Study On The Ultrastructure Of Human Unfertilized Oocytes And Embryos By Rescue ICSI

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.