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目的观察钙离子载体A23187联合嘌呤霉素对卵母细胞质内单精子注射(ICSI)后未受精卵母细胞的激活作用。方法将体外成熟(IVM)-ICSI和常规ICSI后未受精卵母细胞,按行ICSI后体外培养的时间,分为IVM-ICSI22h组(33个)、IVM-ICSI44h组(18个)、ICSI44h组(37个)、ICSI68h组(25个),分别采用钙离子载体A23187联合嘌呤霉素进行激活处理。应用荧光原位杂交(FISH)技术,对来源于双原核合并第二极体合子的激活胚胎进行性染色体分析。结果钙离子载体A23187联合嘌呤霉素能激活行ICSI后22~68h未受精的卵母细胞。其中IVM-ICSI22h组卵母细胞激活率为88%(29/33)、总卵裂率为62%(18/29)、4细胞阶段胚胎发育率为28%(5/18),1个胚胎发育到桑椹胚阶段;而IVM-ICSI44h组、ICSI44h组、ICSI68h组的未受精卵母细胞激活率分别为56%(10/18)、65%(24/37)、52%(13/25);总卵裂率分别为20%(2/10)、42%(10/24)、46%(6/13),仅ICSI44h组有1个胚胎发育到4细胞阶段。FISH对激活胚胎的分析显示,4个胚胎为XX,9个胚胎为XY。结论钙离子载体A23187联合嘌呤霉素能有效激活行ICSI失败的卵母细胞;行ICSI后22h内,是对未受精卵母细胞进行辅助激活较为理想的时机。激活的双原核合并第二极体胚胎中有雄原核的存在。
Objective To investigate the effect of calcium ionophore A23187 and puromycin on the activation of unfertilized oocytes after intracytoplasmic sperm injection (ICSI) in oocytes. Methods The IVM-ICSI22h group (33), IVM-ICSI44h group (18), ICSI44h group were divided into IVM-ICSI22h group (IVM-ICSI44h group) and ICSI44h group (37), ICSI68h group (25), respectively, using calcium ionophore A23187 combined with puromycin for activation. Fluorescence in situ hybridization (FISH) was used to analyze the sex chromosomes of the activated embryos derived from the dihydrobase and the second polar zygotes. Results Calcium ionophore A23187 combined with puromycin could activate unfertilized oocytes 22 ~ 68h after ICSI. Among them, the rate of oocyte activation in IVM-ICSI22h group was 88% (29/33), the total cleavage rate was 62% (18/29), the rate of embryo development in 4-cell stage was 28% (5/18), one embryo The rate of unfertilized oocyte activation in IVM-ICSI44h group, ICSI44h group and ICSI68h group were 56% (10/18), 65% (24/37), 52% (13/25) ; The total cleavage rate was 20% (2/10), 42% (10/24), 46% (6/13) respectively. Only one embryo in ICSI44h group developed into 4-cell stage. FISH analysis of activated embryos showed that 4 embryos were XX and 9 embryos were XY. Conclusion Calcium ionophore A23187 combined with puromycin can effectively activate oocytes failed in ICSI. Within 22 hours after ICSI, it is an ideal time to assist the unfertilized oocytes. Activated dual prokaryotic combined with the presence of prokaryotic male in the second polar body embryo.