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동결보존시 생쥐 전핵배아의 시기에 따른 생존율과 발생율의 비교
김희선,류범용,오선경,서창석,김석현,최영민,김정구,문신용,이진용,Kim, H.S.,Ryu, B.Y.,Oh, S.K.,Suh, C.S.,Kim, S.H.,Choi, Y.M.,Kim, J.G.,Moon, S.Y.,Lee, J.Y. 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1
This study was designed to evaluate the influence of pronuclear age on the survival and post-thawing development after cryopreservation of mouse embryos. Freezing and thawing were performed in the different pronuclear stages of mouse embryos after IVF. Embryos were obtained from $F_1$ hybrid mice and classified into 4 groups according to the pronuclear stage (6hr, 9hr, 12hr and 15hr after insemination). Pronuclear ova were slowly cooled in a biological freezer using 1.5M 1,2-propanediol and 0.1M sucrose as cryoprotectant. Thawing was done at room temperature and 1,2-propanediol was removed by multi-step dilutions. Both frozen-thawed embryos and control fresh embryos were cultured in vitro in Ham's F-10 medium supplemented with 4mg/ml BSA. In control group, the development rate after 48hr was 99.3%, and the complete hatching rate after 144hr was 61.3%. In experimental groups, the survival rate after thawing was 95.4% in 6hr, 88.7% in 9hr, 75.2% in 12hr and 62.4% in 15hr after insemination, the development rate after 48hr was 61.1, 77.0, 67.0 and 79.6%, respectively, and the complete hatching rate after 144hr was 25.7, 43.7, 42.2 and 60.0%, respectively. The survival rate in 15hr was significantly lower (p<0.05) compared with other groups. In vitro development rates after 48hr were similar in all groups, but complement hatching rate was significantly lower (p<0.05) in 6hr group. In conclusion, cryopreservation of mouse pronuclear ova with 2 distinct pronuclei (9hr and 12hr groups) showed better results after thawing compared with early (6hr group) or late pronuclear ova just prior to cleavage (15hr group).
생쥐 모델을 이용한 배아의 할구 생검법과 할구가 생검된 배아의 배양시 공배양 효과에 관한 연구: 인간에서의 착상 전 유전진단 기술 개발을 위한 동물실험 모델의 개발
김석현,류범용,지병철,최성미,김희선,방명걸,오선경,서창석,최영민,김정구,문신용,이진용,채희동,김정훈,Kim, S.H.,Ryu, B.Y.,Jee, B.C.,Choi, S.M.,Kim, H.S.,Pang, M.G.,Oh, S.K.,Suh, C.S.,Choi, Y.M.,Kim, J.G.,Moon, S.Y.,Lee, J.Y.,Chae, H.D.,Kim, C.H. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.1
The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.
난자 세포질내 정자 주입술 후 동결보존 배아이식: 고식적 체외수정시술과의 비교 연구
김석현,지병철,정병준,김희선,류범용,방명걸,오선경,손철,서창석,최영민,김정구,문신용,이진용,Kim, S.H.,Jee, B.C.,Jung, B.J.,Kim, H.S.,Ryu, B.Y.,Pang, M.G.,Oh, S.K.,Shon, C.,Suh, C.S.,Choi, Y.M.,Kim, J.G.,Moon, S.Y.,Lee, J.Y. 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.3
The objective of this study was to compare retrospectively the survival and pregnancy rates(PR) of cryopresered-thawed embryos obtained from intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Ninety-six cycles of cryopresered-thawed embryo transfer (ET) were performed in 79 patients from June, 1996 to September, 1997 and grouped as followings: 20 cycles (16 patients) inseminated by ICSI (ICSI Group) and 76 cycles (63 patients) by conventional IVF (IVF Group). Slow-freezing and rapid-thawing protocol was used with 1.5M propanediol (PROH) and 0.1M sucrose as cryoprotectant. All embryos were frozen-thawed at the two pronuclear (2 PN) stage excluding four cycles in which the early cleavage stage embryos were frozen, and allowed to cleave in vitro for one day before ET. The duration from freezing to thawing was comparable in both groups ($mean{\pm}SD$, $112.1{\pm}80.0$ vs. $124.8{\pm}140.1$ days). The age of female ($31.2{\pm}3.4$ vs. $32.6{\pm}3.3$ years) and the endometrial thickness prior to progesterone injection ($9.4{\pm}2.0$ vs. $9.3{\pm}1.8$ mm) were also comparable in both groups. There was no significant difference in the outcomes of cryopreserved-thawed ET between two groups: survival rate ($85.2{\pm}16.1%$ vs. $82.2{\pm}19.7%$), cleavage rate ($96.9{\pm}6.7%$ vs. $94.7{\pm}13.0%$), cumulative embryo score (CES, $54.5{\pm}31.1$ vs. $49.0{\pm}20.0$), preclinical loss rate (5.0% vs. 5.3%), clinical miscarriage rate (0% vs 29.4%), clinical PR per transfer (35.0% vs. 22.4%), implantation rate (9.9% vs. 5.6%), and multifetal PR (42.9% vs. 17.6%). In conclusion, human embryos resulting from ICSI can be cryopreserved-thawed and transferred successfully, and the survival rate and PR are comparable to conventional IVF.
동결보존된 부고환 정자로 ICSI 시술 후 수정된 수정란의 동결보전 및 배아이식에 의한 임신 1례
문신용,이희선,김희선,류범용,방명걸,오선경,서창석,김석현,최영민,김정구,이진용,Moon, S.Y.,Lee, H.S.,Kim, H.S.,Ryu, B.Y.,Pang, M.G.,Oh, S.K.,Suh, C.S.,Kim, S.H.,Choi, Y.M.,Kim, J.G.,Lee, J.Y. 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.2
This case report describes the pregnancy following the transfer of cryopreserved embryos generated from intracytoplasmic sperm injection (ICSI) using frozen-thawed sperm obtained by microepididymal sperm aspiration (MESA) in patient with congenital absence of the vas deferens (CAVD).
김창근(C . K . Kim),정영채(Y . C . Chung),윤종택(J . T . Yoon),최선하(S . H . Choi),류범용(B . Y . Ryu),정광조(K . J . Chung),김흥율(H . R . Kim),송해범(H . B . Song) 한국축산학회 1990 한국축산학회지 Vol.32 No.1
Effects of adding 10 to 20% fetal calf serum(FCS) and estrous cow serum(ECS), and hormones(GTH or GTH plus E₂) to maturation medium on in vitro maturation(IVM) and in vitro fertilization(IVF) of bovine follicular oocytes and effects of sperm treatment and bull on IVF and embryo development were studied. Finally, developmental potential these in-vitro fertilized embryos at 2-to 8-cell stage(44h postinsemination) was investigated under in vivo(rabbit oviduct) and in vitro(Ham`s Fl0 plus 10% FCS with or without bovine oviduct epithelial cells, BOEC) 4 day-culture system. 1. Addition of 10 to l5% FCS to mKRB and DM(-BSA) and of 20% FCS to Ham`s F10 increased(65%) IVM, respectively. IVM of these media supplemented with hormones was higher(70∼72%) than that of media without hormone. Addition of ECS showed higher IVM at 10% level for mKRB and 10 to 15% for TCM199 and the highest IVM(88%) was obtained in TCM199 containing 10% ECS and LH. 2. Higher IVF rate (based on formation of both pronuclei) in Exp I was obtained from oocytes matured in mKRB plus 10% FCS and HIS-sperm treatment. In Exp 2. IVFrate (based on cleavage. 2-3 cells) of oocytess matured in TCM199 was higher than in other media and IVF using fresh semen was significantly higher but was similar between sperm treatments. In Exr 3, IVM in TCM199 containing 10% ECS and LH and IVF by C+H-sperm treatment significantly increased early cleavage(2-to 8-cell). 3. In vitro-acrosome reaction(AR) among individual bulls in Exp 1 ranged from 16 to 45% and bull group having lower AR showed low IVF rate than group having higher AR. In Exp 3, cleavage rate by sperm treatment with both chemicals(C+H) was higher than that by H alone. 4. In IVF with in vivo-capaciated sperm, development from 2-to 3-cell to above 8-cell was higher under in vivo culture(54.5%) than under in vitro culture(31.3%) and higher in oocytes matured in TCM199 plus to ECS. In IVF with in vitro-capacitated sperm, development into above 8-cell stage was different between initial stage(2-to 3-and 4-to 8-cell) of embryos(26.7% : 41.7%) and significantly lower under in vitro culture from 2-to 3 cell stages(l0% : 29.4%). Under in vitro co-culture with BOEC, embryo development from 4-to 8-cell was significantly improved(53.3%) and 3(20%) of 15 cultured embryos developed into morula. The present results indicate that addition of ECS and hormones can improve IVM, IVF and development of bovine follicular oocytes, sperm factor may be important for IVF and further development, and co-culture with BOEC is an effective means for development into above 16-cell stage.