1

Localsynthesisofsexhormones:

Arethereconsequencesfortheocularsurfaceanddryeye?

Intracrinologyandtheocularsurface

EmmaJ.Gibson(BScOptomHons)ᶦ,FionaStapleton(PhD)ᶦ,JamesS.Wolffsohn(PhD)²,

BlankaGolebiowski(PhD)ᶦ

ᶦSchoolofOptometryandVisionScience,UNSW,Sydney,NSW2052,Australia.

²OphthalmicResearchGroup,SchoolofLifeandHealthSciences,AstonUniversity,

Birmingham,UKB47ET

ABSTRACT

Sexhormonesareassociatedwiththephysiologyandpathophysiologyofalmostall

organsinbody,aswellasmostdiseases.Interestintheassociationsbetweensex

hormonesandoculartissueshasincreasedinrecentyears.Androgensmayhavea

positiveeffectondryeye,whereastheeffectsofestrogenonocularconditionsremain

unclear.Intracrinology,thelocalsynthesisandmetabolismofhormoneswhichis

uniquetohumans,isofrelevancetotheeyeandmayhelptoexplainwhystudiesofthe

relationshipbetweenestrogensanddryeyesignsandsymptomsareinconclusive.

Knowledgeofthepathwaysofhormoneformationandmetabolismiscrucialto

understandingthepathogenesisofoculardiseaseincludingdryeye.Thisreview

examinesthemechanismsofsteroidalsexhormonebiosynthesisandreviewsthe

significanceoflocallyproducedsexhormones,withafocusonocularsurfacetissues.

Muchofthecurrentliteratureisbasedonanimalstudies,whichmaynotbetransferable

tohumansduetotheabsenceofintracrineproductioninanimals.Alargeproportionof

thehumanstudiesinvestigatesystemichormoneslevelsratherthanlocallevels.There

issubsequentlyaneedforadditionalstudiestoprovideabetterunderstandingofthe

localproductionofsexhormoneswithinthehumaneyeandocularsurfaceandto

clarifytherelationshipsbetweenocularlevelsofsexhormonesandconditionsincluding

dryeye.

KEYWORDSintracrinology,androgens,estrogens,dryeyedisease,MGD,ocularsurface

Abbreviationsareprintedinboldfacewheretheyfirstappearwiththeirdefinitions.

2Literaturereview:methods

Areviewwasconductedtoassesswhetherthelocalsynthesisofsexhormoneshas

consequencesforthephysiologyandfunctionoftheocularsurfaceandasubsequent

impactondryeyedisease.Relevantarticleswereidentified,publishedtoJuly2017,

throughsearchesinPubMed,ScopusandMedlinedatabasesaswellasthroughthe

referencelistsofidentifiedpublications.Searcheswereperformedformeeting

abstractsfortheAmericanAcademyofOptometry(AAO),theAmericanAcademyof

Ophthalmology(AAO),theTearFilm,andOcularSurfaceSociety(TFOS)andthe

AssociationforResearchinVisionandOphthalmology(ARVO).Patentsearcheswere

alsocarriedout.

Searchtermsincluded:sexhormones,sexsteroids,intracrinology,estradiol,estrogen,

testosterone,androgenandsteroidogenesis.Additionalsearchtermsforeachsection

include:SectionII:endocrinology;III[A]meibomiangland,lacrimalgland,corneaand

conjunctiva,receptors,mRNA,generegulation,[B]massspectrometry,tears,meibum,

blood,human,[C]ocularimpact,hormonereplacementtherapy,oralcontraception,

estrogentherapy,androgentherapy,treatment,topical,dryeye,keratoconjunctivitissicca,

MGD,symptoms,TBUT,Schirmer,function;[B].

3I. INTRODUCTION:SEXANDDRYEYE

Epidemiology

Approximately3.2millionwomenand1.68millionmenintheUnitedStatessufferfrom

severedryeyesymptomsorclinicallydiagnoseddryeye,withmillionsmore

experiencinglessintensesymptoms.[1,2]Largeepidemiologicalstudieshaveidentified

femalesexandolderageasriskfactorsforthedevelopmentofdryeye.[1–10]Therisk

ofexperiencingdryeyeappearstoincreaseovertwotimeswithmenopause.[11]

Pathophysiology

Dryeyeoccurswhenthetearfilmiscompromised,eitherbyreducedaqueous

productionorincreasedevaporation:tearqualityandstabilityisthusimpaired.The

pathogenesisofdryeyeiscomplexandasyetnotcompletelyunderstood.Itsaetiology

ismultifactorial;inadditiontoinflammatoryprocessesandneuralfeedback

mechanisms,thereisalsostrongevidenceforahormone‐mediatedcontribution.[9,12]

Treatmentofdryeye

Medications,suchasanti‐inflammatoryandanti‐bioticagents,canbeusedfortreating

processesinvolvedindryeye,includingreducingthepresenceofinflammatory

mediatorsorpathogensontheocularsurface.[13]However,manytraditional

treatmentsfordryeyediseaseareonlypalliative,includingartificiallubricantsor

retentionplugs,aimedatincreasingthevolumeoftearsontheocularsurface.A

treatmentdirectedattheunderlyingcausehasthepotentialtoprovideeffectivereliefto

themillionsofpeopleworldwidewhosufferfromdryeyedisease;thisisnotcurrently

possibleduetothecurrentuncertaintyofthepathophysiologyofdryeye.

Sex‐relateddifferences

Sex‐relateddifferencesarefoundinalmosteverycellandtissueinthebody,thusitis

rationaltoexpectthatsex‐differencesalsooccurinoculartissues.[14]Vernal

keratoconjunctivitis(VKC)isawell‐documentedexampleofanocularsurfacedisease

whichshowssignificantsex‐relatedprevalence,withoverthreequartersofpatients

beingmale.[15]VKCalsoshowsatendencytoresolvearoundpuberty,inbothmales

andfemales,whichsuggestsahormonaleffectonVKC.[15]Inaddition,thenumberof

estrogenandprogesteronereceptorsintheepitheliumandsubepitheliumofthetarsal

4andbulbarconjunctivahasbeenshowntoincreasewithdiseaseduration,which

suggestsapossiblerelationshipbetweenthesehormonesandVKC.[16]

Thisreviewexaminesthemechanismsofsteroidalsexhormonebiosynthesisand

considerstheimplicationsoflocalhormoneproductiononinvestigationsofhormone

effectsondryeye.

II. SEXHORMONES

Sexhormones,includingandrogensandestrogens,aresteroidsresponsibleforsomeof

themostprofoundchangeswhichoccurtothebody.Sexhormonesareassociatedwith

thephysiologyandpathophysiologyofalmostallorgans,aswellasmostdiseases.

Figure1showsthesteroidogenicpathways.

Sexhormonebiosynthesis

1. Synthesisofcirculatingsexhormones

Androgensandestrogensarebiologicallyactiveinbothsexesanditiserroneousto

associateestrogenswithfemaleandandrogensexclusivelywithmalesex.Inmenthe

testesprovideanearcontinuoussupplyofandrogensandsmallamountsofestrogens

throughamale’slifetime.[17]Inwomentheovariessecreteestrogens,progesterone

andandrogens.Howeveratmenopauseovariansecretionofestrogenandprogesterone

stops.[18]Womenundergoaslowdeclineintestosteronewithage,whichdoesn’t

appeartobeassociatedwiththefinalmenstrualperiod.[19]

Inadultmen,bloodtestosteronelevelsare100‐150xthelevelofestradiol.[20]Inpre‐

menopausaladultwomen,duringtheearlyfollicularphaseofthemenstrualcycle

(whenestrogenandprogesteronelevelsarelow),circulatingtestosteronelevelsare2‐

3xthatofestradiol,withtheratioincreasingpost‐menopause(>5years)to80timesthe

levelofestradiol.[21]

2. Intracrinology:Synthesisofsexhormonesinperipheraltissue

Aswellasbeingsecretedbythegonadsandadrenals,sexhormonesareproducedin

peripheraltissuesthroughoutthebodyfromcirculatingprecursorsofadrenalor

gonadalorigin.[22]Intracrinologywaspioneeredasanewbranchofendocrinologyby

FerdinandLabrie,inthelate1980’s,describingthemechanismsofthesynthesisof

5activehormonesinperipheraltissuesfromDHEAanditssulfateDHEA‐S(seeFIG1).

Duringintracrinology,hormonesexerttheiractionwithinthesamecellsinwhichthey

aresynthesizedandinwhichtheyaremetabolizedbeforebeingreleasedinto

circulationasinactivecompounds.[22]Theintracellularinactivationofsexhormones

priortoreleaseintoextracellularspaceprotectsneighbouringtissuesfromtheactionof

sexhormonethusavoidingpossibleadverseeffectsoftheirsystemiccirculation.[23]

Akeydifferencebetweenendocrinologyandintracrinologyisthatendocrineorgans,

suchastheovaries,dispersesynthesizedsexhormonesviageneralcirculationtoall

bodilytissues.Conversely,intracrinologyallowsindividualtissuestosynthesizethe

requiredamountofestrogensandandrogenswithoutreleasingsignificantamountsof

biologicallyactivehormonesintocirculation.[24,25]Astheonlyanimalspecieswith

adrenalsthatsecretelargeamountsofDHEAanddehydroepiandrosteronesulfate

(DHEA‐S),humansandotherprimatesareuniqueintheirabilitytoproducehormones

locally.[23]DHEAandDHEA‐Sarereceivedbythelocalcellsfromcirculationthen

convertedintoandrostenedione(4‐dione)andsubsequentlyintoandrogensand

estrogens(FIG1)[26]atlevelsrequiredbythespecificcells.[24]Thereisanabundance

ofDHEAinserum,withserumlevelsofDHEAbeingabout10timeshigherthan

testosteronelevels,and500timeshigherthanestradiollevels.[27]

Inhumans,DHEAlevelsincirculationreachmaximumlevelsbetweenage20‐30years,

decreasingby80%bytheageof70inmalesandfemales.[28],[29]Thisgreatreduction

inadrenalsecretionofDHEAandDHEA‐Sresultsinalargefallintheformationof

androgensandestrogensinperipheraltissueswithage.[30]Iftearproducingtissues

arepresumedtoproducesexhormoneslocallybyintracrinology,thisdropinDHEAand

DHEA‐Scouldbeacontributingfactortotheincreaseofdryeyewithage.[3]

CelecdescribesDHEAasa“humanmolecule”becauselevelsinhumansaresomuch

higherthaninanimalspecies.[31]Theadrenalsofratsandotheranimalsdonotsecrete

significantamountsofDHEArequiredforsynthesisofsexhormonesby

intracrinology.[32]Consequentlyrodents,whosecretesexsteroidssolelyfromthe

gonads,areverydifferenttohumans.Henceasanimalslacktheabilitytomake

hormoneslocally,cautionshouldbetakenwhenapplyingrodent/animalmodelsto

humanmodels.Asaresult,thisreviewfocussesonhumanstudies.

6III. SIGNIFICANCEOFINTRACRINOLOGY

MeasuringtestosteroneandDHTconcentrationsinserumindicatestesticularfunction,

inmen,orovarianfunction,inwomen,anddoesnotincludelocalintracrineproduction.

Almostallandrogensinwomenaremadeinperipheraltissues.Thetotalandrogenpool

inbothmenandwomencanbebetterestimatedbytheserumconcentrationsof

androgenmetabolites:androsteroneglucuronide(ADT‐G),3α‐diol‐Gand3β‐diol‐G.[33]

Inmenitisestimatedthat30‐50%oftotalandrogensaresynthesizedlocallyfrom

inactiveadrenalprecursors.[34]Usingthesumoftheseandrogenmetabolitesinblood,

androgenproductioninpost‐menopausalwomenhasbeencalculatedtobeovertwo

thirdsofthatofmen,muchhigherthanpreviouslythought,[35]yettheirserum

testosteroneconcentrationisonly3%thatofmen(15ng/dLinwomen,461ng/dLin

men).[20,21]Therefore,measuringmetabolitelevelsratherthanlevelsofactive

androgensprovidesamoreaccurateindicationoftheamountofactiveandrogens

withinperipheraltissues.

Estrogensproducedbyintracrineprocessesarealsoimportant.Labrieestimatesthat

pre‐menopause,75%ofestrogensaremadeinperipheraltissuesbyintracrinology,

withthisincreasingto100%aftermenopause(asdepictedinFigure2).[22,26,33]

Althoughthepercentageofestrogensbeingproducedbyintracrinologyincreasespost‐

menopause,thetotalestrogenproducedislowercomparedwithpre‐menopausewhere

therearebothendocrineandintracrinesources.Toachieveabetterestimationofthe

amountofestrogensinthehumanbodymanypublishedstudiesmeasureestrogen

metabolites.Theseinclude,butarenotlimitedto,hydroxylatedmetabolitessuchas2‐

hydroxyestrone,2‐hydroxyestradiol,16α‐hydroxyestroneandestriol[36]orsulphated

metabolites.[37]

Intracrinesynthesisofsexhormonesisvitalformaintainingnormalfunctionin

humans.PeripheraltissuesrequireaccesstohighcirculatinglevelsofDHEAandto

intracrineenzymes,includingaromatase,5α‐reductase,17β‐HSDand3β‐HSD,toenable

biosynthesisofestrogensand/orandrogens.[33,38]Allhumantissues,exceptthe

endometrium,possesstheseenzymes(reviewedbyLabrie)[24]thusintracrinology

allowscirculatinglevelsofestrogenstoremainsubthresholdpost‐menopause.This

avoidsstimulationoftheendometrium,whilstallowingtissuestocreatetheirown

estrogensasneeded.

7

SexhormonesandtheocularsurfaceHumanocularsurfacetissues,likeotherperipheraltissues,arethoughttomakesex

hormonesatlevelsrequiredbyeachtissue,byintracrineprocesses.[22]Itisproposed

thatcirculatingsexhormonesandmetabolitesaresuppliedto,andremovedfrom,the

ocularsurfaceinasimilarwaytoothernutrients:viadiffusionintoandfromthetear

fluid,andbloodvesselsintheconjunctivaandotheroculartissues.During

intracrinology,thesesexhormonesareusedwithinthecellsinwhichtheyare

synthesised,withminimalreleaseofactivehormoneoutofthetissue.Themetabolites

arethenreleasedintocirculationandmayalsobereleasedintothetears.

Table 1: Summary of intracrine machinery identified in human ocular surface tissue. 

1. SteroidogenicenzymesinhumanocularsurfacetissuesThetransformationofadrenalprecursorsteroids,DHEAandDHEA‐S,intoandrogensor

estrogensreliesontheexpressionofsteroidogenicenzymesinperipheraltissues.[30]

Steroidsulfatase,aromatase,glucuronosyltransferase,3β‐HSD‐∆‐5∆4‐isomerasetype1,

17β‐HSDtypes1and3,and5α‐reductasetypes1and2areallrequiredforthe

conversionofDHEA‐Stoandrogensandestrogens,andthenformetabolismintotheir

inactiveglucoronateandsulfatedforms(FIG1).[24]mRNAsforalloftheabove

Intracrinemachinery Cornea Conjunctiva Meibomiangland Lacrimalgland

Receptor

AR +([39],[40]) +([39]) +([39,41,42],) +([39])

ER +([40]) +([43]) +([41,44]) +([45])

PR +([40]) +([43]) +([41,44]) +([45])

ARmRNA +([46],[40]) +([46]) +([46]) +([46])

ERmRNA +([46],[40]) +([46],[43],[47]) +([46]) +([46],[47])

PRmRNA +([46],[40]) +([46],[43]) +([46]) +([46])

EnzymemRNA

5α‐reductase +([39]) +([39]) +([39]) +([39])

steroidsulfatase

3β‐HSD(Type1)

17B‐HSD(Type1+3)

aromatase

glucuronosyltransferase

+([48]) +([48]) +([48]) +([48])

+referstothefindingoftheintracrinemachineryintheoculartissue.

Abbreviations:AR:androgenreceptors;ER:estrogenreceptors;PR:progesteronereceptors;LG:lacrimal

gland;MG:meibomiangland;mRNA:messengerRNA,HSD:hydroxysteroiddehydrogenase

8steroidogenicenzymeshavebeenfoundinhumanlacrimalglandsandmeibomian

glands,aswellasincornealandconjunctivalepithelialcells[39,48](Table2).This

suggeststhattearproducingtissuesareabletoproducesexhormonesby

intracrinology.

2. Possiblelocalactionofsexhormonesproducedbyintracrineprocesses

SexsteroidreceptormRNAshavebeenfoundinhumanocularsurfacetissues,as

summarisedinTable1.[43,46,47,49]IfthesemRNAsaretranslatedintoproteinsforsex

steroidreceptors,ocularsurfacetissuesmaybetargetsitesforsexhormonesandthus

maybesubjecttothelocalactionofthesesexhormones.Thissupportsintracrinology

wheresexhormonesproducedbyintracrineprocessesexerttheiractionlocallywithin

thesamecellsinwhichtheywereformed,beforebeingmetabolisedlocally.[50]

ThepresenceofmRNAaloneforAR/ER/PRdoesnotprovethatthesemRNAsare

translated.However,AR,ERandPRproteinhavebeenfoundintheepithelialcellnuclei

ofhumanmeibomianglands,lacrimalglands,cornea,fornicealandbulbarconjunctiva

whichimpliestranslation.[39–45]Thepresenceofandrogen/estrogen/progesterone

receptorproteinsuggeststhesereceptorsmayfacilitatesexhormoneinfluenceonthe

function,structureandpathologyofthetearproducingtissues.[39]However,no

associationhasbeenreportedbetweenthenumberofestrogenreceptorsinmeibomian

glandbasalcellsanddryeyesymptoms,tearquality(TearBreakUpTime[TBUT]),or

tearvolume(Schirmerscore).[42]

3. Generegulationofocularsurfacetissuesbysexhormones

Toestablishwhethersexsteroidshavesex‐specificorantagonisticeffects,studieshave

examinedtheinfluenceofsexhormonesongeneexpressioninepithelialcellsofhuman

meibomianglandsandconjunctiva,andmicelacrimalglandsandmeibomianglands.

Sexhormoneeffectongeneexpressionisnotlimitedtosexhormonesproducedby

intracrineprocesses,thereforeanimalstudiesareusedtosupportthetwopublished

humanstudiesdiscussedinthissection.

Sexappearstohaveasignificantinfluenceongeneexpressioninocularsurfacetissues,

affectinggenesresponsibleforabroadrangeofprocesses.[51–53]However,the

influenceofsexongeneexpressionseemstobetissue‐specific,withthemajorityofsex

relateddifferencesingeneexpressioninthemousemeibomianglandbeingdifferentto

9thoseinthemouselacrimalgland.[54]Themosthighlyexpressedgenesinhuman

meibomianglandswereuniquetothoseinthehumanconjunctivalepithelia,thusgene

expressionappearstobetissue‐specificinhumansaswellasmice.[52]

Treatmentofimmortalisedhumanmeibomianglandepithelialcells(iHMGEC)and

conjunctivalepithelialcells(iHCEC)inserum‐freemediumwithDHTwasfoundto

influenceexpressionofapproximately3000genes.[53]iniHCECDHTenhanced

expressionofgenesinvolvedinthedevelopmentoftheepithelium,woundhealingand

regeneration,whilstsuppressinggenesrelatedtoimmuneresponseandmitoticcell

cycle.[53]Androgentreatmentinthemeibomianglandincreasedexpressionofgenes

forlipogenesisandsuppressedgenesforkeratinisation.[53]Somegeneswereup/down

regulatedinbothtissues,howeverasdescribedabove,mostgeneregulationwasunique

tothemeibomianorconjunctivalepitheliumcells.[53]ThesefindingsbyKhandelwalet

alsuggestthatandrogensappeartobebeneficialtothephysiologyofmeibomianglands

andconjunctiva.

Theonlystudytolookattheeffectofbothestrogensandandrogensinvitroinhuman

ocularsurfacetissues(sexunknown)isbySchroderetal.52Thisstudyused1nM17β‐

Estradioland10nMDHTtomodulategeneexpressionofmeibomianglanddysfunction

(MGD)associatedmarkersiniHMGEC.[41]iHMGECculturedinserum‐freemedium

werecomparedtothoseculturedinserum‐containingmedium.BothestradiolandDHT

increasedgeneexpressionforkeratinisationinserum‐freemediumand,interestingly,

DHTalsodownregulatedgeneexpressionoflipidsynthesisenzymes.[41]These

findingscontrastwiththepositiveeffectsofandrogensoniHMGEC,foundby

Khandelwal58andinmicestudies.[55–57]Schroderetal52alsofoundestradiolto

increasegenesforproliferationofiHMGEC,whichdisagreeswiththefindingsofa

preliminarystudywhichfoundestradioltodecreaseproliferation.[58]The

contradictoryresultscouldbeexplainedbydifferencesinmethodologybetween

researchgroups,including,butnotlimitedto,theuseofserumcontainingmedium,

hormoneconcentrationsstudiedandtheuseofimmortalizedcelllines.Aswithtopical

orsystemictreatmentinvivo,theeffectofestrogenonhumanoculartissuesremains

unclear.Theeffectofsexhormonesongeneexpressioninhumanoculartissuesis

complex.Withonlytwopublishedstudiesinvestigatingtheseeffectsitisnotyet

possibletoformfirmconclusions.

10Testosteronesignificantlyalteredtheexpressionofthousandsofgenesinthelacrimal

andmeibomianglandsofmice.[57]Interestingly,androgensimpactedexpressionof

genesinvolvedinlipidmetabolismandinhibitionofkeratinizationinthemouse

meibomiangland.[57]ThisagreeswiththestudyofiHMGECbyKhandelwaletal.[53]

Manyofthebiologicalandfunctionaleffectsofandrogenswerethesameinbothmales

andfemales.[57,59]Estrogenandprogesteronealsoinfluencedexpressionofnumerous

genesinthemouselacrimalgland[59]andmeibomiangland.[60]However,their

effectstendedtobeuniquetothoseofandrogensandthenumberofcommongeneswas

limited:estradiol,progesteroneoracombinationofbothsexsteroidssignificantly

influencedlessthan7%ofgenescontrolledbyandrogens.[57]Althoughsexhormones

impactedexpressionofthousandsofgenesinthelacrimalandmeibomianglandsof

mice,testosteroneandestrogenappeartohavedifferenteffects.Testosteroneappears

topromotemeibomianglandfunction,whichagreeswithstudiesofandrogentreatment

(seereviewbyTruongetal).[61]

Theabsenceofestrogen,asaresultofaromataseelimination,wasassociatedwith

up/downregulationofthousandsofgenesinthemousemeibomianglandandlacrimal

gland.[62,63]Morethan90%ofthesearomatase‐linkedgenesweresex‐specificwhich

couldexplainthesexrelateddifferencesofthemousemeibomianandlacrimal

glands.[62,63]Therewasnoeffectontearvolumeinfemales,howeverasignificant

increaseintearvolumeinmalemicewasobserved[62,63]whichsuggestssexspecific

influences.Thusitappearsthatestrogenandaromataseplayanimportantpartingene

regulationinmicemeibomianandlacrimalglands.[62,63]Howeverthiseffectappears

tobesexspecific.

Insummary,thereareveryfewpublishedhumanstudiesoftheeffectsexhormones

haveongeneregulationintearproducingtissues.Micestudiessuggesttestosteroneand

estradiolinfluenceexpressionofthousandsofgenesinthelacrimalglandand

meibomianglandsandthatestradiolhascontrastingeffectstotestosterone’s

stimulatoryeffectofmeibomianglands.Tounderstandthetruenatureoftheroleofsex

hormonesonhumanocularsurfacetissues,furtherstudiesarerequired.

Measurementofsexhormonelevelsattheocularsurface

Manystudiesexamininghormonesareperformedusinganimals;whilstthesehavetheir

logisticalbenefits,includinghavinglargersamplesizesavailableandabilitytocontrol

11environmentalconditions,theoutcomesmaynotbegeneralizabletohumans.Lower

animalspecies,suchasratsandrabbits,aredifferenttohumanssincetheylack

intracrinesynthesisofhormones.[23]Therearemanyothervariableswhichinfluence

theeffectsofhormones,includingspecies,sex,age,experimentalprocedureandstrain

ofhormoneused.[64]

Onedifficultyofinvestigatinglocalsexhormonesinoculartissuesistherequirement

forhumantissue,whichisnotfeasibleinlargescalestudies.Therearestudieswhich

usehumanoculartissuefollowingsurgicalremoval,butthesearerareandsmall

scale.[46]Anotheroptionistomeasuresexhormonesinsamplessuchastears,meibum

orthroughconjunctivalimpressioncytology,which(althoughthisapproachmayhave

otherlimitations)maybeharvestednon‐surgically.

Sexhormonemetabolitesmayenterthetear‐filmasaconstituentoflacrimalgland

secretionsorwithinmeibum,asapossibleresultoftheholocrinenatureofmeibomian

glands.[65,66]Thisspeculationissupportedbythefindingof17β‐Estradioland

progesteroneinhumantears(usingimmunoassaykits,inapreliminarystudy)at

concentrationswhichcorrelatedsignificantlywithserumlevels.[67]

Duetolowconcentrationsofhormonesandthesmallsamplevolumesavailablefrom

collectionoftears,meibumorimpressioncytology,highlysensitive,reliableand

repeatablemethodsarerequired.Liquidchromatographywithtandemmass

spectrometry(LC‐MS/MS)isonesuchmethodandstudiesutilizinganddeveloping

thesemethodshavebeguntobepublished,[68]settingthestandardforfuturestudies.

Theyhavetheadvantageofimprovedreproducibility,accuracyandsensitivityin

comparisontoimmunoassays.[69–71]Toourknowledge,massspectrometrymethods

tomeasuresexhormonesinhumantearsormeibumhavenotbeenpublishedtodate.A

preliminarystudymanagedtosuccessfullydetectpregnenolone,progesterone,DHEA,

androstenedioneandtestosterone,aswellascorticosteroidmetabolites,inhumantears

usingLC‐MS/MS.[72]LC‐MSandGaschromatography–MS(GC‐MS)methodswere

developedtodetect9steroids(testosterone,androstenedione,cortisol,DHEA‐S,DHEA,

cortisone,β‐estradiol,progesteroneandandrost‐5‐ene‐3,17‐diol)andserotoninin

humantears,howeverlevelsweretoolowtoreachtherequiredsensitivity.[73]New

instrumentation,withincreasedsensitivity,providesthepossibilityforfuture

successfulmassspectrometrymeasurementofsexhormonesinhumantears.

12

Sexhormonetreatmentanddryeye.

Duetothedifficultiesofmeasuringlocallevelsofsexhormones,asdiscussedinsection

IIIB,therelationshipbetweenintracrinesexhormonesandclinicalsignsorsymptoms

hasnotpreviouslybeeninvestigated.Truongetalexaminedevidencefortheroleof

systemicsexhormonesintheaetiologyofdryeyeandtheeffectofsystemicsex

hormonesonocularfunction.[61]Theyconcludedthatandrogensmayhaveabeneficial

impactonthelacrimalglandsandmeibomianglands,andthatandrogendeficiencyisa

contributingfactortodryeyeaetiology.Theysuggestedthattheimpactofestrogenis

moreuncertain,withstudiesfindingcontradictoryeffectsofexogenousestrogenon

ocularstructures,shownbytheunresolvedrelationshipbetweenHRTanddryeyesigns

andsymptoms.[61]

Itisimportanttonotethatalthoughstudiesthusfarhavelookedatassociations

betweenserumsexhormonesanddryeye,serumsexhormonesrepresentonlyasmall

portionofsexhormonesmadeinthebody,sinceallestrogensandmostandrogensare

producedandmetabolizedlocallyinperipheraltissuespost‐menopause.[22,26,30]

Topicaltreatmentwithsexhormonesmaybeaviablerouteofadministrationforlocal

treatmentofdryeye.Studieshaveusedtwodifferentroutesoftopicaladministration:

creamappliedtotheeyelidsoreyedrops,summarizedinTable2.

13

Table 2: Summary of the effects of topical androgens and estrogens on symptoms and signs of dry eye. Topical treatment was in the form of eye drops or cream applied to the eyelids.  

Author  No subjects Treatment Dosage Duration Placebo  Effect on Dry eye

Androgens  WO Patent No 1994004155 A1, 1994 

[74] 

F=1  1.5% DHEA drops / 3 weeks N  Improved symptoms, But and lipid layer, reduced 

Schirmer’s 

Worda et al 2001* [75] M=1  3% Testos cream t.i.d. 3 months N  Improved symptoms and lipid layer, restored tear film 

Connor, et al 2001 [Conference][76]  F=1, M=9 1% DHEA drops q.i.d. 2 weeks Y  Improved symptoms, Schirmer’s and TBUT

Connor 2002 [Conference] [77] F=15, M=5 2.5% Testos cream b.i.d. 3 weeks Y  Improved symptoms and Schirmer’s

Connor , et al 2002[Conference] [78]  F=9, M=11 1% Testos, 

1% DHEA drops 

q.i.d. 2 weeks Y  DHEA improved BUT & Schirmer’s

Testos decreased Schirmer’s 

US Patent No 6659985 B2, 2003[79]  F=4, M=1 2.5% DHEA cream t.i.d. 2 weeks N  Improved symptoms & CL WT.  Improved  Schirmer’s 

and TBUT in male subject only  

F=2  2.5% Testos cream b.i.d. 2 weeks N  Improved symptoms , CL WT, Schirmer’s and BUT  

Connor 2003 [Conference][80] F=25, M=3 3% Testos cream b.i.d. 2 weeks Y  Improved symptoms and Schirmer’s

Schiffman 2006[Conference] [81]  179  0.01%, 0.03%, 

0.1% Testos 

/ 6 months Y  Improved MG secretions

Connor 2007[Conference] [82]   F=40, M=10 5% Testos, 

20% Prog cream 

b.i.d. / N  Improved symptoms and TBUT

Connor 2012[Conference][83] F=30  5% Testos cream b.i.d. 1 month N  Improved TBUT

Estrogens 

Lubkin 1992[Conference][84] F=14  E2 drops. 

unknown%  

q.i.d. 2 months Other eye Improved symptoms

US Patent No 6096733, 1998 [85]  F=45  0.1%, 0.25% E2 

drops 

q.i.d. 90 days Y  Improved symptoms and TBUT

F  0.05% E2 drops t.i.d. 10 days Other eye Maturation conjunctival epithelium 

Akramanian et al * 1997 & 1998 [86,87]  F=20  3% E2 ointment / 1 week Y  Improved symptoms, Schirmer’s and TBUT

Sator et al* 1998[88] F=84  0.025% E2 drops q.i.d. 4 months Y  Improved symptoms and Schirmer’s

Abbreviations: *indicates published studies, [Conference]: conference proceedings, F: Female, M: Male, Testos : testosterone, Prog: progesterone, E2: Estradiol, /: unknown, b.i.d: twice daily, t.i.d. three times daily, q.i.d. four times daily, Y: yes, N: no,  CL WT: contact lens wearing time, TBUT; tear break up time, MG: meibomian gland. 

14

Insummary,eyedropsandcreamappliedtotheeyelids,supplementedwith

testosteroneorDHEAhaveabeneficialeffectonsymptoms,tearstabilityand

quantity.[75,77,80,83],[74,78]However,evidenceisweakwithanabsenceofplacebo

controlledpublishedstudieswhicharerequiredtoimproveourunderstandingofthe

possibleapplicationoftopicalandrogensforclinicaltreatmentofdryeye.

Thereareaverylimitednumberofstudiesresearchingtheeffectofestradioltherapyon

dryeye,includingonlytwopublishedcontrolledstudies,oneofwhichhasmany

possibleconfoundingfactors.[88]Evidenceseemstosuggestthattopicalestradiolmay

bebeneficialforthetreatmentofdryeye.Thisisinagreementwithstudieswhichfound

systemicestrogentreatmenttoimprovedryeye,[87–93]butdisagreeswithstudies

whichfoundsystemicestrogentherapytoexacerbatedryeye.[94–96]Although

systemicestrogentherapyhasconflictingresults,thelimitedresultsfromstudiesof

topicalestradioltherapy(includingpreliminarystudies)suggestapositiveeffectof

topicaltreatment,whichmaybemoreassociatedwithlocalsynthesisofsex

hormones.[24]

Topicalestrogenandandrogentherapyappearstohaveapositiveeffectonsignsand

symptomsofdryeye;howeverpublishedevidenceisweakwiththemajorityofstudies

beingpreliminary.

IV. SUMMARY

Thisreviewaimedtoprovideacomprehensivediscussionfocussedontheimpactof

localsexhormonesynthesisondryeye.Itwasnecessarytodiscussthewidertopicof

intracrinologybeforefocussingonocularstructures.Despitetheincreaseinknowledge

oflocalhormonesynthesis,muchoftherecentliteraturefocussesontheeffectsof

circulating,ratherthanlocal,sexhormonesonsignsorsymptomsofdryeye.Thisis

likelyduetotherelativeeaseofsystemicmeasurementincomparisontolocal.

Muchresearchregardingsexhormonesandtheocularsurfacehasbeenperformedon

ratsandmicetoshowthepresenceofsexhormonemRNAin,ocularstructures;the

meibomianandlacrimalglandshavebeenofparticularinterestinpublishedresearchto

date,likelyduetotheirroleintearformation.Labriedescribesintracrinologyas“the

formationofactivehormonesthatexerttheiractioninthesamecellswheresynthesis

15

tookplacewithoutreleaseintothepericellularcompartment.”[22,23]Thepresenceof

steroidogenicenzymesintheocularsurfacetissuessuggeststhathumanocularsurface

tissuesarecapableoflocalformationaswellasinactivationofsexsteroids.The

presenceofAR,ERand/orPRmRNAandproteinsuggeststhattheseoculartissuesare

targetsitesforsexhormonesandthatoncesynthesized,thelocallyproducedsex

steroidsmayexerttheiractionwithinthesametissues.Thisissupportedbyhumanand

micestudieswhichhavedemonstratedthatandrogensandestrogensregulate

numerousgenesinthemeibomiangland,lacrimalglandandconjunctiva.

Theimportantphysiologicalroleofestrogensandandrogensinthefunctionand

structureofoculartissuescallsforanimprovedunderstandingofintracrinehormone

synthesisandtheirmetabolism.Measurementoflocalsexhormonelevelshasits

challenges,includingtheprocurementofhumanocularsurfacetissue.Measuringlevels

ofsexhormonesandmetabolitesintearsormeibumthusprovidesanestimateofocular

surfacetissuelevels,withouttheneedfortissueexcision.Technologicaladvances,

includinginLC‐MSandGC‐MS,willprovidethesensitivityrequiredtomeasurethelow

levelsofsexhormonesandtheirmetaboliteswhicharepresentinhumantearsand

meibum.

Developmentsintechnologynotonlyallowourunderstandingofhowsexhormones

influencetheultrastructureofoculartissues,butalsouncoverthepresenceofthe

biologicalmachineryneededforintracrinehormonesynthesis.Thusitisanticipated

thatwithfurthertechnologicaldevelopments,rapidprogresswillbemadein

understandinghowsexhormonesaffectoculartissuesandcontributetothe

developmentofdryeye.Clarificationoftheactionofsexhormonesonocularsurface

tissuesandtheircontributiontodryeyeisessentialforthedevelopmentofsuitable

hormonebasedtreatmentsfordryeye.

16

FundingStatement:EmmaGibsonissupportedbyaUniversityInternational

PostgraduateAward(UIPA)ScholarshipfromtheUniversityofNewSouthWales

(UNSW).FionaStapleton,none;BlankaGolebiowski,none,JamesWolffsohn,none.

CompetingInterests:Therearenocompetinginterests.Theauthorshaveno

commercialorproprietaryinterestinanyconceptorproductdiscussedinthisarticle.

Contributorstatement:EGandBG:draftingthearticle.Allauthors:criticallyrevisedthe

manuscriptforimportantintellectualcontent.Allauthors:readandapprovedthefinal

manuscript.

17

FIG1:Schematicrepresentationofhumanendocrine(adrenal)andintracrinesteroidogenicpathways.Endocrineprocessesallowcholesteroltobeconvertedintoprogestagensthenandrogens,whicharethentransformedintoestrogens.DHEAandDHEASaresecretedbytheadrenalsandareusedforsteroidogenesisinperipheralintracrinetissues.Corticosteroidsarealsoincludedastheyareformedfromthesameprecursors(progesteroneand17α‐hydroxyprogesterone).Corticosteroidsincludemineralocorticoids(primarybeingaldosterone)andglucocorticoids(primarybeingcortisol).Italicboxesrepresentenzymesinvolved.

AdaptedfromLabrie2007.[28]

Abbreviations:DHEA:dehydroepiandrosterone,DHEAS:dehydroepiandrosterone‐sulphate,DHT:dihydrotestosterone

FIG2:Schematicrepresentationofovarianandadrenalsourcesofsexsteroidsinpremenopausalwomen.HumanshaveadrenalglandswhichsecretelargequantitiesoftheprecursorDHEAwhichisconvertedintoprogestogens,androgensandestrogensinperipheraltissues.Aftermenopauseovarianestradiolsecretionceases,thus100%ofsexsteroidsarethenmadelocallyintissuesbyintracrinepathways.LHstimulatesthesecretionofsexhormonesfromthegonadsandACTHmodulatesadrenalsecretionofDHEA.

AdaptedfromLabrie2003.[30]

Abbreviations:LH:Luteinisinghormone,ACTH:adrenocorticotropin,DHEA:dehydroepiandrosterone.

18

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