人类优生的辅助生育技术-优生.ppt

BETTERASSISTEDREPRODUCTIVETECHNIQUE,ReproductiveMedicalResearchCenterSunYat-SenUniversityofMedicalSciencesZhuangGuang-Lun,PreimplantationGeneticDiagnosis(PGD),HistoryofPGD,Genderdetectionofrabbitblastocystin1968SuccessofIVF(1978)anddevelopmentofmolecularbiologymadePGDpossibleinhumanPGDsuccessinX-linkeddiseaseswasfirstreportedin1990(Handysideetal)40centersin17countrieshavesetupPGD,400healthybabiesbornamong500pregnanciesinabout2000cycles.,IndicationsofPGD,Geneticdiseases,notincludingsexselectionfornon-medicalreasonsHighriskofgeneticdiseases,ChromosomalabnormalitiesBalancedtranslocationDownssyndromeSinglegenediseasesX-linkeddiseasesAutosomalrecessivediseasesAutosomaldominantdiseases,ClinicalandlaboratoryprocedureinPGD,IVFandembryocultureEmbryobiopsyDiagnosis(PCR,FISH)EmbryotransferU/SandprenataldiagnosisFollow-upofnewbornbabies,Invivofertilization,Uterinelavage,囊胚,IVF/ICSI,Biopsy,Transfernormalembryos,PolarbodybiopsyInvitrofertilizationPolarbodydiagnosis,Biopsy,ThewaysofPGD,SpermseparationPolarbodybiopsyEarlycleavageembryobiopsyBlastocystbiopsy,Modifiedflowcytometry,Hoechst33342,IVF,ICSI,400m,X,Y,Polarbodybiopsy,Deduceoocytegenepatternthroughdetectionofthe1stand2ndpolarbodyFordetectionofmaternalgeneticmaterialsonly,notforpaternalChromosomeaneuploidydetectioninagedwomenTheimplantationrateinwomen36yearsoldwas25.6%,thepregnancyratewas12%(Gianaroli,1997).Thepregnancyratewas19.9%.PGDhelptoimproveimplantationrateandpregnancyrate,decreaseabortionrate.,Biopsyofearlycleavageembryo,Aspirate1-2blastomeresin6-8cellstageSimultaneouslydetectbothmaternalandpaternalDNAZonadrillingTyrodesacid(Handyside,1989)Partialzonadissection(Cohenetal,1989)Laser(Montag1998)EvaluationofthesafetyofPGDBlastocystrateHatchingrateImplantationrateFollow-upofnewbornNoevidenceshowsthatPGDincreasestheincidenceofcongenitalabnormalities(Harper,1996),MethodsofPGD,PCRFISHTechniquesderivedfromFISHandPCR,PCR,HighsensitivityEasytodetectlowcopyDNAandspecialnucleicacidsequenceContaminationandamplificationfailure,FISH,Performonintactblastomere,getresultinshorttimeHighspecificityandsensitivitySimultaneouslyshowingmultiplecolorsignalsAfterfixation,nucleushybridizeswithspecificlabeledchromosomeprobe.Diagnosiscanbemadethroughobservationofthenumberoffluorescencesignalsinnucleus,A:NormalmalebicolorFISH.RedsignalshowsXchromosomewhilegreensignalshowsYchromosomeB:SingleXsignalC:SingleYsignal,A,B,C,FluorescencePCR,AnewtechniquewhichdevelopsonthebasisofPCRandDNAhybridizationtechnique.FluorescencePCRcandetectfluorescencesignaldirectlyFluorescencePCRcanbeusedinsexidentificationandsinglegenedetection,OutcomeofPGD,VanSteirteghem2000.May,OutcomeofPGDchildren,VanSteirteghemMay.2000,OutcomeofPGDinSUMS,ApplicationofReconstructedOocyteinART,Cytoplasm-nuclearinteractionduringmaturationisveryimportantforfertilizationandembryodevelopment.,ReconstructedOocyteWhy？,Oocyteagingwithadvancingage！CytoplasmagingAccumulationoffreeradicalandROSMitochondriaDNAmutationATPproductionNuclearagingAbnormalmeioticapparatus-SpindleAneuploidyCytoplasmaging-Keypoint,RoleofMitochondriainCytoplasmAgingEnergyfactorymtDNA:maternalinherited,circular,histone-freeof16.6kbofDNA.Coding13proteinsubunitsrequiredforoxidativephosphorylation.,mtDNAMutationmtDNAmutation401/9（11%）40509/19（47%）5010/11（90%）KitagawaTetal，BiolReprod1993mtDNAdeletionratehigherinadvancedagewomen.KeefeDLetal，FertilSteril1995,WildingMetal，HumReprod2001,Impairedmitochondriaactivityinoldwomen,AccumulationoffreeradicalandROS,mtDNAmutationPhosphorylationdefectATP,OoplasmTransferimproveoocytequalityduetomitochondriadefectInjectionofmitochondriacanpreventoocytefromapoptosisinmice.PerezGIetal，Nature2000Ooplasmtransferhelpfulforagedwomenrepeatreproductivefailduetoimpairedembryodevelopment.CohenJetal，Lancet1997Cohenooplasmtransfer23cycles,12clinicalpregnancy,8delivery,1abortion.BrennerCAetal，FertilSteril2000,Abnormalspindlecauseincreasedaneuploidy.Nondisjunctionoocytechromosomeandaneuploidyratehigherin40yearsthanthatin35years（p0.001）.DaileyTetal，AmJHumGenet1996253435394045AneuploidyrateinMoocyte4.9%11.5%29.8%TsaiMCetal，HumReprod2000,OoplasmtransfercouldnotresolvespindleabnormalityandabnormalychromosomenondisjunctioninMIIoocyteduetoooplasmaaging.Methods：Nuclearreconstruction.,Beforefirstmeiosis,transferGVintonormalooplasmwhichcontentmitochondriamaintainnormalmeiosisZhangJetal，FertilSteril1999,Germinalvesicletransfer（ZhangJ,etal.FertilSteril1999:71:726),Germinalvesicletransfer(ZhangJ,etal.FertilSteril1999;71:726),Manufacturingoocytes(TsaiMC,etal.HumReprod2000;15:988),Manufacturingoocytes(TsaiMC,etal.HumRepr