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

Varicocele has been associated with decrease in seminal parameters. Selenium (Se), copper (Cu), and zinc (Zn) are trace elements essential for normal spermatogenesis of mammals and play a critical role as antioxidant defense system enzymes. Se, Cu, and Zn are associated with sperm quality in fertile and infertile men. However, there is little information about Se, Cu, and Zn concentrations in semen in patients with varicocele and its association with seminal parameters. The purpose of this study was to determine the concentrations of Se, Cu, and Zn in semen of patients with varicocele and the relationship with seminal parameters. Total Reflection X-Ray Fluorescence was used for the fist time in the seminal fluid analysis. The concentration of selenium in men with varicocele was smaller than the normozoospermic group, while no differences were observed for both concentrations of zinc and copper. A significant positive correlation between zinc and selenium concentration was observed. Selenium in seminal plasma correlates with a good spermatozoa concentrations, motility, and morphology. Additionally, a significant positive correlation was observed between zinc levels and sperm count. In conclusion, a decrease in selenium concentration was associated with detriment of seminal parameters. A study should be conducted to evaluate the benefits of both zinc and selenium supplementation to improve seminal parameters in patients with varicocele.

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in seminal fluid, as well as levels of sperm lipid membrane peroxidation, were investigated in fertile and infertile men. Semen samples, obtained by masturbation from 37 infertile and 14 fertile men, were examined for the presence of TNF-alpha and IL-6. The level of lipid peroxidation of the sperm membrane was measured by determining malondialdehyde (MDA) formation. The correlation between the IL-6 and the TNF-alpha concentrations in seminal plasma with the levels of lipid peroxidation of the sperm membranes was statistically evaluated. The IL-6 concentration in seminal plasma of infertile men was significantly higher than that of fertile men (p < .05). Similarly, the level of membrane lipid peroxidation was higher for the semen of infertile men than that of fertile men (p < .001). A significant positive correlation was found between IL-6 levels in seminal plasma and membrane sperm lipid peroxidation (p < .002), but not between this parameter and TNF-alpha levels in seminal plasma. These findings suggest a possible association between IL-6 seminal plasma levels and lipid peroxidation of sperm membrane. Stimulation of reactive species production by human sperm and leucocytes, induced by the high levels of IL-6, could explain these results.

Several pro-inflammatory cytokines at physiological concentrations increase the level of lipid peroxidation of sperm membranes, which could be important for the sperm fecundation process. However, infection-inflammation concentrations of some cytokines, such as IL-8 and TNF-alpha, either alone or in the presence of leukocytes, could drive the lipid peroxidation of the spermatozoa plasma membrane to levels that can affect the sperm fertility capacity.

Objective• To assess the relationship between a marker of epididymal function and both the fragmentation of the sperm nucleus and the integrity and maturity of the sperm membrane in patients with or without varicocele. Patients and Methods• Semen samples were obtained from men with varicocele grades II and III (n = 60) and from a control group with zoospermia defined as normal (n = 30). • Samples were evaluated by a spermiogram, a hypoosmotic swelling test (HOST), neutral α-glucosidase (NAG) enzyme activity, sperm hyaluronan-binding assay (HBA) and DNA fragmentation using a sperm chromatin dispersion (SCD) test. Results• Seminal plasma NAG levels, percentage of sperm bound to hyaluronic acid, HOST-positive cells and sperm quality were significantly lower in the varicocele compared with the control group.• Higher levels of sperm DNA fragmentation, as measured by SCD, were also observed in the varicocele group compared with the control group.• Seminal NAG activity levels showed a strong negative correlation with DNA fragmentation and a significant positive correlation with the HBA test and the HOST. Conclusions• Varicocele causes a reduction in NAG activity by the epididymis that is associated with damage to both the membrane and sperm nucleus and a reduction in the seminal parameters.• NAG levels were correlated with the quality of the sperm membrane and nucleus.• Data suggest that a reduction of fertilization capacity in men during varicocele can result from damage to both the testis and the epididymis.

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Aim: To evaluate the effect of presence, grade and anatomical side of varicocele on semen parameters and to identify age-related modifications in semen quality in men with varicocele. Methods: A prospective clinical study was performed in 363 men with varicocele and 155 normozoospermic men without varicocele. We determined the presence, grade and anatomical localization of varicocele: left (grades I–III), right or bilateral. Additionally, evaluation of semen was done and the hypoosmotic swelling test (HOST) percentage determined. Results: The percentage of spermatozoa with normal morphology, motility, vitality, and HOST was reduced in patients with varicocele. Comparison of semen parameters between the different degrees of left varicocele (I, II or III) shows that the percentage of normal sperm morphology is reduced in men with varicocele grade III, while other parameters are not affected. The percentage of patient normozoospermic was 50 and 24% in men with left varicocele grades I and III, respectively. A negative correlation between increase of age in men and sperm motility was observed only in the varicocele group. Conclusions: This study suggests that both the varicocele grade and an increase of age in men with varicocele could determine the extent of alteration to semen quality.