冷冻保存对人卵母细胞纺锤体及发育潜能的影响

Effects of cryopreservation on the spindles and developmental potentiality of human oocytes

背景:卵子冷冻技术的发展为人类辅助生殖技术带来重大的突破,但卵子冷冻的效果并不理想。目的:研究冷冻保存对不同成熟时期人卵母细胞纺锤体及发育潜能的影响。设计、时间及地点:随机对照细胞学体外实验,于2005-04/2006-05在重庆市妇幼保健院完成。材料:体外受精-胚胎移植和卵胞浆内单精子显微注射治疗周期中剩余的未成熟卵子(GV期和MI期)。方法:将未成熟卵子随机分为对照组、MI/GV卵冷冻组和体外成熟后MII卵冷冻组,用慢冻速融的方法对MI/GV卵和体外成熟的MII卵进行冷冻和解冻,应用Polscope对卵子的纺锤体进行观察,并观察卵子的发育潜能。主要观察指标:不同组卵子的纺锤体可见率,体外成熟情况,冷冻不同时期卵子的复苏情况,及后期的受精、卵裂、可用胚胎情况。结果:对照组纺锤体可见率77.3%,明显高于MII卵冻融组、MI/GV卵冻融组(56.3%,53.8%,P<0.05),冷冻造成了纺锤体的损伤;冷冻后未成熟卵的体外成熟率下降,但无统计学差异;MII期卵、MI期卵和GV期卵复苏率相似;对照组的受精率(81.8%)明显高于MII卵冻融组、MI/GV卵冻融组(62.5%,59.0%,P<0.05),冷冻后的卵子受精能力受到损害;对照组的卵裂率与MII卵冻融组差异无显著性意义(84.4%,72.7%,P>0.05),MII卵冷冻组的卵裂没有受到明显影响;对照组的卵裂率与MI/GV卵冻融组差异有显著性意义(84.4%,52.9%,P<0.05),MI/GV卵冻融后卵裂率明显下降;冷冻组的胚胎质量下降,可用胚胎率明显降低。结论:控制性超排卵周期中剩余的未成熟卵子无论是直接冷冻还是先体外培养成熟后再冷冻,卵子的纺锤体都受到了一定的损伤,对卵子的发育潜能造成了影响。

BACKGROUND： The development of oocytes cryopreservation technique has brought a great breakthrough in Assisted Reproductive Technology, but the result of cryopreserving oocytes remains unsatisfactory. OBJECTIVE： To study the effect of cryopreservation on the spindle and the development potentiality of human oocytes at different mature stages. DEDIGN, TIME AND SETTING： A randomized control cytological in vitro experiment was performed in Chongqing Health Center for Women and Children from April 2005 to May 2006. MATERIALS： The surplus immature oocytes （GV and MI） obtaining from the patients undergoing the treatment of in vitro fertilization-embryo transfer （IVF-ET） or intracytoplasmic sperm injection （ICSI）. METHODS： Immature oocytes were divided randomly into a control Group, a MI/GV frozen group and an in vitro maturation （IVM） MII frozen group. Freezing and thawing of MI/GV and IVM MII oocytes were conducted on the principle of slow-freeze and rapid thaw. Polscope was used to observe the spindle of oocytes, and the developmental potentiality of oocytes was also observed. MAIN OUTCOME MEASURES： The rate of visible spindles and the IVM state of oocytes in each group, the resuscitation rate of oocytes at different stages of cryopreservation, and the fertilization, cleavage, available embryo of oocytes in later cryopreservation phase. RESULTS： The visible spindle rate was 77.3% in the control group, which was obviously higher than in the MII and the MI/GV frozen groups （56.3%; 53.8%, P 〈 0.05）. It showed that the spindle got injured by freezing; The IVM rate of immature oocytes decreased after freezing, but the difference was of no significance; The resuscitation rate was similar between MII and MI/GV oocytes; The fertility rate was obviously higher in the control group （81.8%） than in the MI/GV and MII frozen group （62.5%; 59.0%, P 〈 0.05）, which indicated that the fertilizability of oocytes was injured after freezing; There was no difference of significance in cleavage rate between the control group and MII frozen group （84.4%; 72.7%, P 〉 0.05）, which suggested that cleavage in the MII frozen group was not affected; However, in the MI/GV frozen group, the cleavage rate obviously decreased, and its difference with control group was of significance （84.4%; 52.9%, P 〈 0.05）. The embryos quality of frozen groups decreased, so did their available embryo rates. CONCLUSIONS： No matter performed before or after IVM, cryopreservation affects the spindle and the developmental potentiality of the immature human oocytes surplus from controlled ovarian hyperstimulation cycle.