Leoni G G, Berlinguer F, Succu S, Bebbere D, Mossa F, Madeddu M, Ledda S, Bogliolo L, Naitana S
Department of Physiological, Biochemical and Cellular Sciences, University of Sassari, Sassari, Italy.
Mol Reprod Dev. 2008 Feb;75(2):373-82. doi: 10.1002/mrd.20754.
The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess if in vitro-produced embryo quality could be determined by the timing of blastocoelic cavity re-expansion after vitrification, warming, and in vitro culture using sheep as a model. Blastocysts were produced in vitro, vitrified/warmed, and cultured in TCM-199 plus 10% FCS for 72 hr. Embryos were divided into two groups: re-expanded within 8 hr (A) and from 8 to 16 hr (B) of IVC after warming. Fast re-expanded blastocysts showed higher in vitro hatching rates and total cell number calculated on the hatched blastocysts compared with slow re-expanded ones (P < 0.01). Peroxide status evaluation (P < 0.01) and TUNEL test (P < 0.05) revealed a higher number of positive cells in group B compared with group A. The quantitative analysis of protein synthesis revealed a higher synthesis in fast compared with slow re-expanded embryos (P < 0.05). Quantitative RT-PCR showed that 90-kDa Heat Shock Protein beta was more expressed in group A than in group B (P < 0.05), while the quantity of P34(cdc2), Cyclin b, Aquaporin 3, Na/K ATPase, and Actin did not differ between the two groups. Pregnancy rates after transfer to synchronized recipients were higher in fast compared to slow re-expanded blastocysts (P < 0.05). Our results evidenced that timing of blastocoelic cavity re-expansion after vitrification/warming and in vitro culture can be considered as a reliable index of in vitro produced embryo quality and developmental potential.
准确选择有活力的胚胎对于提高妊娠率和避免徒劳的移植尝试具有重要价值。我们研究的目的是评估以绵羊为模型，通过玻璃化、复温及体外培养后囊胚腔重新扩张的时间，能否确定体外生产胚胎的质量。体外生产囊胚，进行玻璃化/复温处理，然后在添加10%胎牛血清的TCM - 199中培养72小时。胚胎分为两组：复温后体外培养8小时内重新扩张的（A组）和8至16小时重新扩张的（B组）。与缓慢重新扩张的囊胚相比，快速重新扩张的囊胚显示出更高的体外孵化率和基于孵化囊胚计算的总细胞数（P < 0.０1）。过氧化物状态评估（P < 0.０1）和TUNEL检测（P < 0.０5）显示，B组阳性细胞数高于A组。蛋白质合成的定量分析显示，快速重新扩张的胚胎比缓慢重新扩张的胚胎合成更高（P < 0.０5）。定量RT - PCR显示，90 - kDa热休克蛋白β在A组中的表达高于B组（P < 0.０5），而两组之间P34（cdc2）、细胞周期蛋白b、水通道蛋白3、钠钾ATP酶和肌动蛋白的量没有差异。与缓慢重新扩张的囊胚相比，快速重新扩张的囊胚移植到同期受体后的妊娠率更高（P < 0.０5）。我们的结果证明，玻璃化/复温及体外培养后囊胚腔重新扩张的时间可被视为体外生产胚胎质量和发育潜力的可靠指标。
