• 대한생식의학회:학술대회논문집
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• 1999.11a
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• pp.76.1-76.1
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• 1999

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 1998.05a
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• pp.3-7
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• 1998

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.17-23
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• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.58-58
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• 2003

포유동물 체외 수정란의 체외 발육율을 향상시키기 위해서는 배양액내에 생성되는 free radical을 제거하는 것이 한가지 요인으로 지적되고 있다. 체외 배양액내에 생성되는 free radical을 제거하기 위하여 여러 종류의 항산화제 첨가 배양이 많이 이용되어 체외발육성적을 향상시키고 있다. 포유동물의 번식에 있어서 Melatonin은 광주기 조절 작용에 기여하는 호르몬으로서 혈중 Melatonin의 농도는 번식 작용에 중요한 역할을 하며, 또한 수정란의 세포분열과 세포주기에 영향을 미치며 배양액내의 free radical을 제거하여 수정과 초기 수정란 발달에 영향을 미쳐 항산화 기능이 있는 것으로 보고되고 있다.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.207-216
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• 1994

본 연구는 돼지 난포란으로부터 생산된 수정란의 체외발달에 미치는 아미노산, 우태아혈청 (FBS)과 Leukemia Inhibitry Factor의 영향을 조사하였다. 돼지난포란은 도살된 돼지의 난소로부터 회수하여 39$^{\circ}C$, 5% CO2 배양조건하에서 1$\mu\textrm{g}$/ml FSH-p, 1$\mu\textrm{g}$/ml Estradiol-17$\beta$와 10%FBS가 첨가된 TCM-199 배양액내에서 42시간동안 성숙시켰다. 사출된 정자의 수정능 획득은 45와 90% Percoll density gradient법을 통한 원심분리에 의해 얻었으며, 이들 수정능획득된 정자를 성숙된 난포란이 함유된 배양액에 3$\times$105/ml의 농도로 주입하여 10$\pm$1시간동안 배양함으로서 체외수정을 유도하였다. 수정된 난포란은 ; 1) 10% FBS가 함유된 TCM-199, DMEM, mKRB 또는 CR1aa 배양액, 2) 아미노산 또는 10% FBS가 첨가된 CR1 배양액, 3) STO 세포 또는 mLIF (1,000 unit/ml) 첨가된 CR1aa(10%FBS) 배양액, 4) mLIF (1,000 unit/ml)를 수정 직후 또는 8-세포기 이후에 첨가된 CR1aa(10%FBS)의 네가지 배양조건에서 각각 분리 배양하였다. 그결과 체외수정란의 배반포까지 발달율은 아미노산과 10%FBS가 포함된 CR1 배양액에서 다른 배양액에서보다 양호하였고, 특히 8-세포기 이후에 mouse LIF를 첨가한 CR1aa(10% FBS) 배양액에서는 다른 배양조건보다 현저히 높은 결과를 보였으며, 부화 배반포까지도 배발달을 유도할 수 있었다. 따라서 돼지수정란의 발달에 있어서 배양액내에 아미노산과 FBS 및 mouse LIF의 첨가는 효과가 있으며, 특히 8-세포기 이후에 있어서 mouse LIF의 첨가는 돼지의 수정란을 배반포 이후의 단계에까지 발달시킬 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.3
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• pp.173-178
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• 2007

Objective: To compare the outcome of twin pregnancies after in-vitro fertilization (IVF) with that of spontaneously conceived twins. Methods: We analyzed 146 twins retrospectively, who had been delivered between January 2000 and July 2005. After reviewing their obstetric medical records, 72 twins conceived with IVF (IVF group) and 51 spontaneously conceived twins (spontaneous group), whose gestational age was over 24 weeks, were recruited for this study. Results: There were no significant statistical differences of demographic features such as maternal age, gestational age and fetal weight between the two groups. However, the risk of pregnancy-induced hypertension (PIH) and gestational diabetes (G-DM) had significantly increased in the group of IVF (OR 2.59; 95% CI 1.01$\sim$6.66). The risk of fetal weight discordancy rate (>20%) and fetal-sex homology rate in IVF group were decreased (OR 0.37; 95% CI 0.14$\sim$0.96, OR 0.45; 95% CI 0.21$\sim$0.99). Conclusion: In this comparative study, maternal PIH and G-DM risks of IVF group are higher than that of spontaneous group. But, fetal-weight discordancy risk and fetal-sex homology rate were lower in IVF group.

• Neonatal Medicine
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• v.18 no.2
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• pp.197-203
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• 2011

Purpose: The purpose of this study is to compare perinatal outcomes between in vitro fertilization (IVF) twins and naturally conceived twins born to women aged 35 years or older and to provide basic information for taking care of IVF twins born to women aged 35 years or older. Methods: We reviewed the records of perinatal and neonatal outcomes in 288 IVF twins and 220 naturally conceived twins born to women aged 35 years or older between January 2001 and December 2010 at CHA Bundang Medical Center. Results: No difference was observed in the maternal ages of mothers giving birth to IVF twins and those giving birth to naturally conceived twins. Gestational ages and birth weights of IVF twins were not different from those of naturally conceived twins. Various perinatal outcomes, including gestational diabetes mellitus, pregnancy-induced hypertension, placenta previa, premature amniotic membrane rupture, and need for a Cesarean section did not differ between the 2 groups. However, the 1-min and 5-min Apgar scores (P=0.019 and P=0.045, respectively) were different between the 2 groups. The incidence of early-onset sepsis was lower in the IVF twins than in the naturally conceived twins (P=0.02). However, the 2 groups did not show any difference in the incidence of respiratory distress syndrome, bronchopulmonary dysplasia, patent ductus arteriosus, necrotizing enterocolitis, intraventricular hemorrhage, and other congenital anomalies. Conclusion: The perinatal outcomes in IVF twins born to women aged 35 years or older were not significantly different from those of naturally conceived twins.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.115-115
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• 2003

아미노산은 수정란의 체외생산에 있어서 배발달에 유효하게 작용한다. 이러한 이유로 체외배양 단계에 아미노산 첨가에 관한 보고는 많지만, 체외성숙 단계에서의 아미노산 효과와 관련된 보고는 소수이다. 본 실험에서는 먼저 체외성숙 배지에 NEAA와 EAA군들의 첨가 농도가 PB 출현율, 배발달율 그리고 생산된 배반포의 세포수에 미치는 영향과 더불어 새로운 아미노산원으로 LAH의 효과를 검토하였다. 체외성숙을 위한 난포란은 도축 한우 난소에서 2-8mm의 가시난포로부터 회수하였다. 체외성숙용 배지는 10% FBS가 첨가된 CRlaa 용액을 사용하였고, 실험1에서는 NEAA와 EAA(20$\mu l$/ml)군을 각각 $\times$l (NEAA:10$\mu l$/ml, EAA:20$\mu l$/ml), $\times$2 및 $\times$4를, 실험2에서는 LAH를 각각 1, 5 및 10 mg/ml를 첨가하였다. 체외수정은 fer-TALP 용액을, 체외배양은 CRlaa 용액을 배양 2일째까지는 0.3% BSA를, 그 이후에는 10% FBS와 난관상피세포를 첨가하여 사용하였다. 그리고 배양 8일째 확장배반포의 세포수를 조사하기 위하여 이중형광염색을 실시하였다. 통계분석은 $X^2$-test를 이용하였다. 실험 1에서 NEAA와 EAA 첨가 농도에 따른 PB 출현율은 $\times$l 첨가군(46.6%)이 대조군(29.9%)에 비하여 유의적으로 높았다. 수정을, 8세포기 및 배반포까지 발달율은 비슷한 경향이었으나, $\times$l 첨가군이 가장 높았다. 한편 ICM 세포수가 아미노산 첨가 농도의 높을수록 증가하는 경향이었고, 특히 $\times$4 첨가군($23.9 \mu 3.9$)이 대조군($14.8 \mu 2.5$)에 비하여 유의적(P<0.05)으로 높았다. 실험 2에서 LAH 첨가에 따른 핵성숙율은 5mg 첨가군, 배반포까지 발달율은 1mg 첨가군(25.9%)이 각각 가장 높았다. 배양 8일째 배반포의 세포수에 있어서 총세포수와 TE 세포수는 차이가 없었으나, ICM 세포수가 l0mg 첨가군에서 가장 높았다. 본 실험 결과에서 체외성숙 배지에 NEAA와 EAA 첨가가 배발달율에는 효과가 없었지만, 첨가농도의 증가에 따라 ICM 세포수가 증가하였다. 한편 체외성숙 배지에 LAH 첨가는 첨가 농도가 높을수록 배발달율은 낮았지만 ICM 세포수는 증가하였다.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.27-33
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• 2003

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes in vitro maturation of canine oocytes and development of canine IVM/IVF embryos. The results were summarized as follows: 1. The developmental rates to 16 cells of fresh, salts and 4$^{\circ}C$-stored oocytes with and without cumulus cells were 14.3%, 5.0% and 7.5%, 2.8% and 5.7%, 0.0%, respectively. The rate of oocytes with cumulus cells(5.7%~14.3%) was higher than that of denuded oocytes(0.0%~5.0%). 2. The developmental rate to If cells of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 0.0%, 10.7%, 1.5%, respectively. 3. The developmental rate to 16 cells of fresh oocytes with cumulus cell cultured for 24, 32 and 48 hrs in $CO_2$ incubator were 0.0%, 5.3%, 11.8%, respectively. The rate of oocytes cultured for 48 hrs was higher than that oocytes cultured for 24 and 32 hrs. 4. The development to If cells treated activation and non-activation oocytes were 15.0%, 6.7%, respectively. The rate of oocytes treated activation was higher than that oocyte treat non-activation.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.221-227
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• 1999

Successful cryopreservation of bovine immature oocytes can increase availably of oocytes for the in vitro fertilization or nuclear transfer. However, it was not reported successful development to the blastocyst stage following in vitro fertilization of cryopreserved bovine immature oocytes. The objective of this study was to determine the incidence of survival, meiotic maturation, fertilization and in vitro development of cryopreserved bovine immature by ultra rapid cooling methods. The oocytes were adversely affected by brief exposure to EFS40 solution in electron microscope grids and plunged directly into liquid nitrogen. After such ultra-rapid cooled immature oocytes were warmed, 78% of oocytes were matured to the metaphase II stage, 50% of oocytes were fertilized after insemination, and 5% of oocytes were developed to the blastocyst stage. Different sodium concentration of sodium ion in the freezing medium did not affect survival, maturation, fertilization and in vitro development of cryopreserved oocytes. These results suggested that immature bovine oocytes can be cryopreserved by ultra-rapid cooling methods.