Former Abusers of Anabolic Androgenic Steroids Exhibit Decreased Testosterone Levels and

16 Pages·2016·0.7 MB·English

RESEARCHARTICLE
Former Abusers of Anabolic Androgenic
Steroids Exhibit Decreased Testosterone
Levels and Hypogonadal Symptoms Years
after Cessation: A Case-Control Study
JonJarløvRasmussen1*,ChristianSelmer1,PeterBuschØstergren2,Karen
BojePedersen3,MortenSchou4,FinnGustafsson5,JensFaber1,AndersJuul6,
CarolineKistorp1
1 DepartmentofInternalMedicine,CopenhagenUniversityHospital,Herlev,Denmark,2 Departmentof
a11111
Urology,CopenhagenUniversityHospital,Herlev,Denmark,3 DepartmentofInternalMedicine,
CopenhagenUniversityHospital,Slagelse,Denmark,4 DepartmentofCardiology,CopenhagenUniversity
Hospitals,HerlevandGentofte,Denmark,5 DepartmentofCardiology,theHeartCentre,Rigshospitalet,
Copenhagen,Denmark,6 DepartmentofGrowthandReproduction,Rigshospitalet,Universityof
Copenhagen,Denmark
*[email protected]
OPENACCESS
Abstract
Citation:RasmussenJJ,SelmerC,ØstergrenPB,
PedersenKB,SchouM,GustafssonF,etal.(2016)
FormerAbusersofAnabolicAndrogenicSteroids Aims
ExhibitDecreasedTestosteroneLevelsand
HypogonadalSymptomsYearsafterCessation:A Abuseofanabolicandrogenicsteroids(AAS)ishighlyprevalentamongmalerecreational
Case-ControlStudy.PLoSONE11(8):e0161208. athletes.TheobjectiveofthisstudywastoinvestigatetheimpactofAASabuseonrepro-
doi:10.1371/journal.pone.0161208
ductivehormonelevelsandsymptomssuggestiveofhypogonadismincurrentandformer
Editor:DavidJ.Handelsman,UniversityofSydney, AASabusers.
AUSTRALIA
Received:May26,2016 Methods
Accepted:August1,2016
Thisstudyhadacross-sectionalcase-controldesignandinvolved37currentAASabusers,
Published:August17,2016 33formerAASabusers(mean(95%CI)elapseddurationsinceAAScessation:2.5(1.7;
Copyright:©2016Rasmussenetal.Thisisanopen 3.7)years)and30healthycontrolparticipants.Allparticipantswereaged18–50yearsand
accessarticledistributedunderthetermsofthe wereinvolvedinrecreationalstrengthtraining.Reproductivehormones(FSH,LH,testoster-
CreativeCommonsAttributionLicense,whichpermits
one,inhibinBandanti-Müllerianhormone(AMH))weremeasuredusingmorningblood
unrestricteduse,distribution,andreproductioninany
samples.Symptomsofhypogonadism(depressivesymptoms,fatigue,decreasedlibido
medium,providedtheoriginalauthorandsourceare
credited. anderectiledysfunction)wererecordedsystematically.
DataAvailabilityStatement:Theparticipantsinthis
studyareguaranteedtoremaincompletely Results
anonymous.Datacontainexplicitdetailson
FormerAASabusersexhibitedsignificantlylowermedian(25th–75thpercentiles)totaland
demographicsofeachparticipantinthestudy.Ifthe
datasetwouldbemadepubliclyavailable,wefear freetestosteronelevelsthancontrolparticipants(totaltestosterone:14.4(11.9–17.7)nmol/l
someoftheparticipantscouldberecognizedandrisk vs.18.8(16.6–22.0)nmol/l)(P<0.01).Overall,27.2%(13.3;45.5)offormerAASabusers
legalprosecutionsorevenretaliationfromcriminal
exhibitedplasmatotaltestosteronelevelsbelowthelowerreferencelimit(12.1nmol/l)
distributorsofanabolicandrogenicsteroids.Dataare
whereasnocontrolparticipantsexhibitedtestosteronebelowthislimit(P<0.01).Gonado-
availableuponrequestto:JonJarløvRasmussen,M.
D.;email:[email protected],M. tropinsweresignificantlysuppressed,andinhibinBandAMHweresignificantlydecreased
PLOSONE|DOI:10.1371/journal.pone.0161208 August17,2016 1/16
ReproductiveHormonesandHypogonadalSymptomsinAbusersofAnabolicAndrogenicSteroids
D.,PhD;email:caroline.michaela.nervil. incurrentAASabuserscomparedwithformerAASabusersandcontrolparticipants(P<
[email protected]. 0.01).ThegroupofformerAASabusershadhigherproportionsofparticipantswithdepres-
Funding:FundedbyAntiDopingDenmark(no sivesymptoms((24.2%)(11.1;42.2)),erectiledysfunction((27.3%)(13.3;45.6))and
specificgrantnumber,URL:http://www.antidoping.dk/ decreasedlibido((40.1%)(23.2;57.0))thantheothertwogroups(trendanalyses:P<0.05).
),ResearchFoundationofHerlevHospital(no
specificgrantnumber,URL:https://www.
Conclusions
herlevhospital.dk/),DanishHeartFoundation(grant
number:15-R99-A5797-22952,URL:https://www.
FormerAASabusersexhibitedsignificantlylowerplasmatestosteronelevelsandhigher
hjerteforeningen.dk/),andFacultyScholarshipfrom
frequenciesofsymptomssuggestiveofhypogonadismthanhealthycontrolparticipants
UniversityofCopenhagentoJJR(nospecificgrant
number,URL:http://sund.ku.dk/).Thefundershadno yearsafterAAScessation.CurrentAASabusersexhibitedseverelydecreasedAMHand
roleinstudydesign,datacollectionandanalysis, inhibinBindicativeofimpairedspermatogenesis.
decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:JonJarløvRasmussenand
CarolineKistorphavereceivedunrestrictedresearch
grantsfrom‘AntiDopingDenmark’.Thisdoesnotalter Introduction
theauthors'adherencetoPLOSONEpolicieson
sharingdataandmaterials.‘AntiDopingDenmark’ Anabolicandrogenicsteroids(AAS)comprisetestosteroneanditssyntheticderivatives.These
hadnoroleinpreparationofthestudy,datacollection compoundshavebeenusedfordecadesbyprofessionalathletestoenhancemusclestrength
andanalysis,preparationofthemanuscriptor
andperformance[1,2].ThesettingofAASabusehaschangedwithinrecentyears.Arecent
decisiontopublishthefinalversionofthemanuscript
meta-analysisestimatedthelifetimeprevalenceofAASabuseworldwideis6.4%amongmen
and18.4%amongrecreationalathletes[3].Moreover,apreviousstudysuggestedthatprior
AASabusewasthemostfrequentcauseofprofoundhypogonadismamongyoungmen(43%)
[4].ThesefindingsindicateAASabuseisnowprevalentinthebroaderpopulation.
OngoingAASabusecausesdramaticincreasesinplasmaandrogenlevelsthatultimately
facilitateseverehypothalamic-pituitary-gonadal(HPG)-axissuppressionduetonegativefeed-
backmechanismsinvolvingtestosteroneanditsmetabolites[5].HPG-axisinhibitionmay
causelong-lastingspermatogenesisinhibitionandreductionsinbiomarkersofSertoli-cell
function,anti-Müllerianhormone(AMH)andinhibinB.However,informationregardingthe
impactofAASabuseonthesereproductivehormonesisverylimited[6,7].
Anabolicandrogenicsteroid-inducedhypogonadism(ASIH)iscommonamongformer
AASabusersandusuallypresentsashypogonadotropichypogonadismduetoabruptdecreases
inplasmaandrogenlevelsfollowingAAScessation[1,2,5,8].ScientificdataonASIHarelim-
ited,buttheconditionischaracterisedbysymptomsandsignsofhypogonadismsuchas:testic-
ularatrophy,lowplasmatestosteronelevels,impairedspermatogenesis,erectiledysfunction,
fatigue,decreasedlibidoanddepressivesymptoms;andisconsideredtoresolvespontaneously
within6to12months[2,5].Studiesinvestigatingtherecoveryphasesofyoungmenwith
ASIHare,toourknowledge,virtuallynon-existent.Agrowingnumberofstudiesreporting
cases,inwhichASIHmanifestationspersistedyearsafterAAScessation,suggestASIHisa
morepermanentconditioninasubstantialproportionofformerAASabusers[9–16].This
emerginggroupofyoungmenmaybecomeaconsiderablepublichealthconcerninthecoming
years.
Theobjectiveofthisstudywastocomparethereproductivehormonelevelsandsymptoms
suggestiveofhypogonadisminyoungmenwithhistoriesofcurrentandformerAASabuse
withthoseofhealthyage-matchedmen.
ParticipantsandMethods
StudyDesignandParticipants
Weconductedacommunity-basedcross-sectionalcase-controlstudyinthegreaterCopenha-
genareafromNovember2014toDecember2015.Youngmen(18–50years)involvedin
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ReproductiveHormonesandHypogonadalSymptomsinAbusersofAnabolicAndrogenicSteroids
recreationalstrengthtrainingwereenrolledinoneofthefollowingthreegroups:1)current
AASabusers2)formerAASabuserswhohaddiscontinuedAASabuse(cid:1)3monthsbefore
enrollinginthestudyand3)age-matchedhealthycontrolparticipantswhodeniedeverhaving
usedAAS.Wedidnotapplyspecificinclusioncriteriaregardingweeklyhoursofrecreational
strengthtraining,nordidweapplyinclusioncriteriapertainingtotheextentofAASabuse.
Exclusioncriteriaforthethreegroupswere:congenitalhypogonadalconditions,medicallypre-
scribedtestosteronetherapy,knowncardiovasculardiseaseanddiabetesmellitus.Participants
wererecruitedprimarilyfromfitnesscentresinthegreaterCopenhagenareaandbyinternet
advertising.Thefitnesscentresincludedweightliftinggymnasiumswhicharenotundersur-
veillancebytheDanishAntidopingAgencyandareknowntobefrequentedbyAASabusers.
Theadvertisementwasasfollows(inDanish):‘Weareseekingyoungmenforaresearchproj-
ect;inclusioncriteria:age18–50yearsandinvolvedinrecreationalstrengthtrainingorarecur-
rentlyusingAASorhavepreviouslyusedAAS’.Theadvertisementdidnotdisclosethestudy
entailedassessmentsof:androgenlevels,fertilitybiomarkers,libido,erectilefunctionorsymp-
tomsofdepressionorfatigue.
Ethics
ThestudywasperformedinaccordancewiththeDeclarationofHelsinkiandallrelevantlegal
regulationsinDenmark.PermissiontoconductthestudywasgrantedbytheDanishDataPro-
tectionAgency(HEH-2014-095,I-Suite:03250)andethicalapprovalwasgrantedbytheCapi-
talRegionalCommitteeonHealthResearchEthicsinDenmark(H-3-2014-127).Oraland
writteninformedconsentwasobtainedfromallparticipantspriortoinclusion.
Procedures
AllprocedureswereperformedduringonevisitattheCentreofEndocrinologyandMetabo-
lism,DepartmentofInternalMedicine,CopenhagenUniversityHospital,Herlev,Denmark.
Participantsattendedtheresearchlabbetween07:30and09:00a.m.afteraminimumofeight
hoursofovernightfasting.Theywereplacedinthesupinepositionforaminimumof30min-
utes.Allbloodsampleswerethencollectedviaacannulaintherightmediancubitalvein.
Oneinvestigator(JJR)obtainedadetailedAASabusehistory(totalduration,compounds,
doses,useofotherperformanceenhancingdrugs)duringaclinicalinterview,usingastruc-
turedquestionnaire.WeusedtotalnumbersofweeksofAASabuseandtotalnumbersofAAS
compoundsusedasmeasuresoftheextentofAASabuse.Wedidnotcalculateoverallaverage
AASdosesintheAASparticipantsbecausethepharmacodynamicsandpharmacokineticsof
AAScompoundscanvaryconsiderablydependingontheirchemicalstructures[1].Further-
more,AASabusersoftenusenumerousAAScompoundsandalterdosesintermittentlyduring
a‘cycle’[17].Otherinformationsuchasmedicalhistory,illicitdruguse,smokinghabits,alco-
holuse,strengthtraininghistory(totaldurationandweeklyhoursoftraining)anddemograph-
icswerealsoobtained.Testicularsize(ml)wasassessedinallparticipantusingPrader’s
orchidometer,whichhasshownstrongcorrelationswithultrasoundtesticularsizeestimations
[18].
LaboratoryAnalyses
Plasmatotaltestosterone,androstendione,dehydroepiandrosteronsulfate(DHEAS)and
17-hydroxyprogesteronewereallmeasuredusingliquidchromatography-massspectrometry
(LC-MS/MS),accordingtotheCHSMSMSsteroidskit(PerkinElmer,Massachusetts,USA).
Theinter-assaycoefficientofvariation(CV)was<10.0%forallsteroidhormones.Sexualhor-
mone-bindingglobulin(SHBG)wasanalysedbysandwichchemiluminescence-based
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ReproductiveHormonesandHypogonadalSymptomsinAbusersofAnabolicAndrogenicSteroids
immunoassay(Siemens,Munich,Germany)andtheCVwas<7.0%.Freetestosteronewascal-
culatedusingthemethodsuggestedbyBartsch[19].Lowreferencelimits(2.5percentile)for
plasmatotaltestosteroneinyoungmendifferamongstudiesdependingonestimationinsub-
groupsofnonobeseeugonadalhealthyyoungmen(10.4–12.1nmol/l)ormorepopulation-rep-
resentativecohorts(6.6–7.4nmol/l)[20–23].Therefore,weassessedproportionsofthecontrol
groupandgroupofformerAASwhoexhibitedlowtotaltestosteronelevelsusinglowerrefer-
encelimitsforbothasubgroupofeugonadalnonobesehealthysubgroupofyoungmen(12.1
nmol)andapooledpopulation-representativecohort(6.6nmol/l).Gonadotropinsweremea-
suredbyasandwichelectrochemiluminescence-immunoassay(ECLIA)(Cobas,Roche,Swit-
zerland)andtheCVswere<7.0%.SeruminhibinBandanti-Müllerianhormone(AMH)
levelswereusedasmarkersofSertoli-cellfunctionandspermatogenesis[24–26].Serumwas
keptfrozenat-80°Cuntilneededforanalysisonthesamebatch.InhibinBwasmeasuredbya
three-stepsandwich-ELISAassay(inhibinBgenII)(BeckmanCoulter,California,USA).The
intra-assayvariationswere<10.3%,<8.6%and<7.6%atlevelsof36.3,111.4and616.4pg/
ml,respectively.SeruminhibinBisstronglyassociatedwithspermconcentrationsandsperm
countsespeciallyuptoalevelof150pg/ml[25].Weusedareferencelimitof(cid:3)92pg/mlas
cut-offforimpairedspermatogenesisbasedontheresultsofarecentstudy[24].AMHwasana-
lysedbyatwo–stepsandwichelectrochemiluminescenceassay(BeckmanCoulter,California,
USA)andtheintra-assayvariationswere<1.77%,<2.48%and<2.39%atlevelsof7.04,
34.88and105.66pmol/l,respectively.
SymptomsIndicatingHypogonadism
TheBeckDepressionInventory-II(BDI-II)questionnairewasusedtoevaluateparticipants
regardingthepresenceofdepressivesymptoms[27].TheBDI-IIisa21-questionmultiple-
choiceself-reportedpsychometrictest,andeachofitsquestionsisscoredusingascaleranging
from0(minimum)to3(maximum).Atotalscore(cid:3)10isconsiderednormal,andtheBDI-II
isstronglycorrelatedwithothervalidatedpsychometrictestsusedinprimarycaresettings
[28].Questionnumber21oftheBDI-IIwasusedtoassesslibidoamongtheparticipantsand
weconsideredascore<1normal.Erectilefunctionwasevaluatedusingthefive-itemversion
oftheInternationalIndexofErectileFunction(IIEF-5)questionnaire[29].Thequestionnaire
ishighlyvalidatedandconsistsoffivequestionsscoredusingascalerangingfrom1to5.A
totalscore(cid:1)22indicatesnormalerectilefunction.TheShortForm-36(SF-36)questionnaire
wasusedtoassess‘energy/fatigue’.Lowerscoresweresuggestiveofmorepronouncedfatigue
symptoms[30].
StatisticalAnalyses
Categoricalvariableswerecomparedusingachi-squaretestorFisher’sexacttestasappropri-
ate.Thenon-parametricCochran-Armitagetrendtestwasusedtoassesstrendsinhypogona-
dalsymptomsandimpairedspermatogenesisacrossthegroups.Theordinalorderingofthe
groupsfortrendanalyseswasspecifiedapriori.Assumptionsofnormaldistributionswith
respecttonumericalvariableswereevaluatedbyhistogramsandbyassessingthelinearityof
residualsinaquantileplot.Equalityofvariancewasassessedusingresidualsofvariablesdrawn
againstpredictedvaluesandusingLevene’stest.Numericalvariableswerecomparedacross
thegroupsbyanalysisofvariance(ANOVA)andpresentedasmean(standarderror)ifthe
assumptionsofanormaldistributionandequalityofvariancewerefulfilled.Furthermore,
numericalvariableswerecomparedpairwiseamongthethreegroupswithTukey’spost-hoc
test.Thenon-parametricKruskal-Wallis’test(withBoneferroni’spost-hoctest)wasusedto
comparenon-normallydistributedvariableswhichcouldnotbelogarithmicallytransformed
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toanadequatenormaldistribution.Thesevariablesarepresentedasmedians(25th–75thper-
centiles).Weusedpiecewiselinearregression(linearsplines),allowingvaryingslopes,to
modelnonlinearassociations.Missingdatawererare((cid:4)2%forquestionnaires,noneforall
otherdata)andwereaddressedviamultipleimputationsusingthefullyconditionalspecifica-
tionmethod[31].P-values<0.05wereconsideredstatisticallysignificant.Alldatawereana-
lysedusingSASversion9.4(SASInstituteInc.,NorthCarolina,USA).
Results
CharacteristicsofParticipants
Atotalof37currentAASabusers,33formerAASabusersand31controlparticipantsvolun-
teeredtoparticipateinthestudy.Oneparticipantfromthecontrolgroupwasexcludeddueto
cryptorchidismwhichwasdiagnosedduringthestudy,so30controlparticipantswere
includedinthefinalanalyses.Theparticipantsinthethreegroupsdidnotdiffersignificantly
withrespecttoage,smokinghistory,illicitdrugabusehistory,incomeormaritalstatus,but
currentAASabusersperformedstrengthtrainingmorehoursperweekthanparticipantsinthe
othertwogroups(P<0.05)(Table1).
Ahigherproportionofparticipantsinthecontrolgrouphadauniversitydegreethanthe
participantsintheothertwogroups(P<0.01).ThetotaldurationofaccumulatedAASabuse
(geometricmean(95%CI))notedamongcurrentAASabusers(142.3(99.7;203.1)weeks)was
notsignificantlydifferentfromthatnotedamongformerAASabusers(111.8(81.3;153.7)
Table1. Demographiccharacteristicsandanabolicandrogenicsteroids(AAS)abuseinthethreegroups.
Variable Controlgroup CurrentAASabusers FormerAASabusers p-value
n=30 n=37 n=33
Demographiccharacteristics
Age(years)¶ 31.5(1.2) 31.4(1.4) 34.8(1.2) 0.11
Recreationalstrengthtraining(hours/week)¶ 6.5(0.5) 9.2(0.7)a 6.9(0.7) 0.01
Cohabiting(%) 73.3 67.6 57.6 0.41
Income(US$/year) 51,700(5400) 43,000(7700) 60,800(7600) 0.35
Universitydegree(%) 30.0b 0.0 0.0 <0.01
Historyofsmoking(%) 26.7 43.2 51.5 0.10
Alcoholintake<once/week(%) 56.7c 86.5 72.7 0.02
Experiencewithillicitdrugs(%) 56.7 70.3 69.7 0.43
Anabolicandrogenicsteroidsabuse
AccumulateddurationofAASabuse(weeks) - 142.3(99.7–203.1) 111.8(81.3–153.7) 0.32
AASabuseduringelapsedperiod(years) - 5.7(4.5–7.2) 6.3(4.5–8.8) 0.46
ElapseddurationsinceAAScessation(years) - - 2.5(1.7–3.7) -
NumberofAAScompoundsused(n)● 8(4–9) 6(4–9) 0.32
Post-cycletherapy -
RegularlyusedhCG(%) - 48.7 57.6 0.46
Regularlyusedaromataseinhibitors/antioestrogen(%) - 48.7 33.3 0.19
Resultsaregeometricmeans(95%confidenceinterval)unlessotherwisestated.
¶Mean(standarderror)
●Median(25th–75thpercentiles)
Tukey’spost-hoctest(meanandgeometricmean)orBonferroni’spost-hoctest(medians)
asignificantdifferencebetweencurrentAASabusersandtheothertwogroups
bsignificantdifferencebetweencurrentAASabusersandtheothertwogroups
csignificantdifferencebetweencontrolparticipantsandcurrentAASabusers
doi:10.1371/journal.pone.0161208.t001
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ReproductiveHormonesandHypogonadalSymptomsinAbusersofAnabolicAndrogenicSteroids
weeks),andthenumbersofAAScompoundsuseddidalsonotdifferbetweenthetwogroups.
Thetwogroupsreportedpreviousandcurrentexperiencewithvaryingdosesofnumerous
AAScompounds,ofwhichtestosteroneesters,trenbolone,nandrolone,stanozolol,sustanon
andboldenonewerethemostwidelyused(S1Table).Highproportionsofbothcurrentand
formerAASabusersreportedregularlyusinghCGoraromataseinhibitorsfollowingAAS
cycles.TheelapseddurationsinceAAScessation(geometricmean(95%CI))was2.5(1.7;3.7)
yearsamongformerAASabusers.NoneoftheseparticipantsreportedhavingusedAASwithin
sixmonthsandonly15.2%(95%CI)(3.0;27.4)reportedelapsedtimeintervalof6–12months
sinceAAScessation.ElevenformerAASabusershadpreviouslybeenreferredtoanendocrine
clinicforgynaecomastia,butnonehadbeentreatedforgynaecomastia,hypogonadismorinfer-
tility.TheseparticipantsdidnotdifferfromotherformerAASabusersintermsofdemo-
graphiccharacteristics,AASabuse,laboratoryresultsorfrequencyofhypogonadalsymptoms.
ReproductiveHormones
Testicularsizedifferedsignificantlyamongthethreegroups.CurrentAASabusershadthe
smallesttesticularvolume(12.2(0.7)ml)andformerAASabusershadavolumeof17.4(0.8)
mlwhichwas4.8(2.9;6.8)mlsmallerthanthatofthecontrolparticipantswhohadlargesttes-
ticularvolume(Table2).Plasmatotalandfreetestosteronelevelsweresignificantlylower
amongformerAASabusersthanamongcontrolparticipantsandcurrentAASabusers,thelat-
terofwhomexhibitedsignificantlyincreasedplasmatestosteronelevels,asexpected.The
2.5th–97.5thpercentilesfortotaltestosteronerangedfrom12.4–32.3nmol/lamongthecontrol
participantsand5.7–31.4nmol/lamongformerAASabusers.
AhighpercentageofparticipantsinthegroupofformerAASabusers(27.2%(13.3;45.5))
werebelowthelowerreferencelimitforplasmatotaltestosteroneestimatedinnonobeseeugo-
nadalhealthyyoungmen(12.1nmol/l)whereasnoparticipantsinthecontrolgroup(0.0%
Table2. Reproductivehormonelevelsinthethreegroups.
Variable Controlgroup CurrentAASabusers FormerAASabusers p-value
n=30 n=37 n=33
Testicularsize(ml)¶ 22.3(0.6)b 12.2(0.7) 17.4(0.8) <0.01
P-totaltestosterone(nmol/l) 18.8(16.6–22.0)b 98.3(47.4–122.7) 14.4(11.9–17.7) <0.01
P-freetestosterone(pmol/l) 480(420–530)b 3780(1870–5500) 410(320–480) <0.01
P-androstendione(nmol/l)● 2.53(2.27–2.82) 6.92(5.41–8.84)a 2.33(2.06–2.63) <0.01
P-DHEAS(nmol/l)¶ 4805(391) 4929(490) 4348(302) 0.55
P-SHBG(nmol/l)● 33.3(29.1–38.1) 8.4(6.3–11.1)a 26.2(20.7–33.1) <0.01
P-17hydroxyprogesterone(nmol/l)● 2.88(2.49–3.33) 0.14(0.10–0.18)a 2.42(1.86–3.15) <0.01
P-FSH(U/l) 4.2(3.2–5.7) 0.3(0.1–0.4)a 4.4(3.3–6.2) <0.01
P-LH(U/l) 3.1(2.5–3.9) <0.1(<0.1–0.1)a 3.6(2.2–4.3) <0.01
S-inhibinB(pg/ml)¶ 175(9) 81(8)a 170(11) <0.01
S-AMH(pmol/l)● 49.5(41.6–59.0) 21.6(16.3–28.7)a 44.7(37.2–53.7) <0.01
Resultsaremedians(25th–75thpercentiles)unlessotherwisestated.
¶Mean(standarderror)
●Geometricmean(95%confidenceinterval)
Tukey’spost-hoctest(meanandgeometricmean)orBonferroni’spost-hoctest(medians)
asignificantdifferencebetweenthegroupofcurrentAASabusersandthetwoothergroups
bsignificantdifferenceamongallthreegroups
AAS,anabolicandrogenicsteroids,AMH,anti-Müllerianhormone;DHEAS,dehydroepiandrosteronsulfate;FSH,follicle-stimulatinghormone;LH,
luteinizinghormone;P-,plasma;S-,serumSHBG,sexualhormone-bindingglobulin.
doi:10.1371/journal.pone.0161208.t002
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Fig1.AssociationbetweenaccumulateddurationofAASabuse(log2scale)andtestissizeincurrentAASabusers(splinefunction)and
formerAASabusers.Footnote:AAS,anabolicandrogenicsteroids.
doi:10.1371/journal.pone.0161208.g001
(0.0;11.6))werebelowthislimit(P<0.01).Further,amongformerAASabusers3.3%(0.01;
15.8)werebelowthelowerreferencelimitforplasmatotaltestosteroneestimatedinapooled
population-representativecohort(6.6nmol/l).Plasmagonadotropins,SHBG,17-hydroxypro-
gesterone,serumAMHandinhibinBdidnotdiffersignificantlybetweenformerAASabusers
andcontrolparticipants,butweremarkedlydecreasedamongcurrentAASabusers(P<0.01).
TherewerehigherpercentagesofparticipantswithseruminhibinBlevelsbelowthelimitof
impairedspermatogenesis(92pg/ml)amongcurrentAASabusers(56.8%(39.5;72.7))andfor-
merAASabusers(9.1%(1.9;24.3))thanamongcontrolparticipants(3.3%(0.01;17.2))(trend
analysis:P<0.01).
AccumulateddurationofAASabusewasassociatedwithreducedtesticularsizeinformer
abusers(log2coefficient(B)(95%CI):-1.3(-2.4;-0.2),P=0.02)andcurrentAASabusers(dur-
ingtheinitial32weeksofAASabuse,splinefunction,log2coefficient(B):-5.4(-10.8;-0.02),
P=0.049)(Fig1).Wedidnotobserveanysignificantassociationsbetweenplasmatotaltestos-
teronelevelsandaccumulateddurationofAASabuse(log2coefficient(B):0.09(-0.04;0.22),
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Fig2.AssociationbetweenaccumulateddurationofAASabuse(log2scale)andplasmatotaltestosteronelevels(log2
scale)informerAASabusers.Footnote:AAS,anabolicandrogenicsteroids.
doi:10.1371/journal.pone.0161208.g002
P=0.17)(Fig2)orelapseddurationsinceAAScessation(log2coefficient(B):0.05(-0.7;0.17),
P=0.42)(Fig3)amongformerAASabusers.
AmongcurrentAASabusers,increasingaccumulateddurationofAASabusewasassociated
withdecreasingseruminhibinBlevels,whichreachedaplateauafter64weeksofaccumulated
AASabuse(splinefunction,log2coefficient(B):-47.9(-80.3;-15.6),P<0.01)(Fig4).Increas-
ingaccumulateddurationofAASabusewasalsoassociatedwithdecreasingAMHlevels
amongcurrentAASabusers(log2coefficient(B):-0.3(-0.5;-0.04),P=0.03)(Fig5).
SymptomsIndicatingHypogonadism
FormerAASabusersexhibitedthehighestfrequenciesofparticipantswithdepressivesymp-
toms(24.2%(11.1;42.2)),erectiledysfunction(27.3%(13.3;45.6))anddecreasedlibido(40.1%
(23.2;57.0))comparedwiththeothertwogroups(trendanalyses:P<0.05forallthreeparam-
eters)(Fig6,S2Table).FormerAASabusershadalowerscoreontheSF-36questionnairewith
respectto‘energy/fatigue’(58.9(4.3))thanthecontrolgroup(73.5(2.6))andcurrentAAS
abusers(69.2(4.5))indicatingformerAASabusersexhibitedsignificantlymorepronounced
fatiguesymptomsthantheircounterparts(P<0.05).Wedidnotobserveanysignificantasso-
ciationsbetweensymptomsandhormonallevelsorextentofAASabuseamongformerAAS
abusers.
PLOSONE|DOI:10.1371/journal.pone.0161208 August17,2016 8/16
ReproductiveHormonesandHypogonadalSymptomsinAbusersofAnabolicAndrogenicSteroids
Fig3.AssociationbetweenelapseddurationsinceAAScessation(log2scale)andplasmatotaltestosteronelevels(log2
scale)informerAASabusers.Footnote:AAS,anabolicandrogenicsteroids.
doi:10.1371/journal.pone.0161208.g003
Discussion
ThekeyfindingsofthisstudywerethatthegroupofformerAASabusersexhibitedsignifi-
cantlylowerplasmatotalandfreetestosterone,smallertesticularsizes,andfeaturedahigher
proportionofparticipantswithdepressivesymptoms,fatigue,erectiledysfunctionand
decreasedlibidothanthecontrolgroupmorethantwoyearsafterAAScessation.Theseresults
indicatethataconsiderableproportionofformerAASabusersexhibitedpersistentASIHfea-
tures,suchasbiochemicalandfunctionalhypogonadism,yearsafterAAScessation.
WeassessedpercentagesofthegroupsofcontrolparticipantsandformerAASabusers
belowthereferencelimitforplasmatotaltestosteroneusingreferencerangesforbothasub-
groupofnonobeseeugonadalhealthyyoungmen(12.1nmol/l)andapooledpopulation-repre-
sentativecohort(6.6nmol/l).OurfindingswerethatahighproportionofformerAASabusers
werebelowthereferencelimitforeugonadalnonobesehealthyyoungmencomparedwith
noneofthecontrolparticipants,butonly3.3%offormerAASabuserswerebelowthelower
referencelimitusingthepooledpopulation-representativecohortestimation.Thesefindings
suggestaratherhighproportionofformerAASabusersexhibittestosteronelevelsinthelow
areaofthenormalrangeyearsafterAAScessation,whereasonlyasmallproportionofformer
AASabusersexhibitpersistentlymarkedlowtestosteronelevels.
PLOSONE|DOI:10.1371/journal.pone.0161208 August17,2016 9/16
ReproductiveHormonesandHypogonadalSymptomsinAbusersofAnabolicAndrogenicSteroids
Fig4.AssociationbetweenaccumulateddurationofAASabuse(log2scalesplinefunction)andseruminhibinBlevelsincurrentAAS
abusers.Footnote:AAS,anabolicandrogenicsteroids;s-,serum.
doi:10.1371/journal.pone.0161208.g004
SeruminhibinBandAMHweremarkedlydecreasedamongcurrentAASabusers,butwe
didnotobservedifferencesbetweenformerAASabusersandcontrolparticipantsintheseSer-
toli-cellbiomarkers.Nevertheless,accumulateddurationofAASabusewasstronglyassociated
withdecreasinglevelsofinhibinBandAMH,suggestingtheextentofAASabusemaybe
importantwithrespecttospermatogenesisrecoveryandthatitmayincreasetheriskofperma-
nentfertilityimpairmentasshowninpreviouslyreportedcases[10,12–15].Ahigherpercent-
ageofformerAASabusersexhibitedinhibinBlevelssuggestiveofimpairedspermatogenesis
thancontrolparticipants,althoughthedifferencewasnotstatisticallysignificant.Nevertheless,
thisdifferencemayhaveimpactedfertilityamongformerAASabusersatapopulationlevel.
Furthermore,itispossiblethatpost-cycletherapymayhavereducedthefrequencyofimpaired
spermatogenesisinthegroupofformerAASabusers.
WenotedahighproportionofformerAASabusersexhibitingsymptomssuggestiveof
functionalhypogonadism.Wedidnotobserveanyassociationsbetweenthesesymptomsand
reproductivehormonelevels.Previousstudieshaveshownsupraphysiologicdosesoftestoster-
one((cid:1)500mg/week)occasionallyinducehypomaniaormaniainhealthyyoungmenandthat
rapiddecreasesintestosteronelevelscancausedepressivesymptomsanddecreasedlibido[32,
PLOSONE|DOI:10.1371/journal.pone.0161208 August17,2016 10/16