The levels of pre-P2 may provide clues into the pathogenic mechanisms of infertility. The increased proportion of pre-P2 in some patients with increased P1/P2 ratio suggests an involvement of pre-P2 processing. The positive correlation between TUNEL-positive sperm and pre-P2 at low pre-P2/P2 ratios also suggests a link between deficient protamine processing and decreased DNA integrity.

An association between DNA fragmentation in sperm determined by the terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay and the incidence of reproductive failure has been reported, either using flow cytometry or optical microscopy. However, the results obtained using each of these two approaches are different. Since there is a relative lack of studies standardizing these two approaches, the direct comparison of the results described in the different articles is difficult at present. To allow the comparison of the TUNEL results obtained using flow cytometry and optical microscopy, we applied these two approaches in a total of 66 human sperm samples. A positive correlation is detected in the TUNEL results as measured by flow cytometry and optical microscopy (Spearman; r = 0.720, P 5 0.001). The percentage of TUNEL-positive spermatozoa assessed by flow cytometry is 2.6 times higher than that detected in optical microscopy (39.7% + 23.1% versus 15.3% + 10.3%). Although there is a good correlation of the TUNEL results obtained by flow cytometry and optical microscopy, the percentages obtained with either technique are different. Therefore, the TUNEL results described in the present work should be valuable to compare the results described in many independent articles, using either optical microscopy or flow cytometry. ' InternationalSociety for Analytical Cytology Key terms DNA fragmentation; TUNEL; flow cytometry; microscopy; infertility; sperm IT is known that an increased DNA fragmentation is present in the sperm cells of infertile patients (1-4). In addition, an altered DNA integrity in spermatozoa leads to poorly assisted reproduction results (2,5-17). The selection of spermatozoa to perform ICSI is presently based on its morphology and motility, but these parameters do not give information about DNA integrity. Spermatozoa with damaged DNA may also result in increased risk of anomalies in newborns and increased risk of childhood cancer (18-21). Therefore, laboratory techniques to evaluate DNA integrity are important toward a better management of the infertile patients. The etiology of these strand breaks may involve aberrant recombination, defective chromatin packaging, abortive apoptosis, and oxidative stress (22).There are several approaches available to evaluate DNA integrity in human spermatozoa (23)(24)(25). One of the most widely used techniques is the terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay. Using this approach, the DNA fragmentation is assessed through the labeling with dUTP of the double-and single-stranded DNA breaks present in spermatozoa (26-28; and also references cited in Table 2).The TUNEL assay has been considered as a measure of the DNA strand breaks and as a biological assay for sperm quality at the assisted reproduction laboratories (5,6,9,10,(28)(29)(30)(31). However, there are some factors that may result in variation in the