Flow cytometry TUNEL standardization for assaying sperm DNA fragmentation.

We read with great interest Muratori’s paper (Muratoriet al, 2010), because we believe that terminal deoxynu-cleotidyl transferase–mediated dUTP nick end labeling(TUNEL) assay standardization is a matter of greatconcern and that there are certain steps that must befollowed to achieve accurate results.We agree with the authors of the paper but feel theneed to stress several important considerations. As theauthors mention in their introduction, the labeling ofDNA breaks should be carried out by nuclear stainingwith propidium iodide (PI; Muratori et al, 2008). Wehave noted in oligozoospermia samples a clear increasein the percentage of PI-negative events with forwardscatter/side scatter similar to sperm, yielding lowerresults. The upper sperm count limit that could beconsidered a threshold for this phenomenon is difficultto establish; thus, TUNEL/PI should be applied to allsamples, and reference values should be recalculated forfuture clinical use.The authors propose 2 methods to quantify the DNAfragmentation percentage within a sperm sample: athreshold-setting method (Muratori et al, 2000) and ablank subtraction method. The results differed substan-tially between these different methods. The authorschose the first one, stating that it correlated well withsemen quality; in our hands, however, no associationwas found between DNA fragmentation and motility,morphology, and sperm count (Pearson correlation; n 5210). We believe that our data do not invalidate the useof this method because new assays might give differentinformation than previously determined parameters.As the authors report, fixed samples should not bestored at 4uCorat220uC because it affects reproduc-ibility. In our experience, the results obtained using freshcompared with stored samples varied unpredictably by,50% (from 247% to +52%). Other authors suggeststoring the samples (4uC, 220uC) until testing (Stronatiet al, 2006; Ramos et al, 2008), but we do not agree withthem. This point is essential for assay standardization.When the procedure is followed rigorously, the resultsare precise: our intra-assay coefficient of variation is 3%(aliquot processed immediately after fixation), which isin agreement with data reported by Muratori et al(2010). Finally, we congratulate Muratori’s teambecause their comprehensive studies have helped usimprove the analysis of sperm DNA fragmentation.Susana M. Curi, Patricia H. Chenlo, Luis A. Billordo,Pla´cida Baz, Melba L. Sardi, Julia I. Ariagno,Herberto Repetto, Gabriela R. Mendeluk,and Mercedes N. PuglieseLaboratorio de Fertilidad MasculinaDepartamento de Bioqui´mica Cli´nicaINFIBIOC, Facultad de Farmacia y Bioqui´micaUniversidad de Buenos Aires, Argentina