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

由于从受体年轻卵母细胞中去除中期II核的程序以及从供体旧卵母细胞中去除的第一极体的融合/注射程序非常困难，因此不得不改变原始实验计划。我们对这些方法进行了多方面的研究，建立了几种改进的方法。用5 μg/ml细胞松弛素B孵育15分钟后，用两个注射移液管（内径14 μm）抓住卵母细胞，在透明质酸注射部位下方形成更多的卵周空间，以避免卵母细胞被注射移液管尖端破坏。利用压电机械手将一根注射管插入卵周空间，以扩大自由空间。将注射管插入卵周间隙，吸出卵母细胞的第一极体和细胞膜包裹的metapase II染色体。对于从旧供体卵母细胞中取出第一极体，最少需要内径为14 μg的注射管，但当用相同直径的注射管注射极体内容物时，所有受体卵母细胞都被破坏。所有电融合和注射的努力都失败了。因此，建议在将来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.