Critical Aspects of Detection of Sperm DNA Fragmentation by Tunel/Flow Cytometry

Abstract

Despite the many studies documenting an increase of sperm DNA damage in subfertile and infertile subjects, determining whether this parameter is relevant for the clinic is still an open question. Indeed, results of clinical investigations on sperm DNA damage and outcome of ART procedures are often conflicting as many factors affect the predictive power of these tests. The techniques used to reveal such damage is one of these factors. Techniques detecting sperm DNA damage are indeed numerous and heterogeneous. In addition, it is not obvious that they reveal the same type of DNA damage and that their results are equivalent. One of the available methods to detect sperm DNA damage is the TUNEL assay. Since it was developed in the 1990s the TUNEL assay has been widely employed, becoming one of the most popular strategies to investigate the origin, the mechanism, and the clinical meaning of sperm DNA damage. Our group has used TUNEL coupled to flow cytometry to investigate sperm DNA fragmentation for about 10 years. According to our experience, this technique presents some pitfalls and limitations in the accuracy and reproducibility of the measures of sperm DNA fragmentation. In this review, we discuss several technical features of TUNEL and report the solutions adopted by our group to overcome some of its limitations.

Effects of different cryopreservation methods on DNA integrity and sperm chromatin quality in men

“…The variation of the correlation coefficient between the studies may result from the design of the study (the type and number of patients) and the use of different protocols for each assay, especially in the case of the TUNEL assay where no standard clinical protocol exists. Moreover, it was shown that the values obtained when measuring TUNEL‐positive cells manually by fluorescence microscopy were lower than the one obtained using flow cytometry analysis (reviewed in (Muratori et al ., )).…”

The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment

“…A number of different factors such as preparation, fixation and permeabilization of samples negatively affect its clinical application (Muratori et al, 2010). The latter factor is due to the highly compact nature of sperm chromatin by and its reinforced structure in protamines due to inter-and intramolecular bonds (Balhorn et al, 1992;Brewer et al, 2003;Vilfan et al, 2004), which prevent TdT from directly accessing the DNA strand breaks.…”

“…However, this specificity has neither been demonstrated for spermatozoa (Manicardi et al, 1998) nor can this endpoint be used because it can also be reached by other mechanisms such as necrosis. Thus, by using the TUNEL assay, one should rather refer to this DNA damage as 'DNA fragmentation' than 'apoptosis' (Henkel et al, 2004;Muratori et al, 2010).…”

DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters

“…Thus, the influence of semen quality and age on the predictive power of sDF is currently unknown. In addition, the lack of standardization of most methods (22) used to detect sDF hampers the clinical use of the reported threshold values. Our group has developed a new version of flow cytometric terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) combining nuclear staining with propidium iodide (PI) for the detection of sDF and yielding precise, standardized (23), and more accurate measurements compared with simple TUNEL (24).…”