Small variations in crucial steps of TUNEL assay coupled to flow cytometry greatly affect measures of sperm DNA fragmentation

Techniques for assessing sperm DNA damage are numerous and various. There are 2 main types of assay: direct and indirect. The former directly detects the amount of sperm DNA damage, whereas the latter reveals the effects of an exogenous insult on sperm chromatin. In addition, even considering the same type of technique, different strategies to reveal or quantify sperm DNA damage, or both, are used. Finally, these techniques, except for sperm chromatin structure assay (SCSA), lack standardized protocols to which all users can adhere to minimize interlaboratory variations. In this study, we investigated the effects of some of the many ways the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assay is performed when measuring sperm DNA fragmentation by flow cytometry. In addition, by using an established procedure, we determined the precision of the technique by calculating intra-assay coefficients of variation (CVs). We found that concentration of the fixative, the time of storage of fixed samples, the fluorochrome used to label DNA breaks, and the method used to analyze flow cytometric data all greatly affect the measures of sperm DNA fragmentation. In particular, we found that treatment with paraformaldehyde produced additional damage in most samples, suggesting that TUNEL also can be considered an indirect assay when performed in semen samples treated with such a fixative reagent. We also showed that 2 different methods used to analyze data yielded results that, albeit correlating, were different and associated differently to semen quality. On the contrary, the TUNEL assay, as measured here, showed low intraassay CVs, resulting in a quite precise technique when performed in established conditions.