Studies on development of serum-free conditioned media using Vero cells and DMEM with controlled concentration of glucose and pyruvate / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to examine in vitro development of early preimplantation mouse embryos in various kind of serum-free conditioned media (SF-VCM) manufactured from DMEM cultured with Vero Cells. METHODS: A total of 846 two-cell mouse embryos were cultured in different kind of SF-VCM. SF-VCMs were divided into SF-VCM-10, -30 and -50 by media volume using DMEM #1 media, and divided into SF-VCM #1, #2 and #3 by controlled concentration of glucose and pyruvate (manufactured by DMEM #1: mixed three volume of DMEM-G (DMEM with glutamine without glucose and pyruvate) and one volume of DMEM-GGP (DMEM with glutamine, glucose, pyruvate), #2: mixed same volume of DMEM-G and DMEM-GGP and #3: mixed one volume of DMEM-G and three volume of DMEM-GGP, respectively). Experimental groups were mainly added 10% SSS, and 20% hFF was added to only Control group co-cultured with Vero cells. Development of embryos was observed every 24 hours. Results between different groups were analyzed using Chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: In vitro developmental rate by each cleavage stages of mouse embryos cultured in SF-VCMs with a various volumes were significantly (P<0.05) higher in SF-VCM-30 (morula< or =: 97.2%, Blastocyst (BL)< or =: 97.2%, Hatching BL< or =: 82.2%) than other groups. In the rate of development on in vitro co-culture vs. a various SF-VCMs manufactured by DMEM controlled concentration of glucose and pyruvate, Group I (SF-VCM #1) was higher than other groups in each cleavage stages (morula< or =: 98.1%, Blastocyst (BL)< or =: 97.1%, hatching BL< or =: 81.7%, respectively). Moreover, specially, in the developmental rate into the hatching blastocyst < or = after 96 hours in vitro culture, Group I (81.7%) was significantly higher than control group (67.6%, P<0.05). CONCLUSION: SF-VCM #1 manufactured by volume of 30 mL DMEM #1 media cultured in vitro for 48 hours in 250 mL flask was the most effective on in vitro developmental rate of mouse preimplantation embryos. Therefore, it is expected that SF-VCM #1 has application to human IVF-ET.

Effects of Vero Cells Co-culture System on The In Vitro Development of Mouse Preimplantation Embryos in Media with Different Composition of Glucose and Pyruvate / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to examine the effects of vero cells co-culture system on the in vitro development of mouse preimplantation embryos in culture media with different composition of glucose and pyruvate. METHODS: Two-cell embryos were collected from 4-5 weeks old ICR mice. Seven hundreds twenty two embryos were cultured with or without vero cells monolayer in four media with different compositions that was manufactured by two DMEM media with (DMEM-GGP) or without (DMEM-G) glucose and pyruvate. In control, DMEM-G medium which is currently using for human embryo culture in our infertility clinic was used. Group I (DMEM-G(1/4)GP) was cultured in medium which was mixed three volume of DMEM-G and one volume of DMEM-GGP, and group II (DMEM-G(1/2)GP) was cultured in medium which was mixed same volume of DMEM-G and DMEM-GGP, and group III was cultured in DMEM-GGP. All media were added to 20% hFF. Results between different groups were analyzed using a Chi-square test, and considered statistically significant when p value was less than 0.05. RESULTS: The developmental rate into 3-cell

Establishment of Culture System for In Vitro Culture of Mouse Pre-antral Follicles: Effect on In Vitro Growth by Thecal-Stromal Cells in Culture Medium without Hormones / 대한산부인과학회잡지

OBJECTIVE: This present study was conducted to examine the effects on in vitro growth of pre-antral mouse follicles by thecal-stromal cells attached to around the follicular membrane in culture medium without hormones. METHODS: Pre-antral follicles (100-130 micro meter) used in our studies were isolated mechanically by fine 30 G needles attached to 1 ml insulin syringe from mice ovaries of 20-25 days old female ICR strain. Isolated pre-antral follicles were divided into three groups by attached status of thecal-stromal cell layers to follicular membrane. Follicular Initial diameters of group I was 112.5 (mean) +/- 6.2 (SD) micro meter (n=31), group II was 112.8 +/- 7.8 micro meter (n=23), and group III was 115.1 +/- 6.5 micro meter (n=27). Divided pre-antral follicles were washed in fresh Ham's F-10 medium and cultured in 20 micro liter droplets of DMEM with glutamine, glucose and pyruvate under mineral oil on the 60 mm culture dish. All experimental media were supplemented with 10% FBS without hormones. Media were exchanged wholly by fresh media every 2 days in culture. Diameters of intrafollicular oocytes and cultured pre-antral follicles were measured using an precalibrated cross ocular micrometer at X200 every day during 6 days in vitro culture. Results were considered statistically significant when p value was less than 0.05 using Student's t-test. RESULTS: The mean and standard deviation (SD) of initial diameters of pre-antral follicles was 113.5 +/- 2.2 micro meter in three groups. Follicular growth rate of group III was significantly (p<0.05) higher in whole culture periods than group I and group II. Rate of follicular growth between group I and group II were not significant difference in culture for 1 to 3 days. However, group II was significantly higher than group I in culture for 4 days. Diameters of grown follicles for 6 days was 155.2 +/- 18.7 micro meter, 196.9 +/- 24.1 micro meter and 284.2 +/- 47.6 micro meter in group I, group II and group III, respectively. Growth rate of intrafollicular oocytes between three groups were not difference and revealed to continuous growth patterns in whole culture periods. Follicular diameter after culture for 7 days were not measured because of disruption of follicular structure or outgrowth of granulosa cells. CONCLUSION: Pre-antral follicles were grown very well in DMEM medium without hormones. Thecal- stromal cells attached to the surface of follicular membrane has acted effectively in vitro growth of follicles.

The Influences of Different Composition of Glucose and Pyruvate on In Vitro Development of Mouse Preimplantation Embryos in Medium with Glutamine / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to examine the effects on in vitro development of early preimplantation mouse embryos in DMEM medium with glutamine which was controlled by different composition of glucose and pyruvate. METHODS: Four hundred and nineteen mouse 2-cell embryos were cultured in four different media with different composition of glucose and pyruvate for 96 hours. The DMEM-G contained L-glutamine for energy sources was used for control group. Group I embryos were cultured in the medium that mixed one volume of DMEM-GGP contained L-glutamine, D-glucose and sodium pyruvate for energy sources with three volume of DMEM-G, and group II embryos were cultured in the medium that mixed with same volume of DMEM-G and DMEM-GGP, and group III embryos were cultured in DMEM-GGP. RESULTS: At 24 hours, the development into >or=3-cell was significantly higher (por=8-cell was significantly higher in group I (73.1%) than control (44.2%), group II (59.6%) and III (45.8%), and also group II was significantly higher than control and group III. At 48 hours, the development into >or=morula was significantly higher in group I (90.4%) and II (86.5%) than control (73.0%). However, the development into blastocyst, in group III (15.0%) was significantly lower than control, group I and II. At 72 hours, the development into >or=expanded blastocyst was significantly higher in group I (69.2%) than group III (47.7%), and total blastocyst was significantly higher in group I (80.8%) than control (66.3%) and group III (67.3%). At 96 hours, the development into >or=hatching blastocyst was significantly higher in group I (78.8%) than control (61.5%) and group III (57.9%), and also, total blastocyst was significantly higher in group I (85.6%) than control (69.2%) and group III (72.0%). CONCLUSION: The development of early preimplantation mouse embryos cultured in group I medium that mixed one volume of DMEM-GGP with three volume of DMEM-G was better than other groups during the culture period.

Embryonic Developmental Capacity and Pregnancy Rates of Fertilized Oocytes in IVF, ICSI and TESE-ICSI Cycles / 대한불임학회지

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 ( 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.

Effects of Glucose on Blastocyst Formation and Their Cell Numbers of Mouse Embryos / 대한산부인과학회잡지

OBJECTIVE: Mammalian embryos undergo changes of energy environment for transfer from oviduct to uterus. An experimental design was used to examine the effects of glucose on the development in vitro of mouse embryos. METHODS: Two cell embryos were recovered from ICR female mice (3-4 weeks) at 46-50 hrs after 5 IU hCG injection (mated just after hCG injection) and cultured in 50 micro gram MEM droplets supplemented with nothing (control; n=46), 0.5 mM glucose (Group A; n=46) or 3.15 mM glucose (Group B; n=46) under mineral oil. All experimental media were supplemented with 20% human follicular fluid. Results were observed: (i) the number of zona-intact blastocysts (ZiB); (ii) the number of zona-escaped blastocysts (ZeB; hatching~hatched); (iii) the mean cell numbers; and (iv) the proportion of inncer cell mass (% ICM) in the blastocysts. RESULTS: Total blastocyst formation rates were (NS) in glucose groups (group A: 52.2; B: 47.8%) than control group (60.9%). ZiB rates the highest (p<0.05) in control (47.8%) than those in group A (21.7%) and B (28.3%). ZeB rates the highest (NS) in group A (30.4%) than those in control (13.0%) and group B (19.6%). Blastocysts, cultured in group B (50.5), had the highest (NS) mean cell numbers compared with the others (control: 39.2; group A: 45.6). The % ICM in blastocysts cultured in group A (20.6%) was the highest (NS) than those of other tested groups (control: 15.2%; group B: 13.9%). CONCLUSION: This study shows that a low dose of glucose added to culture medium increases the developmental capacity of 2 cell embryos in mice.

The Clinical Outcomes after Embryo Transfer (ET) on Day 2 and Day 5 or Subsequent ET on Day 2-5, 2-6, 2-7, 3-5 and 4-7 in in Vitro FertilIzatIon-ET Cycles / 대한불임학회지

Transfer of human embryos to the uterus at the blastocyst stage has several advantages. There may be an improvement in success rates of pregnancy due to better synchronization of the uterine and embryonic development, self-selection of embryos for transfer along with the possibility of reducing the number of embryos transferred and the risk of multiple pregnancies without altering the overall pregnancy rate would be decreased. However, embryos are transferred to the uterus on day 2 or 3 after insemination before the blastocyst stage is reached in many cases. This may be a reflection of sub-optimal culture conditions although inherent abnormalities like chromosomal anomalies will also contribute to the loss.1~8 Physiologically, the human endometrium prepares itself to its optimum approximately on days 5~7 after ovulation so as to receive a cavitated blastocyst from the fallopian tube for successful implantation.1,9,10 However, conventionally, in most programs offering in vitro fertilization (iVF), 4~8 cell stage embryos are replaced to the uterus.1,9,11 This asynchrony of embryonic stage and preparation of endometrium may be one major contributory cause of increased abortion and low take-home baby rates in infertility patients.1,10~12 Leaving all embryos in extended culture until they develop to the blastocyst stage might result in cancellation of the embryo transfer (ET) procedure if none of the embryos reach that stage. This could have a major adverse psychological impact on the patient, and also denies her the possibility of implantation of early embryos that did not develop to the blastocyst stage in vitro but might have done so in vivo.2 To avoid such events while explore the feasibility of blastocyst transfer, subsequent ET (SET) of early embryos and blastocysts was applied on day 2~3 and 5.2,6,7,13,14 The current study was conducted to investigate the effectiveness of attempted SET of blastocysts on day 5~7 following initial multi-cell embryos transfer on day 2, 3 or 4 in iVF cycles.

Comparison of Embryonic Developmental Capacity by different Co-culture Time of Oocytes in IVF-ET Cycles / 대한불임학회지

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.

Study on Development of Mouse Preimplantation Embryos in Culture Media with Different Composition of Energy Sources / 대한산부인과학회잡지

OBJECTIVE: The objective of this study was to examine the effect on development of mouse preimplantation embryos in culture media with different composition of energy sources in vitro culture. METHODS: Two hundred and seventy one two-cell embryos were cultured in four different culture system for 96 hours. Group I (n=61) was cultured in DMEM-G (DMEM with glutamine) only, groupII (n=64) was cultured in DMEM-GGP (DMEM with glutamine, glucose and pyruvate) only, group III (n=72) was cultured for 48 hours in DMEM-G and then transferred to DMEM-GGP and group IV (n=74) was cultured for 48 hours in DMEM-GGP and then transferred to DMEM-G. Development of embryos in each group was observed every 24 hours. RESULTS: After 24 hours, the rate of development > or = 3-cell was significantly higher in groupII (87.5%) and IV (86.5%) compared with group I (59.0%) and III (62.5%). After 48 hours, the rate of development into > or = morula stage was significantly higher in GroupII (79.7%) and IV (86.5%) compared with group I (34.4%) and III (37.5%). After 72 hours, the rate of development into blastocyst was significantly higher in group IV (74.3%) compared with group I (49.2%) and III (45.8%). After 96 hours, the rate of development into > or = expanded blastocyst was significantly higher in group IV (70.3%) compared with group I (32.8%),II (53.1%), and group III (40.3%). CONCLUSION: Mouse preimplantation embryos development was the most effective in culture system with DMEM-GGP for 48 hours and then transferred to DMEM-G.

Effects of Medium on Blastocyst Formation, Cell Number and Percentage of ICM in Mice / 대한불임학회지

OBJECTIVE: The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. MATERIALS AND METHODS: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values<0.05 were accepted as statistically significant. RESULTS: The blastulation rate in MEM (64.9+/-4.95%) was significantly higher (p=0.0031) than that in TCM (57.2+/-5.22%). No differences were found in the number of ZiB and ZeB between MEM (31.9+/-2.62, 33.0+/-4.58%), and TCM (27.2+/-4.28, 30.1+/-4.58%). A total 314 blastocysts (MEM=166; TCM= 148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM (73.1+/-3.3) than in MEM (61.7+/-2.5). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM (20.9+/-1.3 vs. 17.1+/-1.2%, p=0.0281). The ICM:TE ratio was higher in TCM than in MEM (1 : 4.85+/-0.68 vs. 1 : 3.78+/-0.78, NS). CONCLUSION: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

Effects of Medium on Blastocyst Formation, Cell Number and Percentage of ICM in Mice / 대한불임학회지

OBJECTIVE: The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. MATERIALS AND METHODS: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values<0.05 were accepted as statistically significant. RESULTS: The blastulation rate in MEM (64.9+/-4.95%) was significantly higher (p=0.0031) than that in TCM (57.2+/-5.22%). No differences were found in the number of ZiB and ZeB between MEM (31.9+/-2.62, 33.0+/-4.58%), and TCM (27.2+/-4.28, 30.1+/-4.58%). A total 314 blastocysts (MEM=166; TCM= 148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM (73.1+/-3.3) than in MEM (61.7+/-2.5). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM (20.9+/-1.3 vs. 17.1+/-1.2%, p=0.0281). The ICM:TE ratio was higher in TCM than in MEM (1 : 4.85+/-0.68 vs. 1 : 3.78+/-0.78, NS). CONCLUSION: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

Effects of Exposure of Propidium Iodide and Bisbenzimide on Differential Staining of Mouse Blastocysts / 대한불임학회지

OBJECTIVE: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. MATERIALS AND METHODS: A total 964 blastocysts (early~hatched) was exposed to PI (n=831) (group I:

Antrum Formation and Growth In Vitro of Mouse Pre-antral Follicles Cultured in Media without Hormones / 대한불임학회지

No abstract available.

A Simple Isolating Method of Preantral Follicles from Mouse Ovaries / 대한불임학회지

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 or =131 micrometer, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated 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.

Subsequent Embryo Transfers (SET) on Day 2 and 5: It's Safety and Effectiveness / 대한불임학회지

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.

Effect of Energy Sources (Glucose, Pyruvate and Lactate) Added to Dulbecco's Modified Eagle Medium (DMEM) on the Mouse 2-cell Embryo Development / 대한불임학회지

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.

Human Amniotic Fluid Induces Spontaneous Hardening of the Zona Pellucida of Mouse Immature Oocytes During Maturation In Vitro / 대한불임학회지

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.

Effect of Interleukin-2 on the Surgically Induced Enndometriosis in Rat / 대한면역학회지

It has been shown that wornen with endometriosis have several immunological defects. The effect of interleukin-2 (IL-2) for the treatment of induced endometriosis in rat was studied. The results obtained are as followings: proliferation of epithelium is increased, and the inner surface is undulated with 1.5 nM IL-2. In 7.5 nM IL-2, the epithelial cells are changed to columar ones, and secretory hobs are observed at the apex of individual cell. Secretory activity of epithelium is increased with 0.5 nM IL-2, and apoptosis of the epithelial cell is observed in 15 nM IL-2. The levels of progesterone and estradiol in sera of rat were increased after treatment with IL-2 and were highest in the concentration of 1.5 nM IL-2. The results of this study can be a guide in the development of new therapeutic approaches for the treatment of endometriosis.

The Effects of Oocyte Preparation on the Developing Capacity of Human Oocytesat Intracytoplasmic Sperm Injection (ICSI) / 대한불임학회지

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 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.

Cultivation of the Isolated Bovine Endometrial Stromal Cells and the Effect of Interleukin - 2 on Its Proliferation / 대한면역학회지

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.