Research on improvement of embryonic development by cytoplasmic transfer of oocytes in aged IVF patients

原实验计划必须改变，因为从受体年轻卵母细胞中去除中期II核以及从供体旧卵母细胞中去除第一个极体的融合/注射程序极其困难。我们研究了这些程序的许多方面，建立了几种改进的方法。对于从受者年轻卵母细胞中去除中期II核，我们建立的方法如下。用5μg/mlCytochalasine B孵育15分钟后，用两个注射吸管(内径14μm)夹住卵母细胞，使透明带注射部位下方有更多的卵黄周空间，以避免注射吸管尖端破坏卵母细胞。利用压电式机械手插入一个注射吸管的卵周空间，以扩大自由空间。将注射吸管进一步插入卵周隙，抽吸第一极体和包裹细胞膜的Metapase II染色体，从旧供体卵母细胞中取出第一极体，至少需要14μg内径的吸针，但相同直径的吸针注入极体内容物时，所有受体卵母细胞都被破坏。所有电融合和注射的努力都以完全失败告终。因此，建议在今后的研究中使用仙台病毒。

Original experimental plan had to be changed because the procedures of removals of metaphase II nuclei from recipient young oocytes as well as those of fusions/injections of the first polar body removed from donor old oocytes were extremely difficult. We examined many aspects of these procedures, establishing several improved methods.For removals of metaphase II nuclei from recipient young oocytes, our established methods are as follows. Following incubation with 5 μg/ml cytochalasine B for 15 minutes, oocytes are grasped by two injection pipettes (14 μm in the internal diameter), making more perivitelline space under the injection site of zona pellucida to avoid destruction of oocytes by the injection pipette tip. Piezo manipulator is used to insert one injection pipette perivitelline space to enlarge free space. The injection pipette is further inserted into the perivitelline space and the first polar body and cell-membrane-enclosed metapase II chromosomes are aspirated and removed from oocytes.For removals of the first polar body from old donor oocytes, minimum of 14 μg of pipettes in the internal diameter were necessary, but all recipient oocytes were destroyed when the contents of the polar body was injected by the same diameter of pipettes. All efforts for electrofusion and injections resulted in complete failures. Therefore, usage of Sendai virus may be recommended for investigations in future.