Regional volumes and spatial volumetric distribution of gray matter in the gender dysphoric brain.

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jo u r n al ho me p ag e:w w w . e l s e v i e r . c o m / l o c a t e / p s y n e u e n

Regional

volumes

and

spatial

volumetric

distribution

of

gray

matter

in

the

gender

dysphoric

brain

Elseline

Hoekzema

a,∗,1

,

Sebastian

E.E.

Schagen

c,e,1

,

Baudewijntje

P.C.

Kreukels

b

,

Dick

J.

Veltman

d

,

Peggy

T.

Cohen-Kettenis

b

,

Henriette

Delemarre-van

de

Waal

e,2

,

Julie

Bakker

a,f

a_{NeuroendocrinologyBakkerGroup,NetherlandsInstituteforNeuroscience,Amsterdam,TheNetherlands} b_{CenterofExpertiseonGenderDysphoria,DepartmentofMedicalPsychology,NeuroscienceCampus}

Amsterdam,VUUniversityMedicalCenter,Amsterdam,TheNetherlands

c_{DepartmentofPediatricEndocrinology,NeuroscienceCampusAmsterdam,VUUniversityMedicalCenter,}

Amsterdam,TheNetherlands

d_{DepartmentofPsychiatry,NeuroscienceCampusAmsterdam,VUUniversityMedicalCenter,Amsterdam,}

TheNetherlands

e_{DepartmentofPediatrics,LeidenUniversityMedicalCenter,LeidenTheNetherlands} f_{CentreInterdisciplinairedeGéneoprotéomiqueAppliquée,UniversitéLiège,Liège,Belgium}

Received12September2014;receivedinrevisedform29December2014;accepted21January2015

KEYWORDS MRI; Transsexual; Transsexualism; Genderidentity disorder; Voxel-based morphometry; Sexdifferences

Abstract Thesexual differentiationofthebrain isprimarily driven bygonadal hormones duringfetaldevelopment.Leadingtheoriesontheetiologyofgenderdysphoria(GD)involve deviations herein.Toexamine whethertherearesigns ofasex-atypical braindevelopment inGD,wequantifiedregionalneuralgraymatter(GM)volumesin55female-to-maleand38 male-to-femaleadolescents,44boysand52girlswithoutGDandappliedbothunivariateand multivariateanalyses.Ingirls,moreGMvolumewasobservedintheleftsuperiormedialfrontal cortex, whileboys hadmore volume inthe bilateral superior posterior hemispheresofthe cerebellumandthehypothalamus.RegardingtheGDgroups,atwhole-brainleveltheydiffered onlyfromindividualssharingtheirgenderidentity butnotfromtheirnatalsex.Accordingly, usingmultivariatepatternrecognitionanalyses,theGDgroupscouldmoreaccuratelybe auto-maticallydiscriminatedfromindividualssharingtheirgenderidentitythanthosesharingtheir natalsexbasedonspatiallydistributedGMpatterns.However,regionofinterestanalyses indi-catedlessGMvolumeintherightcerebellumandmorevolumeinthemedialfrontalcortexin female-to-malesincomparisontogirlswithoutGD,whilemale-to-femaleshadlessvolumein

∗_{Correspondingauthor.Tel.:+310204444943.}

E-mailaddress:e.hoekzema@nin.knaw.nl(E.Hoekzema). 1 _{Sharedfirstauthorship.}

2 _Deceased.

http://dx.doi.org/10.1016/j.psyneuen.2015.01.016 0306-4530/©2015ElsevierLtd.Allrightsreserved.

60 E.Hoekzemaetal.

thebilateralcerebellumandhypothalamusthannatalboys.Deviationsfromthenatalsexwithin sexuallydimorphicstructureswerealsoobservedintheuntreatedsubsamples.Ourfindingsthus indicatethatGMdistributionandregionalvolumesinGDadolescentsarelargelyinaccordance withtheirrespectivenatalsex.However,therearesubtledeviationsfromthenatalsexinsexually dimorphicstructures,whichcanrepresentsignsofapartialsex-atypicaldifferentiationofthe brain.

©2015ElsevierLtd.Allrightsreserved.

1.

Introduction

Duringacriticalperiodoffetalandneonataldevelopment, gonadalhormonesarethoughttoprimarilydrivethesexual differentiationofthebrain,sculptingbrainmorphologyinto agender-typicalconfiguration.Ingeneticmales,thetestes secretehighlevelsoftestosteroneduringfetallife, render-ingserumlevelsintheadultmalerange.Testosteronecan beconvertedto17-␤-estradiolby theenzymearomatase, andanimalstudies havedemonstratedthatpre-and peri-natalestradiolactsastheprincipalregulatorofthesexual differentiationoftherodentbrain(BakkerandBaum,2008; McCarthy, 2008). Cerebralmasculinization is precluded in geneticfemales bytheestrogen-binding activityof alpha-fetoprotein,aplasmaproteinsecretedbythefetalliverin highconcentrations(Bakkeretal.,2006;BakkerandBaum, 2008).Manipulatingthe exposuretosexsteroidhormones duringacriticalperiodofprenatalandearlypostnatallife substantiallyperturbsthesex-specificdifferentiationofthe brain(Bakkeretal.,2006;BakkerandBaum,2008).

In humans, pre- and perinatal neuroendocrine factors arealsothoughttoprimarily regulatethesexual differen-tiationofthe brain.Interestingly,individuals(46,XY) with completeandrogeninsensitivity syndrome,whohave non-functional androgen receptor proteins due to a mutation in theAR gene,are born phenotypically female,are sex-uallyattractedtomenandhaveafemalegender identity (Wisniewski et al., 2000; Hines et al., 2003), suggesting thatinhumansmasculinizationmaybedirectedby testos-teroneratherthantestosterone-derivedestradiol.Although the sexual differentiation of the brain is believed to pri-marilytakeplaceduringprenataldevelopment,circulating hormonesduringadolescenceexertactivationaleffectsand furtherpotentiate neural sexualdimorphisms. In addition togonadalhormoneeffects,directeffectsofgenesresiding onthesexchromosomesmayalsocontributetothesexual differentiation of thebrain (Arnold, 2004; Carruthetal., 2002).

Ingenderdysphoria(GD)thereisapersistent incongru-ence between a person’s natal sex and the experienced gender identity. Leading theories on the etiology of this conditioninvolveasex-atypicalcerebralprogrammingthat divergesfromthe sexualdifferentiationofthe restofthe body, postulated to reflect the organizational effects of altered levels of sex steroid hormones during a specific periodoffetaldevelopment(BaoandSwaab,2011; Cohen-KettenisandGooren,1999;Savicetal.,2010;Swaab,2007). Sometwinstudiessuggestaroleforgeneticfactorsinthe developmentofGD,potentiallyinvolvingpolymorphismsin genes encoding elements of the sex steroid signaling or metabolicpathways(Heylensetal.,2012).

Postmortemstudieshaveobservedsex-atypicalvolumes andneuronalnumbersinhypothalamicnucleiinthebrains ofindividualswithGD(Garcia-FalguerasandSwaab,2008; Kruijveretal.,2000;Zhouetal.,1995).Brainstructurein individualswithGDhasalsobeeninvestigatedin vivo.MRI studies examiningwhitematter tractsusingdiffusion ten-sor imaging have shown deviations fromthe natal sex in whitemattermicrostructure inbothfemale-to-males(FM) andmale-to-females(MF) (Ramettietal.,2011a,b).Afew neuroimaging studies have investigated gray matter (GM) in individuals with GD, observing both regional volumet-ricpropertiesin linewiththenatalsexandothersinline withthe gender identity of theGD groups (Luders etal., 2009b,2012;SavicandArver,2011;Zubiaurre-Elorzaetal., 2012;Simonetal.,2013).ArecentstudybyZubiaurre-Elorza etal.(2012)wasthefirsttoexamineGMinFM( Zubiaurre-Elorza et al., 2012). Interestingly, theyobserved signs of subcorticalmasculinizationinfemale-to-maleadultsandof cortical feminization in male-to-female adults, providing the firstinsights intothe developmentalprocesses under-lyingGDinbothsexes.

Inourstudy,wewantedtoexaminebrainanatomyin indi-vidualswithGDduringadolescence,whenpuberty-related sexsteroidhormones aredrivingthefurther‘activational’ sexual differentiation of the brain rather than in adult-hood, when both organizational and activational steroid hormone effects have already sculpted the brain into a sex-specific configuration. Therefore, we acquired high-resolutionanatomicalbrainMRIscansof55female-to-male adolescents and38 male-to-female (MF) adolescentswith GD.Inaddition,toallowcomparisonswithindividuals shar-ingeithertheirgender identityortheirnatalsex,wealso scanned44boysand52girlswithoutGDgoingthrough ado-lescence.

First,we aimedtodefinesexually dimorphicstructures in the adolescent brain by comparing GMvolume in boys and girls without GD. As the principal aim of our study wastoexaminewhethersignsofsex-atypicalcerebral pro-grammingarepresentinthebrains ofGDadolescents,we subsequently comparedtheirregionalGMvolumesto indi-viduals sharing either their gender identity or their natal sex.Toensurethatthedeviationsfromthenatalsexdidnot resultfromhormonetherapy, werepeatedthese analyses in thesubsamplesof GD participantswhohadneverbeen exposedtoanyformofhormonaltreatment.

Finally, considering the recent development of sophis-ticated machine learning algorithms for MRI analyses, we also wanted to explore the data beyond the classical mass-univariatestatisticalapproach.Especiallyrelevantfor the study of individuals with GD is that multivariate pat-ternrecognitionapproachesprovideatooltoquantifythe

predictive value of volumetricGM patternsfor group dis-crimination. Therefore,the final aim of our study wasto examinethesedatausingamultivariateapproachandassess theextenttowhichindividualswithGDcanbediscriminated from either of the control groups based on the spatially distributedpatternsofGMvolumeinthebrain.

2.

Materials

and

methods

2.1. Subjects

Subjects for this study were recruited by the Center of Expertise onGender Dysphoria of the VUUniversity Med-ical Center in Amsterdam as part of a larger research projectinvestigatingbraindevelopment,brainfunctioning, growthandmetabolic aspectsin theclinical management of adolescents withGD (Trial registration number ISRCTN 81574253 (http://www.controlled-trials.com/isrctn/)). All adolescentswithGenderIdentityDisorderwereevaluated according tothestandard protocol at the clinicusing the criteriadescribed inthe Diagnosticand StatisticalManual ofMentalDisorders-IV-TR(DSM-IV-TR)(AmericanPsychiatric Association,2000).Pleasenotethat,inthisversionofthe DSM,‘GenderIdentityDisorder’wasstilltheofficialname ofthediagnosis.Therefore,onlyinthecontextof describ-ing thediagnostic procedure we willuse thisterm rather than the more recent ‘Gender Dysphoria’. When partici-pantswerereferredtotheclinicduringchildhood,thechild andparentsparticipatedinseveralsessionswithamental healthcareprofessionalspecializedinchildrenand adoles-centswithGenderIdentityDisorder.Duringthesesessions, theaimwastodeterminewhetherthecriteriaforaGender IdentityDisorderdiagnosisinchildrenaccordingto DSM-IV-TRweremet,toevaluatethecognitive,psychologicaland socialfunctioningofthechildandfunctioningofthefamily, and,ifindicated, toofferorreferforpsychological treat-mentorcounseling.ThecorecriteriaforaDSM-IV-TRGender IdentityDisorderdiagnosisareastrongandpersistent cross-genderidentificationandapersistentdiscomfortwithhisor hersexorsenseofinappropriatenessinthegenderroleof thatsex.Asaresult,thechildshouldexperienceasignificant amountofdistressorimpairmentinimportantareasoftheir functioning.Inadolescence,theindividuals(re-)appliedto theclinic,wheretheyenteredthediagnosticprocedurefor adolescents.Thisimpliedanevaluationofthepresenceof Gender Identity Disorder in adolescents/adults and other psychopathologyaccordingtoDSM-IV-TRcriteria.

The current analyses were performed several years afterdata acquisition,which allowed ustoascertain that the sample consisted only of individuals with persistent GD. In our study, we included only participants who had been followed at thecenter for many yearsand whohad indeed completed or were in the last phase of cross-sex hormonetreatmentatthetimeoftheanalysesratherthan relyingonparticipants’declarationofintendingtoundergo progressive treatment steps to change their body to fit their gender identity. Two individuals had tobe removed fromthe analyses asit becameclear at some point after the data acquisition that their GD had not persisted into adulthood.Ourfinalsampleconsistedof54female-to-male

adolescents,37male-to-femaleadolescents,44natalmale and52natalfemaleadolescentswithoutGD.

After receiving the diagnosis of Gender Identity Disor-der in adolescence and meeting some additional criteria tobeeligiblefortreatment(KreukelsandCohen-Kettenis, 2011), they were referred for hormonal treatment. The standardfor treatment intheclinic followstheStandards ofCareoftheWorldProfessionalAssociationfor Transgen-derHealth (WPATH,Coleman etal.,2011), a professional organization in the field of transgender care, and the ClinicalPracticeGuidelines of the EndocrineSociety pub-lishedin2009 (Hembreeetal.,2009),comprisingpuberty suppressionwithgonadotrophinreleasinghormone(GnRH) analoguesonceadolescentsstarttodisplaythefirst physi-calandendocrinechangesof puberty(Tannerstage2—3), followed by cross-sex hormone therapy after the age of 16.From18 yearsofageonward, individualswithGDcan be referred for sex reassignment surgery. After gonadec-tomy,GnRH analoguesareno longerprovided. Treatment withcross-sexhormonesiscontinuedaftersexreassignment surgery.Foradditionalinformationregardingthestandards fordiagnosticsandtreatment ofadolescentswithGD,see (Hembreeetal.,2009;KreukelsandCohen-Kettenis,2011). Pleasesee Table1 fordemographicand clinicaldetails of thesample.

Dependingontheageoftheparticipant,intelligencewas estimatedwiththeWechslerIntelligenceScaleforChildren (WISC-III)(Wechsler,1991)ortheWechslerAdultIntelligence Scale(WAIS-III)(Wechsler,1997).Allparticipantsalso under-went a physical examination by a clinician to determine height,weight,bloodpressureandpubertystageaccording toTanner(1962).Tannerstaging,whichdefinestheprogress of puberty based on the development of secondary sex characteristics,isconsideredthegoldstandardforpuberty measurementwhenperformedbyatrainedclinician(Dorn, 2006).

The FM were all gynephilic, whereas the MF were androphilic(i.e.sexuallyattractedtopartnersoftheirnatal sex),withtheexceptionofoneoftheMFwhoreportedtobe indifferentwithregardtothesexofthepartner.Repeating theanalyseswithoutthissubjectdidnotaffectourresults. AllGD participantshadstartedtoexperienceGD symp-tomsinchildhoodandmetcriteriafor‘early-onset’gender dysphoria. Rather than assessing whether symptoms had commencedbeforepubertybyretrospectivequestionnaires, we were able to ensure that our participants’ GD had started in childhoodas theyhad enrolled in our clinic at an early age and had been clinically evaluated for many years.

Noparticipants withanyknownphysical inter-sex con-ditions, neurologicaldisorders, cerebral damageor major medicalconditions were included in this study.To assess psychologicalfunctioninginallgroups,theDutchtranslation oftheChildBehaviorChecklist(AchenbachandEdelbrock, 1983;Verhulstetal.,1996)wasused.In caseof scoresin theclinicalrangeindicatingproblembehavior,participants werenotincludedinthestudy.

The study was approved by the Ethical Review Board of the VU Medical Center. Written informed consent was obtained from the subjects and from the parents or guardiansofthechildparticipantspriortotheir participa-tioninthestudy.

62

E.

Hoekzema

et

al.

Table1 Clinicalanddemographicdata.GnRH:GDindividualsundergoingtreatmentwithGnRHanaloguesbutnotcross-sexhormonesatthetimeoftheMRIacquisition. Cross-sex:GDindividualsundergoingcros-sexhormonetherapy.

Group(N) Treatment stage(N) Age(M±SD) (range) Tanner(M±SD) (range) IQ(M±SD)(range) DurationGnRH (days)(M_±SD) (range) Durationcross-sex (days)(M_±SD) (range) Cumulativedosis cross-sex**_(mg) (M±SD)(range) M(44) 16.42±2.75(10.54) 4.35±1.09(3) 110.5±19.88(85) — — — F(52) 16.29±2.96(11.49) 4.55±0.76(3) 105.19±16.32(60) — — — FM(54) Untreated (17) 15.20±2.76(9.26) 4.12±1.15(3) 101.82±14.65(50) — — — GnRH(16) 15.88_±1.54(4.27) 3.62_±1.41(4) 93.31_±14.71(49) 611.31_±435.22(1435) — — Cross-sex (21)* 19.11±1.29(4.90) 2.86±1.88(4) 105.29±17.27(51) 1595.67±785.92(3328) 1025.25±436.21(1586) 9270.38±9616.43(39,082) Complete sample 16.92±2.84(10.24) 3.47±1.61(4) 100.65±16.24(58) 1170.00±816.39(3796) 1025.25±436.21(1586) 9270.38±9616.43(39,082) MF(37) Untreated (11) 13.77±2.42(7.99) 3.45±1.13(3) 112.45±22.71(70) — — — GnRH(14) 15.29_±0.73(2.22) 3.71_±0.99(2) 93.86_±10.75(38) 692_±247.65(952) — — Cross-sex (12)* 19.04±2.17(7.11) 4.17±0.84(2) 107.83±18.97(58) 1619.75±398.01(1232) 911.76±497.02(1352) 1658.17±1133.68(3867) Complete sample 16.05±2.84(11) 3.78±01.00(3) 103.92±19.02(70) 1137.32±571.89(2141) 911.76±497.02(1352) 1658.17±1133.68(3867)

* _{FouroftheMFandnineoftheFMhadundergonesexreassignmentsurgery.} **_{FM:testosterone(Sustanon),andMF:estradiol(Progynova/Meno-implant).}

2.2. MRIacquisitions

TheMRIimageswereacquiredwitha3TPhilipsIntera scan-ner,equippedwithaSENSE6-channelheadcoil.Toobtaina high-resolutionanatomicalbrainimage,aT1-weightedpulse sequencewasappliedusingthefollowingacquisition param-eters:TR=9ms,TE=3.5ms, matrixsize=256×256, voxel size=1×1×1mm,andnumberofslices=170.

2.3. Univariateanalyses

MRI image processing and statistical analyses were con-ductedwithStatisticalParametricMappingsoftware(SPM8; www.fil.ion.ucl.ac.uk-spm),implementedinMatlab(version 7.8;www.mathworks.com).

Regional GM volume was quantified using a voxel-basedmorphometricapproach,afullyautomatedtechnique designed to evaluate local volumetricproperties of brain anatomy (Ashburner and Friston, 2000). We used the analysis pipeline provided in the Gaser vbm8 toolbox (http://dbm.neuro.uni-jena.de/vbm/). With this toolbox, theanatomicalimagesweresegmentedintodifferenttissue classes using an adaptive maximum a posteriori approach corrected for field inhomogeneities and brought into standardspaceusingaffineandnon-linearnormalization.

Since we worked with a sample of adolescent partic-ipants, we created a customized sample-specific DARTEL templateforourstudy.First,toobtainsuitableinitial age-matched tissue probability maps for import intoDARTEL, wegeneratedacustomizedage-matchedtemplateand tis-suepriors usingtheTOM8toolbox(https://irc.cchmc.org/ software/tom.php)andusedtheseinanaffineregistration. The affine registered gray matter and white matter seg-mentswerethenusedtocreatea sample-specificDARTEL template,whichwasthenimplementedinaDARTEL normal-izationtowarpourdataintoMNIspace.Topreserveoriginal tissuevolumesandallowcomparisonsofvolumesratherthan GMconcentrations,weusedtheGMsegmentsthathadbeen multipliedbytheJacobiandeterminantsextractedfromthe normalizationstep.Finally,an8mmfullwidthathalf maxi-mumsmoothingkernelwasappliedtothedata.Anabsolute thresholdof0.2wasappliedtotheanalyses,andtotalGM volumeofthebrainwasincludedasanuisancecovariate.

As there was a significant difference in IQ between the groups (M±SD, M: 110.50±19.88, F: 105.19±16.32, MF:103.92±19.02,FM:100.66±16.24.F=2.54,p=0.058), IQ was included in the between-group comparisons. We observednocorrelationsbetweenregionalGMvolumesand IQ within the groups. When subgroups of the total sam-plewerecomparedcausingdifferencesinagebetweenthe groupstosurface,thenagewasalsoincludedasacovariate ofnointerest.

The whole-brain results are reported at a threshold of p<0.05 FWE-corrected and an extent threshold of

k>10voxels. Moreover, to examine whether more subtle deviationsfromthenatalsexcouldbeobservedwithin sex-uallydimorphicbrainstructures,regionsof interest(ROIs) werecreatedrepresentingtheseregions.ROIanalyseswere performedinMarsbar(http://marsbar.sourceforge.net/).

ConsideringourhypothesisthatindividualswithGDhave undergone a sex-atypical differentiation ofthe brain that

divergesfromtheir natalsexin thedirectionof the cere-braldevelopmentalprocessestypicalfortheothersex,the ROIoflarger femalevolumewasappliedto thecontrasts ‘F>MF’and ‘MF>M’, andthe ROIsof largermale volume wereappliedtothe contrasts‘MF>F’ and‘FM>F’. The p

valuesfortheROIresultsareBonferroni-correctedforthe numberofROIs.

Inadditiontoanalysesinvolvingthecompletesample,we alsoseparatelycomparedtheGDindividualswhohadnever beenexposedtoanyformof hormonaltherapy (excluding boththeindividualswhosepubertywassuppressedbymeans of GnRH analogues and the GD participants who had ini-tiated cross-sex hormone therapy at the time of the MRI acquisition)toage-matchedcontrolsamplesofbothsexes. Tominimizethecontributionofpuberty-relatedendogenous sexsteroidhormonesandfocusprimarilyonorganizational effects,thestageofpubertaldevelopment(asanindexof thedegreeofpuberty-relatedendogenoussexsteroid hor-monestheindividualhadbeenexposedtountilthetimeof theMRIacquisition)wasincludedasanuisancecovariatein theseanalyses.

Furthermore, we also compareduntreated FMand MF totheGDparticipantswhohadalreadyinitiated cross-sex hormonetherapyatthetimeofMRIacquisitions.

2.4. Multivariateanalyses

Inaddition totheabove-described mass-univariate statis-tical approach, we also performed a pattern recognition analysis.This isamethodthatstemsfrommachine learn-ing,andusesamultivariateapproachtoinvestigatespatially distributed information in MRI data — which is intrinsi-cally multivariate — and hereby allows modeling signal patternsin theimages.Inourstudy,weusedtheanalysis pipelineforpatternrecognitionanalysesprovidedbyPRoNTo (http://www.mlnl.cs.ucl.ac.uk/pronto). This pipeline can beusedtoautomaticallysearchforregularitiesinthedata and train a classifier function that models the relation between spatial signal patterns and experimental factors (inourcase, group) basedonatraining dataset(Schrouff etal.,2013).Thisclassifiercanthenbeusedtopredictthe groupanewimagebelongstousingthespatialdistribution ofthesignalwithintheimage,andcomputetheaccuracy withwhich groups canbediscriminatedfromoneanother basedonwhole-brainspatial signal patterns. Weselected thebinarysupportvectormachine(SVM)anda leave-one-outcross-validationstrategy. Thiscross-validationstrategy computes the accuracy of classifiers by leaving one sub-ject out at a time and predicting the group label of this subjectbasedonatrainingdatasetincludingallremaining subjects,whichisrepeatedforeachsubject.Theresultsof alltheserunsarethenusedtocomputetheaccuracyofthe discriminantfunction.No covariateswereincluded in the multivariateanalyses.Firstwetrainedandtestedalinear SVMpatternclassifierfor the natalboysandgirls without GD,to assess theaccuracy withwhich they couldbe dis-criminatedfromoneanotherbasedonthedistributionofGM volume.Then,werepeatedtheseanalysesfortheGDgroups andindividualssharingeithertheirgenderidentityortheir natalsex. Thesemultivariateanalyses werealsorepeated includingonlythoseMFandFMwhohadneverbeenexposed

64 E.Hoekzemaetal.

Table2 GMgroupcomparisons.Fullresultsofthewhole-braincomparisonsbetweenadolescentboysandgirlsandbetweenthe GDgroupsandadolescentssharingtheirgenderidentity,reportedatawhole-brainstatisticalthresholdofp<0.05FWE-corrected andanextentthresholdof10voxel.

Comparison Brainregion MNIcoordinates Clustersize(mm3_{) T P(FWE-corrected)}

x y z

ComparisonsboysandgirlswithoutGD

M>F LCerebellum −42 −76 −20 591 5.38 0.002 RCerebellum 41 −46 −29 2714 5.71 <0.001 45 −73 −20 5.45 0.002 32 _{−37 −26} 5.32 0.003 Hypothalamus 6 2 −2 243 5.38 0.001 F>M LSuperiorMedial FrontalCortex −8 63 22 206 5.13 0.006 −8 69 15 5.02 0.009

ComparisonsGDtogroupssharinggenderidentity

F>MF RSuperiorMedial Frontal/RSuperior FrontalCortex 18 57 33 164 6.01 <0.001 6 59 37 5.01 0.006 MF>F LCerebellum _{−15 −48 −59} 47 4.80 0.022 M>FM LCerebellum/L Fusiform −42 −78 −18 54 4.86 0.017 FM>M —

ComparisonsGDtogroupssharingnatalsex

M>MF — MF>M — F>FM — FM>F —

toanyhormonaltreatmentandtheirage-matchedcontrols. Balancedaccuracyvalues,whichrepresentthefractionof thesubjectsforwhomacorrectclasslabelwaspredicted usingthetrainedclassifier,areprovidedforeachofthe com-parisons.Permutationtestswereusedtoexaminewhether theclassifierperformedabovechancelevel.

3.

Results

3.1. Univariateresults 3.1.1. Completesample

Toidentifyregionsofsexualdimorphismintheadolescent brain, we first comparedregional GM volumes (corrected for totalGMvolume) betweengirls and boyswithout GD. These whole-brain analyses (thresholded at p<0.05 FWE-correctedandanextentthresholdofk>10voxels)rendered threelocalizedclusters oflargerGM volumesin boysand oneclusterwheregirlshadmoreGMvolume(seeTable2). Morespecifically,ingirls,enlargedvolumewasobservedin asectionoftheleftfrontalcortexextendingfromthe supe-riorpartofthemedialfrontalcortextothesuperiorfrontal cortex.Fortheboys,ontheotherhand,weobserved clus-tersofrelativelylargerGMvolumeinthehypothalamusand inthebilateralcerebellum,coveringprimarilythesuperior posteriorlobesofthecerebellarhemispheres.

Regardingthecomparisons involvingtheGDgroups,we firstcomparedtheadolescentswithGDtoindividualssharing theirgenderidentity.Incomparisontonatalboys, female-to-maleadolescentswerefound tohaverelatively smaller volumesinaclusterextendingfromtheleftfusiformgyrus tothesuperior posteriorcerebellum.When comparingMF tonatalgirls,weobservedsmallerGMvolumesintheright superior frontal and superior medial frontal cortex, and enlargedleftcerebellarvolume.Theresultsofthese com-parisonsareindicatedinTable2andthewhole-brainresults aredepictedinFig.1.

Next,wecomparedtheGDgroupstoindividualssharing theirnatalsex.Forthesecomparisons,nosupra-threshold clusterssurfacedatwhole-brainlevel,suggestingthat over-all the regionalGM volumes of MF and FM arelargely in accordancewiththoseobservedin theirnatalsex. To fur-therevaluatethedegreeofmasculinizationorfeminization of GM in individuals with GD, we compared GM volumes specifically within sexually dimorphic regions, by apply-ing Regions of Interest (ROIs) representing the previously describedregionsofsex-typicalvolume(theROIsoflarger volumeinfemalestothecontrasts‘MF>M’and‘F>FM’and thoseoflargermalevolumeto‘M>MF’and‘FM>F)’.

Forthesecomparisons,wedetected lessvolumeinthe right superior medial frontal cortex and more volume in the right cerebellum in FM relative to girls without GD (see Table 3).Regarding MF, whenexamining GM volumes

Fig.1 Whole-brainresults.Illustrationofallthewhole-brainresults(seeTable2).Forillustrativepurposes,thisfiguredisplays theresultsat p<0.0001 (insteadofp<0.05FWE-corrected)to makethem standout morefrom thebackgroundinthefigure. M=maleswithoutGD;F=femaleswithoutGD;MF=males-to-femaleindividualswithGD;FM=females-to-maleindividualswithGD.

Table3 ROIresults.FullresultsoftheMarsbarROI anal-yses comparing the GD groups and their respective natal sex. Region T Correctedp Completesamples FM>F LCerebellum 1.81 0.1014 RCerebellum** _{3.86 0.0002} Hypothalamus 1.14 0.3364 F>FM Lsuperior medialfrontal cortex** 2.07 0.0198 MF>M Lsuperior medialfrontal cortex* 1.43 0.0776 M>MF LCerebellum** _{2.75 0.0033} RCerebellum** _{2.98 0.0017} Hypothalamus** _{2.40 0.0088} Untreatedsamples FM>F LCerebellum 0.67 0.5850 RCerebellum** _{2.63 0.0172} Hypothalamus 0.09 0.8986 F>FM Lsuperior medialfrontal cortex** 2.10 0.0205 MF>M Lsuperior medialfrontal cortex 0.92 0.8180 M>MF LCerebellum** _{3.15 0.0059} RCerebellum 0.47 0.6851 Hypothalamus* _{2.03 0.0765} * _{trendatp<0.1.} ** _{Significantatp<0.05,}

within these sexually dimorphic regions, we observed a trendforincreasedleftsuperiormedialfrontalvolumeand significantlyless volumeinthebilateral superiorposterior cerebellumandhypothalamusincomparisontoboyswithout GD.

Asasupplementaryanalysis,wealsovolumetrically com-paredthe twoGDgroups totheindividuals withoutGD in ordertoassess whethertherewere anyalterations in GM volumeaccompanyingGD(independentofsex),butno sig-nificantresultssurfacedforthiscomparison.

3.1.2. Untreatedsample

To examine whether these deviations from the natal sex arenotdrivenbyexposuretothehormonetherapyalready providedto a subsample of our GD participants, we also repeatedthesecomparisonsincludingonlyGD adolescents whohadnotyetundergoneanyhormonaltreatmentatthe time of the MRI acquisition and age-matched controls of their natal sex. To minimize the contribution of ‘activa-tional’effectsofpuberty-relatedsexsteroidhormones,the stageofpubertal developmentwasalsoincluded inthese modelsasanuisancecovariateasanindicationofthedegree towhich the bodyhad already been exposed to puberty-relatedsexsteroidhormones.

In line with the findings in the complete sample, we observednodeviationsfromthenatalsexineithertheFM orMFatwhole-brainlevel (p<0.05FWE-corrected). How-ever,whenexaminingGMvolumeswithinsexuallydimorphic brainstructuresusingROIanalyses,weobserveddifferences in both GD groups with respect to their natal sex. More specifically,inFM,lessGMvolumewasobservedintheleft superiormedialfrontalcortexincomparisontoadolescent girls,whilemoreGMvolumewasfoundintheright cerebel-lumthangirlswithout GD.RegardingMF,incomparisonto natalboyswithoutGD,weobservedsmallervolumesinthe

66 E.Hoekzemaetal. leftsuperiorposteriorhemisphereofthecerebellumanda

trendforlessvolumeinthehypothalamus.

We alsocomparedtheuntreatedFMandMFtocontrol subjectssharingtheirgenderidentityratherthantheirnatal sex.Inaccordancewiththeresultsincludingthecomplete sample,whereasnodeviationsinGMvolumeswereobserved at whole-brainlevel (p<0.05 FWE-corrected), when com-paringboth GDgroups toadolescentssharingtheirgender identity we did observe volumetric differences with the natalsexatthiswhole-brainthreshold(F>MF:Rightinferior orbitofrontal cortex: 51 44—14, T=6.80, p=0.006 FWE-corrected.MF>F:—.M>FM:Rightinsula:30237,T=5.87,

p=0.006FWE-corrected.FM>M:—).

To examine the effects of hormone therapy on neu-ral GM, we also directly compared: (a) the untreated FMtoFMundergoingtestosterone treatment, and(b) the untreated MF to MF exposed to estradiol treatment. For these analyses, no results were observed at whole-brain level. To further examine whether there were no differ-encesbetweenthegroupswithinregionscharacterizedby sex differences in the adolescent brain, we also applied the ROIs representing sexually dimorphic brain structures to these comparisons. For FM, there was no significant difference in GM volume between untreated and cross-sex-hormone-treated adolescents (treated FM>untreated FM: Left cerebellum: T=1.13, corrected p=0.346. Right cerebellum: T=1.03, corrected p=0.397. Hypothalamus:

T=1.75, corrected p=0.127. untreated FM>treated FM: Left medial frontal cortex: T=0.08, corrected p=0.468). Regarding the MF, we observed differences between the cross-sexhormone-treatedsample andtheuntreated sam-pleonlyintheleftsuperiormedialfrontalcortex(treated MF>untreated MF: Left medial frontal cortex: T=2.96, corrected p=0.004.untreatedMF>treated MF:Left cere-bellum: T=0.14, corrected p=0.830. Right cerebellum:

T=0.22, corrected p=0.800. Hypothalamus:T=0.43, cor-rectedp=0.705).

3.2. Multivariateanalyses

3.2.1. Completesample

To examine whether GD individuals can be discriminated fromnatalboysandgirlswithoutGDbasedonspatially dis-tributedpatternsofGMvolume,weappliedamultivariate patternclassificationanalysis,incorporatingasupport vec-tormachine algorithm. When usingthis approach for the classificationofboysand girlswithout GD, we obtaineda highlysignificantpredictionaccuracyof88%forthe discrim-inationbetweenthesexes(seeTable4).

Next, we applied this approach to the GD groups and theadolescentssharingtheirgenderidentity.Weobtaineda highaccuracybothfortheclassificationoftheFMandnatal boyswithoutGDandfortheclassificationoftheMFandgirls withoutGD,showingthattheseGDgroupscanbe discrim-inatedfromgroups sharingtheirgender identity basedon spatialGMpatternswithahighaccuracy(seeTable4).

FortheclassificationsinvolvingtheGDgroupsandtheir respectivenatalsex, muchlowerpredictionaccuracy val-ueswere obtained. Onlyfor theMFdid this classification reachsignificance. This groupcould thus bedistinguished fromnatalmalesslightly abovechance levelbasedonGM

Table4 Multivariate results.Prediction accuracyvalues andpvaluesfortheclassifiersobtainedusingamultivariate patternrecognitionapproach.

Classlabels Balanced accuracy(%) pValue Group1 Group2 M F 88.0 <0.0001 MF M 65.5 0.007 F 85.6 <0.0001 UntreatedMF M 45.5 0.61 F 86.4 <0.0001 FM F 52.2 0.32 M 78.4 0.002 UntreatedFM F 50.0 0.55 M 76.5 0.006

anatomy.TheFMcouldnotbediscriminatedfromnatalgirls withastatisticallysignificantaccuracy.

ForbothGD groups,however,theclassification accura-cies for distinguishing them fromindividuals sharing their genderidentitystronglyexceedthoseforthediscrimination fromtheirnatalsexbasedonspatiallydistributedGM.Fig.2 displaystheweightmapsandconfusionmatrixesforeachof theseclassifications.

Althoughamultivariateapproachdoesnotlenditselfto extractingpeakvoxelsbasedontheweightdistribution,a visualinspectionoftheweightmaps,whichdepictthe rela-tivecontributionofeachvoxeltotheclassifier(seeFig.2), suggeststhatvoxelswithahighrelativeweightarecentered onthesexuallydimorphicregionspreviouslyobservedinthe mainmodel.Tofurtherinvestigatethecontributionofthese regions,weappliedtheROIsofthepreviouslydefined sexu-allydimorphicregionsasamasktoreplacethewhole-brain mask commonly used for the analysis. Hence, the analy-ses involving the GD individuals andtheir natalsex were repeatedusingamaskofthesexuallydimorphicstructures. Usingthismask,bothGDgroupscouldbediscriminatedfrom theirnatalsexwithasignificantpredictionaccuracy(FM—F: balancedaccuracy:47.7%,p=0.54.MF—M:balanced accu-racy:61.7%,p=0.042).

3.2.2. Untreatedsample

Werepeatedthesemultivariatecomparisonsincludingonly adolescents with GD who had not been exposed to any hormonal therapy at the time of the MRI acquisition and age-matchedcontrolsubjectssharingeithertheirnatalsex ortheirgenderidentity.Inlinewiththeresultsobtainedfor thecompletesample,weobtainedahighaccuracyforthe classificationofFMandnatalboyswithoutGDaswellasfor theclassificationofMFandnatalgirlswithoutGD.BothGD groupscouldbesignificantlydiscriminatedfromadolescents sharingtheirgenderidentitybasedontheneuraldistribution ofGMvolume(seeTable4).

Fig.2 Multivariateresults.Confusionmatricesandweightmapsfortheclassificationbetween(A)natalboysandgirls,(B)FM andnatalgirlswithoutGD,(C)FMandnatalboyswithoutGD,(D)MFandnatalboyswithoutGD,and(E)MFandnatalgirlswithout GD.PanelsFandGdisplaytheconfusionmatricesfortheFMversusgirlsandtheMFversusboysrespectivelywhenusingamask ofthesexually dimorphicstructures. M=maleswithoutGD; F=femaleswithoutGD; MF=males-to-femaleindividuals withGD; FM=females-to-maleindividualswithGD.

In accordance with the results for the complete sam-ple,muchlowerpredictionaccuracyvalueswereobtained for the classification between the GD groups and their respective natal sex and the classification accuracies for discriminatingMForFMfromindividualssharingtheir gen-deridentitystronglyexceedthosefortheclassificationfrom theirnatalsex.NeithertheFMnortheMFcouldbe discrim-inated from their natalsex witha statistically significant accuracy(seeTable4).

Forcompleteness,we alsorepeatedthesemultivariate analysesusingthemasksoftheROIsofsexuallydimorphic regions(asdescribedabove).Whenusingthismask,theFM couldstillnotsignificantlybediscriminatedfromtheirnatal sex,butasignificantpredictionaccuracywasfoundforthe

classificationbetweentheuntreatedMFand boyswithout GD(FM—F:balancedaccuracy:50.0%,p=0.442.MF—M: bal-ancedaccuracy:77.3%,p=0.016).

4.

Discussion

WhencomparingGMvolumebetweenadolescentboysand girls to identify sexually dimorphic brain structures, we observedclustersofenlargedGMvolumein the hypothal-amus and both superior posterior lobes of the cerebellar hemispheresinboys,whilegirlshadmoreGMvolumeinthe leftsuperiormedialfrontalcortex.Theseclustersare cen-teredonwell-established regionsof sexualdimorphism in thehumanbrain.

68 E.Hoekzemaetal. Thehypothalamus,astructurerichlyendowedwithsex

steroid receptors, plays an essential role in reproduction and sexual behavior, and is a primary site of neural sex differencesin animals (Dohler etal., 1982; Gorskietal., 1980;MatsumotoandArai,1983).In humans,postmortem studies have also discovered several sexually dimorphic nuclei with higher cell numbers and larger volume in males within this region (Allen et al., 1989; Swaab and Fliers, 1985). Clusters of enlarged hypothalamic volume in men as well as in adolescent boys have also been detectedin vivo(Goldstein etal.,2001; Lombardoetal., 2012).

Likethehypothalamus, the cerebellumalsorepresents a site of sexdifferences in the animal and human brain. Itis well establishedthat adult menhave largerabsolute andrelativecerebellarvolumesthanwomen(Chungetal., 2005;Escalonaetal.,1991;Filipeketal.,1994;Goodetal., 2001; Raz et al., 2001; Tiemeier et al., 2010). In accor-dancewiththesevolumetricdifferences,menwerefound tohaveahighernumberofPurkinjecellsthanwomen(Hall etal.,1975).In children andadolescents,cerebellar vol-umeisbetween10to13%largerinboysthangirls(Tiemeier etal.,2010).Interestingly,thesuperiorposteriorlobeofthe cerebellum, which was bilaterally enlarged in adolescent boysin comparisontogirls inour study,representsasite ofespeciallyrobustsexdifferencesinvolumetricproperties anddevelopmentaltrajectoryacrossadolescence(Tiemeier etal.,2010).

TheregionoflargerGMvolumeingirlswaslocatedinthe frontalcortex, which harborssome of the primarysexual dimorphismsofthistype.Previousstudiesinvestigatingsex differencesinthehumanbrainhavealsodetectedincreased volumeorcorticalthicknessintheGMcomprisingthe supe-riorfrontalandmoremedialsectionsofthefrontalcortex inwomencomparedtomen(Goldsteinetal.,2001;Good etal., 2001; Lombardo etal., 2012; Luders etal., 2006, 2009a).Itshouldbenotedthatinthisstudywedidnotobtain informationregardingthemenstrualcycleinthegirls,and thereforewecannotexcludesubtlecycle-relatedeffectson GMvolumes.

In line with theories of sexual differentiation, all the regions of sexual dimorphism identified in our study rep-resentbrainstructuresknowntoexpresshighlevelsofsex steroidreceptors duringearly braindevelopmentin other animalspecies(Goldsteinetal.,2001),suggestingthatbrain regionshomologouswiththoseidentifiedbyanimalstudies ashavingahighsexsteroidreceptordensityduringcritical periodsofbraindevelopmentaremorelikelytoretain sex-ualdimorphismsthroughoutlife.Accordingly,thevolumetric differencesobserved betweentheGDgroupsandthe con-trolgroupsarealsoexclusivelycenteredonbrainstructures richinsexsteroidreceptorsduringfetalandearlypostnatal development.

In the GD groups, when examining the complete sam-ples—i.e.thesamplesincludingGDindividualsatdifferent stages of treatment — we observed regional GM volumes andvolumetricpatternsof GMthatwereconcordantwith theirnatalsex.Volumetricdifferenceswerefoundbetween individuals with GD and adolescents sharing their gender identityinseveralregionsacrossthebrain,centeredon sex-uallydimorphic structures.However,when comparingthe GDgroupstoboysandgirlsoftheirnatalsex,nodifferences

wereobservedinlocalGMvolumesatwhole-brainlevelfor eitherFMorMF.

Furthermore,multivariatepattern recognition analyses showed that for both GD groups the accuracy for distin-guishingthemfromadolescentssharingtheirgenderidentity basedonspatiallydistributedGMpatternswasgreaterand more statistically significant than the accuracy for sep-arating them from their natal sex. Thus at whole-brain level no signs of sex-atypical cerebral development were observed. However, when specifically examining sexually dimorphicstructuresinGDparticipantsusingROIanalyses, inbothmale-to-femaleandfemale-to-maleadolescentswe detectedsubtledeviationsinGMvolumefromtheir respec-tive natalsex inthe direction of individuals sharing their gender identity. More specifically,FMhadmore volumein the right cerebellum and less volumein the left superior medial frontal cortex in comparison to girls without GD, while MFhad lessvolume inthe bilateral cerebellumand hypothalamusandatrendformoremedialfrontalvolume thanboyswithoutGD.

WhenseparatelyexaminingtheuntreatedsampleofMF andFM,asimilarpatternwasobserved.Atwhole-brainlevel (p<0.05 FWE-corrected), differences in GM volume were onlyfoundinbothGDgroupswithrespecttosubjects shar-ingtheirrespectivegenderidentity,butnotincomparison to their natal sex. Likewise, using multivariate analyses, GDadolescentscouldonlybediscriminatedfromindividuals sharingtheirgenderidentitybasedonspatiallydistributed neuralGMpatternswithasignificantaccuracy.

The ROIanalyses separately examining the subsamples of GD adolescents whohad not yet been exposed to any hormonaltreatment alsorenderedsimilarresultstothose obtained using the complete GD samples, showing subtle deviationsinGMvolumefromthenatalsexwithinseveralof thesexuallydimorphicstructuresinthedirectionof individ-ualssharingtheirgenderidentity.WhileuntreatedFMhad lessGMvolumeintheleftsuperiormedialfrontalcortexbut morevolumeintherightcerebellumincomparisontonatal girlswithoutGD,MFwerefoundtohavesmallervolumesin therightcerebellarhemisphereandatrendforlessvolume inthehypothalamuscomparedtonatalboyswithoutGD.

Inlinewiththesefindings,multivariatepattern classifi-cationanalysesdemonstratedthatbothGDgroupscouldbe successfullydiscriminatedfromtheirnatalsexbasedonthe cerebral GMvolumedistributionwhentakingintoaccount onlysexuallydimorphicpartsofthebrain.Intheuntreated samples,astatisticallysignificantpredictionaccuracywas onlyobtainedfortheclassificationbetweentheMFandtheir natalsexwhenapplyingamask of thesexually dimorphic structures.

ThepreviousstudiesevaluatingGMinadultswithGDhave renderedmixedresults,some arguingagainstandsomein supportofasex-atypical differentiation ofthebrain.One factorthatlikelycontributestothisdiscrepancyisclinical heterogeneity.According totheBlanchardtypology,there are two fundamentally different types of transsexualism (Blanchard etal., 1987).In the group commonly referred to as ‘homosexual’ or ‘early-onset’ individuals with GD, the sexual orientation is directed toward the natal sex, symptoms usually start early in childhood (‘early onset’) andphysical differenceshave been observedwithrespect toindividuals sharing theirnatalsex(e.g. MFwerefound

tobeshorterandlighterthanmenwithout GD(Blanchard et al., 1995), although this could not be replicated by other groups (Smith et al., 2005)). ‘Non-homosexual’ or ‘late-onset’transsexuals,ontheother hand,showamore variable sexual orientation (for instance directed at the othernatalsexorasexual)andsymptomsusuallystartduring orafteradolescence.Inthepresentstudy,weinvestigated ‘homosexual’/’early-onset’ individualswithGD. Moreover, onlyindividualswhoarrivedattheclinicatanearlyageand werefollowedat thecenterformanyyearswereincluded inthisstudy,allowingustoverifytheir‘early-onset’status ratherthanrelyonretrospectivequestionnaires.Thissetup ensuredaveryreliablesampleof ‘early-onset’individuals withGD.

SavicandArver(2011)examinedGMin‘non-homosexual’ MFusingVBMandvolumetricdelineationanddidnotobserve anysignsoffeminization,andLudersetal.(2009b)detected onlyalocalizedfeminizationoftherightputamenin primar-ily‘non-homosexual’MF.Ludersetal.(2012)alsoexamined corticalthicknessinMF,observingsomedeviationsfromthe natalsexinseveralregions,althoughthelackofafemale controlsampledidnotfacilitatedeterminingwhetherthese deviationsreflectsignsoffeminization.Ontheotherhand, Zubiaurre-Elorzaetal.(2012)andSimonetal.(2013) inves-tigated GM in ‘homosexual’ participants with GD. Simon etal.(2013) foundthat,inseveralbrainregions,GD indi-vidualsdifferedfromcontrolssharingtheirnatalsexbutnot fromthosesharing theirgender identity. Zubiaurre-Elorza et al. (2012) performed the first study investigating GM in ‘homosexual’/’early-onset’ participants with GD. They observeddifferencesinvolumeoftheputamenbetweenFM andwomenwithoutGDwhilecorticalthicknessdidnot dif-ferbetweenthesegroups.MF,ontheotherhand,deviated frommenwithoutGDincorticalthicknessinseveralareas across the brain but not from women. This lead them to concludethatsignsofsubcorticalmasculinizationcouldbe observedinFMwhileMFexhibitedsignsoffeminizationin thecerebralcortex(Zubiaurre-Elorzaetal.,2012).

Consideringthesefindings,itisinterestingtonotethat, inourstudy,whensearching forsexually dimorphic struc-tures in natal boys and girls without GD, we observed subcortical regions of male-dominant volume, while the locusoffemale-biasedvolumewaslocatedwithinthe cere-bralmantle.Thesefindingssuggestthatthereisadistinction between the degree towhich feminization and masculin-izationprocessesaffectsubcorticalandcorticalsectionsof the brain,although in themale-to-female and female-to-malegroupswedidnotobserveaselectivepredilectionfor corticalversussubcorticalalterations,respectively.

The sex-atypicaldifferentiationof thebrainintegralto mosttheoriesonthepathogenesisofGDiscommonly pos-tulated to reflect exposure to an altered neuroendocrine climateinutero.However,besidestheearlyorganizational effectsofgonadalhormones,exposuretosexsteroidslater inlife,e.g.viapuberty-relatedendogenoushormonesurges orcross-sexhormonetherapy,canexertactivationaleffects onthecentralnervoussystem. Althoughpubertyis avery interestingperiodtostudythebrainaspubertalsexsteroid hormones areexerting activational effects onthe central nervoussystem,it isalsoaperiodcharacterizedbyahigh variabilityinthedegreeofpubertalmaturation.Inourstudy, we also examined GM volumes in the subsamples of GD

adolescentswhohadnotyet beenexposedtoanyformof hormonetherapy,andweobservedgender-atypicalvolumes inseveralofthesexually dimorphicstructures.Inorderto further minimize the contribution of activational effects of sexsteroid hormones during adolescence and enhance thefocusonorganizationaleffects,wealsocontrolledfor thestageofpubertaldevelopmentintheseanalyses,taken asan index of the body’s degree of exposure to endoge-nouspuberty-relatedsexsteroids.Ourresultssuggestthat, before the manifestation of contingent neuroanatomical changesinducedbyendogenouspuberty-relatedsexsteroid surgesor treatment withexogenouscross-sexsteroid hor-mones,therearealreadysubtledeviations fromthenatal sex in individuals with GD, which may reflect signs of a partialsex-atypicalbraindifferentiationduringearly devel-opment.Thefactthattheanalysesinvolvingthecomplete samplesandthoseinvolvingtheuntreatedsamplespointin thesamedirectionandthatnoorverylittledifferenceswere observedbetween thetreatedanduntreatedsamplesalso pointtoorganizationalratherthanactivationaleffects.

However, although the localization of the significant clusters in brain structures expressing high levels of sex steroidreceptorsduringfetaldevelopmentcarefullypoints toan endocrine effect, the current study does not allow determining whether these deviations reflect endocrine, environmental (e.g.differences in upbringing, interest or activities)or geneticinfluencesor acombination ofthese factors. It should also be noted that the sample sizes of theGDindividualswhohadneverbeenexposedtoany hor-monaltreatmentarequitesmall(N=17fortheFMandN=11 fortheMF).Therefore,thesefindingsshouldbeinterpreted withcaution.

Anotherlimitation of the present study is that it does notprovidetheidealframeworkforinvestigatingtheeffects ofcross-sex hormonetherapyonbrainstructureduetoits cross-sectional setup. We also did not obtain sex steroid hormonelevelsat thetimeofthe MRIacquisitions,which mighthavebeenusedforcorrelationanalyses.Afew previ-ousstudieshaveexaminedthistopicinindividualswithGD usingasensitivelongitudinalapproach.Interestingly,these studiesindicatedthatandrogentreatment inFMrendered increasesintotalbrainvolume,regionalcorticalthickness andhypothalamicvolume,while estrogen therapy accom-paniedby anti-androgen treatment in MF was associated withreductionsintotalbrainvolume,corticalthicknessand subcorticalgraymatter volume(Hulshoff Poletal.,2006; Zubiaurre-Elorzaetal.,2014).These studiessuggestthat, althoughsexuallydimorphicstructuresseemtohavealready partiallybeenprogrammedinasex-atypicalmannerinGD childrenwhohavenotyet been exposedtocross-sex hor-monetherapy,sexsteroidhormonesadministeredaspartof cross-sexhormonetherapyinadulthood canfurthersculpt the brain into a morphological configuration that fits the individual’sgenderidentityratherthanthenatalsex.

Insum,sexualdimorphismsinGMvolumewereobserved in several structures of the adolescent brain known to express high levels of sex steroid receptors during fetal andearlypostnataldevelopmentinrodents.Regardingthe comparisonsinvolvingthe GDgroups, atwhole-brainlevel theydifferedfromadolescents sharingtheir gender iden-tity while there were no deviations in GM volume with respect to the natal sex, arguing against a sex-atypical

70 E.Hoekzemaetal. differentiation of the brain in gender dysphoria.

Like-wise,multivariatepatternrecognitionanalysesshowedthat classification accuracies for discriminating the GD groups fromindividuals sharing their gender identity exceed the accuraciesobtained for theclassificationbetween theGD adolescentsandtheirnatalsex.

However, when examining GM volumes within sexually dimorphic structures using ROI analyses, within both GD groupswealsoobservedsubtledeviationsfromthenatalsex whereGMvolumesdivergedfromthenatalsextowardthe groupssharing theirgender identity. Multivariate analyses alsodemonstratedthattheindividualswithGDcould signifi-cantlybediscriminatedfromtheirnatalsexwhentakinginto accountonlytheGMdistributionwithinsexuallydimorphic structures.

Similar results were observed in the subsamples of untreatedFMandMFwhohadnotbeenexposedtoanyform ofhormonaltherapy—whilealsocontrollingfor thestage ofpubertaldevelopment—withinseveraloftheseregions, suggestingthattheGDbrainmayhavealreadypartiallybeen programmedinasex-atypicalmannerduringearly develop-ment.

Taken together, the results of this study indicate that theneuroanatomicalcharacteristicsofadolescentswithGD areprimarilyconcordantwithindividualsoftheirrespective natalsex. However,withinsexually dimorphicbrain struc-tureswe observedsubtlesignsofa volumetricdivergence fromthenatalsexinthedirectionofindividualssharingtheir gender identity,which maypoint toapartial sex-atypical differentiationofthebrain.

Role

of

the

funding

source

Thefundingsourcesdidnotplayaroleinanycomponentof thisstudy.

Conflict

of

interest

statement

Nonedeclared.

Acknowledgements

Wewouldlike tothankalltheparticipants andtheir par-ents.Thisworkwassupportedbyaneducationalgrantfrom FerringBV,Hoofddorp,andbyaVICIgrant(453-08-003)from theDutchScienceFoundation(NederlandseOrganisatievoor WetenschappelijkOnderzoek)toJB.JBisaseniorresearch associateoftheBelgianFondsNationaldelaRecherche Sci-entifique.

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