1、BETTER ASSISTED REPRODUCTIVE TECHNIQUEPreimplantation Genetic Diagnosis(PGD)History of PGDo Gender detection of rabbit blastocyst in 1968o Success of IVF(1978)and development of molecular biology made PGD possible in humano PGD success in X-linked diseases was first reported in 1990(Handyside et al)
2、o 40 centers in 17 countries have set up PGD,400 healthy babies born among 500 pregnancies in about 2000 cycles.Indications of PGDo Genetic diseases,not including sex selection for non-medical reasonso High risk of genetic diseases s Chromosomal abnormalities?Balanced translocation?Downs syndromes S
3、ingle gene diseases?X-linked diseases?Autosomal recessive diseases?Autosomal dominant diseasesClinical and laboratory procedure in PGDo IVF and embryo cultureo Embryo biopsyo Diagnosis(PCR,FISH)o Embryo transfero U/S and prenatal diagnosiso Follow-up of new born babiesIn vivo fertilizationUterine la
4、vage囊胚囊胚IVF/ICSIBiopsyTransfer normal embryos Polar body biopsy In vitro fertilization Polar body diagnosisBiopsyThe ways of PGD Sperm separation Polar body biopsy Early cleavage embryo biopsy Blastocyst biopsyModified flow cytometryHoechst 33342IVFICSI400 mX YPolar body biopsy Deduce oocyte gene pa
5、ttern through detection of the 1st and 2nd polar body For detection of maternal genetic materials only,not for paternal Chromosome aneuploidy detection in aged women The implantation rate in women 36 years old was 25.6%,the pregnancy rate was 12%(Gianaroli,1997).The pregnancy rate was 19.9%.PGD help
6、 to improve implantation rate and pregnancy rate,decrease abortion rate.Biopsy of early cleavage embryo Aspirate 1-2 blastomeres in 6-8 cell stage Simultaneously detect both maternal and paternal DNA Zona drilling Tyrodes acid(Handyside,1989)Partial zona dissection(Cohen et al,1989)Laser(Montag&Boad
7、a,1998)The safety of embryo biopsy Decrease of blastomeres numbers results in delayed development after biopsy,however,it will not influence the potential of further development(Viville;1998)Evaluation of the safety of PGDv Blastocyst ratev Hatching ratev Implantation ratev Follow-up of newborn No e
8、vidence shows that PGD increases the incidence of congenital abnormalities(Harper,1996)Methods of PGD PCR FISH Techniques derived from FISH and PCRPCRu High sensitivity u Easy to detect low copy DNA and special nucleic acid sequence u Contamination and amplification failureFISHo Perform on intact bl
9、astomere,get result in short timeo High specificity and sensitivityo Simultaneously showing multiple color signals o After fixation,nucleus hybridizes with specific labeled chromosome probe.Diagnosis can be made through observation of the number of fluorescence signals in nucleusA:Normal male bicolo
10、r FISH.Red signal shows X chromosome while green signal shows Y chromosomeB:Single X signalC:Single Y signalABCFluorescence PCRo A new technique which develops on the basis of PCR and DNA hybridization technique.Fluorescence PCR can detect fluorescence signal directlyo Fluorescence PCR can be used i
11、n sex identification and single gene detectionOutcome of PGD Van Steirteghem 2000.May Total per cycle(%)per transfer(%)cycle 309transfer cycle 250 80hCG(+)74 24 30 pregnancy(12 W)61 20 24Outcome of PGD childrenVan Steirteghem May.2000Ongoing(12 week)61Termination of pregnancy 1 misdiagnosis for MDNo
12、t delivered 16Delivered 42 12 twin pregnancyBorn 54Stillborn 3 2 MZ twin 1 singletonDied 2 2 from 2 twinAlive 49Outcome of PGD in SUMS case cycle transfer hCG(+)pregnancy deliveredY chrom.abnormality 6 7 7 3 1 1X-linked disease 2 3 3 2 2 2 (hemophilia-A)Chrom.abnormality 2 2 2 1 1 -thalassemia 6 6 6
13、 1 0 0-thalassemia 1 1 1 1 1 1 Total 17 19 19 8 5 4Application of Reconstructed Oocyte in ARTCytoplasm-nuclear interaction during maturation is very important for fertilization and embryo development.Reconstructed OocyteWhy？Oocyte aging with advancing age！Cytoplasm aging Accumulation of free radical
14、 and ROS Mitochondria DNA mutation ATP productionNuclear aging Abnormal meiotic apparatus-Spindle Aneuploidy Cytoplasm aging-Key point Role of Mitochondria in Cytoplasm AgingEnergy factorymtDNA:maternal inherited,circular,histone-free of 16.6 kb of DNA.Coding 13 protein subunits required for oxidati
15、ve phosphorylation.mtDNA Mutation mtDNA mutation 40 1/9 （11%）4050 9/19（47%）50 10/11（90%）Kitagawa T et al，Biol Reprod 1993 mtDNA deletion rate higher in advanced age women.Keefe DL et al，Fertil Steril 1995 Wilding M et al，Hum Reprod 2001Impaired mitochondria activity in old women Accumulation of free
16、 radical and ROSmtDNA mutationPhosphorylation defectATPDonorPatientOoplasm Transferimprove oocyte quality due to mitochondria defectInjection of mitochondria can prevent oocyte from apoptosis in mice.Perez GI et al，Nature 2000Ooplasm transfer helpful for aged women repeat reproductive fail due to im
17、paired embryo development.Cohen J et al，Lancet 1997Cohen ooplasm transfer 23 cycles,12 clinical pregnancy,8 delivery,1 abortion.Brenner CA et al，Fertil Steril 2000 Abnormal spindle cause increased aneuploidy.Nondisjunction oocyte chromosome and aneuploidy rate higher in 40 years than that in 35 year
18、s（p0.001）.Dailey T et al，Am J Hum Genet 1996 2534 3539 4045 Aneuploidy rate in Moocyte 4.9%11.5%29.8%Tsai MC et al，Hum Reprod 2000Ooplasm transfer could not resolve spindle abnormality and abnormaly chromosome nondisjunction in MII oocyte due to ooplasma aging.Methods：Nuclear reconstruction.Before f
19、irst meiosis,transfer GV into normal ooplasm which content mitochondria maintain normal meiosis Zhang J et al，Fertil Steril 1999Germinal vesicle transfer（Zhang J,et al.Fertil Steril 1999:71:726)Germinal vesicle transfer(Zhang J,et al.Fertil Steril 1999;71:726)Manufacturing oocytes(Tsai MC,et al.Hum
20、Reprod 2000;15:988)Manufacturing oocytes(Tsai MC,et al.Hum Reprod 2000;15:988）Manufacturing oocytes(Tsai MC,et al.Hum Reprod 2000;15:988)Denudation and in vitro maturation impede development of reconstructed GV oocyte.Sequential transfer can improve development potent.Liu H et al，Hum Reprod 2000Sequential Transfer
