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대한생식의학회지 제27권 제1호 2010년

착상 전 유전진단 기술 개발의 동물실험 모델로서 할구 생검된 생쥐 배아에서 동결보존 융해 후 배아 발생 양상과 공배양 효과에 관한 연구

서울대학교 의과대학 산부인과학교실, 의학연구원 인구의학연구소;서울대학교 의학연구원 인구의학연구소;서울대학교 의학연구원 인구의학연구소;서울대학교 의학연구원 인구의학연구소;한국과학기술원 의과학연구센터;서울대학교 의학연구원 인구의학연구소;서울대학교 의과대학 산부인과학교실, 의학연구원 인구의학연구소;서울대학교 의과대학 산부인과학교실, 의학연구원 인구의학연구소;서울대학교 의과대학 산부인과학교실, 의학연구원 인구의학연구소;서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실, 의학연구원 인구의학연구소;서울대학교 의과대학 산부인과학교실;울산대학교 의과대학 산부인과학교실;울산대학교 의과대학 산부인과학교실;

김석현;김희선;류범용;최성미;방명걸;오선경;지병철;서창석;최영민;김정구;문신용;이진용;채희동;김정훈;,

Developmental competence and Effects of Coculture after Crypreservation of Blastomere-Biopsied Mouse Embryos as a Preclinical Model for Preimplantation Genetic Diagnosis

Kim, Seok-Hyun;Kim, Hee-Sun;Ryu, Buom-Yong;Choi, Sung-Mi;Pang, Myung-Geol;Oh, Sun-Kyung;Jee, Byung-Chul;Suh, Chang-Suk;Choi, Young-Min;Kim, Jung-Gu;Moon, Shin-Yong;Lee, Jin-Yong;Chae, Hee-Dong;Kim, Chung-Hoon;

Department of Obstetrics and Gynecology, College of Medicine, Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Biomedical Research Center, Korea Advanced Institute of Science and Technology;Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Institute of Reproductive Medicine and Population, Medicine Research Center, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Ulsan University;Department of Obstetrics and Gynecology, College of Medicine, Ulsan University;

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

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