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소 정자에 있어서 활성산소계가 정자 기능과 지방산화 및 DNA 절편화에 미치는 영향
류범용,정영채,김창근,신현아,한정호,방명걸,오선경,김석현,문신용,Ryu, Buom-Yong,Chung, Yung-Chai,Kim, Chang-Keun,Shin, Hyun-A,Han, Jung-Ho,Pang, Myung-Geol,Oh, Sun-Kyung,Kim, Seok-Hyun,Moon, Shin-Yong 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.2
Objective : To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine (X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. Materials and Methods: ROS were produced using a combination of 1000 uM X and 50 mU/ml XO. The ROS scavengers: superoxide dismu tase (SOD) (200 U/ml) and catalase (500 U/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at $37^{circ}C$ under 5% $CO_2$ incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Caionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. Results: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. Conclusion: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.
남성 불임의 진단 및 체외수정의 예후인자로서 정자 형태의 정밀 분석과 정자 첨체반응 및 햄스터 난자 침투 분석의 비교 연구
문신용,류범용,방명걸,오선경,이재훈,서창석,김석현,최영민,김정구,이진용,Moon, Shin-Yong,Ryu, Buom-Yong,Pang, Myung-Geol,Oh, Sun-Kyung,Lee, Jae-Hoon,Suh, Chang-Suk,Kim, Seok-Hyun,Choi, Young-Min,Kim, Jung-Gu,Lee, Jin-Yong 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.1
Objective : This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. Materials and Methods: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. Results: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. Conclusion: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.
인간정자의 처리에 있어서 Percoll과 Sil-Select 방법의 비교
문신용,류범용,신현아,오선경,서창석,김석현,최영민,김정구,최규홍,이진용,Moon, Shin-Yong,Ryu, Buom-Yong,Shin, Hyun-Ah,Oh, Sun-Kyung,Suh, Chang-Suk,Kim, Seok-Hyun,Choi, Young-Min,Kim, Jung-Gu,Choi, Kyu-Hong,Lee, Jin-Yong 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.4
Objective: To evaluate silane-coated silica particles (Sil-select) as an alternative to polyvinylpyrrolidone-coated particles (Percoll) for gradient separation of spermatozoa, for use in assisted reproduction. Methods: 20 normal semen based on WHO criteria were included in this study. Recovery of motile and morphologically normal spermatozoa after using two-layer Percoll and Sil-select gradient respectively was recorded. Motility, HOST (hypoosmotic swelling test) and the detection of malondialdehyde for LPO (lipid peroxidation) after 24 h of incubation at $37^{\circ}C$ in a 5% $CO_2$ incubator were compared. Results: Percoll (78.5%) and Sil-select (79.1%) showed a significant increase in the motility compared to ejaculate (60.9%) but no difference between Percoll and Sil-select. Normal sperm morphology significantly increased after Percoll (57.6%) and Sil-select (53.7%) compared to ejaculate (35.8%) but no difference between Percoll and Sil-select. No differences in the recovery of motile spermatozoa and motility, HOST and the production of malondialdehyde after 24 h incubation were found when comparing the use of Percoll and Sil-select. Conclusion: Sil-select seems to be an attractive alternative to Percoll for sperm separation in assisted reproduction.
인간 정자의 생식력 평가에 있어 첨제반응율과 햄스터 난자 침투 분석법의 비교연구
문신용,류범용,오선경,서창석,김석현,최영민,신창재,김정구,장윤석,이진용,Moon, Shin-Yong,Ryu, Buom-Yong,Oh, Sun-Kyung,Suh, Chang-Suk,Kim, Seok-Hyun,Choi, Young-Min,Shin, Chang-Jae,Kim, Jung-Gu,Chang, Yoon-Seok,Lee, Jin-Yong 대한생식의학회 1995 Clinical and Experimental Reproductive Medicine Vol.22 No.2
This study was designed to determine the relationship between sperm acrosome reaction following ionophore challenge(ARIC) and hamster ovum sperm penetration assay(SPA) as assessment of fertilizing capacity of male. ARIC test and SPA were performed in 23 fertile and 19 subfertile men. The results were as follows; Sperm concentration was significantly higher in fertile group compared with subfertile group: $114.6{\pm}64.40$ vs $61.3{\pm}46.50{\times}10^6/ml$. However, there were no significantly differences in seminal volume, motility and motility index, respectively. There was a significantly correlation between spontaneous and induced AR in fertile and subfertile group, respectively. ARIC value was significantly higher in fertile group, compared with subfertile group: $12.0{\pm}5.57%$ vs $2.6{\pm}4.96%$. Both Penetration rate(PR) and Penetration index(PI) were significantly higher in fertile group, compared with subfertile group: $97.4{\pm}7.40%$ vs $64.9{\pm}36$. 20% and $5.4{\pm}2.88$ vs $1.5{\pm}1.47$, respectively. The Positive predictive value(PPV), Negative predictive value(NPV), sensitivity and specificity of ARIC test (cut-off: 8.5) and SPA(PI cut-off : 3.0) in predicting fertility were 95.0%, 81.8%, 82.6%, 94.7% and 95.2%, 85.7%, 87.0% and 94.7%, respectively. There was no significantly difference in predicting fertility between ARIC test and SPA. In conclusion, ARIC test was shown to have a predictive value for fertilizing capacity comparable to that of the hamster ovum sperm penetration assay. Therefore, ARIC test may be a simple and cost-effective addition to existing semenology instead of SPA.
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.
미수정 및 저수정율의 기왕력이 있는 체외수정시술 환자에서의 난자 세포질내 정자 주입술 : 중증 남성인자 환자와의 비교
문신용(Shin Yong Moon),최영민(Young Min Choi),김석현(Seok Hyun Kim),오선경(Sun Kyung Oh),서창석(Chang Suk Suh),이진용(Jin Yong Lee),정병준(Byeong Jun Jung),김희선(Hee Sun Kim),류범용(Buom Yong Ryu),방명걸(Myung Geol Pang),김정구(Jung 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.2
Objective: The purpose of this study was to determine whether intracytoplasmic sperm injection(ICSI) could overcome the defects of oocytes in IVF-ET patients with previous fertilization failure by conventional fertilization technique. Design: Retrospective study Materials and Methods: A total of 119 ICSI cycles in 57 IVF-ET patients performed from May, 1995 to December, 1997 was enrolled. Subjects were divided into two groups: FR group included 66 ICSI cycles in 35 patients with normal sperm who underwent ICSI due to past history of failed or poor fertilization in the previous IVF-ET cycles, and OAT group included 53 ICSI cycles in 22 patients with severe oligoasthenoterato- zoospermia(OAT) which was defined as sperm concentration < 20 million/ml, mo#dlity < 30% and normal morphology < 4% by strict morphologic criteria. The outcomes of ICSI were analyzed and compared in both groups. Results: The age of female patients, basal serum FSH level, and the numbers of oocytes retrieved and metaphase II oocytes were all comparable in both groups. The fertilization rate after ICSI was similar in both groups(68.7+-25.3% vs. 67.7+-24.5%), as were the cleavage rate of normally fertilized oocytes(93.1+-21.4% vs. 89.3+-21.6%), the number of embryos transferred(4,00+-1.98 vs. 4.64+-2.10), and cumulative embryo score(CES) indicating the quality of embryos(47.3+-33.2 vs. 54.1+-33.2). The implantation rate(4.3+-10.5% vs. 3.8+-11.0%) and the clinical pregnancy rate per cycle(15.2% vs. 13.2%) were also comparable in both groups. Conclusions: Although it has been shown that there is a higher risk of chromosomal abnormalities in oocytes from IVF-ET patients with pevious failed or poor fertilization, higher implantation and clinical pregnancy rates wer#e not observed in patients with OAT following ICSL Therefore, the functional defect of sperm such as loss of capacitation, defect of aaasome reaction, and abnormality of nucleus decondensation should be also considered in patients with previous failed or poor fertilization.
고식적 체외수정시술시 3 일째 배아이식술의 효용성에 관한 연구
문신용(Shin Yong Moon),최영민(Young Min Choi),김석현(Seok Hyun Kim),오선경(Sun Kyung Oh),서창석(Chang Suk Suh),이진용(Jin Yong Lee),정병준(Byeong Jun Jung),김희선(Hee Sun Kim),류범용(Buom Yong Ryu),김정구(Jung Gu Kim),지병철(Byung Ch 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3
Objective: To investigate the positive or negative effect of delaying embryo transfer(ET) one day in IVF-ET. Methods: From May to July, 1997, a total of 64 patients was emolled in this prospective randomized case-controlled study. When the timing of oocyte retrieval was decided, random allocation of patients was made to one of the two groups: day 2 transfer or day 3 transfer. In day 3 transfer group, embryos were cultured in M3 media(Medi-Cult) for further 24 hours. Results: There were no significant differences in age of patients, infertility factor, basal serum FSH level, and serum E2 level on hCG day between two poups, but number of previous IVF-ET cycles was significantly higher in day 3 transfer group(p 0.042). Number of oocytes retrieved, fertilization rate, cleavage rate, and number of embryos transferred had no significant difference, but cumulative embryo score(CES) was significantly higher in day 3 transfer group(p 0.0001). Clinical pregnancy and implantation rates were bigher in day 3 transfer group, but without significance(34.4% vs. 21.9%; 8.7% vs, 5.4%). There were also no significant differences in spontaneous miscarriage and multiple pregnancy rates. Especially in patients over 35 years of age, clinical pregnancy and implantation rates were more higher in day 3 transfer group, but without significance(41.7% vs, 8.3%; 8.5% vs. 1.6%). Conclusion: Considering the higher number of previous cycles in day 3 transfer group, it is at least likely that delaying ET one day may be clinically beneficial in IVF-ET, especially in patients with old age or repeated failure of previous cycles.
폐쇄성 무정자증 환자에서 정자 채취 후 난자 세포질내 정자 주입술의 결과 : 결핵성 부고환염의 영향
문신용(Shin Yong Moon),최영민(Young Min Choi),김석현(Seok Hyun Kim),오선경(Sun Kyung Oh),서창석(Chang Suk Suh),이진용(Jin Yong Lee),정병준(Byeong Jun Jung),김희선(Hee Sun Kim),류범용(Buom Yong Ryu),김정구(Jung Gu Kim),지병철(Byung Ch 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3
Objective: To investigate the intluence of previous tuberculous epididymitis in infertile males with obstructive azoospermia on the outcome of sperm retrieval and intracytoplasmic sperm injection(ICSI) in IVF-ET propam. Methods: Retrospective analysis was paformed in 26 patients with obstructive azoospermia undergoing sperm retrieval and ICSI at Seoul National University Hospital from January, 1996 to August, 1997; 12 cycles in 5 patients with tuberculous obstructive azoospermia(Group A), and 40 cycles in 21 patients with non-tuberculous obstructive azoospermia(Group B). Results: There was no significant difference in the clinical pregnancy rate(PR) per fresh transfer between Groups A and B(20.0%[2/10] vs. 26.8%(11/41)). The rates of embryo implantation and clinical miscarriage were also comparable in both groups(6.3% vs. 11.1%, 50.0% vs. 9.1%). This tendencies were also similar after including five cryopreserved-thawed transfer cycles. Conclusion: Embryo quality and pregnancy outcome in sperm retrieval and ICSI were comparable in both the tuberculous and non-tuberculous obstructive azoospermia patients. Our results suggest that previous history of tuberculous epididymitis in patients with obstructive azoospermia does not affect the outcome of sperm retrieval and ICSI.
국산 Fluorescence in Situ Hybridization 시스템을 이용한 다양한 검체에서의 염색체 분석
문신용,방명걸,오선경,류범용,황도영,정병준,최진,손철,장준근,김종원,김석현,최영민,Moon, Shin-Yong,Pang, Myung-Geol,Oh, Sun-Kyung,Ryu, Buom-Yong,Hwang, Do-Yeong,Jung, Byeong-Jun,Choe, Jin,Sohn, Cherl,Chang, Jun-Keun,Kim, Jong-Won,Kim, Se 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.3
Fluorescence in situ hybridization (FISH) techniques allow the enumeration of chromosome abnormalities and from a great potential for many clinical applications. In order to produce quantitative and reproducible results, expensive tools such as a cooled CCD camera and a computer software are required. We have developed a Chromosome Image Processing System (Chips) using FISH that allows the detection and mapping of the genetic aberrations. The aim of our study, therefore, is to evaluate the capabilities of our original system using a black-and-white video camera. As a model system, three repetitive DNA probes (D18Z1, DXZ1, and DYZ3) were hybridized to variety different clinical samples such as human metaphase spreads and interphase nuclei obtained from uncultured peripheral blood lymphocytes, uncultured amniocytes, and germ cells. The visualization of the FISH signals was performed using our system for image acquisition and pseudocoloring. FISH images were obtained by combining images from each of probes and DAPI counterstain captured separately. Using our original system, the aberrations of single or multiple chromosomes in a single hybridization experiment using chromosomes and interphase nuclei from a variety of cell types, including lymphocytes, amniocytes, sperm, and biopsied blastomeres, were enabled to evaluate. There were no differences in the image quality in accordance with FISH method, fluorochrome types, or different clinical samples. Always bright signals were detected using our system. Our system also yielded constant results. Our Chips would permit a level of performance of FISH analysis on metaphase chromosomes and interphase nuclei with unparalleled capabilities. Thus, it would be useful for clinical purposes.