M540 bodies interfere with TUNEL analyses in human semen samples

Sir, We read with interest the paper by Aitken et al. (2010). In their study, the percentages of TUNEL were compared in selected [with densitygradient centrifugation (DGC)] versus unselected and in live versus total sperm. A further aim of the study was to define a diagnostic threshold value by comparing donors and patients. The study was conducted by flow cytometry without any strategy to exclude M540 bodies. M540 bodies are non-sperm elements that occur in semen, especially from subfertile men (Muratori et al., 2004; Marchiani et al., 2007). Indeed, M540 bodies are located in the same FSC/ SSC (Forward/Side Scatter) region as sperm (Muratori et al., 2004, 2005). Hence, the gate on FSC/SSC does not imply that ‘only spermatozoa were assessed’ (Aitken et al., 2010). In our opinion, omitting to exclude M540 bodies will have significantly affected the results of the paper, as following: