• Clinical and Experimental Reproductive Medicine
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• v.16 no.1
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• pp.93-101
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• 1989

71 cycles of 67 women were treated for superovulation induction in our IVF & ET program from May to September in 1988. Endogenous LH surges were occurred in 21 cycles out of 71 cycles. And then, we selected 50 cycles without endogenous LH surge treated in the same period as control group. We compared egg recovery rate, egg maturity, fertilization rate, cleavage rate and pregnancy rate of study group with those of control group. We were able to detect more than 90% of endogenous LH surge by commencing daily LH monitoring on MCD 9. The egg recovery rate, egg maturity, fertilization rate, and pregnancy rate of the study group were not statistically different from those of the control group. Significantly lower cleavage rate was seen in the study group compared with that of control group. Above results suggested that the cycles with endogenous LH surge do not have to be abandoned and can be treated continuously to achieve successful pregnancy.

• Clinical and Experimental Reproductive Medicine
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• v.22 no.3
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• pp.273-278
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• 1995

Mammalian, including human, spermatozoa undergo morphological and physiological changes during sperm maturation. There were, these changes may affect the fertilization and embryonic development. In this study, we examined the pronucleus formation, pronucleus disappearance and embryonic development in the human oocytes fertilized by intracytoplasmic sperm injection (ICSI). The injected spermatozoa were grouped into ejaculated, epididymal and testicular by the collecting region. Among 363 metaphase II injected oocytes, 287(79.1%) oocytes were normally fertilized and displayed two pronuclei. There were no difference in the fertilization rates and in the pronucleus formation and pronucleus disappearance at 16, 20 and 24 hr after ICSI, among the each spermatozoa group. Also, at 64 hr, the appearance of embryonic development was similar. From these results it can be concluded that there was no difference of maturity among the sperm collected from ejaculated, epididymis and testis in the pronucleus formation and embryonic development. Therefore, testicular spermatozoa are successfully used with ICSI in IVF-ET program.

• 대한생식의학회:학술대회논문집
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• 1999.11a
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• pp.66-67
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• 1999

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.148-152
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• 2011

Objective: Laser-assisted intracytoplasmic sperm injection (LA-ICSI), also known as micro-opening or thinning of the zona pellucida (ZP) prior to ICSI, may help to reduce mechanical damage to the oocyte during the procedure. The aim of the present study was to evaluate and analyze the efficacy of our institutional LA-ICSI program, which features laser-assisted ZP thinning prior to ICSI, in comparison with conventional ICSI (C-ICSI), performed on patients with different clinical characteristics. Methods: Patients undergoing a total of 212 ICSI cycles were randomly divided into an LA-ICSI group (106 cycles) and a conventional ICSI group (106 cycles). To reduce tissue damage, we thinned the ZP by approximately 70%, using a laser, before ICSI. Patients thus treated formed the LAICSI group. Comparisons included the morphological quality of transferred embryos, blastocyst development of the remaining embryos, and clinical pregnancy, in terms of ICSI method and patient characteristics. Results: Fertilization, development of remaining embryos, and pregnancy rate were significantly higher in the LA-ICSI group compared with the C-ICSI group. Fertilization, embryonic development, and the pregnancy rate were all improved in younger patients (<38 years of age) and in those who underwent a low number of IVF-ET attempts (<3 trials). In addition, the pregnancy rate was increased in older patients. Conclusion: LA-ICSI may be useful in improving the chance of pregnancy in all ICSI patients.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.367-372
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• 2000

Objective: This study was performed to evaluate whether vitrification method could be used for the cryopreservation of human blastocysts derived from IVF program. Methods: Surplus embryos were obtained from consented IVF patients. Controlled ovarian hyperstimulation was done with midluteal GnRH agonist, gonadotropin and hCG. After oocyte retrieval and insemination, fresh embryo transfer was done at $4{\sim}8$ cell stage. The surplus embryos after ET were cultured in blastocyst medium up to 6 days after oocyte retrieval. Obtained blastocysts were cryopreserved with our vitrification method. Blastocysts were exposed to 1.5 Methylene glycol (EG) in phosphate buffered saline (PBS) for 2.5 minutes, followed by 5.5 M EG plus 1 M sucrose for 20 seconds. Then 1 to 3 blastocysts were mounted on electron microscope (EM) grid and the grid was plunged into liquid nitrogen for storage. For thawing, blastocyst-containing EM grids were sequentially transferred in 1.0 M, 0.5 M, 0.25 M, 0.125 M and 0 M sucrose solution at the intervals of2.5 minutes. And blastocysts were cultured for about 6 hours and only re-expanded blastocysts were transferred to uterus of the patients on 4 to 5 days after ovulation in natural cycle or on 18 to 19 day of artificial cycle. Results: From Oct. 1998 to Jul. 1999, 34 patients were agreed to participate in this study. The mean age and duration of infertility of the patients were 31.6 years and 4.1 years, respectively. Among 34 cycles. replacements could be done in 20 cycles (58.8%). A total 93 blastocysts were thawed and 48 (51.6%) of them survived. Thirty-eight blastocysts, mean 1.9 embryos per patient, were transferred, resulting in 5 clinical pregnancies which consisted of 1 triplet, 2 sets of twins and 2 singleton pregnancies. The pregnancy rate per transfer was 25% and implantation rate was 23.6%. Five patients delivered 7 healthy babies including 2 sets of twins at term. Conclusion: Successful pregnancies and deliveries were established after transfer of vitrified human blastocysts. Vitrification using ethylene glycol as cryoprotectant and electron microscope grid is a rapid and simple method that can be effectively applied for the cryopreservation of human blastocysts.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.229-236
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• 2002

Objective : The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. Methods : Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at $37^{circ}C$ for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at $4^{circ}C$ overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. Results: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05). Conclusion: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.

• Development and Reproduction
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• v.7 no.1
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• pp.49-56
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• 2003

There have been reports that administrated high-dose gonadotropin-releasing hormone-agonist(GnRH-Ag) suppresses endogenous gonadotropin production and inhibits function of ovary. In human IVF-ET program, however, GnRH-Ag is employed in large amounts during superovulation induction resulting to luteal phase defects which must be supported with progesterone. To elucidate the reason of luteal phase defects by GnRH-Ag, the aim of this study was to investigate the apoptosis changes in the ovary and the hormonal changes in the serum after GnRH-Ag and PMSG administration in adult mice in a method similar to human superovualtion induction. GnRH-Ag(10 ${\mu}$g) or saline was injected every 12h beginning 48h prior to PMSG injection until 48h at)or PMSG injection when blood sampling and ovary collection was performed. In results, the ovary weight in the GnRH-Ag only injection group was significantly lower when compared with the other two groups, PMSG only or PMSC + GnRH-Ag injection. The ratio of preantral follicles in the ovary are increased in the GnRH-Ag only group, while the ratio of antral follicles are decreased and the corpus luteum ratio is increased in the PMSG + GnRH-Ag group. The proportion of all follicles showing apoptosis in the GnRH-Ag only in.iection group was seen to be more than twice the proportion seen in the PMSC only injection group, and such increased apoptosis is decreased after addition of PMSC. The serum levels of both estradiol and progesterone were significantly lower in the CnRH-hg only group compared to those in the other two groups. When the administration of GnRH-Ag were followed by PMSG in;ection, however, estradiol concentration was completely recovered compared to the serum level of PMSG group, but not progesterone level. In conclusion the use of GnRH-Ag in human IVF-ET program may induce the apoptosis and the suppression of hormone production by ovary leading to luteal phase defects, thus adequate progesterone support seems to be necessary against them.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.211-216
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• 1997

The present study was carried out to evaluate whether the coculture system of human embryos with Vero cells can improve the quality of embryo or overcome the repetitive implantation failures in order to obtain pregnancy. From January to December 1996, a total 202 cases which patients with the problems of repetitive implantation failures (group I) or those with the poor embryonic quality in their previous cycles (group II) was analysed. The quality of cocultured embryo, pregnancy, on-going and implantation rates between coculture and control groups were compared. Of 93 cases in group I, coculture was performed in 34 cases and conventional IVF for the rest. Of 109 cases in group II, 36 for coculture and 73 for conventional IVF. In group I, pregnancy, on-going and implantation rates in coculture group (14/34 (41.2%), 9/34 (26.5%), 16/81 (19.8%), respectively) were higher than those of control (11/59 (18.6%), 8/59 (13.6%), 12/152 (7.9%), respectively). There is significance in the pregnancy and implantation rates (p=0.028 and p=0.015). In group II, pregnancy, on-going and implantation rates in coculture group (8/36 (22.2%), 5/36 (13.9%), 8/87 (9.2%), respectively) were higher than those of control (5/73 (6.8%), 3/73 (4.1%), 3/158 (1.9%), respectively). Like the result of group I, there is significance in the pregnancy and implantation rates (p=0.028 and p=0.022). Coculture system with Vero cells works well in the groups of the two indications. Although the case of 3 day-coculture was small as 15 cases in group II, 3 day-coculture improved pregnancy rate (4/15 (26.7%)). Therefore, 3 day-coculture with assisted hatching is recommended to the patients with poor embryonic quality. In conclusion, coculture system with Vero cells can be suggested as an effective method which improves pregnancy rate in those who have repetitive implantation failures or whose embryonic quality was poor in their previous cycles.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.2
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• pp.111-117
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• 2004

Objectives: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. Materials and Methods: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)$\leq$3ea), Group II (n=80) is normal responder (retrieved oocytes>3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by $\beta$-actin. Statistical analysis was performed by using $X^2$ test, Student's t-test and Pearson correlation. Results: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). Conclusion: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.33-40
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• 2001

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.