생쥐 배아 동결시 전핵의 발생시기가 생존률과 발생률에 미치는 영향

양현원,강희규,최규완,차영범,이승재,박종민,Yang, Hyun-Won,Kang, Hee-Kyoo,Choi, Kyoo-Wan,Cha, Young-Beom,Lee, Seung-Jae,Park, Jong-Min 대한생식의학회 1993 Clinical and Experimental Reproductive Medicine Vol.20 No.1

The effects of freezing and 1,2-propanediol on early and late pronucleate stage mouse ova were investigated in terms of survival after thawing and development in vitro. The samples were divided into two groups according to different age in pronucleate ova: ova in(1) early pronuclear stage with two distant pronuclei at 18h after hCG injection, and (2) late pronuclear stage with adjacent pronuclei at 30h. Zygotes in the late pronuclear stage have been proven to be more resistant to 1,2-propanediol, showing a significantly higher developmental rate than zygotes in early stage (80.3 versus 66.3%, <0.05), but survival rate was similar in the two groups (91.0 versus 93.5%). After freezing and thawing, survival and developmental rates were decreased in both groups when compared to the control group (54.3 versus 92.3%, 47.7 versus 73.3%. respectively). And developmental rate in the late pronuclear stage zygotes showed significantly higher than in early (55.4 versus 40.0%) after thawing. In conclusion, early pronucleate mouse ova have a lower developmental capacity in vitro and a lower survival rate after freezing and thawing than late ova. These findings suggest that the timing of freezing could be important for survival and further development in vitro in cryopreservation of human pronucleate ova.

인간 체외수정 및 배아이식에 있어서 과배란 유도 과정에 사용한 GnRH Agonist가 배란 전 난포내 과립 세포의 세포자연사에 미치는 영향

양현원,권혁찬,황경주,박종민,오기석,윤용달,Yang, Hyun-Won,Kwon, Hyuck-Chan,Hwang, Kyung-Joo,Park, Jong-Min,Oh, Kie-Suk,Yoon, Yong-Dal 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.1

There have been many reports to date regarding the role of GnRH as a local regulatory factor of ovarian function as studies of human and rat ovaries revealed GnRH and its receptor. In recent studies it has been shown that GnRH directly causes apoptosis in the granulosa cells of the rat ovary, and such results leads to the suggestion that the use of GnRH agonist for more stable long term ovarian hyperstimulation in human IVF-ET programs causes granulosa cell apoptosis which may lead to follicular atresia. Therefore this study attempts to determine if granulosa-luteal cell apoptosis occurs in patients during IVF-ET programs in which GnRH agonist is employed for ovarian hyperstimulation. The quality of oocyte-cumulus complexes obtained during ovum pickup procedures were assessed morphologically and then the fertilization rate and developmental rate was determined. Apoptotic cells among the granulosa-luteal cells obtained during the same procedure were observed after staining with Hematoxylin-eosin. The fragmentation degree of DNA extracted from granulosa-luteal cells was determined and comparatively analyzed. There was no difference in the average age of the patients, the number of oocytes retrieved, and fertilization and developmental rates between the FSH/hMG group and GnRH-long group. There was also no difference in the apoptosis rate and pyknosis rate in the granulosa-luteal cells between the two groups. However, when the oocyte-cumulus complexes were morphoogically divided into the healthy group and atretic group without regard for the method of hyperstimulation, the results showed that the number of oocytes obtained averaged $11.09{\pm}8.75\;and\;10.33{\pm}4.53$ per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental rate (65.96%, 41.51%, respectively, p<0.01) was significantly increased in the healthy group when compared to the atretic group. The degree of apoptosis in the granulosa-luteal cells showed that in the healthy group it was 2.25% which was not significantly different from the atretic group (2.77%), but the pyknosis rate in the atretic group (27.81%) was significantly higher compared to the healthy group (11.35%, p<0.01). The quantity of DNA fragmentation in the FSH/hMG group was 32.22%, while in the GnRH-long group it was 34.27%, showing no significant difference. On the other hand the degree of DNA fragmentation was 39.05% and 11.83% in the healthy group and atretic group, respectively, showing significantly higher increase in the atretic group (p<0.01). The above results suggest that death of granulosa-luteal cells according to the state of the oocyte-cumulus complex is more related to pyknosis rather than apoptosis. Also, the GnRH agonist used in ovarian hyperstimulation does not seem to directly affect the apoptosis of retrieved oocytes and granulosa-luteal cells, and which is thought to be due to the suppression of the apoptogenic effect of GnRH agonist as a result of the high doses of FSH administered.

전핵 시기 및 2-4 세포 시기에 동결 보존된 배아의 발생률 및 임신률

양현원,최규완,전한식,차영범,이승재,박종민,Yang, Hyun-Won,Choi, Kyoo-Wan,Cheon, Han-Sik,Cha, Young-Beom,Lee, Seung-Jae,Park, Jong-Min 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.1

The survival and pregnancy rates were compared between non-frozen embryos and cryopreserved embryos at either pronucleate or 2-4 cell stages using the freezing and thawing techniques being identical in both groups were compared with fresh embryos. 496 embryos were frozen with 1, 2-propanediol and sucrose and 117 2-4 cell stages embryos had been thawed and 79.6 and 66.0% of them respectively were survival. Clinical pregnancy rate was 19.2% for embryos frozen at the pronucleate stage and 19.0% for embryos frozen at the 2-4 cell stages while the pregnancy rate of non-frozen embryos was 21.3%. There were no significant difference in the survival and pregnancy rates of embryos frozen at pronucleate and 2-4 cell stages. The current cumulative pregnancy rate per retrieval in all cycles with frozen zygotes is 35.4 %, consid~ erably higher than observed in single transfers of embryos without cryopreservation(21.3%); predicted pregnancy rate after transfer of all frozen embryos is 43.3 %. It is concluded that firstly, the survival and pregnancy rate of cryopreserved embryos at pronucleate or 2-4 cell stages are very similar to those from their fresh embryos and non-frozen embryos and secondly, cryopreservation substantially enhances pregnancy attainment from in vitro fertilization.

임파선 전이 및 자궁방 침윤이 동반된 임상병기 IB 자궁경부암에서 Cyclooxygenase - 2 단백의 과발현

양현원(Hyun Won Yang),오기석(Kie Suk Oh),유희석(Hee Sug Ryu),정태영(Tae Young Chung),장기홍(Ki Hong Chang),권혁찬(Hyuck Chan Kwon),김명신(Myoung Shin Kim) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3

N/A Objective: the enzymes cyclooxygenase(COX)-1 and -2 are necessary for the synthesis of prostaglandins. COX-2 is usually absent in normal cells and is upregulated and expressed as a product of the immediate early gene during inflammatory processes. In previous studies, the expression of COX-2 has been shown to be induced by prointlammatory cytockines, and suggestions have been made that overexpression of COX-2 supresses apoptosis and is directly related to tumor growth. We the authors have attempted to determine a relationship between the tumor invasion and metastasis of uterine cervical cancer and COX and apoptosis by comparing the protein expression of apoptosis and COX-I and COX-2 in tumor tissues confirmed with cytokeratin, and therefe determine the clinicopathologic risk factors. Materials and methods: The subjects were 18 patients who were FIGO stage IB uterine cervical cancer patients who underwent surgery at the Ajou University Medical Center. The 18 cases were comprised of 12 cases of squamous cell carcinoma, 3 cases each of adenocarcinoma and adenosquamous carcinoma. There were 9 cases with lymph node or prarametrial involvement and 13 cases with lymphvascular space involvement. All tissues obtained from the cases were subject to immunohistochemical staining for COX-1, -2 and TUNEL method for apoptosis detection, and the following results were obtained. Results: Tumor tissues confirmed by cytokeratin wae separated into tumor surface, tumor stroma, and invasion site portions, and in which increased apoptosis was observed in the tumor surface and tumor stmma, but not in the invasion sites. COX-2 expression was observed in all tumor tissues, which was especially strong in the tumor invasion site. Therefore, it is suggested that COX-2 expression may supress cell apoptosis at the site of tumor invasion. When COX-2 expression was investigated when the cases were divided into groups with regard to the presence or absence of lymph node or parametrial involvement, there was statistically significant (Mann-Whitney U test) COX-2 expression seen microscopically in the tumor stroma (p-value 0.028) and tumor invasion site (p-value 0.040) compared to the tumor surface (p-value 0.499). In other words, in surgically treated stage IB cervical cancer patients, COX-2 was significantly expressed when lymph node or parametrial involvement was present. Conclusions: These results suggest that the expression of COX-2 in stage IB cervical cancer patients may downregulate apoptosic processes and thus enhances tumor invasion and metastasis.

인간 자궁내막의 생리주기에 따른 성호르몬 수용채 , integrin , cyclooxygenase

양현원(Hyun Won Yang),오기석(Kie Suk Oh),김세광(Sei Kwang Kim),김영아(Young Ah Kim),주희재(Hee Jae Joo),유희석(Hee Sug Ryu),권혁찬(Hyuck Chan Kwon),김진영(Jin Yeong Kim),이영돈(Young Don Lee),조동제(Dong Jae Cho) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3

N/A STUDY DESIGN: Tissues were obtained from the endometrium of the posterior hmdus in 42 women (proliferative phase-25 cases, secretory phase-17 cases) with normal menstrual cycles(28-32days interval). The specimens were stained with H&E stain and classified according to the method by Noyes et al(1950) into early proliferative phase(-5~-10days from ovulation), late proliferative phase(-4days~ovulation), early secretory phase (ovulation ~5days), mid-secretory phase(6~10days from ovulation), and late secretory phase(11-14days from ovulation). Immunohistochemical staining of integrin a1, a4, b3, COX-1,-2, ER, PR expression was performed. RESULT: The expression of ER was high in the proliferative phase and low during the secretory phase. The late proliferative phase showed the highest intensity(p<0.05). On the other hand, the expression of PR in stromal cells was relatively uniform during the entire menstrual cycle. However, in epithelial cells, there was a characteristic peak intensity in the late proliferative phase and low intensity in the secretory phase.The expression of integrin a1, a4, b3 in epithelial cells showed no particular pattern in the proliferative phase but showed specific findings in the secretory phase. In the epithelial cells, the intensity of a I staining was increased after the early proliferative phase and sustained during the whole secretory phase(p<0.05), a4 was increased in the early and mid-secretory phases, b3 was increased in the mid-secretory phase to late secretory phase. But the strumal cells were weakly expressed in the whole menstrual cycle but showed no particular pattern, In glandular epithelial cells and stromal cells, COX-1 showed a cyclic pattem according to menstrual cycle; it was strongly expressed in the mid-secretory phase in glandular epithelial cells and mid-secretory and menstrual phase in stromal cells(p<0.05). But in luminal epithelial cells, COX-1 was expressed in the entire menstrual cycle but had no particular pattern. In glandular epithelial cells, stromal cells, and luminal epithelial cells, COX-2 was not expressed during the secretory phase but strongly expressed in the mid-secretory phase(p<0.05). CONCLUSION: The expression of a1, a4, b3, and COX-2 showed as stonng staining during the mid secretory phase which represents the implantation period. The PR expression in epitbelial cells was decreased during same period. These characteristic findings will provide helpful information far histological methods of endormetrial dating and will be useful in the measurement of endometrial maturation during the implantation period.

체외 배양에 있어서 5% 산소 환경과 superoxide dismutase ( SOD ) 가 착상전 생쥐 배아 발달에 미치는 영향

양현원(Hyun Won Yang),이치형(Chi Hyeong Lee),오기석(Kie Suk Oh),유희석(Hee Sug Ryu),송승규(Seung Kyu Song),박동욱(Dong Wook Park) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3

N/A Objective: In the human body the embryo initially gmws in the fallopian tube which is maintained in an 3-8% O2 concentration environment, and various substances such as growth factors and antioxidants present in tbe tubal fluid assists in maintaining a healthy environment for embryo development. But in IVF programs embryo cultures are conducted in incubators with 21.9% O2 and 5% CO2 condition, and such high oxygen concentrations have been reported to increase the production of oxygen free radicals within the embryo and is detrimental to the growth and development of the embryo. The objective of this study, therefore, is to determine the culture conditions which will decrease oxygen free radical production and thereby minimize the injury to the embryo. Methods: Six to eight week old ICR strain mice embryos were cultured in 5% or 21.9% O2 conditions and in culture media to which inaement concentrations of superoxide dismutase (SOD) had been and the H2O2 concentration within the embryo, embryo developmental rate, and degree of fragmentation of the embryos was investigated. Results: The control gmup embryos which were cultured in 21.9% O2 condition without addition of SOD showed developmental arrest at the 2-cell stage or fragmentation, while those cultured in 21.9% O2 condition with addition of SOD showed development to the blastocyst stage with deaeased fragmentation. In particular, the blastulation and fragmentation rates were the lowest in the group to which 500 IU/ml of SOD was added, but in the 5% O2 enviranment group many embryos reached the blastocyst stage and with no difference in frapnentation with or without addition of SOD. The HO relative intensity (120.5+-20.2) within the embryos cultured in 21.9% O2 environment without SOD was significantly higher than that (56.8+-10.8) of group with SOD (p<0.05). As showing that in the 5% O2 environment group without SOD it was 43.8+-7.8 and in the group with SOD it was 37.3+-5.4, the H2O2 concentration within embryos cultured in 5% 02 condition was significantly lower those that of 21,9% 02 environment regardless of SOD addition (p<0.05). Conclusion: The optimal oxygen concentration in incubator for mice embryo cultures is that which is similar to the 5% 0 concentration in vivo. When 20% 02 incubators are routinely used, the addition of SOD to the culture media will decrease the H2O2 concentration within the embryos with subsequent improvement in development. The optimal concentration which should be used is thought to be 500 IU/ml. It is suggested that the use of the above method in human IVF-ET programs will lead to improved embryo quality and enhanced pregnancy rates.

Apoptosis in human pre-implantation embryo associates with increased Lipid droplets by reduction of the pripheral-type benzodiazepine receptor (PBR) and steroidogenic acute regulatory (StAR) proteins

양현원 ( Hyun Won Yang ),( Cheol Hong Park ),( Hyang Heun Lee ),( Dong Hoon Kim ),( Won Il Park ),( S. Samuel Kim ),( Sei Kwang Kim ) 대한산부인과학회 2002 대한산부인과학회 학술대회 Vol.88 No.-

Gonadotropin-releasing hormone agonist induces apoptosis in the Luteal Cells via caspase-3 activation and PARP cleavage by releasing of cytochrome c from mitochondria

양현원 ( Hyun Won Yang ),( S. Samuel Kim ),( Seo Yoo Hong ),( Dae Woon Kim ),( Jung Hwan Shin ),( Won Il Park ),( Eun Joo Park ),( Yong Dal Yoon ) 대한산부인과학회 2002 대한산부인과학회 학술대회 Vol.88 No.-

인간의 착상 기전을 연구하기 위한 3차원적 자궁내막 모델 확립

박동욱,양현원,권혁찬,장기홍,김세광,조동제,오기석,Park, Dong-Wook,Yang, Hyun-Won,Kwon, Hyuck-Chan,Chang, Ki-Hong,Kim, Sei-Kwang,Cho, Dong-Jae,Oh, Kie-Suk 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.1

In order to study the implantation mechanism various methods for culture of endometrial cells in vitro have been attempted. However, a disadvantage is that primary cultures of stromal and epithelial cells do not have the ability to differentiate, and therefore cannot be reproduced in the same manner as in vivo endometrium. The object of this study is to establish a three dimensional culture of endometrial cells which are both morphologically and functionally identical to in vivo endometrium. Endometrial tissues obtained after hysterectomies were cut into thin slices and treated with collagenase and trypsin-EDTA. The stromal cells and the epithelial cells were separated by centrifugation and cultured for 24 hours in DMEM media containing 10% FCS, 100 nM progesterone, and 1 nM estradiol. The cultured stromal cells were mixed with collagen gel and solidified, after which it was covered with matrigel. Epithelial cells were inoculated on the top and then cultured for 3 days. The three dimensionally cultured endometrial cells were stained for integrin ${\alpha}1,\;{\alpha}4,\;{\beta}3$, and cyclooxygenase-l, -2 by immunohistochemistry, which all showed strong expression. The cultured epithelial cells showed the formation of microvilli, tight junctions and pinopodes by electron microscopy. Studies are currently under way utilizing this three dimensional culture model to ascertain the interaction between the embryo and human endometrial cells at the time of implantation, and it is thought that further studies into a new culture environment which would allow longer periods of culture will be necessary.

인간 자궁내막에서 Cyclooxygenase-1과 -2의 주기적 발현 양상

박동욱,양현원,권혁찬,황경주,유정현,이치형,김세광,조동제,오기석,Park, Dong-Wook,Yang, Hyun-Won,Kwon, Hyuek-Chan,Hwang, Kyung-Joo,Yoo, Jung-Hyun,Lee, Chi-Hyeong,Kim, Sei-Kwang,Cho, Dong-Jea,Oh, Kie-Suk 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1

Cyclooxygenase (COX) is an enzyme involved in the conversion of arachidonic acid to prostaglandins (PGs), and exists in two forms, COX-1 and COX-2. COX has been reported to be involved in early implantation by secretion of PGs which causes permeability of vessels and reaction of decidual cells around the implantation site. Recently, in mice and sheep studies, COX-1 and COX-2 expression in the endometrium has been reported to be different according to implantation and stages of the estrous cycle, but expression of COX-1 and COX-2 in human endometrium during the menstrual cycle has not yet been established. The purpose of this study was to observe the variances of COX-1 and COX-2 expression by immunohistochemical staining in endometrial samples obtained from human hysterectomy specimens and biopsies of women of reproductive age according to different stages of the menstrual cycle. Also, we attempted to observe COX-1 and COX-2 expression in the epithelial and stromal cells of the endometrium obtained during the mid-secretory phase, which were cultured separately. COX-2 showed a cyclic pattern of expression according to the different stages of the menstrual cycle and was strongly expressed particularly at the mid-secretory phase which corresponds to the time of implantation. However, COX-1 tended to be increased in the early proliferative, and mid- and late secretory phases, but was also expressed in the whole menstrual cycle showing no particular pattern. In the separately cultured cells COX-1 was expressed in epithilial cells and COX-2 in the stromal cells. The above results suggest that since COX-2 is expressed at the same time as implantation and cultured cells display a specific secretory pattern, COX-2 has inductive endocrine enzyme properties and has an important effect on endometrial cells during implantation. Also, COX-2 expression in endometrial cells may be utilized as a useful marker of endometrial maturation.