• Clinical and Experimental Reproductive Medicine
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• v.42 no.2
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• pp.62-66
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• 2015

Objective: To evaluate the effect of a gonadotropin-releasing hormone (GnRH) antagonist protocol using corifollitropin alfa in women undergoing assisted reproduction. Methods: Six hundred and eighty-six in vitro fertilization-embryo transfer (IVF)/ intracytoplasmic sperm injection (ICSI) cycles were analyzed. In 113 cycles, folliculogenesis was induced with corifollitropin alfa and recombinant follicle stimulating hormone (rFSH), and premature luteinizing hormone (LH) surges were prevented with a GnRH antagonist. In the control group (573 cycles), premature LH surges were prevented with GnRH agonist injection from the midluteal phase of the preceding cycle, and ovarian stimulation was started with rFSH. The treatment duration, quality of oocytes and embryos, number of embryo transfer (ET) cancelled cycles, risk of ovarian hyperstimulation syndrome (OHSS), and the chemical pregnancy rate were evaluated in the two ovarian stimulation protocols. Results: There were no significant differences in age and infertility factors between treatment groups. The treatment duration was shorter in the corifollitropin alfa group than in the control group. Although not statistically significant, the mean numbers of matured (86.8% vs. 85.1%) and fertilized oocytes (84.2% vs. 83.1%), good embryos (62.4% vs. 60.3%), and chemical pregnancy rates (47.2% vs. 46.8%) were slightly higher in the corifollitropin alfa group than in the control group. In contrast, rates of ET cancelled cycles and the OHSS risk were slightly lower in the corifollitropin alfa group (6.2% and 2.7%) than in the control group (8.2% and 3.5%), although these differences were also not statistically significant. Conclusion: Although no significant differences were observed, the use of corifollitropin alfa seems to offer some advantages to patients because of its short treatment duration, safety, lower ET cancellation rate and reduced risk of OHSS.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.289-292
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• 2009

Mongolia has 80% livestock of total agriculture industry, 170,000 farms are engaged, 2,500,000 of cows that were beef and dairy cows are raised. Despite of Mongolian has great application with milk, there are not clear differences between cow and dairy cattle, and the production of milk is also low. But the milk suppliers are varied (horse, sheep, goat, etc), so that the total milk production is 500 thousand ton per year. It's really considerable to improve the breed of owing to many problems with big differences among milk qualities. For carrying out for first year project, artificial insemination project was operated with 3rd grade Holstein semen that were imported from S. Korea, and initiation and field training were also carried out through appropriate AI technique we developed for Mongolia environment. Local information research and MOU conclusion were done with professor D. Altangerel in May $10^{th}{\sim}13^{th}$, 2009, and development for AI technique and AI equipments were supplied for Mongolia breeding and natural environment in July $10^{th}{\sim}17^{th}$ in 2009. All cows were treated by synchronization for AI. To do this, $PGF_{2\alpha}$ injection were treated for luteal phase cow, if it wouldn't work, try again after 11 days. After confirmation of estrus, AI and AI training were carried out with sperm injection in the uterus or cervix by rectum-vagina method which is common worldwide, the most effective artificial insemination technique. If cows were return to next estrus cycle, second AI was carried out about approximately 21 days after artificial insemination. After 2 months, all cows not showing return estrus should be taken pregnancy test. Every pregnant cow will be cared thoroughly. Total 48 cows administrated by $PGF_{2\alpha}$ for synchronization and after 48 hours 45 cows (93.8%) showing estrus were detected and then artificial inseminate them within who 8 cows (27.8%) showed return estrus. Therefore, Using $PGF_2{\alpha}$ for synchronization is effective to use for Mongolia breeding conditions. There are possibility of base for food production after all, including increase of livestock production in Mongolia by improvement of breeding cow with AI and embryo transfer project.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.167-176
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• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.89-96
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• 1999

Some of the information concerning sexual function in the male diabetes has been focused upon the problems of endocrine or semen parameters. However, the characteristics of acrosome reaction and spermatozoa concentration at the epididymis and vas deferens have scarcely been studied, and the causes of the infertility has not been critically identified. So, we designed to inspect the spermatozoa concentration and the characteristics of acrosome reaction at epididymis and vas deferens of diabetic Wistar rat induced by streptozotocin (STZ, 70 mg/kg, ip). Experimental animal was sacrificed at 3 days and 14 days after the STZ injection. In the diabetes-induced rat, the levels of insulin and glucose had a pattern of inverse proportion. The spermatozoa concentrations in caput and corpus epididymis were significantly decreased in all diabetic condition. In cauda epididymis, however, there was significant decrease in sperm concentration at 14 days onward. In diabetic rat, the spontaneous reaction rate of spermatozoa of cauda and vas deferens were significantly higher than the control group. The ARIC (acrosome reaction to ionophore challenge) value of caudal sperm was 28.7 at control, 22.1 at 3 days, and 8.3 at 14 days. In the present study the spermatozoa concentration was decreased and the spontaneous reaction rate was increased by diabetes. In ARIC-test, it is revealed that the fertility of spermatozoa of 14 days group was lower than control or 3 days group. Diabetes mellitus may be provoke the decreased fertilization rate and subsequent infertility and subsequent infertility.

• Toxicological Research
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• v.21 no.2
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• pp.167-173
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• 2005

Bisphenol A (BPA) is a monomer used in the manufacture of a multitude of chemical products, including epoxy resins and polycarbonate. The objective of this study was to evaluate the effects of BPA administration on reproductive characteristics and blood hematological and chemical values in offspring of pregnant dams treated with BPA. BPA was administrated to pregnant mice by intraperitoneally injection with 0, 0.05, 0.5 and 5.0 mg/kg B.W. for 5 times at 3 days interval on gestation days 1-16. There were no treatment-related effects of BPA on reproductive organ weight in male offsprings at 45 days-of-age, but body weight was the lowest in 5.0mg BPA group when compared to other groups (P<0.05). No differences in semen characteristics (sperm concentration, viability, motility and abnormality) were observed between the control and BPA treatment groups. The WBC, HB, HT, MCV, MCH, MCHC, albumin, BUN and total protein of blood hematological and chemical values in male offsprings were not difference for any treatment groups, but RBC value in BPA groups was significantly increased comparing to the control group (P<0.05). The PLT value was slightly higher in 5.0mg BPA groups than in any other group, but not significantly difference among the experimental groups. In female offsprings, the effects of BPA didn't affect to the body and ovary weight, but the uterus weight in 5.0mg BPA group was slightly heavier than that of control group (P>0.05). No statically significant difference in blood hematological values in female offsprings were observed between the control group and BPA groups, but the concentration of albumin and BUN were significantly higher in 0.5mg BPA group when compared to control and other BPA treatment groups (P<0.05). The histological evaluation of testis and ovary in growing offspring at 45 days-of-age was not difference between the control group and BPA groups, but endometriosis of the uterus in female offspring was dramatically increased in 0.5 and 5.0mg BPA groups. These founding suggest that low concentration of BPA might not have a important role on reproductive ability or blood metabolite in offspring of pregnant dams treated with BPA.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.113-119
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• 2012

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.103-108
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• 1996

Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.225-234
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• 2004

Objectives: To evaluate the efficacy of GnRH antagonist cetrorelix in women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) and to determine changes in serum hormone concentrations during cetrorelix administration. Methods: We performed a clinical trial on 30 patients undergoing COH with highly purified follicular stimulating hormone (HP-FSH) and gonadotropin releasing hormone antagonist (GnRHant), cetrorelix. FSH was administrated from day 2 or 3 of cycle with fixed dose and adjusted according to individual response. 0.25 mg of cetrorelix was injected daily subcutaneously from stimulation day 5 until the day of hCG administration. Daily ultrasound monitoring was performed for growing follicles and serum levels of luteinizing hormone (LH), estradiol ($E_2$) and progesterone were measured daily during cetrorelix administration. Up to 4 embryos were transferred. Results: Mean age of enrolled patients was $32.0{\pm}3.4$ years (mean $\pm$ S.D.). All of 30 patients underwent oocyte pick-up, and embryo transfer was done in 28 patients. The total and mean numbers of received oocytes were 196 and $6.5{\pm}4.7$, the number of fertilized eggs was 111, and the fertilization rate was 56.6%. Total duration of FSH administration was $9.2{\pm}2.2$ days and mean of $24.3{\pm}7.7$ ampules of HP-FSH was administered. Total duration of cetrorelix administration was $5.7{\pm}1.9$ days. Serum LH and progesterone levels were maintained in the range of $1.4{\sim}2.9\;mIU/mL$ and $0.3{\sim}0.6\;ng/mL$, which respectively reflected effective prevention of premature LH surge. Clinical pregnancies were achieved in 9 patients, and overall clinical pregnancy rate was 30.0% per oocyte retrieval, and 32.1% per embryo transfer. Conclusion: GnRH antagonist is safe and convenient for COH for IVF-ET and effective with optimal pregnancy rate.

• Environmental Analysis Health and Toxicology
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• v.15 no.4
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• pp.123-130
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• 2000

2-Bromopropane, important industrial chemical, specially in electronic industry at Yangsan in Korea has been reported to cause amenorrhea for female and azoospermia, oligozoospermia or reduced sperm motility for male. 2-BP was investigated through 21 days of repeated dose in male Sprague-Dawley rats. The dose levels per body weight were 0 (control), 250,500 and 1,000 mg/kg. 2-BP dissolved in vehicle olive oil was injected into the intraperitoneum 6 times per week for 3 weeks, but 1,000 mg/kg dose group was 2 weeks because of serious illness. Male rats showed significant decreases in body weight and right and left testis showed typical weight losses depending on the 2-BP. The number of white blood cell and red blood cell , percentage of monocytes, and hemoglobin decreased significantly in high dose (P< 0.05). Red cell volume distribution width increased significantly in the high dose (P< 0.05). Histopathological findings of testes showed a decrease of spermatogenic cells, exfoliation of spermatid and spermatocyte, vacuolization of Sertoli cells and hyperplasia of Leydig cells. Protein band density between 113,000 dalton ($\beta$-galactosidase) and 53,900 dalton (ovalbumin) has decreased in 250 mg/kg dose group, but it has gradually increased to the higher density in 1,000 mg/kg dose group than in control group.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.228-233
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• 2011

Objective: To investigate the effectiveness of GnRH antagonist multiple-dose protocol (MDP) with oral contraceptive pill (OCP) pretreatment in poor responders undergoing IVF/ICSI, compared with GnRH antagonist MDP without OCP pretreatment and GnRH agonist low-dose long protocol (LP). Methods: A total of 120 poor responders were randomized into three groups according to controlled ovarian stimulation (COS) options; GnRH antagonist MDP after OCP pretreatment (group 1), GnRH antagonist MDP without OCP pretreatment (group 2) or GnRH agonist luteal low-dose LP without OCP pretreatment (group 3). Patients allocated in group 1 were pretreated with OCP for 21days in the cycle preceding COS, and ovarian stimulation using recombinant human FSH (rhFSH) was started 5 days after discontinuation of OCP. Results: There were no differences in patients' characteristics among three groups. Total dose and days of rhFSH used for COS were significantly higher in group 3 than in group 1 or 2. The numbers of mature oocytes, fertilized oocytes and grade I, II embryos were significantly lower in group 2 than in group 1 or 3. There were no significant differences in the clinical pregnancy rate and implantation rate among three groups. Conclusion: GnRH antagonist MDP with OCP pretreatment is at least as effective as GnRH agonist low-dose LP in poor responders and can benefit the poor responders by reducing the amount and duration of FSH required for follicular maturation.