| Preimplantation genetic diagnosis (PGD) is a technique to detect the genetic composition in the oocytes or embryos before the pregnancy is established. Therefore, the termination of pregnancy decided by the abnormal result from in prenatal genetic diagnosis could be avoided by PGD. At present, PGD is mainly used in the infertility couple with high risk of transmission of genetic disease or chromosome abnormality to the offspring.Histocompatibility leukocyte antigen (HLA) is the strongest human homologous antigen, which is distributed in cell surface with germ line specificity in responsible for activating immunologic cells. Hemopoietic stem cell transplantation (HSCT) includes bone marrow transplantation (BMT), peripheral blood stem cell transplantation(PBSCT), cord blood stem cell transplantation (CBSCT), fetal hepatocytes transplantation and CD34+ cells transplantation etc. Cord blood, an alternative source of hemopoietic tissue for transplantations, has been successfully applied in the treatments of the diseases such as leukemia, lymphoma, aplastic anemia,heredity immunodeficiency and mediterranean anemia etc. However, the main problem for CBSCT application is the sources of HLA matching cord blood because a full HLA compatibility is desirable in transplantation. The chance of sibling HLA matching is 25% theoretically, but it is no more than 15% in a small family. On average, 10-12 embryos can be achieved in one cycle of in-vitro fertilization and embryo transfer (IVF-ET). Therefore, the theoretical chance of HLA matching embryos is 25% and at least one embryo might be matched. Using PGD to detect HLA antigens in preimplantational embryos and selecting the HLA antigen matched embryos for uterus transfer will open a new approach to the source of cord blood for patient sibling who needs CBSCT.Multiplex nested-PCR (MN-PCR) can analyze multi-loci with high sensibility and specificity while primer extension preamplification (PEP) could amplify the whole genome by a mixture 15 base random oligonucleotides as primers. Successful application of MN-PCR and PEP for detection of Y chromosomal microdeletion, Tay Sschs Disease, Cystic Fibrosis, Hemophilia A, Duchenne muscular dystrophy, RhD blood type and some short tandem repeat (STR) have reported. Although Verlinsky has matched HLA-A,B typing by PGD for CBSCT of Fanconi Anemia, there is so far no reports simultaneous detecting HLA-A,B and DR typing in a single cell or apply the technique of WGA in this field.For investigation of an approach to detect HLA-A,B,DR typing in single cell and the possibility to apply these techniques in clinic, single lymphocyte or single blastomere MN-PCR and PEP for detecting HLA typing were carried out and the sensibility and specificity were examined.1. HLA typing analysis in a single cell by MN-PCR Objective To establish the technique of HLA typing in a single cell by MN-PCR,examine the accurate rate of this technique and investigate whether PGD by MN-PCR could be used for preimplantational HLA matching. Methods Single-lymphocyte and single-blastomere were donated by infertility couples bearing ICSI. Using inter and outer primers, HLA-A,B,DR typing are carried out by MN-PCR. Results: 1. MN-PCR detecting HLA-A, B, DR typing in single-lymphocyte and single-blastomere was successfully established. 2. The accurate amplification rates in single lymphocytes and single blastomeres are 97.25%(671/690) and 98.13%(157/160) respectively. 3. There are no significant differences between single-lymphocyte and single-blastomere in amplification rate, false positive rate, false negative rate and accurate rate (P>0.05).2. HLA typing analysis in a single cell by PEP followed by MN-PCRObjective To establish the technique of HLA typing analysis in a single cell by PEP followed by MN-PCR, examine the amplification rate of this technique and investigate whether PGD by PEP-MN-PCR could be used for preimplantational HLA antigen matching. Methods Single-lymphocyte and single-blastomere were donated by infertility couple... |
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