【作者】 张晓慧；

【导师】 曹云霞；

【作者基本信息】 安徽医科大学 ， 妇产科学， 2009， 硕士

【摘要】 第一部分ICSI后未受精人卵母细胞的人工辅助激活研究目的分别采用乙醇、乙醇+6-二甲基氨基嘌呤(6-DMAP)、离子霉素、离子霉素+6-DMAP、6-DMAP五种激活方法激活处理行卵胞浆内单精子注射(ICSI)后24h未受精人卵母细胞,观察和比较几种不同的人工辅助激活方法对行ICSI后24h未受精人卵母细胞的激活作用及效果。方法收集ICSI后24h仍无受精征象的卵母细胞共计200个,分别采用上述5种方法进行激活处理。16-18h后观察第二极体(PB)排出及原核(PN)形成情况。激活后正常受精的胚胎继续培养3-5天。统计五组的激活率、原核形成情况、卵裂率及后期胚胎发育情况。采用荧光原位杂交(FISH)的方法对激活处理后正常受精发育而来的胚胎进行分析,排除孤雌激活可能。结果五种激活方法处理后的卵母细胞激活率分别为65.6, 75.6, 83.7, 88.2和6.9%,其中离子霉素+6-DMAP组激活率最高,两种激活剂联合激活的激活率要明显高于一种激活剂单独激活(P<0.05);除离子霉素+6-DMAP组激活处理后的一原核形成能力上要明显高于6-DMAP组之外,其他组之间在各种类型原核形成能力上均无明显统计学差异(P>0.05)。FISH分析了35个从2PN发育而来的胚胎,其中有12个胚胎显示为XX信号,10个胚胎显示为XY信号。结论通过人工辅助激活方法是可以使得ICSI后未受精的人卵母细胞恢复正常受精,并且有继续发育的潜能;离子霉素+6-DMAP组的激活率最高,但在发育的潜能上各组之间并没有统计学上的差异。第二部分补救ICSI中人工辅助卵母细胞激活的探索研究目的探索补救ICSI中人工辅助卵母细胞激活技术的应用,是否可以提高受精率和改善胚胎后期的发育情况,并最终可应用于临床的可行性。方法收集常规IVF后无受精征象的人成熟卵母细胞共计148个,补救ICSI后,一组92个卵母细胞采用离子霉素+6-DMAP进行人工辅助激活处理,另一组56个未采用任何激活处理的作为对照组。16-18h后观察第二极体排出及原核形成情况。正常受精的胚胎继续培养3-5天。统计两组的受精率、原核形成情况、卵裂率及后期胚胎发育情况。采用荧光原位杂交(FISH)的方法对激活处理后2PN发育而来的胚胎进行分析,排除孤雌激活可能。结果实验组和对照组补救ICSI后受精率分别为89.1%(82/92)和78.6%(44/56),两组之间受精率比较有显著的统计学差异(P<0.05),正常受精(2PN)率分别为51.2%(42/82)和29.5%(13/44),也有显著的统计学差异(P<0.05)。但两组之间正常受精发育的胚胎在卵裂率、优质胚胎形成率上并没有显著差异(P>0.05)。FISH分析了实验组中12个从2PN发育而来的胚胎,其中4个为XY杂交信号,5个为XX杂交信号,性染色体正常的胚胎比例为75%。结论采用卵母细胞人工辅助激活可以提高补救ICSI的受精率,激活后受精的胚胎有进一步发育的潜能,FISH分析排除了孤雌激活,故人工辅助激活在改善补救ICSI的受精率上有一定的临床应用价值。更多还原

【Abstract】 Part I Study on the Effect of Activation on human unfertilized oocytes after intracytoplasmic sperm injectionObjectiveTo investigate the effect of different activation methods on human unfertilized oocytes after intracytoplasmic sperm injection (ICSI).DesignOocytes allocated to this study were those that did not show any signs of normal fertilization (extrusion of the second polar body, pronuclei formation) after ICSI for 24 hours, and that were still with no signs of degeneration or fragmentation. 200 oocytes were randomly subjected to one of five activation treatment, ethanol(ET), ethanol + 6-dimethylaminopurine (6-DMAP), ionomycin ( IO ) , ionomycin + 6-dimethylaminopurine (6-DMAP), 6-dimethylaminopurine (6-DMAP). Fluorescence in situ hybridization (FISH) was employed to analyze chromosomes using X and Y dual-colored probes.ResultsFertilization occurred in 138(69%) of 200 oocytes which were allocated to the chemical activation treatment groups. The activation rates of the oocytes in the ET, ET+6-DMAP, IO, IO+6-DMAP, 6-DMAP groups were 65.6%, 75.6%, 83.7%, 88.2% and 6.9%, respectively, showing that the activation rates of combining activation treatment groups were significantly higher than those of single activation treatment groups. Except the 1PN formation rate show a significantly difference with pronuclei formation between IO and IO+6-DMAP group, there was no other significantly differences with pronuclei formation among the groups. Sex chromosome analyses indicated 12 embryos with XX and 10 embryos with XY in 35 embryos derived from activated oocytes containing 2PN.Conclusions(1) It is possible to activate unfertilized oocytes after ICSI with chemicals, and the normal fertilized oocytes have further developmental potential; (2)ionomycin + 6-DMAP group showed the best activation rate among the groups, however we did not find any difference in developmental potential between the activation groups.Part II Study on the application of Oocytes Activation in Recue intracytoplasmic sperm injectionObjectiveTo investigate the application effect of Oocytes Activation in Rescue ICSI.DesignOocytes allocated to this study were those that did not show any signs of normal fertilization (extrusion of the second polar body, pronuclei formation) by 18h after IVF, and that were still with no signs of degeneration or fragmentation. 148 oocytes were randomly subjected to the study. The experimental group was that has total 92 oocytes which received chemical activation treatment after rescue ICSI , the control group has total number of 56 oocytes, without receiving chemical activation treatment after rescue ICSI. Fluorescence in situ hybridization (FISH) was employed to analyze chromosomes using X and Y dual-colored probes on the embryos that displayed two pronuclei in the experimental group.ResultsThe Fertilization rates in the experimental group and the control group were 89.1%(82/92)and 78.6%(44/56),the result showed that the fertilization rate of combining activation treatment after rescue ICSI was higher than the control group. The normal fertilization rate of the two groups were 51.2%(42/82)and 29.5%(13/44)respectively, which also had a significantly difference between the experimental group and the control group. Based on the embryos formed from normal fertilized oocytes of the two groups, no significantly difference were found in the cleavage rates and blastocyst rates. Sex chromosome analysis indicated 5 embryos with XX and 4 embryos with XY in 12 embryos derived from activated oocytes containing 2PN after rescue ICSI.ConclusionsThe combination of chemical oocytes activation treatment after rescue ICSI could effectively improve the fertilized rate after Rescue ICSI. and the normal fertilized oocytes have further developmental potential. However, we did not find any difference in developmental potential in the embryos formed from normal fertilized oocytes between two groups, showing that the oocytes activation can be applicated in unfertilized oocytes which receive rescue ICSI treatment.更多还原