Results Of the 202 patients, 48 (23.8%) had normal standard sperm parameters, while 154 (76.2%) had an abnormality in one or more of these parameters. DFI in infertile men with normal sperm parameters was significantly higher than in fertile donors ( p = 0.03), but not significantly different from infertile men with abnormal sperm parameters ( p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperm from infertile men with abnormal and normal semen parameters, but not in fertile donors ( r = −0.26, p = 0.001 and r = −0.48, p = 0.0001, respectively). Conclusion Sperm from infertile men with normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to levels in infertile men with abnormal sperm parameters. Sperm DNA fragmentation analysis is an independent test of sperm quality and has an important diagnostic value in the evaluation of male infertility. Keywords Sperm DNA fragmentation Flow cytometry Male infertility Sperm Introduction Male-factor infertility plays a role in approximately 50% of infertile couples [1] . Standard semen analysis consists of measuring a variety of semen parameters, including volume, pH, sperm concentration, vitality, and morphology, the presence of leukocytes and immature germ cells, and sperm motility [2] . This classical analysis is used to determine whether all or some of the parameters of an ejaculate fall within the range of those of normal fertile men. However, diagnosing defective sperm function by conventional semen analysis is difficult because the spermatozoon is a highly specialized cell that expresses a diverse array of biological properties to achieve fertilization [3] . Additionally, in at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results [1] . When investigations of the female partner are also normal, the couple are classified as unexplained or idiopathic [4] . There is ongoing research into the development of new methods to complement the conventional semen analysis routinely used to assess infertility. One endpoint of interest is sperm DNA integrity, which can be assessed by a variety of assays, such as the sperm chromatin structure assay (SCSA) [5,6] , the sperm DNA integrity assay (SDIa) [7,8] , the terminal deoxynucleotidyl-transferase-mediated dUDP nick-end labeling assay [9] , the in situ nick translation (ISNT) assay [10] , and the single cell microgel electrophoresis (COMET) assay [11] . Methods focusing on the characterization of sperm chromatin condensation and stability are able to reveal hidden anomalies in the structural organization of sperm DNA and have been receiving growing attention. In particular, flow cytometric approaches allow rapid measurements to be carried out with a high level of precision, objectivity, and consistency with reliable results [12] . Both the SCSA and SDIa are flow cytometric methods that may identify spermatozoa with abnormal chromatin packaging, which could be the result of DNA breaks and/or derailments in protamine quantity and composition and/or an insufficient level of disulfide groups [6–8,13] . Acridine orange staining is used, after a low pH challenge, to distinguish between native DNA and fragmented DNA. Furthermore, in various studies, men with sperm DNA fragmentation (>27–30%) were placed into a statistical group taking a longer time to pregnancy, more in vitro fertilization (IVF) cycles, increased miscarriages or no pregnancy [7,14–17] . The objective of this study was to evaluate levels of sperm nuclear DNA fragmentation in a population of infertile men with normal standard semen parameters and to compare the results with those from men who had abnormal semen parameters, as well as with a control group of men with proven fertility. Subjects, materials and methods Study population All infertile couples included in this study had had at least 1 year of regular unprotected intercourse with no pregnancy achieved. A medical history was obtained from the infertile patients, and an experienced urologist performed a genital examination. Study participants were infertile men with normal standard semen parameters on repeated analyses and infertile men with an abnormality in one or more of the standard semen parameters on repeated analyses. The female partners of these men had no history of untreated female-factor infertility and had a normal reproductive and sexual history as well as normal investigations (e.g. pelvic ultrasound, endometrial biopsy, and hysterosalpingogram). Infertile men with azoospermia or couples with untreated female-factor infertility were excluded from the study. A group of healthy donors ( n = 30) with proven fertility (i.e. fathered a child within the last 12 months) and with normal genital examinations were included as controls. Semen samples Semen samples were obtained from infertile men ( n = 202) and from fertile donors ( n = 30) during March 2007 through April 2008, after informed consent. All samples were collected by masturbation, after a period of 3–5 days of sexual abstinence. After 30 min from the time of ejaculation (for semen liquefaction at room temperature), 500 μl of the raw semen sample was frozen into a LN 2 tank at −196 °C for later SDIa. Standard semen analysis Following liquefaction, semen specimens were evaluated for semen volume, appearance, pH, and viscosity. Manual semen analysis was performed according to World Health Organization (WHO) guidelines [1] to determine sperm concentration and motility. Samples (5 μl) of liquefied semen was loaded on a Cell-VU counting chamber (Millennum Sciences, Inc., NY, USA) and examined under x200 magnification. Sperm concentration was expressed as 1 × 10 6 /ml semen, while motility was expressed as a percentage. Smears of the raw semen were stained using the Spermac stain kit (FertiPro...