Journal of Andrology
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Published-Ahead-of-Print December 3, 2009, DOI:10.2164/jandrol.109.008508

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Monica Muratori
Lara Tamburrino
Valentina Tocci
Antonietta Costantino
Sara Marchiani
Claudia Giachini
Ilaria Laface
Csilla Krausz
Maria Cristina Meriggiola
Gianni Forti
Elisabetta Baldi
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Small Variations in Crucial Steps of Tunel Assay Coupled to Flow Cytometry Greatly Affect Measures of Sperm DNA Fragmentation

Monica Muratori *, Lara Tamburrino , Valentina Tocci , Antonietta Costantino , Sara Marchiani , Claudia Giachini , Ilaria Laface , Csilla Krausz , Maria Cristina Meriggiola , Gianni Forti , and Elisabetta Baldi

* To whom correspondence should be addressed. E-mail: m.muratori{at}dfc.unifi.it.

Techniques assessing sperm DNA damage are numerous and heterogeneous. There are two main types of assays: direct and indirect ones. The former directly detect the amount of sperm DNA damage whereas the latter reveal the effect of an exogenous insult on sperm chromatin. In addition, even considering the same type of technique, different strategies to reveal and/or quantify sperm DNA damage are used. Finally, these techniques, except SCSA (Sperm Chromatin Structure Assay), lack standardized protocols to which adhere to minimize inter-laboratories variations. In this study we investigated the effects of some of the many variants by which TUNEL assay is performed on the measures of 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 the concentration of the fixative, the time of storage of fixed samples, the fluorochrome used to label DNA breaks and the method to analysis flow cytometric data, all greatly affect the measures of sperm DNA fragmentation. In particular we found that treatment with paraformaldehyde produced an additional damage in most of samples, suggesting that also TUNEL can be considered an indirect assay when performed in semen samples treated with such fixative reagent. We also showed that two different methods to analyse data yielded results that, albeit correlating, were different and differently associated to semen quality. On the contrary, TUNEL assay, as measured here, showed low intra-assay CVs, resulting in a quite precise technique when performed in established conditions.


Key words: Infertility • Semen Analysis • Sperm • DNA fragmentation • TUNEL assay




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