• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.122-128
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• 2018

Objective: To determine whether fragment removal on in vitro fertilization (IVF) day 2 improved the subsequent development and pregnancy outcomes of fragmented embryos compared to similar-grade embryos without fragment removal. Methods: This study was a retrospective analysis involving 191 IVF cycles in which all embryos had over 10% fragmentation (grade 3 or 4) on day 2 of the IVF-embryo transfer cycle from March 2015 to December 2017. IVF cycles were divided into the fragment removal group (n = 87) and the no fragment removal group (n = 104) as a control cohort. Before fragment removal, embryos with fragmentation on day 2 were incubated in $Ca^{2+}$- and $Mg^{2+}$-free biopsy medium under paraffin oil for 30 minutes. Microsurgical fragment removal was performed with later-assisted hatching and a handmade suction micropipette that had an outer diameter of $30{\mu}m$. Results: There were no significant differences in the characteristics of the patients between the control and the fragment removal groups. After fragment removal and subsequent in vitro culture for 24 hours, the number of blastomeres ($7.1{\pm}1.7$ vs. $6.9{\pm}1.6$) was comparable between the transferred embryos in the two groups, but the morphological grade of the embryos in the fragment removal group ($1.9{\pm}0.7$) was significantly higher than that of the control group ($3.1{\pm}0.5$, p< 0.01). The clinical pregnancy (43.7%) and implantation rates (25.8%) in the fragment removal group were significantly higher than those in the control group (28.8% and 14.0%, respectively; p< 0.05). Conclusion: Early fragment removal on day 2 significantly improved the subsequent development and pregnancy outcomes of fragmented embryos.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.6 no.4
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• pp.265-273
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• 2001

The sea urchin has been used as sea food in many countries. This species has also been an important organism of embryological studies for more than a century. In recent years, sea urchin embryos are being used as testing materials for toxicity of pollutants and toxins. Usefulness of sea urchin embryos as experimental models comes from the easiness in obtaining sea urchin samples and a lot of gametes, in rearing embryos in the laboratory, in observing the cellular movement and organ formation during the embryogenesis and in manipulating blastomeres and genetic maferials. The sea urchin in itself is a key organism for the understanding of deuterostome evolution from the protostomes and of indirect development of marine invertebrates which undergo the planktotrophic larval stage. A fertilized sea urchin egg goes through rapid cleavage and becomes a 60 cell embryo 7hr after fertilization. It then develops into a morula, a blastula, a gastrula and finally a pluteus larva approximately 70 hr after fertilization. At the 60 cell stage, the embryo comprises of five territories that express territory-speciflc genes and later form different organs. Micromeres at the vegetal pole ingress into the blastoceol and become the primary mesenchyme cells(PMCs). PMCs express genes involved in skeletogenesis such as SM30, SM37, SM50, PM27, msp130. Among the genes, SM37 and SM50 are considered to be members of a gene family which is characterized by early blastula expression, Glycine-Proline-Glutamine rich repeat structures and spicule matrix forming basic proteins. Genetic studies on the sea urchin embryos help understand the molecular basis of indirect development of marine invertebrates and also of the biomineralization common to the animal kingdom.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.29-39
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• 2004

Objectives: This study was performed to evaluate the laboratory system for successful PGD using fluorescence in situ hybridization (FISH) and the clinical outcome of PGD cycles in five years experiences. Methods: A total of 181 PGD-FISH cycles of 106 couples were performed, and diagnosed chromosome normality in the preimplantation embryos. The laboratory and clinical data were classified by the following optimization steps, and statistically analyzed. Phase I: Blastomere biopsy with two kinds of pipettes, removal of cytoplasmic proteins without treatment of pepsin and culture of biopsied embryos with single medium; Phase II: Blatomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with single medium; Phase III: Blastomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with sequential media. Results: A total of 3, 209 oocytes were collected, and 83.8% (2, 212/2, 640) of fertilization rate was obtained by ICSI procedure. The successful blastomere biopsies were accomplished in 98.6% (2, 043/2, 071) of embryos, and the successful diagnosis rate of FISH was 94.7% (1, 935/ 2, 043) of blastomeres from overall data. Embryo transfers with normal embryos were conducted in 93.9% (170/181) of started cycles. There was no difference in the successful rate of biopsy and diagnosis among Phase I, II and III. However, the pregnancy rate per embryo transfer of Phase III (38.8%, 26/67) was significantly (p<0.05) higher than those of Phase I (13.9%, 5/36) and Phase II (14.9%, 10/67). Conclusions: The laboratory optimization and experience for the PGD with FISH procedure can increase the pregnancy rate to 38.8% in the human IVF-ET program. Our facility of PGD with FISH provides the great possibility to get a normal pregnancy for the concerned couples by chromosomal aberrations.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.253-260
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• 2004

Objectives: Klinefelter syndrome is the most common genetic cause of male infertility and presents with 47, XXY mainly or 46, XX/47, XXY mosaicism. It is characterized by hypogonadism and azoospermia due to testicular failure, however, sporadic cases of natural pregnancies have been reported. With the development of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI), sperm can be retrieved successfully and ART is applied in these patients for pregnancy. It has been suggested that the risk of chromosome aneuploidy for both sex chromosome and autosome is increased in the sperms from 47, XXY germ cells. Considering the risk for chromosomal aneuploidy in the offspring, preimplantation genetic diagnosis (PGD) could be applied as a safe and more effective treatment option in Klinefelter syndrome. The aim of this study is to assess the outcome of PGD cycles by using FISH for sex chromosome and autosome in patients with Klinefelter syndrome. Materials and Methods: From Jan. 2001 to Dec. 2003, PGD was attempted in 8 cases of Klinefelter syndrome but TESE was failed to retrieve sperm in the 3 cases, therefore PGD was performed in 8 cycles of 5 cases (four 47, XXY and one 46, XY/47, XXY mosaicism). In one case, ejaculated sperm was used and in 4 cases, TESE sperm was used for ICSI. After fertilization, blastomere biopsy was performed in $6{\sim}7$ cell stage embryo and the chromosome aneuploidy was diagnosed by using FISH with CEP probes for chromosome X, Y and 17 or 18. Results: A total of 127 oocytes were retrieved and ICSI was performed in 113 mature oocytes. The fertilization rate was $65.3{\pm}6.0%$ (mean$\pm$SEM) and 76 embryos were obtained. Blastomere biopsy was performed in 61 developing embryos and FISH analysis was successful in 95.1% of the biopsied blastomeres (58/61). The rate of balanced embryos for chromosome X, Y and 17 or 18 was $39.7{\pm}6.9%$. The rate of aneuploidy for sex chromosome (X and Y) was $45.9{\pm}5.3%$ and $43.2{\pm}5.8%$ for chromosome 17 or 18, respectively. Embryo transfer was performed in all 8 cycles and mean number of transferred embryos was $2.5{\pm}0.5$. In 2 cases, clinical pregnancies were obtained and normal 46, XX and 46, XY karyotypes were confirmed by amniocentesis, respectively. Healthy male and female babies were delivered uneventfully at term. Conclusion: The patients with Klinefelter syndrome can benefit from ART with TESE and ICSI. Considering the risk of aneuploidy for both sex chromosome and autosome in the sperms and embryos of Klinefelter syndrome, PGD could be offered as safe and more effective treatment option.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.151-169
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• 1993

The present study was carried out to investigate the best condition for nuclear-cytoplasm fusion and in vitro culture of nuclear transplanted embryos and to investigate the production of nuclear transplanted offsprings. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulses at output voltage of 1.0, 1.5 and 2.0 kV/cm for 100, 150 and $200{\mu}$ sec to induce cell fusion. 1. The culture of intact or zona cut 2-cell embryos in the medium supplemented with cytochalasin B($5{\mu}g/m{\ell}$) and colcemide($0.1{\mu}g/m{\ell}$)for 30 and 60 minutes did not affect the development to later stage. 2. The in vitro developmental rates of group A(a nucleus from one of the blastomeres was removed) and B(electrofusion of group A) were significantly lower than that of control group(p<0.01). 3. When nuclear transplanted embryos were submitted to electrofusion, the significantly higher fusion rates of 2-cell donor nuclei were achieved at the electric field strength of DC 1.5kV/cm for 100 and $150{\mu}$ sec, DC 2.0 kV/cm for $100{\sim}200{\mu}$ sec than DC 1.0 kV/cm for 100 and $150{\mu}$ sec(p<0.01). The significantly higher fusion rates of 4-cell donor nuclei were achieved at DC 2.0 kV/cm for 100 and $150{\mu}$ sec than DC 1.0kV/cm for $100{\sim}200{\mu}$ sec(p<0.01). These fusion rates in 8-cell donor nuclei were 88.7~99.3%. 4. The developmental potency to blastocyst in 2- and 4-cell donor nuclei was significantly higher in DC 1.0 and 2.0 kV/cm for $100{\sim}200{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group (p<0.01). The developmental potency to blastocyst in 8-cell donor nuclei was significantly higher in DC 2.0 kV/cm for $100{\mu}$ sec treated group than in DC 1.0 kV/cm for $100{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group(p<001). 5. The developmental potency to blastocyst after nuclear transplantation was significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 6. The success rate of nuclear injection into enucleated 2-cell embryos was significantly higher in 2-cell donor nuclei than in 4- or 8-cell donor nuclei(p<0.01). 7. The culture time taken for the nuclear transplanted 2-cell embryos to blastocyst stage was significantly longer in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 8. There was no significant difference in the developmental potency of nuclear transplanted embryos within the concentration of EGF at 0 to 15 ng per $m{\ell}$ of BMOC-3 solution. 9. The production rates of offspring after transfer of nuclear transplanted embryos to recipient mouse were significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01).

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.2
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• pp.155-168
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• 1986

In Korea, lots of Israeli carp (Cyprinus carpio) are cultured by means of net cages in dams and lakes, but many carp have been subjected to heavy infestation of the cestoid, Bothriocephalus opsariichthydis. Nevertheless the parasitic state and life history of the cestoid are not yet reported. This reason led the author to study the parasitic state and life history of B. opsariichthydis parasitized in the carp in order to take effective control measures against its damage. Israeli carp were sampled from two fish farms, in Taech'on and Kyongch'on. After dissection of the specimens, the cestoid were obtained and the parasitic rates were examined. After taking the eggs from adult worms, the development of the eggs were observed. Coracidia were exposed to four kinds of crustaceans in order to investigate the infection rate and development of the larva. Finally, tile development of the larva in the final host was investigated. The fully mature eggs were in the cleavage stage, when they are released, and the size ranged 47.5 to 55.0 $(50.9){\times}30.0$ to 32.5 (31.1) um, in the state of under-developed coracidia and blastomeres. The parasitic rate of the cestoid in Israeli carp from Taechon was $55.5\%$ in 1984 and $21.6\%$ in 1985, that from Kyongchon was $64.7\%$ in 1984, and color carp from Kusan was $14.9\%$ in 1984. The eggs were hatched to coracidia within 48 hours under 26 to $28^{\circ}C$. The cestoid showed a strong affinity to Thermocyclops hyalinus and Paracyclops fimbriatus and the infection rates were $93.5\%$ and $75.5\%$, respectively. At 14 days after the infection to Thermocyclops hyalinus and Paracyclops fimbriatus, the larvae of the cestoid grew into fully developed procercoids; 207 to $226 (214){\times}90$ to 102 (94) um in size. Sixty days after carp have ingested the Thermocyclops hyalinus infected with the fully developed procercoids, the larvae of the cestoid matured into adult worms in the intestines of the carp.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.207-214
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• 1996

To examine the developmental capacity of manipulated embryos after ultrarapid refreezing and thawing, mouse embryos were biopsied at 4-cell stage, frozen twice at 4-cell and morula stages, respectively, and then transferred to rec-ipients. Single blastomeres were biopsied from 4-cell embryos by a modified aspiration method. Biopsied 4-cell embryos were equilibrated into freezing medium at room temperature for 2.5 min, loaded into 40 $\mu$I of freezing medium in 0.25 ml plastic straw and then directly immersed into liqiud nitrogen. Freezing medium for 4-cell embryos consisted of 4.0 M ethylene glycol and O.25 M sucrose in dPBS supplemented with 6 mg/lm BSA. Morulae were frozen into freezing medium containing 5.0 M glycerol instead of ethylene glycol. Thawing was conducted by agitating each straw in 3TC water for 20 sec. The c content of each straw was expelled into 0.5 ml of dilution medium, which consisted of 0.25 M sucrose and 3 mg/ml BSA in dPBS. The thawed embryos were rehydrated in dilution medium for 10 min, washed 3 times with dPBS and then cultured in M16 medium at 37$^{\circ}C$, 5% CO$_2$ in air. Blastocysts that developed from frozen or refrozne biopsied embryos were transferred to recipients on Day 3 of pseudopregnancy, respectively. In vitro and in vivo developmental rates of the biopsied and intact 4~cell embryos after freezing and thawing were 78 (10l/130) and 25% (10/40), and 91 (114/125) and 30% (12/40), respectively. Although the rates of in vitro development of biopsied and intact embryos to blastocyst stage were significantly different after freezing and thawing (P

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.61-68
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• 2002

The present study was examined effects of aesculetin and $O_2$concentrations on in vitro development of Hanwoo (Korean Native Cattle) embryos derived from in-vitro matured and fertilized (IVM-lVF) oocytes. The oocytes were cultured for the first 40~44 h after in vitro fertilization, then embryos of 2 to 8 cell stages were cultured under the different culture conditions for another 6 days. In experiment 1, the higher rates of morulae and blastocysts were produced in 5% $O_2$, than in 20% $O_2$(P＜0.05). There was significantly (P＜0.05) higher in embryos cultured with 1 $\mu\textrm{g}$/$m\ell$ than with 0, 5 and 10 $\mu\textrm{g}$/$m\ell$ of aesculetin. In experiment 2, the proportions of embryo developed with blastocysts and morulae plus blastocysts in 5% $O_2$, again was significantly (P＜0.05) higher in 20% $O_2$, during the culture with aesculetin and/or taurine. In the 5 and 20% $O_2$atmosphere, the inclusion of 1 $\mu\textrm{g}$/$m\ell$ aesculetin or 2.5mM taurine increased significantly (P＜0.05) the percentages of blastocysts and morulae plus blastocysts. In experiment 3, in medium with aesculetin plus PDGF and taurine plus EGF than other treatment groups, significantly (P＜0.05) higher developmental rates were obtained. Number of blastomere in balstocyst stage were also higher in medium with that than without aesculetin. However, there were no significant differences in all culture conditions. In experiment 4, the proportions of embryo developed to the morulae and blastocyst stages were significantly (P＜0.05) higher rates in medium with natural and commercial aesculetin than in control medium. No significant differences, however, were observed in between natural (71%) and commercial (70.0%) aesculetin. Number of blastomere in blastocyst stage were also higher in medium with natural and commercial aesculetin than in control medium. However, there was no effect on the number of blastomeres by these treatment. These data indicate that preimplatation embryos are very sensitive to condition that can cause oxygen concentration and show that efficiency role of aesculetin for improving bovine embryo development in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.269-278
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• 2002

Objective s: Chromosome aneuploidy is associated with recurrent abortion and congenital anomaly and genetic diseases occur repeatedly in the specific families. Preimplantation genetic diagnosis (PGD) can prevent aneuploidy or genetic disease by selecting normal embryos before implantation and is an alternative to prenatal diagnosis. The aim of this study is to assess the outcome of PGD cycles by using FISH or PCR, and to determine the clinical usefulness and values in patients with risk of chromosomal aneuploidy or genetic disease. Materials and Methods: From 1995 to Apr. 2001, a total of 108 PGD cycles in 65 patients with poor reproductive outcome were analyzed. The indications of PGD were translocation (n=49), inversion (n=2), aneuploidy screening (n=7), Duchenne muscular dystrophy (n=5) and spinal muscular atrophy (n=2). PGD was applied due to the history of recurrent abortion, previous birth of affected child or risk of aneuploidy related to sex chromosome aneuploidy or old age. Blastomere biopsy was performed in 6$\sim$10 cell stage embryo after IVF with ICSI. In the single blastomere, chromosome aneuploidy was diagnosed by using FISH and PCR was performed for the diagnosis of exon deletion in DMD or SMA. Results: The FISH or PCR amplification was successful in 94.3% of biopsied blastomeres. The rate of transferable balanced emb ryos was 24.0% in the chromosome translocation and inversion, 57.1% for the DMD and SMA, and 28.8% for the aneuploidy screening. Overall hCG positive rate per transfer was 17.8% (18/101) and clinical pregnancy rate was 13.9% (14/101) (11 term pregnancy, 3 abortion, and 4 biochemical pregnancy). The clinical pregnancy rate of translocation and inversion was 12.9% (11/85) and abortion rate was 27.3% (3/11). In the DMD and SMA, the clinical pregnancy rate was 33.3% (3/9) and all delivered at term. The PGD results were confirmed by amniocentesis and were correct. When the embryos developed to compaction or morula, the pregnancy rate was higher (32%) than that of the cases without compaction (7.2%, p<0.01). Conclusions: PGD by using FISH or PCR is useful to get n ormal pregnancy by reducing spontaneous abortion associated with chromosome aneuploidy in the patients with structural chromosome aberration or risk of aneuploidy and can prevent genetic disease prior to implantation.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.4
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• pp.339-348
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• 2010

Objective: To investigate the beneficial effect of fragment removal on the subsequent cell division and clinical outcome of the fragmented human embryos. Methods: A prospective study was performed in Hanna Women's Clinic and Mizmedi Hospital. Sixty couples undergoing In vitro fertilization-embryo transfer (IVF-ET) program were participated in the present study. The microsurgical fragment removal was performed in 106 fragmented embryos of 29 patients before the transfer. As a control group, 122 fragmented embryos of 31 patients were transferred without the fragment removal. Effects of fragment removal on morphological changes and clinical outcomes of fragmented embryos were investigated. Results: Mean morphological grade (G2.79) of fragmented embryos was significantly improved after the fragment removal(G1.63, p<0.001). Most of the fragmented embryos did not show a regeneration of fragments after the fragment removal during the subsequent development, and a beneficial effect of fragment removal on the development of the fragment removed embryos was observed. Implantation and pregnancy rates of fragment removed embryos were 12.3% and 31.3%, whereas the rates of control group embryos were 6.6% and 22.5%, respectively. There was no statistical significance in the rates between the two groups because of the low number of trials. Conclusion: Microsurgical fragment removal improved the subsequent development as well as the morphological grade of fragmented embryos. The fragment removal may be beneficial for neighboring blastomeres by repairing the intercellular communication and removing the secretion of the potential toxic materials by fragments.