A METHOD FOR ANALYZING BLOOD VESSELS

TECHNOLOGICAL FIELD

Thepresentdisclosurerelatestomethodsforanalyzingtissueand bloodvessels.

BACKGROUND ART

Referencesconsidered to berelevantasbackground to thepresently disclosed subjectmatterarelistedbelow:

Acosta,A.A.,Eiberger,L.,Borghi,M.,Calamera,J.C.,Chemes,H. ,Doncel,G. F.,Kliman,H.,Lerna,B.,Lustig,L.,& Papier,S.(2000).Endometrialdating and determination ofthe window ofimplantation in healthy fertile women.Fertility and Sterility,73(4),788-798.

Bashkatov,A.N.,Genina,E.A.,& Tuchin,V.v.(2011).Opticalpropertiesof skin,subcutaneous,andmuscletissues:A review.JournalofInnovativeOpticalHealth Sciences,4(1),9-38.

Chappell,J.C.,Wiley,D.M.,& Bautch,V.L.(2011).How bloodvesselnetworks aremadeandmeasured.CellsTissuesOrgans,795(1-2),94-107.

Chappell,J.C.,Wiley,D.M.,& Bautch,V.L.(2012).How Blood Vessel NetworksAreMadeandMeasured.CellsTissuesOrgans,795(1-2),94-10 7.

DuCheyne,C.,Smeets,M.,& DeSpiegelaere,W.(2021).Techniquesusedto assessintussusceptive angiogenesis:A systematic review.DevelopmentalDynamics, 250(12),1704-1716.

Dubowy,R.L.,Feinberg,R.F.,Keefe,D.L.,Doncel,G.F.,Williams ,S.C., McSweet,J.C.,& Kliman,H.J.(2003).Improvedendometrialassessmentusingcyclin E andp27.FertilityandSterility,80(1),146-156.

Gambino,L.S.,Wrefordm,N.G.,Bertram,J.F.,Dockery,P.,Lederm an,F.,& Rogers,P.A.W.(2002).Angiogenesisoccursbyvesselelongationinpr oliferativephase humanendometrium.HumanReproduction,77(5),1199-1206. Girling,J.E.,Lederman,F.L.,Walter,L.M.,& Rogers,P.A.W.(2007). Progesterone, But Not Estrogen, Stimulates Vessel Maturation in the Mouse Endometrium.Endocrinology,148(11),5433-5441.

Jacques,S.L.(2013).Opticalpropertiesofbiologicaltissues:a review.Physics inMedicine& Biology,5S(11),R37.

Logsdon,E.A.,Finley,S.D.,Popel,A.S.,& MacGabhann,F.(2014).A systems biologyview ofbloodvesselgrowthandremodelling.JournalofCellularandMolecu lar Medicine,18(8),1491-1508.

Martinat-Botte,F.,Renaud,G.,Madec,F.,Costiou,P.,& Terqui,M.(2000). Ultrasonographyandreproductioninswine(INRA).Intervet.

Murray,M.J.,Meyer,W.R.,Zaino,R.J.,Lessey,B.A.,Novotny,D.B .,Ireland, K.,Zeng,D.,& Fritz,M.A.(2004).A criticalanalysisoftheaccuracy,reproducibility, and clinicalutility ofhistologic endometrialdating in fertile women.Fertility and Sterility,81(5),1333-1343.

Noyes,R.W.,Hertig,A.T.,& Rock,J.(1950).DatingtheEndometrialBiopsy. FertilityandSterility,1(1),3-25.

Acknowledgementoftheabovereferenceshereinisnottobeinferre dasmeaning thattheseareinanywayrelevanttothepatentabilityofthepresently disclosedsubject matter.

BACKGROUND

BloodVesselsaregenerated,grow anddieregularlywithinorganismsaspartof theirperpetualchange:growthprocess,injury,occurrencesofpatho logicalconditions, apoptosis,necrosisandmore.Hence,monitoringofbloodvesseldistr ibutioncouldbeof valueinvarioustissuethatarecharacterizedbyincreasednumberofb loodvessels,for example,skin,eye,intestineanduterineendometrium.

Measurementofdifferentfeaturesofbloodvessels,suchasdiamet er,direction, tortuosity,spectrum,andaveragecolor,arefeasiblebyimagingbloo dvesselsatvarious wavelengthsandtechniques. GENERAL DESCRIPTION

Thepresentdisclosureprovidesinaccordancewith someaspects,amethodfor characterizingatleastonefeatureofbloodvesselsatdifferentdept hswithinatissueofa mammaliansubject,themethodcomprising:

(i)identifyingbloodvesselsinanimageobtainedfrom asurfaceofsaidtissue, and

(ii)determining forthe identified blood vessels atleastone feature ofsaid identifiedbloodvessels,acolorindex(5?)oracombinationthereof, whereinthecolor indexdiffersindifferentdepthswithinthetissue.

Thepresentdisclosureprovidesinaccordancewith someaspects,amethodfor characterizingatleastonefeatureofbloodvesselsatdifferentdept hswithinatissueofa mammaliansubject,themethodcomprising:

(i)identifyingbloodvesselsinanimageobtainedfrom asurfaceofsaidtissue,

(ii)determining forthe identified blood vessels atleastone feature ofsaid identifiedbloodvessels,acolorindex(5?)oracombinationthereof, and(iii)repeating step(i)and/orstep(ii)inatleastonetemporarilyseparatedtimepoi nt,whereinthecolor indexdiffersindifferentdepthswithinthetissue.

BRIEFDESCRIPTION OFTHE DRAWINGS

In ordertobetterunderstand thesubjectmatterthatisdisclosedherein andto exemplifyhow itmaybecarriedoutinpractice,embodimentswillnow bedescribed,by way ofnon-limiting exampleonly,with referenceto the accompanying drawings,in which:

Figure1isaschematicrepresentationoftheendometrium,andthet issuesabut it,alongwithabasicmorphologyofbloodvessels.

Figure2isaschematicview oftheuterustissuelayersandcoordinates,withz=0 indicating the uterus surface,the endometrium tissue is considered to include the functionallayerandthebasallayer.

Figure3isatheoreticalgraphshowingpenetrationdepthinendome trialtissue (wherethesignaldropsbye ^-2 )asfunctionofwavelengthofvisiblelight. Figure4isatheoreticalgraphshowingendometrium bloodvesselscolorratioas function of blood vessel depth for classical epithelial/mucousaltissue attenuation parameters.

Figures5A to5F aregraphsshowingtheoreticalsetsofbloodvesseldiameter Probability Distribution Functions (PDFs) as function ofdepth for two competing evolutionmodels(tracks)ofbloodvessels,Figures5A-5C show bloodvesselsevolution ledprimarilybyelongation,followedbycapillarygrowththroughspl ittingangiogenesis, Figures5D-5Fshow amodelbywhichbloodvesselsevolutionofnewlyfoundlayersis primarily governed by sprouting ofnewly formed capillaries,Figures.5A and 5D representshallow layers(i.e.,closetothetissuesurface)andFigures.5B,5C,5E and 5Frepresentdeeperlayers(tissuedepth).

Figures6A-6Iaregraphsshowingtwodifferentevolutionmodelsfo rindividual recordedbloodvesseldiameterPDFsasfunctionoftissuedepth,Figur es6A-6C show anexemplary schematicrepresentation ofblood vesselsdiameterPDFsasfunctionof tissuedepthatday "n"ofthestandardizedcycleday,Figures6D-6Fshow anexemplary theoreticalbloodvesselsdiameterPDFsasfunctionoftissuedepthat day "n+m"ofthe standardized cycle day forthe above-mentioned recorded individual(on day n)and Figures6F-6Ishow acompeting,exemplarytheoreticalbloodvesselsdiameterPDFsas function oftissue depth atday "n+m"ofthe standardized cycle day foradifferent evolutiontrack.

Figures7A and7B areimagesoffreshex-vivoendometialtissue(from swine), overlaid with the computerized identification ofblood vessels,Fig.7A shows all identifiedvessels,Fig.7B showsidentifiedvesselsontheupperleftcorneronlytoallow assessmentoftheidentificationaccuracy,eachimagelongsideequal sabout4.9mm of tissueslab.

Figures8A to8F arehistogramsshowing normalized Probability Distribution Functions (PDFs)ofblood vesseldiameters within depth measure ((B+G)/R - an exemplary color index) in human endometrialtissues,calculated from 48 samples extractedfrom adultwomen,Figure8A showsPDF forthedeepestendometriallayer, Figures8B-8E show PDFforintermediateendometriallayers,Figure8FshowsPDFfor themostsuperficiallayer,therelativenumberdensityofthevessels (Nbv)andthelog- normalfunctionmodelparametersareshown,dataprocessed from 344 imagesof2X magnification,eachcoversafew mm ¹

Figures9A to9F arehistogramsshowing normalized Probability Distribution Functions (PDF) of blood vesseldiameters within depth measure ((B+G)/R - an exemplary color index) in human endometrialtissues,calculated from 48 samples extractedfrom adultwomen,Figure9A showsPDF forthedeepestendometriallayer, Figures9B-9E show PDFforintermediateendometriallayers,Figure9FshowsPDFfor themostsuperficiallayer,therelativenumberdensityofthevessels (Nbv)andthelognormalfunctionmodelparametersareshown,dataproc essedfrom 625imagesof4X, eachcoversafew mm ¹

Figure10isagraph showingchangeofthelognormalfittingparameterco in unitsoflog(pm)forbloodvesseldiameterPDFasfunctionoftissuedep thwithindepth (colorratio)bins,the X axis shows the average (B+G)/R values representing the endometriallayerdepth,whereassmallervalues(onthex-axis)repre sentlayersmore distantfrom thelumen oftheuterinecavity ("deeper"),distributionscalculated from human2X (X symbols)and4X (squaresymbols)imagesamples,verticalerrorbarsareof theorderofthesymbolsize.

Figures11A and11B aregraphsshowinglog-normalfittingparameterratiosof thebloodvesseldiameterPDFs(w inlogspace,Figure11A)andratioofthe "width"(o', in log,Figure 11B)asafunction ofthe cycle day,two symmetrial(in colorratio) populationsofdeep and superficialbloodvesselsPDFswerefittedforeach cycleday clusterofpatientssuch thatforeach cycleday theratio oftheresultand log-normal varianceandstandarddeviationwerecalculated,longlinesareregre ssionlinesforthe entirecycleduration;shortlinesareregressionlinesfortheprolif erativeandsecretory phases separately,cycle daysare inferred by histology,the binscentralvalues are (B+G)/R=0.5and1.5(deepandsupferficial,respectively).

Figures.12A to12E arehistogramsshowingnormalizedProbabilityDistribution Functions (PDF) of blood vesseldiameters within depth measure ((B+G)/R - an exemplarycolorindex)infouradultsowsprovenfertile,Fig.12A showsthePDFforthe deepestendometriallayer,Figures 12B-8D show PDF forintermediate endometrial layersFig.12E showsPDF forthemostsuperficialcrediblelayer,and also involves artifactsofmisinterpretedfreshbloodonthesurface,therelativen umberdensityofthe vesselscanbereadfrom thetotalnumber(Nbv,ontheleft)andthelog-normalfunction modelparametersarewrittenontherightofeachpanel,dataprocessse dfrom 73images altogether,eachofafew mm ² at2X magnification.

Figure.13isagraphshowingcofittingparameterinunitsoflog(pm )ofthelognormaldistributionmodelforthebloodvessldiameterofsw ineaasfunctionofthetissue depth(deepissmallerx-axisvalue),Y-errorsareoftheorderofthesy mbolsize.

DETAILED DESCRIPTION OFEMBODIMENTS

Bloodvesselsplay animportantroleinvariousphysiologicalandpathological conditions,including forexample,tissue development,tissue formation,and wound healing.Therefore,monitoringathree-dimensional(3D)bloodvesse lnetworkintissues aswellasthechangesovertimeinthe3D network,mayprovidevaluableinformation regardingtissuecondition,forexample,growth,orrecoveryphaseas wellasinsightsas totheangiogenesisfunctionalityofthebloodvessels.

Thepresentdisclosureisbasedonthefindingsthatdifferencesin colorsofblood vessels,asobservedinimagesobtainedfrom atissuesurface,canbeusedtodetermine thedepthcoordinatesofthetissueandspecificallythedepthofblood vesselsinthetissue.

Specifically,itwasfound thatextracting colorbandinformation from images, including,interalia,colorvaluesofred,blue,andgreenchannels,f orbloodvesselsmay beusedtorevealthree-dimensionalspatialdistributionofbloodves sels.Asshownherein, thez-coordinateofthebloodvesselswasusedtoexaminetherelations hipbetweenthe depthofthebloodvesselsandit’scharacteristics,including,int eralia,bloodvesselwidth ordiameter.

Tothatendandasdescribedherein,inordertoobtainphysiologica linformation ofthetissueandthebloodvesselswithinthetissue,theentirebloodv esselpopulation wastreatedasastatisticalensemblethatwascharacterizedbyitspro babilitydistribution functions (e.g., size, tortuosity, length between bifurcations, spatial correlation, directionalcorrelation,etc.).Asshownherein,bytreatingthebloo dvesselpopulationas astatisticalensemble,itwaspossibletodifferentiatebetweendiff erentdepthsofblood vessels,using theircolorattribute asobtained in visuallightimages.Thisapproach differentiatesbetweenevolutionpathswhenbloodvesseldiameterdi stributionistaken intoaccount.Theuseofvisible-light,non-destructiveimagingalso allowedmonitoring ofthebloodvesselpopulationevolutionoveraperiodoftimewithinth esametissue.

Basedonthesefindings,itwassuggestedthatitispossibletochar acterizevarious featuresofthetissue,including,interalia,featuresrelatedtoblo odvessels,atdifferent depth ofatissueand henceobtaining information on three-dimensionalblood vessel network.Inaddition,itwassuggestedthatitispossibletomonitorch angeswithtimein one ormore features ofthe three-dimensionalblood vesselnetwork.This may be beneficialinthecharacterizationofphysiologicalprocess,condit ionsanddiseasethatare associatedwithchangesinbloodvessels,including,interalia,grow thofbloodvessels anddestructionthereof.

AsshownforexampleinFigures8A-8F,Figures9A-9F andFigures12A-12E, by employing the methodsdescribed herein,itwaspossible to monitor/characterize changesinbloodvesseldiameterasfunctionoftissuelayer'sdepth.

Hence,inaccordancewithitsbroadestaspect,thepresentdisclos ureprovidesa method fordistinguishing differentdepthswithin a tissue.The method comprising analyzingcolorbanddistributionofbloodvesselsinoneormoreimage sobtainedfrom thesurfaceofthetissuetotherebydistinguishbetweendifferentdep thsofthetissue.

In accordancewith someaspect,thepresentdisclosureprovidesamethod for characterizingthethree-dimensionalnetwork ofbloodvessels,themethodcomprising analyzingbloodvesselsforthecolorvaluesdistributioninoneormor eimagesobtained from thesurfaceofthetissuetotherebycharacterizethethree-dimension alnetworkof bloodvessels.

Insomeexamples,themethodcomprisesobtainingoneormorecolori mages.In someexamples,themethodcomprisesobtainingoneormoreInfrared(IR )images.

Inaccordancewithsomeotheraspects,itisprovidedamethodforch aracterizing three-dimensionalnetworkofbloodvessels,themethodcomprisingan alyzinginoneor moreimagesobtainedfrom thesurfaceofthetissue,thecolorvaluesdistributionofred, blueandgreenchannelsofbloodvesselsidentifiedintheimages,toth erebycharacterize thethree-dimensionalnetworkofbloodvessels. Characterizingthethree-dimensionalnetworkofbloodvesselsinati ssueasused hereinreferstocharacterizingoneormorefeaturesofbloodvessels, thatmayinturn provideinformationonphysiologicaland/orpathologicalcondition softhetissue.

Hence,inaccordancewithsomeaspects,thepresentdisclosurepro videsamethod forcharacterizingatleastonefeatureofbloodvesselsatdifferentd epthswithinatissue ofamammaliansubject.

Themethodcomprisesastepofidentifyingbloodvesselsinanimage obtained from thesurfaceofthetissue.

Themethodfurthercomprisesastep ofanalyzingtheidentified bloodvessels. Analyzingtheidentifiedbloodvesselscomprises(i)determiningatl eastonefeatureof theidentifiedbloodvessels,(ii)determiningacolorindexfortheid entifiedbloodvessels or(iii)acombinationthereof.

ThecolorindexisdenotedhereinasSI.Asshownherein,itwasfound thatthe colorindexdiffersindifferentdepthswithinthetissue.

In someembodiments,thecolorindexisdifferentatthetissuesurfacean data differentdepthwithinthetissue,i.e.adeeperlayerofthetissue.He nce,itwassuggested thatthecolorindexmaybeusedtoindicatethetissuedepthandaccordi nglytoprovide informationontheoneormoreofthebloodvesselsfeaturesindifferen ttissuedepth.

Hence,inaccordancewithsomeaspects,thepresentdisclosurepro videsamethod comprising:(i)identifyingbloodvesselsinoneormoreimages,and(i i)determiningone ormoreofatleastonefeatureoftheidentifiedbloodvessels,acolori ndex(5?)forthe identifiedbloodvesselsoracombinationthereof.

Hence,inaccordancewithsomeaspects,thepresentdisclosurepro videsamethod comprising:(i)identifyingbloodvesselsinoneormoreimages,and(i i)determiningat leastonefeatureoftheidentifiedbloodvesselsandacolorindex(5?) fortheidentified bloodvessels.

Asdescribedherein,themethodallowstoexaminetherelationship betweenthe z-coordinateofabloodvesselasdeterminedbythecolorindex(5?)and thebloodvessels characteristics/features.In someembodiments,themethodcomprisesobtainingoneor moreimagesofthetissue. Asnotedherein,therearedifferentimagingtechniquesthatallow monitoringthe bloodvessels,including,forexampleacameraoranopticfiber.

Thepresentdisclosureisnotlimitedto aspecificimaging method and can be applicabletoanyspectroscopymethod,providedthatitallowsaspati altwo-dimensional imageresolutionthatallowstoresolvethevascularbloodvessels,na mely-10-100pm.

Asappreciated,theincidentlightspectrum isgivenbytheselectedillumination means.Itmayalsobeinconjunctureorreplacedbytheexcited/spontan eouslightemitting processescontributedbythebloodvesselsandthecontainedbloodins idethesevessels. Artificial light-emitting elements may also serve as illumination means (e.g., fluorophores, Q-dots, etc.). The wavelength dependent absorption and scattering mechanismsappliedtotheincident/emittedlightaredeterminedbyth e(partial)tissue slabitcrossesuntilithitsthebloodvessel(ifnotself-emitted)and onitswaybacktothe collecting device(e.g.,camera,fiber,fiberbundle).In caseofan illumination source residinginsideornearbythebloodvesselitself,onlyhalfthelightp athtravelistaken intoaccount.

In caseofreflection,thereflection (magnitude,spectrum,polarization)offthe targetbloodvesselisdictatedbythebloodvesselandthecontainedbl oodreflectance characteristicsand thedetection sensitivity isdetermined by thefilters(including the camera,e.g.,the RGB filtersofthe Bayerpattern),the camera opticsand response functionofitssensor.

Asusedherein,obtainingoneormoreoftheimagesfrom thesurfaceofthetissue refersto imagesthatare obtained withoutphysically penetrating thetissue.In other words,themethodcomprisesobtainingtheoneormoreoftheimagesfrom thesurface ofthetissuereflectscapturingavisualrepresentationofthetissue withoutpenetrationor insertionorincisionintothebody such thattheimagesareobtainedfrom outsidethe tissue.Insomeotherexamples,themethodcomprisesobtainingoneorm oreimagesfrom thesamedistancetosurfaceofthetissue.

Hence,themethodprovidesinsomeexamples,anon-invasivemethod thatallows capturing(obtaining)imagesofatissuesurfacewithoutcausingdama georinjurytothe tissue. Insomeexamples,oneormoreimagesareobtainedbyvisiblelightorinf ra-red oranycombinationthereof.

Insomeotherexamples,oneormoreimagesareobtainedbyvisibleli ght.

In somefurtherexamples,oneormoreimagesarecolorimagesobtained by visiblelightspectroscopy.

In someembodiments,themethodcomprisesobtainingoneormoreimagesin digitalformat.

Hence,inaccordancewithsomeaspects,thepresentdisclosurepro videsamethod forcharacterizingatleastonefeatureofbloodvesselsatdifferentd epthswithinatissue ofamammaliansubject,themethodcomprising:(i)obtainingoneormor eimagesofthe tissuefrom asurfaceofthetissue,(ii)identifyingbloodvesselsintheoneormor eimages, and(iii)determiningoneormoreof(a)atleastonefeatureoftheident ifiedbloodvessels (b)acolorindex(5?)fortheidentifiedbloodvesselsor(c)acombinat ionthereof.

Whenreferringtoimagesobtainedfrom thetissuesurface,itshouldbeunderstood thatthetissuesurfaceisconsideredtoform aplanarsurface.Thethreespatialdimensions ofthetissueareconsideredsuchthatthez-coordinaterepresentthev esseldepthorits averagedepth,withregardtothetissueoutersurfaceandtheplanarx- ydimensionsis representedbyx-andy-coordinates.

Theterm “depthofthetissue”isusedhereintorefertodistancebetweenla yersof atissueinthebodyandinthecontextofthepresentdisclosureisdefin edasadistance betweenthesurfaceofthetissueandaparticularlayerbeneathit.

Thebloodvesseldistancedefinedasthedistancefrom thetissuesurfacemaybe determinedstatisticallybythedifferentialchangeofthespectrum itreflectsoremitsdue toabsorptionandscatteringprocessesthelightundergoesduringits passagethroughthe tissuelayers.

Thedepthofthetissuemaydepend(beaffected)onvariousparamete rsincluding, inter alia,the tissue characteristics (for example degree of tissue transparency or complexity)andthedynamicrangeoftheimagecapturingdevice(forex ampleacamera).

Insomeexamples,thedepthofthetissueisatmostabout10cm,attim esatmost about9cm,attimesatmostabout8cm,attimesatmostabout7cm,attimes atmost about6cm,attimesatmostabout5cm,attimesatmostabout4cm,attimes atmost about3cm,attimesatmostabout2cm,attimesatmostabout1cm from thetissue surface.

Insomeexamples,thedepthofthetissueisatmostabout1cm,attime satmost about0.9cm,attimesatmostabout0.8cm,attimesatmostabout0.7cm,a ttimesat mostabout0.6cm,attimesatmostabout0.5cm,attimesatmostabout0.4 cm,attimes atmostabout0.3cm,attimesatmostabout0.2cm,attimesatmostabout0 .15cm from thetissuesurface.

Insomeexamples,thedepthofthetissueisbetweenabout0.001cm toabout10 cm,attimesbetweenabout0.001cm toabout5cm,attimesbetweenabout0.001cm to about3cm,attimesbetweenabout0.001cm toabout1cm,attimesbetweenabout0.001 cm toabout0.7cm,attimesbetweenabout0.001cm toabout0.5cm,attimesbetween about0.001cm toabout0.1cm.

Insomeexamples,thedepthofthetissueisbetweenabout0.005cm toabout10 cm,attimesbetweenabout0.007cm toabout10cm,attimesbetweenabout0.01cm to about10cm,attimesbetweenabout0.03cm toabout10cm,attimesbetweenabout0.07 cm toabout10cm,attimesbetweenabout0.1cm toabout10cm,attimesbetweenabout 0.3cm toabout10cm,attimesbetweenabout0.7cm toabout10cm,attimesbetween about1cm toabout10cm.

Insomeexamples,thedepthofthetissueisabout0.005cm,attimesa bout0.01 cm,attimesabout0.03cm,attimesabout0.05cm,attimesabout0.07cm, attimesabout 0.1cm,attimesabout0.3cm,attimesabout0.5cm,attimesabout0.7cm, attimesabout 1cm,attimesabout3cm,attimesabout5cm,attimesabout7cm,attimesa bout10 cm.

Asdescribedherein,themethodcomprisesidentifyingoneormoreb loodvessels. Asusedhereintheterm bloodvesselsencompassesanartery,avein,acapillaryorany combinationthereof.Theterm oneormorebloodvesselsasusedhereinmaybedenoted astimeasbloodvesselpopulation.

ThetypicaldiameterrangeofbloodvesselsisshownintheTablebel ow:

Bloodvesselscanbeidentifiedbyemployingdifferentmethods.Fo rexample,the bloodvesselscanbeidentifiedbythemethoddescribedintheexamples below andas showninFigures7A and7B.

Insomeexamples,themethodcomprisessegmentingoneormoreblood vessels from animagetoidentify saidoneormorebloodvessels.Inotherwords,identifying bloodvesselscomprisessegmentingthebloodvesselsinanimage.Segm entationcanbe donebyanymethodknownintheart,including,forexample,commercial software,such asMATLAB.

Theidentifiedoneormorebloodvesselsmaybesubjecttoanalysisa sdescribed herein.

In some embodiments,the method comprises analyzing the identified blood vesselsinordertodetermineatleastonefeatureofthebloodvessels. Asdescribedherein and in accordance with some examples,determining atleastone feature comprises calculating aprobability distribution function (PDF)foratleastonefeature.In some examples,determiningtheatleastonefeaturecomprisesobtainingas tatisticalensemble fortheatleastonefeatureinapopulationofbloodvessels.

Insomeembodiments,thepresentdisclosureprovidesamethodforc haracterizing atleastonefeatureofatissueofamammalian subjectatvariousdepthsofatissue, whereinthetissuedepthisasdescribedherein,atmostabout10cm beneaththetissue surface.Hence,themethodallowscharacterizationofatleastonefea tureofthetissuein differentdepthsofthetissuerangingbetweenthetissuesurfaceanda sdescribedherein, atmostabout10cm beneaththetissuesurface.

Theselectedfeaturemaybeanyfeaturethatisrepresentativeofab loodvessel, forexample,afeaturethatdiffersindifferentdepthsofatissue,i.e .,abloodvesselfeature thatisdifferentatatissuesurfaceandinashallow layer.

In someembodiments,thebloodvesselfeatureisorcomprisesofbloodves sel structure.As appreciated,blood vessels are considered to have tube-like structures, typically made up of three layers.In some embodiments,the method comprises determiningatleastonefeatureofbloodvesselstructure.

Insomeembodiments,thebloodvesselfeatureisorcomprisesblood vesselsize. Asappreciated,bloodvesselsvaryindiameter,rangingfrom largearteriesandveinsthat maybeseveralcentimetersindiameter,totinycapillariesthatareon lyafew micrometers indiameter.Insomeembodiments,themethodcomprisesdeterminingat leastonefeature ofbloodvesselsize.

In some embodiments,the blood vesselfeature isorcomprisesblood vessel elasticity.Asappreciated,thethree typesofblood vesselsmay be characterized by differentelasticity,forexample,arterieshaveahigherdegreeofel asticitythanveins, henceallowingthem toexpandandcontractinresponsetochangesinbloodpressure.In some embodiments,themethod comprisesdetermining atleastone feature ofblood vesselelasticity.

Insomeembodiments,thebloodvesselfeatureisorcomprisesblood flow inthe bloodvessels.Asappropriated,bloodvesselsareresponsibleforthe distributionofblood throughoutthebody such thattheflow ofblood isregulated by thecontraction and relaxationofthesmoothmusclesinthewallsofthebloodvessels.Inso meembodiments, themethodcomprisesdeterminingatleastonefeatureofbloodflow.

Insomeembodiments,thebloodvesselfeatureisorcomprisesvasoc onstriction andvasodilation.Asappreciated,thediameterofbloodvesselscanbe controlledbythe nervoussystem and varioushormonesthrough theprocessesofvasoconstriction and vasodilation.Vasoconstrictionisthenarrowingofbloodvessels,wh ilevasodilationisthe wideningofbloodvessels.Insomeembodiments,themethodcomprisesd eterminingat leastonefeatureofvasoconstrictionandvasodilation.

In someembodiments,theatleastonefeaturecomprisesatleastoneofblo od vesseldiameter,blood vesselwidth,blood vessellength,blood vesseldegree of tortuosity,oxygensaturationlevel,flow patternoranycombinationthereof.

Insomeembodiments,atleastonefeaturecomprisesbloodvesselwi dth(w).

Asdescribedherein,itmaybeassumedthatthebloodvesselhasacir cularshape andhencethebloodvesselwidthisasthebloodvesseldiameter. Asusedherein,bloodvesselwidthreferstothebloodvesseldiameter, whichis themeasurementofthedistanceacrossthebloodvesselinoneormorepo intsoftheblood vessel,forexample,atitswidestpoint.

Insomeexamples,thebloodvesselwidth(w)iscalculatedas:w = w^l'^dl'.

Thediameterofbloodvesselsmayplayanimportantroleindetermin ingtherate ofbloodflow throughthevessel.Hence,thediametermayprovideinformationonthe bloodflow.

Asdescribed herein,the depth ofa tissue correlated with colorinformation obtainedfrom thebloodvesselsandspecificallywithacolorindex.

Insomeembodiments,thecolorindexiscalculatedfrom thebloodvesselscolor bands.

Insomeembodiments,themethodcomprisesextractingcolorinform ationfrom oneormoreimages.

In someembodiments,thecolorindexiscalculatedfrom acombinationofthe bloodvesselsredcolor,greencolorandredcolor.

Insomeotherembodiments,themethodcomprisesextractioncolori nformation from oneormoreimagesusingred-green-blue(RGB)colormodel.

Asappreciated,inaRGB colormodel,eachcolorisrepresentedasacombination ofthreeprimarycolors:red,green,andbluevalues,usuallyranging, forexample,from 0 to255suchthatforexample,redisrepresentedas(255,0,0),greenis( 0,255,0),and blueis(0,0,255).

Insomeembodiments,themethodcomprisesdeterminingthedistrib utionofred colorvalue,greencolorvalueandbluecolorvalueinoneormorebloodv essels.

Insomeembodiments,themethodcomprisesapplyingapixel-by-pix elanalysis ontheidentifiedbloodvessels.

Asappreciated,apixel-by-pixelanalysisreferstoexamination/ interpretationof animageatanindividualpixellevel.Inotherwords,apixel-by-pixel analysisinvolves analyzingeachpixelinanimageandconsideringoneormoreofitscolor ,intensityand texture. Insomeembodiments,themethodcomprisesapplyingapixel-by-pixela nalysis oftheredcolorvalue,thegreencolorvalueandthebluecolorvalueval uesineachone oftheoneormorebloodvessels.

In someembodiments,themethodcomprisesextractingaredcolorcompone nt (R _p )foreachpixelintheidentifiedbloodvesselstoobtainasetof redcolorvalues.

In some otherembodiments,the method comprisesextracting a green color component(G _P )foreachpixelintheidentifiedbloodvesselstoobtainasetof greencolor values.

In some furtherembodiments,the method comprisesextracting a blue color component(B _p )foreachpixelintheidentifiedbloodvesselstoobtainasetof bluecolor values.

Insomeembodiments,themethodcomprisesgeneratinganoutputind icatingthe red colorcomponentvaluesand theircorrespondingpixellocationswithin theoneor moreimages.

Insomeembodiments,themethodcomprisesgeneratinganoutputind icatingthe bluecolorcomponentvaluesandtheircorrespondingpixellocationsw ithinoneormore images.

Insomeembodiments,themethodcomprisesgeneratinganoutputind icatingthe greencolorcomponentvaluesandtheircorrespondingpixellocations withinoneormore images.

In someembodiments,themethodcomprisesdetermining anaveragevaluefor eachoneofred,greenandbluevaluesineachoneoftheoneormorebloodv essels.

Insomeembodiments,themethodcomprisescalculatingameanvalue oftheset ofredcolorvaluestodetermineanaverageredcolor(Ravg)oftheident ifiedbloodvessels. Insomeotherembodiments,themethodcomprisescalculatingameanval ueofthesetof green colorvaluesto determinean averagegreen color(G _avg )oftheidentified blood vessels.Insomefurtherembodiments,themethodcomprisescalculati ngameanvalueof thesetofbluecolorvaluestodeterminean averagebluecolor(B _avg )oftheidentified bloodvessels. Asdescribedherein,thecolorindex(denotedhereinas isanumericalvalue calculatedforeachbloodvessel(identifiedbloodvessel)andiscons ideredhereintobe indictiveofthetissuedepth.

Inaccordancewithsomeembodiments,thecolorindexiscalculated from oneor morecombinationsofR _avg ,GavgandBavg.

In some examples,thecolorindex isadimensionlessquantity and hencethe combinationofR _avg ,G _aV gandB _aV gisgivenasanyratioofR _avg ,G _avg andB _aV g.

In accordance with some embodiments, the color index is calculated as (Bavg-i-Gavg)/Ravg(FormulaI).

Inaccordancewithsomeotherembodiments,thecolorindexiscalcu latedasB _avg /Ravg(FormulaII).

Inaccordancewithsomeotherembodiments,thecolorindexiscalcu latedasG _aV g /Ravg(FormulaIII).

Asdescribedherein,thecolorindexhasadifferentvalueindiffer entdepthsof thetissue.Figure4 showsamodel-basedgraph showingthechangesin acolorindex calculatedbyFormula(I)asafunctionofthebloodvesseldepthsugges tingthatthecolor indexisasensitivemeasurethatisassociated(correlated)with thedepthoftheblood vessel.Inotherwords,itwassuggestedthatthecolorindexhasadiffe rentvalueatthe tissuesurfaceandatadeepertissuelayer.

Bydeterminingthetissuedepth,i.e.,thezcoordinate,thepresen tmethodmay providespatialcoordinatesofbloodvesselsinthetissue.

Insomeexamples,themethodisfordeterminingspatialcoordinate softheblood vesselswithinthetissue.

Thespatialcoordinatesofbloodvesselsasusedhereinrefertothe locationofthe bloodvessel(s)inthebodyandspecificallyintheexaminedtissue,op tionally,relativeto otherstructures.Asappreciated,spatialcoordinatesofbloodvesse lsmaybedefinedusing aCartesian coordinatesystem,which consistsofthreeaxes:x,y,and z,such thatas describedherein,thez-axisrepresentsthedepthordistancefrom asurfaceoftheissue.

Theinformation obtained by themethodsdescribed herein may beusefulfor determiningvariouscorrelations,includingoneormoreofspatialco rrelation,directional correlation,morphologicalcorrelation,functionalcorrelationor anycombinationthereof. Typically,suchcorrelationsmaybedeterminedusingstatisticalmet hods.

Insomeexamples,themethodisfordeterminingspatialcorrelatio nofatleastone featureofthebloodvesselsand/orofthespatialcoordinatesofthebl oodvessels.

Asusedherein,theterm spatialcorrelationreferstotheexcess(overrandom) probabilityoffindingatleastonefeatureofthebloodvesselsand/or spatialcoordinates ofthebloodvesselsgiventhelocationandatleastonefeatureofanoth erbloodvessel.

Inotherwords,itcharacterizesthepatternofthebloodvessels's patialdistribution withinthetissueorthesameforatleastonefeaturethereof.

Insomeexamples,themethodisfordeterminingdirectionalcorrel ationofatleast onefeatureofthebloodvesselsand/orofthespatialcoordinatesofth ebloodvessels.

Asusedherein,theterm directionalcorrelationreferstotheexcess(overrandom) probabilityoffindingatleastonefeatureofthebloodvesselsand/or spatialorientation ("direction")ofthebloodvesselsgiventhelocationandatleastonef eatureofanother bloodvesselinagivenorientation("direction").

Insomeexamples,themethodisfordeterminingmorphologicalcorr elationofat leastonefeatureofthebloodvessels.

Asusedherein,theterm morphologicalcorrelationreferstothedegreetowhich theshapeorstructureofthebloodvesselsasdeterminedbyatleastone featureofthe bloodvesselsiscorrelatedwithotherbloodvessels(orotherfeature s).

Insomeexamples,themethodisfordeterminingfunctionalcorrela tionofatleast onefeatureofthebloodvessels.

Asusedherein,theterm functionalcorrelationreferstothedegreetowhichthe functionofthebloodvesselsasdeterminedbyatleastonefeatureofth ebloodvesselsis correlatedwithotherbloodvessels(orotherfeatures).

Asnotedherein,theabilityto distinguish betweendifferentdepthsofatissue allowsgatheringinformationononeormorefeaturesofthebloodvesse lsindifferent depthsofthetissueinordertoprovideinformationonthetissuephysi ology.

Hence,inaccordancewithsomeembodiments,themethodisforchara cterizing physiologicalprocessin the tissue.The physiologicalprocessmay be any process associatedwith bloodvesselsaswellasanyprocessassociatedwith changesinblood vesselscharacteristics.

Insomeembodiments,thephysiologicalprocessisoneormoreofblo odpressure regulation,bloodclotting,differentiation,neovascularization, angiogenesisandapoptosis oranyrelatedprocesses.

Insomeembodiments,thephysiologicalprocessisbloodpressurer egulation.

In someembodiments,themethod isforcharacterizing blood pressureorany relatedprocessinthetissue.

Bloodpressureregulation asusedhereinrefersto aphysiologicalprocessthat controlstheforceorpressureofbloodagainstthewallsofarteriesas itcirculatesthrough thebody.Bloodpressureisregulatedbyvariousprocesses,suchasadj ustingthediameter ofbloodvessels.Relatedprocessesassociatedwithbloodpressurein clude,forexample, vasoconstriction.Vasoconstrictionisaphysiologicalprocessasso ciatedwithdecreased widthornarrowingofbloodvessels.

Insomeembodiments,thephysiologicalprocessisbloodclotting.

In someembodiments,themethod isforcharacterizing blood clotting orany relatedprocessinthetissue.

Bloodclotting,alsoknownascoagulation,asusedhereinrefersto aprocessby which thebody stopsbleedingbyforming aplugoffibrin andplateletsatthesiteof injury.

Insomeembodiments,thephysiologicalprocessisdifferentiatio n.

In someembodiments,themethod isforcharacterizing differentiation orany relatedprocessinthetissue.

Insomeembodiments,thephysiologicalprocessisneovasculariza tion.

Insomeembodiments,themethodisforcharacterizingneovascular izationorany relatedprocessinthetissue.

Insomeembodiments,thephysiologicalprocessisangiogenesis.

In some embodiments,the method isforcharacterizing angiogenesisorany relatedprocessinthetissue. Angiogenesisasusedhereinreferstoaprocessbywhichnew bloodvesselsform from existing blood vessels.Generation ofnew blood vesselsmay beviasprouting, intussusception(splittingangiogenesis)orelongation.

Neovascularizationasusedhereinreferstoapathologicaltypeof angiogenesis, such as in diseases like cancer, diabetic retinopathy, and age-related macular degeneration,wherethegrowthofnew bloodvesselscancontributetotissuedamageand diseaseprogression.

Insomeembodiments,themethodisforcharacterizingtissuegrowt handrepair.

Insomeembodiments,themethodisforcharacterizingsproutingan dbranching ofnew bloodvesselsfrom preexistingbloodvessels.

Insomeembodiments,thephysiologicalprocessisapoptosis.

Insomeembodiments,themethodisforcharacterizingapoptosisin thetissue.

Insomeembodiments,themethodisforcharacterizingwoundhealin g.

Insomeembodiments,themethodisforcharacterizingtumorprogre ssion.

Characterization ofphysiologicalprocessesassociated with blood vesselsand specificallychangesinbloodvesselsfeaturesmayberelevantfordia gnosisofvarious conditions/disease.

Insomeembodimentsthatmaybeconsideredasaspectsofthepresent disclosure, themethod isamethod fordiagnosisacondition oradiseaseassociated with blood vessels.

In someembodiments,themethod isfordiagnosisacondition associatedwith tissuegrowthandrepair.

In someembodiments,themethod isfordiagnosisacondition associatedwith sproutingandbranchingofnew bloodvesselsfrom preexistingbloodvessels.

In someembodiments,themethod isfordiagnosisacondition associatedwith apoptosis.

In someembodiments,themethod isfordiagnosisacondition associatedwith apoptosisinthetissue. In someembodiments,themethod isfordiagnosisacondition associatedwith woundhealing.

In someembodiments,themethod isfordiagnosisacondition associatedwith tumorprogression.In some embodiments,the method isfordiagnosisproliferative disorder.

As appreciated,analysis ofthe 3D blood vesselsnetwork and consequently obtaininginformationonaphysiologicalprocessandoptionallyrega rdingaconditionor diseaseassociatedwiththisprocessovertime(i.e.,inatleasttwose paratedtimepoints) isalsoimportantasitallowscomparisonofthe3D bloodvesselsnetworkand/oratleast onefeatureasdescribedhereinatdifferenttimesandhencemeasureth eirevolution.

Hence,thepresentdisclosureprovidesamethodforcharacterizin gbloodvessel evolutionovertime.

Asusedherein,bloodvesselevolutionreferstotheprocessbywhic hbloodvessels aredevelopedandchangedovertimethroughnormalaswellasabnormalm echanisms. As appreciated,while in some cases,evolution of blood vessels contributes to developmentofahighlyefficientcirculatorysystem,capableofdeli veringoxygenand nutrientstoallpartsofthebody,insomecasessuchevolutionmaybeas sociatedwith improperdevelopmentandevendiseasesandotherhealthproblems.

In someembodiments,thephysiologicalprocessisassociated with changesin bloodvesselsandthebloodvessels’spatialdistributionovertime .

Insomeembodiments,themethodisforcharacterizingchangesinbl oodvessels withtime.

Hence,thepresentdisclosureprovidesdynamicmeasurementsofth eleastone featureinthetissue.

Asusedhereintheterm dynamicmeasurementsrefertotheabilitytomeasure changesinthetissueandspecificallytoatleastonebloodvesselfeat ure,overtime.As furtherdescribedherein,suchmeasurementsmaybeusedtomonitortis suefunctionand todiagnoseand/ortreatdisease. Insomeembodiments,themethodcomprisesdeterminingtheatleastone feature inatleasttwotemporarilyseparatedtimepointstomonitorchangesin bloodvesselsin thedifferentdepthswithinthetissueovertime.

AsshowninFigures11A and11B,changeswereobservedinthecalculatedratio atdifferentdaysofthecycle.Specifically,theslopechangeofthera tioevolutionshows apivotalpointatornearday14("ovulation"),whichseparatedthetwo cyclephases.

Hence,inaccordancewithsomeaspects,thepresentdisclosurepro videsamethod forcharacterizingatleastonefeatureofbloodvesselsatdifferentd epthswithinatissue ofamammaliansubject,themethodcomprising:(i)identifyingbloodv esselsinanimage obtainedfrom asurfaceofsaidtissue,(ii)determiningoneormoreofatleastonefea ture oftheidentified blood vessels,acolorindex forthe identified blood vesselsorany combinationthereof,whereinthecolorindexdiffersindifferentdep thswithinsaidtissue and(iii)repeatingsteps(i)and/or(ii)inatleastonemoretemporall yseparatedtimepoint.

Thetwotemporarilyseparatedtimepointsasusedhereinencompass twodistinct momentsintimethatareseparatedbyaperiodoftime.

Theimagesobtainedatthetwotemporarilyseparatedtimepointsma yprovidea continuousmonitoringofthetissueovertimeoranintermittentmonit oringofthetissue overtime.

Insomeexamples,themethodprovidesacontinuousmonitoringofth etissue.In someexamples,continuousmonitoringallowscharacterizationofblo odflow.

Insomeexamples,themethodprovidesanintermittentmonitoringo fthetissue. Intermittentmonitoring,i.e.anon-continuousmonitoringofthetis suereferstoamethod ofmeasuringorobservingatissueatsetintervalsorspecifictimes,r atherthanconstantly orinreal-time(refershereinascontinuous).

In someembodiments,employingthemethodofthepresentdisclosureatle ast twoofthetemporarilytimepointsallow acomparisonbetweentwostatesofthetissue andspecificallyoftheatleastonefeatureoftheidentifiedbloodves selsasdeterminedat differentdepthswithinthetissue.

Inaccordancewiththepresentdisclosure,thechangethatoccursb etweenthetwo timepointsmaybesignificantorrelativelyminor,butthecomparison betweenthem may provideinsightintochanges/developmentsin3D bloodvesselsnetworkovertime. Thetemporarilyseparatedtimepoints,eitherprovidingcontinuousm onitoringor intermittent monitoring, may be any amount of time apart suitable to obtain characterizationofbloodvessels.

Forexample,the temporarily separated time pointsmay be a few seconds, minutes,hours,daysandevenmore.

Insomeembodiments,thegapbetweenthetwotemporarilyseparated timepoints is about 1 millisecond, about 5 milliseconds, about 10 milliseconds, about 50 milliseconds,about100milliseconds,about500milliseconds.

Insomeembodiments,thegapbetweenthetwotemporarilyseparated timepoints isabout1second,about5seconds,about10second,about50second.

Insomeembodiments,thegapbetweenthetwotemporarilyseparated timepoints isabout1minute,about5minutes,about10minutes,about50minutes.

Insomeembodiments,thegapbetweenthetwotemporarilyseparated timepoints isabout1hour,about5hours,about10hours,about24hours.

Insomeembodiments,thegapbetweenthetwotemporarilyseparated timepoints isabout1day,about5days,about10days,about14days.

Insomeembodiments,thegapbetweenthetwotemporarilyseparated timepoints isabout1month,about5months.

Thepresentdisclosureisnotlimited to a specific tissue and isapplicableto varioustissuesinasubject.

Insomeembodiments,thetissueisassociatedwithchangesinblood vesselswith time.

Insomeembodiments,thechangesinbloodvesselscomprisedestruc tionofblood vessels.

Asused herein theterm destruction of blood vesselsrefersto theprocessof damaging orbreaking blood vessels,forexample the wallsofblood vessels.These processesmayinturnresultinbleedingandimpairedbloodflow totissuesandmayoccur indifferentconditions,suchastrauma,disease,ormedicalprocedur es. In someembodiments,destruction ofblood vesselsisassociated with uterine fibroids,atherosclerosis,aneurysms,vasculitis,and hemorrhagic stroke.Hence,the methodsdescribedherein areapplicableformonitoring (diagnosis)ofoneormoreof uterinefibroids,atherosclerosis,aneurysms,vasculitis,andhemo rrhagicstroke.

Insomeembodiments,thechangesinbloodvesselscompriseenhance dgrowthof bloodvesselsinsaidtissue.

Asusedhereintheterm enhancedgrowthofbloodvessels,encompassesanormal physiologicalprocessofbloodvesselsgrowthorapathologicalproce ssofbloodvessels growth.

The enhanced growth may be abnormalgrowth ofblood vesselsleading to damagetothetissue.

As noted herein above, an example process of blood vessel growth is angiogenesis.While in some tissues,angiogenesisisessentialfortissue growth and repair,as itallows for the delivery ofnutrients and oxygen to tissues,in certain pathologicalconditionssuch ascancer,angiogenesiscanbecomeexcessive,leadingto theformationofabnormalbloodvesselsthatcanpromotetumorgrowtha ndmetastasis.

Insomeembodiments,thetissuecomprisesatleastaportionoftheb loodvessels thatcanbeviewedfrom outsidethetissue.

Insomeembodiments,thetissueisselectedfrom thegroupconsistingof:liver, kidneys,lungs,brain,heart,intestine,muscles,skin,aretina,ute rineand acancerous tissue.

Insomeembodiments,thetissueisuterine.

A schematicrepresentationoftheuterineisshowninFigure1.Inadditi on,Figure

2 showsaschematicrepresentation oftheuterinetissuecomprising theendometrium tissuethatiscomposedofafunctionallayerandabasallayer.Asfurth ershowninFigure

3representingamodel-basedgraph,avisiblelightcanpenetratet heendometrium tissue andhenceprovideinformationondifferentdepthswithinthistissuea sdiscussedherein.

Insomeembodiments,thetissueistheendometrium tissue.Endometrium refers totheinnerliningoftheuterus.Intheendometrium tissue,bloodvesselsdistributionand evolution play a distinctive role in the tissue characterization.During the feminine menstrualcycleofhealthy,fertilewomen(ormammalsingeneral)theb loodvesselthree- dimensional network changes at a relatively short period of time.Most of the endometrium tissue isbeing generated and washed outduring the cycle within the timescaleoftensofdays.Wellaccepted istheparadigm thatangiogenesiswithinthe endometrium iscloselyrelatedtothesuccessorfailureof,e.g.,embryoimplantat ion.

Figures 5A-5F show two different theoretical scenarios of blood vessels evolution,with Figures 5A-5C showing blood vessels evolution led primarily by elongation,followedbycapillarygrowththrough splittingangiogenesis.Inthismodel, on the shallowerstrata (Figure 5C)relatively big diameterblood vesselsare found. Figures5D-5Fshow amodelbywhichbloodvesselsevolutionofnewlyfoundlayersis primarilygovernedbysproutingofnewlyformedcapillariesandassuc hshallow strata (Figure5F)donotcontainbloodvesselswithlargediameter.Itshould benotedthatin casetimepermits,bloodvesselswithlargediametermaybeformedatth edeeperlayers.

In humans,blood supply to and within the endometrium ismaintained by a cascadeofbloodvesselsofvariousdiameters.Theuterineartery,com ingofftheovarian one,suppliesblood to thearcuate arteriesfrom which theradialarteriesrun radially towardstheuteruslumen and splitintothebasaland spiralarteries(arterioles)which reside within the endometrium.The smallestdiameter vessels in the endometrium undergovascularizationandform capillaries.This3D bloodvesselnetworkpromotesand controlsthetissuegrowth,nourishesitandkeepsupwithitsstructur ebuild-up.Blood vesselsofsmallerdiametersemergefrom themainarteriesinever-decreasingsizesand then,totheoppositedirectionreconnectwiththemainveins.Allbloo dvesselswithinthe endometrium aregeneratedasvesselsofsmalldiameterandgrow totheirfinaldiameter (beforetheendometrialshedding)inacontrolledprocess.

AsshowninFigures8A-8F,Figures9A-9F andFigures12A-12E,acleartrend wasobservedbetweenthebloodvesseldiameterandthedepth.

Interestingly,Figures 8A-8F and Figures9A-9F providing data from human samplesoftheendometrium tissue,show atrendofashorteningdistributiontailsinblood vesselsthatareclosertothetissuesurface.Incontrast,Figures12A -12Eporividingdata from swinesamples,atailofrelativelylargediametervesselswasobserve dinthedeeper layers.Thedifferenceinbloodvesselsasobservedinthehuman samplesvs.thenonhumansamples,suggestthatthemethodsdescribedh ereinmaybeconsideredasreliable methodforfollowingblood vesselevolution (such aschangesin diameter)invarious depth.

Itwassuggestedthatmonitoringchangesofthe3D bloodvesselsnetworkintime andspace(i.e.,indifferentdepthsoftheendometrium tissue),mayprovideinformation regardingtheendometrium statusandassistsinevaluatingtheuterusreadinessfore.g., embryoimplantation.

Hence,inaccordancewithsomeaspects,thepresentdisclosurepro videsamethod forcharacterizing atleastone featureofblood vesselsatdifferentdepthswithin an endometrium tissueofamammaliansubject,themethodcomprising:

(i)identifyingbloodvesselsinanimageobtainedfrom asurfaceoftheuterine,

(ii)determiningatleastonefeatureoftheidentifiedbloodvesse ls,acolorindex fortheidentifiedbloodvesselsoracombinationthereof,whereinthe colorindex differsindifferentdepthswithintheendometrium tissue.

Inaccordancewithsomeotheraspects,thepresentdisclosureprov idesamethod forcharacterizingthediameterbloodvesselsatdifferentdepthswit hinanendometrium tissueofamammaliansubject,themethodcomprising:

(i)identifyingbloodvesselsinanimageobtainedfrom asurfaceoftheuterine,

(ii)determiningthediameteroftheidentifiedbloodvessels,aco lorindexforthe identifiedbloodvesselsoracombinationthereof,whereinthecolori ndexdiffers indifferentdepthswithintheendometrium tissue.

Asnotedherein,thedevelopmentofbloodvesselsintheuterusisac ontrolled processandhenceitisvaluabletocharacterizeoneormorefeaturesof thebloodvessels, including,interalia,thediameterbloodvesselsatdifferentdepths withinanendometrium tissueofamammaliansubjectindifferenttimepoints.

In someembodiments,themethodcomprisesrepeatingsteps(i)and/or(ii )ata temporallyseparatedtimepointasdescribedherein.

Figures6A-6Ishow twodifferenttheoreticalrepresentationsthatmayrepresent differentindividualsubjectsandtheirrecordedbloodvesselsdiame terPDFsasfunction oftissuedepth.BothindividualshavethesamePDFonsamedayshowninF igures6A- 6C,yetafterm days,itwaspossibletodistinguishbetweentwodifferentpatternsin the developmentofthe blood vessels.Specifically,the blood vesselsofthe theoretical individualrepresentedinFigures6D-6Fdidnotevolvetothedegreeth eyshouldinorder to fittheir calendar day,whereas the blood vessels of the theoreticalindividual representedinFigures6G-6I,evolvedatthestandardpacetoshow thediameterPDFsas functionoftissuedepth,exactlythewaytheyshouldbeatthecalendar time.

Itwassuggestedthatbyobtaininginformationonthe3D bloodvesselnetworkin theuterine tissue and specifically in the endometrium tissue atdifferentdepthsand differenttimespoints,itwouldbepossibletocontributetoprecisee ndometrialdatingand may assistin more accuratedetermination ofembryo transfertiming within in vitro fertilization(IVF)treatments.

Itwasfurthersuggested thatby obtaining informationon the3D bloodvessel networkintheuterinetissueandspecificallyintheendometrium tissueatdifferenttimes points,itwouldbepossibletoassesschangesoccurringintheuterusd uringthemenstrual cycle.Suchinformationmaybevaluableinconnectionwithvariouscli nicalsituations.

Insomeembodiments,themethodisformonitoringmenstrualcyclei nasubject.

Themenstrualcycleisanaturalreproductiveprocessoccurring in femalesof reproductive age thatinvolvesa seriesofhormonaland physiologicalchangesthat preparetheuterusforpregnancyeachmonth.Themenstrualcycletypic allylasts28days, butitcanrangefrom 21to35daysinsomeindividuals.Duringthemenstrualcycle,the bodyundergoesseveralphases,includingthemenstrualphase(whenth euterusshedsits lining),thefollicularphase(whentheovarypreparestoreleaseaneg g),ovulation(when aneggisreleasedfrom theovary),andthelutealphase(whentheuteruspreparesfora possiblepregnancy).Thesechangesareaccompaniedbychangesintheb loodvessels, whichsupplythetissuewithoxygenandnutrients.

Itwassuggestedthatmonitoringchangesinthe3D bloodvesselsnetworkusing themethodsofthepresentdisclosuremayprovidevaluableinformatio naboutthehealth oftheendometrialtissueandhelpdiagnoseconditionssuchasendomet rialhyperplasia, whichcanincreasetheriskofendometrialcancer.

Hence,thepresentdisclosureprovidesinaccordancewithsomeasp ects,amethod formonitoringmenstrualcycleinasubject,themethodcomprises:(i) identifyingblood vesselsinanimageobtainedfrom asurfaceoftheuterine,(ii)determiningatleastone featureoftheidentifiedbloodvessels,acolorindexfortheidentifi edbloodvesselsora combination thereof,wherein the colorindex differsin differentdepthswithin the endometrium tissue.

Inaccordancewithsomeembodiments,themethodisformonitoringm enstrual cycleinasubjectandtheatleastonefeatureisthediameteroftheiden tifiedbloodvessels.

Inaccordancewithsomeembodiments,themethodisformonitoringm enstrual cycleinasubject,theatleastonefeatureisthediameteroftheidenti fiedbloodvessels andmethodcomprisesrepeatingsteps(i)and/or(ii)atatemporallyse paratedtimepoint asdescribedherein.

Insomeembodiments,themethodisfordiagnosisofinfertilityand /orinfertility- relatedconditionsinasubject.

Itwassuggestedthatmonitoringchangesinthe3D bloodvesselsnetworkusing themethodsofthepresentdisclosuremayprovidevaluableinformatio naboutinfertility and/orinfertility-relatedconditions.

Hence,thepresentdisclosureprovidesinaccordancewithsomeasp ects,amethod fordiagnosisofinfertility and/orinfertility-relatedconditionsin asubject,themethod comprises:

(i)identifyingbloodvesselsinanimageobtainedfrom asurfaceoftheuterine, (ii)determiningatleastonefeatureoftheidentifiedbloodvessels, acolorindexforthe identifiedbloodvessels,oracombinationthereof,whereincolorind exdiffersindifferent depthswithintheendometrium tissue.

In accordancewith someembodiments,themethodisforevaluatinginfertility infertility-relatedconditionsinasubjectandtheatleastonefeatu reisthediameterofthe identifiedbloodvessels.

In accordancewith someembodiments,themethodisforevaluatinginfertility infertility-relatedconditions,theatleastonefeatureisthediame teroftheidentifiedblood vesselsandmethodcomprisesrepeatingsteps(i)and/or(ii)atatempo rallyseparatedtime pointasdescribedherein.

The term infertility-related conditions asused herein refers to any medical conditionthatmayleadtodifficultyinconceivingorcarryingapregn ancytoterm. Insomeembodiments,theinfertility-relatedconditionscompriseon eormoreof the following:ovulation disorders,tubalblockage,endometriosis,uterineorcervical abnormalitiesorage-relatedinfertility.

Insomeembodiments,themethodisforselectingthetimingofembry otransfer andimplantationinasubject.

Hence,thepresentdisclosureprovidesinaccordancewithsomeasp ects,amethod forselectingthetimingofembryotransferandimplantationinasubje ctinasubject,the methodcomprises:(i)identifyingbloodvesselsinanimageobtainedf rom asurfaceof theuterine,(ii)determiningatleastonefeatureoftheidentifiedbl oodvessels,acolor indexfortheidentifiedbloodvessels,oracombinationthereof,wher einthecolorindex differsindifferentdepthswithintheendometrium tissue.

Inaccordancewithsomeembodiments,themethodisforselectingth etimingof embryotransferandimplantationinasubjectinasubjectandtheatlea stonefeatureis thediameteroftheidentifiedbloodvessels.

Inaccordancewithsomeembodiments,themethodisforselectingth etimingof embryotransferandimplantationinasubject,theatleastonefeature isthediameterof theidentified blood vesselsand method comprisesrepeating steps(i)and/or(ii)ata temporallyseparatedtimepointasdescribedherein.

In some embodiments, the method is for evaluating receptivity of the endometrium toembryoimplantationinasubject.

Hence,thepresentdisclosureprovidesinaccordancewithsomeasp ects,amethod forevaluatingreceptivityoftheendometrium toembryoimplantationinasubjectina subject,themethodcomprises:(i)identifyingbloodvesselsinanima geobtainedfrom a surfaceoftheuterine,(ii)determiningatleastonefeatureoftheide ntifiedbloodvessels, acolorindexfortheidentifiedbloodvessels,oracombinationthereo f,whereinthecolor indexdiffersindifferentdepthswithintheendometrium tissue.

Inaccordancewithsomeembodiments,themethodisforevaluatingr eceptivity oftheendometrium toembryoimplantationinasubjectinasubjectandtheatleastone featureisthediameteroftheidentifiedbloodvessels.

Inaccordancewithsomeembodiments,themethodisforevaluatingr eceptivity oftheendometrium toembryoimplantationin asubjecttheatleastonefeatureisthe diameteroftheidentifiedbloodvesselsandmethodcomprisesrepeati ngsteps(i)and/or (ii)atatemporallyseparatedtimepointasdescribedherein.

In some embodiments,the method isfordetermining suitability forembryo transferandimplantationintheuterineofasubject.

Itwassuggestedthatmonitoringchangesinthe3D bloodvesselsnetworkusing themethodsofthepresentdisclosuremayprovidevaluableinformatio nabouttimingof embryo transferand implantation in asubject,thereceptivity oftheendometrium to embryoimplantationinasubjectand/orsuitabilityforembryotransf erandimplantation inauterineofasubject.

Hence,thepresentdisclosureprovidesinaccordancewithsomeasp ects,amethod forevaluating suitability and timing ofembryo transferand implantation in asubject and/orreceptivityoftheendometrium toembryoimplantationinasubject,themethod comprises(i)identifyingbloodvesselsinanimageobtainedfrom asurfaceoftheuterine, (ii)determiningatleastonefeatureoftheidentifiedbloodvessels, acolorindexforthe identified blood vessels,oracombination thereof,wherein thecolorindex differsin differentdepthswithintheendometrium tissue.

In accordancewith someembodiments,themethodisforevaluatingsuitability and timing ofembryo transferand implantation in asubjectand/orreceptivity ofthe endometrium to embryo implantation in asubjectand the atleastonefeatureisthe diameteroftheidentifiedbloodvessels.

In accordancewith someembodiments,themethodisforevaluating timing of embryotransferandimplantationinasubjectand/orreceptivityofth eendometrium to embryoimplantationinasubject,theatleastonefeatureisthediamet eroftheidentified blood vessels and method comprisesrepeating steps (i)and/or(ii)ata temporally separatedtimepointasdescribedherein.

Insomeembodiments,thesubjectisafemalesubject.Insomeembodi ments,the subjectisconsideredtoundergoorintheprocessofIVF.

Insomeembodiments,inwhichthemethodemploysrepeatingmethods tepsata temporallyseparatedtimepoint,thetwotimespointsareselectedbet weenday 14and day22ofthecycle,whereinthecycleisnormalizedtoproducea28daysc ycle. AsshownintheExamplesbelow,theinventorsusedimagesoffresh,ex-v ivo, endometrialsamplesofdifferentcycledaystoobtainthestatistical evolutiontrackofthe bloodvesselpopulationinbothhumanandanimal(swine)samples.

Insomeembodiments,themethodisanex-vivomethod.

Insomeembodiments,themethodisaninvivomethod.

Forthepurposeofdiagnosis,theatleastonefeatureand/oranycha ngeoftheat leastonefeatureovertimemaybecomparedtothesamefeatureand/orth echangeofthe atleastonefeatureovertimeinafertilefemale.Fertilefemaleasuse dhereinreferstoa womanwhohasthephysicalabilitytoconceiveandcarryapregnancytot erm.

Asnotedherein,themethodsdescribedhereinareapplicableforav arietyoftissue andhencemaybeapplicablefordeterminingapathologicalconditiono fasubject.

Itwassuggestedthatmonitoringchangesinthe3D bloodvesselsnetworkusing the methods of the presentdisclosure may provide valuable information about a pathologicalconditionofthesubject.

Hence,thepresentdisclosureprovidesinaccordancewithsomeasp ects,amethod fordeterminingapathologicalconditionofasubjectinasubject,the methodcomprises:

(i)identifyingbloodvesselsinanimageobtainedfrom asurfaceofatissue,(ii) determining atleastonefeatureoftheidentified blood vessels,acolorindex forthe identified blood vessels,oracombination thereof,wherein thecolorindex differsin differentdepthswithinthetissue.Insomeembodiments,themethodco mprisesrepeating steps(i)and/or(ii)atatemporallyseparatedtimepointasdescribed herein.

In accordance with some embodiments,the method is for determining a pathologicalcondition ofasubjectand theatleastonefeatureisthediameterofthe identifiedbloodvessels.

Insomeembodiments,themethodisfordeterminingapathologicalc onditionof asubject,theatleastonefeatureisthediameteroftheidentifiedblo odvesselsandthe subjectisdiagnosedwithaconditionassociatedwithenhancedgrowth ofbloodvessels in thetissue.In some otherembodiments,the subjectissuffering from atleastone proliferativedisorder. As used herein, "proliferative disorder" is a disorder displaying hyper proliferation.Thisterm meanscelldivisionandgrowththatisnotpartofnormalcellular turnover,metabolism,growth,or propagation of the whole organism.Unwanted proliferationofcellsisseenintumorsandotherpathologicalprolif erationofcells,does notservenormalfunction,andforthemostpartwillcontinueunbridle datagrowthrate exceeding thatofcellsofa normaltissue in the absence ofoutside intervention.A pathologicalstatethatensuesbecauseoftheunwantedproliferation ofcellsisreferred hereinasa "hyperproliferativedisease"or "hyperproliferativedisorder." Itshouldbe notedthattheterm “proliferativedisorder”, “cancer”, “tumor”and “malignancy”allrelate equivalentlytoahyperplasiaofatissueororgan.

Thepresentdasyurealsoprovidesinaccordancewithsomeotherasp ect,amethod for diagnosing a pathological condition in a subject,the method comprising (a) determiningatleastonefeaturecharacteristicsofbloodvesselpopu lationfrom oneor moreimagesobtainedfrom atissueofsaidsubject,saidoneormoreimagesisobtained bylightimagingand(b)determiningifthesubjectissufferingfrom saidpathological disorder,wherein saidpathologicalcondition associatedwith enhancedgrowth and/or formationofbloodvesselsinsaidtissue.

Thepresentdasyurealsoprovidesinaccordancewithsomeotherasp ect,amethod ofassessing responsiveness to a treatmentregimen for a subjectsuffering from a pathologicaldisorderand monitoring diseaseprogression ofsaid subject,themethod comprising:(a)determiningatleastonefeaturecharacteristicsofb loodvesselpopulation from oneormoreimagesobtainedfrom saidsubject,saidoneormoreimagesisobtained byvisiblelightimagingand(b)determiningifthesubjectisresponsi veornon-responsive tothetreatmentregimen.

Asdescribedherein,themethodscomprisingrepairingthestepsof determiningat leastonefeaturecharacteristicsofblood vesselpopulation from oneormoreimages obtainedfrom atissueofsaidsubjectinatleastonetemporarilyseparatedtimepoin t.In someexamples,themethodcomprisescomparingtheatleastonefeature intheatleast twotemporarilyseparatedtimepoint.

Thepresentinventionrelatesto subjects,individualsorpatients.By “patient”, “individual”, “individuals “or“subject”itmeansanyorganism whomaybeaffectedby the above-mentioned conditions,and to whom methodsherein described isdesired, including humans.More specifically,the methodsofthe invention are intended for mammals.By “mammalian subject” ismeantany mammalforwhich the proposed therapyisdesired,includinghuman,equine,canine,andfelinesubje cts,mostspecifically humansandmorespecificallyafemale.

Theterm "about"asusedhereinindicatesvaluesthatmaydeviateupto1%,more specifically5%,morespecifically10%,morespecifically15%,andin somecasesupto 20% higherorlowerthan thevaluereferred to,thedeviationrangeincluding integer values,and,ifapplicable,non-integervaluesaswell,constituting acontinuousrange.In someembodiments,theterm "about"refersto± 10%.

Itshouldbenotedthatvariousembodimentsofthisinventionmaybe presented inarangeformat.Thedescriptionofarangeshouldbeconsideredtohav especifically disclosedallthepossiblesubrangesaswellasindividualnumericalv alueswithinthat range.Forexample,descriptionofarangesuchasfrom 1to6orbetween1and6should beconsideredtohavespecificallydisclosedsubrangessuchasfrom 1to3,from 1to4, from 1to5,from 2to4,from 2to6,from 3to6etc.,aswellasindividualnumberswithin thatrange,forexample,1,2,3,4,5,and6.

Asusedherein,theforms "a", "an"and "the"includesingularaswellasplural referencesunlessthecontextclearlydictatesotherwise.

Itisappreciated thatcertain featuresofthe invention,which are,forclarity, describedinthecontextofseparateembodiments,mayalsobeprovided incombination in asingleembodiment.Conversely,variousfeaturesoftheinvention,w hich are,for brevity,describedinthecontextofasingleembodiment,mayalsobepr ovidedseparately orinanysuitablesubcombinationorassuitableinanyotherdescribed embodimentof theinvention.Certainfeaturesdescribedinthecontextofvariousem bodimentsarenot to be considered essentialfeaturesofthose embodimentsunlessthe embodimentis inoperativewithoutthoseelements.

Itshouldbenotedthatthevariousembodimentsandexamplesdetail edhereinin connectionwithvariousaspectsoftheinventionmaybeapplicabletoo neormoreaspects disclosedherein.Itshouldbefurthernotedthatanyembodimentdescr ibedherein,for example,related to method,may be applied separately orin variouscombinations. Variousembodimentsandaspectsofthepresentinventionasdelineate dhereinaboveand asclaimed in the claims section below find experimentalsupportin the following examples.Thephrases “inanotherembodiment”oranyrefencemadetoembodimentas used herein do notnecessarily referto differentembodiment,although itmay.Thus, variousembodimentsoftheinventioncanbecombined(from thesameorfrom different aspects)withoutdepartingfrom thescopeoftheinvention.

Variousembodimentsandaspectsofthepresentinventionasdeline atedherein above and asclaimed in the claimssection below find experimentalsupportin the followingexamples.

Disclosedanddescribed,itistobeunderstoodthatthisinvention isnotlimitedto theparticularexamples,methodsstepsdisclosedhereinassuchmetho dsstepsmayvary somewhat.Itisalsotobeunderstoodthattheterminologyusedhereini susedforthe purposeofdescribingparticularembodimentsonlyandnotintendedto belimitingsince the scope ofthepresentinvention willbelimited only by the appended claimsand equivalentsthereof.

The following examples are representative of techniques employed by the inventorsincarryingoutaspectsofthepresentinvention.Itshouldb eappreciatedthat whilethesetechniquesareexemplaryofpreferredembodimentsforthe practiceofthe invention,thoseofskillintheart,inlightofthepresentdisclosure ,willrecognizethat numerousmodificationscanbemadewithoutdepartingfrom thespiritandintendedscope oftheinvention.

NON-LIMITING EXAMPLES

Materialsandmethods

Human clinicaltrial

Forty-nine samples of endometrialtissue were collected from 37 recurrent implantationfailure(RIF)patientsthatwereabouttoundergoinvitr ofertilization(IVF) treatmentsinthesubsequentcycle.

Thekeyinclusioncriteriafortheparticipantswere(i)IVFpatien tsdiagnosedwith RIF whowereregularlyovulating;(ii)Age:18-40yearsand (iii)bothpatientswhose fertility status was unknown and patients who had proven to be fertile (previous successfulpregnancy). Key exclusion criteria were (i) patients with known existing endometrial pathology,(ii)patientswith aknown history ofinfertility due to oligo-ovulation or anovulation,(iii)patientswithamedicalhistoryofmalignanttumor sintheirreproductive system,(iv)patientsthatwere on any hormonalmedicationsorhormonaltreatment (excluding hormonalcontraception in previous cycles),(v) patients who were on hormonalcontraceptiontreatmentintheircurrentcycle,(vi)patien tscarryingIUD and (vii)patientsmenstruatingonthedayofthebiopsycollection.

Foreachparticipantthecycledatingwasperformedutilizingthef ollowingfourmethods:

1. Testimony

Participant'sreportofthefirstdayofherlastmenstrualcycle,h eraveragecycle andmensesduration,andthevarianceinboth.

2. Histology

Histopathologicalevaluation by two expertGyneco-histopathologists.Thetwo hadtoreachconsensus.Thisiscommonlyregardedasthegoldstandardm ethodforcycle dating.

3. Hormonelevels

Bloodsampleforhormoneslevelanalysis(LH,FSH,ER,PR,Access2a nalyzer (Beckman& Coulter);AdviaCentaurXP(Siemens).

4. Ultrasound

Endometrial morphology and thickness measured by vaginal ultrasound examination.

Allmethodswerenormalized toproducea28day "standard"cycle,using the patientreportorthehormonemanufacturer(i.e.,Abbott)data.

Thefresh ex-vivo samples(<3 hourspostcollection)wereexamined undera stereoscope(MoticSMZ-171-TL)equippedwitharing144-Ledwhitelig htillumination sourceandamountedcamera(MoticMotiCam3).Imagesat2X and4X magnifications ofendometrialsurfaceareaswerecaptured.

A proprietarydedicatedimageanalysissoftware(underMatlabVer.R20 16aof MathWorks)identified blood vesselson theimageofthetissue surface samplesand ascribedattributes(e.g.,averagediameter,averagecolor,etc.)to eachidentifiedblood vessel.Specifically,anaveragevaluewascalculatedforR (red),G (green)andB (blue) forallpixelsinabloodvessel,toobtainaR_avg,G_avg,B_avgforeach modeledblood vessel.Taken together,foreach modeled blood vesseltheaveragediameter(denoted hereinas “w”)wascalculatedtogetherwiththeaverageR value,averageG valueand averageB value.

Swinepre-clinicaltrial

Fourfemaleswine(Age=l.8+0.6years,Bodymass=215±25kg)wered esignated from ananimalresearchinstitute("LahavCRO”,Israel).Keyinclusionc riteriawere:(i) healthy,proven-fertile,non-pregnantfemaleswine;(ii)age 1-4 years;(iii)noton any medicationinthetwomonthspriortoselection.

Exclusioncriteriaincluded:(i)irregulardiet,(ii)harmedorda mageduterusor menstruation during the surgicalexperiment.The study,using common laboratory animals,wasapprovedbytheIsraeliNationalEthicsCommittee(#IL-1 4-11-292).

Theaveragelengthofaregularmenstrualcycleinthesow is21days((Martinat- Botteetal.,2000)).Cycledayisexpressedfrom day0today20.Here,day0wasdefined asthebeginningoftheLH surge.

Thecycleday foreach oneofthesowswasdetermined using threedifferent conventionalmethods:

1. Behavioralanalysis

Estrusdate(-cycleday 2)wasdetermined by an experienced veterinarian basedonstandingheatandstandardbehavioralcharacteristicsofthe femalesow and thatofamaleswineinitsproximity.

2. Hormonelevels

Threebloodsamplesweredrawnfrom thesows:onesampleontheselection date,twoothersonthesurgicalexperimentday,approximatelyhalfwa ybetweenthe two dates. Blood sampleswereanalyzed (AML Israel,Ltd.,Herzliya,Israel)for Estradioland Progesterone levels.Progesterone levels were obtained from two machines[Cobase601(RocheDiagnostics)and Immulite2000 (Siemens)forthe latter,dueto oneuncertain measurementon theCobasmachine]. Cycleday was determinedbyatwo-term Chi-squareanalysis.

3. Histology

Followingex-vivoimagingoftheendometrium,abiopsywasextract edfrom each imaged uterinelocation.Foreach biopsy,5 pm histology H&E slideswere preparedwith severallongitudinalsections(L.E.M.Ltd.). Pathology analysiswas performedinPharmaseedLtd.(both:NesTziona,Israel)byanexpertin veterinary pathology,in consultation with aworld-renowned expertin swinehistology and endometrium dating.

Analysismethod

A schematicview oftheendometrium andthetissuesabutit,alongwiththebasic morphologyofthemainarteriescascadeisshowninFigure1showing,fo rexample,the endometrium andthemyometrium.Ascanbeseenfrom theschematicrepresentation,the endometrium ismadeoftwomorphologicallyundistinguishablelayers:thefunctio nal layer(functionalis)andthebasallayerbelow itwhichresidesontopofthemyometrium.

ThedifferentlayersandcoordinatesareschematicallyshowninFi gure2.

Thedepthcoordinatesaredepictedasthedistancefrom theendometrium surface levelfacingtheuteruslumen,bythecoordinate -

Typicalz-dimensionsfortheabovementioned tissuestrataare 1-2 mm forthe basallayerand0-6mm forthefunctionallayer.Thez-dimensionofthefunctionalisgrows andshedsregularlyinthecourseofthefemininecycle.

Thex-andy-coordinatesaretakentobethe(arbitrarilyoriented) coordinatesas registeredonaplanarimageoftheendometrium surface,takenfrom thelumenside.

Aninfinitesimalvolumeelement V,isthusdefinedas T = AxAyAz.

AssumingthatD _e (t)betheendometrium totaldepth (functional& basallayer together)asfunctionoftime,t;Z) _m (t)themyometrium totaldepthasfunctionoftime andD _ue (t)= £> _m (t)+ O _u (0 theentireuterustissuedepthasfunctionoftime(asshown inFigure2).

Recording blood vessels(BV),may depend on variousattributes.Among these attributesonemaylist,non-exhaustingly,thebloodvesslaverage(o rmin/max)diameter, thebloodvesslaveragelength,oritslengthbetweenbifurcationsors plits,thebloodvessl degreeoftortuosity,theoxygensaturationlevelwithinthebloodves sl,theflow pattern andtheflow magnitudeprofileswithinthebloodvessl,etc.

Foreach oneoftheseattributes,OQ,onemayconstructaprobabilitydistribut ion function(PDF)thatprovidesoneoftwoprobabilitydensityfunctions ([0-1]range):

1.The absolute probability offinding aBV within the tissuevolume AF,that possessestheattributeocwithintherange[oc/,oCj+ Aoc/]denotedbyP(BV,oCj)

2.TheconditionalprobabilityforagivenBV tohaveitsattributeoCjwithinthe range[oc/+ Aoc/]denotedbyP(oCj|BV).

ThefirstPDF isnaturally themultiplication ofthesecondPDF bytheabsolute probability offinding a BV within the volume AF. Thislatterprobability isnon- homogeneouswithintheendometrium noritisconstantintime.Itshouldthereforebe actually defined asP(BF)= P _BV = P(z,t),wherespecifichomogeneity in x-y (ata largeenoughROIandspecificzandt)isassumedandtheprobabilityisd erivedfrom the BV numberdensityasfunctionofdepthandtimewithintheinfinitesimalt issuevolume AF.

One may also have such PDFsformore than a single attribute,eitherasfree probabilityorconditional,i.e.,oneofthefollowingcombinations: a. P(BV,a _r ,...,a _n ) b. P(a _lt ...,a _n \BV) c. ...,a _m ),wherethetwosetsofattributesarestrangeto eachother

Inordertodemonstratetheproposedmethod,focuswasmadeonaspec ificattribute oc,whichisthewidthoftheBV,w,asmeasuredonthetissueimage.

Sincethediametermay changealong theBV length,the “averagewidth ofthe recordedBV alongitslength”isreferredasthe "BV diameter"or,width.

Hence,w isdefinedtobetheaveragewidthofabloodvesseloflength I(onthe image).Alternatively,itispossibletousethemaximum valueofthewidthoranyother attributethereof.Fortheaveragewidthwehave

AssumingacircularBV cross-sectiontheaveragewidthcanbereadilytranslated to theaveragediameteroftheBV in spiteoftheprojectioneffectsoftheimageand regardlessoftheBV three-dimensionalorientationwithrespecttotheimageplane.

Similarly,theBV z-coordinatemaychangealongtheBV length,andthuscanbe refertoitsaveragez-coordinate.

Ataspecifictime,t,andanarbitrarydepth,z,assumeaPDFoftheBV swidth, w.A good exemplary choice ofsuch PDF functionalform could be a Log-normal distribution,i.e.,forthevariable,P(w,z\BV),theprobabilityden sityofagivenBV to beofwidthbetweenw andw + Aw isexpressedviathelogofw,a>= log(w)tobe

Inm ( ) ² .... _T ... .. . ..

P(w,z|BV) ,whichisaLog-normaldistributionwith ώ = ώ(z) being themean valueoflog(w)and σ = σ(z) thestandard deviation of log(w).

Theintensityoftheincidentlight(ofamplitude:Iz= 0,A)= I _o (λ))atdepthz below thelumen(withintheendometrium,namelyforz< D _e )experiencesattenuation accordingtotheequation where (λ) _e istakentobethetotal (effective)attenuationcoefficient,consideringbothabsorptiona ndscattering,including theanisotropyfactor,g.

Theabsorptioncoefficientoftissuesvariessignificantlyinthe visiblespectrum, whereasthescatteringcoefficientdecreasesmonotonicallyasthewa velengthincreases. ThelattercanbemodeledasacombinedcontributionofRayleighandMie scatteringand ismostlyresponsiblefortherelativehigherpenetrabilityoflonger wavelengths(“redvs. blue”)inthevisiblespectrum.

A goodapproximationtothescatteringdependenceonwavelengthisgive nby

Where f is the relative fraction between the Rayleigh expression (left)for scatteringcontributionduetoparticlesofsizemuchsmallerthanthe lightwavelengthand theMiescattering(rightexpression)forscatterbyparticlescompar ableinsize. Jacques,2013providesausefulapproximationforthetotalextinctio ncoefficient, alongwithacompilationofexperimentalvaluesfor a',fandbforvarioustissues.

Atthetransitionendometrium-myometrium andfora blood vessel located at depth z> D _e below the lumen, I(z>D _e ,A = I _Q (A^e~^ ^e ^ ^De e~^ ^m ^( ^z ~ ^De \ We do notexpectvisible lightpenetration beyond the myometrium,though otherwavelengthsmayreach deeper.Theformalism wepresent hereinshouldnotbelimitedtoanydepthortissuestructure.

Forbloodreflectioncoefficient,5?(A),(assumingtissuerefrac tiveindex~1here), theintensityofthereflectedlight,afterreflectingfrom abloodvessellocatedatz _BV ,is

Andafteritsjourneybackitbecomes

Itwasassumedthatblooditselfisresponsibleformostoftherefle ctionwitha minor,negligiblecontribution from thevesseltissueitself.Thishasbeenjustified by measurementsofthebloodspectrum in-vivothroughitscarryingvessels.Underextreme circumstances(e.g.,pathologicalvessels),thisassumptioncan bereplacedby alinear combinationofthereflectionofthebloodandthevessel.

Figure 2 also showsarepresentative system forcollecting data,such thata collectingdevice(forexampleacamera)capturestheupperlayerofth eendometrium from withintheuteruslumen..AsshowninFigure2,thereisasourceofillum ination which providetheincidentlightofaknown spectrum from the same direction (but possiblyatadifferentincidentangle).

Thez-coordinateismeasured from thetissuesurfaceinwards(i.e.down thez axis).Thelightfirstcrossesthetissuesurface,atadistanceDeitcr ossesthejunction between the endometrium and the myometrium.The inner division between the endometrium functionalandbasallayersinunnoticeablebythelight.Thelightmay hita bloodvesselfrom thatreflectsitbackwards(tothenegativez-direction).Thecolorra tio ofthebackscatteredoutcominglightisdictatedbythetissue,thetar get(BV)depth(z- coordinate)andthereflectionindexofthevesselandmostlytheblood .

Asnotedabove,theendometrium ismadeoftwolayers:functionallayer(sheds every menses) and basallayer (thin,survives the menses).The layers cannotbe distinguishedbyregularhistology and therefore,foropticalpracticalpurposes,itwas approximatedthattheendometrium formsasinglelayer.Sincethebasallayerisbetween 1to2.5mm thick [4],inthefollowing demonstration,only theendometrium willbe referredandhenceneglectingthemyometrium asvisiblelightwillnotgettoit.

As shown in Figure 3,the penetration depth of visible lightthrough an endometrium tissue justified the assumption thatvisible lightwillnotgetto the myometrium.

Forabloodvesselofwidthw thereisamaximum zbeyondwhichitcannolonger bedetected dueto thelightlow (attenuated)intensity,thenoiseleveland thespatial resolution oftheimaging device.To afirstapproximation,the wavelength intensity dependencewasneglected,andreferencewasmadetothepeakofthebloo dreflection spectrum only.Alsothespatialresolutiondependencewasneglectedandasharp width cutoffwasappliedinstead(theminimalbloodvesseldiametertobedet ected,regardless ofitszcoordinate.).Thiscanbejustifiedpostfactafterthin(small w)bloodvesselsare foundatthedeepestpossiblelayersidebysidewithlargediameterves sels.Thezcutoff istakentobe whichisequivalenttoattenuationoftheorder~e

Taking into account the optical parameters (attenuation coefficients) of endometrium-related tissues,e.g.,mucous tissue [5][3] with a'= 18.8cm ^-1 ;f = 0.0;b = 1.62andaflatspectrum incidentlight(“whitelight”),thisz _cutO ffturnsoutto be atthe range of400-800pm depending on the wavelength.Figure 3 showssuch wavelength dependence,whereafactor~44existsbetweentheBluelightpenetrati on depthandtheRedone. Next,amodelforthe BV width distribution function evolution in time was developed.

Fortheendometrium,inparticularforitsfunctionallayer,there isnotenoughtime togrow BVsbeyondcapillariesduringthecycle(ofupto30days).Thereisthus avery sharpdescendofthedistributionfunctiontowardthecapillaryupper diametervalue,as longasthefunctionallayerisconsidered.

Sincearteriolesandvenuleshaveatmostw~100pm,andmostcapilla riesareat iv~5-10pm,these valuesdetermine the two log-normalparameters: ofthe BV distributionwithintheendometrium,withaslightincreasetowardth estratabassalisthat may,ormaynotbesignificantinthedistributionalteration.

Themyometrium,ontheotherhandincludesBVsthataremuchlargert hanthe ~100pupperlimitanditspopulationremainsrelativelyconstantthro ughoutthecycle, namely the distribution time-dependence is much weaker than the one of the endometrium.However,duetoitslocationbelow thebasallayerandthez _cut0 ^ layer, visuallightwon’tgetreflectedoffofBVsresidinginit.Otherligh tbands(IR)shouldbe exercisedforthatmatterattheexpenseoflowerspatialresolution.

For evaluating the endometrium evolution,one would relate the z (depth) coordinateofastratato itsformation time,namely to its “age”.Thisshouldprovide information with regardsto theendometrialtissueevolutionpaceand itsmaturity,as reflectedbytheBV PDF,incomparisontoits “calendar”age.

AssumingA(z)denotethe “Age”oftheendometrium layeratdepth [z,z+ Az] measuredfrom thelumenwallsurfacewherez= 0.If£> _e (t)isknownthenforagivent weobtain

IftheBVswidthdistributionfunctionisafunctionoftheageofthe endometrium tissuelayer,obtainP(w\BV,z)maybeobtainedbyascribingtherelati onbetweenzand A(z).

A sharptransitionoftheP(w\BV,z)isexpectedattheborderlinewhere z= D _e , sincethemyometrium BV populationismuchmoremature.

In ordertoutilizethisPDF evolution formalism,oneneedsto estimatethez- coordinate ofagiven image-identified BV.To thatend,itwasproposed to use the statisticalchangeinbroadbandcolorsofthebloodvessels,asobtain edduetothetissue differentialattenuationinwavelength.

Sincelongerwavelengthsarelessattenuatedthanshortwavelengt h(inthevisual spectrum),onemayuse,e.g.,thestandardRGB camerachannelstocalculatebroadband ratiossuchas

B (B + G)

— or- .

R R

These ratios change mainly due to the Mie scattering by the tissue.Even though conclusionregardingtheBV depthcoordinateforindividualBV,basedonthatratiois nottrivial,the overalleffectfor the entire detected BV population can be clearly ascertained.

Bloodmainlyreflectsinthelongwavelengthsandmostlycontribut estotheRed channelandthusthemereBV detectionisfeasibleevenatrelativelylargez-coordinate.

Figure 4 showsthe actualcalculation ofone ofthese colorratios.The BV reflectioncoefficients,5?(A),arecalculatedbasedonthecompleme ntaryvaluesofthe well-documented oxygenated blood absorption spectrum.Sincebroadbandfilterswas used,theOxy-Deoxybloodratioisnegligibleinsuchcalculation.Not icethatatabout 200 pm,the (G+B)/R ratio levelsand doesnotallow differentiationsbetween BVs residingdeeperthanthisvalue.Other(e.g.IR)wavelengthmaysomewh atalleviatethis limitation.Theratiosmay contain information atdeeperlayersthan 200pm formore accuratetransmissioncoefficientoftheendometrium.Heretheparam etersfrom general epithelialor mucous layers were taken,butthe endometrium,due to its relative transparency,maybearratioinformationdownto400-500pm.Thesedep thsareindeed smallerthanthebasallayerbutprovidedetectionofolder(deeper)z- layers.

Figures5A and5B show schematicsetsofBV diameterPDFsasfunction of depthfortwocompetingevolutionmodels(tracks)ofBVs.Thetwocolum nsrepresent tissuesonthesamestandardcycledaythatobeyeddifferentevolution models.Figure 5A,BV evolutionisledprimarilybyelongation,followedbycapillarygrowt hthrough splittingangiogenesis.Therefore,evenontheshallowerstrata(low estpanel)relatively big diameterBVsarefound.Figure5B,show thatshallow stratado notcontain big diameterBVs,theBV evolutiononnewlyfoundlayersisprimarilygovernedbysprouting ofnewlyformedcapillaries.Inthedeeperlayer,wheretimepermits,b iggerdiameterBVs maybefound.

Uterimay differ in the three “clocks” they exhibit:the calendar time,the “standard”or “average”thicknessversustimefunctionandthefunctionoftheB V PDF versus-Thislatterdependenceshouldalsohavea “standard”(namelyaverageoverthe population)behavioranddeviationsfrom thisaveragemayhintorrelatetodeviationsin theothertwo “clocks”.

Figures6A-6Ishow twodifferenttheoreticalrepresentationsthatmayrepresent differentindividualsubjectsandtheirrecordedbloodvesselsdiame terPDFsasfunction oftissue depth.Schematically,both individualsare characterized by the same PDF (Figures6A-6C)andstartedfrom thesameday "n"oftheirstandardizedcycleday.

However, "m"dayspostthestartingpointofday "n",thePDFoftheindividual representedinFigures6D-6Fdidnotevolvetothedegreetheyshouldin ordertofittheir calendarday. ThePDF oftheindividualrepresentedinFigures6G-6I,evolvedatthe standardpacetoshow thediameterPDFsasfunctionoftissuedepth,exactlythewaythey shouldbeatthecalendartime.

Forexample,bloodvesselsPDF thatdoesnotevolveasrapidlyasexpected,in spiteofthicknessgrowth,mayalludetoa “retarded”uterusthatshallbecomereadyfor e.g.,implantationatalater-than-“standard”timeasshownfore xampleinFigures6D-6F.

Thebloodvesselnetworkwithintheendometrialsurfaceimageswas statistically analyzed the,using allofthe sample'sacquired images(2X and 4X separately),by treatingtheBV populationasastatisticalensemble.Anexemplifyingimageisshowni n Figures7A and7B,wherebloodvesselcomputerizedidentificationisoverlaid(f ullyas showninFig.7A andpartiallyasshowninFig.7B).Assuch,probabilitydistribution functions(PDFs)and thecumulativePDFs(ePDFs)werederived to describetheBV population.

ThefocuswasontwoBV features:

(i) w -theoftheBV averagewidth(alongitslength),whichisequivalenttoits diameter

(ii) theBV averagecolorratio,ascalculatedfrom itsimageRed-Green-Blue (RGB)colors. Thecolorratiowasused:

(B + G)

5?- _R .

Thisratio,JI,isconsidered torepresentz -theBV averagedepth beneath the tissue surface thatfaces the lumen ofthe uterus.Such representation is based on differentialabsorptionandscatteringmodelsfordifferentlightwa vebands(Bashkatovet al.,2011;Jacques,2013).

ThePDFsoftheBV'sdiameterand colorratio may befunctionsofthecycle dating,t,orconditionalprobabilitysuchasthediameterdistributi ongivenacertaintissue depth(colorratio).Thesefunctionsmayormaynoteliminatethenorma lizationfactorof theprobabilityoffindingaBV (ofanydiameter)withintheunitvolumeAT(72)oftissue surfaceunitareaAS.

TheBV PDFwasmodeledataspecifictimeanddepth(tandz)withaLog-normal distributionwiththemean(log)ofitsdiameter aj= a>(z) anditsstandarddeviation c = <J(Z).

Results

Humansamples

Figures 8A-8F depict the blood vessel diameter distribution function,as calculatedfrom asampleof3442X magnificationimages.Allimagesofallpatients, regardlessoftheircycleday,werecollectedtogethertoyield thegeneralbehavioror bloodvesseldiameterdistributiondifferencesasafunctionoftissu edepth.

Thedeepesttissuelayer(smallestcolorratio,JI,denotedherein ascolorindex)is shownonFigure8A having aJI(z)valueof0.16667 and abloodvesseldiameterof 3.5772(w),deeperlayersbytheorderoftheirJI,binvalues,areshown onFigure.8B to Figure.8E.Fig.8Fshowsthemostsuperficialtissuelayerwhichabuts theuterinecavity havingaJI(z)valueof1.833andabloodvesseldiameterof3.0863(w).

Theraw histogramsoftheBV diameterdistribution,asdrawn from the total numberofidentifiedBVsinthelayer'sdepth(N _BV )arealsoshown.Thesolidcurveline is the overlaid log-normal function model of P(w \JZ,< JI< 7?j ₊₁ ) and its corresponding fitting parameters(a>,o'),aswellasthe average layer'svalue (5?bin averagevalue).

Figures9A-9Fshow theresultsoftheanalysisasobtainedfrom 625imagestaken under4X magnification,wherethe5?binlimitsandaveragevalueswithineachd epthbin (layer)areidenticaltothoseinthe2X figure(Figures8A-8F).

InbothFigures8A-8FandFigures9A-9Facleartrendofincreaseda) ("average diameter")asfunction oflayer'sdepth exists,whereasthechangein thedistribution "width"(o)asfunction ofdepth islessevidentdueto theimageresolution,i.e.,the narrowestidentifiableBV.Specifically,ascanbeseenfrom Figures8A-8F,theaverage diameterwasthehighestinthedeeperlayer(Figure8A).

Figure10showsthechangeinthew fittingparameterofthePDF fortheBV diameterdistributionasafunctionofthetissuelayer'sdepth,namel y,byinterpretation, itsdistancefrom thelumenoftheuterinecavity.Theindependentresultsfrom the2X magnification sample (X symbol)and 4X (square symbols)are also shown.The w parameterisinthelogandhencethesmallvaluesonthey-axis.Thediff erenceinthe narrowest identifiable BV, due to the higher spatial resolution under the 4X magnification,causesthesharperdescentofthew valuesathereisanincreaseinthe layersunderthe4X magnification.Followingourrealizationofadistinctglobaltrendf or BV diameterdistributionasfunctionoftheendometriallayer,weturned totime-evolution oftheBV diameter-colorrelationship.Whenassessedindividually,eachoneo fthetwo features(diameter,color)ensemble distribution,did notshow clearevidence foran evolution track along the cycle time course.However,the combination ofthe two characteristics,namelytheconditionalprobabilitydoesclearlydi splayanevolutionline. We splitthecolorspaceintotwobinsonly,asopposedtothesixcolorbins inFigures 8A-8F andFigures9A-9F.Thiswasdoneinordertoreducesamplingerrorsduet othe smallernumberofcapturedimagesforeachcycleday.

Allpatientimagesofthe same cycle day (asdetermined by histology)were collatedtogethertoobtaintheBV diameterPDFswithinthetwo-colorbins.EachPDF wasthenmodelled by thelog-normaldistribution andfittingparameterswerederived alongwiththeirconfidencelevel.Foreachoneofthetwofittingparam eters(to,o)we plottedtheratiooftheparameterascalculatedbythedeephalfofthet issue(atthatcycle day)versusthesuperficialhalf.Figure11A depictstheresultsoftheseratiosasafunction ofthecycleday such thattheratioofthedistribution "average"(a>in thelog space), showsaclearcutevolutionline.Theglobalregressionline(longline )doesnotadequately describetheratioevolutionintime,butthetworegressionlines,one foreachcyclephase, providesagood modelling oftheparameterratio evolution.Interestingly,the slope changeoftheratioevolutionshowsapivotalpointatornearday14("ov ulation"),which separatedthetwocyclephases.

ThesameslopechangeappearsonFigure11B,wheretheratioofthe "width"(o', inlog)fittingparameterevolutionisdepicted.

Itisimportanttonotethattheparameterratiowithinthetwo-colo rbins,israther constantthroughoutday 14,and thereafteritchanges its slope.In general,as the endometrium progressesthroughthesecretoryphase,theBV diameterdistributionofthe superficiallayerisnarrower("tighter")thatthatofthedeeplayer.

Swinesamples

Allsamplestakentogether,regardlessofthecycledayyieldedthe color(depth) -diameterdistributionasdepictedinFigures12A-12E.

Thediameterdistribution(log-normal)average,a),exhibitsave rycleartrendas functionofdepthandassummarizedinFigure13.

Thepremisebehindtheaforementionedresultsforthehumanendome trium isthat theyarenottheonlypossibleconfigurationfortheBV networkasfunctionofdepth,nor oftheprogressionintimethereof.Inordertocorroboratetheseobser vations,werepeated thecalculationforthefourswinewehaddatafor.Byhistologicalasse ssment,theswine wereontheircycledays2,8,14,16(ina21-daycycle).Seventy-threee x-vivoendometrial surfaceimagesofthefourswineunder2X magnificationwereusedforthefollowing analysis.Figures12A-12Eshow theBV diameterPDFofalltheswineregardlessoftheir cycleday.Theuppermostlayer(thelowermostpanel)wassomewhatcont aminatedby freshsurfaceblood.

Incontrasttothehumandata,the "average"ofthelogdistribution(to)becomes smallerasthelayerdeepens. Figure13showsthistrendofthelog-average(to)dependenceontissue depthwith thepositiveslopeasthelayerbecomesmoresuperficial.Deeperlayer s(smaller(B+G)/R valueshaveasmalleraveragewidthwithrespecttomoresuperficialla yers.

ThisobservationprovestherobustnessofthemethodsincetheBV distributionof othermammalsisknowntobedifferenttothatofhumans(seefurtherint hediscussion).

The statisticalmethod presented here,is suggested to resolve some ofthe uncertaintyintheendometrialangiogenesisprocessandestablish clarityinthefaceof competinghypotheses.

ItwasdemonstratedthatbytreatingtheBV populationasastatisticalensemble, one can differentiate between differentdepthsofBVs,using theircolorattribute as obtained in visuallightimages.Thisapproach differentiatesbetween evolution paths when BV diameterdistribution istaken into account.Theuse ofvisible-light,nondestructiveimagingalsoallowsmonitoringoft heBV populationevolutionoveraperiod oftimewithintheverysametissue.TheidentificationofaBV depthwithitsgeneration time("age"),mayormaynotbejustifiedaccordingtotheangiogenesis processesthat haveledtoitscreation.

Inordertomaximizethedifferentiatingpowerofthemethod,thePD Fsofthe BVs (alltogether or within tissue layers) was splitinto a multiplication of their normalizationfactor,namelytheirtotaldensity,andtheirdiameter distribution.These functionscan then bedetermined with variousmathematicalmodels(Chappelletal., 2011)(Logsdonetal.,2014)thatattempttodescribeangiogenesis.Fo rinstance,their shape teaches whether new endometrial BVs are generated through sprouting, intussusception(a.k.a.splittingangiogenesis)orelongation.Itw asshownelsewhere(Or etal.,2022)thattwo-dimensionalBVD alone(withoutsplittingintodepthlayers)does notsufficientlycharacterizetheendometrialevolutiontrack.

There isa closerelationship between the extracted PDFsand theunderlying processesleadingtotheirconstruction.Forinstance,anew endometriallayerhasmore sproutingorsplittingpotentiallocationsthananolder(deeper)end ometriallayerandone may thereforeexpectadiameterdistribution forthenew endometriallayertohavea biggernormalizationfactor("morevessels")inadditiontoitsshape tobeheavilyinclined towardssmallerdiametervalues. Alternatively,onemayarguethatolderendometriallayerbyvirtueof itsage,had a longer time to generate its BVs and therefore should be denser,with a higher normalizationfactor,butinkeepingwiththepreviousscenario-more inclinedtoward biggerdiametervalues.

Analternativescenariomayarguethattheendometrialtissuegrow thrateismuch fasterthantheBV diametergrowthrateandthereforethediameterdistributionshouldb e similarandnarrow (withsmalldiameters)throughoutthedepthoftheendometrium.

A good example ofthe differentstatisticalexpression ofprocesses can be demonstrated if we compare (1) elongation (Gambino et al., 2002) and (2) intussusception,whereeachonerepresentsasinglemechanism ofangiogenesis.

In the caseofelongation,theBV diameterdistribution should stay relatively constant,andifelongationoccursatallendometriallayers,deeperl ayersshouldhavea differentnormalization factor(namely BVD)yetaconsistentnormalized distribution ("shape").On the contrary,ifintussusception rules,a deeperlayerthathasalready undergonesplittingandthediameterdistributionwouldleantowards smallerdiameter valuesincomparisontothenewlygeneratedupperlayerwiththickerBV s.

In reality,probably allfourpossible processesdescribed in the introduction contributeto theBV plexusbuttheremay wellbedifferentweightingsfordifferent processesthroughoutthevariousphasesofthemenstrualcycle.Theme thodwepresented heremaybeusedtoidentify andtrack intussusceptiveangiogenesis,whichhassofar beenbeyondreachinhumanstudiesandmostlyobservedinanimaldata(D uCheyneet al.,2021).Thecurrentstatistical,time-evolution approach cannow becombinedwith othermethodsto quantify itscontribution to the overallangiogenesisprocess.The differentialweightingsmaybethereasonforthecontrastingbehavio roftheswineBV PDFasafunctionofdepthversusthehumandataasshowninthispaper.Th isdifferential PDF behavioraddstothebodyofknowledgeregardingdifferencesbetweend ifferent mammalianspecies(mice,rhesusmacaques,ewes)whenitcomestoangio genesis(see e.g.,(Chappelletal.,2012;Girlingetal.,2007)andreferencesther ein).

Examination ofFigure 8,Figure 9 and Figure 12 revealed acleartrend that shouldn'tnecessarilyexista-priori.Itisonlyduetothecorrectide ntificationofthe "color" fortheBV withthe "depth"coordinatethatlendsmeaningtoit. IntheswinecaseofFigure12deeperlayers(toppanels)show atailofrelatively largediametervessels.Movingtomoresuperficiallayers(downwardo ntheplot)this "tail"diminishesandthus,both theaveragesizediametergoesto smallervalues(plot maximum goestotheright)andthestandarddeviationbecomessmaller(plotsbe come narrower).

Thehumansampleexhibitsthesametrendofashorteningdistributi on "tail"as BVsclosertothetissuesurfaceareconsidered.However,unlikethesw inesamples,the tailcontinuestomuchhigherdiameters.Inview ofthefactthathumancycleislonger (28days)thantheswine's(21days)thismakeslogicalsense.Itmayref lect,forinstance, thedifferencebetweenthehumanmenstrualcycleandananimalestrous one.

From the measurementperspective,even if the tissue effective absorption coefficientsaredifferentforswineandhuman,ortheutilizedlights ourcesaredifferent, themethod isstillself-referenced.Thedifferencesin absorption and illuminationwill globallyaffecttheattenuationforeach "color/depth"layerbutwillnotaffectthetrend.

The immediate practicaluse,therefore,of the proposed method could be identificationofabnormalevolutionoftheBV networkthatleadstovariouspathologies. Thus,asdemonstrated here,even endometrialdating may be achieved through the identification oftherelativeBV population atdifferentendometrialtissuedepths.In combinationwithotherdigital,in-vivo,imagingandcalculations,s uchendometrialdating may bemore accuratethan thetraditionalhistologicalmethods(Acostaetal.,2000; Dubowyetal.,2003;Murrayetal.,2004;Noyesetal.,1950).