Conflicting results are reported by recent studies comparing flow cytometry (FCM) and fluorescence microscopy (FM) for detecting sperm DNA fragmentation by TUNEL assay. Each of the two technologies has specific peculiarities and limitations, but whereas the limitations of FM observation are well known, the biases due to the inability of FCM to recognize morphologically analyzed cells are less explored. In particular, so far, FCM analysis of sperm DNA fragmentation have included in the analyses M540 bodies, round semen structures exhibiting FSC/SSC properties similar to sperm. Semen M540 bodies, altogether with the occurrence of two sperm populations with different nuclear staining, concur to an underestimation of values of sperm DNA fragmentation by FCM. However, even considering such bias, the observed discrepancies between the performance of FM and FCM are not fully explained. We discuss here the possible variables that may affect the results of each of the two technologies and the necessary efforts to be taken to address this issue.