6 182

T H Johannsen and others Peptide hormones and DSD 182:6 P1–P15 Consensus Statement

Peptide hormone analysis in diagnosis and treatment of Differences of Sex Development: joint position paper of EU COST Action ‘DSDnet’ and European Reference Network on Rare Endocrine Conditions T H Johannsen1,2, A-M Andersson1,2, S F Ahmed3, Y B de Rijke4, R F Greaves5,6,7, M F Hartmann8, O Hiort9, P-M Holterhus10, N P Krone11, A Kulle10, M L Ljubicic1,2, G Mastorakos12, J McNeilly13, A M Pereira14, A Saba15, S A Wudy8, K M Main1,2 and A Juul1,2 on behalf of Working Group 3 ‘Harmonisation of Laboratory Assessment’ of the European Cooperation in Science and Technology (COST) Action BM1303 ‘DSDnet’ and Work Package 5 ‘Diagnostics and Laboratory Analysis’ of the European Reference Network on Rare Endocrine Conditions 1Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 2International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 3Developmental Endocrinology Research Group, School of Medicine, Dentistry and Nursing, University of Glasgow, Glasgow, UK, 4Department of Clinical Chemistry, Erasmus MC, University Medical Center, Rotterdam, Netherlands, 5Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Parkville, Victoria, Australia, 6School of Health and Biomedical Sciences, RMIT University, Melbourne, Victoria, Australia, 7Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia, 8Steroid Research & Mass Spectrometry Unit, Laboratory for Translational Hormone Analytics, Division of Pediatric Endocrinology and Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen, Germany, 9Division of Pediatric Endocrinology and Diabetology, Department of Paediatrics and Adolescent Medicine, University of Luebeck, Luebeck, Germany, 10Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, Christian- Albrechts-University, Kiel, Germany, 11Academic Unit of Child Health, Department of Oncology and Metabolism, University of Sheffield, Sheffield, 12UK, Unit of Endocrinology, Diabetes Mellitus and Metabolism, Aretaieon Hospital, Correspondence Faculty of Medicine, National and Kapodistrian University of Athens, Athens, Greece, 13Department of Biochemistry, should be addressed Queen Elizabeth University Hospital, Glasgow, UK, 14Department of Medicine, Division of Endocrinology, Leiden to T H Johannsen University Medical Centre, Leiden, Netherlands, and 15Department of Surgical, Medical and Molecular Pathology and Email Critical Care Medicine, University of Pisa, Pisa, Italy trine.holm.johannsen@ regionh.dk European Journal of Endocrinology

Abstract

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.

European Journal of Endocrinology (2020) 182, P1–P15

https://eje.bioscientifica.com © 2020 European Society of Endocrinology Published by Bioscientifica Ltd. https://doi.org/10.1530/EJE-19-0831 Printed in Great Britain Downloaded from Bioscientifica.com at 10/02/2021 04:05:13AM via free access

-19-0831 European Journal of Endocrinology https://eje.bioscientifica.com conditions isillustratedherein. and managementofthese specificandrareendocrine the qualityofpeptidehormone analysesinthediagnosis and monitoringofpatients with DSD.Theimportanceof assurance ofpeptidehormoneassaysusedforthediagnosis this paper, we discusstheaspectsthatunderliequality hormones inpatientssuspectedofDSDconditions.In present apositionpaperonthequantificationofpeptide Analysis) therefore Endo-ERN (DiagnosticsandLaboratory Assessment)inDSDnetand WorkLaboratory Package5in in relationtoDSD.Working Group3(Harmonisation of is currentlynopositionpaperaddressingpeptidehormones addressing diagnosticgeneticapproachesinDSD( analysis indiagnosisandtreatmentofDSD( published twopositionpapers,oneonsteroidhormone predominantly steroidhormones.DSDnetrecently distinct groups:peptideandnon-peptidehormones, requires biochemicalandgeneticassessment. assessment ofpatientswithknownorsuspectedDSD laboratories with knowledgeofDSD. The laboratory the necessityofnetworksconsistinghighlyspecialized in theinitialevaluationofchild( specialists, thereby resultingina substantial variation of havingDSDisinfluenced by appropriateaccess to reported that the diagnostics of a newborn suspected patient organizations.Arecentlarge,internationalsurvey ERN isanetworkcomposedofhealthcareprovidersand to clinicians,aswellpeoplewithDSD,whileEndo- of allpeopleinterestedinDSD,fromleadingscientists endo-ern.eu on rareendocrineconditions(Endo-ERN; in collaborationwiththeEuropeanReferenceNetwork active between 2013 and 2018.Thistask was performed DSDnet (BM1303)( Cooperation inScienceandTechnology (COST)Action assessment for DSD was one of the tasks of the European crucial tooptimizediagnosisandtreatmentmonitoring. age-adjusted sex-specificreferencevaluesaretherefore analyses withhighsensitivityandspecificityaswell Due tothecomplexityoftheseconditions,laboratory chromosome DSD,allwithawidephenotypicspectrum. (1.)46,XXDSD,(2.)46,XYand(3.)sex : are medicalconditionsthatclassifiedaccording to chromosomal, gonadal,oranatomicalsex( variety ofcongenitalconditionscharacterizedbyatypical Differences ofSexDevelopment(DSD)comprisea Introduction Consensus Statement Biochemical hormone analysis comprises two Biochemical hormoneanalysiscomprisestwo Development ofrecommendationsonlaboratory ), formed in 2017. DSDnet was a network http://www.dsdnet.eu T HJohannsenandothers 2 ). Thisunderlines ), whichwas http://www. 3 ) and one ) andone 1 4 ). DSD ). There ). There plasma concentrationsofAMH andInhibinBinboys and InhibinBbetweensexes ( is awell-knowndifference inconcentrationsofAMH Inhibin Bisthedownregulation ofFSHsynthesis.There a partoftheTGF consisting oftwosubunits( Inhibin Bisadimeric disulphide-linked glycoprotein uterus, andtheupperpartofvaginaaredeveloped. In females,intheabsenceofAMH,Fallopiantubes, β and amemberofthetransforminggrowthfactorbfamily which resembleDSDconditions. with micropenisandbilateralcryptorchidism atbirth, andCHHmaypresent A newbornwitha46,XYkaryotype 45,X/46,XY ( reported forchildrenduringthefirsthoursoflife( dimorphism in concentrations has been LH andlowerconcentrationsofFSHthangirls.Thissex granulosa cells. in both the testicular Sertoli cells and the ovarian cells. FSHstimulatesAMHandInhibinBproduction testicular Leydigcellsandintheovarianthecainterna Postnatally, LHstimulatestestosteroneproductioninthe to the FSH-receptor and LH-receptor, respectively. of a common glandandareglycoproteins consisting anterior pituitary (AMH), andInhibinB.FSHLHaresynthesizedinthe (FSH), luteinizinghormone(LH),anti-Müllerian include thegonadotropinsfollicle-stimulatinghormone testing andduringmonitoringofDSDconditions Analysis ofpeptidehormonesisimportantinfirst-line Clinical applicationofpeptidehormones fetuses firstisinitiatedinthe36 the Müllerian ducts, whereas AMH production in female testicular differentiationandisessentialforregression of congenital hypogonadotropichypogonadism(CHH)( (e.g.45,X( high, asseeninprimary of diagnosticvaluebothwhentheirconcentrationsare reproductive function.Additionally, gonadotropinsare reflects anearlywindowofopportunityinwhichtostudy in boysandlowgirls( the sexes ( of LHdividedbyFSHisanexcellentclassifierinseparating (HPG)axisininfancyoccurs–theratio pituitary-gonadal a transient, postnatal activation of the hypothalamic- ( the first month of life( Peptide hormonesandDSD 7 (TGF ). Thus, especially in mini- – the period in which During infancy, boyshavehigherconcentrationsof AMH isahomodimericdisulphide-linkedglycoprotein β ). In male fetuses, AMH is synthesized soon after 8 ). At this point in time, the LH/FSH ratio is high 10 α ), 47,XXY( -subunit and aunique β proteinfamily. Themainroleof 6 ), and duringthe first years oflife Downloaded fromBioscientifica.com at10/02/202104:05:13AM Fig. 1 11 α )), andwhenlow, asseenin and ). Mini-pubertytherefore th Figs 1 weekofgestation( β 182 ) andis,likeAMH, , β :6 -subunit binding 2 and 5 3 ), during ). Thus, P2 13 12 9 via freeaccess ). ). ),

European Journal of Endocrinology Marseilles, France).Modifiedfrom ( immunometric assay(Immunotech, BeckmanCoulterLtd., y-axis. AMHwasmeasuredusing adoubleantibodyenzyme- ± red linesmarkmaleandfemale referenceranges(mean, values duringinfancyareconnectedviablacklines.Blueand and 926healthyfemales(red)throughoutlife.Longitudinal Serum concentrationsofAMHin1027healthymales(blue) Figure 2 and may, togetherwiththegonadotropins, be usedinthe in girls reflecting ovarian function and follicle reserve and InhibinBconcentrationsareviablebiomarkers absence offunctioningtesticulartissue( conditions, aslowconcentrationsaremarkersforthe in theevaluationofgonadaltypeandfunctionDSD and Inhibin B therefore represent important biomarkers higher thantheconcentrationsseeninmen( mini-puberty, theInhibinBconcentrationsinboysare ( have beenreportedtobe,respectively, upto100times shown onalog10-transformedy-axis(dottedlines:limitofdetection).Modifiedfrom( Müllerian hormone(AMH)in1041healthymales(blue)and799females(red)duringmini-puberty.Theconcentrationsare Serum concentrationsofluteinizinghormone(LH),follicle-stimulating(FSH),theLH/FSH-ratio,InhibinB,andanti- Figure 1 14 2 Consensus Statement s ) and10timeshigher( . d . ). Theconcentrationsareshown onalog10-transformed 15 24 ) thaningirls.During T HJohannsenandothers ) and( 25 ). 16 , 17 15 ) ). AMH . AMH later namedSerotec,Oxford,UK ( immunometric assay(OxfordBio-Innovation, Oxfordshire,UK; Inhibin Bwasmeasuredusing a doubleantibodyenzyme- The concentrationsareshown onalog10-transformedy-axis. lines markmaleandfemalereference ranges(mean, and 1344healthyfemales(red)throughoutlife.Bluered Serum concentrations of Inhibin B in 1161 healthy males (blue) Figure 3 cell function,ahumanchoriogonadotropin(hCG)testcan cell function,whiletestosteroneproductionreflectsLeydig failure. Additionally, asAMHproduction reflects Sertoli orprematureovarian diagnostics of,forexample,primary a normalAMHconcentrationbeforehCGadministration by Ahmed response shouldbedonecautiouslyasdescribedindetail administration, buttheinterpretationoftestosterone low. The test evaluates the testosterone response to hCG until puberty, wherethegonadotropinconcentrationsare the testisusefultoHPGaxisafterinfancyand stimulates Leydig cells directly to produce testosterone, offer furtherinformationongonadalfunction.AshCG Peptide hormonesandDSD et al. ( 18 8 ). ). Inarecent,largestudyofhCGtesting, Downloaded fromBioscientifica.com at10/02/202104:05:13AM 73 https://eje.bioscientifica.com )). 182 :6 ± 2 s . d P3 . ). via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com has recentlybeensuggested byseveralgroupsasanovel LH concentration concentrations in serum. Thus, urinary gonadotropin releasinghormone (GnRH)-stimulatedLH signs ofpuberty( increase inLHconcentrations occursbeforetheclinical acceptable. Additionally, infirstmorningvoided urine,an stable andfrequentlyanalyzedbylaboratories,serum is in plasma,butasthesegonadotropinsarerelatively to centrifugation).FSHandLHcanalsobemeasured (i.e. eliminatingthewaitingtimeforclottingprior facilitates bothstabilityandthespeedofprocessing of choiceforAMHandInhibinB.Theuseplasma of thesehormones.Plasmaandserumarethematrices consulted forthepreferredmatrixandstabilityeach method standardoperatingprocedurethereforeshouldbe to transport.Thepackageinsertand/ortheanalytical and durabilityofthesampleaswellcorrectway for sampleintegrity. Suchknowledgeincludesstorage from collectiontofreezingormeasuringisimportant knowledgeofthetimelyprocessing device), thenecessary technique (includingknowledgeoftypecollection of thesamples.Inadditiontoanappropriatesampling correct patientpreparationandpre-analyticalhandling aware ofthelaboratories’recommendationsregarding research settings.Itisrecommended thatcliniciansbe haveprovenvaluableinpreliminary matrix forpeptidehormoneanalyses,whileurinary Blood (serumandplasma)isthemostcommonlyused Impact ofmatrices relevant steroidhormonesandgeneticanalyses. based onclinicalexaminationandsupplementedwith B. Therequestforthesepeptidehormonesshouldbe of patientswithDSDincludeFSH,LH,AMH,andInhibin Position 1 measurements ofsteroidhormonesandgenetictests. of peptidehormonesmostlyneedtobesupplementedby initial evaluationofthepatientwithDSD.Theanalyses failureintheHPGaxis andsecondary primary a rapidandpowerfultooltodifferentiatebetween gonadotropins, AMH,and/orInhibinBthereforeprovides hCG test( on the testicular function, although it cannot replace the concentration providesimportantadditionalinformation a subnormal testosterone response ( subnormal AMHconcentrationdidnotalwayspredict was correlatedtoanormalhCGresponse.However, a Consensus Statement In summary, thecombinationofmeasuring 18 : Peptidehormonesofinterestforthediagnosis ). 20 ) andiscorrelatedwithbasal and T HJohannsenandothers 19 ). Thus, the AMH laboratories isprovidedinSupplementary Table1. within Endo-ERN.Afulldescription ofmethodsand are fromaquestionnairesurvey amongReferenceCenters Network on Rare Endocrine Conditions (Endo-ERN). The replies Sex Development&Maturation hormone (AMH, (FSH, Reported analyticalplatformsforfollicle-stimulatinghormone Figure 4 on theiruseofbiochemicalanalyses.Indevelopment among its associated 71 Reference Centers based survey The Endo-ERNrecentlyconductedaquestionnaire- Analytical methods settings. Furthermore, LHcanbemeasuredinurineexperimental for thedeterminationofFSH,LH,AMH,andInhibinB. Position 2 available inthiscase. been reported( 25 prematurity ( in referencerangesforgonadotropinsextreme useful for peptide hormonemeasurements, for example, 22 non-invasive method to evaluate pubertal progress ( fluorescent-labelled ligands.Thebenefitsimmunoassays using, for example, chemiluminescent, enzymatic, or All laboratoriesreportedtheuseofimmunoassays. materials supplementary 14 replies( resulted in29replies,AMH26andInhibinB Development &Maturation reporting a platform/method within the topic this position statement, the 33replies from 13 countries Peptide hormonesandDSD ). TheuseofsalivaforFSHandLHdeterminationhas ). Furthermore,theuseofumbilicalbloodhasproved In immunoassays, the analytes are typically measured n = 29), luteinizinghormone(LH, Fig. 4 : Serumorplasmaisthepreferredmatrix 23 n 26 and Supplementary Table andSupplementary 1,seesectionon ) andforAMHinhealthynewborns( = 26), andInhibinB( ), butthereislittlevalidationevidence Downloaded fromBioscientifica.com at10/02/202104:05:13AM givenattheendofthisarticle). wereincluded oftheEuropeanReference n n = 29), anti-Müllerian = 14) withinthetheme 182 :6 . FSHandLH P4 24 21 Sex via freeaccess , , European Journal of Endocrinology improvements inspecificity andthesimultaneous (LC-MS/MS)methodstoallowfor mass-spectrometry laboratories towardliquid chromatography-tandem for interpretation, which have led to the move by some assay, sothismaydifferbetweenmanufacturers aswell. guidelines differonhowtocalculatethelowerlimitofan Tablefor InhibinB(Supplementary 1).Furthermore, from 0.07to2.5pmol/LforAMH,and110pg/mL from 0.02to0.5IU/LforFSH,0.05LH, ranged used. The‘lowervalue’reportedfromthesurvey laboratories, evenwhenthesamemanufacturer’s kitwas survey, the‘lowervalue’that wasreportedvariedbetween of the sensitivity of these methods. In the Endo-ERN entirely comparable.Nevertheless,theygiveanindication quantification, orsomethingelse,andthustheymaynotbe analytical sensitivity, thelimitofdetection, Table 1,thelisted‘lowervalues’maycorrespondto limit ofanassaydiffer. In to reach 20%. However, guidelines to calculate the lower or thepointatwhichlevelofimprecisionisfound sensitivity isusually a functionof the lowestcalibrator relative RPtowhichthisappliesto. guidelines and publications, it isimportant to include the of characterizingreferencevaluesanddecisionpointsin decision points will need adjusting ( calibration slopeandthereforethereferencevalues peptide hormoneRPcancauseadramaticchangeinthe 575.pdf code: 83/575, NIBSC Standard Follicle Stimulating Hormone, Pituitary the referenceinformation(e.g.WHOInternational calibration, andtheirpackageinsertsroutinelyprovide the basisformanufacturers’peptidehormoneassaykit on BiologicalStandardization.Thesepreparationsprovide which are endorsed through the WHO Expert Committee developing peptidehormonereferencepreparations(RPs), of Organization (WHO)providesaninvaluableservice challenge. Within thislimitation,theWorld Health so metrologicaltraceabilitytoa‘true’valueisanongoing only oneanalyteismeasuredatatime. (or higherdetectionlimit).Additionally, inimmunoassays reactivity and/ormatrixeffectsand,thus,lowspecificity exist. However, immunoassaysmaybesubjecttocross- AMH ( For some of the analytes, for example, gonadotropins ( gained withthemandtheiravailabilitytoalllaboratories. offer includetheextensiveexperiencethathasbeen Consensus Statement The specificityofimmunoassays createschallenges The lowerlimitofquantitationorfunctional Peptide hormonescanexistinmanymolecularforms, 28 ). Exampleshaveariseninwhichchangetoa ), and Inhibin B ( https://www.nibsc.org/documents/ifu/83- 29 Table 1 ), highly sensitive methods T HJohannsenandothers and Supplementary andSupplementary 30 ). Hence, as part 27 ),

Position 3: clinical application. the futurechallengesliewithinvalidationfor the LC-MS/MS techniques for peptide quantification, above the needed range for the diagnostic peptides. With analysis ( the detectionoflessabundantcomponentsbyLC-MS/MS concentration rangeofproteinsinplasmamayhinder approach for analysis. In addition, the large dynamic and thechoiceofusingabottom-uportop-down relevant standardsandinternalareavailable, molecular weight(s)toincludeinquantitation,whether for peptide analysis include defining the peptide MS, clinicalapplicationsarestilllacking.Considerations while there is an increasing interest in the use of LC-MS/ as steroids.However, withinthefieldofproteinanalysis, the quantificationofsmall,well-definedmoleculessuch techniques havenowbeensuccessfullyintroducedfor determination ofmultipleanalytes( in which17-hydroxyprogesterone andandrostenedione monitoring, as exemplified in acceptably treated CAH, asoptimaldisease limits maynotinallcases serve value of a compound within the corresponding reference and adolescents. Furthermore, the aim of getting the values isoftenachallenge,especiallyinyoungchildren for the establishment of sex- and age-related reference range ( the LH/FSH-ratiohasbeenreportedtoliewithinmale healthy boys and girls, and in infants with complete AIS, infants, theLH/FSH-ratiohasbeenshowntoseparate may addintheevaluationofDSD;forexample,healthy reference datawithcut-offvaluesforratiosofhormones addition tothereferencedataforhormonesindividually, Inhibin Barepreviouslyreported(e.g.( 3 This is illustrated with AMH and Inhibin B in concentrations accordingtoageanddevelopmentalstage. to thesexdimorphismandphysiologicalfluctuationsin importance, especiallyforreproductivehormonesdue Sex- and age-related reference values are of paramount availability ofreferencedatafromhealthyvolunteers. measured concentrationsofanybiomarkeristhe An importantbasisintheclinicalevaluationof Reference values immunoassay isthecurrentandrecommendedmethod. be implementatedinadiagnosticsetting,measurementby measurement ofFSH,LH,AMH,andInhibinBhasyetto Peptide hormonesandDSD , respectively. ReferencerangesforFSH,LH,AMH,and 8 ). However, therecruitmentofhealthyvolunteers 32 ), therebyresultingindetectionlimitsthatare AsLC-MS/MStechnologyforthe Downloaded fromBioscientifica.com at10/02/202104:05:13AM https://eje.bioscientifica.com 182 24 :6 , 31 25 ). MS-based , 33 Figs 2 , 34 )). In and P5 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com using absoluteconcentration values,thetransformation despite adequateclinicalcontrol ( concentrations generallyare high-normaltoelevated Assessment ServiceforPeptideHormones (Distributionno.:445,February21,2017). calibrator value(exceptthe0-calibrator),whateverpresent;RUO,Research use-onlyanalysis;UKNEQAS,UnitedKingdomNationalExternalQuality Determinations B(Distributionno.:2017/03);Low,Lowestreportablevalue; i.e.ananlyticalsensitivity,limitofdetection,quantification,lowest (exact valuemaydependonthekit),whateverpresent;Labquality(AMH): TumourMarkers(Distributionno.:2017/02);Labquality(FSHandLH):Hormone Bio-Rad: Immunoassay(Monthly)Program(Distributionno.:Cycle14.Dec 2016–Dec2017,SampleNo.8);High,Highestreportablevaluewithoutdilution EQA organizers but dataonnumberofRocheElecsysandCobase411areadded from LabQuality;Permissiontopublishthisinformationhasbeengiventhe † EQA-programs currentlyexistforInhibinB.ForBisshownthemost commonlyusedInhibinB-assaysfoundbyaninternetsearchatPubMed. assurance programs(Bio-Rad,Labquality,andUK-NEQAS)arelistedwith numbersofparticipants(percentagesinbrackets).Nowidelyavailable For FSH,LHandAMH,theplatformgroupsthatconstitutedatleast1%of thetotalnumberofplatformssigneduptoatleastoneexternalquality ANSH LABSInhibinBELISAAL-107(RUO) Oxford Bio-InnovationLtd.** Serotec** DSL** Beckman CoulterInhibinGenIIELISA INHIBIN B,pg/mL Beckman CoulterAMHGenII Beckman CoulterAccess Roche Elecsys/Cobas AMH, pmol/L Perkin ElmerAutoDelfia DiaSorin Liaison Tosoh Vitros Systems bioMérieux VidasGroup Beckman CoulterAccess&UnicelDxI Abbott Architect Siemens Dimension&Vista Siemens Immulite Siemens AdviaCentaur Roche Cobas6000&8000 Roche ModularE&Cobase601-e801 Roche Elecsys&Cobase411* LH, IU/L Perkin ElmerAutoDelfia DiaSorin Liaison Tosoh Vitros Systems bioMérieux VidasGroup Beckman CoulterAccess&UnicelDxI Abbott Architect Siemens DimensionVista Siemens Immulite Siemens AdviaCentaur Roche Cobas6000&8000 Roche ModularE&Cobase601-e801 Roche Elecsys&Cobase411* FSH, IU/L (LH), anti-Müllerianhormone(AMH),andInhibinBnumbersofinstrumentsaccordingtoanalyticalplatforms. Table 1 Instruments forAMHsigneduptoUKNEQAS;**Nolongercommercially available; n n n Consensus Statement Lowest andhighestmeasurementrangeswithoutdilutionoffollicle-stimulatinghormone(FSH),luteinizing T HJohannsenandothers 35 ). Thus,insteadof 0.1 (e801:0.3) 0.10 0.05 0.25 0.1 0.66 0.1 0.2 0.05 0.2 0.1 0.3 0.1 0.1 (e801:0.3) 0.10 1.6 15 15 7 7 0.57 0.14 0.071 0.05 0.2 0.1 0.216 0.1 0.2 0.09 0.2 0.05 0.1 0.10 Measurement range Low term monitoringofTurner syndromepatients,forwhich management inthefuture. Forexample,inthelong- of absolutevaluesinto Peptide hormonesandDSD ‡ measurement rangeisprovidedinpg/mL;*RangeforRocheElecsys, 160.7 164.2 1390 1000 1000 1000 1000 High 200 200 256 400 250 200 110 200 150 200 170 200 200 200 200 171 250 250 250 200 100 250 250 150 200 200 200 260 (16) 168 (11) 305 (19) 300 (19) 291 (18) 269 (17) 167 (10) 299 (19) 304 (19) 301 (19) 18 (1) 15 (1) 27 (2) 49 (3) 80 (5) 53 (3) 19 (1) 24 (24) 28 (28) 44 (44) 14 (1) 26 (2) 50 (3) 81 (5) 52 (3) Downloaded fromBioscientifica.com at10/02/202104:05:13AM s Bio-Rad 1 (0.1) 5 (0.3) 1 (0.1) 6 (0.4) . 1601 1600 d 99 Number ofinstruments . -scores mayimprovepatient † 182 :6 35 (37) 15 (16) 11 (11) 17 (18) 35 (36) 16 (89) 14 (15) 12 (13) 16 (17) NA NA NA NA NA 94 96 18 5 (5) 1 (1) 1 (1) 1 (1) 2 (2) 1 (1) 7 (7) 5 (5) 2 (11) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 7 (7) Labquality (%) P6 via freeaccess European Journal of Endocrinology in whichtraceabilityisensured toareferencesystemthat as appliedtoserumtestosterone) and(2.)harmonization, (SI) and‘trueness’isreached byatop-downapproach(e.g. traceability isensuredtothe InternationalSystemofUnits metrological traceabilityare (1.) standardization,inwhich reference materials ( reference systemcomprisingmethodsand calibration of these procedures are traceable to a shared procedures measurethesamequantityandthat is, theprocessbywhichitisensuredthatmeasurement attained byestablishingmetrologicaltraceability;that The comparability of results across laboratories can be Harmonization oflaboratorytests the analyticalmethod. and decisionlimitsshouldincludeinformationrelatedto and InhibinB.Publicationscontainingreferencevalues in theevaluationofpeptidehormonesFSH,LH,AMH, stage, sex-andage-relatedreferencevaluesaremandatory fluctuations in concentrations with age and developmental Position 4: laboratories ( comparable evaluation of measurements from different across laboratories. This will present the opportunity for is, common reference values can be developed and used differences between laboratories can be minimized; that With tests,systematic thealignmentoflaboratory up tosixtimesthebasallevelshasbeenreported( with AISinmid-childhood,asanexaggeratedLHresponse may beusefulevenintheevaluationofpatientspresenting controversy surrounding this issue exists. Furthermore, it permanent hypogonadotropic hypogonadism, although value inseparatingconstitutionaldelayofpubertyfrom A GnRHtestadditionallymayhavesomediagnostic may increase to 10 IU/L in healthy prepubertal girls ( GnRH testresultsingirlsaged1to3yearswhomLH However, cautionshouldbeconsideredwhenevaluating ( ( before andafterGnRH.AbasalLHofatleast0.3IU/L standard GnRHtestwithgonadotropinmeasurements would simplifyandadvanceclinicalmanagement. therapy usinggonadotropins( for decidingthetimeanddoseofestrogenreplacement as predictivetoolsforovarianfunction( measurements ofgonadotropinsandAMHaresuggested 37 37 Consensus Statement , ) oramaximalGnRH-stimulatedLHabove5IU/L Evaluation ofcentralprecociouspubertyincludesa 38 ) isconsideredasignofcentralHPGactivation. Duetosexdimorphismandphysiological 41 ). 42 ). The two approaches to reach 9 T HJohannsenandothers ), theuseof 9 , 36 ) aswell s . d . -scores 39 40 ). ). peptides. Instead, laboratory comparisonsareachieved peptides. Instead,laboratory laboratories performreferencemeasurementsforthese as referencemethodsarenotdefinedandno Inhibin B),trueabsolutevaluescannotbedetermined, values havebeenestablished. (imprecision) errors, and sex- and age-specific reference determination ofbothsystematic(bias)andrandom for whichatleastparameterslikeaccuracy, including mustuseanalyticalvalidation, The individuallaboratory medicineiscrucial. and thereforeagreementinlaboratory diagnostics and treatmentfollow-up of theseconditions, toimprove so internationalcollaborationisnecessary in supporting agreement ( external qualityassuranceprogramsplayacentralrole among routinemeasurementprocedures). via abottom-upapproach(e.g.inter-methodcomparison is nottraceabletoSI,butinsteaddecidedbyagreement Tumour Markers(AMH)(Labquality, Helsinki, Finland), programs HormoneDeterminations B(FSHandLH) LH) (Bio-Rad Laboratories, CA, USA),theLabquality Bio-Rad Immunoassay(Monthly) Program(FSHand (used inCopenhagen,DK), forFSH,LH,andAMH:the depicts anexampleoffourrepresentativeEQAprograms comparability intheresultsacrosslaboratories. (EQA) programsshouldbeaprerequisitetoachieve Coulter AMHGenIIELISAassay. was becauseRochestandardizedagainsttheBeckman consistency overawiderangeofconcentrations( and BeckmanCoulterAccess2)yieldedhighdegrees of two widelyavailableautomatedassays(RocheElecsys calibrations ( based onvariousantibodycombinationsand confusing due to the implementation of different assays routine measurements of AMH has been complicated and the measurementofAMH.Thus,introduction B, while no international standard currently exists for International standardsexistforFSH,LH,andInhibin method comparisonalsoforthesepeptidehormones. does notmimictheclinicalsamples)maychallenge non-commutability (i.e.thatthereferencematerial to, for example, matrix effects in the reference material, forclinicalsamples( relation observed forareferencematerialandthe the relationobserved measurement procedures’closenessofagreementbetween for areferencematerialiscommutability, whichistwo Standards) thatareuntraceabletoSI.Aprerequisite using referencematerials(e.g.WHOInternational Peptide hormonesandDSD For many peptides (including FSH, LH, AMH, and With bothharmonizationandstandardization, Participation inexternalqualityassurance 44 ). However, recentlyitwasshownthat Downloaded fromBioscientifica.com at10/02/202104:05:13AM 43 ). DSD are rare conditions, https://eje.bioscientifica.com 182 42 :6 ). However, due 44 Table 1 ). This P7 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com 1 in2000live-bornfemale infants( chromosomal abnormalities, occurringinapproximately Turner syndromeisoneofthemostcommon Sex-chromosome DSDconditions within eachconditionareshownin corresponding peptidehormoneconcentrationlevels be viewedfromtheclassificationofDSD( The useofpeptidehormonesasmarkersforDSDcan to DSD Peptide hormoneanalysisinrelation sample exchangeforInhibinB. aim toparticipateinactivitiesofpeercomparison,suchas When EQAprogramsareunavailable,laboratoriesshould aim toparticipateinEQAprograms,whenavailable. relevant peptide hormones in blood. Laboratories should assurance (EQA)programsarewidelyavailableforthese for InhibinBisrecommended. achieve agreementacrosslaboratories,anEQAprogram exist, ofwhichtheassaybyAnshLabsislisted.Thus,to available. Furthermore,several Gen IIELISAandthereforearenolongercommercially assays are former versions of the Beckman Coulter Inhibin the oneslistedin and serum with thefollowingcriteria:(1.)InhibinB on PubMed( commonly usedInhibinB-assays,weperformedasearch Italy), andthereisnoprogramforurineLH( exist currently(ProBioQual,France,andQualimedlab, our knowledge,onlytwoEQAprogramsforInhibinB (SKML, theNetherlands)forFSHandLH.To thebestof Stichting KwaliteitsbewakingMedischeLaboratoria programs existforpeptidehormones,example, or theUK-NEQASprogram.Importantly, otherEQA of platformsparticipatingintheBio-Rad,Labquality, groups thatconstitutedatleast1%ofthetotalnumber UK NEQAS,Edinburgh,UK). forPeptideHormones(AMH; Quality AssessmentService respectively, andtheUnitedKingdomNationalExternal Consensus Statement 2017.Themostcommonlyusedassaysreportedwere Position 5: With ofthemost theaimofobtaininganoverview and 2016and(2.)InhibinB https://www.ncbi.nlm.nih.gov/pubmed/ ExceptforInhibinB,externalquality Table 1 . However, threeofthelisted Table 1 research use-only T HJohannsenandothers and Table 2 liststheplatform human 45 ). Theclassical and . Table 1 human and versions 1 ). The serum ). and )

to low-levelconcentrations of AMHandInhibin B have of gonadalfailure.Duringmini-puberty, undetectable inhealthycontrols,maybeasaconsequence observed levels inearlychildhoodandduringpubertythan Turner syndromepatientsexhibithighergonadotropin largest overlapinmid-childhood.However, asagroup, values fromchildhoodthroughadolescence( The gonadotropin concentrationsoverlap with reference in earlychildhoodandfrompubertyonward( biphasic patternwithincreasedgonadotropinsecretion and adolescents,girlswithTurner syndromeexhibita within normalfemalerange( above femalereferencerange,butwithLH/FSHratios puberty, gonadotropinlevelshavebeenreportedtobe with (45,X/46,XX)Turner syndrome.Inmini- pubertal developmentismorefrequentinpatients development ofstreakgonads( with aprenataldegenerationofovarianfolliclesand X-monosomic Turner variantismostlyassociated high LHandFSH,lowAMHInhibinB)( from normaltolevelsindicativeofgonadalfailure(i.e. in thegonadotropins,AMH,andInhibinBthatallvary gonadal function and androgenization, which is reflected greatlyintermsof vary section). Maleswiththiskaryotype considered tohaveTurner syndrome(seeprevious genitalia. FemaleswithY-chromosome materialare predominantly maleorfemale,withambiguous ‘Turner’-cell line,thesemaypresentasphenotypically While patientswitha45,X/46,XYconstitutionhave 45,X/46,XY mosaicism premature ovarianfailure( ≤ been reported( infertility, and variable degrees of learning difficulties. testes, hypergonadotropichypogonadism, gynecomastia, or mosaicims( , structurally abnormal Xchromosomes, 10–20% ofKlinefeltercases arecausedbyhigher-grade chromosomes, constitutes 80–90% of the cases, while andresultsfromtheaneuploidy ofthesex karyotype ,whichisdefinedbythe47,XXY to 1:1500inmalenewborns( abnormality, with an estimated frequency of 1:1000 Klinefelter syndromeisthemostcommonchromosomal Klinefelter syndrome Peptide hormonesandDSD 3 pmol/Lhasbeenreportedasapredictorofimminent 8 51 ). Furthermore,anAMHconcentrationof ). Thecardinalstigmatainclude small Downloaded fromBioscientifica.com at10/02/202104:05:13AM 36 ). 8 50 ). Asinhealthychildren 46 ). Theclassicalformof ), whilespontaneous 182 :6 10 9 ), withthe , 48 9 , , 49 P8 47 ). via freeaccess ). ). European Journal of Endocrinology and atestisoranovotestis ontheotherside.In46,XX ononeside or theremaybeacombination ofanovary contain both ovarian andtesticular tissue (ovotestis) rare ovotesticular DSD, the gonads may In the very 46,XX ovotesticularDSD Disorders ofgonadal(ovarian)development 46,XX DSDconditions again dependsontheageofboy. depend ontheextentoftesticularabnormality, which However, may the likelihoodofabnormalbiochemistry gonadotropin concentrationsarealsodescribed( male reference ranges( B, andtheLH/FSH-ratiohavebeenreportedwithin puberty, concentrationsofgonadotropin,AMH,Inhibin due toanabnormalSertolicellfunction( abnormal Leydigcellfunction,whileAMHisdecreased Most often,thegonadotropinsareincreasedduetoan 17-hydroxylase deficiency(CYP17A1),3 biosynthesisdeficiencies,steroidacuteregulatoryproteindeficiency(StAR),StAR,P450sidechaincleavageenzyme(CYP11A1), AIS, androgeninsensitivitysyndrome;F,female;M,male;PMDS,persistentMüllerianductsyndrome.*geneticmutationsresultinginthefollowing phenotypical features,regardlessofchromosomalconstitution. As DSDcoverspatientswithawidespectrumofphenotypesincludingambiguousgenitalia,maybeevaluatedbasedonthemostpredominant Congenital hypogonadotropic Other PMDS (testiculardysgenesis) PMDS (AMHresistance) PMDS (impairedAMHproduction) Leydig-cell hypoplasia Partial AIS Complete AIS 5 StAR, CYP11A1,CYP17A1,HSD3B2 46,XY gonadaldysgenesis(partial) 46,XY (complete) 46,XY DSDconditions 21-hydroxylase deficiency 46,XX gonadaldysgenesis 46,XX ovotesticularDSD 46,XX DSDconditions Klinefelter syndrome 45,X/46,XY mosaicism Turner syndrome Sex-chromosome DSDconditions Inhibin BinDifferencesofSexDevelopment(DSD). Table 2 Consensus Statement α hypogonadism and HSD17B3* -reductase deficiency Concentration levelsofluteinizinghormone(LH),follicle-stimulating(FSH),anti-Müllerian(AMH),and 8 ), although higher-than-normal β -hydroxysteroid dehydrogenasedeficiency (HSD3B2),and17 T HJohannsenandothers Decreased forsex Elevated forM Normal forM Normal forM Elevated forM Elevated forM Elevated forF Normal forM Elevated forsex Variable Elevated forF Normal forsex Elevated forF Variable Elevated forM Variable Elevated forF LH 24 ). Inmini- 11 Normal toElevated Normal forM Normal toElevated Normal forM Elevated forsex Variable Elevated forF Normal forsex Elevated forF Variable Elevated forM Variable Elevated forF FSH Decreased forsex Elevated forM Elevated forM Elevated forM ). ). for M for F commonly low. elevated, whileAMHandInhibin Bconcentrationsare Thus, concentrationsofgonadotropins arecommonly of theuterus,andhypergonadotropic hypogonadism. or absentpuberty, amenorrhea, hypoplasia primary for example, in Different causativemutationsingeneshavebeenreported, ovarianfailure. by streakgonadsthatleadtoprimary heterogenous groupofphenotypicalfemalescharacterized 46,XX gonadaldysgenesis(46,XXGD)isagenetically 46,XX gonadaldysgenesis ofgonadalfunction. provide anoverview information onthegonadsseparately, thepeptidesmay AMH andInhibinBmeasurementscannotcontribute Therefore, phenotypicalpresentationvaries.Although and/or regression of Wolffian and Müllerian structures. testosterone andAMHsecretionthusthestimulation DSD, thegenitaldevelopmentdependsonextentof Peptide hormonesandDSD FSHR Normal forM Normal forM Elevated forF Decreased tonormal Normal forsex Variable Decreased forF Normal forsex Decreased forF Variable Decreased forM Variable Decreased forF AMH Decreased forsex Decreased forM Elevated forM Decreased forM β -hydroxysteroid dehydrogenase(HSD17B3); for M ( 52 ) Downloaded fromBioscientifica.com at10/02/202104:05:13AM . The phenotype includes delayed https://eje.bioscientifica.com 182 Normal forM Normal forM Elevated forF Decreased tonormal Normal forsex Variable Decreased forF Normal forsex Decreased forF Variable Decreased forM Variable Decreased forF Inhibin B Decreased forsex Decreased forM Normal forM Normal forM for M :6 P9 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com but mutations in alistofgenes(e.g. development ( heterogeneous groupcharacterizedbydeficientgonadal 46,XY gonadaldysgenesis(46,XYGD)isagenetically 46,XY gonadaldysgenesis Disorders ofgonadal(testicular)development 46,XY DSDconditions sex andage. puberty. AMHandInhibinBaremostoftennormalfor the startoftreatmentinpatientswithcentralprecocious they may occasionally be high at presentation or upon not displayaninitialelevationofgonadotropins,while tumor). Patients with pseudo-precocious puberty may central) oradulthood(menstrualdisorders,benigntestis present inchildhood(precociouspuberty, pseudo-,or with ambiguousgenitalia.However, patientsmayalso thereby excludingotherformsofDSDinnewborns distinguishing between46,XXand46,XYindividuals, and InhibinBprovideimportantinformationwhen before CAHisgeneticallyconfirmed,FSHLH,AMH, by theexcessiveandrogenexposure seen in CAH neonatesisdue to prenatal programming the notionthatgonadotropinsecretionpattern prior totreatment( LH, lowFSH),albeitbothgonadotropinswerelower been reportedtoresemblethoseinhealthyboys(high during infancybeforeandafteronsetoftreatmenthave literature isscarce, gonadotropinlevelsinCAHfemales deficiency inthefirst-linetesting.Furthermore,although Δ4-androstenedione areindicativeof21-hydroxylase 17-hydroxyprogesterone, 21-deoxycortisol,and genitalia withoutpalpablegonadsininfancy. Elevated 46,XX individualstypicallypresentwithambiguous overproduction of , the majority of DSD in46,XXindividuals( 21-hydroxylase deficiencyisthemostcommoncauseof Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency Androgen excess 46,XY GDincludeshypergonadotropic hypogonadism the gonads.Thus,hormonal patternincomplete the timingandamountof sexhormonesproducedby 46,XY GD ( development, although abnormal, is presentinpartial 46,XY GD, there is no testis development, while testis SOX9 Consensus Statement ) areassociatedwith46,XYGD( 57 55 ). The resulting phenotype depends on ). The primary etiology is unknown, ). The primary 54 ). Thisresemblancesupports T HJohannsenandothers 53 ). Becauseofthe SRY 56 in utero , ). Incomplete WT1 , SF1 . Thus, and

deficiency (CYP17A1),3 chain cleavageenzyme(CYP11A1)deficiency,17-hydroxylase Steroid acuteregulatoryprotein(StAR)deficiency,P450side Androgen biosynthesisdefects levels havealsobeen reported from puberty onwards ( hypergonadotropic hypogonadismanddecreasedAMH while patientswithpartial46,XYGDwhomayexhibit varying degreesdependentonthetestosteronesynthesis, varying production, whileWolffian derivativesarepresentin Müllerian structuresarepresentbecauseofAMH synthesis. 46,XYindividualshavetestes,thusno enzymatic defectsresultinginimpairedandrogen These areautosomalrecessiveconditionscausedby (HSD17B3) deficiency deficiency (HSD3B2),and17 undetectable levelsofAMHisreportedfrominfancy( which hasbeenreportedfrompubertyonward( (PAIS) resistanceoftheandrogenreceptorto circulating 46,XY individualswithcomplete (CAIS)orpartial by normalandrogenproduction andmetabolismin Androgen insensitivitysyndrome (AIS)ischaracterized Androgen insensitivitysyndrome Defects inandrogenaction Inhibin concentrationsarelowtonormal( concentrations withinthereferencerange,whileAMHand The hormonalpatternischaracterizedbygonadotropin degrees ofunder-virilizationtocompletefeminization. Thephenotypethereforeranges fromvarying embryogenesis. to masculinize external genitalia in geneticmales during to the more potent dihydrotestosterone that is required 2 deficiency, thereisaninabilitytoconverttestosterone condition due to mutationsin 5 5 are low, whereasAMHlevelsarenormaltohigh. contribute tothediagnostics;thatis,testosteronelevels between steroidhormonesandpeptidemay ( AMH ishighwithinorabovethenormalmalerange 46,XY individuals.However, inthefirstmonthsoflife, Inhibin Bconcentrationswithinthenormalrangefor have hypergonadotropichypogonadismandAMH resulting invariablemasculinization.Patientstypically Peptide hormonesandDSD 60 α α -reductase type 2 deficiency is an autosomal recessive -reductase type2deficiencyisanautosomalrecessive -reductase deficiency ). Inandrogenbiosynthesisdefects,thediscrepancy β -hydroxysteroid dehydrogenase Downloaded fromBioscientifica.com at10/02/202104:05:13AM β -hydroxysteroid dehydrogenase SRD5A2 182 . In5 :6 60 α -reductase type -reductase type , 61 ). 58 P10 ) and 58 59 via freeaccess ). ), European Journal of Endocrinology result ofmutationsinthe Persistent Müllerianduct syndrome(PMDS)isthe Persistent Müllerianductsyndrome Disorders ofAMHand receptor AMH iswithinorabovemalereferencerange( are withinmalerange.Particularly, inthefirst yearoflife, within orabovemalerange,andAMHInhibinBlevels genitalia. LHlevelsaretypicallyincreased,FSH hypogonadism overgenitalambiguitytofemaleexternal the degreeofinactivationandvariesthereforefrommale of testosterone( due tothescarcity ofLeydigcellsandconsequentlyalack hypoplasia, thereisafailureinpre-andpost-natalvirilization receptor that is,the mutations inthegeneencodingLeydigcellreceptor, Leydig cellhypoplasiacanbetheresultofinactivating Leydig-cell hypoplasia LH-receptor defects in PAIS infants( above malerangeinCAISinfantsand ( B concentrationsingeneralarewithinthemalerange As theSertolicellfunctionisintact,AMHandInhibin situ elevated LHconcentrations( tohighlightthese stimulation testcanfurtherserve level( at thehypothalamic-pituitary elevated LHconcentrationsduetoandrogenresistance ( levels arelowinCAISinfants,buthighPAIS infants gynecomastia ( with cryptorchidism, micropenis,hypospadias,and labial fusiontoapredominantlymalephenotype female phenotypewithmildclitoromegalyandsparse in PAIS, this phenotype ranges from a predominantly some residualbindingaffinityoftheandrogenreceptor due toregressionoftheMüllerianducts.Causedby genitalia, butinternalfemalegenitaliafailtodevelop lack of masculinization of theWolffian ductsand external females ( AIS isreportedtohaveaprevalenceof4.1per100000 constitution, thephenotypevariesfrommaletofemale. androgens. Thus,despiteamalechromosomal 60 64 Consensus Statement , ). Frompubertyonward,AISischaracterizedby havenormaltohighconcentrationsofFSH( 67 ). Inthefirstyearoflife,AMHlevelsarewithinor (LHCGR) 62 ). In CAIS, the phenotype is female due to the l uteinizing hormone-chorionic gonadotropin uteinizing hormone-chorionicgonadotropin 63 68 60 gene. In 46,XY DSD due to Leydig cell gene.In46,XYDSDduetoLeydigcell ). Duringthefirstmonthsoflife,LH ). The resulting phenotype depends on ). Theresultingphenotypedependson ). 40 AMH T HJohannsenandothers ). Adultswithtestes gene ortheAMH 65 ), andanLHRH 59 ). 66 in in ). ). it mediates the intra-abdominal testicular descent, and it is controlling thegubernacular growthanddifferentiation, within routineDSDdiagnostics. Insl-3isthemajorfactor males and females and is thus expected to be acandidate This peptide is mainly produced in gonadal tissues in testicular impairment. both lowerthannormalanddependentonthedegreeof dysgenesis, the AMH- and Inhibin B concentrations are affected, LHconcentrationsarenormal.Intesticular AMH concentrations.Astestosteroneproductionisnot AMH-receptor resistancewhenaccompaniedbyhigh when accompaniedbylowAMHconcentrationsand concentration of Inhibin B indicates AMH deficiency external genitalambiguity( both SertoliandLeydigcellsareaffected,resultingin external phenotypes.Intesticulardysgenesis,typically with PMDS due to mutations thus have normal male present, while external virilization is normal. Patients structures, sotheFallopiantubesanduterusare only interfere with the regression of the Müllerian of testiculardysgenesis.Thegeneticabnormalities AMH-receptor resistance,respectively, ortheresult receptor type2gene,resultinginAMHdeficiencyor the peptidehormoneInsulin-likefactor3(Insl-3)( developed forclinicalapplicationisquantification of example ofaLC-MS/MSanalysisthatrecentlyhasbeen within therequirementsforclinicalapplication.An determination ofmultipleanalytes,thechallengeslie technique, asmentioned,alsoallowsforsimultaneous detection limitthanobtainedbyimmunoassays.Asthis because ofitspotentiallyhigherspecificityandlower hormones maybeanalyzedusingLC-MS/MS-technique In thenearfuture,itisexpectedthatalsopeptide Future directions life ( of AMHandInhibinBhavebeenreported at thistimeof the lackofstimulationtestis,lowconcentrations as gonadotropinconcentrationsarelowinCHH.Dueto window ofopportunitytoexplorethegonadotropicaxis, CHH, thepostnatalgonadotropicsurgeprovidesauseful under-virilized andtherebyqualifyasaDSDphenotype.In hypogonadism (CHH),theexternalgenitaliamaybe In somecasesofcongenitalhypogonadotropic Congenital hypogonadotropichypogonadism Other Peptide hormonesandDSD 12 ). Downloaded fromBioscientifica.com at10/02/202104:05:13AM 69 https://eje.bioscientifica.com ). InPMDS,thenormal 182 :6 P11 70 via freeaccess ). European Journal of Endocrinology https://eje.bioscientifica.com submitted manuscriptandapproved submission. this of content entire the for responsibility accepted have authors the All Author contributionstatement Framework (CHAFEA Programme Health by Partnership Agreementnumber:739527–Endo-ERN). co-funded Union’s Network European Reference Third European the a is Endo-ERN BM1303). Action Technology) and Science (Reference 2020 Horizon EU-program the under funding received in DSDnet Cooperation (European COST The Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthispositionpaper. no is there that declare authors The Declaration ofinterest EJE-19-0831 at paper the of version online the to linked is This Supplementary materials in EQA. Comparability betweenlaboratoriesrequiresparticipation for normative data to optimize diagnostics and treatment. establishment ofcommonsex-andage-relatedreferences suspected of DSDconditions. This paper will support the the alignmentofpeptidehormoneanalysisinpatients position statementshavebeendevelopedtosupport hormone physiologyandstructureisessential.Five andknowledgeofpeptide approach ismandatory a diagnosticsetting,bothclinicalandbiochemical constitute a heterogenous group of rare conditions. In with expertiseinDSD.DifferencesofSexDevelopment of highlyspecializedendocrinereferencelaboratories One of the aims of Endo-ERN is to establish a network Conclusions platforms( spectrometry and asthismethodiscompatiblewithexistingmass- simultaneous analysisofseveralpeptidesatonetime with respecttofuturepeptideanalyses,asitallowsfor undetectable withtheabsenceofLeydigcells. to beamarkeroffunctionalLeydigcellsandwill,thus, the causeofcryptorchidism inhumans.Insl-3isthought may leadtocryptorchidism, butsuchmutationsarerarely ( involved in the transinguinal and inguinoscrotal descent 71 Consensus Statement ). In animals, mutations in the gene encoding Insl-3 Furthermore, targetedproteomicsseemsattractive . 72 ). T HJohannsenandothers https://doi.org/10.1530/

References replying tothesurvey. The authors would like to thank the Reference Centers within Endo-ERN for Acknowledgements

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