• Journal of Embryo Transfer
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• v.14 no.3
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• pp.195-201
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• 1999

This study was carried out to improve a technique of cloned animal prodcution by preactivation of nuclear recipient oocytes with ionomycin and 6-dimethylaminopurine (6-DMAP) in rabbits. The oocytes were collected from the oviduct of superovulated rabbit at 19∼20 hours post hCG injection. The collected oocytes were preactivated and self-enucleated by treating 5 uM ionoycin for 5 min, and 2.0 mM 6-DMAP for two hours. Microsurgical removel of the chromation complex in the second polar bodies was effectively performd and single blastomere separated from 32-cell stage rabbit embryos was injected into the perivitelline space of the enculeated recipient oocyts. Follwoing electrofusion and in vitro culture for 18 hours, the nuclear transplant(NT) embryos were transferred into the uterine horns of naturally mated or synchronized recipient does. When 32 NT embryous reconstituted with preactivated oocytes were transferred to 2 recipient does, one foster doe delivered two offspring (6.3%), while not a offspring was delivered from three foster does which received 17 NT embryos reconstituted with non-preactivated oocytes. A total of 68 NT embryos reconstituted with preactivated oocytes were transferred into the uterine horns of 7 synchronized ecipient does. Among them, two recipients were pregnant and delivered three offspring(5.9%).

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.136-136
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• 2003

Mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses, the overall success rate achieved by cloning techniques to date is low. This present study compared the incidences of DNA fragmentation during development of IVF, parthenotes (PT), nuclear transfer (NT) and transgenic (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counter staining was used for determination of DNA fragmentation and total number, respectively. TG and NT donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM+15% FCS until confluent, for 5 days. At 19 h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24 hpm with the combinations of ionomycin (5 M, 5 min) and cyclo-heximide (10 g/ml, 5 h) after electric fusion by a single DC pulse (1.6 KV/cm, 60 sec). Parthenotes were produced by the same activation protocol at 24 hpm. (중략)

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.281-284
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• 1994

Sex determination in genomic DNA from human blood leucocytes was performed by amplification of human Y chromosome-specific DNA sequences using PCR technique. A clear DNA fragment(154 nucleotides long) was appeared only in the male genomic DNA, but no specific band was observed in the case of female genomic DNA and negative control. To know the sensitivity of this method, the amplification reaction was performed in genomic DNA diluted to 2pg equivalent to the amonut present in the single human cell, and clear band also observed. The PCR amplification was so succesfully performed in the single leucocyte separated from human blood using micromanipulator that this techniqe is assumed to be applied to single blstomere before embryo transfer.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.1
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• pp.47-51
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• 2017

Objective: This study was designed to report the status of assisted reproductive technology (ART) therapy in South Korea between January 1, 2012 and December 31, 2012. Methods: A localized online survey, originally developed by the International Committee Monitoring Assisted Reproductive Technologies, was first launched and provided to all available ART centers via email in 2015. Fresh embryo transfer (FET) cases were categorized as standard in vitro fertilization, intracytoplasmic sperm injection (ICSI), or half-ICSI. Thawed embryo transfer (TET) and other related procedures, including surgical sperm retrieval, were surveyed. Results: Data from 33,956 ovum pick-up procedures were provided by 75 clinics in 2012. Of the 33,088 cycles in which ovums were retrieved, a complete transfer was performed in 90.5% (29,932 cycles). In addition, 10,079 FET cycles were confirmed to have resulted in clinical pregnancy, representing a pregnancy rate of 30.5% per ovum pick-up and 33.7% per ET. The most common number of embryos transferred in FET was 2 (41.6%), followed by 3 (34.0%), and non-elective single ETs (10.0%). Of the 10,404 TET cycles in which transfer was completed, 3,760 clinical pregnancies (36.1%) were confirmed by ultrasonography. Conclusion: The overall clinical pregnancy rate for FET and TET cycles in 2012 was higher than in 2011 (33.7% vs. 33.2% and 36.1% vs. 31.1%, respectively). The most common number of embryos transferred in FET cycles was 2, unlike in 2011.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.219-222
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• 2007

These study was carried out to investigate the effects of the collection time, culture time and activation of canine oocytes on in vitro maturation rates. The activated oocytes were cultured in 10% FCS+TCM-199 media containing hormonal supplements (10 IU/ml HCG, 10 IU/ml PMSG, 10 ug/ml gonadotropin) at 5% $CO_2$, 95% air, $38^{\circ}C$. 1. IVM rate of in vitro cultured cumulus-attached oocytes recovered from ovaries that collected at follicular and luteal stages of the reproductive cycles were 11.4% and 5.7%, respectively. IVM rate of oocytes recovered from ovaries that collected at follicular stages of the reproductive cycles was significantly higher than that of luteal stage (p<0.05). 2. When IVM was carried out at different periods of 40, 48, and 70 hrs, the IVM rates of oocytes matured in vitro were 2.9%, 8.6%, 5.7%, respectively. These results indicate that the IVM time between $48{\sim}70$ hrs gives the highest maturation rate for the oocytes matured at the different stages. 3. IVM rate of oocytes matured in vitro for 10 hrs after single and combined activation treatment by ET, IP and CH and Ca+DMAP, CH+DMAP, ET+CH were $11.5{\pm}1.2%,\;10.8{\pm}1.0%,\;9.6{\pm}1.2%\;and\;12.4{\pm}1.5%,\;11.8{\pm}1.5%,\;11.2{\pm}1.4%$ respectively. This was higher than that in both single and combined stimulated groups compared to control group ($6.2{\sim}7.2%$).

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.112-112
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• 2002

Under normal conditions, women produce a single dominant follicle that participates in a single ovuation each menstrual cycle. But Polycystic ovary syndrome(PCOS) conditions, folliculogenesis does not proceed normally. This condition leads to the accumlation of large numbers of small graffian follicles in which the theca interstitial cells (TIC) produce abnormally large amounts of androgen. PCOS is probably the most common endocrine disorder, affecting women of reprodutive age with 5-10% prevalence estimate. Chronic anovulation, hyperandrogenism, hirsutism, obesity, infertility and polycystic ovaries are clinical hallmarks of women with PCOS. Its etiology remains unknown. To investigate the gene expression pattern of ovary in PCO-induced rat, we used cDNA expression analysis. Total RNA was extracted from the ovary of PCO-induced rat and reverse-transcribed in the presence of［$\alpha$$^{32}$P］-dATP Which were hybridized to Atlas$^{TM}$ Rat Toxicology 1.2 array (Clontech) representing approximately 1176 rat genes. We compared gene expression between ovary of pco-induced immature female rats and control. Differential gene expression profiles were revealed (LIFR-alpha, ADRA1A, Heat shock 90-kDa protein A, PDGFRA). Reverse transcription-polymerase chain reaction(RT-PCR) was used to validate the relative expression pattern obtained by the cDNA array. The precise relationship between the altered expression of genes and PCO is a matter of further investigation. This study was supported by Korea Science and Engineering Foundation(KOSEF)

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.127-132
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• 2013

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.75-80
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• 2007

The objective of this study was to examine the optimal concentration and the exposure time of ethanol, Ca-ionophore, and strontium to achieve massive recipient oocytes in porcine. The cleavage (51.4% vs. $21.3{\sim}44.3%$) and embryo development rates (45% vs. $13.3{\sim}29.9%$) were significantly higher (p.0.05) in oocytes treated with 10% ethanol for 10 min than other treatments. The oocytes treated with 25mM Ca-ionophore for a minimum of 2min and 20mM strontium for a minimum of 6h showed significantly higher cleavage and embryo development rates than those of other treatments (P<0.05). Cleavage rate with duplicated ethanol treatment was significantly lower than those with ethanol alone (P<0.05). The cleavage rate and embryo development rates were significantly lower in duplicated strontium treatment than those in both alone and combination (P<0.05). But the cleavage and embryo development rates in treatment with Ca-ionophore were significantly higher in combined treatment (Ca-ionophore and cycloheximide) than those in single or duplicated treatment (P<0.05). These results might induce establishment of the optimal concentration and the exposure time on activation media to build up activation condition of porcine oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.261-265
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• 2000

Objective: This study was performed to evaluate the influence of maternal age on embryo quality and the frequency of multiple pregnancy in IVF-ET program. Method: 86 conventional IVF-ET cycles were divided into three groups according to the age by 5 year (group A: 26-30, group B: 31-35, group C: 36-40 yrs). The in vitro fertilization and development outcome (fertilization, cleavage and high quality embryo rate) and the pregnancy outcome (pregnancy, implantation, G-sac/high quality embryo and multiple pregnancy rate) were examined. And then, these results were compared among the groups. Results: The rates of fertilization (62.7, 68.5 and 65.4%, respectively) and cleavage (95.6, 97.6 and 98.0%, respectively) were not different among the groups. And the high quality embryo (HQE) rate also was not different among the groups (61.8, 62.9 and 62.8%, respectively). The pregnancy rate of group C (23.3%) was significantly lower than that of group A (41.2%) and B (48.7%). And the implantation rate was significantly decreased with advance in maternal age (group A; 17.3%, B; 12.6% and C; 6.0%). The G-sac/high quality embryo rate was significantly higher in group A (70.8%) when compared to group B (32.2%) and C (40.0%). On the other hand, the multiple pregnancy rate was significantly lower in group C (14.3%) when compared to group A (71.4%) and B (36.8%). Conclusion: The pregnancy rate was significantly decreased over 35 years. The G-sac/HQE and multiple pregnancy rate were significantly high below 31 years. Thus, these results suggest that the number of high quality embryo transferred should be limited by the age and another criteria for embryo quality evaluation were required for single embryo transfer.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.133-139
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• 1997

Large scale production of cloned embryos requires the technology of multiple generational nuclear transfer(NT) by using NT embryos itself as the subsequent donor nuclei. In this work we investigated comparatively the effects of enucleated oocytes treated with ionomycin and 6-DMAP on the electrofusion rate and in vitro developmental potential in the first and second NT embryos. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 15 hours after hCG injection. The enucleated oocytes were pre-activated by 5 min incubation in 5$\mu$M ionomycin and 2 hours incubation in 2 mM 6-DMAP at 19~20 hours post-hCG before microinjection. In the first and second generation NT, the unsynchronized 16-cell stage embryos were used as nuclear donor. The separated donor blastomeres were injected into the enucleated activated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of single pulse for 60 $\mu$sec at 1.25kV/cm in $Ca^2$+, $Mg^2$+ - free 0.28 M mannitol solution. In the non-preactivation group, the electrofusion and electrical stimulation was given 3 pulses for 60 $\mu$sec at 1.25 kV/cm in 100$\mu$M $Ca^2$+, $Mg^2$+ 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in TCM-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The results obtained were summarized as follows: 1. In the first generational NT embryos, the electrofusion rate of preactivated and non-activated oocytes(80.4 and 87.8%) was not significantly different, but in the second generational NT embryos, the electrofusion rate was significantly(P<0.05) higher in the non-activated oocytes(85.7%) than in the preactivated oocytes(70.1%). 2) In the first and second generational NT embryos, the developmental potential to biastocyst stage was significantly(P<0.05) higher in the preactivated oocytes(39.3 and35.7%) than in the non-preactivated oocytes(16.0 and 13.3%). No significant difference in the developmental potential was shown between the first and second generational NT embryos derived from the preactivated oocytes. In conclusion, it may be efficient to use the oocytes preactivated with ionomycin and 6-DMAP for the multiple production of cloned embryos by recycling nuclear transfer.