【作者】 唐志霞；

【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】 第一部分人类受精失败的卵母细胞超微结构的研究目的比较体外受精(IVF)或单精子卵胞浆内注射(ICSI)后受精失败的卵母细胞超微结构的差异,进一步探讨卵母细胞受精失败的原因,为临床治疗提供理论依据,协助患者选择下一周期的最佳治疗方法。方法收集IVF/ICSI受精失败的MⅡ期卵母细胞为实验组,对照组为正常的MⅡ期卵母细胞。将收集的受精失败及正常的卵母细胞进行单细胞固定、脱水、包埋、超薄切片和染色,透射电子显微镜下观察,拍照,观察其超微结构,并比较三组间超微结构的差异。结果IVF组透明带内层致密,外层十分松散,凹凸不平,内部有孔洞及电子密度较大的小颗粒;ICSI组透明带结构疏松,呈网格状,内见高密度的电子沉积物。IVF组透明带厚度(14.7±2.4μm)明显高于正常组(10.1±2.1μm),且有统计学意义(P<0.05)。ICSI组透明带厚度(12.2±3.0μm)高于正常组,但无统计学意义(P>0.05)。IVF组透明带厚度高于ICSI组,无统计学意义(P>0.05)。IVF组皮质颗粒广泛分布于皮质区,部分呈线状排列于质膜下;ICSI组和对照组皮质颗粒线状排列于质膜下。IVF组异常线粒体所占比例为41.2%,明显高于其余两组,有统计学意义(P<0.05)。IVF组可见成熟及未成熟的高尔基复合体,还可见正在形成皮质颗粒的高尔基复合体;其余两组内未见高尔基复合体。三组内卵周间隙均狭窄透亮,有卵泡细胞的残留物,内含微绒毛细长,倒伏或竖起在胞质膜的表面。结论1、受精失败的卵母细胞内细胞器发生变化;2、卵母细胞胞质中细胞器的异常分布与出现,导致胞质不成熟,卵母细胞的透明带增厚、变硬导致精子穿透障碍,从而导致IVF组卵母细胞受精失败;3、受精失败的卵母细胞内脂滴数量少,体积小可能与受精失败有关。第二部分人类受精失败的卵母细胞补救ICSI后胚胎超微结构的研究目的比较受精失败的卵母细胞补救ICSI后胚胎发育的各阶段与正常ICSI胚胎发育的各阶段超微结构的差异,进一步探讨IVF受精失败的原因,以期提高人类辅助生殖技术的成功率。方法实验组为受精失败的卵母细胞补救ICSI后各个发育阶段胚胎(2-cell、4-cell、8-cell、致密桑椹);对照组为正常的卵母细胞ICSI后各个发育阶段胚胎(2-cell、4-cell、8-cell、致密桑椹)。将收集的胚胎进行固定、脱水、包埋、超薄切片和染色,透射电子显微镜下观察,拍照,观察其超微结构,并比较两组内胚胎各个发育阶段超微结构的差异。结果研究发现,实验组内2-、8-细胞组透明带厚度与其余两组相比,均有统计学意义(P<0.05);对照组内2-细胞组透明带厚度明显高于其余三组(P<0.05)。实验组内2-细胞、4-细胞组异常线粒体所占比例与其余两组相比,均有统计学意义(P<0.05);对照组内致密桑椹组异常线粒体所占比例均高于其余三组(P<0.05)。实验组内各期胚胎异常线粒体所占比例均高于对照组(P<0.05)。对照组内脂滴直径从4-细胞期开始显著增加,各组间均有显著性差异(P<0.05);对照组内4-、8-细胞及致密桑椹期脂滴直径均显著大于实验组(P<0.05);实验组内致密桑椹期时脂滴直径显著高于其他时期(P<0.05)。结果还显示,对照组2-细胞期胚胎未见明显的高尔基复合体;实验组2-细胞期、4-细胞期胚胎均未见到明显的高尔基复合体,两组内不同胚胎高尔基复合体随着胚胎的发育逐渐成熟。两组内胚胎各时期都存在滑面内质网,与线粒体关系密切。对照组2-细胞、4-细胞及8细胞胚胎细胞核为圆形或椭圆形,核膜完整;实验组2-细胞、4-细胞及8细胞胚胎细胞核形状为椭圆形,似核膜有断裂。各期胚胎核内均可见电子密度较高、呈颗粒状的染色质结构。2-细胞期两卵裂球之间距离较远,微绒毛少;实验组4-细胞期、8细胞期卵裂球之间部分位置连接紧密,部分位置细胞内间隙较大;致密桑椹期以及对照组的8细胞期卵裂球之间几乎没有细胞内间隙,相互接触的绝大部分是间隙连接。结论1、受精失败组补救ICSI后形成的各期胚胎透明带厚度先变薄后又变厚;2、两组内线粒体、脂滴及内质网在各期胚胎分布基本相似,但受精失败组补救ICSI后形成的各期胚胎异常线粒体所占比例均高于对照组;3、受精失败组补救ICSI后形成的各期胚胎细胞核形状不规则,似核膜有断裂;4、两组内早期胚胎发育中卵裂球之间空隙较大,以微绒毛连接为主,对照组内8-细胞以上,卵裂球之间几乎没有细胞内间隙,相互接触的绝大部分是间隙连接;5、已初步探讨了人类受精失败的卵母细胞补救ICSI后形成的各期胚胎的超微结构的改变,但如何提高其成功率仍需进一步研究。更多还原

【Abstract】 PartⅠStudy on the Ultrastructure of Human Unfertilized OocytesObjectives To study the ultrastucture of human unfertilized oocytes and to investigate why human MII oocytes failed to fertilize after in vitro fertilization (IVF). At the purpose of this study was to provide the basic and clinical rational for IVF-ET and may be helpful to select the best clinically assisted fertilization method for the next cycle.Methods Collecting unfertilized oocytes after IVF/ICSI and normal matured oocytes handled with fixed, dehydrated, embedded, ultrashined and observed by transmit electron microscopy (TEM). To compare the ultrastuctural differences among the group IVF, ICSI and normal group.Results In IVF group, the endothecium structure of zona pellucida was compact, while the outer layer structure was very loose and uneven. Many pores and high electron density particles were found in the interior of zona pellucida. In ICSI group, the structure of zona pellucida is loose and grids, in which high density electron deposition were also found. The thickness of zona pellucida of group IVF, ICSI and normal group was 14.7±2.4μm, 12.2±3.0μm and 10.1±2.1μm, respectively. There was significant difference between group IVF and normal (P<0.05). There was no difference between other two groups(P>0.05). In IVF group, cortical granules were dispersed in the oocytes cortex, only a part of which were beneath the oolemma .In other two groups , there are one row of dense cortical granules. The rate of abnormal mitochondria of group IVF was significantly higher than those of other two groups (P<0.05). The oocyte cortex contained matured and immatured Golgi complex in group IVF, the presence of Golgi complex still forming cortical granules. The Golgi complex was not found in the other two groups. The perivitelline space (PVS) was narrow, light space between the oolemma and inner surface of the ZP in the three groups, which contained numerous cytomembranse derived from the feet of follicular cells.Conclusions1、Great changes had happened to the organelles of unfertilized oocytes.2、Organelles appeared in abnormal position were related to oocytes cytoplasmic maturation, which led to fail in fertilization. Failed fertilization in conventional IVF was due to absence of acrosome reaction and abnormal dense of the zona pellucida.3、In the unfertilized oocytes, the quantity of lipid droplet decreased and the volume of lipid droplet is smaller than normal oocytes, which may be related to failure of fertilization in IVF/ICSI.PartⅡStudy on the Ultrastructure of Embryos by Rescue ICSI of Unfertilized OocytesObjectives To study the ultrastructure of embryos by rescue ICSI of unfertilized oocytes and the embryos by conventioanal ICSI. At the purpose of this study was to compare the differences of all embryo development stages and to know the reason of fertilization failure and furthermore to improve the clinical pregnancy rate of assisted reproduction technology (ART).Methods collecting embryos after rescue ICSI of unfertilized oocytes and embryos by conventional ICSI handled with fixed, dehydrated, embedded, ultrashined and observed by transmit electron microscopy (TEM). To compare the differences of ultrastucture among all embryo development stages including 2-cell, 4-cell, 8-cell and compact molula stage.Results As shown by transmission electron microscopy, the thickness of zona pellucida was significantly differences between embryos at 2-cell, 8-cell stage and other two embryo stages in test group (P<0.05). The thickness of zona pellucida of embryos at 2-cell stage was significantly higher than those of other embryo development stages in control group (P<0.05). The rate of abnormal mitochondria was significantly difference between embryos at 2-cell, 4-cell stages and other two embryo stages in test group (P<0.05). The rate of abnormal mitochondria of embryos at compact molula stage was significantly higher than those of other embryo development stages in control group (P<0.05). The rates of abnormal mitochondria of embryo development stages in test group were all higher than those of control group (P<0.05). In control group, the diameter of lipid droplet began to increase from 4-cell stage on, and there were significantly differences at all embryo development stages (P<0.05).The diameter of lipid droplet of embryos at 4-cell,8-cell and compact molula stages in control group was significantly higher than those of test group (P<0.05). In test group, the diameter of lipid droplet of embryos at compact molula stage was significantly higher than those of other embryo development stages (P<0.05). We also found that there was no conspicuous Golgi complex of embryos at 2-cell stage in control group. We could not find conspicuous Golgi complex of embryos at 2-cell and 4-cell stages in test group. At all development stages, the Golgi complex became gradually matured. Smooth endoplasmic reticulum existed at all development stages, which was related to mitochondria. In embryos at 2-cell, 4-cell and 8-cell stages in test group, the shape of nucleus was round or oval and the nuclear membrane was integraty. However, in control group, the shape of nucleus was oval and it seemed that nuclear membrane had broken.We could also find high density electron deposition and chromoplasm in nucleus of all development stages. There were gap junctions in embryos at 2-cell stage and less microvilli, but these junctions became compacter between the part of the blastomere, and looser connection was also found in embryos at 4-cell and 8- cell stages . In contrast, the gap junction became tight without intercellular space since embryos at compact morula and 8-cell stages in control group.Conclusions1、The thickness of zona pellucida was thinner firstly and then became thickening at all embryo development stages by rescue ICSI of unfertilized oocytes;2、The distribution of mitochondria, lipid droplet and endoplasmic reticulum looked like the same at all embryo development stages in these two group, but the rates of abnormal mitochondria of all embryo development stages by rescue ICSI of unfertilized oocytes were all higher than those of control group;3、The nucleus appeared irregular nucleoli and nuclear membrane may have faults in embryos by rescue ICSI of unfertilized oocytes;4、Early embryonic development, the blastmeres of embryos had junctions through enchased microvilli; the gap junction became tight without intercellular space from 8-cell stage of embryos to compact morula in control group.5、Ultrastructural changes of Embryos by Rescue ICSI has initially studied, but how to improve the rate of clinical pregnancy, we still need a furthermore study.更多还原