Braz. j. . vol.82 número5

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www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

Imbalance

in

the

diurnal

salivary

testosterone/cortisol

ratio

in

men

with

severe

obstructive

sleep

apnea:

an

observational

study

夽

Cristina

Mihaela

Ghiciuc

a,1

,

Lucia

Corina

Dima-Cozma

b,1

,

Raluca

Mihaela

Bercea

c,1

,

Catalina

Elena

Lupusoru

a

,

Traian

Mihaescu

c

,

Sebastian

Cozma

d

_,

_Francesca

_Romana

_Patacchioli

e,∗

a_University_of_Medicine_and_Pharmacy_Grigore_T._Popa,_Department_of_{Pharmacology,}_Iasi,_Romania

b_University_of_Medicine_and_Pharmacy_Grigore_T._Popa,_School_of_Medicine,_Department_of_Internal_Medicine,

Iasi,Romania

c_University_of_Medicine_and_Pharmacy_Grigore_T._Popa,_Clinic_of_Pulmonary_Diseases,_Iasi,_Romania d_University_of_Medicine_and_Pharmacy_Grigore_T._Popa,_Department_of_{Otorhinolaryngology,}_Iasi,_Romania e_Sapienza_University_of_Rome,_Department_of_Physiology_and_Pharmacology_‘‘V._{Erspamer’’,}_Rome,_Italy

Received24June2015;accepted8September2015 Availableonline17December2015

KEYWORDS

Obstructivesleep apnea;

Saliva; Cortisol; Testosterone

Abstract

Introduction:Thecomplexrelationshipbetweensleepdisordersandhormonescouldlead to

alterations inthe productionofcortisol and testosteroneinobstructive sleep apnea (OSA)

patients.

Objective: Thepurposeofthisstudywastodeterminethediurnaltrajectoriesofsalivary

free-testosterone,free-cortisolandtheirratio(T/C).

Methods:TensubjectsnewlydiagnosedwithOSA,basedonnocturnalpolysomnography

eval-uation and excessive daytime sleepiness, and seven matched controls were consecutively

recruited.Cortisolandtestosteroneweremeasuredinsalivarysamplescollectedupon

awaken-ing,atnoonandintheevening.Thepsychometricevaluationofanxiety/depressionandreferred

sexualfunction disturbanceswasperformedtoevaluatethepresenceofneuropsychological

comorbidities.

夽 _Please_cite_this_article_as:_Ghiciuc_CM,_Dima-Cozma_LC,_Bercea_RM,_Lupusoru_CE,_Mihaescu_T,_Cozma_S,_et_al._Imbalance_in_the

diur-nal salivary testosterone/cortisolratio in men withsevere obstructive sleepapnea: anobservational study. Braz JOtorhinolaryngol. 2016;82:529---35.

∗_{Corresponding}_author.

E-mail:[email protected](F.R.Patacchioli). 1 _These_authors_contributed_equally_to_this_work.

http://dx.doi.org/10.1016/j.bjorl.2015.09.004

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Resultsandconclusion: ThemainfindingwasthatOSAsubjectsdisplayedhypocortisolismupon

awakeningandasignificantreductionintestosteroneconcentrationintheeveningin

compar-isonwiththecontrolgroup,whichhasmaintainedthephysiologicaltestosteroneandcortisol

diurnalfluctuation,withhigherhormoneconcentrationsinthemorningandlower

concentra-tionsintheevening.Theuseofdatafrommultiplediurnalmeasurementsratherthanasingle

pointallowedthedetectionofT/Cratiochangesofoppositesignsatthebeginningandendof

theday:theOSAsubjectshadahigherT/Cratiothanthecontrolsinthemorning,whiletheir

T/Cratiowassignificantlylowerthanthatofthecontrolsintheevening.Theimbalancesin

theanabolic-catabolicdiurnalequilibriumsuggestthatOSAisassociatedwithadysregulation

ofthehypothalamic---pituitary---adrenalandhypothalamic---pituitary---gonadalaxes,potentially

anunderlyingcauseofsomeoftheneuropsychologicalcomorbiditiesobservedinOSApatients.

by Elsevier Editora Ltda. This is an open access article under the CC BY license (http://

creativecommons.org/licenses/by/4.0/).

PALAVRAS-CHAVE

Apneiaobstrutiva dosono;

Saliva; Cortisol; Testosterona

Desequilíbrionaproporc¸ãosalivardiurnadetestosterona/cortisolemhomens comapneiaobstrutivadosonograve:umestudoobservacional

Resumo

Introduc¸ão:A relac¸ão complexa entre os distúrbios do sono e os hormônios pode levar a

alterac¸õesnaproduc¸ãodecortisoletestosteronaempacientescomApneiaobstrutivadosono

(AOS).

Objetivo:Oobjetivodesteestudofoideterminarascurvasdiurnasdetestosteronaecortisol

livresnasalivaesuaproporc¸ão(razãoT/C).

Método: Dezindivíduosrecém-diagnosticadoscomAOScombasenaavaliac¸ãopor

polissono-grafia noturna e sonolência diurna excessiva e sete controles pareados foram recrutados,

consecutivamente.Cortisoletestosteronaforammedidosemamostrasdesalivacoletadasao

acordar,aomeio-diaeànoite.Aavaliac¸ãopsicométricadosdistúrbiosdeansiedade/depressão

efunc¸ãosexualmencionadosfoirealizadaparadetectarapresenc¸adecomorbidades

neurop-sicológicas.

Resultados: OachadoprincipalfoiqueosindivíduoscomAOSapresentamhipocortisolismoao

acordareumareduçãosignificantenaconcentraçãodetestosteronaànoite,emcomparação

comogrupo controle, quemanteve avariac¸ãofisiológica diurna detestosterona e cortisol

comconcentrac¸õeshormonaismaiselevadaspelamanhãeconcentrac¸õesmaisbaixasdurante

anoite.Ousodedadosde váriasmensurac¸ões diurnas,em vezdeuma únicamensurac¸ão,

permitiudetectar asalterac¸ões narazão T/Cdesinais opostos noinícioe nofinal dodia:

osindivíduos comAOS apresentaram razãoT/Cmaiorqueoscontrolesnapartedamanhã,

enquantoquearazãoT/Cfoisignificantementeinferioràdoscontrolesduranteanoite.

Conclusão:Osdesequilíbriosnobalanc¸oanabólico-catabólicodiurnosugeremqueaAOSestá

associada auma desregulac¸ão dos eixoshipotálamo-hipófise-adrenal e

hipotálamo-hipófise-gonadal,potencialmenteacausasubjacentedealgumasdascomorbidadesneuropsicológicas

observadasempacientescomAOS.

por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http://

creativecommons.org/licenses/by/4.0/).

Introduction

Obstructive sleep apnea (OSA) is a chronic respiratory disturbance that can be diagnosed with an overnight polysomnography(PSG).Itisaserioushealthproblemwith prevalence greater than 26% in the general middle-aged population.1---3

Excessive daytime sleepiness and snoring are often associated with neuropsychological symptoms, includ-ing concentration difficulties and cognitive disturbances. Hypoxia,sleep fragmentation,obesity, and ageing in OSA

patientswerefoundtobeassociatedwithlowserum testos-terone levels.Luboshitzkyet al.4 _demonstrated_that _men

withsleepapneahaddecreasedlevelsoftestosteroneand luteinizinghormoneduringnightlysleep,whichispossibly causedbythecombinedeffectsofhypoxiaandsleep frag-mentation.Morerecently,Berceaandco-workersreported thatthemorningserumtestosteroneconcentrationinobese patientswithsevereOSAwasassociatedwithincreased anx-iety/depressionandsleepefficiencydisturbances.5

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could lead toalterations in the production of cortisol.6---9

Testosterone and cortisol, major end products of the hypothalamic---pituitary---gonadal (HPG) axis and the HPA axis, respectively, are part of a biological balance that modulatespsychologicallyandphysicallyintegratedhuman responses.Theinterrelationshipbetweensexandstress hor-mones has been extensively investigated in the context ofsports medicineby measuringthe testosterone/cortisol (T/C) ratio as a marker of anabolic/catabolic activities connectedwithathleteperformanceandovertraining.10---11

Furthermore,theT/Cratiohasbeenproposedasahormonal markerofpsychopathologies,12,13_and_a_specific_association

betweenareductionoftheT/Cratioandischemicdisease hasalsobeenreported.14

The purposeofthisstudywastodeterminethediurnal fluctuation of salivary free-testosterone (T) and free-cortisol(C)andtostudytheirinterrelationshipbymeasuring diurnal variationsintheT/C ratioofpatients withsevere OSA. This analysis will expand upon previous studies that providedonlypartialdatafromsingle-pointhormonal mea-surements.

Interestingly,sexhormoneimbalanceisoftenassociated withdepressiveand anxiousmood disordersin men15 _and

bidirectionallyinteractswitherectilefunction.16_Therefore,

toassessthepresenceofcomorbidities,theHamilton Inven-toryQuestionnairesforDepression(HDS)andAnxiety(HAS) andtheInternationalIndexforErectileFunction(IIEF) ques-tionnairewereadministeredtothestudypopulation.

Materials

and

methods

Studypopulation

This prospective study was conducted over 6 months betweenMay 2011andDecember2012 intheSleep Labo-ratoryintheClinicofPulmonary Diseases(Iasi,Romania). ThestudywasformallyapprovedbythelocalEthics Commit-tee(Protocoln◦_14,_April_29th,_2011)._All_Caucasian_subjects

wererecruitedamongpatientsvisitingtheCentreforSleep Disturbancesandprovided theirwritten informedconsent beforethestartofthestudy.

We previously estimated that at least 14 subjects (7 per group) were required to detect a mean absolute dif-ferencecorrespondingtoa 50%variationontheexpected peakof T/C ratioin healthy subjects(40.00±20.00 arbi-trary unit) witha two-tailed ˛of 0.05 and 80% of power analyses.Thus,10patients(OSAgroup)wereselectedbased onthefollowinginclusioncriteria:male, 40---60yearsold, non-smoker,withbodymassindex(BMI)>30kg/m2_(obese)

and newly diagnosed with severe OSA based on noctur-nal polysomnography (PSG) evaluation (apnea-hypoapnea index, AHI≥30h−1₎ _and _excessive _daytime _sleepiness

(EpworthSleepinessScale,ESS≥10).17 Theexclusion

crite-ria included the following: acute or chronic associated diseases,smoking,useofanychronicmedicationand non-cooperative attitude. Patients with suspected OSA were hospitalized for 4 consecutive days. On the morning of admittance,bloodsampleswerecollectedfor biochemical-hematological parameter determinations, and pulmonary functionassessment (spirometry)and psychometric evalu-ationswiththeHamiltonRatingScaleforDepression,18_with

theHamiltonAnxietyRatingScaleandwiththeInternational Index Erectile Function questionnaire (IIEF)19 _were

per-formed.Abaselinerestingelectrocardiogram(ECG),heart rate(HR)(BTL-08USA)andsystolicanddiastolicblood pres-sure were recorded (M3 --- Omron, Japan). Conventional

sleep recordings (SOMNOlab V2.01, Weinmann, Germany) were obtained from 10:00 pm to 7:00 am (day 2) and AHI,arterial oxygensaturation (SpO2),desaturationindex

(ODI)andmicroarousalindex,werescoredapplyingstandard criteria.20_According_to_American_Academy_of_Sleep_Medicine

2007,theAHIwascalculatedasthemeannumberofapneas plushypopneasperhourofsleep;apneaswerescoredwhen theabsenceofairflowwaslastingatleast10sand hypop-neas whenthere wasat least a 30% drop in the oronasal airflow,withcoincidentoxygendesaturationofatleast4%, lastingatleast10s.7,20

Theparticipantswereinstructedonhowtocollectsaliva andaskedtoavoid food,coffeeandalcohol consumption, teethbrushingandanyphysicalexerciseforatleast30min beforeeachsalivacollection.21 _Thus,_on_the_day_after_(day

3),salivawascollecteduponawakening(between6:30hand 7:30h),atnoon(beforelunch)andintheeveningat19:00h beforedinner,for measuringthe dailycortisoland testos-teronefluctuation. The exacttimeof salivasampling was monitoredbyastaffmember.Amongthepatients hospital-izedwithsuspectedOSA,weselected7adultmalesubjects forthecontrolgroupwhohadthesamesomatic characteris-ticsoftheOSAsubjectsbutdidnotsufferfromsleepapnea (AHI<5h−1_,_ESS_<_10).

Salivarysamplingprocedure,testosteroneand cortisolassay

Saliva was collected using the Salivette (Sarstedt, Italy) samplingdevice,whichallowsforquickandhygienicsaliva recoverythroughcentrifugationat3000rpmfor15min.22

For each sample, duplicate measurements were per-formed for the direct assay of testosterone on 100␮L of saliva (inter-assay coefficient of variation was <10%, and intra-assay coefficient of variation <7% with a minimum detectable concentration of 3.5pg/mL at the 95% confi-dencelimitusingcommercialimmunoenzymatickitsandfor thedirect assayof cortisolon25␮Lof saliva (inter-assay coefficientofvariationwas<10%,andintra-assaycoefficient ofvariation<7%withaminimumdetectableconcentration of0.5ng/mLatthe95%confidencelimit(Diametra,Italy).

Dataanalysis

Alldatawerereportedasthe mean±SEM. The statistical

analyseswereperformed andthe graphicswereproduced using the SigmaPlot 11 software package (SxST.it, Italy). Normal distribution of the data was tested using the Kolmogorov---Smirnovtest.Whereappropriate,Student’st -test or the Mann---Whitney Utest was usedas parametric andnon-parametrictests,respectively,forthecomparisons betweengroups.

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Table1 Somatic,polysomnographic,andpsychometricvariablesinthestudypopulation.

Control(n=7) OSA(n=10) Statistics p-Value

Age(years) 51±3 53±3 T=58.000 0.66

BMI(kg/m2₎ _32.2_±_0.6 _32.3_±_0.7 _T₌_69.000 _0.591

Waistcircumference(cm) 105±2 109±2 t=−1.623 0.125

Polysomnographyparameters

AHI(h−1₎ _2.57_±_0.48 _63.5_±_9.3 _T₌_28.000 _<0.001

MinimumSpO2(%) 87.6±1.49 68.5±4.27 T=95.000 0.002

ODI(h-1) 4.86±0.83 58.4±9.35 T=28.000 <0.001

Sleepduration(min) 419.3±8.5 395.3±7.5 t=2.088 0.054

Sleepefficiency(%) 79.9±0.7 78.6±0.7 t=1.370 0.191

Microarousal(h-1) 9.9±0.94 46.7±6.31 T=28.000 <0.001

ESS 4.0±0.83 12.9±1.23 t=−4.910 <0.001

SystemicbloodpressureandHR

SBP(mmHg) 117±3 127±2 t=−2.744 <0.05

DBP(mmHg) 69±4 76±2 t=−1.657 0.118

Heartrate(beats/min) 67±1 75±2 T=48.000 0.156

Psychometricscores

HDS 5±1 10±0.54 t=−5.073 <0.001

HAS 2±1 5.3±1 T=38.000 <0.05

EF 27.9±0.7 16.0±0.9 T=98.000 <0.001

OF 9.0±0.2 6.9±0.3 T=97.000 <0.001

SD 9.1±0.1 6.9±0.2 T=98.000 <0.001

IS 14.0±0.2 6.5±0.2 t=18.190 <0.001

OS 10.0±0 8.5±0.2 T=98.000 <0.001

Dataareexpressedasthemean±SEM.BMI,bodymassindex;AHI,apnea-hypopneaindex;minimumSpO2,minimumoxygensaturation; ODI,oxygendesaturationindex;ESS,EpworthSleepinessScale;SBP,systolicbloodpressure;DBP,diastolicbloodpressure;HDS,Hamilton DepressionScore;HAS,HamiltonAnxietyScore;EF,erectilefunction,OF,orgasmicfunction;SD,sexualdesire;IS,intercoursesatisfaction; OS,overallsatisfaction.

T,C,and theT/C ratiomeasured in both groups at 7:00, 12:00,and19:00onthesamplingday.

Theareaunderthecurve(AUC)wascalculatedfromthe AUCdeterminedforeachsubjectbythetrapezoidalmethod usingthethreesalivarytestosteronevaluesmeasuredduring thesamplingday(morning,noon,andevening).23

Statisticalsignificancewassetatp<0.05.

Results

Characteristicsofthestudypopulation

As reportedin Table1, thetwogroups were matched for age,and there wereno significant differences in theBMI values;themeanwaistsizewassimilarforbothgroupsand washoweverabovethenormalrangeformen.

Theresultsofthepulmonaryfunctiontests(spirometry), forcedexpiratoryvolume(FEV),andvitalcapacity(FEV/VC ratio)were withinthe normal limits for subjects in both groups (data not reported). As shown in Table 1, all PSG parameterswerewithinnormalrangesinthecontrolgroup. Incontrast,theOSAgrouphadsignificantlyhigherAHIvalues andasignificantdecreaseintheminimumSpO2,whichisa

characteristicofsevereOSA.TheODIwasalsosignificantly increasedintheOSAgroup.Theshorterdurationofsleepin OSAsubjectsdoesnotreachstatisticalsignificanceandno changeshave been detectedin the sleepefficency. More-over, thenumber of microarousal events wassignificantly

higherintheOSAgroupthaninthecontrolgroup.In addi-tion,excessivedaytimesleepiness,asevaluatedbytheESS, wassignificantlyhigherintheOSAgroupthaninthecontrol group.

Table1alsoshowsthatthemeansystolicbloodpressure wasslightlyelevatedintheOSAgroupcomparedtothe con-trol group,with nochange in diastolic blood pressure. In addition, the OSA patients showed a tendency toward an increasedHR.

TheHamiltonInventoryScoresforDepression(HDS)and Anxiety(HAS)andtheIIEFresultsforthestudypopulation arealsoreportedinTable1.OSApatientshadsignificantly higherpsychometricscoresforanxietyanddepressionthan thecontrols.Thepsychometricscoresofthecontrolgroup indicatednosignsofdepressionoranxiety.Furthermore,the OSApatientsweresignificantlymorelikelytohaveerectile functiondisordersthanthecontrols.

Individualhematologicalandserumbiochemicalmarkers of glycemiaandlipids(totalcholesterol andtriglycerides) were within the normal ranges for both groups (data not shown).

Diurnaltrajectoriesofsalivaryfree-testosterone andsalivaryfree-cortisolinthestudypopulation

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160

120

80

40

0

8

6

4

0 2

Saliv

ar

y cor

tisol (ng/ml)

Saliv

ar

y testosterone (pg/ml)

Awak ening

(6.30-7.30am)

12pm 7pm

Day time

Control (n = 7) OSA (n = 10)

Figure 1 Diurnal trajectories of salivary free-testosterone and salivary free-cortisol in the study population. The data arepresentedasthemean±SEM.Statistical analysis:a two-wayANOVAfollowedbypost-hoctestformultiplecomparisons: * and**: p<0.05 andp<0.01, respectively, vs. thevalue at 07:00; †_, ††_, _and†††_:_p_<_0.05, _p_<_0.01, _and_p_<_0.001,

respec-tively,vs. the value at12:00;§ _and§§_:_p_<_0.05_and_p_<_0.01,

respectively,vs.thecontrol.

Atwo-wayANOVAanalysisshowedthattherewere signif-icantdifferencesinthesalivarytestosteroneconcentrations measuredatdifferenttimesoftheday(GROUP:F1,50=0.184,

p=0.670; TIME: F2,50=15.350, p<0.001; GROUP×TIME

interaction: F2,50=3.701, p=0.032). The post-hoc test for

multiplecomparisonsshowedthattheTestosterone concen-tration measured in the evening for the control group (91±4pg/mL)wassignificantlylowerthanthevalueinthe

morning(124±10pg/mL, p<0.05).Thesamediurnal fluc-tuation was detected in the OSA group; the Testosterone in theevening(54±5pg/mL) wassignificantlylower than

that in the morning (136±14pg/mL, p<0.001). In addi-tion, the Testosteroneconcentration in the OSA group at 19:00wassignificantlylower(p<0.01)thantheTestosterone concentration in the control group. Finally, no signifi-cant differencewas detected between the control group and the OSA group in the AUC computed from the diur-nal total Testosterone production (1240±53pg/mL/h and 1222±108pg/mL/h,respectively).

TheCconcentrationsinthecontrolandOSAgroupsare reportedinFig.1.Atwo-way ANOVAdemonstrated signif-icant differences between and within the subject groups (GROUP: F1,50=9.594, p=0.003; TIME: F2,50=33.152,

p<0.001; GROUP×TIME interaction: F2,50=3.753,

p=0.031).

50

40

30

20

10

0

T/C r

atio

Awak ening

(6.30-7.30am)

12pm 7pm

Day time

Control (n = 7) OSA (n = 10)

Figure2 Diurnaltrajectories oftheT/Cratiointhe study population.Thedataarepresentedasthemean±SEM. Statis-ticalanalysis:atwo-wayANOVAfollowedbypost-hoctestfor multiplecomparisons,*p<0.001vs.thevalueat7am;†_p_<_0.05

vs.thevalueat12pm;§_p_<_0.05_vs._the_{corresponding}_control.

In both the control and OSA groups, the salivary Cortisol concentration in the morning (7.2±0.6ng/mL

and4.6±1.0ng/mL, respectively)wassignificantlyhigher

than that in the evening (2.5±0.4ng/mL, p<0.001 and 2.1±0.1ng/mL, p<0.001, respectively). Furthermore, in themorning,theCortisolconcentrationintheOSAgroupwas significantlylowerthanthatinthecontrolgroup(p<0.05).

DiurnaltrajectoriesofsalivaryT/Cratiointhe studypopulation

Fig. 2 depicts the diurnal trajectories of the T/C ratio in the study population. A two-way ANOVA demonstrated significant differences in the T/C ratio measured dur-ing the sampling day (GROUP: F1,50=0.633, p=0.431;

TIME: F2,50=3.880, p=0.028; GROUP×TIME interaction:

F2,50=5.347, p=0.008). In the control group,there wasa

progressiveandstatisticallysignificant increaseintheT/C ratioduringthesamplingday(7:00=18±9;12:00=27±3;

19:00=43±5).Incontrast,therewerenosignificantdiurnal

fluctuationsintheT/CratiointheOSAgroup(7:00=28±3;

12:00=34±4;19:00=27±4).Additionally,theT/Cratioin

theOSAgroupwassignificantlyhigherthanthatinthe con-trolgroupin themorning(p<0.05)andsignificantlylower thanthatinthecontrolgroupintheevening(p<0.05).

Discussion

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disturbances.The majorfindingof thisstudy isthat male obeseOSA patientshaveareducedTconcentrationinthe eveningandareducedCconcentrationinthemorning com-paredwithnon-apneicobesecontrols. Furthermore,male obeseOSApatientshaveanimbalanceintheT/Cratio mea-suredinthemorningandintheevening.

Sexhormonesarebelieved tobeinvolvedinthe patho-genesis of OSA in adult male subjects who present with reducedlevelsof circulatingandrogens.5,16 _In_the _present

study,therewerenodifferencesbetweenthecontroland OSAgroupsinthefluctuationofthediurnalTtrajectory;the eveningTconcentrationwastypicallylowerthanthe morn-ingconcentrationsinbothexperimentalgroups.24,25_Salivary

testosteroneconcentrationlowerthan controlsalso trans-latesin an increase incomorbidities (fatigue, depression, general reduction of the well-being sense, sleep distur-bances,etc.)16_affecting_our_OSA_subjects._Since_BMI_above

thenormalrangewaspresentinbothexperimentalgroups, OSA itself, rather than obesity, seems to be the cause of the drop of salivary testosterone concentration in the presentstudy.Ithasbeen suggestedthatthemorningrise intestosterone productionis derived fromanendogenous testosterone circadian rhythm and is not directly related toanyofthesleepstages.26 _However,_the_OSA_patients_in

thisstudy experienceda decreasein thelead tostressful modificationsofthesleeppatternswithadecreasedsleep efficiency.26

Inthisstudy,wedemonstratedthatOSAsubjects main-tainphysiologicalcircadianactivityintheHPAaxis,withthe highestcortisolconcentrations presentinthemorningand thelowestpresentintheevening.Themaintenanceof reg-ularcircadian cortisolproductionin OSAsubjects hasalso beenreportedinapreviousstudy27_;_however,_these_authors

didnotdetectanydifferencebetweentheOSApatientsand controlsinthemorningcortisolconcentration.This discord-ancemayberelatedtothefactthatinthepresent study is detected in the saliva only the ‘‘bioactive’’ free frac-tion,whichcorrespondstotheunboundfreeplasmafraction of thehormone.7 _Indeed, _morning_{hypocortisolism} _in _OSA

patientshas been recentlyconfirmed.8 _The _highest _T_and

Cconcentrations occurat thebeginning ofthe day, while thelowestlevelsoccurintheevening.25,28_In_our_study,_we

useda three-pointdiurnal sampling schedule for thefirst timeandwereabletodetectagradualincreaseintheT/C ratiothroughouttheday.Achangeinthebalancebetween thetwosteroidhormonesmayputanindividualatriskfor abreakdowninhomeostasis.Inthepresentstudy,theOSA subjectshadahighermorningT/Cratiothanthecontrols; however, in the evening, the T/C ratio was significantly lowerintheOSAsubjectscomparedwiththecontrolgroup. Thisimbalanceintheanabolic-catabolicequilibriumcould beattributedtothe morninghypocortisolismthat charac-terizessevere OSA7,8_;_the _resultant _flattened _response _to

chronicallyrepeatednocturnalchallengesreflectsa dysreg-ulationoftheHPAaxis.29_In_contrast,_the_decrease_in_the_T/C

ratiointheOSAgroupintheeveningcouldbearesultofthe sharpandmarkeddropintheTconcentrationat19:00.

Obesity in men is associated with reduced androgen secretion.30 _However,_in_the_present_study,_obesity_was_not

considered a confounding factor in the T concentration reductionbecauseobesity occurredin bothgroups. Treat-mentof moderatetosevere OSA withcontinuouspositive

airway pressure (CPAP) does not reliably increase testos-terone levels in most studies. In contrast, a reduction in weightdoessopredictablyandlinearlyinproportiontothe amountofweightlost.16

Compared with the control group, the OSA patients demonstrated either a mild anxious-depressive disorder or excessive daytime sleepiness. Recently, a relationship between a depressive mood and obesity wasdescribed.31

However,thecontributionoftheT/Cratioimbalancetothe development of anxious-depressive symptoms in patients withsevere OSAandthekeyrolethatobesityplaysinthe pathogenesisofOSAcomorbiditiesneedstobefurther stud-ied.

OSA is a recognized cause of sexual disturbances in men.16,32,33 _In _this _study,_we _confirmed _that _OSA _patients

sufferfrommildtomoderatesexualdysfunctionasassessed bytheIIEFquestionnaire.19

Conclusions

ThemainfindingofthisstudyisthatmaleobeseOSApatients havesignificantchangesintheTandCdiurnaltrajectoriesin comparisonwithnon-apneicobesecontrols.Hypocortisolism was particularly pronounced in the morning. Conversely, the Tconcentrationwaslowest in theevening. Moreover, althoughOSApatientsmaintainedthephysiologicaldiurnal T/C ratiofluctuation, they exhibitedan imbalance in the diurnalT/Cratiotrajectory.

Thisstudy improved onother previous studies byusing salivaryhormonemeasurementstodetectthefreefraction ofsteroidhormonesintheplasma34_and_by_employing_a

tar-getedprotocol.Inparticular,theuseofdatafrommultiple diurnal measurements rather than a single point permit-ted the detection of T/C ratio changes of opposite signs at the beginningand end of theday, allowing for a more accuratestudyoftheHPGandHPAaxes.Finally,itislikely thatinOSAsubjects,poorsleepquality,tiredness,and psy-chological distress areamong the comorbidities affecting theresponsescrucialtothemaintenanceofthe physiolog-icalanabolic-catabolichormonalbalance.However,wedid notestablish a causalrelationship between thesefactors. In fact, a limitationof this study is that we only demon-stratedtheco-existenceofimbalancedTandCproduction andOSA comorbidities.To fullyaddress thispossible rela-tionship,furtherresearch,withsamplesizesadequatefora correlativestudydesign,isnecessary.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

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ScientificBilateralAgreementbetweenSapienzaand Grig-oreT.PopaUniversities.

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