研究開発・学会発表
診療・治療
第9回 環太平洋不妊会議
- Developmental potency of human embryos vitrified with carboxylated poly-L-lysine.
- 2013年11月13日（水）～14日（木） 神戸国際会議場
- 第9回 環太平洋不妊会議
- *Y. Tsuji1, Ph.D, H. Ogata1, Ph.D, K Furuhashi1, A. Kajiwara1, MD, Y. Tokura1, MD, S. Yamada1, MD, S.
Ogata1, MD, Ph.D, Y. Mizusawa1, MD, Ph.D, E. Okamoto1, MD, Ph.D, Y. Matsumoto1, S. MD, Kokeguchi1, MD, Ph.D, M. Shiotani1, MD, Ph.D, K. Matsumura2, Ph.D SH. Hyon3, Ph.D
1. Hanabusa Women’s Clinic, Kobe, Japan
2. Japan Advanced Institute of Science and Technology, Nomi, Japan
3. Center for Fiber and Textile Science, Kyoto Institute of technology, Kyoto, Japan
【Objective】
In vitrification of embryos, a combination of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) has been widely used as cryoprotectant agents (CPA). Although DMSO is the most effective CPA, it also has cytotoxicity to embryos. Recently, carboxylated poly-L-lysine (PLL) is shown to be effective for the cryopreservation of cultured cells, such as fibroblasts and iPS cells. In this study, we evaluated the efficacy and toxicity of PLL if it could be an alternative to DMSO.
【Design and method】
Experiment 1; Determination of optimal PLL concentration in mouse embryos
167 mice (ICR) embryos (8 cell) recovered from were vitrified with 15% DMSO, 5%, 10%, 15%, 20% or 25% PLL containing 20% serum substitute supplement (SSS), 15% EG and 0.5M sucrose. These embryos vitrified with each solution were thawed and then cultured to the blastocyst stage. The survival rate and the developmental rate into blastocysts were assessed in each group. In addition, the blastocysts were incubated with a fluorescein probe for Caspase 3/7 to detect apoptotic cells.
Experiment 2; Effect of PLL on human embryos.
Day 2 human embryos (day 0 means a fertilization day) donated by patients who had completed fertility treatment were used in this study. Forty human embryos were vitrified with 15% DMSO or 10% PLL containing 20% SSS, 15% EG and 0.5 M sucrose. These embryos vitrified by each solution were thawed and cultured to the blastocyst stage for evaluation in a same manner as Experiment 1.
【Result】
In Experiment 1, there was no significant difference in the survival rate (96.4% vs 93.1%, 93.1%, 96.3%, 92.6% and 96.3%) and the developmental rate into blastocysts (85.2% vs 81.5%, 85.2%, 80.8%, 84.0% and 80.8%) between each group. In addition, the ratio of apoptotic cells in total cells was 7.3%, 6.2%, 7.2%, 7.7%, 8.2% and 7.6%, respectively. There was no significant difference between groups.
In experiment 2, the survival rate vitrified with DMSO or PLL was 100%, respectively. In addition, there was no difference in the developmental rate of blastocysts (65% vs 60%) in both groups. Finally, the ratio of apoptotic cells in both DMSO and PLL groups showed similar percentage (22% vs 18%).
【Conclusion】
Our results indicate that PLL has an equivalent potency to DMSO in effectiveness and safety for the vitrification of human embryos.
