钙离子载体A23187联合嘌呤霉素激活行卵母细胞质内单精子注射后未受精卵母细胞的研究

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-ICSI 22h组（33个）、IVM—ICSI 44h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。结论钙离子裁体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。

Oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection

OBJECTIVE: To investigate the effect of assisted oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection (ICSI). METHODS: All 113 discarded oocytes that showed no evidence of fertilization at 16 - 18 hours after in vitro maturation (IVM)-ICSI cycles and conventional ICSI were assigned to four groups according to the time after ICSI: IVM-ICSI 22-hour group (n = 33), IVM-ICSI 44-hour group (n = 18), ICSI 44-hour group (n = 37) and ICSI 68-hour group (n = 25). All unfertilized oocytes were exposed to calcium ionophore A23187 (5 micromol/L) for 5 minutes and subsequently incubated with puromycin (10 microg/ml) for 4 hours. After incubation, the oocytes were cultured in vitro for 3 - 5 days. The activation rate, proportion of oocytes that showed pronucleus formation and cleavage rate were calculated after activation. Sex chromosomal analysis was performed by dual color fluorescence in situ hybridization (FISH) on the embryos that displayed two pronuclei and a second polar body. RESULTS: The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 - 68 hours after ICSI. Best results were achieved in IVM-ICSI 22-hour group, which elicited 88% (29/33) of activation rate, 62% (18/29) of cleavage rate and 28% (5/18) of 4-cell embryos. One embryo in this group developed to the morular stage. The activation rate and developmental potential of the activated embryos in IVM-ICSI 44-hour group, ICSI 44-hour group and ICSI 68-hour group decreased. FISH analysis showed 4 embryos with XX and 9 embryos with XY in 16 embryos. CONCLUSIONS: The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 - 68 hours after ICSI. The cultured time of unfertilized oocytes after ICSI affects activation efficiency and developmental potential of the activated embryos. The activated zygotes that display two pronuclei and a second polar body can develop normally.