Correlation of Clusterin Expression and Apoptosis in Prostate Cancer and Benign Hyperplastic Tissues / 대한비뇨기과학회지

PURPOSE: We studied the apoptotic index in prostate cancer tissues and investigated the relationship of apoptosis and clusterin expression. MATERIALS AND METHODS: Forty-two archival prostatectomy specimens of varying grades of prostate cancer and 10 of benign prostatic hyperplasia were subjected to immunohistochemical clusterin staining with anti- clusterin antibody. Staining intensities were classified from 0 to 3. Apoptotic index was calculated with TUNEL positive cells under fluorescence microscope. We performed double staining for clusterin and TUNEL using immunofluorescence technique to determine the relationship between apoptosis and clusterin expression. RESULTS: Immunohistochemistry of clusterin showed a weak intensity in all benign tissues. Clusterin was localized mainly in the epithelial cells. Staining intensity was increased according to Gleason grade of cancer. Apoptotic indices of cancer were 0.86+/-0.8%, 0.76+/-1.0%, 0.39+/-0.4% and 0.14+/-0.09% in grades 2, 3, 4 and 5, respectively. In immunofluorescence localization study, apoptosis was not detected in the cancer cells stained with clusterin. Conversely, clusterin was not expressed in the cells showing apoptosis. CONCLUSIONS: These results more clearly show that clusterin acts as a survival protein protecting from apoptosis in prostate cancer. In addition, our findings revealed that the apoptotic index is lower in high grade prostate cancer. These findings have significant clinical implications for identifying the value of apoptotic index and clusterin expression in prostate cancer. Further study is needed to define the role of clusterin in the development and progression of prostate cancer.

GnRH-agonist Induces Apoptosis of Human Granulosa-luteal Cells Via Caspase-3 and -9 and PARP Cleavage / 대한산부인과학회잡지

OBJECTIVE: GnRH-agonist (GnRH-Ag) used in controlled ovarian hyperstimulation (COH) for IVF-ET has been known to affect directly on apoptosis of human ovarian cells, but its mechanism is not clearly understood. Therefore, the purpose of the present study was to investigate whether caspase-3 and -9 activation and poly-(ADP-ribose)-polymerase (PARP) cleavage are involved in the mechanism(s) by which GnRH-Ag induces apoptosis in human granulosa-luteal cells. METHODS: Human granulosa-luteal cells collected from IVF-ET patients were cultured and treated with 10(-6) M GnRH-Ag or saline as a control. To access apoptosis in the cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay and DNA fragmentation analysis were preformed 24 h after the treatment. Activity of caspase-3 and -9 in the cells was examined by using a fluorogenic substrate. Caspase-3 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage were analyzed by Western blotting. RESULTS: DNA fragmentation in the cells increased in the higher concentration over 10(-6) M GnRH-Ag. In the result of TUNEL assay, the rate of apoptotic cells in GnRH-Ag treatment increased significantly compared with that of saline treatment (p<0.05). The activity of caspase-3 and -9 investigated by using a fluorogenic substrate increased only in the apoptotic cells. In Western blot analysis, the cells treated with GnRH-Ag revealed an increase in active forms of caspase-3 and -9 compared with those of the saline treatment. In addition, cleavage of PARP also increased in the cells treated with GnRH-Ag. CONCLUSION: These results suggest that activation of caspase-3 and -9 and cleavage of PARP might be involved in apoptosis of human granulosa-luteal cells induced by GnRH-Ag.

Expression of Steroidogenic Acute regulatory (StAR) Protein during Oocyte Apoptosis in the Gonadotropin-Stimulated Human Ovary / 대한산부인과학회잡지

OBJECTIVE: To determine the distribution and expression of steroid acute regulatory (StAR) protein in human oocyte and embryo in relation to apoptosis. METHODS: Immuno-labelling and confocal microscopy were applied to examine the localization of StAR protein in human oocytes and embryos. Western blot analysis was also used for qualitative and quantitative assessment of StAR protein expression. RESULTS: There were lipid droplet accumulation in fragmented human oocytes and embryos. StAR protein (30 kDa) expression was detected in human oocytes and embryos. The level of StAR protein expression was lower in the fragmented group than the normal group. CONCLUSION: The present study provides evidence for involvement of StAR protein in the apoptosis of fragmented oocytes and embryos during in vitro fertilization (IVF) program as well as in the normal development of human oocytes and embryos.

The Study on Apoptosis and Expression of Fas, Fas-ligand, Bax, and Bcl-2 in Human Fragmented Embryos / 대한불임학회지

OBJECTIVE: The present study was performed to investigate whether apoptosis occur in human embryos by annexin staining and detect the expression of Fas, Fas-ligand (FasL), Bax, and Bcl-2 in human fragmented embryos derived from IVF-ET by immunofluorescence and Western blot analysis. MATERIALS AND METHODS: Using annexin staining, immunofluorescence and Western blot analysis on normal and fragmented embryos, we were able to detect apoptotsis and apoptotic gene products in fragmented embryos. RESULTS: Phosphatidylserine (PS) translocation, the marker for apoptosis, were detected frequently in fragmented embryos. Bcl-2 and Bax protein were detected in both fragmented and non-fragmented embryos. When fragmented embryos compared to normal embryos, immunofluorescent intensity of Bcl-2 tended to be lower in fragmented embryos. Bax gene expression increased in the fragmented embryos compared to the normal embryos. This result supports a model in which the molar ratio of Bcl-2 to Bax determines whether apoptosis induced or inhibited in human embryo. Fas was highly expressed in human preimplantation embryos but not FasL. It suggests that embryo may undergo apoptosis by binding with FasL produced by follicular or immune cells. CONCLUSION: The over expression of Bax and Fas will trigger apoptosis to lead embryo fragmentation and change embryo to be nonviable.

Expression of Cyclooxygenase-1, -2 in Human Uterine Cervix of Preterm and Term Pregnancies / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to determine whether cyclooxygenase [COX]-1 and COX-2 are expressed in the pregnant human uterine cervix and if they are expressed differentially between preterm and term pregnancies. METHODS: Fourteen patients delivered between 29 and 41 weeks of gestation were matched for obstetrical history and maternal age were divided into a preterm group who delivered between 29 and 36 weeks [n=7], and a term group who delivered between 37 and 41 weeks of gestation [n=7]. Immediately after vaginal delivery cervical biopsy samples were obtained and immunohistochemicaly stained for COX-1 and COX-2 and the degree of staining was evaluated by H-scoring system. RESULTS: Expression of COX-1 and COX-2 was found in all epithelial and stromal cells of uterine cervical tissues of pregnancy. The expression of COX-1 and COX-2 was significantly stronger in the term group compared to the preterm group in epithelial cells [HSCORE : 2.14+/-0.69 vs. 1.14+/-0.38 ; 3.71+/-0.76 vs. 1.86+/-0.90, p0.05]. CONCLUSION: In the pregnant human uterine cervix, COX-1 and COX-2 are found to be expressed, and both are strongly expressed in the cervical epithelial cells of term pregnancies compared to preterm pregnancies. It is suggested that the uterine cervix, under the control of prostaglandins, is actively involved in the process of labor, and that the role of COX-2 in epithelium is particularly significant in term pregnancies compared to preterm pregnancies.

The Effects of 5% Oxygen Condition and Superoxide Dismutase ( SOD ) on the in - vitro Development of Preimplantation Mouse Embryos / 대한산부인과학회잡지

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.

High Cyclooxygenase - 2 Expression in Stage IB Cervical Cancer with Lymph Node Metastasis or Parametrial Invasion / 대한산부인과학회잡지

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.

Expression pettern of Sex Hormone Receptors , Integrins , Cyclooxygenases ( COX ) in Human Endometrium During Menstrual Cycle / 대한산부인과학회잡지

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.

3-Dimensional Culture System of Endometrial Cells for Studying the Human Implantation Mechanism / 대한불임학회지

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 alphal, alpha4, beta3, and cyclooxygenase-1, -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 studied into a new culture environment which would allow longer periods of culture will be necessary.

Effects of GnRH Agonist Used for Ovarian Hyperstimulation in Human IVF-ET on the Apoptosis of Preovulatory Follicular Cells / 대한불임학회지

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-rosin. 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+/-8.75 and 10.33+/-4.53 per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental ,ate (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.

Cyclic Expression of Cyclooxygenase-1 and -2 in Human Endometrium / 대한불임학회지

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 stuffy was to observe the variances of COX-1 and COX-2 expression by immunohistoehemical 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.

Effects of Low Oxygen Condition on the Development of Mouse Embryos Cultured In Viro / 대한산부인과학회잡지

OBJECTIVE: It is known that mouse embryos before implantation develop in a low oxygen environment of 3- 8% concentration and with antioxidant materials such as vitamins, antioxidant enzymes, ferrous binding proteins, and albumin in follicular and tubal fluids. However, the 20% oxygen culture condition with chemically defined media might be produce an abundance of ROS, and leads to developmental delay or developmental block in vitro. In this study, we attempt to elucidate the relationship between intracellular H2O2 production and embryo development in different oxygen culture conditions of mouse embryos. METHODS: Prenuclear embryos from C57BL/CBA Fl hybrid and ICR mouse were cultured in incubators which provided 5% carbon dioxide, 20% oxygen and 5% carbon dioxide, 5% oxygen. Measurement of H2O2 level in a embryo was performed with DCHFDA(2, 7 -dichlorodihydroflourescein diacetate)and analyzed with Quanti-cell 700, and the number of blastomeres was counted with DAPI( 4, 6'-diamidino-2-phenylindole). RESULTS: Oxygen concentration of the culture medias was significantly higher in the 20% oxygen environment compared to that of 5% oxygen environment. Culture of mice embryos in high oxygen condition leads to high HO concentrations at 2 cell stage and developmental delay or ""2-cell block"" regardless of the strain. But in a 5% oxygen environment, which is similar to in-vivo conditions HO production was suppressed continuously through out culture and development of embryos was definitely improved. CONCLUSION: These results suggest that there is a difference in the production of ROS or protective mechanism according to the mouse strains and stage of development, and it is thought that in-vitro culture in 5% oxygen environment provides stable in vivo equilibrium but in a 20% oxygen environment there is production of ROS which overcome the protective mechanism which leads to cellular damage and embryo developmental delay.

The Effects of Free Radical Scavenger, Rutin, on the Development and the Cell Number of Blastocyst in Mouse Early Embryos / 대한산부인과학회잡지

It is well known that developmental delay or arrest occurs before implantation inmammals, which have undergone in vitro culture. Recently, these phenomenon are being attributedto oxygen free radicals, and successful cell culture are being obtained by lowering theoxygen environment of in vitro culture. This is due to the fact that the oxygen concentrationin the fallopian tube is around 5%, which is lower than the room air 20% concentrationfor in vitro culture.Rutin, which is a free radical scavenger, was added to early embryo mice culture andcompared the free radical level at blastocyst stage with that of different culture conditions,and found that free radical level was markedly decreased. Also, there was increased embryodevelopment with decreasing free radical levels in the experimental group, and there wassignificant increase in the blastulation rate and blastomere count.This study therefore suggests the possibility of improved in in-vitro embryo culture.

The Effects of Free Radical Scavenger, Rutin, on the Development and the Cell Number of Blastocyst in Mouse Early Embryos / 대한산부인과학회잡지

It is well known that developmental delay or arrest occurs before implantation inmammals, which have undergone in vitro culture. Recently, these phenomenon are being attributedto oxygen free radicals, and successful cell culture are being obtained by lowering theoxygen environment of in vitro culture. This is due to the fact that the oxygen concentrationin the fallopian tube is around 5%, which is lower than the room air 20% concentrationfor in vitro culture.Rutin, which is a free radical scavenger, was added to early embryo mice culture andcompared the free radical level at blastocyst stage with that of different culture conditions,and found that free radical level was markedly decreased. Also, there was increased embryodevelopment with decreasing free radical levels in the experimental group, and there wassignificant increase in the blastulation rate and blastomere count.This study therefore suggests the possibility of improved in in-vitro embryo culture.

Experience of Simplified Protocols in In-Vitro Fertilization and Embryo Transfer Program / 대한산부인과학회잡지

During the past few years much effort has been put into simplifying the clinical man-agement of in-vitro fertilization/embryo transfer cycles. One important step was the intro-duction of transvaginal ultrasound-guided oocyte collection, as previously described. This study describes further simplifications in the clinical management of ovarian stimulation and luteal support, and in-vitro fertilization procedure. During the period from October 1994 to September 1995, two major simplification steps were introduced. All cycles were administe-red with a gonadotrophin-releasing hormone agonist according to a long or short protocol preventing premature LH surge. During period I (Group I, n=62 cycles), closer monitoring by several pelvic ultrasound scans and serum oestradiol was used for monitoring the ovarian stimulation ; HTF media with fetal cord serum was used for insemination, growth and tran-sfer media in IVF-ET procedure ; progesterine in oil was daily used by intramuscular injec-tion for luteal support. During period II(Group II-I, n=71 cycles), only several ultrasound scans were used for monitoring the ovarian cycle ; Medi-cult IVF media containing synthetic serum replacement was used for insemination, growth and transfer media; Progesterine in oil was used daily by intramuscular injection for luteal support. During period III(Group II-II, n=16 cycles), further simplification of the clinical management was introduced by using a intravaginal micronized progesterone(Utrogestan) for luteal support. Retrospective analysis between Group I and Group II showed no differences in the number of oocyte(13.2+/-0.8/14.6+/-1.0), fertilization rate(71.5 %/60.7 %), cleavage rate(63.6 %/57.9 %), number of embryos transfered(5.0+/-0.5/4.5+/-0.5). Ongoing pregnancy rates obta ined from the three groups(Group I, Group II-1, Group II-II) were 25.8 %, 25 % and 40 %, respectively(p=ns). But introduction of minimal monitoring gave a significant reduction in the average number of US measurements in the simplified groups(Group II) compared with the group using the conventional monitoring protocol(Group I)(3.8+/-1.0/8.7+/-2.8, p<0.05). In the above groups, five patients developed severe OHSS but there was no differenc e in the distribution. Conclusively, simplified protocols including minimal follicle monitoring only by US, IVF-ET with Medi-cult IVF media containing synthetic serum replacement and the luteal support with intravaginal micronized progesterone gave a increased efficacy of the clinical phase of IVF treatment without a reduction in the success rate.

Effects of Oxygen Free Radical on the In-vitro Development of Human Embryos / 대한산부인과학회잡지

INTRODUCTION: In human IVF-ET only a few oocytes develop to be good quality embryos depending on the incubation conditions and the quality of ovum and sperm, while the rest show abnormal morphology due to unequal cell division or fragmentation of the cell. Fragmented embryos limit developmental potential and rarely result in implantation. Such abnormal embryo development have been reported to be due to genetic defects or inadequate culture environment. This in-vitro environment differs from in-vivo conditions in that the oxygen concentration is higher(20 %) and in such conditions, the mouse embryos show a higher ROS in simple culture media. ROS is believed to cause damage to the cell membrane and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of ROS within embryos and morphological features of cell damage induced by ROS. MATERIALS AND METHODS: A total of 53 human oocytes and embryos(31 fragmented and 10 nonfragmented embryos, 12 unfertilized oocytes) were obtained in IVF-ET programs. The relative intensity of ROS within embryos was reassured using 2,7-dichlorodihydrofluorescein diacetate (DCHFDA ; Molecular Probes, USA) by Quanti cell 500(Imaging Co. UK), and DNA fragmentation was observed via TEM(Zeiss EM 902A, Germany) and ApopTag kit(Oncor Co. USA). RESULTS: The ROS levels was significantly higher in fragmented embryos(72.21 +/- 9.62) compared to nonfragmented embryos(31.30 +/- 3.50, p<0.05) and unfertilized oocytes(30.75 +/- 2.67, p<0.05). Apoptosis was only observed in fragmented embryos, and was absent in nonfragmented embryos. Also the DAPI staining showed that the nuclei of fragmented embryos were broken up into irregular shapes, and the EM showed confirmation of apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. CONCLUSION: We conclude that there is a direct relationship between increased ROS and apoptosis, and that further studies should be undertaken.

Expression of CD44v6 in Cervical Cancer / 대한부인종양콜포스코피학회잡지

CD44 is a cell-surface glycoprotein postulated to play a role in tumor cell metastasis. Aberrant expression of the cell adhesion molecule CD44 has been detected in human tumors and the expression of specific isoforms(splice variants) has been shown to be associated with metastasis and poor prognosis in human malignancies. We used variant exon sequence-specific monoclonal antibody to epitope encoded by exon v6 of human variant CD44 to study the expression of CD44 splice variant by immunochemistry in fifty nine samples of human cervical cancer. twenty seven tissue samples of cervical intraepithelial neoplasia(CIN) and normal cervix were included in this study. CD44v6 was stained positive in the basal and parabasal layer of normal epithelial cells homogenously but was absent in the stromal cells. The intensity of CD44v6 staining was the strongest in invasive squamous cell carcinoma followed by normal cervical epithelium, CIN, adenocarcinoma. In the malignant samples, heterogeneity in staining intensity among different clusters of tumor cells was observed. Furthermore the intensity of staining was stronger in proportion to stage, depth of invasion, lymphovascular invasion(p<0.05), and lymph node metastasis(p=NS). This study suggest that the expression of CD44v6 adhesion molecule may be useful value in predict the high stage, depth of invasion, lymphovascular invasion and lymph node metastasis probably.

Transcervical GIFT & ZIFT by tactile sensation / 대한산부인과학회잡지

No abstract available.

Effects of the age of pronucleate ova on survival and development in cryopreservation of mouse embryos / 대한불임학회지

No abstract available.