Dynamic aspects concerned with the mechanism of separating motile sperm from nonmotile sperm, leukocytes, and debris with the use of high-density Percoll gradients

OBJECTIVE: To investigate the dynamics and the efficacy of separating motile from nonmotile sperm with the use of Percoll gradients as a function of centrifugation time. DESIGN: In vitro laboratory experiments on human sperm. SETTING: Male Infertility Institute. PATIENT(S): Fresh sperm specimens collected from donors and patients. INTERVENTION(S): Aliquots of sperm specimens, spread on top of 40% and 80% Percoll layers in test tubes, were centrifuged for 5-60 minutes. MAIN OUTCOME MEASURE(S): After centrifugation, samples were aspirated from the bottom, interface, and top layers for microscopic analysis. RESULT(S): Within 5 minutes, only fast-moving sperm reached the bottom as a result of both centrifugation force and their own tail movements. From 10 minutes onward, nonmotile sperm, slowed down by Percoll, joined them and gradually reversed the separation process. Leukocytes, debris, and abnormal sperm remained in the upper levels and never reached the bottom. CONCLUSION(S): Separation of motile sperm with the use of Percoll gradients is strongly dependent on centrifugation time. The separation is the gap formed between down-swimming sperm, accelerated by centrifugation, and nonmotile sperm, decelerated by Percoll. This gap gradually disappears with extended spinning.