Clinical Conditions of Exacerbated Oxidative Stress That Lead to Sperm DNA Damage Varicocele

Clinical varicocele has been associated with an increase in free radical production including both oxygen and nitrogen-derived radical species [34, 35]. Nitric oxide production is proportional to the severity of varicocele [36] and patients with varicocele have increased levels of 8-oxo-guanosine (8-OHdG) in leukocyte DNA indicating oxidative damage [37] . Saleh et al., using the Sperm Chromatin Structure Assay (SCSA) test, reported a significant increase in the extent of sperm DNA damage in infertile patients with varicocele [38] and Smith et al., reported that the presence of varicocele was associated with high levels of sperm DNA damage, as assessed by the SCSA and TUNEL tests, even in the presence of normal sperm parameters. Furthermore, DNA damage was correlated with oxygen radical levels [39]. In addition, the fact that varicocelectomy is associated with a significant decrease in both oxygen radical production and sperm DNA damage, this further supports the role of oxidative stress in the pathophysiology of varicocele [40, 41]. Enciso et al. found that mean sperm DNA fragmentation levels in patients with varicocele were 32.4%. This value was 2.6 times higher than that observed in fertile patients. In addition, an even more significant difference was found when the different patterns of sperm DNA damage were evaluated, i.e. the contribution of spermatozoa with massive DNA-nuclear damage displaying the so called "degraded sperm" pattern. This particular sperm subpopulation, visualized using the sperm chromatin dispersion test (SCD) and characterized by a highly protein depletion, retaining very low amounts of DNA after massive protamine removal (Fig. 11.2a. c) when

Fig. 11.3 Sperm DNA fragmentation levels in patients with leukocytospermia and varicocele. Box-and-whisker plots to compare sperm DNA fragmentation (a) and sperm degradation (b) in two different cohorts of patients (leukocytospermia and varicocele) and of sperm donors

compared with spermatozoa with fragmented DNA (Fig. 11.2b) , are frequently found in patients with varicocele. Patients diagnosed with varicocele tend to present higher basal levels of SDF (Fig. 11.3a) and also higher levels of degraded sperm (Fig. 11.3b) when compared with donors. The ratio of the degraded sperm pattern to the total of sperm population containing fragmented DNA in these patients was 1-4.2 or higher. This proportion is significantly lower in normozoospermic patients (1-10 or lower). The observed increase in this degraded sperm subpopulation may be related to an increase in ROS and the resulting oxidative stress, which causes lipid peroxidation of sperm plasma membrane and nuclear DNA damage. Nitric oxide released by the endothelial cells from the dilated spermatic veins and per-oxynitrite generated by its reaction with the superoxide anion are probably responsible, at least in part, for the observed oxidative damage [34]. DNA fragmentation could be either a direct expression of this damage or a consequence of the triggering of an apoptotic-like process by ROS overproduction. These sperm nuclear abnormalities, such as the degraded sperm pattern, were reproduced in an experimental model of varicocele in rats with enhanced ROS production and an increase in sperm DNA damage. Thus, the degraded sperm pattern most likely corresponds to the highest degree of nuclear damage also compromising the nuclear matrix as a consequence of an intense and prolonged exposure to DNA nuclear-damaging factors where not only the DNA but also the proteins, especially those from the nuclear matrix, are affected, leading to an advanced lytic stage. The nature of the nuclear damage in the degraded type deserves further investigation although it is anticipated that it represents a specific sperm subpopulation collapsed by the massive presence of single and double strand DNA breaks.

An enhancement in oxidative stress, in addition to be determined by an increase in ROS production, may also be determined by a decrease in antioxidant defences [42] or both [43], as it has been reported in men with varicocele. Moreover, the increase in seminal ROS levels seems to be correlated with varicocele grade [44]. This is a well-known factor that may induce DNA fragmentation either in vivo or in vitro [45]. Nitric oxide and peroxynitrite are produced in high concentrations in the dilated spermatic veins thus contributing to the high level of oxidative stress observed in men with varicocele [34, 35, 46]. In addition to the dilated veins, ROS may be released into the seminiferous tubules by immature spermatozoa with proximal cytoplasm retention [1, 24, 26], which are frequently found in semen samples from patients with varicocele. Leukocytospermia

Leukocytospermia is defined as a concentration of leukocytes in semen above one million/mL. Leukocytospermia is often the result of an inflammatory process in the male reproductive tract including orchitis, epididymitis, prostatitis, or inflammation of the seminal vesicles. It is also associated with infertility and reduced semen quality affecting sperm motility and sperm morphology. Leukocytospermia is frequently associated to bacteriospermia. In general, leukocytospermia leads to an oxidative stress environment where ROS are largely present as a result of leukocyte activation [32]. As previously mentioned, co-incubation of sperm suspensions with phytohae-magglutinin-activated polymorphonuclear leukocytes in vitro results in a significant increase in the production of oxygen radicals by immature sperm from infertility patients [32]. One of the possible explanations for this effect is that proinflamma-tory factors released by activated leukocytes amplify oxygen radical production by immature sperm by 2-3 orders of magnitude.

Are there any differences in the pattern of sperm DNA damage in patients with varicocele or leukocytospermia compared to those observed in normozoospermic patients or sperm donors? In order to throw some light into this particular issue and assuming that in both clinical situations sperm are exposed to high levels of oxida-tive stress, we have reanalyzed some of our existing data compiling 51 donors, 90 varicocele patients and 72 patients with leukocytospermia. The results for sperm DNA fragmentation distribution and the presence of degraded sperm are shown in Fig. 11.3 . Patients diagnosed with leukocytospermia tend to present higher basal levels of SDF (Fig. 11.3a) and also higher levels of degraded sperm (Fig. 11.3b) when compared with donors. There were significant differences in the level of sperm DNA damage when the three groups were compared (Kruskal-Wallis test Chi-square: 103.3; P < 0.0001). However, for whole sperm DNA fragmentation, these differences are not so clear when leukocytospermia is compared with varicocele (U de Mann-Whitney, Z -2.049: P = 0.04). Although there were significant differences in the prevalence of the degraded sperm pattern when the three groups were compared (Kruskal-Wallis test Chi-square: 126.04; P < 0.0001) the prevalence of the degraded sperm pattern was significantly higher in the varicocele compared to the leukocytospermia group (U de Mann-Whitney, Z -7,632: P=0.0001). This implies that, even assuming that there are similar levels of DNA damage in both groups of patients, the pattern of DNA damage is different and more aggressive in the case of the clinical varicocele. Subsequently, the effects of oxidative stress on sperm DNA integrity render different results in both clinical situations suggesting that in the particular case of the varicocele either the intensity or the type of damage is different compared to the case of leukocytospermia.

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