At the time in vitro fertilization and embryo transfer, patients with unsuitable endometrium recovered by hormone. However, the overtreatment of hormone causes indispositionly the uterus internal secretion and finally induces endometriosis. Therefore, this study was done to inverstigate the effects of interleukin-2, which was known to differentiator and proliferator of T cells, on proliferation of the endometrial stromal cells in vitro. We have exammined the effects of interleukin-2, on the proliferation of bovine endometrial stromal cells in vitro, assessed by ['H]-thymidine incorporation and MTT assay methods. Results indicate that we isolated endometrial stromal cells from bovine uterus and established in vitro culture system. And interleukin-2 showed distint stimulatory effect on proliferation of the established stromal cells. These stimulative effects were not affected by estrogen and progesterone indirectly. In conclusion, these data imply that interleukin-2 may proliferate bovine endometrial stromal, cells, and it provides clue for understanding of direct actions of cytokines on the endometriat cells.
Cytokines ; Embryo Transfer ; Endometriosis ; Endometrium ; Estrogens ; Female ; Fertilization in Vitro ; Humans ; Interleukin-2 ; Interleukins* ; Progesterone ; Stromal Cells* ; T-Lymphocytes ; Uterus

OBJECTIVE: This study was performed to evaluate and compare the embryonic developmental capacity and pregnancy rates in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with ejaculated sperm or testicular sperm cycles. MATERIALS AND METHODS: Fertilization was examined in the following morning after IVF (group I), ICSI (group II) or TESE-ICSI cycles (group III). Fertilized oocytes were co-cultured with Vero cells until embryo transfer (ET). On day 2 and 5~7, grades of embryos (<4- or > or =4-cell) and blastocysts (BG1, 2, 3 or early) were evaluated. Clinical pregnancy rate was determined by detecting G-sac with transvaginal ultrasonogram. We analyzed the results bychi2 and Student's t-test and considered statistically significant when P value was less than 0.05. RESULTS: Fertilization rate was significantly higher (p<0.05) in group I (79.0+/-21.2%) than in group II and III (56.8+/-21.6% and 36.7+/-25.3%). Cleavage and blastulation rate of group I (95.8+/-13.8% and 59.5+/-25.3%) were significantly higher (p<0.05) than those of group III (83.4+/-18.6% and 40.4+/- 36.5%). Clinical pregnancy rate was significantly higher (p<0.05) in group I and II (40.7% and 41.7%) than that in group III (12.5%). No differences were found in the rates of multiple pregnancy and abortion among three groups. Embryonic implantation rate was higher in group I (15.1+/-20.2%, p<0.05) and II (14.7+/-20.6%, NS) than that in group III (5.1+/-15.6%). However, embryonic implantation rate was increased in ET with blastocyst(s) among three groups. CONCLUSIONS: Fertilized oocytes obtained from TESE-ICSI were harder to be successfully cultured to blastocyst stage for 5~7 days than that from IVF cycles. However, all blastocyst(s) ET increased the embryonic implantation rate equally in IVF, ICSI and TESE-ICSI cycles.
Blastocyst ; Embryo Transfer ; Embryonic Development* ; Embryonic Structures ; Female ; Fertilization ; Fertilization in Vitro ; Oocytes* ; Pregnancy ; Pregnancy Rate* ; Pregnancy* ; Pregnancy, Multiple ; Sperm Injections, Intracytoplasmic* ; Spermatozoa ; Ultrasonography ; Vero Cells

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: Our present studies were conducted to examine more effective isolating method of preantral follicles from mouse ovaries. METHODS: ICR mice (3-6 weeks old) were sacrificed through cervical dislocation and their ovaries were removed and put into watch glasses containing Hams F-10 supplemented with 10% fetal bovine serum (FBS). Preantral follicles were isolated by three different methods; 1) enzymatical method and 2) mincing method, and 3) scraping method. Enzymatical method was carried out as following. Ovaries were bisected with a pair of fine 30G needles. Bisected ovaries were incubated at 37degrees C and 5% CO2 incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 IU/ml. After 20 min.,follicles were isolated by repeated pipetting. Isolated preantral follicles were collected, and the remnant of tissues was placed in incubator and previous procedure was repeated. Mincing method was carried out with a pair of fine 30G needles attached to 1 ml syringes and mined ovary. Scraping method was carried out wit a pair of fine 30G needles and scratched to surface of ovary. The differences between isolating methods were analyzed using Student's t-test and Chi-square. Results were considered statistically significant when P value was less than 0.05. RESULTS:In handling time, mincing or scraping method (28+/-3.42 min or 16+/-1.58 min) were significantly (p<0.00001) shorter than enzymatical method (72+/-1.69 min), and scraping method was significantly (p<0.01) shorter than mincing method. Total number of isolated follicles was significantly (p<0.0001) higher in enzymatical method (49.8+/-3.91) than in mincing or scraping method (25.3+/-2.33 or 20.5+/-1.75). Isolated follicles in < or =90 micrometer were significantly (p<0.005) higher in enzymatical method (15+/-1.71) than in mincing or scraping method (7.8+/-0.98 or 8.1+/-1.31). In 91~130 micrometer, isolated follicles were significantly (p<0.0005) higher in enzymatical method (33+/-3.27) than in mincing or scraping method (16.3+/-1.82 or 10.7+/-1.38). In > or =131 micrometer, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in < or =90 micrometer was highest in scraping method (39.6% vs. enzymatical method:30.1%, p<0.05; mincing method: 30.9%, p=0.11719, NS). Rate of follicles in 91~130 micrometer was significantly (p<0.05) lower in scraping method (52.7%) than in emzymatical or mincing method (66.3% or 64.5%). Rate of follicles in > or =131 micrometer was highest in scraping method (8.3% vs. enzymatical or scraping method: 3.6%, p<0.05)or 4.6%, p=0.19053, NS). CONCLUSIONS: This study suggests that scraping method is simple and useful for isolation of preantral follicles, because this method reduced handling time and recovered enough follicles. The recovered rate of isolated follicles in diameter of 91~130 micrometer was highest in all methods.
Animals ; Collagenases ; Dislocations ; Eyeglasses ; Female ; Glass ; Incubators ; Mice* ; Mice, Inbred ICR ; Needles ; Ovary* ; Syringes

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

This study was conducted to investigate the effects of Tissue Culture Medium 199 (TCM) and Dulecco's Modified Eagle Medium (DMEM) on the blastulation and grade of human oocytes on Vero cells in vitro. A cohort of 79 and 93 oocytes in metaphase II stage were used in TCM 199 and DMEM respectively. No differences were found in the nurser of oocytes showing two-pronuclei between TCM (82.3%) and DMEM (86.0%). The number of fertilized oocytes reaching the blastocyst was not significant in TCM (60.0%) and DMEM (63.1%).4 total of 89 blastocysts were categorized into the four grades (BG1, BG2, BG3 and early) depending on their morphology. The number of embryos achieving the blastocyst grade 1 (BG1) was significantly higher (p<0.05) in DMEM (50.8%) than TCM (15.0%). It is concluded that cultured oocytes in DMEM with glutamine on Vero cells should be significantly increased BG1.
Blastocyst ; Cohort Studies ; Eagles ; Embryonic Structures* ; Glutamine* ; Humans* ; Metaphase ; Oocytes ; Vero Cells*

No abstract available.
Animals ; Mice*

OBJECTIVE: To evaluate whether co-culture of oocytes on vero cell monolayers from Day 0 (Day 0 group) after egg retrieval results in an increase in developmental capacity such as fertilization rate, embryo quality, blastulation and clinical pregnancy rate compared with co-culture of oocytes from Day 1 (Day 1 group). METHODS: Sperms were treated with Hams F-10 supplemented with 10% human follicular fluid (hFF). Vero cells for co-culture were prepared in TCM-199 with 10% FBS. Oocytes were co-cultured from Day 0 and fertilized oocytes were co-cultured from Day 1 on vero cell monolayers in DMEM with 10% and 20% hFF, respectively after egg retrieval. On day 1, 2 and 5, fertilization rate and grade of embryos and blastocysts were evaluated. Results (fertilization rate, cleavage rate, grade of embryos and blastocysts and pregnancy rate) were considered statistically significant when p value was less than 0.05 using t-test and ci2. RESULTS: In sibling oocytes of same cycles, no differences were found in fertilization rate (94.6 vs. 91.4%), cleavage rates (94.6 vs. 91.4%), embryo grade (on day 2 and 3) and blastulation (65.6 vs. 57.0%) and their grade. In different oocytes of different cycles (patients), no differences were found in fertilization (79.8 vs. 78.3%), cleavage rates (77.7 vs. 76.4%) and blastulation (56.0 vs. 45.3%), but pregnancy rate was higher in the Day 0 group than in the Day 1 group (60.0 vs. 42.9%). CONCLUSIONS: This study revealed that the embryonic development capacities were not affected by the different co-culture time in the sibling oocytes of same cycles. Although no statistical significance, because of small size of study, there was a trend for higher pregnancy rates in Day 0 group compared to Day 1 group in different oocytes of different cycles.
Blastocyst ; Coculture Techniques* ; Embryonic Development* ; Embryonic Structures ; Female ; Fertilization ; Follicular Fluid ; Humans ; Oocytes* ; Ovum ; Pregnancy ; Pregnancy Rate ; Siblings ; Spermatozoa ; Vero Cells