Current status of assisted reproductive technology in Korea, 2009

Great advances have been made in the field of assisted reproductive technology (ART) since the first in vitro fertilization (IVF) baby was born in Korea in the year of 1985. However, it deserve to say that the invaluable data from fertility centers may serve as a useful source to find out which factors affect successful IVF outcome and to offer applicable information to infertile patients and fertility clinics. This article intended to report the status of ART in 2009 Korean Society of Obstetrics and Gynecology surveyed. The current survey was performed to assess the status and success rate of ART performed in Korea, between January 1 and December 31, 2009. Reporting forms had been sent out to IVF centers via e-mail, and collected by e-mail as well in 2012. With International Committee Monitoring Assisted Reproductive Technologies recommendation, intracytoplasmic sperm injection (ICSI) and non-ICSI cases have been categorized and also IVF-ET cases involving frozen embryo replacement have been surveyed separately. Seventy-four centers have reported the treatment cycles initiated in the year of 2009, and had performed a total of 27,947 cycles of ART treatments. Among a total of 27,947 treatment cycles, IVF and ICSI cases added up to 22,049 (78.9%), with 45.3% IVF without ICSI and 54.7% IVF with ICSI, respectively. Among the IVF and ICSI patients, patients confirmed to have achieved clinical pregnancy was 28.8% per cycle with oocyte retrieval, and 30.9% per cycle with embryo transfer. The most common number of embryos transferred in 2009 is three embryos (40.4%), followed by 2 embryos (28.4%) and a single embryo transferred (13.6%). Among IVF and ICSI cycles that resulted in multiple live births, twin pregnancy rate was 45.3% and triple pregnancy rate was 1.1%. A total of 191 cases of oocyte donation had been performed to result in 25.0% of live birth rate. Meanwhile, a total of 5,619 cases of frozen embryo replacement had been performed with 33.7% of clinical pregnancy rate per cycle with embryo transfer. When comparing with international registry data, clinical pregnancy rate per transfer from fresh IVF cycles including ICSI (34.1%,) was comparable to clinical pregnancy rate per transfer in European Society for Human Reproduction and Embryology report was 32.5% though lower than 45.0% for USA data. There was no remarkable difference in status of assisted reproductive technology in Korea between the current report and the data reported in 2008. The age of women trying to get pregnant was reconfirmed to be the most important factor that may have impact on success of ART treatment.

Gene Expression Profiles of Uterine Normal Myometrium and Leiomyoma and Their Estrogen Responsiveness In Vitro

BACKGROUND: Uterine leiomyomas are common benign smooth muscle tumors among the reproductive aged-women. The research has been aimed to identify the differentially expressed genes between normal myometrium and leiomyoma and to investigate the effects of E2 on their expression. METHODS: Gene microarray analysis was performed to identify the differentially expressed genes between normal myomerium and leiomyoma. The data was confirmed at protein level by tissue microarray. RESULTS: Gene microarray analysis revealed 792 upregulated genes in leiomyoma. Four genes (tropomyosin 4 [TPM4], collagen, type IV, alpha 2 [COL4alpha2], insulin-like growth factor binding protein 5 [IGFBP5], tripartite motif-containing 28 [TRIM28]) showed the most dramatic upregulation in all leiomyoma samples. Tissue microarray analyses of 262 sample pairs showed significantly elevated expression of TPM4, IGFBP5, estrogen receptor-alpha, and progesterone receptor (PR) protein in leiomyoma from the patients in their forties, COL4alpha2 in the forties and fifties age-groups, and TRIM28 in the thirties age-group. PR, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R) and IGFBP5 were induced by E2 in in vitro culture of tissue explants from which cells migrated throughout the plate. Among these, PR, IGF-1, IGFBP5 genes showed higher expression in tissue compared to cells-derived from tissue in leiomyoma and IGF-1R in leiomyoma cell. CONCLUSIONS: This observation implies the importance of the whole tissue context including the cells-derived from tissue in the research for the understanding of molecular mechanism of leiomyoma. Here, we report higher expression of TRIM28 in leiomyoma for the first time and identify E2-responsive genes that may have important roles in leiomyoma development.

Comparison of Gene Expression Profile in Eutopic Endometria with or without Endometriosis: A Microarray Study / 대한불임학회지

OBJECTIVE: Pathogenesis of the endometriosis is very complex and the etiology is still unclear. Our hypothesis is that there may be some difference in gene expression patterns between eutopic endometriums with or without endometriosis. In this study, we analyzed the difference of gene expression profile with cDNA microarray. METHODS: Endometrial tissues were gathered from patients with endometriosis or other benign gynecologic diseases. cDNA microarray technique was applied to screen the different gene expression profiles from early and late secretory phase endometria of those two groups. Each three mRNA samples isolated from early and late secretory phase of endometrial tissues of control were pooled and used as master controls and labeled with Cy3-dUTP. Then the differences of gene expression pattern were screened by comparing eutopic endometria with endometriosis, which were labeled with Cy5-dUTP. Fluorescent labeled probes were hybridized on a microarray of 4,800 human genes. RESULTS: Twelve genes were consistently overexpressed in the endometrium of endometriosis such as ATP synthase H transporting F1 (ATP5B), eukaryotic translation elongation factor 1, isocitrate dehydrogenase 1 (NADP+), mitochondrial ribosomal protein L3, ATP synthase H+ transporting (ATP5C1) and TNF alpha factor. Eleven genes were consistently down-regulated in the endometriosis samples. Many extracellular matrix protein genes (decorin, lumican, EGF-containing fibulin-like extracellular matrix protein 1, fibulin 5, and matrix Gla protein) and protease/protease inhibitors (serine proteinase inhibitor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1), and insulin like growth factor II associated protein were included. Expression patterns of selected eight genes from the cDNA microarray were confirmed by quantitative RT-PCR or real time RT-PCR. CONCLUSION: The result of this analysis supports the hypothesis that the endometrium from patients with endometriosis has distinct gene expression profile from control endometrium without endometriosis.

A case of severe hydronephrosis due to ureteral stricture from endometriosis / 대한산부인과학회지

Endometriosis is a relatively common disease, affecting 5-10% of women of reproductive age. But, endometriosis affecting the urinary tract is very rare entity. Involvement of urinary tract by endometriosis occurs in about 1% of women with pelvic endometriosis. Ureteral endometriosis is mostly asymptomatic for a long time, and associated with nonspecific symptoms at clinical presentation and difficult preoperative diagnosis. The involvement of the ureter is rarely intrinsic by implantation of endometrial tissue in the wall of the ureter, but rather due to external compression by adjacent endometriosis and its attendant inflammation and fibrosis. We have experienced a case of right severe hydroureteronephrosis due to ureteral stricture from endometriosis. Laparoscopic nephrectomy was done due to renal atrophy. At the same time, laparoscopic total hysterectomy with right salpingo-oophorectomy was performed because of the uterine adenomyosis and right ovarian endometrioma. So, we report that with a brief review of literatures.

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 Effect on IVF-ET Outcomes of Intramural Myomas / 대한산부인과학회잡지

OBJECTIVE: The aim of this study was to assess the effect of intramural uterine myoma in patients with a normal endometrial cavity on IVF-ET cycle outcome. This study was also investigated to find out whether medical supression or myomectomy needs to precede to IVF-ET. METHODS: The subjects were 300 infertile patients who got IVF-ET from January 1999 to December 2002 at the Department of Obstetrics and Gynecology in Kyungpook National University Hospital, and they were divided into myoma group of 97 patients and non-myoma group of 203 ones. This study group did not include patients found to have large myoma (>or= 7 cm), or myoma distorting the uterine cavity. RESULTS: Compared with the results of IVF-ET according to the presence of intramural uterine myoma, pregnancy rate and implantation rate between two groups had no statistically significant differences (p>0.05). Further comparisons within the myoma group showed no difference in pregnancy rate by number of myomas or site of myomas (p>0.05). CONCLUSION: Intramural uterine myoma not distorting the uterine cavity is considered to have no effect on the outcome of IVF-ET and no difference in the pregnancy rate according to the number of myomas or site of myomas. Therefore, these findings suggest that medical treatments or myomectomy before IVF-ET is not necessary to those infertile patients with intramural uterine myomas not distorting the uterine cavity.

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.

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:

Hysteroscopic management of intrauterine adhesion and their reproductive outcomes / 대한산부인과학회잡지

One hundred sixty three patients were hysteroscoped for various reasons at our department over 10 year period. Ninety eight patients out of 163 who were diagnosed and treated for their intrauterine adhesions(IUAs) by hysteroscopy were analysed retrospectively. Patients were classified as having mild, moderate or severe stage of IUAs according to American Fertility Society classification. Twenty nine patients(29.6%) had mild IUAs, 43 patients (43.9%) had moderate IUAs, and 26 patients(26.5%) were classified as having severe stage IUAs. The predisposing causal factors of IUAs were abortion, full-term delivery, H. mole, pelvic tuberculosis and myomectomy in order of frequency but in eight cases no cause was identified. After hysteroscopic surgery, although high rate(83.9%) of restoration of normal menstruation was attained but term pregnancy rate was only 53.3% and rather high rate (32%) of preterm or abortion were identified and those pregnancy outcomes were correlated with the stage of IUAs. Complications of surgery consisted of uterine or bladder perforation, pulmonary edema and hemorrhage.

Peritoneal Fluid levels of various Cytokines in Patients with Ovarian Lesions / 대한산부인과학회잡지

OBJECTIVE: Recent studies have shown that multifunctional cytokines have been implicated in the pathogenesis and pathophysiology of various ovarian lesions including ovarian cancer and endometriosis. This study was performed to determine the possibility of the clinical usefulness of peritoneal cytokines in patients with various ovarian lesions. METHODS: Peritoneal fluid was obtained from patients with benign cystic adenoma(n=11), benign cystic teratoma(n=10), endometriosis(n=23), malignant ovarian tumor(n=11), and women without evidence of any pathology(n=7) at the time of laparotomy or operative laparoscopic surgery and were examined for the levels of IFN-gamma, IL-1beta, IL-5, IL-6, IL-10 and TNF-alpha using enzyme-linked immunosorbent assay(ELISA). Statistical analysis was performed with the one way ANOVA, Scheffe test or Mann-Whitney test. RESULTS: The mean concentration of IL-6 and IL-10 was significantly higher in peritoneal fluid specimens with ovarian cancer than other benign pathology(p<0.05). Both IL-6 and IL-10 levels in peritoneal fluid specimens with endometriosis tended to be higher than normal, and the levels of IL-6 and IL-10 were significantly higher in peritoneal fluid of women with severe endometriosis compaired to women with mild endometriosis (p<0.05). CONCLUSION: This study supports the concept that regional immunologic dysfunction might be involved in the disease process of various ovarian pathology such as ovarian cancer and endometriosis. A larger study would help in evaluating the potential use of local peritoneal cytokines in differentiating ovarian cancer from other benign pathology, and demonstrating any association between concentrations of cytokines and severity of endometriosis.

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

No abstract available.

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.

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.

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.

Patterns of Cytokine mRNA Expression of Various Endometriosis Lesions / 대한산부인과학회잡지

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.