Epidermal Growth Factor(EGF)가 생쥐 초기배아의 발생에 미치는 영향

변혜경,이호준,김성례,김해권,김문규,Byun, Hye-Kyung,Lee, Ho-Joon,Kim, Sung-Rye,Kim, Hae-Kwon,Kim, Moon-Kyoo 대한생식의학회 1995 Clinical and Experimental Reproductive Medicine Vol.22 No.2

Growth factors (GFs) produced by the embryo or by the maternal reproductive tract have been reported to regulate the embryonic development and differentiation. Among GFs, EGF as a mitogen plays a role in mitosis and functional differentiation of trophectoderm cells in mouse. The present study was carried out to investigate the effect of EGF on development of mouse embryos and to localize EGF in the mouse oocytes and embryos, which has been reported to be detected in the reproductive tract in mammals. To investigate the effect of EGF on the development of the embryo, mouse 2-cell embryos were cultured to blastocysts stage in Ham's F10 medium, treated with EGF(10-50 ng/ml) for 72 hrs. Immunocytochemistry was performed from oocyte to blastocyst stage with anti-EGF and anti-Mouse IgG, in order to determine the stage which EGF would be expressed in mouse. Exogenous EGF (more than 10 ng/ml) in the culture medium improved the developmental and hatching rates in the mouse embryos. As a result of immunocytochemistry, the embryonic EGF was expressed after the late 4-cell stage. EGF is thought to enhance preimplantation embryonic development and hatching. Exogenous EGF in the culture medium is thought to activate EGF receptor in the late 4-cell embryos and to enhance blastulation and hatching in mouse embryos. It is concluded that EGF enhances the developmental and hatching rates in the mouse embryos.

Efficacy of Coculture System in the Patients with Poor Prognoses on Human IVF-ET Program

변혜경,염혜원,궁미경,손일표,강인수,이호준,Byun, Hye-Kyung,Youm, Hye-Won,Koong, Mi-Kyung,Son, Il-Pyo,Kang, Inn-Soo,Lee, Ho-Joon The Korean Society for Reproductive Medicine 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.2

본 연구는 Vero cell을 이용한 사람 배아의 공동배양술이 배아의 질을 향상시킬 수 있거나 또는 반복적 착상 실패를 극복하여 임신을 가능케 할 수 있는지 알아 보고자 시행되었다. 1996년 일년 동안, 반복적 착상 실패를 경험한 환자 (group I)와 이전 주기에서 배아의 질이 나빴던 환자 (group II)를 포함한 총 202례를 분석하여 대조군과 공동배양군 간의 배아의 질, 임신률, 임신유지율 및 착상률을 비교하였다. Group I 93례 가운데 34례는 공동배양을, 나머지 59례는 기존의 체외수정을 시행하였다. Group II 109례에서는 공동배양 36례, 기존의 체외수정 73례를 시행하였다. Group I에서 공동배양군의 임신률, 임신유지율 및 착상률은 각각 14/34 (41.2%), 9/34 (26.5%), 16/81 (19.8%)로 대조군 (11/59 (18.6%), 8/59 (13.6%), 12/152 (7.9%))에 비하여 높았으며, 특히 임신률과 착상률은 유의한 차이를 나타내었다(p=0.028, p=0.015). Group II에서는 공동배양군의 임신률과 임신유지율 및 착상률이 각각 8/36 (22.2%), 5/36 (13.9%), 8/87 (9.2%)로 대조군 (5/73 (6.8%), 3/73 (4.1%), 3/158 (1.9%))에 비하여 높았고, group I의 결과에서와 마찬가지로 임신률과 착상률의 유의한 차이를 나타내었다(p=0.028, p=0.022). 이상에서 Vero cell을 이용한 공동배양술은 위의 두가지 주소를 가진 환자군에서 좋은 결과를 나타내었다. 또한 group II에서 3일-공동배양군의 임신률 역시 향상되어 (4/15 (26.7%)), 보조부화술을 겸비한 3일-공동배양이 이전 주기에서 배아의 질이 나빴던 환자군에 적용될 수 있음을 알 수 있었다. 결론적으로 Vero cell을 이용한 공동배양술은 반복적 착상 실패를 경험한 환자나 또는 이전 주기에서 배아의 질이 나빴던 환자에게 적용하여 임신률을 향상시킬 수 있을 것으로 사료된다. The present study was carried out to evaluate whether the coculture system of human embryos with Vero cells can improve the quality of embryo or overcome the repetitive implantation failures in order to obtain pregnancy. From January to December 1996, a total 202 cases which patients with the problems of repetitive implantation failures (group I) or those with the poor embryonic quality in their previous cycles (group II) was analysed. The quality of cocultured embryo, pregnancy, on-going and implantation rates between coculture and control groups were compared. Of 93 cases in group I, coculture was performed in 34 cases and conventional IVF for the rest. Of 109 cases in group II, 36 for coculture and 73 for conventional IVF. In group I, pregnancy, on-going and implantation rates in coculture group (14/34 (41.2%), 9/34 (26.5%), 16/81 (19.8%), respectively) were higher than those of control (11/59 (18.6%), 8/59 (13.6%), 12/152 (7.9%), respectively). There is significance in the pregnancy and implantation rates (p=0.028 and p=0.015). In group II, pregnancy, on-going and implantation rates in coculture group (8/36 (22.2%), 5/36 (13.9%), 8/87 (9.2%), respectively) were higher than those of control (5/73 (6.8%), 3/73 (4.1%), 3/158 (1.9%), respectively). Like the result of group I, there is significance in the pregnancy and implantation rates (p=0.028 and p=0.022). Coculture system with Vero cells works well in the groups of the two indications. Although the case of 3 day-coculture was small as 15 cases in group II, 3 day-coculture improved pregnancy rate (4/15 (26.7%)). Therefore, 3 day-coculture with assisted hatching is recommended to the patients with poor embryonic quality. In conclusion, coculture system with Vero cells can be suggested as an effective method which improves pregnancy rate in those who have repetitive implantation failures or whose embryonic quality was poor in their previous cycles.

생쥐 초기배아의 Glucose Transporter유전자 발현 양상에 관한 연구

염혜원,변혜경,송견지,김해권,이호준,Youm, Hye-Won,Byun, Hye-Kyung,Song, Gyun-Ji,Kim, Hae-Kwon,Lee, Ho-Joon 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

동결수정란 이식주기에서 수정란 융해 후 생존율과 임신율에 영향을 미치는 요인

김정욱,변혜경,염혜원,전진현,박용석,송인옥,송지홍,최범채,궁미경,전종영,강인수,Kim, Jeong-Wook,Byun, Hye-Kyung,Youm, Hye-Won,Jun, Jin-Hyun,Park, Yong-Seog,Song, In-Ok,Song, Ji-Hong,Choi, Bum-Chae,Koong, Mi-Kyoung,Jun, Jong-Young,Kang, 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.1

Objective: The purpose of this study was to determine the important factors affecting survival and pregnancy rate in frozen-thawed embryo transfer cycles. Methods: we performed retrospective analysis in 738 cycles of frozen-thawed embryo transfers, in relation to the insemination methods, the freezing stage of embryo, patient's age, infertility factors and the origin of injected sperm in ICSI cycles. After conventional IVF or ICSI, the supernumerary PN stage zygotes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant. Results: The survival rates of thawed embryos were 69.3% (1585/2287) in conventional IVF group and 71.7% (1645/2295) in ICSI group. After frozen-thawed embryo transfers, 27.0% (92/341) and 32.0% (109/341) of pregnancy rates were achieved in conventional IVF and ICSI group, respectively. There were no significant difference in the survival and pregnancy rates according to the insemination methods, the freezing stage and patient's age. However, the pregnancy rate (36.2%) of male factor infertility was significantly higher than the tubal (27.2%) and other female factor infertility (22.9%). In ICSI group, the origin of injected sperm did not affect the outcome of frozen-thawed embryo transfer cycles. Conclusion: The present study demonstrates that acceptable clinical outcomes can be achieved after the transfer of frozen-thawed embryos regardless of the stage of embryos for freezing, the patient's age and the origin of injected sperm.

생쥐 초기배아와 사람의 수정란의 발생에 미치는 생식수관 상피세포의 영향에 관한 연구

이호준,변혜경,김정욱,황정혜,전종영,김문규,Lee, H.J.,Byun, H.K.,Kim, J.W.,Hwang, J.H.,Jun, J.Y.,Kim, M.K. 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.3

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

폐쇄성 무정자증 환자의 신선고환조직 정자와 동결고환조직 정자의 운동성이 임신율에 미치는 영향

박용석,이형송,변혜경,염혜원,송상진,임천규,이유식,윤종민,서주태,송지홍,강인수,궁미경,Park, Yong-Seog,Lee, Hyoung-Song,Byun, Hye-Kyung,Youm, Hye-Won,Song, Sang-Jin,Lim, Chun-Kyu,Lee, You-Sik,Yun, Jong-Min,Seo, Ju-Tae,Song, Ji-Hong,Kang, I 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.2

Objective: ICSI with testicular sperm could achieve optimal fertilization and pregnancy. This study was performed to observe the influence on fertilization and pregnancy of motility of fresh testicular sperm and sperm extracted from frozen-thawed seminiferous tubules in obstructive azoospermia. Materials and Methods: We analysed clinical outcome of ICSI using fresh testicular sperm and sperm extracted from thawed seminiferous tubules. The presence of motility were compared to determine the factor for optimal fertilization and pregnancy rates. Results: In 316 cases of TESE-ICSI in obstructive azoospermia, ICSI with fresh testicular sperm (fresh sperm group) were 163 cases and ICSI with sperm testicular sperm extracted from frozen-thawed seminiferous tubule (thawed sperm group) were 153 cases. The fertilization rates were 71.3% and pregnancy rates were 32.5% in fresh sperm group, in thawed sperm group, 65.1% and 33.3% respectively. The fertilization and pregnancy rates of motile and non-motile testicular sperm were 72.9% and 33.6%, 50.0% and 18.2%, respectively (p<0.05). The fertilization and pregnancy rates of motile and non-motile sperm extracted from the thawed seminiferous tubule were 67.8% and 34.7%, 55.1% and 28.1%, respectively (p<0.05). The comparative of the results of ICSI using motile fresh testicular sperm and motile sperm extracted from thawed seminiferous tubule, fertilization and pregnancy rates were not significantly different (72.9% and 33.6%, 67.8% and 34.7%, respectively). Conclusion: These results suggest that successful pregnancy in TESE-ICSI treatment is influenced by the motility of fresh testicular sperm and sperm extracted from thawed seminiferous tubule in obstructive azoospennic patients.

일반적인 체외수정 방법과 세포질내 정자주입술로 얻어진 배아의 동결-융해 후 이식의 결과

김정욱,한미현,변혜경,전진현,손일표,궁미경,백은찬,강인수,이호준,Kim, Jeong-Wook,Han, Mi-Hyun,Byun, Hye-Kyung,Jun, Jin-Hyun,Son, Il-Pyo,Koong, Mi-Kyoung,Paik, Eun-Chan,Kang, Inn-Soo,Lee, Ho-Joon 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.1

Intracytoplasmic sperm injection (ICSI) recently has been utilized widely as the most successful technique to overcome the unfertilization problem in cases of severe male infertility in couples who could not be treated by conventional IVF. Recently, indications of ICSI have been extended further and more fertilized oocytes become available. Thus, it is necessary to examine the efficiency of freezing the surplus embryos obtained from ICSI. We compared the survival rate and the future outcome of cryopreserved embryos obtained either after conventional IVF or ICSI during the same period. After ICSI or IVF, five best-quality embryos from each patient were transferred in the stimulation cycle and the surplus pronuclear (PN) stage oocytes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant. A total of 792 embryos from ICSI trial were thawed and 65.2% (516/792) survived. The survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 63.5%, 68.2%, 64.0%, respectively. After 111 transfers, 34 pregnancies were achieved, corresponding to a clinical pregnancy rate of 30.6% per transfers. We thawed 1033 embryos from IVF trials and 57.5% (594/1033) survived. In IVF cycle, the survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 58.2%, 65.2%, 40.2%, respectively. Thirty eight clinical pregnancies were established after 134 transfers, corresponding to a pregnancy rate of 28.4% per transfer. The cleavage rate of thawed PN stage oocytes from ICSI trial (61.3%) was significantly higher than those from conventional IVF (53.4%). The developmental rates of good embryo (${\geqq}$ grade II) in thawed PN stage oocytes obtained from conventional IVF and ICSI were 63% and 65%, respectively. We concluded that PN stage oocytes, multicellular embryos resulting from ICSI procedure can be successfully frozen/thawed with reasonable clinical pregnancy rates comparable to those of IVF.

인간의 체외수정배아이식술에서 보조부화술이 임신률에 미치는 영향에 관한 연구

이호준,김정욱,변혜경,전진현,손일표,전종영,Lee, H.J.,Kim, J.W.,Byun, H.K.,Jun, J.H.,Son, I.P.,Jun, J.Y. 대한생식의학회 1995 Clinical and Experimental Reproductive Medicine Vol.22 No.2

In human IVF-ET, the development and morphology of the embryo have been known to affect implantation and pregnancy rates(PRs). Recently, pregnancy has been reported to related to the embryos with thick zona-pellucida, high levels of fragmentation, poor blastomere development and zona hardening. Although the mechanism of implantation is unclear, it is thought that the hatching process precedes implantation and that the hatching is related to implantation and PRs. This study was carried out to investigate the effect of assisted hatching(AHA) on the improvement of PRs in human IVF-ET. The results were as follows; 1. The PRs of the AHA group (40.8%) was significantly higher than that of control group(27.2%)(P<0.01). 2. According to the age of patients, the PRs of control and AHA groups were 33.9%(20/59), 44,4%(12/27) in <30 yrs, 26.1%(30/115), 38.3%(18/47) in 31-35 yrs, 22.4%(13/58), 41.4%(12/29) in >36 yrs, respectively. 3. According to the factors of infertility in AHA group, unexplained(immunologic factor) (40.0%) and male factors(41.9%) were higher than female(tubal obstruction, endometriosis, adhesion) factor (28.9%). As a result, it is suggested that AHA technique improve the PRs in poor prognosis patients. It is concluded that AHA method can be used to improve the PRs in human lVF-ET.

고환조직 정자채취술과 세포질내 정자주입술을 이용한 고환조직 정자의 수정률과 임신율

박용석,서주태,전진현,변혜경,김종현,이유식,손일표,강인수,이호준,Park, Yong-Seog,Seo, Ju-Tae,Jun, Jin-Hyun,Byun, Hye-Kyung,Kim, Jong-Hyun,Lee, You-Sik,Son, Il-Pyo,Kang, Inn-Soo,Lee, Ho-Joon 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.1

This study was carried to determine the possibility of finding motile spermatozoa and fertilization, pregnancy rate after testicular sperm extraction(TESE) with ICSI in obstructive and non-obstructive azoospermic patients. In 154 cases(132 patients), obstructive azoospermia was 77 cases and non-obstructive azoospermia was 77 cases. In obstructive azoospermia, patients generally showed normal spermatogenesis and included vas agenesis(n=8), multiple vas obstruction(n=7), epididymal obstruction (n=54). Total of 982 retrieved oocytes were obtained and 84.4% were injected. The fertilization rates with 2 PN and cleavage rate were 72.5% and 62.3%, respectively. 30 pregnancies(38.9%) were achieved and the ongoing pregnancies were 22 cases (28.6%). In non-obstructive azoospermia, patients showed hypospermatogenesis(n=49), maturation arrest(n=4), Sertoli cell only syndrome (n=24). The various stages of spermatogenic cell could be retrieved by TESE and could be reached normal fertilization and embryo development with ICSI. Total of 1072 retrieved oocytes obtained and 80.2% were injected. The fertilization rates with 2 PN and cleavage rate were 52.8% and 68.9%, respectively. 22 pregnancies(30.1%) were achieved and the ongoing pregnancies were 19 cases(26.0%). Conclusively, the combination of TESE with ICSI using testicular spermatozoa can achieve normal fertilization and pregnancy rate and effective method in obstructive and non-obstructive azoospermic patients.

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.