Abstract: Objective: To study the effects of centrifugation and pipette on human motility and intracellular ROS, so as to optimize the sperm handling procedure. Methods：Seven sperm samples with normal seminal parameters were treated with various centrifugation force （200 g，600 g) and centrifugation duration (5 min，15 min） and different pipetting times （2，6，10 times）. Motility parameter and intracellular ROS were measured. Results：For the centrifuge procedure, the centrifugation duration could significantly influence the sperm motility and intercellular ROS level （P＜0.05）, while centrifugation did not significantly influence the percentage of progressive motile sperm （ＰＲ） and the velocity of average path（VAP）（P＞0.05）. The centrifugation duration more than 5 minutes could significantly decrease the sperm motility and intracellular ROS level（P＜0.05）. For the pipetting procedure, the pipetting more than 2 times would significantly decrease the sperm motility （P＜0.05），while the pipetting over 6 times would significantly decrease the PR and VAP （P＜0.05）. However, the intracellular ROS level was not significantly changed by the repeated pipetting. Conclusions：The special causion is that the centrifuge over 5 minutes and pipetting over 2 times should be avoided during in vitro handling human sperm sample.

Key words: Spermatozoa, Sperm motility, Centrifugation, density gradient, Suction, Oxidative stress, Fertilization in vitro