• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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• pp.303-310
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• 2002

Objective s: To estimate the frequency of Y chromosome microdeletions in the Korean population of infertile men and to evaluate the relationship between microdeletion on the Y chromosome and clinical phenotypes of infertile men with idiopathic azoospermia and oligozoospermia. Materials and Methods: Genomic DNA was extracted from blood samples collected from 330 infertile men attending the Infertility Clinic at Samsung Cheil Hospital, Korea. Six sequence tagged sites (STSs) spanning the azoospermia factor (AZF) regions of the Y chromosome were amplified by polymerase chain reactions (PCRs). Results: Microdeletions on Y chromosome were detected in 35 (10.6%) of the 330 infertile men. Most of the microdeletions (91.4%) involved AZFb or AZFc. The high incidence of microdeletions were found in AZFc region (57.1%), but the low in AZFa (8.6%) and AZFb (5.7%). Larger microdeletions involving two or three AZF regions were detected in 28.6% of cases. All patients (6 patients) with deletion of AZFa region showed no germ cell phenotypes, Sertoli cell only syndrome or Leydig cell hyperplasia in histopathologic examinations. Conclusion: Microdeletions on the Y chromosome, especially, at AZFc/DAZ regions may be the major cause of azoospermia and severe oligozoospermia. We suggest that idiopathic infertile men have genetic counselling and microdeletion analysis on the Y chromosome before IVF-ET and ART program.

• Clinical and Experimental Reproductive Medicine
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• 제23권3호
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• pp.367-377
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• 1996

Genes on the long arm of Y chromosome, particularly interval 6, are believed to playa critical role in human spermatogenesis. The objective of this study was to validate a sequenced-tagged site(STS)-mapping strategy for the detection of Yq microdeletion and to use this method to determine the proportion of men with Yq microdeletions in idiopathic, obstructive, nonobstructive azoospermia, severe OATS and in normal males. We analyzed three STS markers mapped to interval 6 within long arm of the Y chromosome from 106 nonobstructive, 30 obstructive azoospermia, 15 severe OATS patients, and normal 42 males in Korean men. By PCR, we tested leukocyte DNA, for the presences of STS markers(DAZ, sY129 and sY134) and SRY gene as internal control. And PCR results were confirmed by Southern hybridization, and were investigated by SSCP analysis for DAZ gene mutation. None of 42 normal males and 30 obstructive azoospermia had microdeletions, Of the 15 severe OATS typed with DAZ, sY129 and sY134, 3(20.0%) patients failed to amplify 1 or more STS markers, and of the 106 nonobstructive azoospermia typed with DAZ, sY129 and sY134, 12(11.3%) patients failed to amplify 1 or more STS markers. From these results, high prevalence(12.4%) of Yq deletion(DAZ, sY129, sY134) in men with nonobstructive idopathic azoospermia and severe OATS were observed in Korean infertility patients. To avoid the infertile offspring by assisted reproductive technique using ICSI or ROSI, genetic diagnosis will be needed in IVF-ET program.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.1-12
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• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• Clinical and Experimental Reproductive Medicine
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• 제33권1호
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• pp.25-33
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• 2006

목 적: 본 연구에서는 착상전 생쥐 배아에서 분리한 할구를 이용하여 배아줄기세포주를 확립하고 그 효용성과 특성을 살펴보고자 하였다. 연구방법: 생쥐 (C57BL/6J)의 2- 또는 4-세포기 배아에서 투명대를 제거하고 할구를 분리하여 지지세포와 공동배양한 후 할구로부터 형성된 내세포괴를 분리하여 계대배양을 실시하였다. 계대배양 중인 세포주의 특성을 확인하기 위해 alkaline phosphatase 활성도와 표지 인자 및 관련 유전자 발현을 세포면역화학적 염색과 RT-PCR 방법으로 살펴보았다. 또한, 계대배양 중인 배아줄기 세포주의 염색체 분석을 실시하였다. 결 과: 전체적으로 2-세포기에서 분리한 할구와 4-세포기에서 분리한 할구에서 각각 3.0% (1/33)와 4.0% (1/25)의 효율로 배아줄기세포주를 확립할 수 있었다. 이는 4-세포기의 배아를 사용하였을 때의 16.7% (5/30)에 비해 현저하게 낮았다. 분리된 할구로부터 확립된 배아줄기세포주에서 SSEA-l 과 Oct-4의 발현을 관찰하였고, 이들에서 분화된 배아체에서 삼배엽성 분화 관련 유전자들의 발현도 확인할 수 있었다. 결 론: 본 연구에서는 동물모델을 이용하여 착상전 초기 배아에서 분리한 할구를 이용하여 배이줄기세포를 확립할 수 있음을 확인하였다. 지속적인 관련 연구를 통해 인간의 체외수정 및 배아이식술에서 배아의 파괴 또는 발생 능력에 손상을 주지 않고 새로운 인간 배아줄기세포주를 생산할 수 있는 방법을 개발하고 실용화할 수 있을 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.29-39
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• 2004

Objectives: This study was performed to evaluate the laboratory system for successful PGD using fluorescence in situ hybridization (FISH) and the clinical outcome of PGD cycles in five years experiences. Methods: A total of 181 PGD-FISH cycles of 106 couples were performed, and diagnosed chromosome normality in the preimplantation embryos. The laboratory and clinical data were classified by the following optimization steps, and statistically analyzed. Phase I: Blastomere biopsy with two kinds of pipettes, removal of cytoplasmic proteins without treatment of pepsin and culture of biopsied embryos with single medium; Phase II: Blatomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with single medium; Phase III: Blastomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with sequential media. Results: A total of 3, 209 oocytes were collected, and 83.8% (2, 212/2, 640) of fertilization rate was obtained by ICSI procedure. The successful blastomere biopsies were accomplished in 98.6% (2, 043/2, 071) of embryos, and the successful diagnosis rate of FISH was 94.7% (1, 935/ 2, 043) of blastomeres from overall data. Embryo transfers with normal embryos were conducted in 93.9% (170/181) of started cycles. There was no difference in the successful rate of biopsy and diagnosis among Phase I, II and III. However, the pregnancy rate per embryo transfer of Phase III (38.8%, 26/67) was significantly (p<0.05) higher than those of Phase I (13.9%, 5/36) and Phase II (14.9%, 10/67). Conclusions: The laboratory optimization and experience for the PGD with FISH procedure can increase the pregnancy rate to 38.8% in the human IVF-ET program. Our facility of PGD with FISH provides the great possibility to get a normal pregnancy for the concerned couples by chromosomal aberrations.

• Clinical and Experimental Reproductive Medicine
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• 제15권2호
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• pp.103-117
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• 1988

Ovulation induction was done with 3 different regimens as clomid combined with HMG, HMG only, and FSH combined with HMG in 28 menstrual cycles for IVF-ET and GIFT program. The appearance of endogenous LH surge, estradiol plateau, atypical LH surge, and time from initiation to peak of LH surge in serum and urine were observed and compared in 3 groups. 1. The estradiol concentration of serum LH surge day was similar in three groups but 1st group (Clomiphene Citrate+Sequential HMG) was slightly higher at $1924.0{\pm}865.1\;pg/ml$. In regards to the existence of serum estradiol plateau, 3rd group (FSH+Sequential HMG) was highest at 60%, and 1st group and 2nd group (HMG only) were similar at 33% and 44% respectively. 2. The number of ovarian of ovarian follicle which was more than 18mm in diameter was $4.1{\pm}2.0$, $4.2{\pm}2.1$ respectibely for 2nd group and 3rd group. Although the numbers were slightly higher thean 1st group for each ovarian follicle, serum estradiol value per follicle was higher for 1st group at $583.0{\pm}261.2pg/ml$. 3. When measuring the urine LH surge according to Hi-Gonavi and according to the standard set by three different types of surge, simultameous satisfaction for 1st group, 2nd group, 3rd group was two cases, five cases, four cases respectively at 40%, and the remained cases were composed of numorous type combination which satisfied the two definition, simultaneously in this study, the LH surge starting time was determined only in the cases tow combination were satisfied simultaneously at first, but there are something to study more. In one case of the 3rd group. 4. The concentration of LH surge start in urine and serum of 2nd group was highest at306. $0{\pm}287.2IU/l$ and $34.0{\pm}9.9mIU/ml$ and 1st group was low at $116.6{\pm}66.1IU/l$ and 7.4mIU/ml. The urine and serum value of LH was highest at $1644.4{\pm}988.8IU/l$, $65.9{\pm}15.0mIU/ml$ for 2nd group, 1st group was low at urine, and 3rd group was low of serum. With pregnancy established, the LH concentration of urine was relatively high but on the contrary the LH concentration of serum was low compared to urine concentration. 5. Time from LH surge start to the maximun of urine and serum value was highest at 15. $7{\pm}9.1$ hrs and $10.8{\pm}4.9$ hrs for 1st group and 3rd group. With pregnancy established, time was shortened for urine but on the contrary serum showed an increase in time. 6. The concentration of LH which increases with time on urine was highest at 2nd group ($194.6{\pm}76.8\;IU/hour$). The lowest increase for serum was at 3rd group (2.1mIU/hour). With pregnancy established, urine showed more increase than control group ($266.5{\pm}47.4\;IU/hour$) and for serum there was similar increase ($3.4{\pm}0.8\;mIU/hour$). 7. There were two examples of non-typical surge from 1st group and 3rd group each. Among these three cases showed a reestablishment of LH surge after 10-24 hours. 8. Endogenous spontaneous Lh surge occurs once for each 2nd group and 3rd group.

• Clinical and Experimental Reproductive Medicine
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• 제34권2호
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• pp.107-115
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• 2007

목 적: ISO 9001:2000 품질경영시스템은 국제적인 인중 기준에 의해 제품 및 서비스의 품질을 적절하게 판정하고 이를 향상시키기 위해 시행되고 있다. 본 연구에서는 이러한 품질경영시스템을 보조생식술 센터에 성공적으로 도입하고 적용하는 과정에서 확인된 효용성에 대해 기술하고자 한다. 연구방법: 제일병원 아이소망센터는 2004년 1월부터 ISO 9001:2000 인증을 위한 활동을 시작하였으며, 고객만족도조사와 주기적인 내부심사와 사후심사를 실시하였다. 심사과정에서 확인된 부적합 사항에 대한 시정 및 예방 조치와 이에 따른 효과성을 검증하였으며, 고객불만사항에 대한 지속적인 관리와 개선을 위한 프로젝트를 진행하였다. 결 과: 본 센터는 초일류 불임센터로 성장하고 최적의 품질경영시스템을 확립하기 위한 품질방침을 설정하여, 2004년 6월에 한국품질재단 (Korean Foundation for Quality)으로부터 "체외수정 및 배아이식 시술에 관련된 연구"에 대하여 ISO 9001:2000 인증을 받았다. 이러한 품질방침을 실현하기 위해 품질경영시스템에 적합한 품질매뉴얼, 프로세스, 절차서, 지침서 등에 대한 문서화를 완료하였다. 삼 년 동안의 내부심사와 사후심사에서 각각 140건과 7건의 부적합이 확인되었으며, 이에 대한 시정조치를 실시하였다. 결 론: 본 센터에서는 ISO 품질경영시스템의 도입과 운영을 통해 고객만족도의 향상, 체계적인 문서화를 통해 업무의 투명화와 효율화가 가능하였다. 보조생식술 센터에서의 ISO 품질경영시스템은 보조생식술에 대한 국가적인 관리시스템을 효율적으로 운영하기 위한 기본적인 기관별 경영시스템으로서 활용이 가능할 것으로 생각된다.