Antisperm antibodies detection by flow cytometry is affected by aggregation of antigen-antibody complexes on the surface of spermatozoa

Flow cytometry (FCM) analysis of live antibody-coated spermatozoa subjected to immunofluorescence staining (FCM test) is considered an objective metho d for the quantitative detection of antisperm antibodies (ASA). But the cro ss-linking of cell surface antigen (Ag) with bivalent antibodies and/or ant igen-antibody (Ag-Ab) complexes with second antibodies may induce the reorg anization of surface components (patching and capping) and result in their shedding from the sperm surface. The present study estimates the relationsh ip between aggregation of Ag-Ab complexes on the sperm surface and the resu lts of indirect FCM test, Swim-up spermatozoa of normozoospermic men were i ncubated with ASA-positive sera from infertile patients and with second ant ibodies fluorescein isothiocyanate (FITC)-labelled goat anti-human IgG poly clonal antiserum under different conditions and then analysed by FCM and fl uorescence microscopy. It was shown that low temperature, cytochalasin B, e xcess or lack of the primary and/or secondary antibodies and sperm fixation by paraformaldehyde may inhibit aggregation and shedding of Ag-Ab complexe s and dramatically increase ASA quantity determined on the sperm surface. H owever, inhibition of aggregation on the live sperm surface was observed on ly in a minority of ASA-positive samples and was poorly reproducible using semen of different donors. A high probability of Ag-Ab complex shedding fro m the sperm surface during experimental manipulation limits the use of indi rect FCM test for quantitative ASA determination.