OBJECTIVES: The aims of this study were to evaluate the interaction of topotecan with adriamycin, etoposide, 5 fluorouracil (5 FU) and mitomycin C in the established four ovarian cancer cell lines and three cervical cancer cell lines and to establish whether the combination of topotecan with other antitumor drugs would be a synergism for chemotherapy in patients with ovarian and cervical cancer, METHODS: Five antitumor drugs were tested for synergism and antagonism in combination studies in four ovarian cancer cell lines (A2780, A2780 cisplatin resistant variant, A2780 taxol resistant variant, SKOV3) and three cervical cancer cell lines (HeLa, SiHa, ME180). Cytotoxic effects were determined by MTT assay. Synergic interaction was calculated by the median effect principle in which combination index (CI) of less than one suggest a synergic interaction. RESULTS: Dm value of topotecan against SKOV3 (2.07 ug/ml), HeLa (3.32 ug/ml), SiHa cell lines (2.5 ug/ml) were above peak plasma concentration of topotecan (0.5 ug/ml) but most antitumor drugs tested in combinations index were within clinically relevant range. Combination with topotecan showed a synergic effect (CI<1) in seven cancer cell lines at a intermediate or high level of cytotoxicity especially with mitomycin C (6/7), etoposide (6/7), 5 FU (6/7) and adriamycin (4/7). Most striking findings were that a synergic effect was shown in all ovarian cancer cell lines to topotecan/mitomycin C, topotecan/5 FU and topotecan/esoposide combination showed a synergic effect in all cervical cancer cell lines. Topotecan/adriamycin combination showed synergism at an intermediate or high level of cytotoxicity in cisplatin or Taxol resistant ovarian cancer cell lines (A2780CP, A2780TX, SKOV3). CONCLUSION: These results suggested that topotecan showed a synergic effect with a wide range of antitumor drugs: adriamycin, etoposide, 5 FU, mitomycin C in ovarian and cervical cancer cell lines. Combinations of topotecan/mitomycin C, topotecan/5 FU and topotecan/adriamycin for ovarian cancer and a combination of topotecan/etoposide for cervical cancer seemed worthy of consideration for clinical application.
Antineoplastic Agents* ; Cell Line ; Cisplatin ; Doxorubicin ; Drug Therapy ; Etoposide ; Fluorouracil ; Humans ; Mitomycin ; Ovarian Neoplasms ; Paclitaxel ; Plasma ; Strikes, Employee ; Topotecan* ; Uterine Cervical Neoplasms

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Culture requirements for in vitro development of human preimplantation embryos have not been fully defined. Helper cells in coculture would pave the way for repro-ducible embryo culture system of in vitro fertilization-embryo transfer(IVF-ET) in human. The purpose of this study is to evaluate the influence of BRL cells in coculture on human embryo development in vitro. Supernumerary 2-8 cell stage embryos from IVF-ET patients were used in this experiment. The embryos were assigned to two treatments, one for conventional embryo culture in 2 ml of Ham's F10 + 15% huamn serum(control), and the other for the coculture trial. Monlayer for the coculture of embryos was prepared by plating 1X 10(5) viable BRL cells per well in 4-well tissue culture plate 48 hours prior to the onset of coculture. In twenty four hours after plating, all wells were washed out and 0.5 ml of the medium was placed into the well and then preincubated. Embryos were scored according to embryo quality, assigned to each treatment and further cultured for 5 days. A total of 63 embryos from 10 patients were randomized(23 controls, 40 coculure). With grade I embryos, higher percentage of embryos in coculture group developed to blastocyst stage(61.3%) than in control group(30.7%, p < 0.05). With grade II and III embryos, no differences in the rates of development to morula and blastocyst sta-ge were found between control and coculture groups. The overall rates of development to morula and blastocyst stage were 65.2% and 21.7%, 77.5% and 50.0% for control and coculture, respectively. Differences in the development to blastocyst stage were found between control and coculture groups(p < 0.05). The data indicate that BRL cell coculture improves human embryo development to balstocyst stage in vitro.
Animals ; Blastocyst ; Buffaloes* ; Coculture Techniques* ; Embryonic Development ; Embryonic Structures* ; Female ; Humans* ; Morula ; Pregnancy ; Rats* ; T-Lymphocytes, Helper-Inducer

No abstract available.
Animals ; Mice*

Pregnancy is a fundamental and evolving process to sustain life in universe. In human, the reproduction is a more complex and highly regulated process. The optimal maturation and the successful fertilization of gamates lead to the successful development of an embryo. Synchronization between embryo development to the blastocyst and differentiation of the endometrium to receptive state is essential to implantation. Greater understanding of sperm and egg development and fertilization is one of the major basics of the clinical application of the assisted reproductive technologies (ART). The abnormalities of these unique processes result in difficult conceiving or infertility. The prevalence of infertility reaches 10~15% of reproductive age couples all over the world, and KIHASA (Korea Institute for Health and Social Affairs) reported in 2003 that it was about 13.5% in Korea. Because most infertile couples, except some specific instances, have difficulties in conceiving, they can be pregnant with infertility treatment such as IVF (in vitro fertilization) or ICSI (intracytoplasmic sperm injection). Generally female fecundability decreases with increasing age. So it is important to try to conceive in earlier ages. Recently, highly developed ART makes it possible to overcome almost all infertility problems.
Blastocyst ; Embryonic Development ; Embryonic Structures ; Endometrium ; Epidemiology* ; Family Characteristics ; Female ; Fertility ; Fertilization ; Humans ; Infertility* ; Korea ; Ovum ; Physiology* ; Pregnancy* ; Prevalence ; Reproduction ; Reproductive Techniques, Assisted ; Sperm Injections, Intracytoplasmic ; Spermatozoa

OBJECTIVE: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes of our study was to investigate the effect of a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. METHODS: HAF was obtained from patients undergoing amniocentesis at 16~20 weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at 4 8~52 hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrf. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at 37degrees C for 10 min. RESULTS: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). CONCLUSIONS: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.
Amniocentesis ; Amniotic Fluid* ; Animals ; Chymotrypsin ; Digestion ; Epididymis ; Female ; Fertilization ; Herpes Zoster* ; Humans* ; Male ; Mice* ; Mice, Inbred ICR ; Oocytes* ; Ovum ; Pregnancy ; Sperm-Ovum Interactions ; Spermatozoa ; Zona Pellucida*

OBJECTIVE : Mammalian embryos undergo changes of energy environment for transfer from oviduct to uterus. Also, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration (oviduct - glucose: 0.5 mM, pyruvate: 0.32mM, lactate: 10.5 mM; uterus - goucose: 3.15 mM, pyruvate: 0.1mM, lactate: 5.87 mM, respectively). This study was conducted to examine the effect of these energy sources added in DMEM with glutamine on the mouse embryo development. METHODS: There was used ICR female mouse. Two cell embryos of mouse are collected by method of 'flushing'. Flushing fluid was used Ham's F-10 added to 20% FBS. The collected 2 cell embryos were cultured in media such as Control (only DMEM), group A and B (DMEM supplemented with 0.5 mM and 3.15 mM glucose), and group C and D (DMEM supplemented with 0.1 mM and 0.32 mM pyruvate), and group E and F (DMEM supplemented with 5.87 mM and 10.5 mM lactate). All experimental media supplemented with 20% hFF, respectively. Pattern of embryo development was observed to interval at 24hr during 96hr. RESULTS : The media with glutamine added glucose (group A: 51.0%; group B: 48.4%) was significantly (p<0.05) higher than other experimental group in development into the morula stage after 24 hr in culture, but not significantly different compared with control and the rate of development into the blastocyst was significantly (p<0.05) low in the both of pyruvate (group C: 7.9% group D: 6.8%) and lactate (group E: 7.1%, group F: 7.1%) treatment group after 48 hr in culture. Development into the hatched and attached balstocyst after 96hr in culture revealed similarly in control (81.9%) and glucose treatment group (group A: 83.3%, group B: 82.8%). However, development into the hatched and attached blastocyst after 96hr in culture revealed significantly (p<0.05) development in the glucose treatment group (group A: 82.3%, group B: 78.5%) than control (63.2%), and its of pyruvate (group C: 34.1%, group D: 34.1%) and lactate (group E: 25.9%, group F: 33.3%) treatment group were significantly (p<0.05) lower than control similar to previous observations. CONCLUSION : The glucose added to the DMEM with only glutamine, as energy source, was highly to the rate of development compared with control, but the other energy sources were not, synthetically. Above refer to, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration. Thus, further studies are will examine continuously to effects by interaction of different energy sources in the mouse embryo culture.
Animals ; Blastocyst ; Eagles* ; Embryo, Mammalian ; Embryonic Development* ; Embryonic Structures* ; Female ; Flushing ; Glucose ; Glutamine ; Humans ; Lactic Acid ; Mice* ; Morula ; Oviducts ; Pregnancy ; Pyruvic Acid* ; Uterus

OBJECTIVE: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. METHODS: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). Oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blastocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistically significant when rho value was less than 0.05. RESULTS: No differences was found in the fertilization between Group I(81.0%, 98/121) and Group II(81.8%, 180/220). In case of cleavage rate, no difference was found in Group I(95.9%, 94/98) and Group II(66.7%, 12/18) than in Group (26.3%, 5/19). CONCLUSION: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.
Blastocyst ; Coculture Techniques ; Eagles ; Embryo Transfer* ; Embryonic Structures* ; Female ; Fertilization ; Fertilization in Vitro ; Follicular Fluid ; Humans ; Oocytes ; Pregnancy ; Pregnancy Rate ; Spermatozoa ; Vero Cells

OBJECTIVE: In the preparation of ICSI, cumulus and corona cells should be removed from the oocytes by using a combination of enzymatic (hyaluronidase) and mechanical (pipetting) methods. But little is known about the effects of different degrees of oocyte denudation and incubation time between denudation and sperm injection on the outcomes of ICSI. The aim of this study was to evaluate the effects of varying the degrees of oocyte denudation and the lengths of incubation time from denudation to sperm injection on the outcomes of ICSI. METHODS: In experiment 1, patients (oocytes) were grouped into group A and B according to the degree of denudation, complete and partial, respectively. In experiment 2, patients (oocytes) were grouped into group I, II and III according to the length of incubation time of denuded oocytes until sperm injection as < 1, 1~2 and >2 hours, respectively. RESULTS: There was no significant difference between the degree of oocyte denudation on the survival, fertilization and development rates after ICSI procedure. In case of the incubation time of denuded oocytes until ICSI, survival rates was higher in group III (83.1%) than in group I (61.5%, p<0.05) or group II (64.3%). However no statistically significant differences were found between incubation time and fertilization or development rates. CONCLUSIONS: This study reveals that the outcomes of ICSI are not affected by the degree (complete or partial) of oocyte denudation. However the denuded oocytes with incubation period of more than 2 hours show better outcomes of ICSI than those with the incubation period of less than 2 hours.
Fertilization ; Humans* ; Oocytes* ; Sperm Injections, Intracytoplasmic* ; Spermatozoa ; Survival Rate

OBJECTIVE: The pathogenesis of endometriosis is generally accepted that retrograde menstruation and alterations in the local pelvic immune environment. This study was performed to help elucidate what kind of role various cytokines might play in the pathogenesis of endometriosis. METHOD: Concentrations of peritoneal fluid cytokines were compared in 7 women with normal pelvic finding and 23 women with endometriosis by enzyme-linked immunosorbent assay(ELISA). The patterns of cytokine mRNA expression in 8 ovarian endometrioma and 12 superficial pelvic endometriosis lesions were investigated by reverse transcription-polymerase chain reaction(RT-PCR) amplification method. RESULT: Both IL-6 and IL-10 levels in peritoneal fluid specimens with endometriosis tended to be higher than normal. However, there were no significant differences between peritoneal fluid concentrations of IFN-gamma, TNF-alpha, IL-1beta, and IL-5 of women with and without endometriosis. The levels of IL-6 and IL-10 were significantly higher in peritoneal fluid of women with severe endometriosis compared to women with mild endometriosis. IL-1beta mRNA was expressed in all of 8 deep and 12 superficial endometriosis lesions. IL-5 and IL-6 mRNA were expressed in only two black lesions respectively, however, both were not expressed in the all deep lesions. Expressions of IL-10 mRNA occurred in one red and one black lesion while this was expressed in only one of the deep lesions. TNF-alpha mRNA was expressed in one red and one black lesion of 12 superficial lesions compared with four of the deep lesions. There was the difference between kinds of increased cytokines in the peritoneal fluid and those of expressed cytokines in the endometriotic lesions of patients with endometriosis. CONCLUSION: This study supports the concept that local immunologic factors may be important in the pathogenesis and pathophysiology of endometriosis. The pattern of cytokine mRNA expression of endometriotic lesions would seem to indicate that proinflammatory cytokines such as IL-1beta and TNF-alpha are responsible for the development or progression of endometriosis.
Ascitic Fluid ; Cytokines ; Endometriosis* ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunologic Factors ; Interleukin-10 ; Interleukin-5 ; Interleukin-6 ; Menstruation Disturbances ; RNA, Messenger* ; Tumor Necrosis Factor-alpha