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

김석현,김희선,류범용,최성미,방명걸,오선경,지병철,서창석,최영민,김정구,문신용,이진용,채희동,김정훈,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 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.1

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.

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.

체외수정시술시 배아의 보조부화술을 이용한 임신율 향상에 관한 연구

김석현,김광례,채희동,이재훈,김희선,류범용,오선경,서창석,최영민,김정구,문신용,이진용,Kim, Seok-Hyun,Kim, Kwang-Rye,Chae, Hee-Dong,Lee, Jae-Hoon,Kim, Hee-Sun,Ryu, Buom-Yong,Oh, Sun-Kyung,Suh, Chang-Suk,Choi, Young-Min,Kim, Jung-Gu,Moon, S 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.1

In spite of much progress in vitro fertilization and embryo transfer (IVF-ET) program, the pregnancy rate remains at 20-30%, and the endometrial implantation rate per embryo transferred at 10-15%. As a result, about 90% of embryos may fail to implant to the endometrium, and many attempts such as optimization of follicular development, improvement of in vitro culture system including coculture, and micromanipulation of zona pellucida have been made to improve embryonic implantation after IVF-ET. Recently, several procedures of assisted hatching (AH) using micromanipulation have been introduced, and pregnancies and births have been obtained after AH. To develop and establish AH as an effective procedure to improve embryonic implantation, AH with partial zona dissection (PZD) was performed in 116 cycles of 89 infertile couples who had previous repeated failures of standard IVF-ET more than two times (Group I: 71 cycles in 54 patients), or who had implantation failure of embryos with good quality (Group II: 15 cycles in 13), or who had undergone AH without specific indication (Group III: 30 cycles in 22) from January, 1995 to Februry, 1996, and the outcomes of AH were analyzed according to pregnancy rate. The number of oocytes retrieved after controlled ovarian hyperstimulation (COH) was $9.9{\pm}7.1$ in Group I, $11.5{\pm}4.5$ in Group II, and $7.9{\pm}6.4$ in Group III. The number of embryos transferred after AH was $4.7{\pm}1.8$ in Group I, $5.3{\pm}1.3$ in Group II, and $3.5{\pm}2.4$ in Group III. The mean cumulative embryo score (CES) was $56.8{\pm}30.0$ in Group I, $76.1{\pm}35.9$ in Group II, and $38.5{\pm}29.9$ in Group III. The overall clinical pregnancy rate per cycle and per patient was 12.7% (9/71) and 16.7% (9/54) in Group I, 33.3% (5/15) and 38.5% (5/13) in Group II, and 6.7% (2/30) and 9.1% (2/22) in Group III, respectively. There were significant differences in the numbers of oocytes retrieved and embryos transferred, CES, and the clinical pregnancy rate per cycle among three groups. There was a significant inverse correlation between basal serum FSH level and CES, and no pregnancy occurred in patients with CES less than 20. In conclusion, AH of human embryos with PZD prior to ET has improved the implantation and pregnancy rates in IVF-ET patients with the past history of repeated failures, especially in spite of transfer of embryos with good quality, and AH will provide a range of novel techniques which may contribute much to effective management of infertile couples.

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.

다태임신에서의 선택적 유산술시 복식 천자와 질식 천자의 비교 연구

김석현,문신용,이진용,Kim, S.H.,Moon, S.Y.,Lee, J.Y. 대한생식의학회 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.1

The number of multifetal pregnancies has increased dramatically as a result of the widespread clinical use of ovulation induction and assisted reproductive technology(ART) in infertile patients. In multifetal pregnancies, the adverse outcome is directly proportional to the number of fetuses within the uterus, primarily because of an increased predisposition to premature delivery. It is extremely difficult to counsel patients about the expected outcome of pregnancies involving three or more fetuses. To increase the chances of delivering infants mature enough to survive without being irreversibly damaged by the sequelae of marked prematurity, selective fetal reduction(SFR) to the smaller number of fetuses should be considered in multifetal pregnancies. From January, 1991 to December, 1992, transabdominal SFR in multifetal pregnancies was performed in 22 patients including 13 triplet, 7 quadruplet, 1 quintuplet and 1 heptuplet pregnancies. Transabdominal SFR using intracardiac KCI injection and aspiration of amniotic fluid was carried out in 8-13 weeks of gestation. After procedure, 20 patients were remained as twin pregnancies, and 2 patients as triplet pregnancies. There have been 11 sets of twin delivery including 2 stillbirths, 2 sets of triplet delivery including 1 stillbirth, and 1 singleton delivery. Six cases were delivered after 37 weeks of gestation, 4 cases in 33 - 37 weeks, and 1 case in 30 weeks. Unfortunately, 3 stillbirths occurred in 20-24 weeks of gestation, and 4 cases were aborted. As 7 losses of pregnanancy including 1 case of septic abortion occurred, the delayed fetal loss rate was 38.9%(7/18) in transabdominal SFR. All babies born after 30 weeks of gestation were healthy, and no fetal anomaly directly related to the procedure was encountered. From July, 1993 to February, 1995, transvaginal SFR was performed in 20 patients including 15 triplet, 4 quadruplet and 1 quintuplet pregnancies. Transvaginal SFR using the same method as transabdominal SFR was carried out in 8-11 weeks of gestation. After procedure, 19 patients were remained as twin pregnancies, and 1 patient as singleton pregnancy. There have been 13 sets of twin delivery including 2 stillbirths, and 1 singleton delivery. Six cases were delivered after 37 weeks of gestation, 5 cases in 36-37 weeks, and 1 case in 30 weeks. Unfortunately, 2 still-births occurred in 20 weeks and 21 weeks of gestation, respectively, and 2 cases were aborted. As 4 losses of pregnancy including 1 case of septic abortion occurred, the delayed fetal loss rate was 25.0%(4/16) in transvaginal SFR. No fetal anomaly directly related to the procedure was encountered. It is suggested that transvaginal SFR could be performed more easily and earlier with the lower fetal loss rate as compared with transabdominal SFR. In conclusion, SFR is a rather safe and ethically justified procedure that may improve the outcome of multifetal pregnancies.

쉽게 배우는 특허 Q & A - 직무발명

김석현,Kim, Seok-Hyeon 한국광학기기산업협회 2010 光學世界 Vol.128 No.-

자갈하천에서 서식처 교란이 어류 군집구조에 미치는 영향

김석현,이완옥,조강현,Kim, Seog Hyun,Lee, Wan-Ok,Cho, Kang-Hyun 응용생태공학회 2014 Ecology and resilient infrastructure Vol.1 No.2

본 연구는 전형적인 자갈하천인 가평천에서 하천 환경과 서식처 교란이 어류 군집구조에 미치는 영향을 파악하기 위하여, 13개 조사지점에서 미국 환경청의 간편 생물평가법을 적용하여 하천환경을 평가하고 어류 군집구조를 조사하였다. 하천환경 평가 자료를 이용하여 주요인분석을 실시한 결과, 하상 경사와 관계가 있는 하상 서식환경, 유속/수심 체제, 유사 퇴적이 주요한 평가항목으로 판별되었다. 가평천에서 출현한 어종은 12과 46종이었고, 우점종이 참갈겨니 (Zacco koreanus), 아우점종이 피라미 (Z. platypus)이었다. 종별 개체수 자료를 사용하여 계층적 군집분석의 결과, 조사지는 상류, 중류 및 하류의 세 개의 집단으로 구분되었다. 비모수다차원척도법 결과에 의하면, 어류는 하천환경 평가 항목 중 하상 서식환경, 하상 매몰, 유속/수심 체제, 유사 퇴적, 하도 개수, 여울 빈도 항목과 유의한 상관관계가 있었다. 따라서 자갈하천인 가평천에서 어류 군집구조는 일차적으로 하상의 종적 환경 변화에 의하여 영향을 받고, 인위적 교란에 의하여 군집구조에 변화가 나타났다. Fish assemblages play an integral role in stream ecosystem and are influenced by stream environmental conditions and habitat disturbances. Fish community structures and habitat parameters of U.S. EPA rapid bio-assessment protocol were surveyed to investigate the effect of stream environment and habitat disturbance on fish communities at 13 study sites in the Gapyeong Stream, a typical gravel-bed stream. Principal component analysis (PCA) based on data from habitat assessment at each study site indicated that the study sites were differentiated by habitat parameters such as embeddedness, velocity/depth regime and sediment deposition, which were related with bed slope. A total of 46 species belonging to 12 families were collected in the Gapyeong Stream. A dominant species was Zacco koreanus, subdominant species was Z. platypus. Hierarchical cluster analysis based on species abundance classified fish communities into the three main groups along the stream longitudinal change. Non-metric multidimensional scaling (NMDS) portrayed that fish community structures were related to major habitat parameters, i.e., epifaunal substrate/available cover, embeddedness, velocity/depth regime, sediment deposition, channel alternation and frequency of riffles. These results suggested that fish community structures were primary affected by the longitudinal environmental changes, and those were modified by habitat disturbance in the Gapyeong Stream, a gravel-bed stream.

기후변화 및 수자원 분야 연구를 위한 인공위성 원격탐사의 역할

김석현,Kim, Seok-Hyeon 한국수자원학회 2022 물과 미래(한국수자원학회지) Vol.55 No.3

쉽게 배우는 특허 Q&A: 특허심사하이웨이

김석현,Kim, Seok-Hyeon 한국광학기기산업협회 2011 光學世界 Vol.131 No.-

한국인 남성을 대상으로 한 햄스터 난자 침투 분석법의 정상 가임역 설정

김석현,방명걸,신창재,김정구,문신용,이진용,장윤석,Kim, Seok-Hyun,Pang, Myung-Geol,Shin, Chang-Jae,Kim, Jung-Gu,Moon, Shin-Yong,Lee, Jin-Yong,Chang, Yoon-Seok 대한생식의학회 1991 Clinical and Experimental Reproductive Medicine Vol.18 No.1

To establish the normal fertile range in the results of the sperm zona-free hamster ova penetration assay (SPA) in Korean male, SPA using the low temperature ($4^{\circ}C$) capacitation in TEST-yolk buffer (TYB) was performed in 67 fertile and 26 infertile men. Sperm parameters in routine semen analysis were also checked and compared with the results of SPA. Sperm concentration, motility and motility index (MI) were significantly higher in fertile group compared with infertile group: $96.0{\pm}46.6$ vs $43.6{\pm}31.9{\times}10^6/ml$, $65.5{\pm}14.8%$ vs $45.8{\pm}23.6%$ and $46.31{\pm}13.29$ vs 27.40{\pm}17.98$, respectively. In fertile group, the hamster ova penetration rate (PR) was $98.5{\pm}5.0%$ (80%-100%), and the penetration index (mean penetrations per ovum, PI) was $9.59{\pm}6.35$(3.1-29.0). All the fertile men showed PI>3.0. In infertile group, PR was $24.6{\pm}24.8%$ (0%-70%), and PI was $0.40{\pm}0.42$ (0-1.3). Both PR and PI were significantly lower in infertile group. There was a significant correlation beween PI and sperm motility or MI, respectively, in fertile group whereas there was no correlation in infertile group. These data suggest that SPA using the low temperature capacitation in TYB can be a valuable diagnostic tool for the assessment of male fertility in vitro and provide an important supplement to the traditional tests of sperm quality.