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OBJECTIVE: We evaluated the effectiveness of sperm washing using the discontinuous two-layer density gradient centrifugation method to remove antisperm antibodies attached to the sperm surface. METHOD: We prospectively enrolled sixty-six men with unexplained infertility who were seeking evaluation. Each patient delivered one semen specimen for the study. We determined antisperm antibody levels using the direct immunobeads test. Specimens were classified into two groups according to the pre-washing levels of antibody-bound spermatozoa: group 1 (low antisperm antibodies levels, immunobeads test ,20%; n ¼ 54), and group 2 (high antisperm antibodies levels, immunobeads test $20%; n ¼ 12). Sperm washing was carried out using the discontinuous two-layer colloidal density gradient centrifugation method. Pre- and post-wash levels of antisperm antibodies were compared in the groups. RESULTS: The pre- and post-wash percentage of spermatozoa with antisperm antibodies attached to their surface was 11% and 6.5% in group 1 (mean difference ¼ 40%; p , 0.01), and 30% and 19.5% in group 2 (mean difference ¼ 36.8%, p ¼ 0.02), respectively. The effectiveness of density gradient centrifugation in removing antisperm antibodies was not different between the groups, but individual variations from 252.3% to 23.9% were observed. CONCLUSIONS: Sperm washing by density gradient centrifugation is an overall effective method to remove antibodies bound to sperm membranes, regardless of the levels of antisperm antibodies in the neat semen. Due to an inter-individual variation in the effectiveness of the method, we recommend that each patient be tested before applying sperm washing by density gradient centrifugation in intrauterine insemination.

Effectiveness of sperm washing by discontinuous density gradient centrifugation to remove antibodies bound to the sperm membrane