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European Journal of Endocrinology 10.1530/EJE-17-0776 handle laboratoryinvestigationofandrogenexcessdisordersinwomen. discussed inthisreview, includingthoseofadrenalandrogenexploration, withtheaimofhelpingclinicianstobetter assays thatareyettobedeveloped,ismeasureSHBGandcalculatefreetestosterone.Afewotherpitfallswill be normal values,whichwillmaskandrogenexcessstatus.Onewaytoavoidthispitfall,awaitingdirectfreetestosterone are typicallylowinoverweighthyperandrogenicpatients.Thus,SHBGmaydecreasecirculatingtestosterone to laboratory. Circulatingtestosterone isstronglyboundtosex-hormone-bindingglobulin(SHBG),andSHBGlevels quality controlprocessesandstandardization,soastoensureappropriateinterpretationirrespectiveoftheparticular immunoassay andmassspectrometryneedtooperatewithinaqualityframeworkbeactivelyengagedinexternal well identifiedandmustberecognizedsystematicallyaddressed.Ingeneral,laboratoriesusingdirecttestosterone testosterone, providingoptimalaccuracywithalowlimitofdetection.Yet, thepitfallsofthesetwotechniquesare Liquid chromatographycoupledtotandemmassspectrometry(LC–MS/MS)willbecommonlyusedformeasuring achieved byimmunoassayusingaspecificanti-testosteronemonoclonalantibody, ideallyafteranextractionstep. androgens andtestosterone,requiringaccuratetechniqueswithhighspecificitysensitivity. Thesegoalscanbe pitfalls inmeasuringtestosteronerelatetoitslowconcentrationandthestructuralsimilaritybetweencirculating Measuring totaltestosteronelevelisthefirst-lineapproachinassessingandrogenexcesswomen.Themain Abstract Bron, France d’Endocrinologie MoléculaireetdesMaladiesRares,GroupementHospitalierEst,HospicesCivilsdeLyon, Bernard Lyon 1,Lyon, France, 1 Henri Déchaud Michel Pugeat laboratory diagnosis Hyperandrogenic statesinwomen:pitfalls MANAGEMENT OFENDOCRINEDISEASE Fédération d’Endocrinologie,GroupementHospitalierEst,HospicesCivilsdeLyon, Bron,France, https://doi.org/10.1530/EJE-17-0776 www.ej Review Michel Pugeat Invited Author’s profile arousal andtheirdeviancein males of diseaserelatedtoenvironment. Hiscurrentinterestisontheinfluenceofandrogens onsexual and indevelopingahighlysensitive BPA immunoassay thathasbeenausefultoolforinvestigation testosterone. Hehasundertaken prospectiveresearch ontheendocrinedisrupterbisphenolA(BPA) syndrome(PCOS)emphasizingthat SHBGdoesinfluencetheclinicalinterpretationoftotal ovary chronic diseases.Hisresearch investigationofpolycystic specificallyfocusesonthelaboratory SHBG/CBG levels are associatedwith metabolic syndromeanditsinherentrisksfordeveloping original descriptionofphenotypesassociatedto areas of Prof. Pugeat’s research is on sex steroid hormones and their bindingproteins with the e-online.org 1 1 , 2 ,
2 , is currentlyEmeritProfessor, UniversityClaudeBernard,Lyon, France.Themain 3 and , Ingrid Plotton Véronique Raverot 3 INSERM U1060InstitutCarMen,Lyon, France,and 4 © 2018EuropeanSociety ofEndocrinology M Pugeatandothers 2 , 4 , Aude Brac de la Perrière . Printed inGreatBritain 4 CBG/SHBG genemutationsandtheevidencethat hyperandrogenic states Laboratory diagnosisof 4 Published byBioscientifica Ltd. Laboratoire d’Hormonologie, 1 , Gérald Raverot 2 Université Claude 1 , 2 , Downloaded fromBioscientifica.com at09/25/202110:48:19AM
(2018) Endocrinology European Journal of [email protected] Email to MPugeat should beaddressed Correspondence 178
178 :4 , R141–R154
R141 –R154 via freeaccess European Journal of Endocrinology www.eje-online.org diagnosis ( investigationsshouldbeconductedforlaboratory worldwide ( of hyperandrogenic states is significant, at 6–12% ( androgen receptormachinery active intracellularmetabolismand/orenhanced increased androgen activity within the cell, through and bioactivity( and clearancethatenhanceandrogencellavailability include increasedperipheralandrogenmetabolism (PCOS) ( syndrome commonpolycysticovary and thevery genetic diseasesthatimpairadrenalsteroidogenesis, with exceptionalandrogen-secretingtumors,rare encompass excessandrogenproductionassociated of excessandrogendisorder in women.They Hyperandrogenic statesaretheclinicalmanifestation Introduction succeed indiagnosis. pitfalls and how to circumvent them so as finally to hyperandrogenic statesin females, byidentifyingthe investigation canaccuratelyidentifytheorigin of women ( to evaluate gonadotropin profile in androgen excess puzzling forconsensualrecommendationsonhow regulationofprogesterone havebeenon LHinhibitory effect ofandrogens(LH) regulationandtheinhibitory feedback ofestradiolregulationonluteinizinghormone the menstrualcycle,positiveratherthannegative androgen disorder:changinghormonalprofileduring (TSH) data, it is less consistent when applied to female hormone (T4/T3)andthyroid-stimulating thyroidinvestigation,balancing laboratory However, althoughthisparadigmiswelladaptedto blood, salivaorurinesamplesevenwithincells. factors,in regulatory and theirspecificpituitary hormones fromthyroid,adrenalandgonadalsources to identifymostendocrinedisordersbymeasuring feedback regulationassumesthatitisusuallypossible the considered hormone ( limiting theconsequencesofsurplusordeficiencyin maintaining endocrinefunctionhomeostasisand/or systemactivation orinhibitionofthespecificregulatory or deficientendocrineglandactivityisassociatedwith deficient hormonalsecretionand/oraction.Excessive symptoms, acombinationofwhichsuggestsexcessor Review This reviewwillchallengetheconceptthatandrogen Endocrine diseasesaretypicallysuspectedonclinical 16 1 , 12 , 2 17 10 , , 3 13 ). , , 11 4 , 7 , 14 , ) andraisesthequestionofhow 5 8 ). , ). Finally, theymayalsoinvolve 6 ). Hyperandrogenicstatesalso 15 ). This robust paradigm of M Pugeatandothers 9 ). Overallprevalence Paradigm foridentifyingandrogen excessinwomen. Figure 1 Society ofEndocrinology( and the PCOSSpecialInterestGroupof European Society ( workshop group( Rotterdam ESHRE/ASRM-sponsoredPCOSconsensus 1990 NIH-sponsoredconferenceonPCOS( hyperandrogenic states( total testosteroneasfirst-lineinvestigationof secreting tumorsandmustbepromptlyidentified( and clitorisenlargementaresuggestiveofrareandrogen- voice masculinization,abnormalmuscledevelopment alopecia ( patients withrecurrentacneseborrheaorandrogenetic growth withamalepattern(hirsutism)andissuspectedin Hyperandrogenic stateisidentifiedonexcessivehair approach tohyperandrogenic states Consensus andrecommendations forthediagnostic SHBG-bound complexis57%( 2%, whilethealbumin-boundcomplexis41%and for targetcells;inwomen,thisfractionislessthan small steroidfractionisprotein-unboundandavailable globulin (SHBG).Accordingtothelawofmassaction,a tightly andspecificallyboundtosexhormone-binding circulates intheblood, looselyboundtoalbuminbut identifying hyperandrogenicstates?Testosterone hyperandrogenic states Laboratory diagnosisof T >3.5-7.0nm Ovarian androgen Is totaltestosteronetherelevanthormonefor Most consensusstatementsrecommendmeasuring DHEA DHEAS S Adrenal ca 4 or hyperthecosi 9 ), theFrenchEndocrineSocietyConsensus( DHEAS ). Rapidonsetofvirilizingsymptomssuchas ol > 16000nmol/l (>2–3SD norm rc se in creting om al 3 ) s a ), theAndrogenExcessandPCOS Tota tumor l te Downloaded fromBioscientifica.com at09/25/202110:48:19AM stos iue 1 Figure 18 terone ). 19 ). Thereisnodefinitive ). Theseincludethe ACTH test 178 T <3.5 17OHP <1000ng/d 17OH :4 of 21 Non
nm classical fo P > OH PCOS 2 ol defi 1000 ng/d , 14 ciency R142 5 rm ), the , l l 9 13 ). via freeaccess ) European Journal of Endocrinology without increasedhairgrowth orrecurrentacne. ultrasound mayshowincreased testosteronelevels that patientswithpolycystic ovarianmorphologyon Conversely, the Rotterdamconsensus( androgen sensitivityorincreased testosteroneclearance. may havenormaltestosteronelevels,suggestinghigh reference testosteroneassay( of hirsutism. This point has been documented using a Testosterone levelispoorlycorrelatedwith theseverity direct immunoassay Pitfalls inmeasuringtotaltestosterone by 1200 weakly toSHBG,withmuchhigherMCRsof2200and androstenedione showlowbindingaffinityandbind day respectively( to theirrespectiveMCRs,whichare315,485and720 decreasing orderofmagnitude( to dihydrotestosterone,testosteroneandestradiol(in concentrations. ThestrongbindingaffinityofSHBG time, isthemainprocessdecreasingcirculating androgen as thevolumeofbloodclearedirreversiblyperunit Conversely, themetabolicclearancerate(MCR),defined maintains circulating testosterone concentration. metabolism fromboth ovarian andadrenal precursors, including directovariansecretionandperipheral adipose tissue.Theproductionrate(PR)oftestosterone, ( dehydroepiandrosterone (DHEA) from the adrenal cortex precursor: androstenedionefromthecalcellsand physicians. Itisprimarilyproducedasanandrogen has astrong‘mediaprofile’forthepublic,studentsand is themainactivecirculating androgen.Testosterone essentially based on theassumptionthat testosterone the free-hormonehypothesis(seebelow). ( blood intothestromalmatrixofuterineendometrium a matrix-associated protein, can be sequestered from the prostate cancercells( Moreover, humanSHBGisfoundinbreastand to theplacentafor aromatization duringpregnancy. might beanimportantpathwayforandrogentransport This hasbeenreportedfortrophoblasticcelllinesand biologically activeSHBG-boundandrogensandestrogens. ( interacting withmegalin,themainendocyticreceptor complex, althoughithasbeenshownthatSHBG,by evidence foraspecifictransitoftheSHBG-boundsteroid 23 22 20 Review ), which are converted into testosterone, especially in ). Taken together, theseintriguingfindingschallenge ), mayprovideaspecificpathwayforcellularuptakeof The rationaleformeasuringtestosteroneis L/day respectively( 23 , 21 24 ) and,byinteractingwithfibulin, , Table 1 25 , 27 26 ). Severelyhirsutepatients 19 ). ). Incontrast,DHEAand M Pugeatandothers )) isinverselycorrelated 3 ) pointedout L/ *K Dehydroepiandrosterone Androstenedione Estradiol Testosterone Dihydrotestosterone ( binding affinity constantat37°CaccordingtoLongcope Table 1 testosterone. time ofdayorpointinthecycleiseffectiveformeasuring in practice,bloodsamplingfastingconditionsatany that testosteronebemeasuredintheearlyfollicularphase, cycle arefairlyinsignificant.Althoughitisrecommended the advantagesofastable non-radioactivetracer( immunoassays using125-iodine-labeled steroids,with tritiated or enzyme-conjugated tracers, and challenge Technology) usually exhibithighersensitivitythan resolved fluorometricdetection ofeuropium(Delfia chemiluminescent. Steroidimmunoassaysusingtime- enzymatic (HRPandPAL), fluorescent(europium)and The chiefmarkersareradioactive(iodine125andtritium), and the system used to immobilize the immune complex. immunoassay is in part dependent on the labeled probe testosterone complex. and on the technique used to reveal the antibody-bound the quality of the antibodies used to capture testosterone SHBG). mainly relatedtosteroidtransportproteins(albuminand extraction alsoeliminatesmuchofthematrixeffect, and/or insufficient antibody specificity ( immunological reaction because of theirsimilar structure LH20), eliminatessteroidslikelytointerferewiththe the extract by chromatography (Celite, HPLC, Sephadex step, usinganorganicsolvent,followedbypurificationof to thatofothercirculating androgens.Anextraction specificity, astheconfigurationoftestosteroneissimilar that matchitsparticularstructure,atthepriceoftheir molecules. To assay testosterone, antibodies are developed on theinherentabilityofanantibodytobindsmall be achievedbycompetitiveimmunoassaysthatrely require accurate and sensitive techniques ( similarity betweencirculating androgensandtestosterone hyperandrogenic states Laboratory diagnosisof 23 a : 10 , 24 The lowlimitofdetectionorsensitivitythe Precision andspecificityaremainlydependenton Low testosteroneconcentrationandthestructural Variations in testosterone levels over the menstrual − ), Mahoudeau 9
× M Half-life ( − 1 at37°C. T 1/2 et al ), metabolicclearancerate(MCR)and . ( Downloaded fromBioscientifica.com at09/25/202110:48:19AM T 25 1/2 ND 60 50 34 53 ) andDunn
(min)
MCR 2040 2200 178 720 485 315 (L/day) et al
www.eje-online.org :4 12 . ( 28 , 19 SHBG binding 13 ). ). This can affinity* 0.07 0.03 0.7 1.2–1.3 5.5 ). Steroid R143 et al 29 via freeaccess . ). European Journal of Endocrinology www.eje-online.org reported ( false highresultsfortestosterone andDHEAShavebeen medicine.Intheseconditions, biotin asacomplementary and diabetesmellitus, patients maysometimesuse biotin treatment in various diseases, including alopecia in assayskits( efficiently eliminatesbiotininterference,whenincluded of biotin in magnetic streptavidin-coated microparticles overcome thispitfall,ithasbeenshownthatadsorption signaling system,leadingtoover-orunder-estimation.To microparticles inthesolidphaseandinterferewith on thesystemused,willbindtostreptavidin-coated hormone orthebiotinylatedspecificantibody, depending sample, bycompetitionwiththetraceofbiotinylated multiple sclerosis. An excess of biotin in the blood outcome and quality of life in patientswith progressive times the RDI) iscurrently indicated to improveclinical 000 order ofmilligramsperday. High-dosebiotin(10 vitamin with a recommended daily intake (RDI) in the ( diagnosis haslongbeenrecognizedforthyroiddiseases biotin, leadingtoariskofmisdiagnosis.Thisfalse system canbeaffectedbyhighlevelsofcirculating Hormone immunoassaysusingthestreptavidin-biotin hormone immunoassay Novel pitfallsassociatedwithadvancesin testosterone bydirectimmunoassayarelistedin main advantages, disadvantages and pitfalls of measuring whereas radioactive systems are less widely used.The major detectionprinciplesinmodernautomatedanalyzers, the precisionofimmunometricassays. phase matrix.Thisapproach has considerably enhanced exploited toimmobilizeimmunecomplexesinthesolid- streptavidin forbiotinandbiotinylatedcompoundsis be measured.Thehighbindingaffinity( biotin molecule in enzymes or in the hormone to streptavidin-biotin systembyincorporatingthesmall In addition,immunoassaysexploitthehigh-affinity • • • • • Advantages Table 2 30 Review Increased effort bymanufacturerstocalibratetheirassays Precision: goodintra-andinter-assay reproducibility human error Suitable forautomatedplatformsthatreducetheriskof Easy touse,requiringsmallsamplevolume,shortassaytime widely usedmethods( RIAs andchemiluminescenceimmunoassaysarethemost , Nowadays, chemiluminescence and fluorescence are the Nowadays, chemiluminescenceandfluorescencearethe 31 , Main advantages,disadvantagesandpitfallswithdirectimmunoassay. 32 33 , ), butthisremainstobefurther documented. 33 34 ). Biotin(vitaminB7)isawater-soluble ). Apotentialbenefithasbeenclaimedfor > 80% oflabs) M Pugeatandothers
K Table 2 a =10 15 ) of . for measuringtestosterone( mass-selective detectionandisthereferencemethod the initial analytical approach for simple molecular- components andsoftware. principles, for moreinformationonmassspectrometry 37 This chapterreferstocomprehensivereviews( a must Measuring testosterone bymassspectrometry: assessing androgenstatusin womenandchildren( low limits of detection argue for it being the reference for ( now enhancing analysis speed, sensitivity and resolution automated analysisplatformsareavailable. decrease, becoming analternativetoimmunoassay when in thenearfuturebesuitableforroutineuse,ascosts procedure makelargeseriesofassaysimpractical,itmay the specialexpertiserequiredandtime-consuming in Europe,becausethehighcostofequipment, deficiency ( steroid metabolismdisorders,including21-hydroxylase current approachtoandunderstandingofcongenital steroid measurement.Ithassubstantiallyimprovedour LC–MS. Presently, LC–MS/MSoffersthebestqualityfor target steroids,hasgreatlyincreasedtheselectivityof collision celltorevealthefragmentationpatternof coupling 2quadrupolemassfilterswithaninterposed carbonyls thatarereadilyionizedwithoutderivatization. steroids, including testosterone, which possess unsaturated for measuringmost making derivatization unnecessary remarkable progress, since the eluent phase is liquid, direct couplingofLCandMS.LC–MSconstitutesa ionization andofelectrosprayhasallowed (LC) andtheemergenceofatmosphericpressurechemical derivatization. The development of liquid chromatography large bloodsample( hyperandrogenic states Laboratory diagnosisof 38 • • • • • Disadvantages andpitfalls , ). Theprecisionandaccuracy ofLC–MS/MSandits Unexpected interference:e.g.,biotin specificity oftheantibodiesused Potential interferenceandmatrixeffects, despitegood Potential interferencewithbindingproteins testosterone antibodies Lack ofspecificity, whichdependsonthequalityof Low accuracy Gas chromatography (GC) coupled with MS was Gas chromatography(GC)coupledwithMSwas Ultra-performance liquid chromatography (UPLC) is Although LC–MS/MSisnotwidelyusedinlaboratories The introductionoftandemMS,whichinvolves 38 , 39 , 37 40 ). ) that readers are encouraged to consult > 2 mL) for a step of extraction and mL) forastepofextractionand Downloaded fromBioscientifica.com at09/25/202110:48:19AM 35 , 36 ). GC–MS requires a ). GC–MSrequiresa 178 :4 28 35 R144 , , 36 36 via freeaccess ). , European Journal of Endocrinology • • • • Advantages Table 3 goal’,becausetheavailabilityofMS islimited unnecessary by MStechnologywouldbe‘anunrealisticand enemy of good’, arguing that replacing all immunoassays they equallyechoVoltaire’s maxim that ‘Thebestisthe However, pitfallsmustberecognizedandaddressed, highly accurate analyses with enhanced specificity. is acceptable’.With thisaiminview, LC–MS/MSallows philosophy: ‘Startwithwhatisrightratherthan was defendedbyTaylor hyperandrogenic statesinwomen.Thisrealisticapproach for measuringtotaltestosteronethediagnosisof methods immunoassay andmassspectrometry-based The currentpositionadvocatesappropriateuseofboth Current recommendations fortotaltestosterone assay LC–MS/MS for measuring testosterone are listed in reliability. The main advantages and drawbacks of in clinicallaboratoriesbutalsoessentialtoincrease for morewidespreaduseofthispowerfultechnology MS/MS-based analyzer systems, is not only a prerequisite with thefinalgoalofdevelopingfullyautomated heterogeneous. AutomatingtheLC–MS/MSprocesses, and theirinstrumentconfigurationsareextremelystill commercial LC–MS/MS assaykitsare presently available, analyzers.Onlyafew by automatedclinicalchemistry human error, theriskofwhichcanlikelybeminimized based sampleworkupbeforeMSanalysis. inaccuracy canbecontrolledbysufficientLCseparation- internal standard compounds. Most potentialsources of effects withdifferentialinfluenceontargetanalytesand may berelatedtotheionizationprocessandmatrix overestimated. The inaccuracy of LC–MS/MS methods 39 must be recognized and systematically addressed ( measurement oftotaltestosteronelevels.Yet, pitfalls Application ofLC–MS/MSshouldprovideaccurate Pitfalls inmeasuringtestosteronebymassspectrometry Review ). TheselectivityofMS/MSdetectionwasoriginally of severaldifferent steroids Potential forsimultaneousmeasurement Sensitivity (daughter ions) the parentionintospecific,smaller ions interest (parention)andtofragment select forthemassofcompound High specificityowingtoitsability Accuracy Finally, importantsources ofunreliableresultsinclude Main advantagesvsdisadvantagesandpitfallswithLC–MS/MS formeasuringtestosterone. et al . ( 40 M Pugeatandothers ), echoingFranzKafka’s • • • • Disadvantages andpitfalls isobaric congeners,matrixcompounds sharingmasstransitions,etc. standard, interferencefromin-source transformationofconjugatemetabolites, standards, differential impact ofmatrixeffects onanalyteandisotopeinternal pressure ionization,standardization mandatory, ‘isotopeeffects’ ofinternal Technical pitfalls:highdegreeofvariation intheefficiency ofatmospheric Variability between labs (riskofhumanerror, automataarenotyetavailable) Technical demands Expensive equipment Table 3 38 , .
which modulatestheirbioactivitybyrestrictivediffusion the maintransportsystemfortestosteroneandestradiol, pregnancy andunderoralcontraception. hormone-binding globulin(TBG)level,notablyduring of thyroid function, to allow for changes in thyroid has been widely developed for routineinvestigation accurately than total plasma hormone level. This concept concentration reflectstheclinicalsituationmore is bioactive.Theregoodevidencethatfree-hormone protein-unbound orfreecirculating hormonefraction The free-hormonehypothesis( The free-hormone hypothesis ( MS equipmentbecomesavailableallaroundtheworld this attitude. An ultimate consensus will be possible when measuring testosterone can realistically be founded on by costandtechnicaldemands.Recommendationsfor any negativefeedbackregulationofLHsecretion( the normalrange. gonadotropin maintainsfreetestosteronelevelswithin to humanmalephysiology, wherefeedbackregulationof to regulatecellbioavailability. Thismodelistranslatable function ofSHBGistomaintaintestosteronelevelsand strongly substantiatetheconceptthatakeyphysiological cells, asshownusingtritium-labeledprobes.Theseresults to lowbioavailabilityofsexsteroidhormonetarget a mildhypogonadalphenotypethatwaspossiblydue in LHlevel.Nevertheless,maletransgenicmiceexhibited adaptive feedbackregulationwithasignificantincrease was essentiallyunchanged,likelytheconsequenceof testosterone concentration.Incontrast,free of SHBG-boundtestosteroneandconsequentlyincreased circulating SHBGwasassociated withprolongedhalf-life h been re-validated on a mousemodeloverexpressing into target tissue. Thefree-hormone hypothesis has hyperandrogenic states Laboratory diagnosisof 3 SHBG ( , 4 Similar considerations should be applied to SHBG, In women,however, androgensshowminimalif , 13 , 43 18 ). Inthismodel,aremarkableincreasein ). Downloaded fromBioscientifica.com at09/25/202110:48:19AM 41 , 178 42 www.eje-online.org ) statesthatthe :4
R145 44 ). In ). In via freeaccess
European Journal of Endocrinology www.eje-online.org of freetestosteroneinthe dialysate,usingareliable It alsoallowsmeasurement oftheactualconcentration 2–3% ofthetotaltestosterone circulating intheblood. the percentage offree tube or chamber. This method allows measurement of testosterone throughamembraneinsmalldialysis testosterone fractions can be separated from the free incubation time at 37°C, the SHBG- and albumin-bound 3 despite technicaldifficulties( best estimateof free testosterone plasma concentration, prior toquantification( method toseparateprotein-boundfromfreetestosterone testosterone. the freeconcentration,whichismuchlowerthantotal and accuratehighlysensitiveassaysformeasuring equilibrium betweensteroidsandbindingproteins, develop amethodologythatdoesnotdisturbthebinding biological activity( a better index of overall production and a marker of the protein-unboundfractionoftestosteroneprovides According tothefree-hormonehypothesis,measuring testosterone concentration Measuring orcalculatingfree subjects, withdecreasedLHpulsatility( some progesteronefeedbacksensitivityinadolescentPCOS gonadotropin regulationinnormalwomen( androgen receptorblocker, hasessentiallynoeffecton noteworthy thatadministrationofflutamide,anon-steroid in turn perpetuates the hyperandrogenic state ( promotes abnormalneuroendocrineLHsecretion,which Samuel Yen ( production, in a vicious circle that was early described by increased LHsecretionmaintainshighovarianandrogen or increasingovarianandrogenproduction( causing rapidLHpulsesecretionandfurthermaintaining hormone pulse frequency normally exerted by progesterone, excess reducestheinhibitionofgonadotropin-releasing gene. Inaddition,itisalsodocumentedthatandrogen well-identified mechanismintheliverexpressionofSHBG and/or inflammation( documented thatinsulinresistance( contributes This is especially the case in patients with low SHBG, which ( inthelatefollicularphase secretion byestradiol,observed contrast, the positive feedback regulation of LH pulsatile 44 H-testosterone totheserumsample,withanappropriate Review ), may increase LH levels and maintain androgen excess. ), mayincreaseLHlevelsandmaintainandrogenexcess. Equilibrium dialysis has long been the standard per se 49 ). The current concept is that androgen excess ). Thecurrentconceptisthatandrogenexcess to clinical hyperandrogenism. It is well toclinicalhyperandrogenism.Itiswell 41 , 46 52 42 3 H-testosterone, whichisusually , ) andisconsideredtoprovidethe ). Thetechnicalchallengeisto 47 ) decrease SHBG levels by a ) decreaseSHBGlevelsbya 53 M Pugeatandothers ). Byaddingatraceof 45 51 ), liver lipogenesis ), liverlipogenesis ). 50 ) but restores ) butrestores 48 48 ). Thus, ). Thus, ). It is ). It is hormone fraction( disturb SHBG-binding sites andconsequently the free- binders, suchasdrugsor endocrinedisrupters,may ligands ( model ofthebindingequilibriumnaturalendogenous on apparentaffinity( and onestudyreportedaneffectofSHBGconcentration change inSHBG-bindingaffinity, hasbeen identified ( recognized. SHBGgenepolymorphism,associatedwith well withequilibriumdialysisresults( general, calculated values for free testosterone correlate is thecaseinwomenbutnotadultmen( the molar ratio of total testosterone to SHBG is low, which Theoretically, FTIcorrespondstofreetestosteronewhen total testosteronetoSHBG,expressedasapercentage. free testosteroneindex(FTI),definedastheratioof free testosterone have been proposed, including a Various derivedtestosteronecalculationstoestimate testosterone from SHBG-binding sites is negligible. with endogenous ligands that may significantly displace testosterone are constant. It also considers that interaction concentrations andalbumin-bindingaffinityfor variability inSHBG-bindingaffinityandthatalbumin The calculationassumesthatthereisnointer-individual SHBG concentrationsusingaccurateassayshasbeen Calculation of free testosterone from testosterone and testosterone concentration Surrogate methodstocalculatefree show inadequateaccuracyandprecision( commercially availableimmunoassaysforfreetestosterone techniques, cliniciansmustbearinmindthatthecurrent requirements whencoupledwithLC–MS/MS( in analytical sensitivity, convenience and sample recently developed ( coupling ultrafiltrationtoGC–MSdetection,hasbeen than equilibrium dialysis ( and foundtobefaster, withfewertechnicalrequirements 3H-testosterone astracer( testosterone assay. Itrequireshighlypurifiedradioactive and SHBG-boundtestosterone andhasbeenclaimedto but is dependent on the equation that is used( a surrogatefordirectfreetestosteronemeasurement, practice ( claimed tobeareliableapproachforroutineclinical hyperandrogenic states Laboratory diagnosisof Awaiting routineavailabilityofthesepromising Centrifugal ultrafiltration dialysis hasbeen validated Bioavailable testosteroneencompasses albumin-bound Pitfalls incalculatingfreetestosterone should be 60 19 ). Usingthelawsofmassaction,itprovides ) providesevidencethatpotentialSHBG 66 ). 65 57 Downloaded fromBioscientifica.com at09/25/202110:48:19AM ). Moreimportantly, theDunn ) with further improvement 54 , 56 55 ). Areference method, ).
178 60 :4 , 63 59 ). ). 58 61 ). , 61 62 R146 , ). In 62 64 via freeaccess ). ) European Journal of Endocrinology was associatedwithanadverse metabolicphenotype. testosterone, unlikeisolated androstenedioneelevation, Lerchbaum the severityofandrogen phenotype.Interestingly, high incidence of glucose intolerance correlating with serum androstenedioneandinsulinsensitivity, anda also reported a strongnegative association between sensitive markerinPCOS-relatedandrogenexcess.They index; however, highandrostenedionelevelwasamore correlated withhighandrostenedioneandfreeandrogen the Rotterdamcriteria,thathightestosteronelevels O’Reilly approach:Alternative measuringandrostenedione discriminating ( or in combination to elevated free testosterone, was more for PCOSdiagnosis,butthatestroneconcentration,alone reported thatfreetestosteroneconcentrationwasuseful ( sensitivity andspecificity(87%)fordiagnosisofPCOS study showedthatserumSHBGlevelsgood for PCOSdiagnosis,butnotsuperior. Interestingly, one androgen; freetestosteronewassimilarlydiscriminating total testosterone being the most commonlyelevated on theRotterdamcriteriaintwo-thirdsofpatients, of hirsute patients, Carmina had normalandrogenlevels.Inaconsecutiveseries increased in55.5%;thisstudy, one-fifthofpatients elevated in38%ofcases,whileforfreetestosteronewas al et population ofclinicallyhyperandrogenicpatients,Azziz according toVermeulen testosterone fromtotaltestosterone,SHBGoralbumin, Most studiesreportedintheliteraturecalculatedfree Free vstotaltestosteroneinhyperandrogenicpatients routine evaluationofandrogenexcess( SHBG-bound testosterone isareliable instrument for assay variability. With sometechnicalprecautions,non- including SHBG,whichmayincreaseintra-andinter- assay method is incompleteprecipitation of globulins, major limitationoftheammoniumsulfateprecipitation reported tobeeffectiveinafewlaboratories( from albumin-boundplusfree centrifugation toseparateSHBG-bound to precipitate the globulins,including SHBG, before target cells( be thebioavailablefractionofcirculating testosteronefor 73 Review ). UsingaMS-basedtechnique,Stener-Victorin . ( 71 ) reportedthatoveralltotaltestosteronewas t al et 67 t al et ). Theuseofsaturatedammoniumsulfate . ( 74 75 ). . ( ) reported,inPCOSpatientson 76 ) reportedthatelevated free t al et t al et . ( 3 H-testosterone, hasbeen M Pugeatandothers 53 . ( ). Inaconsecutive 72 69 ) reported PCOS , 3 H-testosterone 70 ). 68 t al et ). A . relatively stablethroughouttheday( ( slowrateof12.8 very bound toalbuminandisclearedfromthecirculation ata action ofDHEAsulfotransferase( ( half-life of ~50 ACTHsecretionbyDHEA.DHEAhasashort of pituitary (ACTH), withnoevidenceofnegativefeedbackregulation under thecontrolofadrenocorticotropichormone The adrenalcortexproduces Adrenal androgen excessinvestigation risk inPCOSneedsfurtherinvestigation( ( of PCOSanditshyperandrogenemicsub-phenotypes and free testosterone provides good accuracy for diagnosis with orwithoutdilution, depending onthemethod subjects), immunoassay isperformed on plasma sample plasma inlargequantities (around 6 Since DHEASisawater-soluble antigenpresentinhuman Immunoassay forDHEASmeasurement adrenocortical causeofandrogenexcess( increased androgenlevels.Thisrepresentsonemonogenic low DHEAS levels but been reported in patients with very gene encodinghumanPAPS synthase2( for androgensynthesis.Bycontrast,mutationsofthe DHEA sulfationmaylimittheamountofavailable pathway of DHEA metabolism,suggesting that increased and PAPSS2. SulfationofDHEAtoDHEASisthemajor synthesized by the two isoforms of PAPS synthase, PAPSS1 required forsulfotransferaseactivity. Inhumans,PAPS is the adrenalglandsandliver. ThesulfatedonorPAPS is is themajorenzymeresponsibleforDHEAsulfationin ( (CYP17), inthereticulariszoneofadrenalgland activities, suchascytochrome-b5-dependent17,20lyase be associatedtodeclineactivityinselectedenzymatic secretion. Thisagingprofileofandrogensecretionmight ( decreasing after30 years prior formationoftestosterone( once convertedtoandrostenedione,withoutrequiring converted intodihydrotestosteroneinperipheraltissues ovarian follicles to synthesize testosterone ( precursor ofactiveandrogensandcanbetakenupby hyperandrogenic states Laboratory diagnosisof 80 80 76 85 ). Consequently, inwomen,DHEASconcentrationis ). AsignificantportionofDHEAissulfatedthroughthe , ), butremainstobefurtherinvestigated.SULT2A1 DHEA productionvarieswithagingandstarts The combination of total testosterone, androstenedione 77 , 78 ). However, application for predicting metabolic min, with high clearance of 2040 L/day, withalonghalf-lifeof~17 Downloaded fromBioscientifica.com at09/25/202110:48:19AM 85 DHEA ), withnochangeinACTH 84 81 ). inthezonareticularis ). DHEASisstrongly 178 82 µmol/L inyoung www.eje-online.org :4 86 ). DHEASisthe 79 ). PAPSS2 ). 83 ) and even R147 ) have L/day via freeaccess h
European Journal of Endocrinology www.eje-online.org 10 Moran negative results.Thelargest multicenter trial,reportedby after anyglucocorticoidtreatmentthatmightcausefalse 17OHP levelshouldbeassessedintheabsenceoforwell optimally sampledduringthefirststageofcycle.Basal morning plasma17-hydroxyprogesterone(17OHP)levels, should beruledoutinhirsuteandPCOSpatientsbasedon Most if not all guidelines suggest that diagnosis of NCAH hyperplasia (NCAH)inhyperandrogenicstates Screening fornonclassicalcongenitaladrenal excess AAP( associated with insulin resistance may be associated with MEDIGENE studysupporttheconceptthatsomegenes adrenocortical function ( particularly testosterone, have only limited impact on in PCOS,itwasconcludedthatovarianandrogens,and is controversialinboth secretion ofadrenalandrogens,theeffecttestosterone are alsosomecontrastingresultsinthefield( monkeys agreewiththesefindings( hyperinsulinemia inPCOSpatients( in correlation with decreased insulin resistance and and thiazolidinedione treatment decrease DHEAS levels vitro to ACTHinPCOS( insulin amplifiesadrenalsteroidogenesisinresponse axis dysfunction( production with no evidence of hypothalamic–pituitary display ageneralizedincreaseintheadrenalsteroid contrast, many PCOSpatientsunder ACTH stimulation small portionofpatientswithincreasedDHEA/DHEAS.In for abnormalsteroidogenesisaccountonlyavery phenotype. Inheriteddefectsintheenzymesresponsible patients withhighDHEASlevelsmayshowaPCOS-like when DHEASisusedasamarkerofAAP( women showexcessadrenalandrogenproduction(AAP) Approximately 20–30%ofhyperandrogenicand/orPCOS DHEAS inhyperandrogenicstates unlikely, giventhetypicalconcentration( antibodies renders interference from othersteroidsvery a non-radioactivemarker, etc.).Usinghighlyspecific used (radioimmunoassay, automatedimmunoassay with Review ng/mL is asensitive criterion for NCAH diagnosis; in Regarding theinfluenceofovarianandrogenson data( et al . ( 90 98 99 , ). 91 ), showedthatbaseline17OHP levelabove ). Inanoppositeway, metformin( 88 88 ). , In vivo 89 87 in vivo ), whichisconsistentwith ). Preliminary results from the ). Preliminary studieshavereportedthat and M Pugeatandothers in vitro 95 93 ), althoughthere , 12 94 studies( ). ). Studiesin 96 87
). ). Hirsute 97 92 in ); ) NCAH screening in hyperandrogenic women ( accuratecut-offvalueisroutinelyusedfor This very regardless ofthetypemutation,couldbeidentified. nearly 100%ofpatientswithgeneticallyprovenNCAH, a test hasbeendeveloped,using250 NCAH. To enhance screening efficiency, an ACTH challenge 8% falsenegatives:i.e.,patientswithgenetically proven morning baseline 17OHP metabolic dysfunctionsinwomen ( establishing themutualinfluence ofandrogenexcesson in PCOSwomenopennew fieldsofinvestigationfor together withthelipogeniceffectsofandrogensreported aldo-ketoreductase type1C3(AKR1C3)expression, insulin thatincreasestheandrogen-activatingenzyme insulin andHomaindex).Thedirectinfluenceeffect of 11OHA4 and metabolic disorder markers (BMI, fasting obese PCOSwomen,whoshowclosecorrelationbetween obese PCOSwomenhavehigher11OHA4levelsthannon- of circulating androgensinwomenwithPCOSandthat that 11-oxygenatedandrogensconstitutethemajority excess disorders( opened upnewperspectivesforunderstandingandrogen (UPC2-MS/MS) for their analysis, have spectrometry performance convergencechromatography-tandemmass of 11-oxygenatedsteroids,andthedevelopmentultra- 11 In addition,theabundanceofadrenalC19steroid by 5 Dihydrotestosterone, whichisgeneratedfromtestosterone The identificationofandrogenmetabolites Perspectives inassessinghyperandrogenic states of the sequencing platformsshouldenableroutine 104 agreement with a more recent single-center study ( testosterone isthemainpotentnaturalandrogen( dihydrotestosterone, challengestheparadigmthat characterization of11-keto-testosteroneand11-keto- with androgenicactivity, andespeciallytherecent androgen inPCOSpatients( to beastrongindependentpredictorofglucuronidated be furtherinvestigated.Interestingly, DHEASwasfound provide significantcomprehensiveprofilesthatmeritsto metabolites havebeenmeasuredbyMS( hyperandrogenic states Laboratory diagnosisof > β 10 -hydroxyandrostenedione (11OHA4)asaprecursor , The identificationof11-oxygenatedsteroids α 105 -reductase, anditsmajorglucuronidatedandrogen ng/mL cut-off,MorelandTardy ( CYP21A2 , 106 ). Inthenearfuture,developmentof gene. 108 ). O’Reilly Downloaded fromBioscientifica.com at09/25/202110:48:19AM < 2 74 ng/mL was associatedwith et al ). . ( 110 109 µg cosyntropin.With 178 ). ) elegantly showed 101 :4 74 ) showedthat ). Theycould 102 R148 , 107 100 103 via freeaccess ). ), , European Journal of Endocrinology concentrations comparable to thosefoundinmen( control sampleconcentration andbecomesacceptablefor concentration levels.Scatter decreaseswithincreasing and tothelackofprecision ofmostassaykitsatthese This variationisduetodifferencesbetweenassaykits exhibiting concentrations close to those found in women. There isconsiderablescatterincontrolserumvalues immunoassays withoutpriorextractionorpurification. out total testosterone are using inroutine various carrying Thevastmajorityoflaboratories (over95%) observations. of resultsobtainedovertimehaspromptedanumber of of numerousanalytes,includingtestosterone.Analysis receive lyophilizedcontrolsera6timesperyear, forassay of biologists(ProBioQual)( including France since 1977 by a Lyon-based association ( central importanceforthediagnosisofendocrinedisease the riskofinaccuratehormoneassessmentthatare that canidentifytheimprovementsrequiredtoreduce external quality assessment (EQA). EQA is anaudit tool these professionalgroupsthereforehaveaninterestin of diagnosticsystemsandhealthcareregulators.All staff,manufacturers involving clinicians,laboratory responsibility implementation isamultidisciplinary national policies and accreditation system. Ensuring correct accordingto hospitals andhealthcaresystemsvary validate theanalyticalperformanceoftheseassays. calibration as well as for the hospital laboratories to on manufacturerstoensurereliableassaydesignand automated assays,andthisplacesaspecialresponsibility been forin-houseassaystobesupersededbycommercial assay designandcalibration.Sincethe1980s,trendhas special responsibilityonmanufacturerstoensurereliable to besupersededbyautomatedassays,andthisplacesa Since the1980s,trendhasbeenforin-houseassays External qualityassessment(EQA)ofhormoneassays free ofovarianormetabolicdisorder( ranges and deviations in a population of normal women Interpretation requiresknowledgeofnormalconcentration results. of steroidhormonesdonotprovidesatisfactory However, manyautomatedmethodsfordetermination introduction ofhomogeneoushormonestandards. of results.Progresshasbeenachievedthroughthe Assay standardizationiscrucialforcorrectinterpretation of hyperandrogenic states Main recommendations forlaboratoryinvestigation 112 Review Obviously, theinfrastructureandmanagementof ). EQA has been undertaken in several countries, 13 ). Participatinglaboratories M Pugeatandothers 111 , 112 ). 13 ). promoted ( and aCDCHormoneStandardizationProjecthasbeen ovarian androgen-secretingtumorandhyperthecosis. hormone (GnRH)agonistcouldbehelpful,inidentifying a functional adrenal source and gonadotropin-releasing dexamethasone testtosuppressandrogensarisingfrom with hypercorticism). Inunusualcircumstances, androgen-secreting adrenalcarcinoma (oftenassociated must bemeasured. DHEAS level of an androgen-secreting tumor. In this case, DHEAS limit (or Where testosteroneis2-foldhigherthantheuppernormal excess foundedonasingletotaltestosterone assay Decision-tree forevaluatingtheoriginofandrogen of directtestosteroneimmunoassay. in apositionstatementbytheEndocrineSociety( The limitationsoftestosteroneassaysweresummarized one ( homodimer bindstwotestosterone moleculesratherthan data showing that each SHBG affinity and crystallography binding toSHBG assuming variabilityof SHBG-binding multi-step dynamicallostericmodeloftestosterone’s free hypothesisanditsinterpretationproposed a state. However, arecentreviewchallengedthehormone- insulinresistanceandmoderate inflammatory overweight, protein, islowinmanycircumstances, including of the fact that SHBG, the main testosterone transport testosterone, whichhastheadvantageoftakingaccount testosterone. Thereisdebateonhowtomeasurefree providesthebesttool formeasuring mass spectrometry hyperandrogenic statesinwomen.Byitshighaccuracy, Increased total testosterone level is the main indicator of Remarks inconclusion essentially associatedtometabolicsyndrome. particularly incaseofreducedSHBGconcentrationthatis of androstenedione but noelevation of testosterone been reportedinpatientshavingisolatedelevation comparatively withtestosterone,anddissociationshave be ruledout.Lastly, depending ontheclinicalsetting,Cushingdiseaseshould assay on basal or ACTH-stimulated conditions) and, deficiencies shouldbeperformed(17OH-progesterone screening forthenonclassicformof21-hydroxylase limit, themostlikelydiagnosisisPCOS.However, hyperandrogenic states Laboratory diagnosisof Where testosteroneisjustabovetheuppernormal 116 ). Thisconceptisyettobeindependently verified. > 2 113 s . d . , ofthemeannormalrange),itissuggestive 114 , ∆ 115 4-androstenedione hasbeenstudied Downloaded fromBioscientifica.com at09/25/202110:48:19AM ). Table 2 liststheprosandcons > 178 gd indicates 600 µg/dL www.eje-online.org :4 R149 28 via freeaccess ), European Journal of Endocrinology www.eje-online.org References the public,commercialornot-for-profit sector. This researchdidnotreceiveanyspecificgrantfromfundingagencyin Funding perceived asprejudicingtheimpartialityofthisreview. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest excess disordersinwomen. domain inthecomprehensionandtreatmentofandrogen urine andvarioustissues.Thisfuturewouldopenanew performances ofMS,notonlyinthebloodbutalso of androgenprecursorsaswellmetabolitesbyusing perspective couldbetoaccessthecompleteprofile in thepathophysiologyofPCOS.Themostexciting a markerforthesedisorders,whichplaypart,notably 8 7 6 5 4 3 2 1 Review Vermeulen A, Verdonck L, Straeten MV&Orie N.Capacityof the Rosenfield RL. 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