• Clinical and Experimental Reproductive Medicine
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• 제29권3호
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• pp.167-178
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• 2002

Objective : The present study was performed to investigate whether apoptosis occur in human embryos by annexin staining and detect the expression of Fas, Fas-ligand (FasL), Bax, and Bcl-2 in human fragmented embryos derived from IVF-ET by immunofluorescence and Western blot analysis. Materials and Methods: Using annexin staining, immunofluorescence and Western blot analysis on normal and fragmented embryos, we were able to detect apoptotsis and apoptotic gene products in fragmented embryos. Result: Phosphatidylserine (PS) translocation, the marker for apoptosis, were detected frequently in fragmented embryos. Bcl-2 and Bax protein were detected in both fragmented and non-fragmented embryos. When fragmented embryos compared to normal embryos, immunofluorescent intensity of Bcl-2 tended to be lower in fragmented embryos. Bax gene expression increased in the fragmented embryos compared to the normal embryos. This result supports a model in which the molar ratio of Bcl-2 to Bax determines whether apoptosis induced or inhibited in human embryo. Fas was highly expressed in human preimplantation embryos but not FasL. It suggests that embryo may undergo apoptosis by binding with FasL produced by follicular or immune cells. Conclusion: The over expression of Bax and Fas will trigger apoptosis to lead embryo fragmentation and change embryo to be nonviable.

• Animal cells and systems
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• 제15권3호
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• pp.211-218
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• 2011

Fragmentation in human pre-implantation embryos has been suggested as the process of apoptosis. We have previously demonstrated a direct relationship between the increased reactive oxygen species (ROS) and apoptosis in human pre-implantation embryos. ROS is known to suppress the function of mitochondria in which steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) are presented. Therefore, the purpose of this study was to examine the expression of StAR and PBR in human pre-implantation embryos and to evaluate whether reduction of these proteins is associated with apoptosis. Apoptosis was detected by annexin V-fluorescein isothiocyanate (FITC) and mitochondrial membrane potential was measured by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1). Immunofluorescence staining and Western blotting were applied to examine the expression of StAR and PBR in the embryos. Lipid droplets in the embryos were stained with Oil Red O. The fragmented pre-implantation embryos were stained with annexin V-FITC, but not the normal ones. The mitochondria with active membrane potential were present less in the fragmented embryos compared with the non-fragmented embryos. We also confirmed that both StAR and PBR were expressed in the embryos and their expression levels were lower in the fragmented ones. In addition, the number and size of lipid droplets were increased in the fragmented embryos. The present study provides evidence that reduction of StAR and PBR in human pre-implantation embryos is associated with an increase in the lipid droplets leading to apoptosis.

• Clinical and Experimental Reproductive Medicine
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• 제22권3호
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• pp.279-285
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• 1995

Despite the frequent incidence of embryo fragmentation in early human embryos, the reason of the embryo fragmentation has not been known yet. This study was conducted to investigate the histological difference(s) between fragmented (FR) and non-fragmented (NFR) human embryos focusing on comparison of mitochondrial distribution and protein synthesis. Multi-pronuclei zygotes (MPZ) such as three or more pronuclei containing in human in vitro fertilization and embryo transfer (IVF-ET) program were used for this study. MPZ were cultured in TCM-199 supplemented with 10% of human fetal cord serum (hFCS) in 5% $CO_2$ incubator at $37^{\circ}C$ for 24 hours. The cleaved embryos to 2-4 cells after 24 hours were grouped by their grade of fragmentation. Embryos were stained with Rhodamine123 (Rh123) and fluorescence was evaluated under the fluorescence microscope through PB 450-490 filter (Leitz). Regarding to protein synthesis during early human embryogenesis, there is no significant difference in the amount of synthetic proteins between FR and NFR embryos. Distribution of cytoplasmic organelles in embryos was evaluated by transmission electron microscope (TEM). The cytoplasmic distribution of mitochondria was different between FR and NFR embryos. The mitochondrial distribution was even in NFR, whereas severely aggregated in FR. It is not able to clarify in the present study whether this uneven mitochondrial distribution in FR embryo is the reason for embryo fragmentation or is the result from fragmentation. Physiological disparity related to the mitochondrial distribution may be one of the reasons for embryo fragmentation. Further studies should be addressed to investigate the physiological differences between FR and NFR embryos.

• Clinical and Experimental Reproductive Medicine
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• 제37권4호
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• pp.339-348
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• 2010

목적: 본 연구는 체외수정 및 배아이식술 (In vitro fertilization-embryo transfer, IVF-ET)에서 인간의 파편화된 배아를 대상으로 수행한 파편제거술이 배아의 발달과 임상적 결과에 미치는 유용한 결과를 조사하고자 수행하였다. 연구방법: 본 연구는 전향적 연구로서 한나여성의원과 미즈메디병원에서 수행되었으며 IVF-ET 시술을 받는 60명의 환자를 대상으로 하였다. 실험군으로서 29명 환자의 106개의 파편화된 배아를 대상으로 이식하기 전 미세수술적 파편 제거술을 수행하였고 대조군으로서 31명의 환자의 122개의 파편화된 배아의 파편을 제거하지 않고 이식하였다. 파편 제거술이 파편화된 배아의 형태학적 변화와 임상적 결과에 미치는 영향을 조사하였다. 결과: 실험군 배아의 평균 형태학적 등급은 G2.79였으나 파편제거술 이후 G1.63 (p<0.001)로 유의하게 향상되었다. 대부분의 파편화된 배아는 파편제거 후 이어지는 배양과정 동안 파편화 현상이 재 발생하지 않았으며 파편이 제거된 배아의 발달에 파편제거술이 유용한 효과를 미치는 것이 관찰되었다. 실험군의 착상률과 임신율은 각각 12.3%와 31.3%이었으나 대조군은 각각 6.6%와 22.5%를 나타내었다. 이러한 두 군간의 결과의 차이는 낮은 시술 건수로 인해 통계학적 유의성은 없었다. 결론: 미세수술적 파편제거술은 파편화된 배아의 형태학적 등급뿐만 아니라 지속적인 발달능력을 향상시켰다. 파편제거술은 파편에 의해 손상된 세포간 전달체계의 복원과 파편에 의한 해로운 물질의 생성 가능성을 제거함으로써 주위의 할구들에게 이로운 효과를 나타낸 것으로 생각된다.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 1996년도 추계 학술대회 및 정기총회
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• pp.46.2-47
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• 1996

• Clinical and Experimental Reproductive Medicine
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• 제45권3호
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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.