생쥐 초기 배아에서 분리한 할구를 이용한 배아줄기세포주 확립

임천규,성지혜,최혜원,조재원,신미라,전진현,Lim, Chun Kyu,Sung, Ji Hye,Choi, Hye Won,Cho, Jae Won,Shin, Mi Ra,Jun, Jin Hyun 대한생식의학회 2006 Clinical and Experimental Reproductive Medicine Vol.33 No.1

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. Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

Isolation and Culture of Human Embryonic Stem-like Cells from Abnormal Blastocysts

임천규,성지혜,박종혁,김선종,윤현수,궁미경,전진현,Lim, Chun-Kyu,Sung, Ji-Hye,Park, Jong-Hyuk,Kim, Sun-Jong,Yoon, Hyun-Soo,Koong, Mi-Kyoung,Jun, Jin-Hyun The Korean Society for Reproductive Medicine 2003 Clinical and Experimental Reproductive Medicine Vol.30 No.4

목 적: 인간의 배아줄기세포는 전분화능과 영속성을 가지고 있어 발생 및 분화에 관련된 기초 연구 뿐 만 아니라 재생의학, 약물검색 등에서도 매우 유용한 재료로 이용될 수 있다.본 연구에서는 유전체의 변형이 배아줄기세포주의 확립 효율에 미치는 영향을 살펴보고자 비정상적인 포배기 배아에서 내세포괴를 분리하여 배양하였다. 연구 방법: 인간의 체외수정 및 배아이식술에서 공여 받은1개 또는3개의 전핵이 관찰되는 비정상 수정란 (n=20)과 착상전 유전진단에서 이수성이 확인된 배아 (n=27)를 대상으로 하였다. 일반적인 immunosurgery 방법으로 영양배엽세포들을 제거하고 내세포괴를 분리한 후 PMEF 혹은 STO feeder 세포위에서 배양하였다. 배아줄기세포의 배양시스템을 검증하기 위해서 이미 확립된 Miz-hES1 cell line을 동시에 같은 조건 하에서 계대배양하였다. 결 과: 비정상 수정란에서 발생된 포배기 배아에서 분리한 1개의 내세포괴가 배아줄기세포와 유사한 colony를 형성하였으나, 계대배양에는 실패하였다. 이수성 배아에서 발생된 포배기 배아의 내세포괴 배양에서는 두개의 colony가 계대배양 중에 영양배엽세포의 형태로 분화되어 미분화 상태를 유지하지 못하였다. 동일한 시기와 조건 하에서 계대배양된 Miz-hES1 cell line이 미분화상태로 유지됨을 karyotyping (46, XY)과 immunophenotyping (positive in SSEA-3 and -4)으로 확인하였다. 결 론: 본 연구의 결과에서 비정상 수정란과 이수성 배아에서 발생된 포배기 배아에서 유래한 내세포괴는 배아줄기세포주 확립 및 미분화 상태 유지 능력이 매우 저조한 것으로 여겨진다. 따라서, 인간의 배아줄기세포주를 확립하는데 있어 배아의 정상여부가 중요한 요소로 작용할 것으로 생각된다.

형광직접보합법을 이용한 착상전 유전진단 기법의 최적화와 경험 축적에 의한 임신율의 향상

임천규,민동미,이형송,변혜경,박소연,류현미,김진영,궁미경,강인수,전진현,Lim, Chun-Kyu,Min, Dong-Mi,Lee, Hyoung-Song,Byun, Hye-Kyung,Park, So-Yeon,Ryu, Hyun-Mee,Kim, Jin-Young,Koong, Mi-Kyoung,Kang, Inn-Soo,Jun, Jin-Hyun 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.1

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.

착상전 배아의 분리된 할구에서 중기염색체 상을 획득하기 위한 효율적인 방법의 개발에 대한 연구: 미세소관 형성 저해제의 효과

임천규,민동미,이형송,김진영,궁미경,강인수,전진현,Lim, Chun-Kyu,Min, Dong-Mi,Lee, Hyoung-Song,Kim, Jin-Young,Koong, Mi-Kyoung,Kang, Inn-Soo,Jun, Jin-Hyun 대한생식의학회 2003 Clinical and Experimental Reproductive Medicine Vol.30 No.2

Objectives: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. Materials and Methods: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. Results: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were $1.0{\mu}M$ of vinblastine (20.3%), $5.0{\mu}M$ of nocodazole (28.1%) and $1.0{\mu}M$ colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine ($1.0{\mu}M$) and nocodazole ($1.0{\mu}M$). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. Conclusions: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.

복막 투석 환자에서 colistin으로 치료한 imipenem resistance Acinetobacter baumannii에 의한 복막염

임규성 ( Kyu Seong Lim ),박지나 ( Ji Na Pak ),문주영 ( Ju Young Moon ),정경환 ( Kyung Hwan Jeong ),이상호 ( Sang Ho Lee ),이태원 ( Tae Won Lee ),임천규 ( Chun Gyoo Ihm ),김명재 ( Myung Jae Kim ) 대한신장학회 2008 Kidney Research and Clinical Practice Vol.27 No.3

Peritonitis in continuous ambulatory peritoneal dialysis (CAPD) is a major cause of technical failure in peritoneal dialysis. The major pathogen is gram positive bacteria, and other main pathogens include gram negative bacteria, mixed microorganisms and fungi. The case of imipenem resistance Acinetobacter baumannii (IRAB) peritonitis are not common. We report a case of peritonitis by IRAB that was not responsive to the empirical antibiotics for CAPD-associated peritonitis. A 56-year-old male with a CAPD catheter inserted 2 weeks before visited our hospital for abdominal pain and turbid peritoneal fluid. He had been diagnosed as having an end stage renal disease (ESRD) about a month before. White blood cell and neutrophil count were elevated at the initial peritoneal fluid analysis, so we diagnosed him as having CAPD-associated peritonitis. Antibiotic therapy was initiated with intraperitoneal injections of ceftazidime/cefamezine which were soon changed to vancomycin/ceftazidime. However, vancomycin/ceftazidime regimen proved ineffective. On the fifth and sixth hospital day, IRAB was cultured from the CAPD catheter exit site swab and peritoneal fluid sampled on the first visiting day. Accordingly, we changed the antibiotics to colistin and removed the CAPD catheter, which led to clinical and laboratory improvement. In the cases of CAPD associated peritonitis in patients who have a history of ICU stay, exposure to the 3rd generation cephalosporin or imipenem, or who are elderly, we must suspect unusual pathogen or multi-drug resistance pathogen such as IRAB.

유전질환 및 염색체 이상의 예방을 위한 착상전 유전진단의 결과

김진영,임천규,송인옥,유근재,양광문,한국선,허걸,송지홍,전진현,민동미,박소연,전종영,궁미경,강인수,Kim, Jin-Yeong,Lim, Chun-Kyu,Song, In-Ok,Yoo, Keun-Jai,Yang, Kwang-Moon,Han, Kuk-Sun,Hur, Kuol,Song, Ji-Hong,Jun, Jin-Hyun,Min, Dong-Mi,Park, So- 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Objective s: Chromosome aneuploidy is associated with recurrent abortion and congenital anomaly and genetic diseases occur repeatedly in the specific families. Preimplantation genetic diagnosis (PGD) can prevent aneuploidy or genetic disease by selecting normal embryos before implantation and is an alternative to prenatal diagnosis. The aim of this study is to assess the outcome of PGD cycles by using FISH or PCR, and to determine the clinical usefulness and values in patients with risk of chromosomal aneuploidy or genetic disease. Materials and Methods: From 1995 to Apr. 2001, a total of 108 PGD cycles in 65 patients with poor reproductive outcome were analyzed. The indications of PGD were translocation (n=49), inversion (n=2), aneuploidy screening (n=7), Duchenne muscular dystrophy (n=5) and spinal muscular atrophy (n=2). PGD was applied due to the history of recurrent abortion, previous birth of affected child or risk of aneuploidy related to sex chromosome aneuploidy or old age. Blastomere biopsy was performed in 6$\sim$10 cell stage embryo after IVF with ICSI. In the single blastomere, chromosome aneuploidy was diagnosed by using FISH and PCR was performed for the diagnosis of exon deletion in DMD or SMA. Results: The FISH or PCR amplification was successful in 94.3% of biopsied blastomeres. The rate of transferable balanced emb ryos was 24.0% in the chromosome translocation and inversion, 57.1% for the DMD and SMA, and 28.8% for the aneuploidy screening. Overall hCG positive rate per transfer was 17.8% (18/101) and clinical pregnancy rate was 13.9% (14/101) (11 term pregnancy, 3 abortion, and 4 biochemical pregnancy). The clinical pregnancy rate of translocation and inversion was 12.9% (11/85) and abortion rate was 27.3% (3/11). In the DMD and SMA, the clinical pregnancy rate was 33.3% (3/9) and all delivered at term. The PGD results were confirmed by amniocentesis and were correct. When the embryos developed to compaction or morula, the pregnancy rate was higher (32%) than that of the cases without compaction (7.2%, p<0.01). Conclusions: PGD by using FISH or PCR is useful to get n ormal pregnancy by reducing spontaneous abortion associated with chromosome aneuploidy in the patients with structural chromosome aberration or risk of aneuploidy and can prevent genetic disease prior to implantation.

배아줄기세표의 인슐린 분비세포로의 유도 분화에 대한 연구

성지혜,임천규,최혜원,이형송,신현상,전진현,윤현수,궁미경,Sung, Ji-Hye,Lim, Chun-Kyu,Choi, Hye-Won,Lee, Hyoung-Song,Shin, Hyeon-Sang,Jun, Jin-Hyun,Yoon, Hyun-Soo,Koong, Mi-Kyoung 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.4

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

인간 배아줄기세포로부터 분화된 세포에서 MACS 방법을 이용하여 분리한 세포의 특성에 대한 연구

조재원,임천규,신미라,방경희,궁미경,전진현,Cho, Jae Won,Lim, Chun Kyu,Shin, Mi Ra,Bang, Kyoung Hee,Koong, Mi Kyoung,Jun, Jin Hyun 대한생식의학회 2006 Clinical and Experimental Reproductive Medicine Vol.33 No.3

목 적: 인간 배아줄기세포는 재생 의학이나 조직공학에 있어서 큰 잠재적인 능력을 가지고 있는 것으로 알려져 있다. 이들은 다양한 growth factors 처리나 유전자 발현을 변화시켜 특정 세포로 유도 분화 및 분리가 가능하지만 그 효율성은 아직까지 낮은 상태이다. 본 연구에서는 인간 배아줄기세포로부터 비특이적으로 분화된 세포들을 특정 세포 표면 항체를 이용한 magnetic cell sorting (MACS) 방법으로 분리, 배양하여 그들의 특성을 살펴보았다. 연구방법: 미분화 배아줄기세포주(Miz-hESC4)를 물리적인 방법으로 계대 배양하였으며, 부유 배양법으로 배아체 형성을 유도하였다. 배아체의 자발적인 분화를 위해 DMEM에 10% FBS를 첨가하여 2주 동안 배양하였다. 이렇게 분화된 세포들을 CD34, human epithelial antigen (HEA), human fibroblast (HFB)에 대한 항체를 이용한 MACS system으로 각각의 항체에 대한 양성 또는 음성 세포를 분리하였다. 이러한 MACS 분리 세포를 4주 동안 배양하면서 형태적인 변화를 관찰하고 특이 유전자의 발현 양상을 분석하였다. 결 과: 분리 배양한 CD34 양성 세포들은 배양 초기에는 둥근 형태를 나타내다가 배양 후기에는 작은 다각형의 형태로 관찰되었으며, HEA 양성 세포들은 큰 다각형의 형태를 나타내었고, HFB 양성 세포들은 전형적인 방추체 형태로 관찰되었다. 특이 유전자에 대한 RT-PCR 결과에서, CD34 양성 세포들과 HFB양성 세포들에서는 내배엽과 중배엽 관련 유전자의 발현하는 것을 확인할 수 있었고, HEA 양성 세포들에서는 외배엽 관련 유전자인 NESTIN과 NF68KD의 발현을 관찰할 수 있었다. 배양기간이 경과함에 따라 CD34 양성 세포의 특이 유전자 발현 양상이 변화되었다. 결 론: 이상의 결과는 비특이적으로 분화된 인간 배아줄기세포로부터 특이 세포를 MACS 방법을 이용하여 성공적으로 분리할 수 있음을 보여주었다. 따라서, MACS 방법과 특이 세포에 대한 항체는 인간 배아줄기세포의 유도 분화와 특이 세포의 분리에 매우 유용할 것으로 생각된다. Objective: Human embryonic stem (ES) cells have a great potential in regenerative medicine and tissue engineering. The human ES cells could be differentiated into specific cell types by treatments of growth factors and alterations of gene expressions. However, the efficacy of guided differentiation and isolation of specific cells are still low. In this study, we characterized isolated cells from differentiated human ES cells by magnetic activated cell sorting (MACS) system using specific antibodies to cell surface markers. Methods: The undifferentiated hES cells (Miz-hESC4) were sub-cultured by mechanical isolation of colonies and embryoid bodies were spontaneously differentiated with DMEM containing 10% FBS for 2 weeks. The differentiated cells were isolated to positive and negative cells with MACS system using CD34, human epithelial antigen (HEA) and human fibroblast (HFB) antibodies, respectively. Observation of morphological changes and analysis of marker genes expression were performed during further culture of MACS isolated cells for 4 weeks. Results: Morphology of the CD34 positive cells was firstly round, and then it was changed to small polygonal shape after further culture. The HEA positive cells showed large polygonal, and the HFB positive spindle shape. In RT-PCR analysis of marker genes, the CD34 and HFB positive cells expressed endodermal and mesodermal genes, and HEA positive cells expressed ectodermal genes such as NESTIN and NF68KD. The marker genes expression pattern of CD34 positive cells changed during the extension of culture time. Conclusion: Our results showed the possibility of successful isolation of specific cells by MACS system from undirected differentiated human ES cells. Thus, MACS system and marker antibodies for specific cell types might be useful for guided differentiation and isolation of specific cells from human ES cells.

클라인펠터 증후군 환자에서 착상전 유전진단의 결과

김진영,임천규,전진현,박소연,서주태,차선화,궁미경,강인수,Kim, Jin-Yeong,Lim, Chun-Kyu,Jun, Jin-Hyun,Park, So-Yeon,Seo, Ju-Tae,Cha, Sun-Hwa,Koong, Mi-Kyoung,Kang, Inn-Soo 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.4

Objectives: Klinefelter syndrome is the most common genetic cause of male infertility and presents with 47, XXY mainly or 46, XX/47, XXY mosaicism. It is characterized by hypogonadism and azoospermia due to testicular failure, however, sporadic cases of natural pregnancies have been reported. With the development of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI), sperm can be retrieved successfully and ART is applied in these patients for pregnancy. It has been suggested that the risk of chromosome aneuploidy for both sex chromosome and autosome is increased in the sperms from 47, XXY germ cells. Considering the risk for chromosomal aneuploidy in the offspring, preimplantation genetic diagnosis (PGD) could be applied as a safe and more effective treatment option in Klinefelter syndrome. The aim of this study is to assess the outcome of PGD cycles by using FISH for sex chromosome and autosome in patients with Klinefelter syndrome. Materials and Methods: From Jan. 2001 to Dec. 2003, PGD was attempted in 8 cases of Klinefelter syndrome but TESE was failed to retrieve sperm in the 3 cases, therefore PGD was performed in 8 cycles of 5 cases (four 47, XXY and one 46, XY/47, XXY mosaicism). In one case, ejaculated sperm was used and in 4 cases, TESE sperm was used for ICSI. After fertilization, blastomere biopsy was performed in $6{\sim}7$ cell stage embryo and the chromosome aneuploidy was diagnosed by using FISH with CEP probes for chromosome X, Y and 17 or 18. Results: A total of 127 oocytes were retrieved and ICSI was performed in 113 mature oocytes. The fertilization rate was $65.3{\pm}6.0%$ (mean$\pm$SEM) and 76 embryos were obtained. Blastomere biopsy was performed in 61 developing embryos and FISH analysis was successful in 95.1% of the biopsied blastomeres (58/61). The rate of balanced embryos for chromosome X, Y and 17 or 18 was $39.7{\pm}6.9%$. The rate of aneuploidy for sex chromosome (X and Y) was $45.9{\pm}5.3%$ and $43.2{\pm}5.8%$ for chromosome 17 or 18, respectively. Embryo transfer was performed in all 8 cycles and mean number of transferred embryos was $2.5{\pm}0.5$. In 2 cases, clinical pregnancies were obtained and normal 46, XX and 46, XY karyotypes were confirmed by amniocentesis, respectively. Healthy male and female babies were delivered uneventfully at term. Conclusion: The patients with Klinefelter syndrome can benefit from ART with TESE and ICSI. Considering the risk of aneuploidy for both sex chromosome and autosome in the sperms and embryos of Klinefelter syndrome, PGD could be offered as safe and more effective treatment option.

세포질내 정자주입을 시행한 인간 난자의 전핵 형성 및 배아 발생에 관한 연구

전진현,임천규,박용석,이호준,김종흡,김문규,Jun, Jin-Hyun,Lim, Chun-Kyu,Park, Yong-Seog,Lee, Ho-Joon,Kim, Jong-Heup,Kim, Moon-Kyoo 대한생식의학회 1995 Clinical and Experimental Reproductive Medicine Vol.22 No.3

Mammalian, including human, spermatozoa undergo morphological and physiological changes during sperm maturation. There were, these changes may affect the fertilization and embryonic development. In this study, we examined the pronucleus formation, pronucleus disappearance and embryonic development in the human oocytes fertilized by intracytoplasmic sperm injection (ICSI). The injected spermatozoa were grouped into ejaculated, epididymal and testicular by the collecting region. Among 363 metaphase II injected oocytes, 287(79.1%) oocytes were normally fertilized and displayed two pronuclei. There were no difference in the fertilization rates and in the pronucleus formation and pronucleus disappearance at 16, 20 and 24 hr after ICSI, among the each spermatozoa group. Also, at 64 hr, the appearance of embryonic development was similar. From these results it can be concluded that there was no difference of maturity among the sperm collected from ejaculated, epididymis and testis in the pronucleus formation and embryonic development. Therefore, testicular spermatozoa are successfully used with ICSI in IVF-ET program.