• Journal of Embryo Transfer
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• v.30 no.3
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• pp.195-200
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• 2015

The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female $F-{_1}$ mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG ($81.1{\pm}15.1$), 1,2-PROH ($88.0{\pm}21.1$) or the control ($99.9{\pm}21.3$) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO ($14.7{\pm}1.3$), EG ($12.1{\pm}1.1$), Glycerol ($15.2{\pm}1.8$), and 1,2-PROH ($11.5{\pm}1.3$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.5{\pm}0.7$, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.193-199
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• 2002

This study was conducted to investigate the optimal physiologic mating time in Hanwoo for protection to decrease of reproductivity and improvement of production of offspring. We observed 32 cows that were devide into 4 parts of treatment : T1(12 months of age and 0.5kg daily gain), T2(12 months of age and 0.8kg daily gain), T3(15 months of age and 0.5kg daily gain) and T4(18 months and 0.5kg daily gain). The first heat of treated cows was 263.3$\pm$6.4 days and average weight was 181.1$\pm$11.3kg. It was revealed the conception rates of first insemination were 25%(T1), 75%(T4) and number of insemination of T3 and T4(both 1.5) was lower than T1 and T2(2.3 and 2.4). In return of estrus after heifer's first parturition, they(T1, T2, T3 and T4) showed 66.2 days, 76.7 days, 62.4 days and 68.5 days respectively and the average was 65.7 days. Plasma progesterone(P4) concentration was nearly the same during the observation periods of treated cows and P4 was released just after 12 months. Only 5 cows (15.6%) in 32 were showed normal estrus cycle and ovulation before 12 months. Before and after parturition, P4 concentration was decreased fastly and then there was no detection of P4 from after parturition to 40 days after milking. P4 would be released again on 45 day after parturition. The results were summarized as that the optimal mating time of Hanwoo heifers was decided by the 14 months of age, 110 cm height and 265kg weight.

• The Korean Journal of Nuclear Medicine Technology
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• v.19 no.1
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• pp.72-80
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• 2015

Purpose Serum estradiol ($E_2$) measurement is requested for diagnosing menstrual cycles, ovulation induction, infertility, and menopause. $E_2$ is measured using several methods and kits including radioimmunoassay (RIA) and chemiluminescece immunoassay (CLIA). The purpose of this study was to evaluate quality of these methods and to compare them with each other. Materials and Methods Seven radioimmunoassay kits and two CLIA methods were included in the analysis. Using standard samples and patient samples, intra-assay precision, inter-assay precision, correlation between other methods, sensitivity, and recovery rate were evaluated. Results For all tested kits and methods, coefficients of variance (CVs) of intra-assay precision test were 10.9~13.6% in low-level samples and less than 10% in medium and high-level samples. CVs of inter-assay precision test were 10.8~12.3% in low-level samples and less than 10% in medium and high-level samples with all tested kits and methods. Recovery rates were $92.7{\pm}12.4%$ for SIEMENS, $101.4{\pm}18.4%$ for DIAsource, $95.1{\pm}11.5%$ for AMP, $108.4{\pm}18.5%$ for BECKMAN COULTER, $104.2{\pm}13.5%$ for BECKMAN COULTER Ultra Sensitive, $101.3{\pm}11.6%$ for CIS Bio, and $93.1{\pm}13.2%$ for MP kits. Sensitivity was 7.5, 6.2, 5.7, 6.2, 5.3, 4.5, and 5.5 pg/mL for SIEMENS, DIAsource, AMP, BECKMAN COULTER, BECKMAN COULTER Ultra Sensitive, CIS Bio, and MP kits, respectively. The measurement by MP kit was slightly higher than those by other kits in low-level samples, and the measurement by E170 was slightly higher than those of other kits in medium and high-level samples. In the measurement of standard sample for external quality control, SIEMENS kit produced relatively lower values whereas E170, Architect, and MP kits produced relatively higher values compared with other kits. Conclusion All tested kits for $E_2$ measurement have satisfactory performance for clinical use. However, correlation between kits should be considered when test kits are to be changed, because some pairs of kits do not have correlations with each other.

• Journal of Practical Agriculture & Fisheries Research
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• v.4 no.1
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• pp.110-118
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• 2002

This study was conducted to investigate the physiology of optimal reproductive age in Hanwoo for protection to decrease of reproductivity and improvement of production of offspring. Thirty two cows were devided into 4 groups of treatments : T1(12 months of age and 0.5kg daily gain), T2(12 months of age and 0.8kg daily gain), T3(15 months of age and 0.5kg daily gain) and T4(18 months of age and 0.5kg daily gain). The days of the first heat of treated cows were 263.3±6.4 days and average weight was 181.1±11.3kg. The conception rates of first insemination were 25%(T1) and 75%(T4), and the number of insemination of T3 and T4(both 1.5) was lower than those of T1 and T2(2.3 and 2.4), respectively. With regard to estrus return after the first parturition, T1, T2, T3 and T4 showed 66.2,76.7, 62.4 and 68.5 days, respectively, indicating the average days of estrus return was 65.7. Plasma progesterone(P4) concentration was nearly the same during the observation periods of treated cows and P4 began to be detected after 12months. Only 5(15.6%) out of 32cows showed normal estrus cycle and ovulation before 12 months. During the peri-parturition period, P4 concentration was rapidly decreased and there was no detection of P4 from parturition to 40 days after milking. P4 would be released again on 45 day after parturition. The results imply that the optimal reproductive age of Hanwoo heifers would be around at the 14 months of age, 110cm height and 265kg weight.

• Korean Journal of Agricultural Science
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• v.13 no.1
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• pp.82-89
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• 1986

This experiment was carried out to determine the optimum freezing and thawing rates of the hamster embryos. The female hamsters were induced to superovulate by intraperitoneal injections of 30 i.u. PMSG and mated with males of the same strain of 4 days the PMSG injection. They were killed and embryos were flushed from the oviduct and uterine horn on 3 days after mating. Embryos were flushed with a modified Dulbecco's phosphate-buffered saline and equilibrated with 1.5 M-dimethylsulphoxide by a 3-step procedure. The freezing rates of the samples were $1^{\circ}C/min$ from room temperature to $-6^{\circ}C$ and the samples were seeded at $-6^{\circ}C$. After being held for 3 min at the seeding temperature, the rates were $0.3^{\circ}C/min$ from $-6^{\circ}C$ to $-35^{\circ}C$. From $-35^{\circ}C$ to $-70^{\circ}C$, the rates were divided into $0.1^{\circ}C/min$, $1^{\circ}C/min$ and $10^{\circ}C/min$, respectively. At $-70^{\circ}C$ the samples were plunged directly into liquid nitrogen. The samples were thawed at $4^{\circ}C/min$ and $12^{\circ}C/min$ from $-196^{\circ}C$ to $37^{\circ}C$, and for 2 min in $37^{\circ}C$ water bath, respectively. The average numbers of ovulation points and embryos recovered were 35.1 and 27.0 appearing 77.0% recovery rates. Eight cell embryos in the embryos recovered were 24.8. The survival rates of embryos according to the freezing rates were 55.5~67.7% at $0.1^{\circ}C/min$, 58.8~64.9% at $1^{\circ}C/min$ and 40.5~44.7% at $10^{\circ}C/min$, respectively. The survival rates at $10^{\circ}C/min$ were significantly low. The survival rates of embryos according to the thawing rates were 53.5% at $4^{\circ}C/min$, 53.7% at $12^{\circ}C/min$ and 59.1% in $37^{\circ}C$ water bath. The survival rates, in $37^{\circ}C$ water bath were slightly higher, but we did not find any differences among them. In conclusion, the best freezing rates of hamster embryos were $1^{\circ}C/min$ from the room temperature to $-6^{\circ}C/min$, $0.3^{\circ}C/min$ from $-6^{\circ}C/min$ to $-35^{\circ}C$ and $-0.1^{\circ}C/min$ or $1^{\circ}C/min$ from $-35^{\circ}C$ to $-70^{\circ}C$. The hamster embryos thawed for 2 min in $37^{\circ}C$ water bath showed the best survival rates.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.1
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• pp.49-56
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• 2010

Objective: To investigate assisted reproductive technology (ART) outcomes in women with WHO class I anovulation compared with control group. Design: Retrospective case-control study. Methods: Twenty-three infertile women with hypogonadotropic hypogonadism (H-H) who undertook ART procedure from August 2003 to January 2009 were enrolled in this study. A total of 59 cycles (H-H group) were included; Intra-uterine insemination with super-ovulation (SO-IUI, 32 cycles), in vitro fertilization with fresh embryo transfer (IVF-ET, 18 cycles) and subsequent frozenthawed embryo transfer (FET, 9 cycles). Age and BMI matched 146 cycles of infertile women were collected as control group; 64 cycles of unexplained infertile women for SO-IUI and 54 cycles of IVF-ET and 28 cycles of FET with tubal factor. We compared ART and pregnancy outcomes such as clinical pregnancy rate (CPR), clinical abortion rate (CAR), and live birth rate (LBR) between the two groups. Results: There was no difference in the mean age ($32.7{\pm}3.3$ vs. $32.6{\pm}2.7$ yrs) and BMI ($21.0{\pm}3.1$ vs. $20.8{\pm}3.1kg/m^2$) between two groups. Mean levels of basal LH, FSH, and $E_2$ in H-H group were $0.62{\pm}0.35$ mIU/ml, $2.60{\pm}2.30$ mIU/ml and $10.1{\pm}8.2$ pg/ml, respectively. For ovarian stimulation, H-H group needed higher total amount of gonadotropin injected and longer duration for ovarian stimulation (p<0.001). In SO-IUI cycles, there was no significant difference of CPR, CAR, and LBR between the two groups. In IVF-ET treatment, H-H group presented higher mean $E_2$ level on hCG day ($3104.8{\pm}1020.2$ pg/ml vs. $1878.3{\pm}1197.7$ pg/ml, p<0.001) with lower CPR (16.7 vs. 37.0%, p=0.11) and LBR (5.6 vs. 33.3%, p=0.02) and higher CAR (66.7 vs. 10.0%, p=0.02) compared with the control group. However, subsequent FET cycles showed no significant difference of CPR, CAR, and LBR between the two groups. Conclusion: H-H patients need higher dosage of gonadotropin and longer duration for ovarian stimulation compared with the control groups. Significantly poor pregnancy outcomes in IVF-ET cycles of H-H group may be due to detrimental endometrial factors caused by higher $E_2$ level and the absence of previous hormonal exposure on endometrium.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.247-268
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• 1996

It has been known for a long time that gonadotropins and steroid hormones play a pivotal role in a series of reproductive biological phenomena including the maturation of ovarian follicles and oocytes, ovulation and implantation, maintenance of pregnancy and fetal growth & development, parturition and mammary development and lactation. Recent investigations, however, have elucidated that in addition to these classic hormones, multiple growth factors also are involved in these phenomena. Most growth factors in reproductive organs mediate the actions of gonadotropins and steroid hormones or synergize with them in an autocrine/paracrine manner. The insulin-like growth factor(IGF) system, which is one of the most actively investigated areas lately in the reproductive organs, has been found to have important roles in a wide gamut of reproductive phenomena. In the present communication, published literature pertaining to the intrauterine IGF system will be reviewed preceded by general information of the IGF system. The IGF family comprises of IGF-I & IGF-II ligands, two types of IGF receptors and six classes of IGF-binding proteins(IGFBPs) that are known to date. IGF-I and IGF-II peptides, which are structurally homologous to proinsulin, possess the insulin-like activity including the stimulatory effect of glucose and amino acid transport. Besides, IGFs as mitogens stimulate cell division, and also play a role in cellular differentiation and functions in a variety of cell lines. IGFs are expressed mainly in the liver and messenchymal cells, and act on almost all types of tissues in an autocrine/paracrine as well as endocrine mode. There are two types of IGF receptors. Type I IGF receptors, which are tyrosine kinase receptors having high-affinity for IGF-I and IGF-II, mediate almost all the IGF actions that are described above. Type II IGF receptors or IGF-II/mannose-6-phosphate receptors have two distinct binding sites; the IGF-II binding site exhibits a high affinity only for IGF-II. The principal role of the type II IGF receptor is to destroy IGF-II by targeting the ligand to the lysosome. IGFs in biological fluids are mostly bound to IGFBP. IGFBPs, in general, are IGF storage/carrier proteins or modulators of IGF actions; however, as for distinct roles for individual IGFBPs, only limited information is available. IGFBPs inhibit IGF actions under most in vitro situations, seemingly because affinities of IGFBPs for IGFs are greater than those of IGF receptors. How IGF is released from IGFBP to reach IGF receptors is not known; however, various IGFBP protease activities that are present in blood and interstitial fluids are believed to play an important role in the process of IGF release from the IGFBP. According to latest reports, there is evidence that under certain in vitro circumstances, IGFBP-1, -3, -5 have their own biological activities independent of the IGF. This may add another dimension of complexity of the already complicated IGF system. Messenger ribonucleic acids and proteins of the IGF family members are expressed in the uterine tissue and conceptus of the primates, rodents and farm animals to play important roles in growth and development of the uterus and fetus. Expression of the uterine IGF system is regulated by gonadal hormones and local regulatory substances with temporal and spatial specificities. Locally expressed IGFs and IGFBPs act on the uterine tissue in an autocrine/paracrine manner, or are secreted into the uterine lumen to participate in conceptus growth and development. Conceptus also expresses the IGF system beginning from the peri-implantation period. When an IGF family member is expressed in the conceptus, however, is determined by the presence or absence of maternally inherited mRNAs, genetic programming of the conceptus itself and an interaction with the maternal tissue. The site of IGF action also follows temporal (physiological status) and spatial specificities. These facts that expression of the IGF system is temporally and spatially regulated support indirectly a hypothesis that IGFs play a role in conceptus growth and development. Uterine and conceptus-derived IGFs stimulate cell division and differentiation, glucose and amino acid transport, general protein synthesis and the biosynthesis of mammotropic hormones including placental lactogen and prolactin, and also play a role in steroidogenesis. The suggested role for IGFs in conceptus growth and development has been proven by the result of IGF-I, IGF-II or IGF receptor gene disruption(targeting) of murine embryos by the homologous recombination technique. Mice carrying a null mutation for IGF-I and/or IGF-II or type I IGF receptor undergo delayed prenatal and postnatal growth and development with 30-60% normal weights at birth. Moreover, mice lacking the type I IGF receptor or IGF-I plus IGF-II die soon after birth. Intrauterine IGFBPs generally are believed to sequester IGF ligands within the uterus or to play a role of negative regulators of IGF actions by inhibiting IGF binding to cognate receptors. However, when it is taken into account that IGFBP-1 is expressed and secreted in primate uteri in amounts assessedly far exceeding those of local IGFs and that IGFBP-1 is one of the major secretory proteins of the primate decidua, the possibility that this IGFBP may have its own biological activity independent of IGF cannot be excluded. Evidently, elucidating the exact role of each IGFBP is an essential step into understanding the whole IGF system. As such, further research in this area is awaited with a lot of anticipation and attention.