Establishment And Application Of Preimplantation Genetic Diagnosis System For Chromosome Translocation Carrier

Objective: (1)To establish a preimplantation genetic diagnostic (PGD)system for chromosome translocation carder by single-cell BAC-FISH(bacterial artificial chromosome-Fluorescent in situ hybridization,BAC-FISH) .(2) Apply the PGD system to clinic practice.Method: (1)Choose a chromosome translocation carrier, who wasconfirmed a karyotype of 46, XY, t(1;12)(q32; q13) by G banding .Heneeded the IVF/ICSI and desired to accept PGD. According to thecarrier's karyotype, we chose suitable BAC clones and made theBAC-probes .Then preliminary experiment was performed in peripheralblood lymphocyte of normal control and the carrier, my lab's lymphocytestrain.Single-cell BAC-FISH was performed in blastomeres from thecouples with normal karyotype alter IVF/ICSI (in-vitro fertilization/intracytoplasmic sperm injection) .(2) PGD was performed in fourchromosome translocation carder couples. Case 1 was a male carrier withthe karyotype of 46, XY, t(1;12)(q32; q13);Case 2 was amale carrierwith the karyotype of 46, XY, t(7; 13) (p10; q10);Case 3 was a femalecarrier with the karyotype of 46, XX, t (1; 5) (p32; q35.1) and Case4 was a male carder with the karyotype of 45, XY, der (13; 14) (q10;q10) .All the spouses of 4 carriers were with normal karyotype.Result: (1) In the PGD system established, the probes averagehybridization efficiency and specificity was 95.9%,100% in peripheralblood lymphocyte respectively.35 lymphocytes were fixed byTH((Tween-20/HC1) method. 34.3% (12/35) lymphocytes were lost.65.7% (23/35) lymphocytes had one intact nucleus. 0% (0/35) lymphocytehad two nuclei. 98 lymphocytes were fixed by TH+MA (Tween-20/HC1+Methanol :Acetic acid). 3.1% (3/98) lymphocytes were lost. 94.9%(93/98) lymphocytes had one intact nucleus. 2%(2/98) lymphocytes hadtwo nuclei.205 blastomeres were fixed . 0% (0/205) was lost. 47.4%(97/205) blastomeres had one intact nucleus. 6.8% (14/205)blastomeres had more than one nucleus. 19.0% (39/205) blastomeres hadnuclear fragmentation. And 26.8% (55/205) blastomeres had no nucleus.Among the 97 nuclei, FISH signals occurred in 93(95.9%) nuclei. (2) The four PGD cycle indicated that six embryos with normal/balancedchromosome constitution (case 1:1 embryo; case 2: lembryo; case 4:4embryos).Five embryos with normal/balanced chromosome constitutionwere transferred (case 1:1 embryo; case 2: lembryo; case 4:3 embryos).Case 3 had two embryos with undetermined results, were transferred. Onepregnancy was achieved (Case 4).Conclusion: (1) We established the PGD system for chromosometranslocation carder by single-cell BAC-FISH. (2) At present, comparedwith the PGD by two commercialized probes in our country, the PGDsystem we established can reduce the cost, and also is more precise. (3)A carrier couple with 45, XY, der(13; 14)(q10; q10)had one pregnancysuccessfully by the PGD system .(4) The system established foundationfor patient with other abnormal chromosome. The consummate PGDwould be provided for more patient and family with abnormalchromosome.