• Clinical and Experimental Reproductive Medicine
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• 제46권4호
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• pp.178-188
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• 2019

Objective: To determine the clinical pregnancy (CP) and live birth (LB) rates arising from frozen embryo transfers (FETs) that had been generated under the influence of in vitro fertilization (IVF) adjuvants given to women categorized as poor-prognosis. Methods: A registered, single-center, retrospective study. A total of 1,119 patients with first FETs cycle include 310 patients with poor prognosis (109 treated with growth hormone [GH], (+)GH group vs. 201 treated with dehydroepiandrosterone, (-)GH group) and 809 patients with good prognosis (as control, (-)Adj (Good) group). Results: The poor-prognosis women were significantly older, with a lower ovarian reserve than the (-)Adj (Good) group, and demonstrated lower chances of CP (p< 0.005) and LB (p< 0.005). After adjusting for confounders, the chances of both CP and LB in the (+)GH group were not significantly different from those in the (-)Adj (Good) group, indicating that the poor-prognosis patients given GH had similar outcomes to those with a good prognosis. Furthermore, the likelihood of LB was significantly higher for poor-prognosis women given GH than for those who did not receive GH (p< 0.028). This was further confirmed in age-matched analyses. Conclusion: The embryos cryopreserved from fresh IVF cycles in which adjuvant GH had been administered to women classified as poor-prognosis showed a significant 2.7-fold higher LB rate in subsequent FET cycles than a matched poor-prognosis group. The women with a poor prognosis who were treated with GH had LB outcomes equivalent to those with a good prognosis. We therefore postulate that GH improves some aspect of oocyte quality that confers improved competency for implantation.

• Perinatology
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• 제29권4호
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• pp.180-184
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• 2018

In assisted reproductive techniques, monozygotic twinning is not influenced by the trial of reducing the number of embryos transferred. We present extremely rare case of monochorionic triamniotic triplet pregnancy following in vitro fertilization and two-embryo transfer. During the first trimester, the crucial ultrasound findings of monochorionic triamniotic triplet pregnancy are three distinct amniotic sacs forming a triangle or ipsilon zone within a single chorionic cavity. After prudential counseling about the potential risk of a monochorionic triamniotic triplet pregnancy with the patient, selective reduction of two fetuses via radiofrequency ablation was performed. We report a case of selective reduction for monochorionic triamniotic triplet pregnancy using radiofrequency ablation at 12 weeks' gestation and obstetric progression after radiofrequency ablation was uneventful until 20 weeks' gestation.

• 한국수정란이식학회지
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• 제10권1호
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• pp.45-51
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• 1995

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 $\mu$g /nl of FSH, 10 $\mu$g /ml of LH and 1 $\mu$g /ml of estradiol-17$\beta$ at 39t in a 5% $CO_2$ incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1$\mu$rn) to 23 hours(58.4$\mu$m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

• Clinical and Experimental Reproductive Medicine
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• 제42권4호
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• pp.163-168
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• 2015

Objective: To investigate the impact of fibroids on the blood flow of the uterine and subendometrial arteries and in vitro fertilization (IVF) outcomes. Methods: In this study, we analyzed 86 IVF/intracytoplasmic sperm injection (ICSI) cycles in which a gonadotropin-releasing hormone antagonist protocol was used for controlled ovarian stimulation between January 2008 and March 2009. The subjects comprised 86 infertile women with (fibroid group, n=43) or without (control group, n=43) uterine fibroids. Results: Patient characteristics were similar between the fibroid and control groups. The IVF/ICSI outcomes in patients with fibroids were similar to those of patients in the control group. The resistance index (RI) and pulsatile index (PI) of the uterine and subendometrial arteries on the day of embryo transfer were also comparable between the two groups. IVF outcomes and uterine hemodynamics in patients with multiple (${\geq}2$) fibroids were similar to those of patients with a single fibroid. However, clinical pregnancy and implantation rates were significantly lower in patients with fibroids who experienced uterine cavity distortion than in patients with fibroids who had a normal uterine cavity (both p<0.05). The RI and PI of the subendometrial artery were significantly higher on the day of embryo transfer in patients with fibroids who experienced uterine cavity distortion than in patients with fibroids who had a normal uterine cavity (both p<0.05). Conclusion: Fibroids which distorting the uterine cavity might impair the subendometrial artery blood flow clinical pregnancy rate and embryo implantation rate in infertile patients undergoing IVF. Otherwise, IVF outcomes were not influenced by the presence of uterine fibroids.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.175-181
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• 2008

This study was conducted to investigate an effective recipient oocyte and culture system for producing of Hanwoo (Korean native cattle) somatic cell nuclear transfer (SCNT) embryos. Hanwoo ear skin fibroblasts were used as donor cells. In vitro matured Hanwoo or Holstein oocytes were enucleated, and single donor cells were transferred into the perivitelline space of the enucleated oocytes. The couplets were subsequently fused and activated. The reconstructed embryos were cultured in a conventional or sequential culture system. In the former, embryos were cultured in CR2aa medium for eight days; in the latter, embryos were cultured in modified CR2aa-A (mCR2-A) for three days and then further cultured in modified CR2aa-B (mCR2-B) for five days. In the experiment with the recipient oocyte, the rate of embryo development to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein ones (48.8% vs 38.9%). BIastocysts derived from Hanwoo recipient oocytes contained significantly (p<0.05) higher numbers of total cells than those derived from Holstein recipient oocytes ($156.0{\pm}68.2$ vs $134.7{\pm}54.8$). There was no difference in the mean proportion of apoptotic cells in blastocysts between the sources of recipient oocytes. In the experiment with the embryo culture system, the blastocyst rate was somewhat higher in sequential system than in conventional system (50.0% vs 43.5%), though there was no significant difference. The numbers of total ($160.0{\pm}69.0$ vs $156.7{\pm}68.4$) and apoptotic cells ($14.0{\pm}10.4$ vs $11.8{\pm}6.4$) were not different between the culture systems. In conclusion, the present study demonstrated that Hanwoo recipient oocytes and the sequential culture system were more effective in supporting the production of Hanwoo SCNT embryos.

• 한국수정란이식학회지
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• 제31권3호
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• pp.235-242
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• 2016

This study tested the association between genotypes of the single nucleotide polymorphism (SNP) marker, rs81437607 and capric acid (FA_C10_0) compositions in longissimus dorsi muscle in pigs. Eighteen fatty acid (FA) compositions were measured in a total of 974 $F_2$ animals among 1,106 $F_2$ progeny produced between Landrace and Jeju Black Pig (JBP). Among FA compositions tested, we identified a cluster of highly significant SNPs for capric acid compositions on 58 Mb position of Sus scrofa chromosome 12 (SSC12) using genome-wide association study (GWAS) with $F_2$ genotypes from SNP panel analysis. GWAS results showed that the rs81437607 was the highest trait-related SNP marker with capric acid levels. Three genotypes (C/C, C/T and T/T) of rs81437607 marker were found in $F_2$ population by further pyrosequencing. Association analysis results showed the significant differences between rs81437607 genotypes and capric acid compositions (P<0.05). The $F_2$ pigs harboring rs81437607 C/C ($0.119{\pm}0.002%$) and C/T ($0.116{\pm}0.002%$) genotypes showed additively higher levels of capric acid content than those of T/T homozygotes ($0.109{\pm}0.002%$) ($P=1.30{\times}10^{-12}$). These results suggested that the genetic variations of rs81437607 may be helpful to find causative variants and assist as molecular genetic markers for improving the capric acid contents in longissimus dorsi muscle in pigs.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.97-97
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• 2002

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2${\times}$10$\^$6//${\mu}\ell$. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6${\times}$10$\^$6//${\mu}\ell$. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.

• 한국수정란이식학회지
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• 제10권2호
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• 한국수정란이식학회지
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• 제22권1호
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• pp.47-51
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• 2007

더러브렛종 암말의 번식 능력을 향상시키기 위해 호르몬 투여 및 인공 조명에 의한 인위적으로 발정 조절을 실시한 결과는 다음과 같다. 발정 주기에 있는 암말의 $PGF_2{\alpha}$ 또는 altrenogst투여에 의한 발정 발현율은 100%이었으며, 수태율은 각각 95.2% 및 71.4%였다. 전년도 공태마에 대하여 altrenogest단독 또는 altrenogest과 estradiol 병행 투여시 3월 이내 100% 발정이 발현되었으며, 발정 발현일은 altrenogest 투여 종료 후 단독 투여군이 4.3일, estradiol 병행 투여군이 3.7일이었고, 배란일은 발정 발견 후 각각 2.7일과 2.5일이었고, 처리군간의 유의적인 차이는 없었다. 그리고 수태율은 altrenogest 단독 투여군이 80.0%를 나타내었다. 인공 조명에 의한 발정 발현율은 92.9%였고 수태율은 76.9%였다. 이상의 결과로 보아 번식 계절에 있는 암말에 대한 발정 동기화는 수태율을 높일 수 있는 수단으로 활용될 수 있고, 전년도 비임신으로 공태중인 암말에 altrenogest 투여는 경주마의 조기 번식 및 수태율 향상에 기여할 수 있을 것으로 사료 된다.

• 한국수정란이식학회지
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• 제27권1호
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• pp.9-14
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• 2012

This study was conducted to analyze the transgenic efficiency and sex ratio in ${\alpha}$-1,3-galactosyltransferase (GalT) knock-out (KO) transgenic pigs according to generation. GalT KO piglets were produced by artificial insemination or natural mating. The transgenic confirmation of GalT KO was evaluated by PCR amplification using specific primers. After electrophoresis, three types of bands were detected such as 2.3 kb single band (Wild), 2.3 and 3.6kb double bands (GalT KO -/+; heterozygote), and 3.6kb single band (GalT KO -/-; homozygote). Transgenic efficiency in F1 generation was 64.5% (23/35) of GalT KO (-/+). In F2 generation, GalT KO transgenic efficiency was 36.4% (21/57, Wild), 47.5% (28/57, GalT KO -/+), and 16.1% (8/57, GalT KO -/-), respectively. Interestingly, no homozygote piglets were born in 6 deliveries among total 11 deliveries, although they were pregnant between male (M) and female (F) $F_1$ heterozygote. In the 5 litters including at least one GalT KO -/- piglet, the transgenic efficiency was 13.3% (2/24, Wild), 51.3% (14/24, GalT KO -/+), and 35.3% (8/24, GalT KO -/-), respectively. The sex ratio of M and F was 40:60 in $F_1$ and 49:51 in $F_2$ generation, respectively. Based on these results, GalT KO transgenic pigs have had a reproductive ability with a normal range of transgenic efficiency and sex ratio.