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RESEARCHARTICLE A new approach for the analysis of facial growth and age estimation: Iris ratio CarlosEduardoPalharesMachado1,2,3*,MartaReginaPinheiroFlores4,La´ıseNascimento CorreiaLima5,6,RachelLimaRibeiroTinoco7,8,AdemirFranco9,AnaCristina BarretoBezerra2,MartinPaulEvison10,MarcoAure´lioGuimarães3 1 NationalInstituteofCriminalistics,BrazilianFederalPolice,MinistryofJustice,Bras´ılia,DistritoFederal, Brazil,2 HealthScienceCollege,UniversityofBras´ılia,Bras´ılia,DistritoFederal,Brazil,3 MedicoLegal Centre,DepartmentofPathologyandLegalMedicine,RibeirãoPretoMedicalSchool,UniversityofSão Paulo,RibeirãoPreto,SãoPaulo,Brazil,4 SchoolofDentistry,UniversityofSãoPaulo,SãoPaulo,São Paulo,Brazil,5 SchoolofDentistry,FederalUniversityofMaranhão,SãoLu´ıs,Maranhão,Brazil, 6 PiracicabaDentistrySchool,StateUniversityofCampinas,Piracicaba,SãoPaulo,Brazil,7 Schoolof Dentistry,UniversitySalgadodeOliveira,Nitero´i,RiodeJaneiro,Brazil,8 DepartmentofAnthropology, NationalMuseum,FederalUniversityofRiodeJaneiro,RiodeJaneiro,RiodeJaneiro,Brazil,9 Department a1111111111 ofOralHealthSciences,KatholiekeUniversiteit,Leuven,Belgium,10 DepartmentofAppliedSciences, a1111111111 CentreforForensicScience,NorthumbriaUniversity,Newcastle,UnitedKingdom a1111111111 a1111111111 *[email protected] a1111111111 Abstract Thestudyoffacialgrowthisexploredinmanyfieldsofscience,includinganatomy,genetics, OPENACCESS andforensics.Inthefieldofforensics,itactsasavaluabletoolforcombatingchildpornogra- Citation:MachadoCEP,FloresMRP,LimaLNC, phy.Thepresentresearchproposesanewmethod,basedonrelativemeasurementsand TinocoRLR,FrancoA,BezerraACB,etal.(2017)A fixedreferencesofthehumanface—specificallyconsideringmeasurementsofthediameter newapproachfortheanalysisoffacialgrowthand ageestimation:Irisratio.PLoSONE12(7): oftheiris(irisratio)—fortheanalysisoffacialgrowthinassociationwithageinchildrenand e0180330.https://doi.org/10.1371/journal. sub-adults.Theexperimentalsampleconsistedofdigitalphotographsof1000Braziliansub- pone.0180330 jects,agedbetween6and22years,distributedequallybysexanddividedintofivespecific Editor:MohammedE.Elsalanty,Augusta agegroups(6,10,14,18,and22yearolds±onemonth).ThesoftwarepackageSAFF-2D® University,UNITEDSTATES (ForensicFacialAnalysisSystem,BrazilianFederalPolice,Brazil)wasusedforpositioning Received:June9,2016 11landmarksontheimages.Tenmeasurementswerecalculatedandusedasfixedrefer- Accepted:June14,2017 encestoevaluatethegrowthoftheothermeasurementsforeachagegroup,aswelltheaccu- mulatedgrowth(6–22yearsold).TheIntraclassCorrelationCoefficient(ICC)wasappliedfor Published:July7,2017 theevaluationofintra-examinerandinter-examinerreliabilitywithinaspecificsetofimages. Copyright:©2017Machadoetal.Thisisanopen Pearson’sCorrelationCoefficientwasusedtoassesstheassociationbetweeneachmea- accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense,which surementtakenandtherespectiveagegroups.ANOVAandPost-hocTukeytestswereused permitsunrestricteduse,distribution,and tosearchforstatisticaldifferencesbetweentheagegroups.Theoutcomesindicatedthat reproductioninanymedium,providedtheoriginal facialstructuresgrowwithdifferenttiminginchildrenandadolescents.Moreover,thegrowth authorandsourcearecredited. allometryexpressedinthisstudymaybeusedtounderstandwhatstructureshavemoreor DataAvailabilityStatement:Allrelevantdataare lessproportionalvariationinfunctionfortheagerangesstudied.Thediameteroftheiriswas withinthepaperanditsSupportingInformation foundtobethemoststablemeasurementcomparedtotheothersandrepresentedthebest files. cephalometricmeasurementasafixedreferenceforfacialgrowthratios(orindices).The Funding:Theauthorsreceivednospecificfunding methoddescribedshowspromisingpotentialforforensicapplications,especiallyaspartof forthiswork. thearmamentariumagainstcrimesinvolvingchildpornographyandchildabuse. Competinginterests:Theauthorshavedeclared thatnocompetinginterestsexist. PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 1/19 Irisratiofortheanalysisoffacialgrowth Introduction Thehumanfacedoesnotgrowhomogenouslyovertime.Eachofthemanyfacialstructures developsindifferentdimensionsanddirections[1,2].Consequently,thefacialanatomy reachesdifferentproportionsdependingonage[1,3,4].Thisphenomenon,knownasallome- try,isthereasonwhyachild’sfacedoesnotcorrespondtoasmallerversionofanadult’sface. Growthandalterationsincraniofacialmorphologyarousesinterestinmanyfieldsofscience, especiallyphysicalanthropology[5–7]andgenetics[8].Inthesefields,morphologyismost oftenstudiedinrelationtoevolutionaryprocess[9,10]andmedicaltherapeutics[11–15]. However,nowadays,alternativeapplicationsofcraniofacialmorphology,suchasinthescope offorensicsciences,hasreceivedmajorattention.Inthisregard,forensicstudiesweredevel- opedinthelastdecadetoinvestigatefacialgrowthasananthropometrictoolforageestima- tionproceduresincasesinvolvingchildpornography[16,17]. Traditionalanthropometryisperformedbytakingmeasurementsdirectlyfromthesubjects usingcalipersormeasuringtapes.Clearly,thisrequiresacontrolledenvironmentaswellas theconsentandcooperationoftheexaminedsubject—whichcanbeachallengingtaskwhen childrenareinvolved[18].Analysisoftwo-dimensional(2D)images,suchascephalograms andphotographs,hasconsequentlyemergedasanalternativemethodforinvestigationsinthis field.Amongthe2Dtechniquesusedforimageacquisitionandanalysisofthehumanface, photo-anthropometryremainsapopularapproachforepidemiologicalandforensicstudies.It consistsoflandmarkingphotographstoenablethemeasurementofdistances,angles,andpro- portions[5].Thesameprincipleisalsoapplicableinthree-dimensional(3D)images[15,19]. Adirectcomparisonbetweenmeasurementstakenfromdifferentphotographsisonlyreli- ableiftheimageswereacquiredstandardlyorwithmetricreferences.Ingeneral,photographs couldhamperanthropometricanalyses.However,anthropometricexamsarefeasibleifthe photographsaretakenfollowingthesameprotocol.Inthiscontext,ratiosandanglesbetween facialdistancescouldbecalculatedtoallowananthropometricexam,evenintheabsenceof metricreferencesintheimage[20].Yet,theratiosobtainedbetweentwolinearfacialdistances culminateinindicesthatmayplayanimportpartinanthropologyastoolsfortheclassification offacialtypes[21]. Ingeneral,understandingthegrowthandalterationsincraniofacialmorphologyrequires long-termlongitudinalstudiesthatregisterhumandevelopmentfromchildhoodtoadulthood withdirectorindirectmeasurementsoftheface.Usinganinnovativeapproach,Ferrarioetal. [15]conductedamixedlongitudinalandcross-sectionalstudyonthequantificationofgrowth alterationsincraniofacialmorphologybyemployinganoninvasive3Dassessmentthatused camerasandinfra-redsensors.Differencesingrowthtimingandfacialproportionsbetween malesandfemalescouldbedetectedfollowingthisapproach.Otherauthorshavesimilarly foundotheranthropologicalapplicationsfortheassessmentofgrowthalterationsincraniofa- cialmorphology,namelytheageestimationofchildreninvolvedinpornography[17]. Ononehand,allometryenablesageestimationinforensicsciences.Ontheotherhand,it limitstheusefulnessoffacialindicesforthedetectionofdifferencesingrowthproportions. Thisisjustifiedsincetheindicesarefoundedontwomeasurementsthataretakenfromfacial structuresthatdevelopwithindifferenttimings.Inotherwords,theuseofindicestoassess growthproportionsissimilartoestimatingsomeoneelse’scarspeedwhiledrivingaseparate car.Inthiscontext,theoutcomeisnottherealcarspeed,butrathertherelativespeed betweenbothcars.Forthehumanface,theuseofindicesresultsinrelativevariationsof facialstructures[2].Despitetheirrelativenature,measurementstakenfromthefaceare closertoreal-lifemeasurementsifmorestablefacialstructuresareconsideredasreferences intheindexequation. PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 2/19 Irisratiofortheanalysisoffacialgrowth Thepresentstudythereforeaimedtoanalyzetheallometryofhumangrowthusingasetof 10facialmeasurementstakenfromchildren,adolescents,andyoungadults.Thediameterof theiriswasusedasreferencetoassessthecraniofacialvariationsintheremainingnine measurements. Materialandmethods Samplingandfacialanalysis Thesampleconsistedof1000photographs,takenstandardly,ofBraziliansubjectsaged between6and22years.Thesamplewashomogeneouslydivided(n=200)intofiveagegroups of6,10,14,18,and22yearolds,withastandarddeviationof1monthineachagegroup.The photographswereselectedfromadatabaseoftheBrazilianFederalPolice.Forinclusioninthe database,thephotographsweretakenfollowingtheInternationalCivilAviationOrganization (ICAO)guidelinesforpassportsandwerestoredin.PNG24-bitformatwithresolutionof 640×480pixels.Inaddition,thesameflashsystemandcameramodelwereusedforallphoto- graphs,andpositioned1.5metersfromthesubject’sface.Onlyphotographsofsubjectswith neutralfacialexpression,closedlips,andheadpositionedstraighttowardsthecamerawere selected.Photographsofsubjectswithheadrotationinthesagittal,axial,orcoronalaxeswere excluded,aswellasthosewithfacialdeformationsorevidentasymmetries.Subjectswithfacial hair,adornments,andmakeupwerealsoexcludedduetoincompletevisualizationoftheface. Acephalometricanalysisofallphotographswasperformedbyasingleexaminer.Toassess intra-examinerreliability,asetof100imageswereexaminedinduplicatebypositioning11 landmarksonpre-establishedreferencepoints(Table1)[22].Atthisstep,anon-commercial softwarepackagedevelopedbytheBrazilianFederalPolicefor2Dfacialanalysis(SAFF-2D1– ForensicFacialAnalysisSystem,DepartmentofFederalPolice,Brazil)wasused.Thesoftware registersCartesiancoordinatesinthex-andy-axesforeachofthepositionedlandmarks.The intra-andinter-examinerreliabilityofthelandmarkingmethodologywasalsoassessedprior tothestudyandconsistedoftheanalysisof10photographsbythreetrainedexaminers,three times,withinanintervalof15days. Table1. Definitionofthecephalometriclandmarksusedinthisstudy[2,22]. Landmark Definition 1.Nasion(n) Theinterceptionofthemidsagittalplaneandthelinecrossingthesuperiorpalpebral creases,abovetheuppereyelids. 2.Subnasale(sn) Thelowestpointofthenoseonthemidsagittalplane. 3.Gnathion(gn) Thelowestpointofthechin,onthemidsagittalplane. 4.Endocanthion Themediallimitoftheeye. (en) 5.Ectocanthion Thelaterallimitoftheeye. (ec) 6.Iridionlaterale(il) Themostlateralpointoftherimoftheiris. 7.Iridionmediale Themostmedialpointoftherimoftheiris. (im) 8.Pupil(pu)a Thecentralpointoftheiris,mathematicallycalculated,betweentheIridionlaterale andtheIridionmedialeofeacheye. 9.Zygion(zy) Thewidestpointintheregionofthezygomaticboneseeninthefrontalview. 10.Chelion(ch) Thelaterallimitofthemouth. 11.Alare(al) Themostlateralpointonthe“wing”ofthenose. aNotdescribed,butcalculatedasthearithmeticmeanbetweentheIridions. https://doi.org/10.1371/journal.pone.0180330.t001 PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 3/19 Irisratiofortheanalysisoffacialgrowth Fig1.Graphicrepresentationofthemeasurementsadoptedinthisstudy.*Theirismeasurementcorrespondedtotheaverage valueoftherightandleftim-il;theimagewasobtainedandillustrativelyusedwiththeconsentofthesubjectandrelatives. https://doi.org/10.1371/journal.pone.0180330.g001 Calculationofabsolutemeasurements Oncelandmarkswereregistered,theircoordinateswereusedtocalculate10measurements expressedinpixels.Amongthe10measurements,ninewereconsideredtobejustifiedforthe presentstudyonthebasisthattheyarecommonlyusedtobuildfacialindicesinanthropomet- ricstudies[17,18,21,22].Theremainingmeasurementcalculatedthediameteroftheirisand wasincludedasanewapproachforanthropometricfacialanalysis(Fig1). RelativeMeasurementGroups(RMG) Inordertoenabletheinvestigationofallometryamongthe10measurements,RelativeMea- surementGroups(RMG)wereestablished(Table2).Thesegroupsallowedtheobservationof growingdifferencesfrom10diverseperspectives.Thisapproachwasfoundedondetermining afixedreferencemeasurementtobuildspecificratios(similartoindices,butwithoutexpress- ingcentesimalvalues)foreachgroup.Variationsbetweenmeasurementswereobservedasa functionofage. Sincethisapproachisfoundedontheanalysisofproportions,thefixedreferencevalue couldbeselectedrandomlyforeachgroup.However,themeanvaluesforthereferencemea- surementsfromthetotalsample(n=1000)wereusedtoreachvaluesclosertotheactualfacial measurements.Oncethereferencevalueswereestablished,theimageswerescaledbasedon thepercentagedifferencebetweenthereferencevalueandatargetvalueforeachgroup(size PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 4/19 Irisratiofortheanalysisoffacialgrowth Table2. RelativeMeasurementGroups(RMG)andfixedmeasurements. RMG FixedMeasurement n RMG1 zy-zy 1000 RMG2 ec-ec 1000 RMG3 en-en 1000 RMG4 pu-pu 1000 RMG5 iris* 1000 RMG6 al-al 1000 RMG7 ch-ch 1000 RMG8 n-sn 1000 RMG9 n-gn 1000 RMG10 sn-gn 1000 *Theiriswascalculatedbythearithmeticmeanofthedistancesbetweenthepointsilandimforeacheye. https://doi.org/10.1371/journal.pone.0180330.t002 normalization);e.g.iftheoriginalreferencemeasurementrequiredenlargementbyafactorof 1.5toreachthetargetvalue,thesameenlargementfactorwasappliedtotheremainingmea- surements.Thisprocedurekeptthemetricproportionsofthephotographsunalteredand enabledthecomparisonofdifferentfacialstructuresfromthesameperspectiveineachRMG. AfterestablishingtheRMG,thevariablesineachgroupwereconvertedtopercentageval- ues(anthropometricratios)ofthereferencemeasurements(Table3).Atthisstage,thesample stratificationbyagerange(6,10,14,18,and22yearsold)wasusedtoassessanthropometric variationsduringageing.Despiteknowingofthedifferencesincraniofacialdevelopment betweensexes[1],thepresentstudydidnotaimtoinvestigatecraniofacialdevelopmentin functionofsexdimorphism.Forthatreason,thesamplewasanalyzedgroupedbysex. CalculationofRelativeGrowth(RG)andAverageRelativeGrowth (ARG) TheRelativeGrowth(RG)wasassessedforeachagegroup,withtheageof6yearsconsidered asthe“zeropoint”,fromwhichpercentagevariationswereinvestigatedasafunctionofagein relationtotheotheragesubgroups.OnceRGwasobtainedforeachmeasurement,Average RelativeGrowth(ARG)wasassessedbytakingintoconsiderationthevaluescalculatedfor eachRMG. Table3. RepresentativeformulasforcalculatingtheproportionalmeasurementsforeachRelativeMeasurementGroupafterimagescaling. RMGa FixedMeasurementb(n=1000) ProportionalMeasurementsc(AnthropometricRatios) 1:zyzy 2:ecec 3:enen ... 10:sngn 1 zyzy zyzy ¼zyzy ecec ¼ecec enen ¼enen ... sngn ¼sngn RMG1 zyzy RMG1 zyzy RMG1 zyzy RMG1 zyzy 2 ecec zyzy ¼ zyzy ecec ¼ecec enen ¼enen ... sngn ¼sngn RMG2 ecec RMG2 ecec RMG2 ecec RMG2 ecec 3 enen zyzy ¼ zyzy ecec ¼ecec enen ¼enen ... sngn ¼sngn RMG3 enen RMG3 enen RMG3 enen RMG3 enen ... ... ... ... ... ... 10 sngn zyzy ¼ zyzy ecec ¼ecec enen ¼enen ... sngn ¼sngn RMG10 sngn RMG10 sngn RMG10 sngn RMG10 sngn aRMG:RelativeMeasurementGroup; bFixedMeasurement:referencemeasurementusedforcalculatingratiosobtainedthroughthemeanreferencevaluesofeachgroup; cProportionalMeasurements:measurementcalculatedforeachimagefromtheratiobetweentheimagemeasurementfoundandthemeanvalueofthe fixed(reference)measurementineachgroup(calculatedforeachimageandeachRMG). https://doi.org/10.1371/journal.pone.0180330.t003 PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 5/19 Irisratiofortheanalysisoffacialgrowth Table4. Quantificationofthemetricvariationofmeasurement1(zy-zy)asafunctionofageandRelativeMeasurementGrouptodeterminethe AverageRelativeGrowth. Measurement Age(years) RMG1 RMG2 RMG3 ... RMG10: ARG zy-zy ec-ec en-en sn-gn 1.zy-zy 6to10 0 D%zyzyRMG2 D%zyzyRMG3 ... D%zyzyRMG10 ARGzyzy 6(cid:0)10 6(cid:0)10 6(cid:0)10 6(cid:0)10 10to14 0 D%zyzyRMG2 D%zyzyRMG3 ... D%zyzyRMG10 ARGzyzy 10(cid:0)14 10(cid:0)14 10(cid:0)14 10(cid:0)14 14to18 0 D%zyzyRMG2 D%zyzyRMG2 ... D%zyzyRMG10 ARGzyzy 14(cid:0)18 14(cid:0)18 14(cid:0)18 14(cid:0)18 18to22 0 D%zyzyRMG2 D%zyzyRMG2 ... D%zyzyRMG10 ARGzyzy 18(cid:0)22 18(cid:0)22 18(cid:0)22 18(cid:0)22 6to22(cumulative) 0 Δ%zyzyRMG2 Δ%zyzyRMG3 Δ%zyzyRMG10 ARGzyzy 6(cid:0)22 6(cid:0)22 6(cid:0)22 18(cid:0)22 https://doi.org/10.1371/journal.pone.0180330.t004 Table4detailstheprocessfollowedtoquantifytheARGwhenusingthecraniofacialmea- surement1(zyzy)asreference.Analysisofthecolumnsallowsforthevisualizationofthe proportionalgrowthofthezyzymeasurementwithineachofthe10RMGs,ineachofthedif- ferentageranges.Zerovaluesinthefirstcolumnareexplainedbythescalingprocess,which madethereferencemeasurementfixedforallagerangesinRMG1(zy-zy).Thecodification Δ%zyzyRMG2canbeinterpretedasthepercentagevariationofmeasurementzy-zy,within 6(cid:0) 10 RMG2,whenconsideringthemeanvaluesobtainedfromthecohortof6to10yearolds (6–10);thecolumnonthefarrightshowstheARGforthezy-zymeasurementineachage rangeobtainedfromthemeanvaluesoftherelativezy-zygrowth,byconsideringallRMGs (i.e.ARGzyzy,referstotheagerangeof6–10years).ThebottomlineofTable4showsthe 6(cid:0) 10 cumulativerelativegrowthofzy-zyfrom6to22yearsofage. AfterARGquantificationforallmeasurements,arankingoftherelativevariationswasper- formedconsideringtheagerangesseparatelyandcombined(cumulativegrowth).Inthispro- cedure,positivevaluesindicatedpositivevariationsofaspecificmeasurementcomparedtothe meangrowthoftheothers,whiletheoppositewasinterpretedfornegativevalues. Themeasurementsthatusedirisdiameterasreference(RMG5)–proposedasanew approachinthepresentstudy—wereusedastheparametertoanalyzevariationintheremain- ingmeasurements.Basedonthisanalysis,anewrankingsystemwasdesignedtoprovideout- comesclosertotherealgrowthofthemeasurementsconsidered. Statisticalanalysis TheKolmogorov-SmirnovtestwasusedtoassessdatanormalityandtheIntraclassCorrelation Coefficient(ICC)appliedtotheevaluationoftheintra-andinter-examinerreliabilityforposi- tioninglandmarksandperformingmeasurements.Descriptivestatisticswereusedforinitial datascreening.Pearson’sCorrelationCoefficientwasusedtoassociatemeasurementswithin eachgroupandage.ThestatisticaldifferencesineachRMGwereassessedbyapplyingthe ANOVAtestasafunctionofage(consideringthedifferentageranges).APost-hocTukeytest wasusedtosearchforstatisticaldifferencesbetweenspecificagerangesandtoverifytheeffi- ciencyofeachfixedmeasurementindetectingtherelativegrowth. AllstatisticaltestswereperformedwiththeSPSS121.0softwarepackage(IBM1,New York,USA),withconsiderationforasignificancelevelof5%andconfidenceintervalof95%. Researchdatabasesusedforstasticalanalysismaybefoundwithinthesupportinginformation filesS1–S6Files. Ethicsstatement ThepresentresearchwasconductedwiththeapprovaloftheCommitteeofEthicsinResearch oftheUniversityofSãoPaulo,underprotocolnumber17017213.0.0000.5440.Theimages PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 6/19 Irisratiofortheanalysisoffacialgrowth Table5. Cephalometriclandmarkmeasurementsexpressedinpixels. Measurement 6years(n=200) 10years(n=200) 14years(n=200) 18years(n=200) 22years(n=200) Totalsample (n=1000) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1.zy-zy 254.84 23.70 249.77 16.78 245.85 16.82 247.15 24.77 250.01 26.23 249.52 22.21 2.ec-ec 169.15 9.20 167.65 8.41 166.74 8.91 166.26 8.55 166.70 7.74 167.30 8.62 3.en-en 62.29 4.92 61.07 4.57 60.27 4.54 59.84 4.43 60.24 4.38 60.74 4.64 4.pu-pu 114.68 6.15 114.58 5.44 114.78 5.76 115.50 5.56 116.17 5.07 115.14 5.63 5.iris 24.96 1.63 23.58 1.64 22.20 1.67 21.67 1.64 21.56 1.45 22.79 2.07 6.al-al 64.86 4.74 65.04 5.69 67.56 6.00 68.04 5.79 67.81 5.85 66.66 5.80 7.ch-ch 80.10 8.29 83.98 7.77 85.27 8.22 85.94 8.35 87.32 6.64 84.52 8.24 8.n-sn 93.67 7.79 96.17 9.02 98.22 8.52 98.32 9.49 99.44 9.50 97.16 9.10 9.n-gn 205.58 13.77 211.15 13.93 216.44 15.92 219.91 15.76 223.73 15.40 215.36 16.27 10.sn-gn 111.97 10.35 115.03 10.07 118.27 11.19 121.64 10.90 124.36 13.76 118.25 12.15 https://doi.org/10.1371/journal.pone.0180330.t005 usedtoillustratethepresentstudywereacquiredandusedwiththesignedconsentofthepho- tographedsubjectandhisrelatives. Results Thedatahereinstudiedpresentedwithanormaldistribution.TheIntraclassCorrelationCoef- ficientrevealedexcellentoutcomesforthelandmarkingperformedintheintra-andinter- examinerreliabilitytests,whichwereperformedpriortoandduringtheresearchproject (ICC>0.90;p<0.001).Themeanandstandarddeviationofeachofthecephalometric measurementsobtainedbeforesizenormalizationarepresentedinTable5.Thevaluesare expressedinpixels,withmalesandfemalesgrouped,consideringthefiveagegroupsandthe completesample(6–22yearsold). TheARGofeachmeasurementasafunctionoftheagerangesarepresentedinTable6. Usingirisdiameter,ARGreachedthelowestvaluesinallagegroupsincomparisontothe othermeasurements.Positivevaluesindicateameasurementwitharelativegrowthhigher thanthemeanvariationofmeasurements,whilenegativevaluesindicatetheopposite.The irisvalueswereconsistentlynegativeandwasthemeasurementleastalteredwithage.Inthe cumulativeanalysis(6–22years),irismeasurementsreachedavalue4.2timeslower(-14.83%) thanthesecondhighestrankedmeasurement(en-en,-4.62%). Table6. AverageRelativeGrowthforthe10measurementsstudiedasafunctionofage. Measurement AverageRelativeGrowth 6to10years 10to14years 14to18years 18to22years 6to22years 1.zy-zy -2.18% -1.90% 0.23% 0.39% -3.29% 2.ec-ec -1.08% -0.85% -0.57% -0.49% -2.80% 3.en-en -2.14% -1.57% -1.05% -0.10% -4.62% 4.pu-pu -0.27% -0.12% 0.33% -0.17% -0.07% 5.Iris -5.74% -6.20% -2.62% -1.23% -14.83% 6.al-al 0.01% 3.52% 0.46% -1.14% 3.00% 7.ch-ch 4.60% 1.23% 0.52% 0.87% 7.54% 8.n-sn 2.39% 1.84% -0.22% 0.51% 4.74% 9.n-gn 2.49% 2.17% 1.31% 0.96% 7.28% 10.sn-gn 2.57% 2.45% 2.58% 1.33% 9.41% https://doi.org/10.1371/journal.pone.0180330.t006 PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 7/19 Irisratiofortheanalysisoffacialgrowth Fig2.AverageRelativeGrowthofthe10measurementsstudiedaccordingtoanage-segmentalanalysis. https://doi.org/10.1371/journal.pone.0180330.g002 GraphicalrepresentationofARGprogressbetweenagegroups(Fig2)enablesaclearinter- pretationofthedatareportedinTable6,showingthatfacialmeasurementshaddifferentrela- tivegrowthbetweentheagegroups.SomeofthespecificmeasurementspresentedARGvalues thatrangedbetweenthepositiveand/ornegativescaleforthedifferentageranges(e.g.width oftheface:zy-zy),whileothermeasurementsconsistentlyremainedpositiveornegative,such astheheightofthelowerthirdoftheface(sn-gn)andthediameteroftheiris.Thecumulative approachforARG(6–22yearsold)enabledtheconstructionofaproportionalgrowthrankfor eachofthemeasurements(Fig3).Theheightofthelowerthirdofthefacepresentedthehigh- estpositivevalue(+9.41%),indicatingagreaterrelativegrowth,whilethediameteroftheiris hadthemostnegativevalue(-14.83%),suggestingitisthemoststablemeasurementofthe study. Table7expressesPearson’sCorrelationCoefficient(r)betweenageandtherespective RMGmeasurements.Amongthe81possibilitiesexpressedintheTable(9×9),onlytwocombi- nationsresultedinverystrongcorrelation(0.8(cid:20)|r|<1)withage:thewidthofthelips(ch-ch) inRMG8(n-sn),andthediameteroftheirisinRMG9(n-gn).Themeasurementsreachedout- comesthatweremoreconsistentlyandstronglycorrelatedtoagewithinRMG5(Irisratios), reportingsevenvalueswithstrongcorrelation(0.6(cid:20)|r|<0.8)andtwowithmoderatecorrela- tion(0.4(cid:20)|r|<0.6),amongninepossibilities.Apartfromthesemeasurements,mostcombi- nationsresultedinweak(0.2(cid:20)|r|<0.4)orveryweak(0<|r|<0.2)correlationswithage. Consideringthemeanvaluesofthecorrelations(bottomline),itispossibletoinferthatall groupshadaweakcorrelationwithage,exceptforthegroupthatusedthediameteroftheiris asreference(RMG5). TheANOVAtestindicatedastatisticallysignificantgrowth(p<0.05)inallRMGs,for almostalltherelativemeasurements,andshowedmetricdifferencesfrom6to22years.On theotherhand,thepost-hoctest(Table8)indicatedthatthedifferencesingrowthmainlytook placeinthetwolowerageranges(6–10,10–14yearsold),whileRGdetectionfelldramatically PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 8/19 Irisratiofortheanalysisoffacialgrowth Fig3.RankingofthecumulativeAverageRelativeGrowthexpressedaspercentagevaluesforthecumulativeagerangeof 6to22years. https://doi.org/10.1371/journal.pone.0180330.g003 Table7. Pearson’sCorrelationCoefficient(r)assessmentofthecorrelationbetweenthe10measurementsandage(6–22years)inrelationtoeach RMG. Measurement RelativeMeasurementGroup—Pearson’sCorrelationCoefficient(r) RMG1: RMG2: RMG3: RMG4: RMG5: RMG6: RMG7: RMG8: RMG9: RMG10: zy-zy ec-ec en-en pu-pu Iris al-al ch-ch n-sn n-gn sn-gn 1.zy-zy .a -.024 .055 -.155** .489** -.265** -.287** -.261** -.400** -.298** 2.ec-ec .047 .a .109** -.445** .677** -.320** -.333** -.327** -.567** -.318** 3.en-en -.058 -.113** .a -.284** .417** -.323** -.331** -.290** -.431** -.320** 4.pu-pu .219** .445** .281** .a .709** -.186** -.237** -.185** -.404** -.234** 5.Iris -.596** -.682** -.421** -.713** .a -.645** -.628** -.696** -.804** -.567** 6.al-al .310** .317** .323** .189** .629** .a -.085** -.008 -.150** -.142** 7.ch-ch .356** .376** .343** .262** .672** .104** .a .081* -.019 -.053 8.n-sn .303** .324** .286** .189** .685** .017 -.067* .a -.132** -.062 9.n-gn .512** .566** .426** .407** .799** .152** .014 .149** .a -.062 10.sn-gn .445** .444** .412** .350** .684** .196** .075* .150** .133** .a MEANb 0.32 0.37 0.30 0.33 0.64 0.25 0.23 0.24 0.34 0.23 a.Notcalculatedsinceoneormorevariablesareconstant. b.Meancalculatedusingabsolutevalues. **p<0.01,levelofsignificance; *p<0.05,levelofsignificance https://doi.org/10.1371/journal.pone.0180330.t007 PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 9/19 Irisratiofortheanalysisoffacialgrowth intheolderageranges(14–18,18–22yearsold).TheRMGthatemployedthediameterofthe irisasthereferencemeasurement(RMG5)wasthegroupwithmoremeasurementscapableof detectinggrowthintheageranges6–10,10–14,and14–18years.Intheagerangeof18–22 years,growthdifferenceswereonlyobservedforsevenmeasurementassociationsoutofthe81 possibilitieswhenconsideringallRMGs(Table8). Oncetheirismeasurementwasobservedtoofferthebestoutcome,itwasusedtoevaluate facialgrowthwithamoreaccurateperceptionofthemetricchangesthatoccurinthefacial structuresaccordingtoage(Fig4).Insubjectsagedbetween6–10years,mouthwidthwasthe facialdimensionwiththehighestgrowthpercentage(10.9%).Yetinsubjectsaged10–14years, thenasalwidthpresentedhighestgrowthpercentage(10.3%),whileinsubjectsaged14–18 and18–22years,theheightofthelowerthirdofthefacewasthefacialdimensionwithhighest growthpercentage—presenting5.5%and2.8%growth,respectively.Theheightofthelower thirdofthefacealsodisplayedthehighestgrowthpercentage(28.8%)inthecumulativeanaly- sis(6–22yearsold).Thisgrowthpercentagewasmorethantwicethatoffacewidth(zy-zy: 13.63%). Discussion Craniofacialgrowthisacontinuousmorphogeneticprocessthatmodifiesasetofrelatedana- tomicstructuresinamorphological,functional,andbalancedmanner.Thisprocessisprimar- ilyexploredinthefieldsofanthropology,genetics,dentistry,medicine,andforensics[1,8]. Allometry,whichisfoundedonthecomparisonsofanatomicproportions,isoneofthemech- anismsforexaminingthecraniofacialgrowthprocess.Despitebeingmorecommonlyusedin comparativeanimalbiologythaninhumananthropology[2],allometryrecentlyemergedasa potentialtoolforforensicageestimation. AccordingtotheStudyGrouponForensicAgeDiagnostics,standardparametersforage estimationarenecessarytoguidebestpracticesinternationally[23–24].Specialattentionis giventoagesofmajorlegalimportance,suchas14,18,and21years[25–28].Asrecom- mendedbytheGroup,estimatedageshouldbebasedonthreeindependentevaluations, namely:1)clinicalexaminationusinganthropologicalaspectsandsexualmaturity;2)hand andwristexaminationusingcarpalradiographs;and3)dentalexaminationusingpanoramic radiographs[29,30].However,underspecialcircumstances,theonlyevidenceavailablefor analysisisaphotograph,inwhichtheabovementionedguidelinesarenotapplicable.Analy- sisofthematurationofsexualtraitsmaybeconsideredwhenfullbodyimagesareobtained; however,ageestimationthroughananalysisofsexualmaturationusingphotographsshould notbeconsideredforforensicpurposes[17,30].Thisisprimarilyjustifiedbythefactthat outcomesmaybebiasedbyintentionalmodifications,suchastheremovalofpubichair[16, 17].Thistypeofevidenceismostcommonincybercrimesinvolvingchildabuse.Inthese crimes,victimsandperpetratorsmaybephotographed[30]andtheirfacialtraitsmaycon- tributetocrimecharacterization.Nevertheless,ageestimationthroughfacialphotographsis achallengingprocedure. ThesearchforageindicatorsthroughfacialtraitsisjustifiedbyCattaneoetal.[16].The authorsprovidedimagesofmodels(aged>18yearsold)fromapornographicwebsiteto23 examiners,consistingofforensicexperts,pediatricians,gynecologists,andlaypersons.The examinerswereaskedtoestimatetheageofthemodelsmentioningwhichphotographicindi- catorofagecontributedmostsignificantlyineachcase.Facialtraitsweredescribedasthe mostcitedindicator,reaching64%oftheforensicexperts’opinions.Thisoutcometriggered furtherresearchinthisfield,includingthedevelopmentofmethodologiesbasedonfacialindi- cesforforensicageestimation[17]. PLOSONE|https://doi.org/10.1371/journal.pone.0180330 July7,2017 10/19
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