Study On Preimplantation Genetic Diagnosis By Fluorescence In-situ Hybridization And Its Prime Clinical Application

Objectives: 1. To erect a safe and feasible strategy of embryo biopsy;2. To establish an exact, reliable and stable fluorescence in-situ hybridization technique for preimplantation genetic diagnosis in earlystage human embryos;3. To analysis the chromosomal abnormality in embryos from multi-pronucleus by fluorescence in-situ hybridization; 4. To study the relationship between the embryo morphologic indicators and the chromosome 21 aneuploidy ; 5. To investigate the clinical application of preimplantation genetic diagnosis by fluorescence in-situ hybridization.Methods: 1. Single blastomere from 8-cells mice embryos was sampled by microbiopsy after acidic Tyrode's zona drilling or mechanical dissection. Mice embryo survival and blastulation rate were compared between the two zona drilling methods; 2. Human D3 embryos with <3 cells were collected, fixed and fluorescence in situ hybridized; 3. Fluorescence in-situ hybridization was undertaken in D2 embryos from multi- pronucleus zygotes were using CEPX probe and CEPY probe. Abnormality of sex chromosome of embryos from conventional in vitro fertilization or intracytoplasmic sperm injection was observed. 4. Abnormality of chromosome 21 in embryos of hypoevolutism and embryos with >40% fragment were analysed by fluorescence in-situ hybridization using chromosome 21 locus specific identifier probe (LSI21); 5. 9 PGD clinical cycles in which couples are at risk of a known genetic defect were carried out.Rusults: 1. Comparison of the acidic Tyrode's group and the mechanical dissection group revealed no significant difference in embryosblastulation rate; the embryo survival rate in acidic Tyrode's group was remarkably higher than in mechanical dissection group ;2 .For D3 embryos, the fixation rate of nuclear is 95.91%, and the hybridization rate is 93.62%; 3 Chromosome X, Y aneuploidy differed in embryos from multi-pronucleus between the IVF group and ICSI group; 4. There is significant difference in abnormality rate of chromosome 21 between the embryos of hypoevolutism and that with >40% fragment and normal embryos; 5. In 9 PGD therapy cycles, embryo biopsy success rate was 96% , and FISH hybridization rate was 95.83%.After embryo transfer,two pregnancies were obtained, the pregnancy rate was 22.22%.Conclusion: 1. Acidic Tyrode's drilling may be an ideal approach for zona drilling in embryos biopsy for high success rate and easy operation Acidic Tyrode's drilling method would be a capital method; 2. The results show that FISH can be used in experimental study and clinical application of PGD; 3. The cause of multi- pronucleus in embryos from IVF or ICSI would be different. Sex chromosomal normality rates in multi- pronucleus embyos from IW and ICSI were 3.17% and 2.94% respectively. Multi- pronucleus embryos should be avoided for embryo transfer; 4. Cleavage velocity and fragment amount of embryo were related to chromosome 21 aneuploidy. For embryo transfer, embryos can be chosen according to normal developing status and cleavage velocity ; 5. 9 PGD cases by fluorescence in-situ hybridization leading to 2 pregnancy illustrate that PGD clinical application in our center is feasible.