—Original Article—

Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturationYolanda SEGOVIA1), Noemí VICTORY1), Irene PEINADO2), Laura M GARCÍA-VALVERDE3), Magdalena GARCÍA1), Jon AIZPURUA3, 4), Ana MONZÓ2) and María José GÓMEZ-TORRES1, 4)

1)Departamento de Biotecnología, Facultad de Ciencias, Universidad de Alicante, Alicante, Spain2)Unidad de Reproducción Humana, Hospital La Fe, Valencia, Spain3)IVF Spain, Medicina Reproductiva, Alicante, Spain4)Cátedra Human Fertility, Universidad de Alicante, Alicante, Spain

Abstract. The development of an effective program that combines in vitro maturation (IVM) and cryopreservation forimmatureoocyteswouldrepresentanoveladvanceforin vitrofertilization(IVF),especiallyasameanstopreservethefertilityofwomeninuniquesituations.Theaimof thisstudywas toanalyze theultrastructuralcharacteristicsofhumanoocytes,obtainedaftercontrolledovarianstimulation,todeterminewhetherIVMisbestperformedbeforeoraftervitrification.Tothisend,weanalyzedthefollowingfeaturesinatotalof22MIIoocytes:size,zonapellucidaandperivitellinespace,mitochondrianumber,M-SER(mitochondria-smoothendoplasmicreticulum)aggregatesandM-V(mitochondria-vesicle)complexes,thenumberofcorticalgranulesandmicrovilli,andthepresenceofvacuolizationusingtransmissionelectronmicroscopy(TEM).Eachoocytepresentedaroundedshape,withanintactoolemma,andwassurroundedbyacontinuouszonapellucidaandperivitellinespace.StatisticalanalysiscomparingoocytesvitrifiedbeforeorafterIVMindicatedthattherewerenosignificantdifferencesbetweenexaminedcharacteristics.Key words: Cryopreservation,Electronmicroscopy,In vitromaturation

(J. Reprod. Dev. 63: 377–382, 2017)

Approximately80%ofoocytesobtainedafterovariancyclestimulation(OS)areinmetaphaseII(MII).Theremaining

oocytes,inmetaphaseI(MI)andprophaseI(PI),areusuallydiscardedduetotheirlowcapacityforembryonicdevelopment[1].Moreover,thereisevidenceforanincreaseintheincidenceofaneuploidyinembryosobtainedthroughOSandin vitromaturation(IVM)[2],withacleardecreaseinsuccessfulembryoimplantation[3].IfasmallnumberofMIIoocytesareobtained,IVMwithoocytesinMIorPIcanhelpincreasethenumberoffertilizedoocytesandhencethenumberofembryosthatcanbetransferredtothepatient[4].Additionally,hormonalcyclesareunnecessaryinordertoobtaintheimmatureoocytes,avoidingovarianhyperstimulationsyndrome(OHS)andallowingwomenwithalowresponsetogonadotropinstimulationtoincreasetheirfertility[5].Moreover,womenundergoingoncologicaltreatmentcanalsobenefit,asitallowstreatmenttobeginimmediately[6].Consequently,itisimportanttodevelopandoptimizeaprotocolthatallowsPIoocytestobesuccessfullyvitrifiedandmaturedviaIVM,greatlyimprovingtheopportunitytopreservefertility[7].Therefore,itiscrucialtoidentifyanysimilaritiesordifferencesinoocytesmaturedin vitrotoestablishobjectivecriteriaforassessing

oocytequalityandpromoteresearchaimedatimprovingmaturationtechniques.Inthisregard,Coticchioet al.2016[8]haveestablishedthatmost,butnotall,oocyteultrastructuralfeaturescandevelopnormallyin vitro.Ontheotherhand,matureMIIoocytescanbedifficulttocryo-

preserveusingcurrenttechniques[9]duetocertainspecificfeatures,suchasarelativelylargevolume.Thisleadstoalowsurface-to-volumeratio,highwatercontent,ahighdegreeofcytoplasmicspecialization(includingcytoskeletalcharacteristics),andprecisechromosomalarrangement[10].Infact,ultrastructuraldamageisoneofthemainadverseeffectsassociatedwithcryopreservationduetothetoxiceffectsofcryoprotectants,theformationoficecrystals,andosmoticstress[11].Themeioticspindleisespeciallysensitivetothecryopreservationprocess.CryopreservationofimmaturePIoocytescouldavoidsomeoftheseissues,especiallythoserelatedtospindleandchromosomecryodamage,astheyareprotectedbythenuclearmembrane.However,PIoocytesstillneedtobematuredin vitro[12]butcurrentprotocolsforoocytecryopreservationandmaturationaresuboptimalandclinicalsuccesshasonlybeenobtainedinalimitednumberofcases[13].Itisthereforeessentialtodefinesomeobjectivecriteriatoestablishhowoocytequalitymaybeaffectedbycryopreservation,withaviewtosupportingorrulingouttheapplicabilityofdifferentprotocolsandassessingthepossiblehealthrisksforchildrenbornfromcryopreservedoocytes[14].Duringoocytematuration,wecandistinguishbetweentwodistinct

processes,nuclearmaturationandcytoplasmicmaturation.Inthenuclearmaturationphase,meioticprocessesrestart,goingfromPItoMII.Cytoplasmicmaturationincludeschangestotheooplasmthatare

Received:January17,2017Accepted:April11,2017PublishedonlineinJ-STAGE:April30,2017©2017bytheSocietyforReproductionandDevelopmentCorrespondence:YSegoviaHuertas(e-mail:Yolanda.segovia@ua.es)This is anopen-access articledistributedunder the termsof theCreativeCommonsAttributionNon-CommercialNoDerivatives(by-nc-nd)License.(CC-BY-NC-ND4.0:https://creativecommons.org/licenses/by-nc-nd/4.0/)

Journal of Reproduction and Development, Vol. 63, No 4, 2017

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necessaryfortheoocyte’sfuturedevelopment[15].Synchronizationofthesetwoprocessesensuresnormalfertilizationandsuccessfulembryonicdevelopment[16].Transmissionelectronmicroscopy(TEM)isavaluableresearchtoolthatcanbeusedtodetermineanoocyte’scytoplasmicmaturationstatus.Withinoocytes,themostabundantorganellesaremitochondria.Theseoftenassociatewiththemembraneofthesmoothendoplasmicreticulum(SER)orsmallvesiclesandtheseassociationsplayanimportantroleintheproductionofusefulsubstancesduringfertilizationandinmembraneneoformationduringearlyembryogenesis[17].TheymayalsoacttoregulatefreecalciumlevelsandATPproduction,andhavearoleinseveralcellularactivitiesatfertilization,includingcalciumsignalmediation[18].Mitochondria-smoothendoplasmicreticulum(M-SER)aggregatesareveryabundantinMIIoocytesandareconsideredamarkerfornormalcytoplasmicmaturation.Incontrast,highnumbersofmitochondria-vesicle(M-V)complexesareanindicatorofcellularagingduetoexceedingtheIVMtime.Thiscanadverselyaffectfertilizationandtheearlystagesofembryonicdevelopment[19].Itisalsonecessarytoanalyzethearrangementofcorticalgranulesandthedegreeofvacuolization[18].Assessingoocytequalityisbasedonmanymorphometriccriteria.

Inordertoevaluatebothstructuralandultrastructuraloocytechar-acteristics,severalparametersshouldbeassessedusingbothlightmicroscopy(LM)andTEM[20].Theseincludeoocyteshapeanddimension,zonapellucida(ZP)texture,perivitellinespace(PVS)appearance,oolemmaintegrityanddensity,mitochondria,M-SERaggregateandM-Vcomplexnumber,quantityofcorticalgranules(CG)andtheirarrangement[10],andthepresenceofooplasmicvacuolization[21].Theaimofthepresentstudywastoevaluateultrastructureoo-

cytecharacteristicsinordertodeterminewhetherIVMshouldbeperformedbeforeoraftervitrification,aslittleisknownabouttheefficiencyorconsequencesofcryopreservationinimmatureandin vitromaturedoocytes.

Materials and Methods

ThisstudywasreviewedandacceptedbytheEthicalandScientificCommitteeoftheLaFeHospital,Valencia,Spain.Signedinformedconsentwasobtainedfromallparticipants.

Oocyte collectionControlledovarianstimulationofpatientswasperformedwitha

shortantagonistprotocolusing(150–300IU/day)rec-FSH(GonalF1050;MerckandCo,Madrid,Spain)andGnRH(Orgalutran®;MSDandCo,Hoddesdon,UK)forpituitarysuppression.Triggeringwasperformedthroughtheadministrationof250mcgofrec-hCG(Ovitrelle,Merck,London,UK)whentherewereatleastthreefollicles>16mmpresent.Oocyteretrievalwasperformedviavaginalpunctureguidedbyultrasound36hlater.Cumulus-oocytecomplexeswereremovedusinghyaluronidase(SynVitro®Hyadase;Origio®,Màlov,Denmark)solutionforamaximumof30secwithadenudingpipette(Flexipet®DenudingPipette,Cook®Medical,Bloomington,IN,USA).Intotal,22MIIimmatureoocyteswereidentifiedthroughthepresenceofagerminalvesicle(PIstage)andincludedinthisstudy.Ofthese,10werevitrifiedbeforeIVM(group

1)and12werevitrifiedafterIVM(group2).

In vitro maturationHealthyoocyteswereplacedinanIVMmediumconsisting

ofblastocystmedium(CCM™,Vitrolife®,Göteborg,Sweden)supplementedwithhumanmenopausalgonadotropin(hMG,Menopur®75U.I.,Ferring®,Madrid,Spain)andserumsubstitute(SSS,IrvineScientific®,SantaAna,CA,USA)underparaffinoilat37ºCina6%CO2humidifiedatmosphere.After24and48hofculture,matureoocyteswereidentifiedbythepresenceofthefirstpolarbodyusinganinvertedmicroscope(Olympus,IX70,Tokyo,Japan).ThegeneraldistributionofcytoplasmicorganellesduringoocytematurationisshowninFig.1.

Oocyte vitrificationOocytes(PIandMII)werevitrifiedinKitazato®(KITAZATO

Vitrification/Thawingmedia,Biopharma,Shizuoka,Japan)mediumusingtheCryotop®system(KITAZATOVitrification/Thawingmedia),accordingtoamodifieddropprotocolproposedbyWanget al.2013[22].Oocyteswereequilibratedina20µldropofbasicsolution(BS)for1minbeforethedropwasmergedwitha20µldropofequilibrationsolution(ES)for3min.Next,asecond20µldropofESwasmergedforafurther3min.Theoocyteswerethenplacedinanew20µldropofESfor6min.Afterequilibration,theoocytesweretransferredtofourdropsofvitrificationsolution(VS)andloadedintotheCryotop®tobestoredinliquidnitrogen.Thetotaltimeforthefinalprocesswas60sec.Allprocedureswereperformedatroomtemperature(22–25ºC).Oocytewarmingwasperformedbasedonproceduresspecific

totheKitazato®Kit.EachCryotop®wasremovedfromliquidnitrogenandquicklysubmergedin1mlofthawingsolution(TS)at37ºCfor1min.Theoocyteswerethentransferredto300µlofdilutionsolution(DS)for3minandthentransferredto300µlofwashingsolution(WS).After5min,theoocyteswerewashedinnewWSmediumandplacedinIVMmedium.Theseprocedureswereperformedatroomtemperature(22–25ºC).Thesurvivalrateafterthawingwasevaluatedmicroscopically2

to3haftercultureandwasbasedonobservationsofthemorphologyandintegrityoftheoocytemembrane.

Electron microscopyTheoocyteswerefixedandprocessedforTEManalysisusing

methodspreviouslydescribedbyNottolaet al.,2007[23].Oocytefixationwasperformedusing2%glutaraldehyde(SIC,Rome,Italy)inphosphatebufferedsaline(PBS).Afterfixationforatleast2daysat4ºC,sampleswererinsedinPBS,postfixedwith1%osmiumtetroxide(ElectronMicroscopySciences,Hatfield,PA,USA)inPBSandrinsedagaininPBS.Theoocyteswerethenembeddedinsmallblocksof2%agar(Sigma-Aldrich,St.Louis,MO,USA)approximately5×5×1mminsize.Thesewerethendehydratedinanascendingseriesofethanolconcentrations,immersedinpropyleneoxide(ElectronMicroscopySciences,Hatfield,PA,USA)forsolventsubstitution,andfinallyembeddedinepoxyresinEPON-812(ElectronMicroscopySciences,Hatfield,PA,USA).Semithinsectionsof1.0µmthicknesswerecutseriallywithaglassknifeonaLeicaLKB-IIIultramicrotomeandthenmountedongelatinizedslides,stained

OOCYTESVITRIFIEDBEFOREANDAFTER IVM 379

with0.5%toluidineblue,andexaminedunderaLeicaDMRBlightmicroscope.PhotomicrographsweretakenwithaLumeneraInfinitymicroscopecamera(Microsercon,SLU,Madrid,Spain).Ultrathinsectionswerecutwithadiamondknifeanddouble-contrastedwithuranylacetate5%andleadcitrate2.5%.ThesewereexaminedunderaJEOLJEM-1400PlustransmissionelectronmicroscopeequippedwithaGatanOriusdigitalcamera(Gatan,Pleasanton,CA,USA)forimagecapture.

Morphometric analysisInthisstudy,onlyoocytesthatwerevisuallyascertainedtobe

ofgoodqualityusingLMwereselectedforultrastructuralanalysis.Featuresusedtoevaluatequalityincludedthepresenceofaregularandroundedshape,aclearandmoderatelygranularcytoplasm,anarrowPVSwiththefirstpolarbody,andanintactandcolorlessZP.Theevaluationoforganelledensitywasperformedthroughthe

collectionofTEMmicrographsofwholesurfaceprofilesat6300×magnificationonthreeequatorialultra-thinsectionsperoocyte(distancebetweenthesectionswas3–4μm).Theseimagesweredigitallyenlargedtoaididentificationoforganelles.ImageJsoftware[24]wasusedtomeasurethedimensionsof

mitochondria,CG,microvilli,andvesicles.Foreachexperimentalgroup,atleastsixoocyteswereselectedforstatisticalanalysis.

Statistical analysisAlldatawereexpressedasamean±standarddeviationand

comparedusingunpairedt-tests(Rsoftwarev3.3.1,https://www.r-project.org/).DifferencesinvalueswereconsideredsignificantifP<0.05.Mitochondriaandvesiclevalueswereexpressedasthenumberofeachper100µm2,whileCGandmicrovillivalueswereexpressedasthenumberper10µmofthelinearsurfaceprofile.

Results

General featuresThroughLMexaminationofsemithinsections,bothgroups

(oocytesvitrifiedbeforeandafterIVM)possessedgoodqualityoocytes,asevidencedbytheirregularroundedshape,amaximumdiameterof90–105µm,thecytoplasmshowingauniformandfinegranulartexture,andweresurroundedbyacontinuousZPandPVS(Figs.2aand2c).TEManalysisatlowmagnification(Figs.2band2d)revealedthatorganellesinbothgroupswereabundantanduniformlydispersedinthehomogeneousooplasm,withslightmicrovacuolizationofthecytoplasm.However,someGolgiapparatuswereoccasionallyobservedinoocytesvitrifiedbeforeIVM(Fig.3a).

MitochondriaUltrastructuralanalysisusingTEMrevealedthatthemostnumerous

andcommonlyidentifiedorganellesweremitochondria.TheseoftenassociatedwiththeSERtoformlargeM-SERaggregatesor,less

Fig. 1. General distribution of cytoplasmic organelles during humanoocyte maturation. Diagram of PI oocytes (a) Diagram ofmetaphase-IIoocytes,(b)MicrographsofPI,(c)andmetaphase-II (d) shown by LM. GV=germinal vesicle, ZP=zonapellucida;PVS=perivitellinespace,PB1=1stpolarbody;M=mitochondria;G=Golgiapparatus;CG=corticalgranules;RER=rough endoplasmic reticulum; SER=smooth endoplasmicreticulum. Note the metaphase II oocyte with chromosomesat the equator (arrow), cortical granules disposed beneath theoolemma, mitochondria uniformly distributed throughout thecytoplasm, fragmented Golgi apparatus, and reorganization ofthecytoskeleton(modifiedfromMao et al.,2014[32]).Bar:20µm(c,d).

Fig. 2. Lightandelectronmicrographsofhumanoocytesvitrifiedbefore(group1)andafter(group2)in vitromaturation.Group1(a,b)andgroup 2 (c, d).Continuous zona pellucida (ZP) and perivitellinespace (PVS)canbeenobserved (a–d).Note the regularpresenceofmicrovilli(mv)(b,d).Arowofelectron-densecorticalgranules(CG)isseenundertheoolemma(O)(b,d).M=Mitochondria,V=vesicle,PB1=1stpolarbody.Bar:10µm(a,c);2µm(b,d).

SEGOVIAet al.380

frequently,associatedwithsmallvesicles(V)toformM-Vcomplexes(Figs.3b–3e).Mitochondriawerelocalizedhomogenouslyacrosstheentirecytoplasmandwereroundedorovalinprofile,withfewtransversalcristaeandamatrixshowingmoderateelectrodensity(Fig.3c).Wealsofoundelongatedmitochondrialformsconfinedtothecenteroftheooplasm(Fig.3d).LargeM-SERaggregateswerelocatedinthecorticalareasoftheooplasm(Fig.3f).Thenumberofmitochondriawasslightlyhigheringroup2.Themeannumber±SDofmitochondriaper100µm2was45.6±7.5and54.33±8.6inoocytesvitrifiedbeforeandafterIVM,respectively.However,therewasnostatisticallysignificantdifferenceinthenumberofmitochondriaamonggroups(P=0.119)(Fig.4a).

VesiclesInallobservedoocytes,typicalnumbersofSERvesiclesand

complexedM-Vwerepresent.Themeannumber±SDofvesiclesper100µm2wasfoundtobe6.69±3.1and7.25±1.8inoocytes

vitrifiedbeforeandafterIVM,respectively.Therewasnosignificantdifferenceamonggroups(P=0.930)(Fig.4b).

Cortical granulesUsingelectronmicroscopy,wewereabletoidentifyuptothree

rowsofsphericalCGimmediatelybelowtheoolemmathroughelectron-densematrixdifferences(Figs.3gand3h).However,afewisolatedCGwereoccasionallydetectedintheinnerooplasmofsomeoocytesingroup1(oocytesvitrifiedbeforeIVM).Morphometricanalysisrevealedthatthemeannumber±SDofcorticalgranulesper10µmofthelinearsurfaceprofilewas5.68±2.5and8.99±3.1inoocytesvitrifiedbeforeandafterIVMrespectively,andtherewasnosignificantdifference(P=0.3535)(Fig.4c).

MicrovilliSimilarultrastructuralfeatureswerefoundinbothgroups.Oocytes

weresurroundedbyaregularoolemma,withnumerousmicrovilliarrangedinatypicalpatternprojectingintoaPVS(Figs.3gand3h).Therewasnosignificantdifferenceinthenumberofmicrovilliper10µmofthelinearsurfaceprofilebetweenthetwogroups(meannumber±SD:15.16±2.2and16.21±2.9inoocytesvitrifiedbeforeandafterIVM,respectively,P=0.8361)(Fig.4d).

Discussion

Inrecentyears,therecoveryofimmatureoocytesandsubsequentIVMhasbeenfoundtobeanattractivealternativetoin vitrofertiliza-tion(IVF)[25].AlthoughIVMmethodsarenotyetfullyoptimizedforhumans[26],therearerecentreportsofhealthyinfantsbeingbornfollowingIVM[27,28].However,littleresearchhasbeencarriedoutintowhetherIVMshouldbeperformedbeforeoraftervitrification.Zhanget al.2011[29]havesuggestedthatvitrificationofimmatureoocytesmaybeabetterwaytopreservemicrotubuleorganizationandreducecytoskeletalspindledamage.Ontheotherhand,otherstudies[30,31]reportthattheIVMprocedureismoreefficientwhenitisperformedbeforeoocytevitrification.Inthepresentwork,wehaveinvestigatedtheultrastructuralchangesthatoccurinthecytoplasmicorganellesofoocytesinbothcases.Cytoplasmicmaturationisacomplexprocess,butelectronmicros-

copyallowsoocytequalitytobeassessedbasedonmorphometriccriteria.ThecriteriausedfortheidentificationofcytoplasmimmaturityarethepresenceofnumerousCGdispersedintheoocytecortexinsteadofbeingpositionedbeneaththeoolemma,thepresenceofGolgicomplexesstillformingcorticalvesicles,theabsenceofmitochondriaintheoocytecortex,theabsenceofSERtubuleaggregates,andthepresenceofSERlargevesicleswithoutassociatedmitochondria.OuranalysisconfirmedthatalloocytesinourstudycouldbeconsideredgoodqualitywhenassessedbyLMandTEM.Inbothgroups,oocyteswereroundinshapeandtheoolemma,ZP,andPVSappearedcontinuous.Infact,CGwerefoundinsimilarnumbersbeneaththeoolemma,withonlyafewisolatedCGlocatedinthesubcortexofoocytesingroup1(oocytesvitrifiedbeforeIVM).Moreover,CGshowedvariationinelectrodensity,whichrepresentsanimportantparametertoevaluatecytoplasmmaturation.SparselyelectrodenseCGcanbeinterpretedasimmatureorganellesorasanearlymorphologicalsignofexocytosis[14,23].However,low

Fig. 3. Microphotographofdifferentorganellesfoundinhumanoocytesvitrifiedbefore(group1)(a,c,e,g)andafterin vitromaturation(group 2) (b, d, f, h). a)Golgi apparatus. b) SomeSER smallvesicles (V) are encircled by flattened, crescent-shapedmitochondria(M).c)Roundedmitochondriaendowedwithfewtransversalcristae.d–e)M-Vcomplexes.Noteelongatedformswithacentralconstriction.f)M-SERaggregates.g,h)Corticalgranules (CG) disposed just beneath the oolemma. SER =smoothendoplasmicreticulum;T=aggregatesoftubules;mv=microvilli;PVS=perivitellinespace.Bar:250nm(a–c);500nm(d–h).

OOCYTESVITRIFIEDBEFOREANDAFTER IVM 381

levelsofvacuolizationhavealsobeenobserved,whichcouldbeconsideredamarkerofhighqualityoocytes[32].InasmallnumberofoocytesvitrifiedbeforeIVM,someGolgi

complexeswereobserved.Previousstudies[33]havereportedthatGolgiapparatusarerarelyfoundinMIIoocytes.ThismaysuggestthatoocytesvitrifiedbeforeIVMareofalowerqualitythenthosevitrifiedafterwards.However,thiscouldalsobeinterpretedasanincreaseinproteinproductionintheooplasm.Thenumberandstatusofmitochondriawerealsosimilarbetween

bothgroups,withacomparablenumber,shape,internalarchitecture,andtexture.Themitochondriashowednosignsofapoptosis,asreportedinotherstudiesthathaveexaminedIVMofoocytes[29].Mitochondria,M-SERaggregates,andM-Vcomplexesplayaroleintheproductionofmaterialsusefulformaturationandfertilization[34].Mitochondrialabnormalitiesmayalsobeassociatedwithembryoincompetence[35,36].Ourresultshaveshownthatthemitochondriainbothgroupsdidnothaveadisorganizedmatrixorthepresenceofdarkvesicles.Withregardstovesicles,SERelementsmaydynamicallyacquire

differentshapes(tubulesorvesicles),althoughtheybelongtothesamesystemofinterconnectedmembranes[21].Inthisrespect,duringthematurationoftheoocyte,theaggregationofSERtubulesappearstobetheearliestobservableevent.Subsequently,thesetubulesbecomesurroundedbymitochondria,formingmitochondria-SERaggregates.Moreover,smallM-Vcomplexesarefoundinbothimmatureandmatureoocytes[12,37].Finally,theultrastructureofthemicrovillishowedanormalpattern

inbothgroups.Thischaracteristicisveryimportantasimpropermicrovillidistributioncontributestofertilizationfailurethroughineffectivespermatozoon-oocytefusion[38].Inconclusion,humanoocytescanbevitrifiedbeforeorafterIVM,

butoocytescryopreservedafterIVMshowslightimprovementsintermsoftheultrastructuralcharacteristicsofcytoplasmmaturation.However,furtherstudiesareneededtoconfirmtheseresultsowingtothelimitednumberofsamplesused.

Acknowledgments

ThisresearchwassupportedbytheUniversityofAlicanteVI-GROB-186andUAUSTI14-04,andbytheChairofHumanFertil-ityoftheUniversityofAlicante.WewishtothankVanessaPinillaforhertechnicalsupport.

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