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

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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.