• Clinical and Experimental Reproductive Medicine
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• v.32 no.2
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• pp.177-185
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• 2005

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.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.287-292
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• 1998

The objective of this study was to confirm whether the vitrification method using EFS35 has detrimental effect for cytoskeleton and chromosome constitution of the mouse oocytes by indirect immunocytochemistry and chromosome analysis. Mouse oocytes were vitrified using EFS35 which consisted of 35% ethylene glycol, 18% ficoll, 0.3 M sucrose and 10% FBS in M2 medium. The results obtained in this experiment were summarized as follows: When the survival rates after being exposed or vitrified in EFS35 were examined, there were not different between two groups (97.7 and 89.3%). Also, when the microtubule morphology and microfilament distribution in vitrified oocytes were examined, normal percentage of two cytoskeleton in vitrified group (95.5 and 100%) was not different from that in control (97.5 and 100%) and exposed group (92.3 and 100%). In addition, the rate of oocytes containing a normal chromosome number in vitrified group (73.5%) after IVF was not different from that in control (79.5%) and exposed group (78.7%). These results indicated that the cytoskeletal morphology and chromosome constitution of mouse oocytes were not affected by cryoprotectant (EFS35) or freezing apd that vitrification methods using EFS35 was suitable for cryopreservation of mouse oocytes.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.171-176
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• 1998

This study was carried out to confirm whether the in vivo developmental potential of mouse hatched blastocysts (HBs) can be obtained by vitrification method using the cryoprotectant EFS35. The HBs ($\theta$ 130$\mu\textrm{m}$) were cultured in vitro until day 5 from zygotes produced in vivo and were equilibrated in 10% ethylene glycol(EG) for 5 min, and then were exposed or vitrified in EFS35 (35% EG, 18% Ficoll and 0.3M sucrose). After 30 min thawing, re-expanding HBs were transferred into one or both uterine horns of pseudopregnant recipients on day 3 (4~6 embryos /horn). Pregnancy rates of recipients and implantation were assessed by autopsy on day 15 of gestation. The results obtained in these experiments were summarized as follows : After thaw-ing, in vitro survival of HBs was not significantly different between exposed (65.5%) and vitrified(54.5%) group. Also, when the in vivo development potential was examined, total implantation was not different between control (58.5% ) and vitrified (41.0%) group, although the live fetus formation of vitrified group (24.0%) was significantly lower than that of control (58.3%) group (p< 0.05). These results suggested that vitrification freezing method of mouse HBs using EFS35 can be used to make wide the utility of embryo transfer of the more embryos produced in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.87-92
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• 1998

This study was carried out to investigate in vitro/in vivo development of vitrified mouse oocytes. Mouse oocytes were vitrified using EFS30, 35 and 40 (30, 35 and 40% ethylene glycol, 18% ficoll and 0.5 M sucrose in M2 medium). After being exposed or vitrified-thawed, oocytes of normal morphology were inseminated in vitro by $1-2\times10^6/ml$ of epididymal sperm. The rates of fertilization, in vitro/in vivo development and cell number (inner cell mass and tropectoderm cell) of blastocysts in each treatment group were examined. The results obtained in these experiments were summarized as follows: The cleavage rates were obtained in EFS35 containing 35% ethylene glycol higher than in EFS30 and EFS40. The development rate of vitrified-thawed oocytes to two-cell stage after in vitro fertilization (51.1%) was significantly different compared to that of exposed to vitrification solution without cooling (60.0%) and control (68.2%) (p<0.05). However, there were no differences in the blastocyst formation from the cleaved embryos among groups (75.0, 73.3 and 80.0%). Also, the mean number of cells per blastocysts of vitrified group $(92.5{\pm}2.9)$ was similar to that of the exposed $(98.5{\pm}5.3)$ and control $(100.9{\pm}4.8)$. In vivo development of the blastocysts derived from vitrified-thawed oocytes resulted in fetal development (50.7%) and implantation rates (80.0%) which are very similar to those of control (58.2, 78.2%). These results suggest that mouse oocytes could be cryopreserved using vitrification solution (EFS35) based on ethylene glycol.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.141-148
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• 1999

This study was to test whether the viability of bovine hatched blastocysts (HBs) can be maintained after vitrification and thawing. The HBs were produced in vitro at Day 9 and Day 10 after IVF, and they were classified to small (S-HBs; ø$\leq$300 ${\mu}{\textrm}{m}$) and large(L-HBs; ø＞300 ${\mu}{\textrm}{m}$) on the basis of embryo diameter using eyepiece micrometer. As freezing solution, we used EFS35 which consisted of 35% ethylene glycol (EG), 18% ficoll, 0.3 M sucrose and 10% FBS added in mDPBS. Vitrification was taken by two-step method, the HBs were equilibrated in 10% EG for 5 minutes and then shortly exposed in EFS35 and plunged into L$N_2$for 30~45 sec. After thawing, the survival rates were assessed by the re-expansion of the blastocoel during 2 h and 16 h of culture. The results obtained in these experiments were summarized as follows; 1) When the blastocysts(40.8%) recovered at Day 8 after IVF were further cultured for 24 h(Day 9 after IVF) and 48 h(Day 10 after IVF), the rates of HBs were 20.5% and 6.7%, respectively. Also, the total cell number of HBs on Day 9 was significantly higher than that of HBs on Day 10 (p＜0.01). 2) When the effects of freezing solution to the survival of Day 9 L-HBs were examined, the rate of vitrified group (75.7%) was significantly lower than 100% of control and exposed group(p＜0.05). 3) When the survival rates of vitrified HBs according to size and developmental age were examined, the data of L-HBs (75.5%) and S-HBs(63.6%) on Day 9 were slightly higher than those of L-HBs(64.3%) and S-HBs(60.7%) on Day 10. 4) Also, when the in vitro survival of Day 9 HBs was evaluated under different culture condition after thawing, the result in culture medium only (79.3%) was significantly higher than 43.2% in co-culture group (p＜0.05). These results demonstrated that bovine HBs can be successfully cryopreserved by two-step vitrification method using EFS35.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.71-76
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• 1998

This study was carried out to examine whether the developmental capacity of bovine immature oocytes frozen ultra-rapidly using electron microscope (EM) grids and EFS30 can be obtained. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. As criterior of oocyte viability, the rates of maturation, fertilization and embryonic development were determined. The results obtained in this experiment were summarized as follows: When ultra-rapidly frozen immature oocytes were thawed, 43.2% of them were survived. The rates of maturation (84.1%) and normal 2 pronuclei formation (57.5%) of frozen immature oocytes were not significantly different when compared to those of control (92.5, 65.0%). In addition, the rates of $\geq2$-cell (65.0%) and blastocyst formation (30.8%) of freezing group were not significantly different when compared to those of control (73.7, 35.7%). These results demonstrate that developmental capacity of frozen-thawed bovine immature oocytes can be successfully obtained when survived from the ultra-rapid freezing method using EM grid and EFS30.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.447-458
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• 1995

This study was carried out to characterize nonadrenergic, noncholinergic(NANC) relaxation of porcine retractor penis(PRP) muscle induced by electrical field stimulation(EFS) and to investigate the actions of niric oxide(NO) and vasoactive intestinal polypeptide(VIP) as candidates for NANC neurotransmitters. Biphasic relaxations of PRP muscle were induced by EFS to NANC nerve. Rapid-phase relaxation was observed at low frequency(0.5-16Hz) and slow-phase relaxation followed during high frequency(8-60Hz). Both relaxations were frequency-dependent and TTX($1{\times}10^{-6}M$)-sensitive. L-NAME($2{\times}10^{-5}M$) inhibited the rapid-phase relaxation, but not the slow-phase relaxation. The inhibition of the rapid-phase relaxation with L-NAME was reversed by L-arginine ($1{\times}10^{-3}M$) but not by D-arginine($1{\times}10^{-3}M$). Methylene blue($4{\times}10^{-5}M$) reduced the rapid-phase relaxation. Exogenous No(ExoNO, $1{\times}10^{-5}-1{\times}10^{-4}M$) induced dose-dependent relaxations of PRP muscle. Oxyhemoglobin($5{\times}1^{-5}M$) blocked the relaxation induced by ExoNO and inhibited EFS-induced relaxation. Hydroquinone($1{\times}10^{-4}M$) also abolished the relaxation induced by ExoNO, but did not affect EFS-induced relaxation. L-NAME resistant slow-phase relaxation to EFS was inhibited by ${\alpha}$-chymotrypsin(2.5 U/ml). Both methylene blue($4{\times}10^{-5}M$) and Nethylmaleimide($1{\times}10^{-4}M$) reduced the slow-phase relaxation by EFS. [4-Cl-D-$Phe^6$, $Leu^{17}$]-VIP($3{\times}10^{-6}M$) inhibited the slow-phase relaxation by EFS. External applications of VIP ($1{\times}10^{-7}M$) caused relaxations that were simillar to the L-NAME resistant slow-phase relaxations induced by EFS, and relaxant effects of exogenous VIP were blocked by ${\alpha}$-chymotrypsin(2.5 U/ml).

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.2967-2971
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• 2013

Background: Extraskeletal Ewing sarcoma (EES)/primitive neuroectodermal tumours (PNET) are rare soft tissue sarcomas. Prognostic factors and optimal therapy are still unconfirmed. Materials and Methods: We performed a retrospective analysis on patients to explore the clinic characteristics and prognostic factors of this rare disease. A total of 37 patients older than 15 years referred to our institute from Jan., 2002 to Jan., 2012 were reviewed. The characteristics, treatment and outcome were collected and analyzed. Results: The median age was 28 years (range 15-65); the median size of primary tumours was 8.2 cm (range 2-19). Sixteen patients (43%) had metastatic disease at the initial presentation. Wide surgical margins were achieved in 14 cases (38%). Anthracycline or platinum-based chemotherapy was performed on 29 patients (74%). Radiotherapy was delivered in 13 (35%). At a median follow-up visit of 24 months (range 2-81), the media event-free survival (EFS) and overall survival (OS) were 15.8 and 30.2 months, respectively. The 3-year EFS and OS rates were 24% and 43%, respectively. Metastases at presentation and wide surgical margins were significantly associated with OS and EFS. Tumour size was significantly associated with OS but not EFS. There were no significant differences between anthracycline and platinum based chemotherapy regarding EFS and OS. Conclusions: EES/PNET is a malignant tumour with high recurrence and frequent distant metastasis. Multimodality therapy featuring wide surgical margins, aggressive chemotherapy and adjuvant local radiotherapy is necessary for this rare disease. Platinum-based chemotherapy can be used as an adjuvant therapy.

• Resources Recycling
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• v.21 no.1
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• pp.30-40
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• 2012

Electric arc furnace slag is made in ironworks during steel refining, it is been increasing chemical and physical resistibility using ageing method of unstable state of melting steel slag for using concrete's fine aggregates. Which is been changing stable molecular structure of aggregates, it restrains moving of ion and molecule. In Korea, KS F 4571 has been prepared for using the electric arc furnace oxidizing slag to concrete aggregates(EFS). In this study, Electric arc furnace oxidizing slag is used in the PRCC(Polymer Rapid setting Cement Concrete) which is applied a bridge pavement of rehabilitation, largely. The results showed that the increment of compressive strength development by 10- 20%. The flexural strength of EFS-Con increased greatly as the electric arc furnace oxidizing slag changed. The compressive strength and flexural strength developed enough for opening the overlayed EFS-Con to the traffic after 4 hours of EFS-Con placement. The permeability of EFS-Con was evaluated as negligible due to its very low charge passed. Thus, EFS-Con could be used at repairing or overlaying the concrete at fast-track job sites.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.75-84
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• 1999

This study assessed development in vitro of pronuclear(PN) stage embryos cryopreserved by the method of either vitrification or slow freezing, by using of different cryoprotectants, and equilibration and cooling rate, in rabbit. Ethyleneglycol- ficoll- sucrose(EFS) or ethyleneglycol- polyvinylpyrrolidone - galactose- (EPG-I) for vitrification, and EPG- II for slow freezing as cryoprotectant were used. The pronuclear embryos were exposed to EFS for 0 to 5 min and diluted with D-PBS and/or pre-dilution with 0.5 M sucrose. To examine the viability of frozen-thawed embryos, PN embryos were co-cultured with bovine oviductal epitherial cell(BOEC) for 5 days to hatching blastocyst stage in 39 $^{\circ}C$ 5% $CO_2$incubator. The results obtained were as follows: The dilution with 0.5 M sucrose and D-PBS after the exposure to EFS for 1.0 min resulted in no significant(P＜0.05) decrease in the development of PN embryos to hatching blastocyst(72.0%), compared with controls. The development of PN embryos cryopreserved to hatching blastocyst was not significantly (P＜0.05) different between EFS for 1.0 min(72.0%), EPG-I for 1.0 min(72.0%) and EPG-II for 30 min(66. 7%). The post-thaw development of PN embryos to hatching blastocyst was similarly very low as 6.1% and 11.5% in vitrification with EFS and slow freezing with EPG-II, respectively. The incidence of post-thaw zona-crack in PN embryos cryopreserved by slow freezing with plunging to liquid nitrogen at －35$^{\circ}C$ was signicantly(P＜0.05) higher(25.0%), compared with －85$^{\circ}C$ (1.9%). These results indicated that the rabbit PN embryos could be cryopreserved with either vitrification or slow freezing procedure, and frozen PN embryos could be successfully developed in vitro to haching blastocyst. but the post-thaw development of cryopreserved PN embryos was still very low under the present conditions.