepatientsamples.
ManythankstoMaryVigilforediting.
Wearealsogratefultoallwomenwhotookpartinthisstudy.
Authordetails1DepartmentofGynecologyandObstetrics,UniversityofDuisburg-Essen,Hufelandstrasse55,45122Essen,Germany.
2InstituteforMedicalInformatics,BiometryandEpidemiology(IMIBE),UniversityofDuisburg-Essen,Hufelandstrasse5545122Essen,Germany.
3DepartmentofGynecologicalEndocrinologyandReproductiveMedicine,UniversityofHeidelberg,Vostrasse9,69115Heidelberg,Germany.
Received:7May2015Accepted:27July2015References1.
FanchinR,SchonuerLM,RighiniC,GuibourdencheJ,FrydmanR,TaiebJ.
SerumAnti-MüllerianhormoneismorestronglyrelatedtoovarianfollicularstatusthanseruminhibinB,estradiol,FSHandLHonday3.
HumReprod.
2003;18(2):323–7.
2.
deVetA,LavenJS,deJongFH,ThemmenAP,FauserBC.
Anti-Müllerianhormoneserumlevels:aputativemarkerforovarianaging.
FertilSteril.
2002;77(2):357–62.
3.
DurlingerAL,VisserJA,ThemmenAP.
Regulationofovarianfunction:theroleofAnti-Müllerianhormone.
Reproduction.
2002;124(5):601–9.
4.
WeenenC,LavenJS,VonBerghAR,CranfieldM,GroomeNP,VisserJA,etal.
Anti-Müllerianhormoneexpressionpatterninthehumanovary:potentialimplicationsforinitialandcyclicfolliclerecruitment.
MolHumReprod.
2004;10(2):77–83.
5.
KelseyTW,WrightP,NelsonSM,AndersonRA,WallaceWH.
AvalidatedmodelofserumAnti-Müllerianhormonefromconceptiontomenopause.
PLoSOne.
2011;6(7),e22024.
6.
KningerA,KochL,EnekweA,BirdirC,Kasimir-BauerS,KimmigR,etal.
Changeofanti-Mullerian-hormonelevelsduringfollicularphaseinPCOSpatients.
GynecolEndocrinol.
2015;31(1):26–30.
7.
LaMarcaA,StabileG,ArtenisioAC,VolpeA.
Serumanti-Mullerianhormonethroughoutthehumanmenstrualcycle.
HumReprod.
2006;21(12):3103–7.
8.
LaMarcaA,BroekmansFJ,VolpeA,FauserBC.
MacklonNS;ESHRESpecialInterestGroupforReproductiveEndocrinology–AMHRoundTable.
Anti-Mullerianhormone(AMH):whatdowestillneedtoknowHumReprod.
2009;24(9):2264–75.
9.
KningerA,KauthA,SchmidtB,SchmidtM,YerlikayaG,Kasimir-BauerS,etal.
Anti-Mullerian-hormonelevelsduringpregnancyandpostpartum.
ReprodBiolEndocrinol.
2013;11:60.
10.
RustamovO,SmithA,RobertsSA,YatesAP,FitzgeraldC,KrishnanM,etal.
Anti-Mullerianhormone:poorassayreproducibilityinalargecohortofsubjectssuggestssampleinstability.
HumReprod.
2012;27(10):3085–91.
11.
HanX,McShaneM,SahertianR,WhiteC,LedgerW.
Pre-mixingserumsampleswithassaybufferisaprerequisiteforreproducibleanti-MullerianhormonemeasurementusingtheBeckmanCoulterGenIIassay.
HumReprod.
2014;29(5):1042–8.
12.
JohnsonU,GustaviiB.
Complementcomponentsinnormalpregnancy.
ActaPatholMicrobiolImmunolScandC.
1987;95(3):97–9.
13.
DerzsyZ,ProhászkaZ,RigóJrJ,FüstG,MolvarecA.
Activationofthecomplementsysteminnormalpregnancyandpreeclampsia.
MolImmunol.
2010;47(7–8):1500–6.
14.
DennyKJ,WoodruffTM,TaylorSM,CallawayLK.
Complementinpregnancy:adelicatebalance.
AmJReprodImmunol.
2013;69:3–11.
15.
Segura-CervantesE,Mancilla-RamirezJ,ZuritaL,ParedesY,ArredondoJL,Galindo-SevillaN.
BloodSC5b-9complementlevelsincreaseatparturitionduringtermandpretermlabor.
JReprodImmunol.
2015.
109:24-30.
16.
BlandJM,AltmanDG.
Statisticalmethodsforassessingagreementbetweentwomethodsofclinicalmeasurement.
Lancet.
1986;1:307–10.
17.
RCoreTeam.
R:Alanguageandenvironmentforstatisticalcomputing.
Vienna,Austria:RFoundationforStatisticalComputing;2014.
availableathttp://www.
R-project.
org/.
18.
IBMCorp.
Released.
IBMSPSSstatisticsforwindows,version20.
0.
ArmonkNY:IBMCorp;2011.
19.
LieFongS,SchipperI,ValkenburgO,deJongFH,VisserJA,LavenJS.
Theroleofanti-Müllerianhormoneintheclassificationofanovulatoryinfertility.
EurJObstetGynecolReprodBiol.
2015;186:75–9.
20.
LutterodtM,ByskovAG,SkoubySO,TaborA,YdingAndersenC.
Anti-Müllerianhormoneinpregnantwomeninrelationtootherhormones,fetalsexandincirculationofsecondtrimesterfetuses.
ReprodBiomedOnline.
2009;18(5):694–9.
21.
RaeK,HolleboneK,ChettyV,ClausenD,McFarlaneJ.
Follistatinserumconcentrationsduringfull-termlabourinwomen—significantdifferencesbetweenspontaneousandinducedlabour.
Reproduction.
2007;134:705–11.
SubmityournextmanuscripttoBioMedCentralandtakefulladvantageof:ConvenientonlinesubmissionThoroughpeerreviewNospaceconstraintsorcolorgurechargesImmediatepublicationonacceptanceInclusioninPubMed,CAS,ScopusandGoogleScholarResearchwhichisfreelyavailableforredistributionSubmityourmanuscriptatwww.
biomedcentral.
com/submitKningeretal.
ReproductiveBiologyandEndocrinology(2015)13:86Page7of7