• 대한생식의학회:학술대회논문집
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• 대한불임학회 1997년도 제34차 추계 학술대회
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• pp.23-29
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• 1997

• 한국가축번식학회지
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• 제22권4호
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• pp.395-403
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• 1998

본 연구에서는 돼지난자내 원형정자 주입후 수정율과 체외 배 발달을 조사하였다. 원형정자 주입 2시간 전에 인위적 전기자극을 주고 원형정자를 주입했을 때 난자들의 수정율이 주입 직후에 전기자극을 준 것들과 전기 자극을 주지 않은 것들보다 수정율이 높았다. 원형정자와 원형 정자핵을 각각 주입 한 후 전핵 형성율과 전핵 이동율을 조사하였으나 유의차를 발견할 수 없었다. 전핵의 형성과 이동중 미세소관의 움직임을 간접형광면역법 및 공춧점 현미경을 사용하여 조사해본 결과, 난활성 직후 난자의 표층에서 미세소관이 발생해서 이것에 의해 웅성 및 자성전핵이 난자 중심부로 이전됨을 볼 수 있었다. 원형정자와 원형정자핵 주입후 배양 6일째에 각각 25% 와 27%의 배반포로 형성되었고, 8 일째 형성된 배반포를 염색하여 관찰한 철과 세포수가 각각 평균 87에서 99개가 형성되었음을 알 수 있었다. 이러한 결과는 체외 성숙된 돼지 난자 내에 원형정자 혹은 원형정자 핵을 미세 주입하는 방법에 의해 정상척인 배발달을 하는 돼지 수정란 생산이 가능한 것을 보여주는 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권3호
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• 한국가축번식학회지
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• 제22권2호
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• pp.195-202
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• 1998

본 연구에서는 돼지 난자 내에 돼지, 사람, 소 및 생쥐의 정자를 미세 주입한 후 전핵형성과ㅏ 전핵의 이동을 관찰하였다. 핵과 미세소관은 정자 주입 후 간접면역 형광염색을 실시한 후 공초점주사현미경으로 관찰하였다. 돼지 난자 내에 돼지정자를 직접 주입하였을 경우 일반적인 수정과정과 동일하게 정자중편부에서 성상체가 형성되었고, 이 성상체에 의해서 웅성 및 자성 전핵의 이동(44%), 유사분열(3%) 및 2-세포기(13%)까지 정상적인 수정이 이루어지는 것을 관찰할 수가 있었다. 반면에 이종(사람, 소 및 생쥐)의 정자를 돼지난자에 직접 주입하였을 경우 단위발생시 난 활성이 유도된 난자와 같이 난자자체에서 형성된 미세소관에 의해 전핵이 이동(47, 30 및 17%)하는 것을 볼 수가 있었다. 하지만, 접합체 형성 및 2-세포기로의 분리되는 과정은 관찰할 수 없었다. 이러한 결과로 돼지 난자 내에 이종의 정자가 주입되었을 때 정자의 핵은 비록이적으로 전핵으로 발달되고 난자 중심부로 이동된다는 것을 보여주는 것인데, 이때 전핵을 움직이는 것은 정자에서 유래된 중심체에 의한 것이 아니라 난자세포질 자체의 미세소관에 의한 것으로 관찰되었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.52-52
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, one group of oocytes was activated with 7% ethanol for 5 min, and second group was not activated. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24-30 hrs in a incubator with 5% CO₂ in air at 38.5℃. (omitted)

• Clinical and Experimental Reproductive Medicine
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• 제41권3호
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• pp.137-139
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• 2014

The effect of artificial oocyte activation (AOA) with a calcium ionophore on intracytoplasmic morphologically selected sperm injection (IMSI) was examined in patients with histories of repeated failed implantation attempts. Four singleton pregnancies and one twin pregnancy were obtained after embryos transfer (5/14, 35.7%). Therefore, AOA combined with IMSI can be considered an option for cycles with a fertilization defect and recurrent implantation failures.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 1996년도 추계 학술대회 및 정기총회
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• pp.66.1-66.1
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• 1996

• 대한생식의학회:학술대회논문집
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• 대한불임학회 1999년도 제38차 추계 학술대회
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• pp.73-74
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• 1999

• Clinical and Experimental Reproductive Medicine
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• 제44권2호
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• pp.90-95
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• 2017

Objective: We investigated whether the insemination method (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]) affected morphokinetic events and abnormal cleavage events in embryonic development. Methods: A total of 1,830 normal fertilized embryos were obtained from 272 IVF and ICSI cycles that underwent ovum retrieval culture using a time-lapse system (Embryoscope) from June 2013 to March 2015. All embryos were investigated by a detailed time-lapse analysis that measured the developmental events in the hours after IVF or ICSI insemination. Results: No significant differences were observed between the two groups regarding clinical outcomes (p>0.05). ICSI-derived embryos showed significantly faster morphokinetics than those derived from conventional IVF, from the time to pronuclear fading to the time to 6 cells (p<0.05). However, no significant differences were found from the time to 7 cells to the time to expanded blastocyst (p>0.05). There were no differences in abnormal cleavage events between the two groups (p>0.05); they showed the same rates of direct cleavage from 1 to 3 cells, 2 multinucleated cells, 2 uneven cells, and reverse cleavage. Conclusion: The morphokinetics of embryo development was found to vary between IVF- and ICSI-fertilized oocytes, at least until the 6-cell stage. However, these differences did not affect the clinical outcomes of the embryo. Additionally, no significant differences in abnormal cleavage events were found according to the fertilization method.

• Clinical and Experimental Reproductive Medicine
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• 제42권3호
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• pp.118-125
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• 2015

Objective: The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. Methods: A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. Results: The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate ($76.1%{\pm}37.3%$ vs. $49.0%{\pm}49.1%$, $66.7%{\pm}48.7%$; group I vs. group II, group III, respectively) and the average number of transferred embryos ($1.5{\pm}0.7$ vs. $1.1{\pm}0.4$, $1.1{\pm}0.6$). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). Conclusion: In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.