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대한생식의학회지 제30권 제2호 2010년
착상전 배아의 분리된 할구에서 중기염색체 상을 획득하기위한 효율적인 방법의 개발에 대한 연구: 미세소관 형성 저해제의 효과
삼성제일병원 생식생물학 및 불임연구실,성균관대학교 의과대학 산부인과학교실
임천규, 민동미,이형송,김진영,궁미경,강인수,전진현
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
Laboratory of Reproductive Biology & Infertility, Department of Obstetrics and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Kore
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 Methods: 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 mM of vinblastine (20.3%), 5.0 mM of nocodazole (28.1%) and 1.0 mM colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 mM) and nocodazole (1.0 mM). 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.
키워드 : Preimplantation genetic diagnosis, Metaphase chromosomes, Microtubule depolymerizing agent (MTDA), F
교신저자 : junjh55@hanmail.net
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