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1、BETTER ASSISTED REPRODUCTIVE TECHNIQUE,Reproductive Medical Research CenterSun Yat-Sen University of Medical Sciences Zhuang Guang-Lun,Preimplantation Genetic Diagnosis(PGD),History of PGD,Gender detection of rabbit blastocyst in 1968 Success of IVF(1978)and development of molecular biology made PGD
2、 possible in human PGD success in X-linked diseases was first reported in 1990(Handyside et al)40 centers in 17 countries have set up PGD,400 healthy babies born among 500 pregnancies in about 2000 cycles.,Indications of PGD,Genetic diseases,not including sex selection for non-medical reasons High r
3、isk of genetic diseases,Chromosomal abnormalities Balanced translocation Downs syndrome Single gene diseases X-linked diseases Autosomal recessive diseases Autosomal dominant diseases,Clinical and laboratory procedure in PGD,IVF and embryo culture Embryo biopsy Diagnosis(PCR,FISH)Embryo transfer U/S
4、 and prenatal diagnosis Follow-up of new born babies,In vivo fertilization,Uterine lavage,囊胚,IVF/ICSI,Biopsy,Transfer normal embryos,Polar body biopsy In vitro fertilization Polar body diagnosis,Biopsy,The ways of PGD,Sperm separation Polar body biopsy Early cleavage embryo biopsy Blastocyst biopsy,
5、Modified flow cytometry,Hoechst 33342,IVF,ICSI,400m,X,Y,Polar body biopsy,Deduce oocyte gene pattern 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
6、 old was 25.6%,the pregnancy rate was 12%(Gianaroli,1997).The pregnancy rate was 19.9%.PGD help 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
7、 drilling Tyrodes acid(Handyside,1989)Partial zona dissection(Cohen et al,1989)Laser(Montag&Boada,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 o
8、f the safety of PGD Blastocyst rate Hatching rate Implantation rate Follow-up of newborn No evidence shows that PGD increases the incidence of congenital abnormalities(Harper,1996),Methods of PGD,PCR FISH Techniques derived from FISH and PCR,PCR,High sensitivity Easy to detect low copy DNA and speci
9、al nucleic acid sequence Contamination and amplification failure,FISH,Perform on intact blastomere,get result in short time High specificity and sensitivity Simultaneously showing multiple color signals After fixation,nucleus hybridizes with specific labeled chromosome probe.Diagnosis can be made th
10、rough observation of the number of fluorescence signals in nucleus,A:Normal male bicolor FISH.Red signal shows X chromosome while green signal shows Y chromosomeB:Single X signalC:Single Y signal,A,B,C,Fluorescence PCR,A new technique which develops on the basis of PCR and DNA hybridization techniqu
11、e.Fluorescence PCR can detect fluorescence signal directly Fluorescence PCR can be used in sex identification and single gene detection,Outcome of PGD,Van Steirteghem 2000.May,Outcome of PGD children,Van Steirteghem May.2000,Outcome of PGD in SUMS,Application of Reconstructed Oocyte in ART,Cytoplasm
12、-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 and ROS Mitochondria DNA mutation ATP productionNuclear aging Abnormal meiotic apparatus-Spindle An
13、euploidy 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 oxidative phosphorylation.,mtDNA Mutation mtDNA mutation 40 1/9（11%）4050 9/19（47%）50 10/11（90%）Kitagawa T e
14、t 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 2001,Impaired mitochondria activity in old women,Accumulation of free radical and ROS,mtDNA mutationPhosphorylation defectATP,Ooplasm Transferimprove oocyte quality due
15、 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 impaired embryo development.Cohen J et al，Lancet 1997Cohen ooplasm transfer 23 cycles,12 clinical pregnancy,8 d
16、elivery,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 years（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
17、et al，Hum Reprod 2000,Ooplasm transfer could not resolve spindle abnormality and abnormaly chromosome nondisjunction in MII oocyte due to ooplasma aging.Methods：Nuclear reconstruction.,Before first meiosis,transfer GV into normal ooplasm which content mitochondria maintain normal meiosis Zhang J et
18、al，Fertil Steril 1999,Germinal 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 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 2000,Sequential Transfer,Thank you！,
