• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.325-333
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• 1997

The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VS11 and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VS11 is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.1-12
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• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• 한국발생생물학회지:발생과생식
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• 제5권1호
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• pp.23-33
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• 2001

포유류의 난자가 수란관내로 배란될 때는 난포액 성분도 같이 수란관내로 들어간다. 본 연구에서는 처음으로 난포액의 일부 성분이 수란관액에 의해서 변화하는 것을 관찰하였다. 사람의 난포액을 gelatin zymogram으로 분석한 결과 621kDa gelatinase 이외에 110kDa gelatinase (GA110) 등의 여러 gelatinase 활성이 나타났다. 이 활성들은 EDTA나 phenanthroline에 의해 억제된 반면 PMSF 처리에 의해서는 아무런 변화가 없었다. 소의 수란관액에서는 62kDa gelatinase의 활성만이 주로 관찰되었다. 소의 수란관 액과 사람의 난포액을 1:1로 섞고 이를 37$^{\circ}$C에서 3시간 동안 둔 결과 사람의 난포액의 GA110 활성은 사라졌다. 사람의 난포액에 APMA를 첨가한 결과 GA110의 활성은 대부분 감소하고 대신 62kDa gelatinase의 활성은 오히려 증가하였다. 반면에 사람의 난포액에 EDTA를 3시간 동안 처리한 결과 GA110의 활성은 오히려 현저히 증가하였고 이 때 다른 gelatinases의 활성은 영향을 받지 않았다. PMSF나SBTI는 난포액내의 gelatinases활성에 아무런 변화를 일으키지 않았다. EDTA, PMTA 혹은 SBTI 등의 proteinase inhibitor를 미리 처리한 사람의 난포액에 소의 수란관 내액을 섞은 경우에도GA110의 활성은 여전히 감소하였다. 사람의 혈청에서도 EDTA에 의해 활성이 현저히 증가하는 GA110이 발견되었다. 사람의 난포액과 유사하게 혈청내의 GA110도 소의 수란관액에 의하여 활성이 사라졌다. 그러나 사람의 난포과립세포의 추출물에서는 단지 92kDa gelatinase만 관찰이 되었다. 마지막으로 anti-human gelatinase A 항체를 사용하여 사람의 난포액과 혈청 그리고 난포과립세포의 추출액을 western blotting한 결과 621kDa과 GA110 만이 항원-항체 반응을 나타내었다. 이 같은 결과로 미루어 사람의 난포액과 혈청에는 gelatinase A의 독특한 isoform인 GA110이 있으며 특히 난포액내의 GA110은 수란관액성분에 의해 선택적으로 분해되는 것으로 여겨진다.

• 한국수정란이식학회지
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• 제26권2호
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• pp.97-104
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• 2011

This study was carried out to confirm the effects of luteotrophin, human chorionic gonadotrophin (hCG), and an anti-luteolytic agent, flunixin meglumin (FM), on pregnancy rates in Hanwoo with in vitro produced (IVP) embryo transfers (ET), and to research the effects on the estrus cycle. Treatments included hCG and FM administration 3~10 minutes prior to ET. Also, pregnancy rates were compared with lidocane treatment and FM treatment prior to ET. The results are shown below. 30-day pregnancy rate was 76.7% in the hCG-treated group and 75.7% in the FM-treated group. Both rates were higher than the 70% rate for the control group. 42-day pregnancy rate was 76.7% in the FM-treated group. This was higher than 66.7% recorded for both the hCG-treated and control groups. The pregnancy rate of the hCG-treated group was high at Day 30 (76.7%) but low at Day 40 (66.7%), and there were no differences from the FM-treated and control groups. The recurrent estrus rate of infertile individuals at 2 weeks after ET was 36.4% in the hCG-treated group, under 71.4% in the FM-treated group and 80.0% in the control group. The non-pregnancy rate of individuals without recurrent estrus was 18.2% in the hCG-treated group, which was higher than the 0% rate in both the FM-treated and control groups. The pregnancy rates were higher in the FM-treated group than the Lidocane-treated group with 72.3% versus 67.5% in the heifers and 48.9% versus 43.6% in the cows. From the above results, the FM treatment proved more effective than the hCG treatment and no treatment whatsoever in increasing pregnancy rates after ET. In addition, hCG treatment was shown to be undesirable due to the deviations it caused in the reproductive physiology of the hCG-treated recipients. Therefore, in our study, the FM treatment resulted in a higher pregnancy rate than either lidocaine treatment or no-treatment in the trials of ET.

• Clinical and Experimental Reproductive Medicine
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• 제42권2호
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• pp.45-50
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• 2015

Objective: Artificial oocyte activation (AOA) is an effective method to avoid total fertilization failure in human in vitro fertilization-embryo transfer (IVF-ET) cycles. AOA performed using a calcium ionophore can induce calcium oscillation in oocytes and initiate the fertilization process. We evaluated the usefulness of AOA with a calcium ionophore in cases of total fertilization failure in previous cycles and in cases of severe male factor infertility patients with non-motile spermatozoa after pentoxifylline (PF) treatment. Methods: The present study describes 29 intracytoplasmic sperm injection (ICSI)-AOA cycles involving male factor infertility at Cheil General Hospital from January 2006 to June 2013. Patients were divided into two groups (control, n=480; AOA, n=29) depending on whether or not AOA using a calcium ionophore (A23187) was performed after testicular sperm extraction-ICSI (TESE-ICSI). The AOA group was further split into subgroups according to sperm motility after PF treatment: i.e., motile sperm-injected (n=12) and non-motile sperm-injected (n=17) groups (total n=29 cycles). Results: The good embryo rate (52.3% vs. 66.9%), pregnancy rate (20.7% vs. 52.1%), and delivery rate (10.3% vs. 40.8%) were lower in the PF/AOA group than in the control group. When evaluating the effects of restoration of sperm motility after PF treatment on clinical outcomes there was no difference in fertilization rate (66.6% vs. 64.7% in non-motile and motile sperm, respectively), pregnancy rate (17.6% vs. 33.3%), or delivery rate (5.9% vs. 16.7%) between the two groups. Conclusion: We suggest that oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles regardless of restoration of sperm motility after PF treatment. AOA may be useful in selected patients who have a low fertilization rate or total fertilization failure.

• Clinical and Experimental Reproductive Medicine
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• 제31권2호
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• pp.111-117
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• 2004

Objectives: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. Materials and Methods: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)$\leq$3ea), Group II (n=80) is normal responder (retrieved oocytes>3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by $\beta$-actin. Statistical analysis was performed by using $X^2$ test, Student's t-test and Pearson correlation. Results: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). Conclusion: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.

• 과학기술학연구
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• 제12권1호
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• pp.185-207
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• 2012

황우석 사태 이후 한국의 국가는 줄기세포 연구를 장려하고 시험관 아기 산업을 장려하겠다는 입장과 "글로벌 스탠다드"에 부합하는 윤리적 규제를 도입하겠다는, 많은 경우 서로 모순될 수밖에 없는 입장을 표명하여 왔다. 줄기세포 연구에 대한 윤리적 규제가 점점 강화되면서 인간배아세포 연구가 위축되면서, 연구 공동체와 바이오산업, 임상의사와 환자, 그리고 국가 자체를 위기로부터 구원해줄 대안으로 떠오른 것은 체세포 줄기세포였다. 그러나 한국 생명공학기술에 대한 연구들은 주로 배아줄기세포에 초점을 맞추고 있으며, 조혈줄기세포나 지방유래줄기세포와 같은 체세포 줄기세포에 대한 연구에는 상대적으로 관심이 적은 것으로 보인다. 배아줄기세포가 흔히 실험적이고 윤리적으로 논란거리로 여겨지는 반면에, 조혈모 혹은 간엽줄기세포와 체세포 줄기세포는 별다른 공적인 논의 없이 대중들의 일상 속으로 들어와 있다. 한국의 많은 일반인들은 조혈모 줄기세포 치료를 통해 백혈병으로부터 생명을 구한 환자들의 사례에 이미 익숙한가 하면, 다른 한편에서 지방유래줄기세포 치료를 선전하는 의사들의 수가 늘고 있고, 지방유래줄기세포의 개념을 활용하여 만든 화장품이 소비자들의 주목을 받고 있기도 한 현실이 이미 진행되고 있다. 이러한 맥락에서, 본 논문은 배아줄기세포나 국가 정책이나 연구 규제에만 집중되어 시장을 놓치고 있는 윤리적 논의는 한국에서 줄기세포 기술의 정치의 전모를 다루기에 한계가 크다는 사실을 주장하고자 한다.

• 한국동물생명공학회지
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• 제37권4호
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• pp.292-297
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• 2022

Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.

• Journal of Yeungnam Medical Science
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• 제12권1호
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• pp.124-134
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• 1995

Ham's F-10 기본 배양액과 이 배양액에 여러가지 농도의 EDTA와 태아제대혈청을 단독 또는 같이 혼합한 배양액을 얻어 각각의 배양액에서 5-10주된 백서를 이용하여 2 세포기 배아의 단계적 분할정도를 96시간 동안 관찰하였다. Ham's F-10 기본 배양액에 비해 태아제대혈청이나 EDTA $50{\mu}M$에서 $100{\mu}M$을 단독 또는 함께 첨가한 배양액에서 2 세포기의 배아의 상실배기 난할률이 매우 높았으며 (p<0.05), 포배기 난할률은 태아제대혈청과 EDTA $50{\mu}M$에서 $100{\mu}M$을 첨가한 배양액에서 유의하게 높았다(p<0.05). 체외수정실에서 흔히 사용되는 태아제대혈청을 첨가한 배양액과 비교시 태아제대혈청과 EDTA $100{\mu}M$을 첨가한 배양액에서 상실기 난할률이 유의하게 높았다(p<0.05). 태아제대혈청과 여러 농도의 EDTA를 첨가한 배양액과 EDTA만 첨가한 배양액의 비교에서 태아제대혈청과 EDTA $50{\mu}M$ 및 $100{\mu}M$을 첨가한 배양액에서 EDTA $200{\mu}M$만 첨가한 배양액에 비해 상실기 난할률이 매우 유의하게 높았고(p<0.05), 포배기 난할률은 태아제대혈청과 EDTA $100{\mu}M$을 첨가한 배양액이 EDTA $200{\mu}M$을 단독 또는 태아제대혈청과 함께 넣은 배양액에 비해 유의하게 높았다(p<0.05). 결론적으로 Ham's F-10 기본 배양액에 태아제대혈청과 적정 농도의 EDTA $50{\mu}M$ 및 $100{\mu}M$을 첨가한 배양액에서 초기 수정란의 분할이 우수함을 알 수 있고, 이는 수정란의 배양시 적절한 배양액을 선택할 수 있는 기초 자료로서 유용할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제34권2호
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• pp.107-115
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• 2007

목 적: ISO 9001:2000 품질경영시스템은 국제적인 인중 기준에 의해 제품 및 서비스의 품질을 적절하게 판정하고 이를 향상시키기 위해 시행되고 있다. 본 연구에서는 이러한 품질경영시스템을 보조생식술 센터에 성공적으로 도입하고 적용하는 과정에서 확인된 효용성에 대해 기술하고자 한다. 연구방법: 제일병원 아이소망센터는 2004년 1월부터 ISO 9001:2000 인증을 위한 활동을 시작하였으며, 고객만족도조사와 주기적인 내부심사와 사후심사를 실시하였다. 심사과정에서 확인된 부적합 사항에 대한 시정 및 예방 조치와 이에 따른 효과성을 검증하였으며, 고객불만사항에 대한 지속적인 관리와 개선을 위한 프로젝트를 진행하였다. 결 과: 본 센터는 초일류 불임센터로 성장하고 최적의 품질경영시스템을 확립하기 위한 품질방침을 설정하여, 2004년 6월에 한국품질재단 (Korean Foundation for Quality)으로부터 "체외수정 및 배아이식 시술에 관련된 연구"에 대하여 ISO 9001:2000 인증을 받았다. 이러한 품질방침을 실현하기 위해 품질경영시스템에 적합한 품질매뉴얼, 프로세스, 절차서, 지침서 등에 대한 문서화를 완료하였다. 삼 년 동안의 내부심사와 사후심사에서 각각 140건과 7건의 부적합이 확인되었으며, 이에 대한 시정조치를 실시하였다. 결 론: 본 센터에서는 ISO 품질경영시스템의 도입과 운영을 통해 고객만족도의 향상, 체계적인 문서화를 통해 업무의 투명화와 효율화가 가능하였다. 보조생식술 센터에서의 ISO 품질경영시스템은 보조생식술에 대한 국가적인 관리시스템을 효율적으로 운영하기 위한 기본적인 기관별 경영시스템으로서 활용이 가능할 것으로 생각된다.