Analyze Abnormal Fertilization Embryo With FISH And Explore A Technique For Karyotyping Embryo Single Blastomere

Background:Chromosome abnormality in human preimplantation embryo is verycommon and often cause arrest of embryo development or spontaneousabortion. Normal zygote possesses two pronucleus, the abnormal zygoteincludes single pronucleate zygotes,tripronuclear zygotes andapronuclear zygotes.The presence of numerical chromosome abnormalities in humanembryos was studied using fluorescence in-situ hybridization with four ormore chromosome-specific probes. However, the major limitation of thetechnique is the limited chromosome analysesed by FISH and the limitedavailability of locus-specific DNA probes, which are needed for eachparticular case of translocation. The development of a reliable method forobtaining a metaphase chromosome plate from a biopsied blastomerewould allow the use of standard probes for whole chromosome painting.Objective:1. To analyze the normal rate of the chromosomes of the abnormalfertilization oocyte.2. To develop a reliable technique for karyotyping single humanblastomeres for preimplantation diagnosis.Methods:1.Isolate the single pronucleate zygote from normal zygotes and goon culturing to the five day, if embryo developing to blastocyst, we willseparate the ICM and analysis the remaining trophoblast cells by FISH; Ifembryo can't develop to blastocyst, we will analysis all blastomeresrespectively by FISH;2. We take out of the embryo's blastomere and carry out a series ofoperation to obtain a metaphase chromosome plate:1) Use colcemid to induce blastomere' chromosome to metaphaseplate directly;2) Enucleating the in-vitro maturation human oocyte and fusing theoocyte with the blastomere;3) Fuse the blastomere with mouse Mâ…¡oocyte and induce PCC;4) Fuse the blastomere with mouse zygote. Results:1. We have analysesed 138 embryos from single pronucleate zygotesby FISH and obtain 87 successful outcomes. Among them, 48 comesfrom IVF, the pencentage of haploid, diploid, chimera and multiploid is18.8%,54.2%,20.8% and 6.2% respectively;39 from ICSI, thepencentage of haploid, diploid, chimera and multiploid is 46.2%,25.6%,23.1% and 5.1% respectively. In 87 successful outcomes, 16embryos develop to blastocyst, twelve of them is diploid (75.0 %), four ischimera (25.0 %);2. The results of karyotyping single human blastomeres as follow:1) 57 blastomeres inducing by colcemid directly, we get 9metaphase and success ratio is 15.8%;2) 26 blastomeres inducing by human IVM oocytes, we get 4metaphase and success ratio is 15.4% ;3) 13 blastomeres inducing by mouse oocytes, but all oocytes areactivated by the electric impulse and success ratio is 0;4) 160 blastomeres inducing by mouse zygotes, we get 25metaphase and success ratio is 15.6 %.Conclusion:1. The FISH results indicate that single pronucleate ICSI zygotesare usually haploid, the possible reason is that the sperm does notde-condensation but the oocyte has been activated; But the singlepronucleate IVF zygotes are usually diploid, the possible reason isasynchrony of the pronuclear appearance or possible male and femalepronuclear fusion. Our research demonstrates that if the embryo fromsingle pronucleate zygote can develop to blastocyst, they are all diploid orchimera. The reason perhaps is that the haploid embryo hasn't thepotential to develop to blastocyst.2. Using four methods, the success rates of preparing the metaphaseplate are all about 15.5%. at present, the efficiency of inducing theblastomere's chromosome to metaphase plate is still low, and thistechnique doesn't fit for PGD now.