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.

Expression of peroxiredoxin I regulated by gonadotropins in the rat ovary / 대한생식의학회지

OBJECTIVE: Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. This report examined the expression of Prx isotype I in the rat ovary after hormone treatment. METHODS: Immature rats were injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) to induce the growth of multiple preovulatory follicles and 10 IU of human chorionic gonadotropin (hCG) to induce ovulation. Immature rats were also treated with diethylstilbestrol (DES), an estrogen analogue, to induce the growth of multiple immature follicles. Northern blot analysis was performed to detect gene expression. Cell-type specific localization of Prx I mRNA were detected by in situ hybridization analysis. RESULTS: During follicle development, ovarian Prx I gene expression was detected in 3-day-old rats and had increased in 21-day-old rats. The levels of Prx I mRNA slightly declined one to two days following treatment with DES. A gradual increase in Prx I gene expression was observed in ovaries obtained from PMSG-treated immature rats. Furthermore, hCG treatment of PMSG-primed rats resulted in a gradual stimulation of Prx I mRNA levels by 24 hours (2.1-fold increase) following treatment, which remained high until 72 hours following treatment. In situ hybridization analysis revealed the expression of the Prx I gene in the granulosa cells of PMSG-primed ovaries and in the corpora lutea of ovaries stimulated with hCG for 72 hours. CONCLUSION: These results demonstrate the gonadotropin and granulosa cell-specific stimulation of Prx I gene expression, suggesting its role as a local regulator of follicle development.

Clinical study of complications in the surgical management of stress urinary incontinence using Transobturator tape (TOT) / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to evaluate the complications of Transobturator tape (TOT) in the surgical treatment for stress urinary incontinence and their management. METHODS: From March 2005 to October 2007, 206 patients diagnosed with stress urinary incontinence were operated using TOT at Chonnam National University Hospital. We reviewed medical records and analyzed the data according to age, parity, menopausal state, concomitant operations and complications. RESULTS: Mean age of the patients was 52.6+/-10.5 years and mean parity was 2.9+/-1.4. 91 patients (44.2%) were in postmenopausal state and 201 patients (97.6%) had other concomitant gynecologic operations. There were no intraoperative complications such as vaginal injury or bladder perforation. Postoperatively, there were 2 cases (1.0%) of vulva hematoma, 6 cases (2.9%) of urinary retention, 4 cases (1.9%) of de novo urgency and 4 cases (1.9%) of vaginal erosion. CONCLUSION: The surgical treatment using TOT is thought to be safe and effective means for the management of stress urinary incontinence. Although rare, complications may occur, therefore surgeons must be aware of the management of each complications.

Black Cohosh and St. John's Wort (GYNO-Plus(R)) for Climacteric Symptoms

PURPOSE: This study was conducted to investigate the efficacy of black cohosh (Cimicifuga racemosa) and St. John's wort (Hypericum perforatum) in women with climacteric symptoms, and to assess their effects on vaginal atrophy, hormone levels, and lipid profiles. MATERIALS AND METHODS: In this double-blind randomized, placebo-controlled, multicenter study, 89 peri- or postmenopausal women experiencing climacteric symptoms were treated with St. John's wort and black cohosh extract (Gynoplus

Control Mechanisms of Ovarian Follicle Development by Follicle Stimulating Hormone and Pituitary Adenylate Cyclase-activating Polypeptide / 대한불임학회지

OBJECTIVE: Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel hypothalamic neuropeptide, has been suggested to play a role in ovarian folliculogenesis. The present study evaluated the effect of PACAP on the growth of preantral follicles. METHODS: Preantral follicles were mechanically isolated from ovaries of 21-day-old rats and cultured in groups for 3 days in serum-free medium in the absence or presence of PACAP-38 (10-6 M). RESULTS: Treatment with PACAP-38 resulted in an increase in follicle diameter by 75% whereas treatment with follicle stimulating hormone (FSH) increased follicle diameter by 65%. PACAP-38 treatment enhanced the granulosa cell proliferation as measured by thymidine incorporation analysis. Furthermore, the production of progesterone by cultured granulosa cells and GFSHR-17 cell line was stimulated by PACAP-38. Interestingly, PACAP enhanced FSH action on stimulation of SF-1 and aromatase gene expression. CONCLUSION: The present results demonstrate that PACAP stimulated preantral follicle growth by potentiating proliferation and by stimulating steroidogenesis.

Effect of low dose estrogen menopausal hormone therapy on nitric oxide and antioxidant activity / 대한산부인과학회지

OBJECTIVE: To evaluate the effects of low dose estrogen menopausal hormone therapy on cardiovascular system METHODS: This study categorized 95 postmenopausal women between March 2004 and August 2004. Thirty patients of estrogen therapy group, fifteen patients of estrogen-progestin therapy group, fifteen patients of low-dose estrogen therapy group, and fifteen patients of low-dose estrogen-progestin therapy group were divided. Remaining 20 patients served as control group which did not receive the hormone treatment. The blood pressure, pulse rate, lipid profile, and NO metabolites and antioxidant activity of plasma and urine were measured. RESULTS: Diastolic blood pressure was lower in hormone treatment group than control group's. Although it was not significant, the total cholestrol and LDL-cholestrol in the plasma of treatment group were lower than those of the control group while HDL-cholestrol were higher. Triglyceride in the plasma of treatment group was higher. Changes of blood pressure, pulse rate and lipid profile in low-dose group were similar to those of conventional standard dose. The plasma concentration of NO metabolites in treatment group was higher. Also, the plasma concentration of NO metabolites in low-dose group was similar to that of conventional dose. CONCLUSION: A low-dose hormone therapy was expected to bring about the improvement of endothelial cell dependent vascular reactivity like conventional dose, resulting in the reduction of diastolic blood pressure, the improvement of lipid profile, and an increase in plasma concentration of the NO metabolites. A low-dose hormone therapy may thus presumably provide beneficial effects on cardiovascular system.

Control Mechanisms of Ovulation by Pituitary Adenylate Cyclase-Activating Polypeptide / 대한불임학회지

No abstract available.

Vitrification of Mouse Embryos in Ethylene Glycol-based Solutions / 대한불임학회지

OBJECTIVE: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. MATERIALS AND METHODS: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. RESULTS: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. CONCLUSION: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

Vitrification of Mouse Blastocyst / 대한산부인과학회잡지

OBJECTIVE: The aim of this study was to compare the survival and developmental rate of two vitrification solutions for the vitrification of mouse expanded blastocysts. METHODS: Mouse embryos were obtained at 2 cell stage and cultured to expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% serum substitute supplement (SSS). The vitrification solutions used were EFS40 and VS. EFS40 consisted of 40% ethylene glycol, 18% ficoll, and 0.5 M sucrose while VS consisted of 20% ethylene glycol, 20% DMSO, and 10% 1,3-butanediol diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% calf serum (CS). Toxicity was tested by exposing expanded blastocysts to vitrification solution. The vitrification procedure used for EFS40 was performed in three steps, after which they were warmed in 5 steps with 0.5 M sucrose. VS was performed in two steps, after which they were warmed with 1.0 M trehalose. Recovery, survival and hatching rate per expanded blastocysts recovered were compared between two groups. RESULTS: In toxicity test, survival and hatching rate of EFS40 group were 95% and 100%, respectively. In contrast, survival and hatching rate of VS group were 100% and 87.5%, respectively. After vitrification and warming in solution, recovery rate for EFS40 group was 73.7% whereas recovery rate for VS group was 66.5%. After 24 h culture, survival and hatching rate were 80.5% and 20.7% for EFS40 and 66% and 0% for VS group, respectively. After 48 h culture, survival and hatching rate were 69% and 33.3% for EFS40 and 58.3% and 1.9% for VS group, respectively. Survival and hatching rate in EFS40 group were significantly higher than those found in VS group. CONCLUSION: The EFS40 solution was better than VS solution for vitrification of mouse expanded blastocysts.

Expression of Apoptotic Genes in Mouse Preimplantation Embryo Development / 대한불임학회지

OBJECTIVES: The aim of this study was to evaluate the influence of three different media on preimplatation embryo development and the expression of Bcl-2, Mcl-1, Bax, and Bok in mouse. MATERiALS AND METHODS: Two-cell embryos were retrieved from iCR female mice (4 weeks old) at 48 hr after hCG injection and cultured in Ham's F-10, HTF, and G1.2 media. The developmental rate of 2-cell embryos was evaluated from 24 hr to 72 hr after culture. RT-PCR was performed for the detection of Bcl-2, Mcl-1, Bax, and Bok gene expression. RESULTS: The rates of morula and blastocyst in HTF and G1.2 media (88%, 98.1%) were significantly higher than those in Ham's F-10 media (39.6%) at 48 hr. Likewise, the rates of hatching and hatched blastocyst in HTF and G1.2 media (21.9%, 52.9%) were higher than those in Ham's F-10 media (3.5%) at 72 hr. Bcl-2 and Bax mRNAs were highly detected in embryos cultured in Ham's F-10 when compared in embryos cultured in HTF and G1.2. in contrast, the expression of Mcl-1 and Bok was not significantly different. CONCLUSION: These results show that HTF and G1.2 culture media increase the rate of blastocyst formation and stimulate Bcl-2 and Bax gene expression in mouse preimplantation embryos.

Expression of Proapoptotic Bcl-2 Family Member in the Mouse Ovary (i) / 대한불임학회지

OBJECTIVES: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. METHODS: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at 21~23 days of age. Ovaries were collected 1~3 days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for 1~2 days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 iU PMSG. Some mice received a single intraperitoneal injection of 10 iU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. RESULTS: Treatment of immature mice with diethylstilbestrol (DES) for 24~48 h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for 24~48 h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at 6~9 h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. CONCLUSION: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.

The Effect of Periovulatory Progesterone Supplementation in In Vitro Fertilization and Embryo Transfer Program / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to investigate whether high dose progesterone intramuscular injections before oocyte retrieval and thereafter increase the implantation and pregnancy rates through improvement of uterine receptivity in patients undergoing in vitro fertilization-embryo transfer (IVF-ET). METHODS: The retrospective randomized analysis was performed in whom undergoing conventional IVF- ET at Chonnam National University Hospital Infertility Clinic from August, 1996 to July, 2001. The study group consisted of 57 patients having intramuscular progesterone injections for 4-5 days from the day of hCG injection to the day of embryo transfer and 60 patients without progesterone supplement (control group). We compared between two groups with respect to age distribution, cause of infertility, blood levels of hormone, number of aspirated ovum, number of fertilized egg, number of cleaved embryo, number of transfered embryo, embryo transplantation, cumulative embryo score, chemical and clinical pregnancy rates. RESULTS: The oocytes retrievals were done at 87 cycles in study group and 82 cycles in control group. There were no significant differences in the average age and distribution of causes of infertility. Tubal factor was the dominant cause of infertility in both groups. There were no significant differences in the number of aspirated eggs, number of fertilized eggs, cleavage rates and number of multinuclear fertilized eggs. The embryo transfer were performed 76 out of 87 cycles in study group, and 64 out of 82 cycles in control group. The average number of transferred embryos to the uterine cavity was not different, in the study and control group (2.72+/-1.64 and 2.39+/-2.03 respectively). The chemical pregnancy rate did not differ significantly (7.89% in study group, and 6.25% in control group). The clinical pregnancy rate was higher in the control group (18.75%) than in the study group (12.84%), but the result was not statistically significant. However, the number of fertilized eggs and cumulative embryo score were significantly higher in study group. CONCLUSION: High dose of progesterone supplementation before and after oocyte retrieval in IVF-ET cycles did not improve pregnancy outcome, instead showed lower pregnancy rate than no supplement group, thus we cannot consider progesterone supplementation improve endometrial receptivity and increase implantation and pregnancy rate. But, since we could improve the fertilization rate and embryo development rate through increase of the number of fertilized eggs and cumulative embryo score, further evaluation is needed in this field and we have to make vigorous efforts to increase implantation rate in IVF-ET cycles.

Expression of Apoptosis Gene Bok in the Rat Ovary / 대한산부인과학회잡지

OBJECTIVE: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the rat ovary during the follicle development. METHODS: We used the female rats of SD strain. Bok mRNA levels in the ovary were determined by Northern blot analysis. In situ hybridization were performed to determine the specific ovarian cell type expressing Bok mRNA. RESULTS: Northern blot analysis of ovaries obtained from immature rats revealed increasing levels of Bok mRNA during postnatal development. The major cell types expressing Bok mRNA were the granulosa cells of preantral and atretic follicles. Treatment of immature rats with diethylstilbestrol (DES) for 24-48 h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in rats removed DES for 24- 48 h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature rats with pregnant mare's serum gonadotropin (PMSG) for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. Interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSG-primed rats with human chorionic gonadotropin (hCG) stimulated strongly ovarian Bok mRNA expression at 6-9 h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. In adult estrus cyclic ovaries, Bok gene expression was higher on proestrus and estrus than metaestrus and diestrus. Moreover, the highly increased expression of Bok mRNA was found in rat ovaries at 48 h after hypophysectomy. CONCLUSION: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.

A Study Comparing In Vitro Fertilization Results After Day 2 and Day 3 Embryo Transfer / 대한산부인과학회잡지

OBJECTIVE: To compare the clinical outcomes of day 2 embryo tranfer with those of day 3 embryo transfer after In Vitro Fertilization (IVF). METHODS: Medical records of 192 patients (217 cases) undergoing IVF-ET at Chonnam National University Hospital from February 1995 to July 2001 were reviewed. The number of cases of the day 2 transfer (D-2) group and the day 3 transfer (D-3) group was 152 and 65, respectively. Clinical characteristics, mean number of fertilized oocytes, frequency of embyo qualities, mean number of embyo transferred, cumulative embryo score (CES) per transfer, pregnancy rate per cycle, clinical pregnancy rate per cycle and implantation rate per transferred embyo were compared between the two groups. RESULTS: In embyo quality, grade I in D-2 group (69.5%) was much more than in D-3 group (52.3%) in number, and grade III and IV in D-3 group (9.7% and 6.2%) was much more than in D-2 group (1.0% and 0.2%). CES was significantly higher in D-3 group than in D-2 group (62.4+/-40.0 versus 49.1+/-34.2). However, there were no statistically significant differences between the two groups in number of fertilized oocytes, number of embryo transferred, pregnancy rate, implantation rate and pregnancy outcome. There were also no significant differences between the two groups in implantation and pregnancy rates according to age, under 35 vs over 35 and the presence or absence of previous IVF-ET failure. CONCLUSION: There were no difference in the pregnancy rate and implantation rate between the two groups. The CES which indicates the quality of embryo was higer in D-3 group than in D-2 group. Since this study did not show the direct effect of better quality of D-3 embryo on implantation rate and pregnancy rate, further investigation should be needed. In addition, further studies about improving culture media for embryonic development and selecting better quality embryos for delayed embryo transfer should be continued.

Regulation of Steroid Thyroid Hormone Receptor 3 (TR3) mRNA Expression by Luteinizing Hormone in Human Early Luteinized Granulosa Cells / 대한산부인과학회잡지

OBJECTIVE: The present study examined the gonadotropin regulation of TR3 gene expression by luteinizing hormone (LH) in cultured human luteinized granulosa cells. METHODS: TR3 mRNA levels were detected by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) method in cultured human luteinized granulosa cells collected from patients undergoing in vitro fertilization. RESULTS: TR3 transcript was transiently induced by LH, reaching maximum levels 1 hr after stimulation, in a dose-dependent manner. LH-stimulated TR3 expression was abolished by actinomycin D, but was superinduced by cycloheximide. Treatment of luteinized granulosa cells with Rp-cAMP, an inhibitor of protein kinase A, as well as, chelerythrin, an inhibitor of protein kinase C, suppressed LH-stimulated TR3 mRNA levels. In addition, forskolin and TPA mimicked the LH action on the induction of TR3 gene, implying the role of protein kinase A and C activation. CONCLUSION: Taken together, the present study demonstrates that TR3 gene was rapidly and transiently induced by LH in human luteinized granulosa cells. The results imply that TR3 may play a role in ovulation by initiating a cascade of ovulation-specific gene expression in response to LH.

Stimulation by EGF, bFGF and GnRH of Ovarian Pituitary Adenylate Cyclase-Activating Polypeptide Gene Expression in Cultured Rat Preovulatory Follicles / 대한불임학회지

No abstract available.

The Experimental Study on Cryopreservation of Mouse Embryo / 대한불임학회지

OBJECTIVES: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). MATERIALS AND METHODS: Two to eight cell embryos were obtained from oviducts of mated F1 hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow- cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. RESULTS: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1%, 79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. CONCLUSIONS: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

Comparison of Intracytoplasmic Sperm Injection and Partial Zona Dissection followed by Insemination in Hamster Oocytes / 대한불임학회지

OBJECTIVES: This study was to investigate the fertilization rate after intracytoplasmic sperm injection (ICSI) or partial zona dissection (PZD) of human and hamster sperm into hamster oocyte in in vitro fertilization (IVF). In addition, the possibility of clinical application was evaluated by the comparison of usefulness and difference of these method. MATERIALS AND METHODS: Hamster immature oocytes were obtained from oviducts superovulated by PMSG and hCG, and hamster sperms were obtained from epididymis. The freezed human sperms were thawed before use. Fertilization were confirmed by two pronuclei, one pronucleus, swollen sperm head or/and two polar bodies at 7~8 hour after ICSI or PZD. RESULTS: The fertilization rates after ICSI and PZD of human sperm to hamster oocyte were 3.6%, 64.2%, 73.6%, and 55.6% for negative control, positive control, ICSI, and PZD respectively, suggesting that ICSI only showed improved fertilization rate (p<0.01). The fertilization rates after ICSI and PZD of hamster sperm to hamster oocyte were 11.1%, 51.2%, 39.6%, and 72.7% for negative control, positive control, ICSI, and PZD respectively, suggesting that PZD only showed improved fertilization rate (p<0.01). PZD showed significantly higher fertilization rate than ICSI (p<0.05). CONCLUSIONS: As for the fertilization rate by ICSI and PZD using hamster oocyte in IVF, ICSI technique was considered to be more useful for human sperm and PZD technique for hamster sperm. Therefore, ICSI technique was considered more appropriate for experimental application using human sperm and hamster oocyte.

Control Mechanisms of Follicle Rupture during Ovulation in Mammals / 대한내분비학회지

No abstract available.

Electron microscopy of the oocyte-cumulus complex and immuncytochemistry on the distribution of fibronectin, tenascin, and laminin / 대한산부인과학회잡지

OBJECTIVE: Immunofluorescence microscopy including confocal laser scanning microscopy and electron microscopy were used to study the production of fibronectin, tenascin, and laminin in the cumulus-corona (CC) cells surrounding mature, unfertilized oocytes after ovulation in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the complex biochemical mechanism between the ovum and the oviduct. METHODS: Mature oocyte-cumulus complex (OCC) was cultured for 24 and 48 hour and fixed in 3.7% formaldehyde. Specimens were incubated with a mixture of primary monoclonal antibodies recognizing different epitopes of fibronectin, tenascin, and laminin, and then with a mixture of secondary antibodies containing FITC, TRITC, and Cy-5 conjugated antibodies. Observation was made by confocal laser scanning microscope equipped with epifluorescece optics. Transmission electron microscopy were used to observe the OCC at 24 and 48 hours after cultrue. RESULTS: The immunocytochemical date demonstrated that CC masses are capable of producing fibronectin and tenascin but their production is heterogeneous in the CC population. Immunoreactivity to fibronectin and tenascin was shown mostly by inner corona cells, and the intensity of immunofluorescence decreased from the central corona cells to the peripheral cumulus cells. Colocalization of fibronectin and tenascin was evident in most CC cells. Moreover, fibronectin and tenascin immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. Whereas, laminin immunofluorescence was found around plasma membrane and extracellular area, but a intracytoplasmic reaction was rarely observed. The distribution of laminin immunofluorescence was similar to that of fibronectin and tenascin, but in some cumulus cells, colocalization between them was not found. Ultrastructurally, cumulus cells projected numerous long, thin microvilli into the intercellular area and some micovilli penetrated into zona pellucida. The inner layer of the cumulus mass was loose arrangement of relatively uniform, small cells with widened intercellular spaces, whereas in the outer layer, cumulus cells are rather larger in size and compact arrangement by narrow, irregular spaces. A small and large linear gap junctions were easily found at cell contacts. The cytoplasm of most cells had abundant organelles typical of steroidogenesis: numerous mitochondrias, a well-developed smooth endoplasmic reticulum, electron dense lipid droplets, and bundles of microtubules and microfilaments. Rudimentary disrupted basal lamina along the cytoplasmic border was rarely seen in a few inner conora cells. CONCLUSION: Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process. Cumulus cells appears to be a heterogeneous and dynamic system for suitable microenviroment of fertilization. And functional differences between corona and cumulus cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins and steroidogenesis-related organelles.