In this study, we examined the effect of different methods of puncturing the cell membrane of human oocytes using Piezo-ICSI on the fertilization rate and oocyte degeneration rate. Three methods were compared. (1) One-pulse method: After piercing the zona pellucida, the pipette is pushed to a position of about 70% of the oocyte diameter. Then the cell membrane was punctured by applying one pulse, and the spermatozoa were injected. (2) Three-pulse method: After piercing the zona pellucida, the pipette was pushed to a position of about 70% of the oocyte diameter, and the cell membrane was punctured with the first pulse; a second pulse was applied after pushing the pipette to about the 80% position; then, after pushing the pipette further to about the 90% position, the spermatozoa were injected by applying a third pulse. (3) Cell membrane puncture by expansion method: the pipette was pushed to a position of about 90% of the oocyte diameter. After the cell membrane had fully expanded, one pulse was applied to puncture the cell membrane and inject the spermatozoa. In membrane puncture with a pulse from Piezo-ICSI, it has been reported that the expandability of the cell membrane is low, and when the pipette pierces the oocyte cytoplasm, the membrane can break before application of the pulse (abnormal rupture of the membrane) increasing the degeneration rate. The incidence rate of oocytes with abnormal rupture of the membrane was also compared among the 3 methods. The normal fertilization rates of the one-pulse method, three-pulse method, and cell membrane puncture by expansion method were 74.7%, 72.9%, and 77.2% respectively, and there were no significant differences among them. The incidence rate of oocytes with abnormal rupture of the membrane was 14.5%, 8.9%, and 20.7% respectively, with significant differences among the three methods (P < 0.05). The total oocyte degeneration rates of the three methods were 3.9%, 2.1%, and 3.2% respectively, and there were no significant differences among them. In abnormally ruptured oocytes, injected spermatozoa into the location of the destruction. In the cell membrane puncture by expansion method, spermatozoa were injected at the position of 90% irrespective of the location of destruction. The respective denaturation rates were 23.8% and 15.7%, with a lower tendency being observed in the cell membrane extension puncture method. There were no differences among the fertilization rates of the three methods. However, the result of the cell membrane extension puncture method suggests that injecting spermatozoa at a position deeper than the membrane rupture position effectively avoids oocyte degeneration.