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Korean Journal of Fertility and Sterility 2003;30(2):111-118.
Published online June 1, 2003.
Development of an Efficient Method for Obtaining Metaphase Chromosomes in Individual Blastomeres of Mouse and Human Preimplantaion Embryos: Effect of Microtubule Depolymerizing Agents.
Chun Kyu Lim, Dong Mi Min, Hyoung Song Lee, Jin Young Kim, Mi Kyoung Koong, Inn Soo Kang, Jin Hyun Jun
1Laboratory of Reproductive Biology & Infertility, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
2Department of Obstetrics and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
OBJECTIVES: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. MATERIALS AND MEHTODS: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. RESULTS: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were 1.0 micrometer of vinblastine (20.3%), 5.0 micrometer of nocodazole (28.1%) and 1.0 micrometer colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 micrometer) and nocodazole (1.0 micrometer). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. CONCLUSIONS: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.
Key Words: Preimplantation genetic diagnosis; Metaphase chromosomes; Microtubule depolymerizing agent (MTDA); Fluorescence in-situ hybridization (FISH)
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