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불임환자와 반복자연유산 환자에서 루프스 항응고인자와 항카디오리핀 항체에 대한 연구
남윤성,차광렬,백진영,김남근,강명서,오도연,Nam, Yoon-Sung,Cha, Kwang-Yul,Baek, Jin-Young,Kim, Nam-Keun,Kang, Myung-Seo,Oh, Do-Yeon 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.1
Objective : To report the prevalence of lupus anticoagulants and anticardiolipin antibodies in patients with recurrent spontaneous abortion and infertility. Material and Method: Lupus anticoagulants and anticardiolipin antibodies were analyzed by Diluted Russell's Viper Venom Test (DRVVT) and solid phase enzyme immunoassay, respectively. Results : In 200 patients with infertility, there were 6 cases (3%) with positive lupus anticoagulants or anticardiolipin antibodies. Of these, 3 patients (1.5%) showed positive lupus anticoagulants and anticardiolipin antibodies, respectively. In 120 patients with recurrent spontaneous abortion, there were 13 cases (10.8%) of positive lupus anticoagulants or anticardiolipin antibodies. Of these, one patient (1%) showed lupus anticoagulants and 12 patients (10%) showed anticardiolipin antibodies. But in two groups, there was no cases with positive lupus anticoagulants and anticardiolipin antibodies. Conclusion: Lupus anticoagulants and anticardiolipin antibodies are definite cause of recurrent spontaneous abortion. There has been a speculation that they might be associated with infertility and repeated IVF failures. But it was found that the role of lupus anticoagulants and anticardiolipin antibodies in these cases are not clear.
반복자연유산 환자에서 Homocysteine과 Methylenetetrahydrofolate Reductase 돌연변이의 상관관계에 대한 분석
남윤성,차광렬,김남근,강명서,김세현,오도연,Nam, Yoon-Sung,Cha, Kwang-Yul,Kim, Nam-Keun,Kang, Myung-Seo,Kim, Se-Hyun,Oh, Do-Yeon 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.3
Objective : To analyze the interrelationship between homocysteine and methylenetetrahydrofolate reductase (MTHFR) mutation in patients with recurrent spontaneous abortion. Material and Method: Homocysteine and MTHFR mutation were tested by fluorescent polarizing immunoassay and PCR-RFLP method, respectively. Results: In patients with homocysteine level less than 5 ?mol/L, there was no case of normal group but there were four cases of heterozygosity and one case of homozygosity. In patients with homocysteine level 5$\sim$10 ? mol/L, the number of normal, heterozygosity and homozygosity group were eleven, eighteen and eight, respectively. In patients with homocysteine level $10{\sim}15$ ? mol/L, the number of normal, heterozygosity and homozygosity group were four, one and one, respectively. In patients with homocysteine level more than 15 ? mol/L, there was no case of normal and heterozygosity group but there were two cases of homozygosity. Conclusions: Hyperhomocysteinemia due to MTHFR mutation is a cause of recurrent spontaneous abortion. And there was a significant relationship between homocysteine and MTHFR mutation.
반복자연유산 환자에서 Antithrombin III 결핍증에 대한 연구
남윤성,차광렬,김남근,강명서,오도연,Nam, Yoon-Sung,Cha, Kwang-Yul,Kim, Nam-Keun,Kang, Myung-Seo,Oh, Do-Yeon 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.4
Objective : To analyze the antithrombin II deficiency in patients with recurrent spontaneous abortion. Material and Method: The blood samples were tested by chromogenic assay to evaluate the activity of antithrombin III. Results: There was only one case of antithrombin III deficiency. This patient experienced one neonatal death after delivery and one FDIU (fetal death in utero). And also this patient showed a lupus anticoagulant and the prolongation of PTT. Conclusions: Women with recurrent miscarriage who have no obvious identified cause should consider hematologic screening. Antithrombin III deficiency could be a cause of recurrent spontaneous abortion. But the incidence is very rare in Korean patients.
반복자연유산에서 Methylenetetrahydrofolate Reductase 돌연변이에 대한 분석
남윤성,차광렬,김남근,김선희,임진우,강금덕,강명서,김세현,오도연,Nam, Yoon-Sung,Cha, Kwang-Yul,Kim, Nam-Keun,Kim, Sun-Hee,Lim, Jin-Woo,Kang, Geum-Duk,Kang, Myung-Seo,Kim, Se-Hyun,Oh, Do-Yeun 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.3
Objective: To analyze the methylenetetrahydrofolate reductase (MTHFR) mutation in patients with recurrent spontaneous abortion. Material and Method: The blood samples of patients with recurrent spontaneous abortion were tested by PCR-RFLP method. Results: Of 51 cases of study group, 14 (27.5%) were normal, 25 (49.0%) were heterozygosity, and 12 (23.5%) were homozygosity. Of 58 cases of control group, 20 (34.5%) were normal, 30 (51.7%) were heterozygosity, and 8 (13.8%) were homozygosity. But the difference between two groups was not significant (p=0.190). Conclusion: Hyperhomocysteinemia due to MTHFR mutation is a cause of recurrent spontaneous abortion. Therefore, the study for MTHFR mutation should be included in the workup of recurrent spontaneous abortion.
Laser Captured Microdissection을 이용한 유전자 발현에 대한 연구 (I): RT-PCR을 위한 난자의 RNA 추출 및 증폭을 위한 최소한도의 확립
박창은,고정재,차광렬,이경아,Park, Chang-Eun,Ko, Jung-Jae,Cha, Kwang-Yul,Lee, Kyung-Ah 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.3
Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.
윤세진,김기령,정형민,윤태기,차광렬,이경아,Yoon, Se-Jin,Kim, Ki-Ryeong,Chung, Hyung-Min,Yoon, Tae-Ki,Cha, Kwang-Yul,Lee, Kyung-Ah 대한생식의학회 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.3
Objective: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Mullerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. Materials and Methods: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in ${\alpha}$-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. Results: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. Conclusion: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.
생쥐 Preantral 난포의 체외배양: FSH의 종류와 농도 및 초자화 냉동보존의 영향
이숙현,신창숙,정형민,고정재,차광렬,이경아,Lee, Sook-Hyun,Shin, Chang-Sook,Chung, Hyung-Min,Ko, Jung-Jae,Cha, Kwang-Yul,Lee, Kyung-Ah 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.4
Objectives: 1) To compare the efficacy of urofollitropin (Follimon) to that of recombinant human FSH (rhFSH) on the growth and maturation of mouse early preantral follicles in vitro, and 2) effect of vitrification on the growth and maturation of preantral follicles and oocytes . Methods: Isolated early preantra1 follicles (100-130 ${\mu}m$ diameter) were cultured for 12 days in 20 ${\mu}l$ ${\alpha}$-MEM media drop under the mineral oil. Follimon or rhFSH was added to the culture medium at various concentrations (0, 10, 100, and 1000 mIU/ml). Results: With Follimon, the dose of 10 mIU/ml showed the best follicle survival, growth, and MIl rate of oocyte than the other concentrations. Whereas the optimal dose of rhFSH was 100 mIU/ml. Despite the different optimal doses, the efficacy of two different FSHs on the follicle growth and maturation was similar. Isolated mouse preantral follicles were cryopreserved by vitrification and cultured in vitro for 12 days with 100 mIU/ml rhFSH. Despite the decreased follicular survival rate after thawing, the follicular growth and maturation rate of its oocyte were comparable to those of the fresh follicle. Conclusion: Results from the present study revealed that 1) the optimal doses of Follimon and rhFSH for in-vitro culture of mouse follicles are different, and 2) the frozen-thawed follicles develop normally after vitrification.
남윤성,정창진,김남근,윤태기,차광렬,Nam, Yoon-Sung,Jeong, Chang-Jin,Kim, Nam-Keun,Yoon, Tae-Ki,Cha, Kwang-Yul 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.3
Objective : To analyze the clinical characteristics of obese infertile women. Material and Method: Height, weight, body mass index, menstrual pattern, glucose, insulin, glucose/insulin ratio, dehydroepiandrosterone sulfate (DHEA-S), testosterone, free testosterone and plasminogen activator inhibitor (PAI-1) of 15 obese infertile women were tested. Results: Of 15 obese infertile women, the number of diabetes mellitus, hyperinsulinemia, and insulin resistance was 2 (13%), 2 (13%), 2 (13%), respectively. The incidence of increased DHEA-S, testosterone, and free testosterone was 7 (47%), 1 (7%), 6 (40%), respectively. Notably, all patients showed increased PAI-1. Conclusions: Obesity is associated with infertility as well as many kinds of health problems. Obesity is closely related to insulin resistance and it also causes hyperandrogenism. Increased PAI-1 is one of the important causes of thrombophilia. Consequently, in the workup of obese infertile patient, many aspects of health problems should be considered.
한국인 남성 불임 환자에서 5,10-Methylenetetrahydrofolate Reductase (MTHFR) 유전자의 1298번의 다형성과의 관련성에 관한 연구
정유미,정태규,김현주,이숙환,박정훈,김남근,김세현,차광렬,이수만,Jeong, Yu-Mi,Chung, Tae-Gue,Kim, Hyun-Joo,Lee, Sook-Hwan,Park, Jung-Hoon,Kim, Nam-Keun,Kim, Se-Hyun,Cha, Kwang-Yul,Lee, Su-Man 대한생식의학회 2003 Clinical and Experimental Reproductive Medicine Vol.30 No.4
Objective: To investigate the association of genetic background between MTHFR A1298C genotype and male infertility. Materials and Methods: We compared 377 infertile males with 396 healthy fertile males with one or more offspring. Infertile males were classified into four subtypes (281 azoospermia, 26 oligoasthenoteratozoospermia (OAT), 59 severe OAT and 11 remnants) by World Health Organization (WHO). Pyrosequencing analysis for MTHFR (methylenetetrahydrofolatereductase) A1298C variation was performed on polymerase chain reaction (PCR) product of study group. To validate pyrosequencing data of A1298C variation for randomly selected 50 samples, we compared the pyrosequencing result with the PCR-RFLP (Restriction Fragment Length Polymorphism) result of MTHFR A1298C genotype. Results: We studied MTHFR A1298C variation by pyrosequencing. A1298C variation data (1298 AC; p=0.2166 and 1298 CC; p=0.5056) of MTHFR gene was no significant difference in between fertile and infertile males. Conclusion: The genetic analysis in MTHFR gene didn't appear genetic difference in Korean fertile and infertile males. We require further study for MTHFR gene in infertile males.
남윤성,김남근,이수만,강명서,오도연,차광렬,Nam, Yoon-Sung,Kim, Nam-Keun,Lee, Su-Man,Kang, Myung-Seo,Oh, Do-Yeon,Cha, Kwang-Yul 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.3
Objective : To evaluate factor XII deficiency in patients with recurrent spontaneous abortion and its relation to aPTT. Material and Method: Factor XII was analyzed by clotting method. Results: Of 70 patients with recurrent spontaneous abortion, there were 35 cases of factor XII deficiency. Among them, there were only 3 cases of prolonged aPTT. Conclusions: It is still unclear whether factor XII deficiency is related to recurrent spontaneous abortion. Molecular approaches should be used to understand further the causal relationship. But based on this result, in the workup of patients with recurrent spontaneous abortion, factor XII should be included. aPTT is not likely to represent the abnormality of factor XII.