title: Small variations in crucial steps of TUNEL assay coupled to flow cytometry greatly affect measures of sperm DNA fragmentation.
abstract: 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.
authors: Department of Clinical Physiopathology, Andrology Unit, University of Florence, Firenze, Italy. m.muratori@dfc.unifi.it
authors: Monica
authors: M
authors: Muratori
authors: Lara
authors: L
authors: Tamburrino
authors: Valentina
authors: V
authors: Tocci
authors: Antonietta
authors: A
authors: Costantino
authors: Sara
authors: S
authors: Marchiani
authors: Claudia
authors: C
authors: Giachini
authors: Ilaria
authors: I
authors: Laface
authors: Csilla
authors: C
authors: Krausz
authors: Maria Cristina
authors: MC
authors: Meriggiola
authors: Gianni
authors: G
authors: Forti
authors: Elisabetta
authors: E
authors: Baldi
mesh_terms: D000328:Adult; D053938:DNA Fragmentation; D005404:Fixatives; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005557:Formaldehyde; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D011108:Polymers; D011309:Preservation, Biological; D013094:Spermatozoa / Q000648:ultrastructure*
chemical_list: D005404:Fixatives; D005456:Fluorescent Dyes; D011108:Polymers; D005557:Formaldehyde; C003043:paraform