Circulating Testosterone as the Hormonal Basis of Sex Differences in Athletic Performance

Abstract

Elite athletic competitions have separate male and female events due to men’s physical advantages in strength, speed and endurance so that a protected female category with objective entry criteria is required. Prior to puberty, there is no sex difference in circulating testosterone concentrations or athletic performance but from puberty onwards sex difference in athletic performance emerges as circulating testosterone concentrations rise in men because testes produce 30 times more testosterone than before puberty with circulating testosterone exceeding 15-fold those of women at any age. There is a wide sex difference in circulating testosterone concentrations and reproducible dose-response relationship between circulating testosterone and muscle mass and strength as well as circulating hemoglobin in both men and women. These dichotomies largely accounts for the sex differences in muscle mass and strength and circulating hemoglobin levels resulting in at least an 8-12% ergogenic advantage in men. Suppression of elevated circulating testosterone of hyperandrogenic athletes results in negative effects on performance, which are reversed when suppression ceases. Based on the non-overlapping, bimodal distribution of circulating testosterone concentration (measured by liquid chromatography-mass spectrometry) and making allowance for women with mild hyperandrogenism including that of polycystic ovarian syndrome, who are over-represented in elite athletics, the appropriate eligibility criterion for female athletic events should be a circulating testosterone of less than 5.0 nmol/L. This would include all women other than those with untreated hyperandrogenic disorders of sexual development (DSD), testosterone-treated female-to-male (F2M) transgender, noncompliant male-to-female (M2F) transgender or androgen doping.

Full text

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CirculatingTestosteroneastheHormonalBasisofSex1
DifferencesinAthleticPerformance2
Shorttitle:HyperandrogenismandFemaleAthletics3
4
DavidJ.Handelsman1,AngelicaLindénHirschberg2,StephaneBermon35
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6
1ANZACResearchInstitute,UniversityofSydneyandDepartmentofAndrology,7
ConcordHospital,Sydney,NewSouthWales,Australia,8
2DepartmentofWomen´sandChildren´sHealth,KarolinskaInstitutetand9
DepartmentofGynecologyandReproductiveMedicine,KarolinskaUniversityHospital,10
Stockholm,Sweden11
3UniversitéCôted'Azur,LAMHESSNice,FranceandInternationalAssociationofAthletics12
Federations,HealthandScienceDepartment,Monaco13
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Keywords:testosterone,sexdifference,athleticperformance,muscle,hemoglobin,disordersofsex15
differentiation16
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Words:13,172  References:161  Tables:5  Figures:818
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Correspondence:21
ProfDJHandelsman22
ANZACResearchInstitute23
SydneyNSW213924
Australia25
E:djh@anzac.edu.au26
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Disclaimer:30
DJHisamedicalandscientificconsultantforInternationalAssociationofAthleticsFederations31
(IAAF)andmemberoftheInternationalOlympicCommittee(IOC)workinggroupon32
hyperandrogenicfemaleandtransgenderathletesandtotheAustralianSportsAnti‐Doping33
AgencyandisalsoamemberofWorldAnti‐DopingAgency(WADA)’sHealth,Medicineand34
ResearchCommittee.HehasreceivedinstitutionalgrantsupportfromBesinsHealthcareand35
Lawleyforinvestigator‐initiatedclinicalstudiesintestosteronepharmacologyandhasprovided36
experttestimonyintestosteronelitigation.37
ALHisamedicalandscientificconsultantfortheSwedishOlympicCommitteeandamemberof38
theIAAFandIOCworkinggroupsonhyperandrogenicfemaleathletesandtransgenderathletes.39
ShehasreceivedgrantsupportfromIAAFforastudyontestosteroneandphysicalperformancein40
women.41
SBisamedicalandscientificconsultantfortheIAAFandamemberoftheIAAFandIOCworking42
groupsonhyperandrogenicfemaleathletesandtransgenderathletes43
Theauthorshavenootherinvolvementwithanyentityhavingafinancialinterestinthematerial44
discussedinthemanuscript.Opinionsexpressedinthispaperarethepersonalviewoftheauthors45
anddonotrepresentthoseoftheIAAF,IOC,WADAorSwedishOlympicCommittee.46
47
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Acknowledgements:TheauthorsaregratefulforhelpfulinsightsandcommentsfromAlan50
VernecandOsquelBarroso(WADA),PeterHarcourt(AFL,FIBA)andRichardBudgett(IOC).51

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Abstract52
Eliteathleticcompetitionshaveseparatemaleandfemaleeventsduetomen’sphysicaladvantagesin53
strength,speedandendurancesothataprotectedfemalecategorywithobjectiveentrycriteriaisrequired.54
Priortopuberty,thereisnosexdifferenceincirculatingtestosteroneconcentrationsorathletic55
performancebutfrompubertyonwardssexdifferenceinathleticperformanceemergesascirculating56
testosteroneconcentrationsriseinmenbecausetestesproduce30timesmoretestosteronethanbefore57
pubertywithcirculatingtestosteroneexceeding15‐foldthoseofwomenatanyage.Thereisawidesex58
differenceincirculatingtestosteroneconcentrationsandreproducibledose‐responserelationshipbetween59
circulatingtestosteroneandmusclemassandstrengthaswellascirculatinghemoglobininbothmenand60
women.Thesedichotomieslargelyaccountsforthesexdifferencesinmusclemassandstrengthand61
circulatinghemoglobinlevelsresultinginatleastan8‐12%ergogenicadvantageinmen.Suppressionof62
elevatedcirculatingtestosteroneofhyperandrogenicathletesresultsinnegativeeffectsonperformance,63
whicharereversedwhensuppressionceases.Basedonthenon‐overlapping,bimodaldistributionof64
circulatingtestosteroneconcentration(measuredbyliquidchromatography‐massspectrometry)and65
makingallowanceforwomenwithmildhyperandrogenismincludingthatofpolycysticovariansyndrome,66
whoareover‐representedineliteathletics,theappropriateeligibilitycriterionforfemaleathleticevents67
shouldbeacirculatingtestosteroneoflessthan5.0nmol/L.Thiswouldincludeallwomenotherthanthose68
withuntreatedhyperandrogenicdisordersofsexualdevelopment(DSD),testosterone‐treatedfemale‐to‐69
male(F2M)transgender,noncompliantmale‐to‐female(M2F)transgenderorandrogendoping.70
71

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1.Background72
73
Virtuallyallelitesportsaresegregatedintomaleandfemalecompetitions.Themainjustificationistoallow74
womenachancetowin,aswomenhavemajordisadvantagesagainstmenwhoare,onaverage,taller,75
stronger,fasterandhavegreaterenduranceduetotheirlarger,strongermusclesandbonesaswellasahigher76
circulatinghemoglobin.Hence,elitefemalecompetitionformsaprotectedcategorywithentrythatmustbe77
restrictedbyanobjectiveeligibilitycriterionrelatedbynecessitytotherelevantsex‐specificphysical78
advantages.Thepracticalneedtoestablishaneligibilitycriterionforelitefemaleathleticcompetitionledthe79
InternationalAssociationofAthleticFederations(IAAF)toestablisharulein2011,endorsedbythe80
InternationalOlympicFederation(IOC)in2012,forhyperandrogenicwomen.ThefirstIAAFregulationstated81
thatforathletestobeeligibletocompleteinfemaleevents,theathletemustbelegallyrecognisedasafemale82
and,unlessshehascompleteandrogeninsensitivity,maintainserumtestosteronelessthan10nmol/L.That83
IAAFeligibilityrulewaschallengedbyanathletetotheCourtforArbitrationinSports(CAS)whichruledin201584
that,althoughaneligibilitycriterionwasjustified,thescientificgroundsfortheoriginalIAAFrulewas85
consideredinsufficient,notablyintheextentofthecompetitiveadvantageenjoyedbyhyperandrogenic86
athleteswhohadcirculatingtestosteronegreaterthan10nmo/L.TheCASsuspendedthehyperandrogenism87
eligibilityrulependingreceiptofsuchevidence.Inthatcontext,thepresentpaperreviewstheavailable88
evidenceonthehormonalbasisforsexdifferencesinathleticperformance.Itconcludesthattheevidence89
justifiedarevisedeligibilitycriterionofathresholdcirculatingtestosteroneconcentrationof5nmol/L90
(measuredbyamassspectrometrymethod).91
92
2.Sex,FairnessandSegregationinSport93
94
Ifsportisdefinedastheorganizedplayingofcompetitivegamesaccordingtorules(1),fixedrulesare95
fundamentalinrepresentingtheboundariesoffairsportingcompetition.Rulebreaking,whetherby96
breachingeligibilityorcompetitionrules,suchasuseofbanneddrugs,illegalequipmentormatchfixing,97
createsunfaircompetitiveadvantagesthatviolatesfairplay.Cheatingconstitutesafraudagainstnotjust98
competitorsbutalsospectators,sponsors,thesportandthepublic.Intheabsenceofgenuinefair99
competition,elitesportwouldloseitswidepopularappealandabilitytocaptivateandinspirewiththe100
authenticattractionofgenuinecontestbetweenhighlytrainedathletes.101
102
Nevertheless,fairnessisanelusive,subjectiveconceptwithmalleableboundariesthatmaychangeover103
timeassocialconceptsoffairnessevolve.Forexample,untilthelate19thcenturywhenorganizedsports104
trainersemerged,trainingitselfwasconsideredabreachoffairnesssincecompetitionwasenvisagedat105
thattimeasacontestbasedsolelyonnaturalendowments.Similarly,sportsoncedistinguishedbetween106
amateursandprofessionals.Theconceptoffairnesshasdeepandcomplexphilosophicalrootsmainly107

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focusedonnotionsofdistributivejustice.Theseconsiderationsimpactonsportthroughtheuniversal108
applicationofanti‐discriminationandhumanrightslegislation.Lessattentionisgiventothephilosophical109
basisoffaircompetitioninelitesportwheretheobjectivesarenotegalitarianbutaimtodiscovera110
hierarchyofachievementderivedfromamixtureofunequalnaturaltalentandindividualtrainingeffort.111
Excellent,insightfuldiscussionofthelegalandmoralcomplexitiesofsexandfaircompetitioninelitesports112
fromalegalscholarandformerelitefemaleathleteisavailable(2).113
114
Thetermssexandgenderareoftenconfusedandusedasifinterchangeable.Sexisanobjective,specific115
biologicaltermwithdistinct,fixedfacets,notablygenetic,chromosomal,gonadal,hormonaland116
phenotypic(includinggenital)sex,eachofwhichhasacharacteristicdefinedbinaryform.Whileallfacets117
ofbiologicalsexarealmostalwaysalignedsothatassignmentofsexatbirthisstraightforward,rare118
instanceswhereanytwoormorefacetsofbiologicalsexconflictconstituteanintersexstate,nowreferred119
toasDisorders(orDifferences)ofSexDevelopment(DSD)(3).Bycontrast,genderisasubjective,malleable,120
self‐identifiedsocialconstructwhichdefinesaperson’sindividualgenderroleandorientation.Prompted121
bybiological,personalandsocietalfactors,volitionalexpressionofgendercantakeonvirtuallyanyform122
limitedonlybytheimaginationwithsomeindividualsassertingtheyhavenotjustasinglenatalgenderbut123
twogenders,none,adistinctthirdgenderorgenderthatvaries(fluidly)fromtimetotime.Hence,while124
genderisusuallyconsistentwithbiologicalsexasassignedatbirth,inafewitcandifferduringlife.For125
example,ifgenderwerethebasisforeligibilityforfemalesports,anathletecouldconceivablybeeligibleto126
competeatthesameOlympicsinbothfemaleandmaleevents.Thesefeaturesrendertheunassailable127
personalassertionofgenderidentityincapableofformingafair,consistentsexclassificationinelitesport.128
129
Thestrongestjustificationforsexclassificationinelitesportisthatafterpubertymenproduce20times130
moretestosteronethanwomen(4‐7)resultingincirculatingtestosteroneconcentrations15timeshigher131
thaninchildrenorwomenofanyage.Age‐gradecompetitivesportingrecordsshownosex‐related132
advantagespriortopubertyonwards,whereasfromtheageofmalepubertyonwardsthereisastrongand133
ongoingmaleadvantage(8).Thestrikingmalepost‐pubertalincreaseincirculatingtestosteroneprovidesa134
major,ongoing,cumulativeanddurablephysicaladvantageinsportingcontestsbycreatinglargerand135
strongerbones,greatermusclemassandstrength,andhighercirculatinghemoglobinaswellaspossible136
psychological(behavioural)differences.Inconcert,theserenderwomen,onaverage,unabletocompete137
effectivelyagainstmeninpower‐basedorendurance‐basedsports.138
139
Sexclassificationinsportthereforerequiresproofofeligibilityasonlywomenshouldcompeteinthe140
protected(female)category.Thisdeceptivelysimplerequirementforfairnessistakenforgrantedbypeer141
femalecompetitorswhoregardparticipationbymales,orathleteswithphysicalfeaturesclosely142
resemblingmales,asunfair.Thismakespolicingofeligibilityinescapableforsportstoavoidunfairmale143

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participationinfemaleevents.Yet,suchpolicinginevitablyintrudesintohighlypersonalmatterssothatit144
mustbeachievedwithrespectfordignityandprivacydemandinguseoftheleastinvasive,scientifically145
reliablemeans.Unsurprisinglythisdilemmahasalwaysbeenhighlycontentioussinceitfirstentered146
internationalelitesportsintheearly20thcenturyandithasbecomeincreasinglyprominentand147
contentiousinrecentdecades;nevertheless,therequirementtomaintainfairplayinfemaleeventswillnot148
disappearaslongasseparatefemalecompetitionsexist.Overrecentdecadestherehasbeenprogressively149
betterunderstandingofthecomplexbiologyofgeneticsexdeterminationandtheimpactofpubertal150
sexualmaturationinestablishingphenotypicsexualdichotomyinphysicalcapabilities.Thesesex151
dichotomousphysicalfeaturesformthebasisof,butremainquitedistinctfrom,adultgenderrolesand152
identity.Overthelastcenturyasknowledgegrewtheattemptstoformalizeascientificbasisforthe153
unavoidablenecessityofpolicingeligibilityforthefemalecategoryhavebeencontinuallychallenged.Most154
recently,theincreasingassertionofgenderself‐identificationasasocialcriterionhasfurtherchallenged155
thehegemonyofbiologyfordetermining“sportssex”,Coleman’saptterm(2).Allowingsubjectivegender156
self‐identificationtobecomethesolecriterionofsportssexwouldallowforgamingandperceptionsof157
systematicunfairnesstogrow.Thecaseforwomen’ssportsbeingdefinedbysexratherthangender,158
includingtheconsequencesofaccedingtogender‐basedclassificationhavebeenoutlined(9)inarguingthe159
importanceofpropermedicalmanagementofathletesintendingtocompeteinfemaleevents.160
161
Separatemaleandfemaleeventsinsportisadominantformofclassificationthatissuperimposedon162
othergraduatedagegroupandweight(e.g.weightlifting,powerlifting,wrestling,boxing,rowing)163
classifications,whichreflectdifferencesinstrength,power,speedtoensurefairnessintermsof164
opportunitytowinand,additionally,safetyincontactsports.Ageandweightclassificationsrelyon165
objectivecriteria(birthdate,weigh‐inweight)foreligibilityasnecessarilyshouldsexclassification.166
Nevertheless,somepowersportsdependentonexplosivestrengthandpower(egthrowingevents,167
sprinting)donotsegregateweightclasses,whileothersportswhereheightisanadvantage(egbasketball,168
jockeys)donothaveheightclassifications.Thesesportsdisproportionatelyattractathleteswithgreater169
weightand/orpower‐to‐weightratiooradvantageousstature,respectively.Ifsexclassificationwere170
eliminatedsuchopenormixedcompetitionswouldbedominatedalmostexclusivelybymen.Ittherefore171
seemshighlyunlikelythatsexclassificationwouldeverbediscardeddespitecallsonphilosophicalor172
sociologicalgroundstoend“gender”classificationinsport(10).173
174
3.Sexdifferenceincirculatingtestosteronelevels175
3.1Testosteronebiosynthesis,secretionandregulationinmenandwomen176
Anandrogenisahormonecapableofdevelopingandmaintainingmasculinecharacteristicsinreproductive177
tissues(notablythegenitaltract,andothertissuesandorgansassociatedwithsecondarysexual178

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characteristicsandfertility)andcontributingtotheanabolicstatusofnon‐reproductivebodytissues(11).179
Thetwodominantbioactiveandrogenscirculatinginmaturemammals,includinghumans ‐‐testosterone180
anditsmorepotentmetabolite,dihydrotestosterone(DHT)‐‐accountforthedevelopmentandmaintenance181
ofallandrogen‐dependentcharacteristics,andtheircirculatinglevelsinmenandnon‐pregnantwomenarise182
fromsteroidssynthesizeddenovointhetestes,ovaryoradrenals(12).183
Thesexuallyundifferentiatedgonadsintheembryodevelopintoeitherovariesortestesaccordingto184
whetheraychromosome(oratleastthesrygene)ispresent.Afterbirthanduntilpubertycommences,185
circulatingtestosteroneconcentrationsareessentiallythesameinboysandgirls,otherthanbrieflyinthe186
neonatalperiodofboyswhenhigherlevelsprevail.Theonsetofmalepuberty,abrain‐drivenprocess187
triggeredbyastillmysterioushypothalamicorhighercerebralmechanism(13),initiatesthehormonal188
cascadeofpuberty.Inmalesthisleadstoenhancedpituitaryluteinizinghormone(LH)secretionthat189
stimulatesthe500millionLeydigcellsinthetestestosecrete3‐10mg(mean7mg)oftestosteronedaily(4,190
6,7,14,15).Thiscreatesaveryhighlocalconcentrationoftestosteronewithinthetestisaswellasasteep191
downhillconcentrationgradientintothebloodstreamthatmaintainscirculatingtestosteronelevelsatadult192
malelevels,whicharetightlyregulatedbystrongnegativehypothalamicfeedbackofcirculating193
testosterone.However,intheabsenceoftestesthesemechanismsdonotoccurinfemales.Ingirls,serum194
testosteroneincreasesduringpuberty(16),peakingatage20‐25yearsbeforedeclininggraduallywithage(17,195
18)butitremainslessthan2nmol/Latallages,asdeterminedbyareliablemethod(seebelow).Inadult196
women,circulatingtestosteroneisderivedfromthreeroughlyequalsources–directsecretionfromtheadrenal197
glandortheovaryaswellasindirectlyfromextra‐glandularconversion(inliver,kidney,muscle,fat,skin)from198
testosteroneprecursorssecretedbytheadrenalandovary.However,incombinationthesedifferentsources199
produceabout0.25mgoftestosteronedailysothatthroughoutlifewomenmaintaincirculatingtestosterone200
levelsoflessthan2nmol/L.Circulatingtestosteroneconcentrationsinwomenaresubjecttolittledynamic201
physiologicalregulation.Asaresult,circulatingtestosteroneconcentrationsinhealthypre‐menopausal202
womenarestable(non‐fluctuating)andnotsubjecttostrongnegativefeedbackbyexogenoustestosterone203
likemen.Eventhesmallrise(50%)atthetimeofthemid‐cycleLHsurgetriggeringovulation(19),remains204
withinthephysiologicalrangeforpre‐menopausalfemales.Insummary,onlywhencirculatingtestosterone205
concentrationsinmaleadolescentsrisesabovethecirculatingpre‐pubertalconcentrationsdoesthe206
virilisationcharacteristicofmencommence,progressandremainthroughoutadultlifeatleastuntiloldage207
(18).208
209
3.2Maleandfemalereferencerangesforcirculatingtestosterone210
211
Areliablethresholdforcirculatingtestosteronemustbesetusingmeasurementbythereferencemethodof212
liquid(orgas)chromatography‐massspectrometry(LC‐MS)ratherthanusingoneofthevariousavailable213
commercialtestosteroneimmunoassays.Thenecessaryrelianceonsteroidmassspectrometryforclinical214

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applicationsinendocrinology,reproductivemedicineandsportsmedicineiswidelyrecognized.Ithasbeen215
standardfordecadesinanti‐dopingscience(20)andthegrowingconsensusisthatitisrequiredforhigh216
qualityclinicalresearchandpracticerecognizedbycognateprofessionalsocieties(21,22)andeditorialsin217
leadingclinicalendocrinology(23)andreproductivemedicine(24)journals.Theinherentlylimitedspecificity218
oftestosteroneimmunoassaysarisesfromantibodycross‐reactivitywithstructurallyrelatedsteroids(suchas219
precursorsandmetabolites)otherthantheintendedtarget.Asaresult,allsteroidimmunoassaysincluding220
fortestosteronedisplaymethod‐specificbiaswhereby,forexample,thelowerlimitofatestosterone221
referencerangeinhealthyyoungmenvariesfrom7.3to12.6nmol/Laccordingtotheimmunoassayused,so222
thatnoconsensusdefinitionofalowerlimitcouldbeobtainedindependentofthecommercialimmunoassay223
methodused(25).Further,testosteroneimmunoassaysareoptimizedforcirculatinglevelsinmenbutdisplay224
increasinginaccuracyatthelower,byanorderofmagnitude,circulatingtestosteroneconcentrationsin225
womenorchildren.Incontrasttoimmunoassays,LC‐MSbasedmethodsarehighlyspecificanddonot226
dependonproprietaryantibodies.UsingLC‐MS‐basedmeasurements,method‐specificbiascanbeavoided227
andafixedconsensuslowerreferencelimitdefined(seetable1).Hence,fortheprecisionrequiredin228
sportsmedicine,whetherforeligibilitycriteriaoranti‐dopingapplications,testosteroneinserummustbe229
measuredbyLC‐MSmethods.230
231
Priortopuberty,levelsofcirculatingtestosteroneasdeterminedbyLC‐MSarethesameinboysandgirls232
(16)aswellasremaininglowerthan2nmol/Linwomenofallages.However,fromtheonsetofmale233
pubertythetestessecrete20timesmoretestosteroneresultingincirculatingtestosteronelevelsthatare234
15timesgreaterinhealthyyoungmenthanagesimilarwomen.UsingLC‐MSmeasurement,circulating235
testosteroneinadultshasastrikingly,non‐overlappingbimodaldistributionwithwideandcomplete236
separationbetweenmenandwomen.Table1summarisesdatafromappropriatereportedstudiesusing237
MS‐basedmethodstomeasureserumtestosteroneinhealthymenandwomen.Basedonanumber‐238
weightedpoolingwithconventional95%two‐sidedconfidencelimitsoftheeightavailablestudiesusingLC‐239
MSmeasurementsofserumtestosterone,thelimitsofthereferencerangeforhealthyyoungmen(18to240
40years)is7.7nmol/Lto29.4nmol/L.Similarly,summarisingthenineavailablestudiesforhealthy241
menstruatingwomenunder40years,the95%(twosided)referencerangeis0to1.7nmol/L.These242
referencelimitsneglectfactorssuchasoralcontraceptiveuse(26,27),menstrualphase(19),SHBG(28,243
29),overweight(30,31),fastingandsmoking(32),aswellasdiet(31)andphysicalactivity(33,34)in244
womenandmen,allofwhichhavesmalleffectsoncirculatingtestosteronebutwithoutmaterially245
influencingthedivergencebetweenthenon‐overlappingbimodaldistributionofmaleandfemale246
referencerangesofcirculatingtestosterone.247
248
Increatingathresholdforeligibilityforfemaleeventsitisalsonecessarytomakeallowancefor249
hyperandrogenicwomenincludingwomenwithpolycysticovarysyndrome(PCOS)andnon‐classical250

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adrenalhyperplasia.PCOSisarelativelycommondisorderamongwomenofreproductiveageswitha251
prevalenceof6‐10%,dependingonthediagnosticcriteriaused(35),inwhichmildhyperandrogenismisa252
keyclinicalfeatureandhashigherthanexpectedprevalenceamongelitefemaleathletes(26,36‐38).Non‐253
classicaladrenalhyperplasiaisamilderandlater(adult)onsetvariantofclassicalcongenitaladrenal254
hyperplasia(39)withamuchhigherbutstillrarepopulationprevalence(1:1000vs1:16,000fortheclassical255
variant(40).Table2summarisesclinicalstudies(n=16,≥40women)reportingserumtestosterone256
concentrationsmeasuredbyLC‐MSinsamplesfromwomenwithPCOS.Thepooleddatarevealsthatthe257
upperlimitofserumtestosteroneinwomenwithPCOSis3.1nmol/L(95%confidenceinterval,onesided)258
or4.8nmol/L(usinga99.99%confidenceinterval,onesided)(table3).Henceaconservativethresholdfor259
circulatingtestosteroneof5nmol/LmeasuredbyLC‐MSwouldidentifyfewerthan1:10,000womenwith260
PCOSasfalsepositives,basedoncirculatingtestosteronemeasurementalone.Circulatingtestosterone261
higherthanthisthresholdislikelytobeduetotestosterone‐secretingadrenalorovariantumors,262
intersex/DSD,badlycontrolledornon‐compliantM2Ftransgenderathletesortestosteronedoping.263
264
3.3Thephysiologicaleffectsoftestosteronedependonthecirculatingtestosterone,notitssource265
(endogenousorexogenous)266
Testosterone,whetherofnaturalendogenousormanufacturedexogenoussource,hasanidenticalchemical267
structureandbiologicaleffects,asidefromminordifferencesinisotopiccompositionwhicharebiologically268
insignificant.Regardlessofitssource,atequivalentdosesandcirculatinglevels,exogenoustestosterone269
exertsthesamebiologicalandclinicaleffectsoneveryknownandrogen‐responsivetissueororgan,apart270
fromeffectsonspermatogenesis,whichasdiscussedbelowisonlyamatterofdegree.Consequently,271
exogenoustestosteroneisafullyeffectivesubstituteforendogenoustestosteroneintherapeuticuse,272
counteringtheeffectsoftestosteronedeficiencyduetohypogonadism(reproductivesystemdisorders).Any273
purporteddifferencesbetweenendogenousandexogenoustestosteroneare,likethedifferencesbetween274
menandwomen,duetocorrespondingdifferencesintheendogenousproductionrateorexogenousdose.275
Suchdifferencesineffectiveexposureleadtocorrespondingdifferencesincirculatingtestosteronelevels276
anditseffectsaccordingtothedose‐responsecurvesfortestosterone.277
Likeallhormonesanddrugs,overtheireffectiverangeofbiologicalactivitythedose‐responserelationship278
fortestosteroneisusuallyasigmoidalcurvewithlowerandupperplateausjoinedbyamonotonicallyrising279
middleregion,whichmaybelinearinthenaturalscalebutmoreoftenlog‐linear(linearonthelogorsimilar280
transformedscale).Inthemiddleportionofthetypicalsigmoidaldose‐responsecurveforthesameincrease281
intestosteronedose(orconcentration),theresponsewouldbeincreasedinsimpleproportional(ielinear)282
butmoreoftenonalogarithmicscale.Bycontrast,atthelowerandupperplateausofdoseorconcentrations,283
changesintestosteroneexposuremayevokeminimalornoresponseontheendpoint.Forexample,in284
womenofanyagecirculatingtestosteroneconcentrationsarealongthelowerplateauofthedose‐response285

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curve,sothatincreasesincirculatingtestosteroneconcentrationswithinthatlowerplateaumayhave286
minimalornoeffect.InfemaleathleteswiththemildhyperandrogenismofPCOS,higherperformancehas287
beenshown(38)withtheirmusclemassandpowerperformancecorrelatingwithandrogenlevels(26).288
However,beyondtheseeffectswhereendogenoustestosteroneconcentrationsareinthehigh‐normaladult289
femalerange,itisonlywhentheincreasesincirculatingtestosteroneconcentrationssubstantiallyand290
consistentlyexceedthoseprevailinginchildhood(<2nmol/L)andamongwomenincludingthosewithPCOS291
(<5nmol/L)thattheeffectswouldreplicaterisingtestosteroneconcentrationsofboy’sinmid‐ tolate292
puberty(typically>8nmol/l)whichcausethemasculinizingeffectsofincreasedmuscle,boneand293
hemoglobincharacteristicsofmen.Asshownabove,thecirculatingtestosteroneofmostwomennever294
reachesconsistentlyabove5nmol/L,alevelwhichboysmustsustainforsometimetoexhibitthe295
masculinizingeffectsofmalepuberty.296
Secondarily,theeffectsoftestosteronearemodulatedinaformoffinetuningbythepatternsofexposure,297
suchaswhetherthecirculatingtestosteroneisdeliveredintheun‐physiologicalsteady‐stateformat(e.g.298
quasi‐steadystatedeliverybyimplantortransdermalproducts)orbythepeak‐and‐troughdeliveryof299
injectionsasopposedtothenaturalstateofendogenousfluctuationsinserumtestosteronearoundthe300
averageadultmalelevels.However,theselatterpatterneffectsaresubtleandthedominanteffectremains301
thatofdoseandaveragetestosteroneconcentrationsinblood,howevertheyarise.Furthermore,thereis302
evidencethattheandrogensensitivityofresponsivetissuesdifferandmaybeoptimalatdifferentcirculating303
testosteroneconcentrations(41).304
Malesexualfunctionismaintainedbyendogenoustestosteroneatadultmalecirculatingconcentrations.305
Theseeffectscanbereplicatedbyexogenoustestosteroneifandonlyifitachievescomparablecirculating306
testosteroneconcentrations.Forexample,inawell‐controlledprospectivestudyofoldermenwithprostate307
cancer(42),androgendeprivationachievingcastratelevelsofcirculatingtestosteronesustainedover12308
monthsmarkedlysuppressedsexualdesireandfunction,whereasthoseeffectsdidnotoccurinage‐matched309
menhavingnon‐hormonaltreatmentforprostatecancerorthosewithoutprostatecancer.Inhealthy310
youngermenwhoseendogenoustestosteroneisfullysuppressed,theirsexualfunctioncompletelyrecovers311
whencirculatingtestosteronewasrestoredtothephysiologicalmalerangebyadministrationofexogenous312
testosterone(43).Similareffectswerealsoobservedinhealthy,middle‐agedmeninwhommalesexual313
functionwasfullymaintained(comparedwithplacebo)during2yearsoftreatmentwithanexogenous314
androgen(DHT)despiteitcausingsustained,completesuppressionofendogenoustestosterone(44).This315
furthersupportsthekeyinterpretationthatthebiologicaleffectsofexogenousorendogenoustestosterone316
arethesameatcomparablecirculatinglevels.317
318
Clinically,exogenoustestosteronereplicatesfullyalleffectsofendogenoustestosteroneonevery319
reproductiveandnon‐reproductiveorganortissue,withthesoleexceptionofthetestis.Spermproduction320

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inthetestisrequiresaveryhighconcentrationoftestosterone(typically100timesgreaterthaninthegeneral321
bloodstream),whichisproducedinnatureonlybytheactionofthepituitaryhormoneLH.LHstimulatesthe322
Leydigcellsintheinterstitialspaceofthetestisbetweenseminiferoustubulestoproducehighintra‐testicular323
concentrationsoftestosterone,whicharenecessaryandsufficienttoinitiateandmaintainspermproduction324
intheadjacentseminiferoustubules.Thishighconcentrationoftestosteronealsoprovidesadownhill325
gradienttosupplytherestofthebody,wherecirculatingtestosteroneactsonandrogen‐responsivetissues326
tomaintainmasculinepatternsofandrogenization.Whenexogenoustestosterone(oranyotherandrogen)327
isadministeredtomen,pituitaryLHissuppressedbynegativefeedbackandthespermproductionhaltsfor328
aslongasexogenoustestosteroneorandrogenexposurecontinues,afterwhichitrecovers(45).However,329
eventhereductioninspermatogenesisandtestissizewhenmenaretreatedwithexogenoustestosteroneis330
onlyamatterofdegree.Itiswellestablishedinrodents(46,47)thatspermatogenesisisinducedby331
exogenoustestosteroneifthetestosteroneconcentrationsinthetestisarehighenoughtoreplicatewhat332
occursnaturallyviaLHstimulation(48).However,directreplicationthathighdosetestosteronealsoinitiates333
andmaintainsspermatogenesisinhumansisnotfeasibleasthesetestosteronedosesare10‐100times334
higherthancouldbesafelygiventohumans.Nevertheless,confirmatoryevidenceinhumansisavailable335
fromrarecasesofmenwithanactivatingmutationoftheCG/LHreceptor(49,50).Thismutationcauses336
autonomoustesticulartestosteronesecretionleadingtoprecociouspubertyarisingfromthepremature337
adultmalecirculatingtestosteroneconcentrationswhichleadtocompletesuppressionofcirculating338
gonadotropin(LH,FSH)secretion.Inthisillustrativecasethetestiswasexposedtonon‐physiologicallyhigh339
testosteroneconcentrations(butwithoutanygonadotropinstimulation)whichinducedspermproduction340
andallowedfornaturalpaternity(49).Thisindicatesthatevenforspermatogenesis,exogenoustestosterone341
canreplicateallbiologicaleffectsofendogenoustestosteroneinaccordancewiththerelevantdose‐response342
characteristics.343
Themostrealisticviewisthatincreasingcirculatingtestosteronefromthechildhoodorfemalerangetothe344
adultmalerangewillhavethesamephysiologicaleffectswhetherthesourceoftheadditionaltestosterone345
isendogenousorexogenous.Thisisstronglysupportedbywell‐establishedknowledgeabouttherelationship346
ofcirculatingtestosteroneconcentrationswiththetimingandmanifestationsofmalepuberty.The347
characteristicclinicalfeaturesofmasculinisation(musclegrowth,increasedheight,increasedhemoglobin,348
bodyhairdistribution,voicechangeetc)appearonlyifandwhencirculatingtestosteroneconcentrationsrise349
intotherangeofmalesatmid‐pubertywhicharehigherthaninwomenatanyageevenaftertherisein350
circulatingtestosteroneinfemalepuberty.Ifandonlyifthepubertalriseincirculatingtestosteronefails,351
themalesaffectedareclinicallyconsideredhypogonadal.Suchafailureofmalepubertymayoccurfor352
geneticreasons(arisingfrommutationsthatinactivateanyofthecascadeofproteinswhoseactivityiscritical353
inthehypothalamustotriggermalepuberty)orasaresultofacquiredconditions,causedbypathological354
disordersofthehypothalamusorpituitaryorfunctionaldefectsarisingfromseveredeficitsofenergyor355

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nutrition(egextremeovertraining,undernutrition),thelatterbeingcomparablewithhypothalamic356
amenorrheaoranorexianervosainfemaleathletes/balletdancers.Ifmalepubertyfails,testosterone357
replacementtherapyisfullyeffectiveinreplicatingtheallthedistinctivemasculinefeaturesapartfrom358
spermatogenesis.359
360
361
3.4ElevatedcirculatingtestosteroneconcentrationcausedbyDSDs362
RaregeneticintersexconditionsknownasDSDscanleadtomarkedlyincreasedcirculatingtestosteronein363
womenand,whencoupledwithambiguousgenitaliaatbirth,appearingasundervirilizedmale,orvirilized364
females.Thiscancauseathleteswhowereraisedandidentifyaswomentohavecirculatingtestosterone365
levelscomparablewithmenandmuchexceedingthatofnon‐DSD(andnon‐doped)women,includingthose366
withPCOS.Keycongenitaldisordersinthiscategoryare46XYDSDsnamely5αreductasedeficiency(51),367
17β‐hydroxysteroiddehydrogenasetype3deficiency(52),androgeninsensitivity(53,54)aswellas368
congenitaladrenalhyperplasia(55),whichisa46XXDSD.Thereisevidencethatthefirstthreeconditions,369
componentsof46XYDSDs,are140timesmoreprevalentamongelitefemaleathletesthanexpectedinthe370
generalpopulation(56).371
Genetic5αreductasedeficiencyisduetoaninactivatingmutationinthe5αreductasetypeIIenzyme(51).372
ThisleadstoadeficitofDHTduringfetallifewhenDHTisrequiredforconvertingthesex‐undifferentiated373
embryonicandfetaltissuetoformthesex‐differentiatedmasculineformexternalgenitalia.Althoughgenetic374
males(46XY)with5αreductasedeficiencywilldeveloptestes,theyusuallyremainundescendedandlabial375
fusiontoformascrotumandphallicgrowthdoesnotoccur.Henceatbirththeexternalgenitaliamayappear376
feminine,leadingtoafemaleassignednatalsex.Thus,individualswith5α reductasedeficiencymayhave377
malechromosomalsex(46XY),gonadalsex(testes),andhormonalsex(adultmaletestosterone378
concentrations),butsuchseverelyunder‐virilizedgenitaliathataffectedindividualsmayberaisedfrombirth379
asfemalesratherthanasunder‐virilizedmales.However,fromtheonsetofmalepuberty,testicularLeydig380
cellsstartproducinglargeamountsoftestosterone,andthesteepriseincirculatingtestosteronetoadult381
malelevels(withthepermissiveroleof5αreductaseactivity)leadstomasculinevirilisation,includingmale382
patternsofmuscleandbonegrowth,hemoglobinlevelsandothermasculinebodyhabitusfeatures(hair383
growthpattern,voicechange),aswellasphallicgrowth(56).Suchchangesofmalepubertypromptaround384
halfaffectedindividualswhohadfemalesexassignedatbirthanddevelopedasgirlspriortopubertytoadopt385
amalegenderidentityandrole(57).Spermareformedinthetestessothat,usinginvitrofertilization,these386
individualsmayfatherchildren(58).387
Seventeenβ‐hydroxysteroiddehydrogenasetype3deficiency(52)hasasimilarnaturalhistoryto5α388
reductasedeficiency.Thisdisorderisduetoinactivatingmutationsinasteroidogenicenzymeexpressedonly389
inthetestisandwhichisessentialfortestosteroneformationinthefetus.Intheabsenceofafunctional390

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enzyme,thetestismakeslittletestosteronebutinsteadsecreteslargeamountsofandrostenedione,the391
steroidimmediatelypriortotheenzymaticblock.Inthecirculation,theexcessofandrostenedioneis392
convertedtotestosterone(mainlybytheenzymeAKR1C3(12)).Althoughthecirculatingtestosteroneisthen393
convertedtocirculatingDHT,insufficientDHTisformedlocallywithintheurogenitalsinustovirilisegenitalia394
atbirth.Thiscausesthesamesevereunder‐virilisationoftheexternalgenitaliaofgeneticallymale395
individuals,leadingtoambiguousgenitaliaatbirthdespitemalechromosomal,gonadalandhormonalsex.396
Whenpubertyarrives,thetestesstartproducingtheadultmaletestosteroneoutputthisleadstomarked397
virilisationandsubsequentassumptionofamalegenderidentitybysomeaffectedindividuals,conflicting398
withafemaleassignednatalsexandchildhoodupbringing.399
Androgeninsensitivity,whicharisesfrommutationintheandrogenreceptor(AR),posesdifferentbut400
complexchallengesforeligibilityforfemaleathleticevents.AstheARislocatedontheXchromosome,401
geneticmales(46XY)arehemizygous,sothataninactivatingmutationintheARcanbepartiallyorfully402
insensitivetoandrogenaction.Affectedindividualshavemaleinternalgenitalia(testesintheinguinalcanal403
orabdomenwithWolffianducts)andconsequentlyadultmalecirculatingtestosteroneconcentrationsafter404
puberty.Thesenon‐lethalmutationshaveawidespectrumoffunctionaleffects,rangingfromfullresistance405
toallandrogenactionincompleteandrogeninsensitivitysyndrome(CAIS)whereindividualshaveafull406
femalephenotypewithnormalfemaleexternalgenitalia,topartialandrogeninsensitivitysyndrome(PAIS)407
wheresomeandrogenactionisstillexertedleadingtovariousdegreesofambiguousgenitalia,ortomild408
androgeninsensitivitywhichproducesaverymild,under‐virilisedmalephenotype(normalmalegenitaland409
somaticdevelopmentbutwithlittlebodyhairandnomalepatternbalding)(53).Testosterone(and410
dihydrotestosterone)havenoconsistenteffectofinducingnormalnitrogenretention(anabolic)responses411
inpatientswithCAIS(59‐62)althoughsomereducedandrogenresponsivenessisretainedbypatientswith412
PAIS(60,63‐66).AthleteswithCAIScanfairlycompeteasfemalesbecausethecirculatingtestosterone,413
althoughatadultmalelevels,hasnophysiologicaleffectsothat,intermsofandrogenactionandtheensuing414
physicalsomaticadvantagesofmalesex,affectedindividualsareindistinguishablefromfemalesandgainno415
benefitsofthesexdifferencearisingfromunimpededtestosteroneaction.Amorecomplexissueariseswith416
athleteshavingPAISreflectingthedegreeofincompleteimpairmentofARfunction.Residualandrogen417
actioninsuchARmutationsishardertocharacterisequantitativelyasthereisnostandardized,objectivein418
vitrotesttoquantifyARfunctionality.Hence,althoughindividualswithPAISmayhaveadultmalecirculating419
testosteroneconcentrationsbutvariableandrogensensitivity,atpresentthisrequiresacase‐by‐case420
evaluation,primarilybasedonthedegreeofvirilisation.Thecurrentbestavailableclinicalapproachto421
determiningthefunctionalimpact(degreeoffunctionality/sensitivity)ofanARmutationisbasedonthe422
degreeofsomatic,primarilygenital,virilisationassessedaccordingtotheQuigleyclassificationofgradeof423
androgensensitivity(67).424
Congenitaladrenalhyperplasia(CAH)isarelativelycommondefectinadrenalsteroidogenesisinthe425

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enzymaticpathwayleadingtosynthesisofcortisol,aldosteroneandsexsteroidprecursors.Thedisease426
variesinseverityfromlife‐threatening(adrenalfailure)tomild(hirsutismandmenstrualirregularity),oreven427
asymptomaticandundiagnosed.ThemostcommonmutationscausingCAHoccurinthe21hydroxylase428
enzyme,accountingfor95%ofcases(55).Thedefectleadstoabottleneck,creatingamajorbackingupof429
precursorsteroidswhichthenoverflowintoothersteroidpathways,leadingtodiagnostichighlevelsof17430
hydroxyprogesteroneand,infemalepatients,excessivecirculatingtestosteroneorotheradrenal‐source431
androgenprecursors(egandrostenedione,DHEA)whichmaybeconvertedtotestosteroneintissues.A432
commonclinicalproblemwithmanagementofCAHisthatglucocorticoid/mineralocorticoidtreatmentisnot433
alwaysfullyeffectivepartlyduetovariablecompliance,whichmayleavehighcirculatingtestosterone,434
includingwellintoorevenabovethenormalmalerange(68).Itisunlikelythatmildnon‐classicalcongenital435
adrenalhyperplasiaisamajorcontributortothemildhyperandrogenismprevalentamongelitefemale436
athletes.TheprevalenceofPCOS(6‐16%)isabout100timeshigherthanmildnon‐classicalcongenitaladrenal437
hyperplasia(0.1%,(40))whileadisproportionatelyhighnumberofelitefemaleathletes(especiallyinpower438
sports)havePCOS(36).Inonestudyofhyperandrogenicfemaleathletes,evenmildNCAHwasruledoutby439
normal17hydroxyprogesterone(26)andinanother(38)reportedserumandrostenedioneandcortisoldid440
notdifferfromcontrols,rulingoutsignificantcongenitaladrenalhyperplasia..441
442
4.Sexdifferenceinmuscle,hemoglobin,boneandathleticperformancerelatingtoadult443
circulatingtestosteroneconcentrations444
445
Followingpuberty,testosteroneproductionincreases(16)butremainsbelow2nmol/Linwomenwhereas446
inmentestosteroneproductionincreases20‐fold(from0.3mgadayto7mgaday)leadingtoa15‐fold447
highercirculatingtestosteroneconcentrations(15vs1nmol/L).Thegreatermagnitudeofsexdifferencein448
testosteroneproduction(20fold)comparedwithcirculatinglevels(15fold)isduetowomen’shigher449
circulatingSHBG,whichretardstestosteroneclearancecreatingaslowercirculatinghalf‐timeof450
testosterone.Thisorderofmagnitudedifferenceincirculatingtestosteroneconcentrationsisthekeyfactor451
tomen’ssuperiorathleticperformanceduetoandrogeneffectsprincipallyonmuscle,boneandhemoglobin.452
453
4.1Muscle454
4.1.1Biology:455
Ithasbeenknownsinceancienttimesthatcastrationinfluencesmusclefunction.Modernknowledgeofthe456
molecularandcellularbasisforandrogeneffectsonskeletalmuscleinvolveseffectsduetoandrogen457
(testosterone,DHT)bindingtotheandrogenreceptorwhichthenreleaseschaperoneproteins,dimerizes458
andtranslocatesintothenucleustobindtoandrogenresponseelementsinthepromoterDNAofandrogen459
sensitivegenes.Thisleadstoincreasesin(a)musclefibrenumbersandsize,(b)musclesatellitecellnumbers,460

Page15of51
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(c)numbersofmyonuclei,and(d)sizeofmotorneurons(69).Additionallythereisexperimentalevidence461
thattestosteroneincreasesskeletalmusclemyostatinexpression(70),mitochondrialbiogenesis(71),462
myoglobinexpression(72)andinsulin‐likegrowthfactor(IGF‐I)content(73)whichmayaugmentenergetic463
andpowergenerationofskeletalmuscularactivity.464
Customizedgeneticmousemodelscanprovideuniquephysiologicalinsightintargetingspecificmoleculesor465
theirreceptorstoprovideexperimentalinsightintomammalianphysiologywhichisunobtainablebyhuman466
experimentation.Thetightevolutionaryconservationofthemammalianreproductivesystemexplainswhy467
geneticmousemodelshaveprovidedconsistent,highfidelityreplicationofthehumanreproductivesystem468
(74,75).Geneticmales(46XY)withandrogeninsensitivitydisplayingsimilarfeaturesoccurthrough469
spontaneouslyoccurringinactivatingARmutationsinallmammalianspeciesstudiedincludinghuman,where470
theyareknownaswomenwithCAIS.Theconverse,geneticfemales(46XX)resistanttoallandrogenaction,471
cannotoccurnaturallyinhumansorothermammals.Thisisbecausefullyandrogenresistantfemalesmust472
havebothXchromosomescarryinganinactivatedAR.InturnthisrequiresacquiringoneXchromosomefrom473
theirfather.However,thepotentialfathersaresterileashemizygousmalesbearingasinglecopyanX474
chromosomewithaninactiveARproducenosperm,asafunctionalARisbiologicallyindispensablefor475
makingsperminanymammal.However,androgenresistantfemalescanbebredbygeneticengineering476
usingtheCre‐Loxsystem(76).Animportantfindingfromsuchstudiesisthatandrogen‐resistantfemalemice477
haveessentiallythesamemusclemassandfunctioncomparedwithwild‐typeandrogensensitivefemales478
bearingnormalARwhereasandrogen‐resistantmalemicehavesmallerandweakermusclemassand479
functionthanwild‐typemalesbutarecomparableinsteadwiththemuscleofwild‐typefemales(77).This480
indicatesthatandrogenaction,representedbycirculatingtestosterone,isthekeydeterminantofthehigher481
musclemassandstrengthcharacteristicofmalescomparedwithfemales.Furthermore,endogenous482
circulatingtestosteronehasminimaleffectsonskeletalmusclemassandstrengthinfemalemice.Although483
theseexperimentscannotbereplicatedinhumans,theirkeyinsightisthatthehighercirculatingtestosterone484
inmalesisthedeterminantofthemale’sgreatermusclemassandfunctioncomparedwithfemales.485
Nevertheless,thereisalsoevidencethathyperandrogenicwomen,mostlywithPCOS,haveincreasedmuscle486
massandstrengththatcorrelateswithmildlyincreasedcirculatingtestosteroneinthehigh‐normalfemale487
range(26,38).488
489
4.1.2Observationaldata:490
Thereisaclearsexdifferenceinbothmusclemassandstrength(78‐80)evenadjustingforsexdifferencesin491
heightandweight(80,81).Onaverage,womenhave50‐60%ofmen'supperarmmusclecross‐sectional492
area(CSA)and65‐70%ofmen'sthighmuscleCSA;andwomenhave50‐60%ofmen'supperlimbstrength493
and60‐80%ofmen'slegstrength(82).Youngmenhaveonaverageaskeletalmusclemassofover12kg494
greaterthanage‐matchedwomenatanygivenbodyweight(80,81).Whilenumerousgenesand495

Page16of51

environmentalfactors(includinggenetics,physicalactivityanddiet)maycontributetomusclemass,the496
majorcauseofthesexdifferenceinmusclemassandstrengthisthesexdifferenceincirculating497
testosterone.498
Age‐gradecompetitivesportsrecordsshowminimalornofemaledisadvantagepriortopuberty,whereas499
fromtheageofmalepubertyonwardsthereisastrongandongoingmaleadvantage.Correspondingtothe500
endogenouscirculatingtestosteroneincreasinginmalesafterpubertyto15‐20nmol/L(sharplydiverging501
fromthecirculatinglevelsthatremain<2nmol/Linfemales),maleathleticperformancesgofrombeingequal502
onaveragetothoseofage‐matchedfemalesto10‐12%betterinrunningandswimmingevents,and20%503
betterinjumpingevents(8)(figure1).CorroborativefindingsareprovidedbyaNorwegianstudythat504
examinedperformanceofadolescentsincertainathleticeventsbutwithoutreferencetocontemporaneous505
circulatingtestosteroneconcentrations(83).Thestrikingpost‐pubertalincreaseinmalecirculating506
testosteroneprovidesamajor,ongoing,cumulativeanddurableadvantageinsportingcontestsbycreating507
atleastgreatermusclemassandstrengthsuchthatthesesexdifferencesrenderwomenunabletocompete508
effectivelyagainstmen,especially(butnotonly)inpowersports.509
Thesefindingsaresupportedbystudiesofnon‐athleticwomenshowingthatmusclemassisincreasedin510
proportiontocirculatingtestosteroneinwomenwithmildlyelevatedtestosteronelevelsduetoPCOS(84,511
85),aconditionwhichismoreprevalentamongelitefemaleathleteswhoexhibitthesefeatures(26,36,38),512
oftenundiagnosed(37),butwhichmayprovideanergogenicadvantage(38),consistentwiththegraded513
effectsofcirculatingtestosteroneonexplosiveperformanceinmenandwomen(86).514
Studiesofelitefemaleathletesfurthercorroboratethesefindings.Onestudydemonstratesdose‐response515
effectsofbetterperformanceinsome(400m,400mhurdles,800mrunning,hammerthrow,polevault)but516
notallathleticeventscorrelatedwithsignificantlyhigherendogenoustestosteroneinfemale,butnotmale,517
athletes.Evenwithinthelowcirculatingtestosteronelevelsprevailingwithinthenormalfemalerange,in518
theseeventstherewasasignificantadvantageof1.8%to4.5%amongthoseinthehighestcomparedwith519
thelowesttertileofendogenoustestosterone(27).Afurtherstudyofelitefemaleathletescorroboratesand520
extendstheseobservationsinthatendogenousandrogensareassociatedwithamoreanabolicbody521
compositionaswellasenhancedmuscularperformance(26).Inthisstudy106SwedishOlympicfemale522
athleteswerecomparedwith117age‐andweight(BMI)‐matchedsedentarycontrolwomenfortheirmuscle523
andbonemass(bydualenergyX‐rayabsorptiometry,DEXA),theirmuscularstrength(squatand524
countermovementjumps),andtestosteroneandDHT,aswellasandrogenprecursors(DHEA,525
androstenedione)andurinaryandrogenglucuronidemetabolites(androsterone,etiocholanolone,3and17526
3α‐diols)measuredbyliquidchromatography‐massspectrometry(26).Theathletesdisplayedhighermuscle527
(andbone)massthanthesedentarycontrolwomen,withstrengthtestscorrelatingstronglywithmuscle528
masswhetherintotalorjustinthelegs.Inturn,musclemassandstrengthwerecorrelatedwithandrogens529
andandrogenprecursors.Consideringthatsuchstudiesmaybeconfoundedbyfactorssuchasmenstrual530

Page17of51

phaseanddysfunction,andheterogeneoussportsdisciplines,whichweakenthepowerofthestudy,these531
findingscanberegardedasquiterobust.532
4.1.3Interventionaldata:533
Dose‐responsestudiesshowthat,inmenwhoseendogenoustestosteroneisfullysuppressed,add‐back534
administrationofincreasingdosesoftestosteronethatproducegradedincreasesincirculatingtestosterone,535
causesadose‐dependent(whetherexpressedaccordingtotestosteronedoseorcirculatinglevels)increase536
inmusclemass(measuredasleanbodymass)andstrength(41,87).Takentogether,thesestudiesprovethat537
testosteronedosesleadingtocirculatingconcentrationsfromwellbelowtowellabovethenormalmale538
rangehaveunequivocaldose‐dependenteffectsonmusclemassandstrength.Thesedatastronglyand539
consistentlysuggestthatthesexdifferenceinleanbodymass(muscle)islargely,ifnotexclusively,dueto540
thedifferencesincirculatingtestosteronebetweenmenandwomen.Thesefindingshavestrongimplications541
forpower‐dependentsportperformanceandlargelyexplainthepotentefficacyofandrogendopinginsport.542
Thekeyfindingsprovidingconclusiveevidencethattestosteronehasprominentdose‐responseeffectsin543
menarereportedinstudiesbyBhasinetalthatprovedamonotonicdose‐response,extendingfromsub‐to544
supra‐physiologicalrangeformenfortestosteroneeffectsonmusclemass,sizeandstrengthinhealthy545
youngmen,findingsthathavebeenreplicatedandconfirmedbyanindependentgroup(41).Bothsetsof546
studiesusedacommondesignoffullysuppressingallendogenoustestosterone(tocastratelevels)forthe547
fulldurationoftheexperimentbyadministeringaGnRHanalog.IntheBhasinstudies,participantswerethen548
randomizedtofivegroupswhoreceivedweeklyinjectionsof25mg,50mg,125mg,300mgor600mgof549
testosteroneenanthatefor20weeks.Ineffectthiswastwosub‐andtwosupra‐physiologicaltestosterone550
doses.Inthesestudies,thelowesttestosteronedoseproducedameanserumtestosteroneof253ng/dl(8.8551
nmol/L)inyoungermenand176ng/dl(6.1nmol/L)inoldermen.Thestudiesshowedaconsistentdose‐552
responseformusclemassandstrengththatwasclearlyrelatedtotestosteronedoseandconsequentialblood553
testosteroneconcentrations(upperpanel,figure2).554
ThestudyofFinkelsteinetalinvolvedthesamedesignandinvolved400healthymenaged20to50yearsof555
agewhohadcompletesuppressionofendogenoustestosteroneforthe16weeksofthestudywith556
testosteroneaddedbackusingdailydosesof0,1.25g,2.5g,5gor10gofatopical1%testosteronegel(41).557
Thisagaincreatedagradeddose‐responsecurveforserumtestosteroneandformusclemassandstrength.558
Theinclusionofazero(placebo)dosealloweddifferentiationbetweenthezeroandlowesttestosterone559
dose.Theplacebo(zero)doseproducedaserumtestosteroneof0.7nmol/L,thetypicalmeanforcastrated560
men,childhood,andwomenofanyage.Meanwhilethelowesttestosteronedose(1.25ggelperday)561
producedaserumtestosteroneof6.9nmol/L,whichisequivalenttothatofamaleinearlytomid‐puberty.562
Akeyfindingforthisreviewisthat,fromthisstudyofmen,theincreaseinserumtestosteronefrommean563
ofnormalfemaleconcentration(0.9nmol/L)tosupra‐physiologicalfemaleconcentrations(6.9nmol/L)564
producedsignificantincreasesof2.3%fortotalbodylean(muscle)mass,3.0%forthighmusclearea,and565

Page18of51

5.5%increaseinlegpressstrength(digitiseddatapoolingbothcohortsfromlowerpanel,figure2).566
Studiesoftheergogeniceffectsofsupra‐physiologicalconcentrationsofcirculatingtestosteronerequire567
studiesadministeringgradeddosesofexogenoustestosteroneformonths.Duetoethicalconcerns568
regardingrisksofunwantedvirilisationandhormone‐dependentcancers,however,fewstudieshave569
administeredsupra‐physiologicaltestosteronedosestohealthywomen.Onewelldesigned,randomized570
placebo‐controlledstudyofpostmenopausalwomeninvestigatedtheeffectsofdifferenttestosteronedoses571
onmusclemassandperformanceandphysicalfunction(88).Sixty‐twowomen(meanage53)allhada572
standardestrogen‐replacementdoseadministeredduringa12weekrun‐inperiod(toeliminateany573
hypotheticalconfoundingeffectsofestrogendeficiency),afterwhichtheywererandomizedtooneoffive574
groupsreceivingweeklyinjectionsoftestosteroneenanthate(doses:0,3mg,6.25mg,12.5mg,and25mg575
respectively)for24weeks.Theincreasingdosesoftestosteroneproducedanexpecteddose‐responsein576
serumtestosteroneconcentrations(byLC‐MS)withthehighesttestosteronedose(25mg/week)produced577
ameannadirconcentrationof7.3nmol/L.Thewomenwhosetestosteroneconcentrationswereincreased578
to7.3nmol/Lachievedsignificantincreasesinmusclemassandstrength(table4),rangingfrom4.4%for579
muscle(lean)masstobetween12%and26%formeasuresofmusclestrength(chestandlegpress,loaded580
stairclimb).Asmusclestrengthmeasurementiseffort‐dependent,theplacebo‐controlleddesignofthe581
Huangstudysupportthefurtherinterpretationthatthehighestdoseoftestosteronealsohadprominent582
mentalmotivationaleffectsintheeffort‐dependenttestsofmusclestrength.Thesefindingsprovidesalient583
directevidenceoftheergogeniceffectsofhyperandrogenisminfemaleathletesconfirmingthatatleastup584
toaveragecirculatingtestosteroneconcentrationsof7.3nmol/L,womendisplayasimilardose‐response585
relationshipasdomenforsupra‐physiologicaltestosteronewithsignificantgainsinmusclemassandpower.586
587
Theseeffectsoftestosteroneadministrationoncirculatingtestosteroneconcentrationsinfemalesmaybe588
comparedwiththeeffectsinmalesfromtheFinkelsteinandBhasinstudies.Inmen,thelowesttestosterone589
dose(1.25g/day)increasedmeanserumtestosteroneto6.9nmol/Lequivalenttoearlytomid‐malepuberty590
resultinginsignificantincreasesoftotalbodylean(muscle)mass(2.3%),thighmusclearea(3.0%),andleg591
pressstrength(5.5%)comparedwiththeplacebodosewhichresultedinaserumtestosteroneof0.7nmol/L.592
IntheHuangstudy(figure3),musclemassandstrengthinpostmenopausalwomendisplayedaflatresponse593
atthe3lowerdoses,whencirculatingtestosteroneconcentrationsremainbelow5nmol/L,anddisplayeda594
significantincreaseonlywhenthemeancirculatingtestosteroneconcentrationproducedbythehighest595
testosteronedosefirstincreasedcirculatingtestosteroneconcentrationsabove5nmol/L.Thispattern,flat596
atlowerdosesandrisingathighestdose,representsthelowerplateauandtheearliestrisingportion,597
respectively,ofthesigmoidaldose‐responsecurveoftestosteroneformuscle.598
DatacorroboratingtheHuangstudyresultscomesfromanotherwell‐controlledstudyinwhichpost‐599
menopausalwomenwhowereadministeredmethyltestosteronefollowingarun‐inperiodofestrogen600

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replacementdisplayedasignificantincreaseinlean(muscle)massaswellasupperandlowerlimbpower601
duringa16‐weekdouble‐blind,parallelgroupstudy(89).602
Similarly,twoprospectivestudiesofthefirst12monthstreatmentoftransmen(F2Mtransgender)showsa603
consistentmajorincreaseinmusclemassandstrengthduetotestosteroneadministration.Inonestudy604
testosteronetreatmentof17transmenachievingadultmalecirculatingtestosteronelevels(mean31nmol/L)605
increasedmusclemassby19.2%(90)whereas,conversely,testosteronesuppression(usinganestrogen‐606
basedtreatmentregimen)in20transwomenreducedcirculatingtestosteronelevels