玻璃化冷冻和程序化冷冻方法对人类卵母细胞骨架及其发育潜能的影响

Effects of vitrification and slow freezing on ultrastructure and developmental potential of human oocytes

目的探讨玻璃化冷冻和程序化冷冻对人卵母细胞纺锤体定位、细胞骨架及其发育潜能的影响。方法将第2日发育为M_II卵母细胞随机分为对照组、程序化冻融组、玻璃化冻融组(解冻0 h、1 h、3 h)。应用液晶偏振光显微镜(Polscope)成像系统观察卵母细胞纺锤体与第一极体(Pb)的夹角、表面积、卵透明带内层光阻值和外层光阻值。采用扫描电子显微镜和透射电子显微镜观察卵母细胞的表面和内部超微结构。统计2种冻融方法对卵母细胞发育潜能的影响。结果对照组、程序化冷冻解冻培养3 h组和玻璃化冷冻解冻后培养0 h、1 h、3 h组中的纺锤体可见率分别为92.4%、56.4%、11.2%、24.8%、61.1%。与程序化冻融组相比,玻璃化冷冻解冻培养3 h后卵母细胞中纺锤体与Pb的夹角更小(37.3°与68°,P=0.023)。对照组、程序化冻融组和玻璃化冻融后培养3 h组中卵母细胞的纺锤体面积、卵透明带内层光阻值和透明带外层光阻值差异无统计学意义(P>0.05)。与程序化冻融组相比,玻璃化冻融后培养3 h组中卵母细胞表面突起丰富,微绒毛形态较为正常,倒伏在细胞表面,卵透明带边界较为清晰,与对照组较为接近。程序化冻融组的正常受精率(65.7%)明显低于对照组(79.2%,P=0.041),而卵裂率和囊胚形成率与对照组和玻璃化冻融组差异无统计学意义(P>0.05)。玻璃化冻融后培养3 h组中正常受精率、卵裂率、囊胚形成率与对照组相比,差异无统计学意义(P>0.05)。结论相比程序化冷冻,玻璃化冷冻对卵母细胞纺锤体和卵透明带的损伤及对卵母细胞的发育潜能的影响都较小,可以作为卵母细胞冷冻的一种有效方法。

Objective To compare the the spindles, cytoskeleton and the developmental potential of human oocytes between vitrification and slow freezing approaches by polscope and electron microscopy. Methods The immature human oocytes were randomly divided into control, slow freezing, and vitrification freezing-thawing groups（0 h, 1 h, 3 h after thawing）. The spindle, the angle of spindle to the first polarbody, the surface area of oocytes and the lining and outer retardance of zona pellucida were observed by Polscope. The surface and ultrastructure of oocytes were observed by scanning electron microscopy（SEM） and transmission electron microscopy（TEM）. Finally, the influences of two freezing methods on developmental capability of human oocytes were analyzed. Results The visible rate of spindle was 92.4%, 56.4%, 11.2%, 24.8%, 61.1% in control group, slow freezing-thawing group, and vitrification freezing-thawing after 0 h, 1 h and 3 h, respectively. Compared with slow freezing, the angle of the spindle to the first polar body in vitrification freezing-thawing after 3 h group was smaller（37.3°, 68°, P=0.023）. No significant differences were observed in the surface area of oocytes, the lining and outer ret of oocytes zona pellucida between vitrification freezing-thawing after 3 h group and slow freezing group. The protrusions of oocyte surface were increased, the microvilli were normal, and laid down on the membrane surface in vitrification freezing-thawing after 3 h group than slow freezing group, and similar results of better recovery of perivitelline space and mitochondria were obtained. The 2 pronucleus（PN） fertilization rate in slow freezing group（65.7%） was decreased compared with control group（79.2%, P=0.041）. No significant differences were observed between vitrification freezing-thawing after 3 h group and control group in 2 PN fertilization rate, cleavage rate and blastocyst formation rate. Conclusion Preliminary results suggest that the vitrification freezing-thawing for oocyte cryopreservation is a better choice than slow freezing-thawing.