Essay: Infertility factors in men

Introduction
It is not possible to find a cause of infertility in nearly 50% of infertile men and this situation has been defined as unexplained or idiopathic (Iammarrone et al. 2003). The role of trace elements in sperm and whole semen quality may have significant influence upon men infertility (Abou-Shakra et al. 1989). Some metals are nonessential xenobiotics that can be measured in most of the general population (Centers for Disease Control and Prevention -CDC 2005), other are essential for good health but may be harmful above certain levels (Agency for Toxic Substances and Disease Registry – ATSDR 2003, 2004, 2005; Greger 1999; Institute of Medicine IOM 2001). Human and animal evidence suggests that these metals may have adverse impacts on male reproductive health at relatively low levels. Chromium in the human organism is allocated quite regularly in each tissue and is necessary for his normal development. Chromium can bind to nucleic acids and acts essential role in metabolism of glucose, some proteins and lipids. Deficiency of chromium occurs rarely (Kabata-Pendias, Pendias 1999). Although, chromium is component of enzymes and stimulates their mutual activity, has been associated with reduced semen quality. The experiment, wherein monkeys (Macaca radiata Geoffrey) were given chromium through drinking water for 6 months, caused at them the decrease of sperm count and sperm forward motility (Subramanian et al. 2006) and negative chromium influence on spermatogenesis process through the induction of oxidative stress were observed (Aruldhas et al. 2005; Subramanian et al. 2006). Whereas, mice which were injected with chromium in CrO3 compound in the dose of 1 mg/kg body weight, it caused additionally increased of the rates of sperm abnormality (Acharya et al. 2006). In men occupationally exposed to Cr(VI) there was found the decrease of sperm count and percentage of sperm motility (Li et al. 2001) as well as the increase of percentage of morphologically abnormal sperms (Kumar et al. 2005). Chromium treatment disrupted spermatogenesis, leading to accumulation of prematurely released spermatocytes and spermatids to the lumen seminiferous tubules. Granulation of chromatin and vacuolation between acrosomal cap and manchette microtubules of elongated spermatids and in Golgi area of round spermatids were also observed. Specific activities of the antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase) and the non-enzymatic antioxidants (glutathione, vitamins A, C and E) decreased, while concentration of H2O2 and hydroxyl radicals in testes increased. Induction of oxidative stress were also noticed in experiences in mice at application of Cr(VI) (Pereira et al. 2005). In men occupationally exposed to Cr(VI) there was found the decrease of concentration of zinc in sperm and the increase of follicle stimulating hormone (FSH) in the blood serum (Li et al. 2001).
Lead presence is found in all tissues and does not caused poisoning at first, because to 90% of received nickel is accumulated in bones (Kabata-Pendias, Pendias 1999). However, Pb may adversely affect sperm shape, motility, and DNA integrity (Eibensteiner et al. 2005, Hernandez-Ochoa et al. 2005, Jurasovic et al. 2004, Telisman et al. 2007). The seminal fluid lead concentration was higher in the infertile (3,6 ?? 3,2 ??g/l) than in fertile men (1,7 ?? 1,0 ??g/l); (Saarenen et al. 1987) and negative impact on motility and viability of spermatozoa and sperm count were observed (Eibensteiner et al. 2005, De Rosa et al. 2003). In rats, which were given intraperitoneal injections of 25 mg/kg of lead acetate or 25 mg/kg of lead acetate with 4 mg/kg of zinc acetate, significant increase of percentage abnormal spermatozoa was found (Piao et al. 2007). Lead is suspected for inexplicable infertility of men through the influence on e.g.: occur of spontaneous premature acrosome reaction, as well as negative impact of lead in seminal plasma on IVF rates was noticed (Benoff et al. 2003). Interactions between lead and other chemical elements can have the essential influence on disturbances of metabolism necessary chemical elements for health. Increase of lead concentration forces the expulsion of iron and copper. Increase of copper level in diet decreases sorption of lead. Antagonism in arrangement of Pb-Zn is conjugate in addition with metabolism of copper. Association between lead and selenium consists in secondary effect of synthesis poorly solvable selenides of lead which are accumulate in kidney. Calcium and phosphorus also have antagonistic influence on receiving of lead (Kabata-Pendias, Pendias 1999). Benoff et al. (2000) also noticed negative lead impact on male fertility in view of its chemical affinity to potassium channels, while Robins et al. (1983) observed the possible damaging effect of lead on chromatin of spermatozoa and spermatogenesis. Most likely high exposure to lead caused negative impact on spermatozoa as a result of increased lipid peroxidation (Kasperczyk et al. 2008).
Cobalt takes part in activation different enzymatic processes, mainly in oxidoreductive reactions (Kabata-Pendias, Pendias 1999). Continuous exposure of male mice to cobalt (400 ppm) via drinking water over a 13-week period resulted in a reproducible, sequential pattern of seminiferous tubule degeneration. Initial changes involved vacuolation of Sertoli cells and formation of abnormal spermatid nuclei, followed by the presence of multinucleated cells and sloughing of cells (Anderson et al. 1992). Chronic exposure of male mice to cobaltous chloride dramatically increased serum testosterone levels and decreased testicular weight, epididymal sperm concentration, and fertility (Pedigo et al. 1988). In adult male rats, which were maintained on a diet containing cobalt, loss of sperm tail filaments and degeneration of sperm mitochondria were found (Mollenhauer et al. 1985). There were also noticed degenerative and necrotic changes in the germinal epithelium and Sertoli cells (Corrier et al. 1985).
Many of the sperm alterations present in idiopathic infertility have recently been related to an alteration of the process of sperm maturation and, in particular, to the presence of high levels of reactive oxygen species (ROS); (Iammarrone et al. 2003). An increased production of ROS results in oxidative damage to cellular lipids, proteins and DNA (de Lamirande, Gagnon 1992, Griveau et al. 1995, Kodama et al. 1997, Fr??czek, Kurpisz 2005). The reactive oxygen species will attack the membranes of spermatozoa, decreasing their viability (Irvine 1996). Catalase (CAT) is an ubiquitous antioxidant enzyme that is present in most aerobic cells. Catalase is involved in the detoxification of hydrogen peroxide (H2O2), a reactive oxygen species, which is a toxic product of both normal aerobic metabolism and pathogenic ROS production. Catalase is tetramer of four identical subunits (approx. 60 kDa each). Each monomer contains a heme ‘ prosthetic group at the catalytic center (Fita and Rossmann 1985). In humans, the highest levels of CAT are found in liver, kidney, and erythrocytes. In many cells of mammals two catalases are present; cytoplasmatic catalase T (Typical) and peroxisomal catalase A (Atypical); (Mayes 1998, Bartosz 2008). The seminal plasma catalase activity was significantly lower in the asthenozoospermic dogs than in the normozoospermic dog. The motility of sperm incubated in solution containing catalase was also significantly higher than that of control sperm incubated in solution without enzyme (Kawakami et al. 2007). In the buffalo the catalase values were highly associated with sperm progressive motility and viability (Alvi-Shoushatri et al. 2009). During incubation of spermatozoa in solution containing xanthine’xanthine oxidase (X-XO) system, catalase prevents against the decline in all motility parameters (Baumber et al. 2000). Catalase activity was also positively correlated with percentage of progressive motility and concentration of spermatozoa in semen of male subjected to a mental stress (students of medical school just before the final examinations – stress period); Eskiocak et al. (2005).
In group of infertile men, an alteration of the sperm parameters is frequently present, such as decreased sperm concentration, diminished motility and morphological abnormalities. The knowledge about the influence of chemical elements upon spermatozoa demonstrated frequently conflicting results and is still incomplete and thus demands complementation. Thus, the aim of the present study was to analyze the level of Co, Cr and Pb in human semen and CAT activity in seminal plasma, and then correlate the results with sperm quality.
Materials and Methods
Human Semen
Semen samples were obtained from men (N=168) undergoing routine infertility evaluation. Each of the subjects was interviewed, and a questionnaire was used to elicit the following information: (1) occupational exposure to agents that are known to affect spermatogenesis, (2) alcoholic consumption, and (3) smoking habit. We have taken account of the principles and criteria of the World Health Organization WHO procedures for sperm collection, analysis, and definitions in our studies. Thus, after 3 to 7 days of abstinence, semen samples were collected into sterile containers. After liquefaction, semen analysis was performed according to the WHO guidelines to obtain volume, sperm concentration, motility, and morphology (Vayena et al. 2002) using the Makler?? Chamber. The study design included two groups based on their ejaculate parameters. So, the group I consisted of males with normal ejaculate (normozoospermia; n=39) and served as the control group. Group II consisted of males with abnormal volume of semen, abnormal concentration, morphology, or motility of spermatozoa; males with more than one abnormal semen variables; and males with no spermatozoa in the ejaculate (n=129).
Cobalt, chromium and lead analysis in semen
Seminal Co, Cr and Pb were measured in 162 of 168 samples (group I, n=38; group II, n=124). Co, Cr and Pb concentrations in semen were determined by inductively coupled plasma-mass spectrometry (ICP-MS), i.e., 7500CE-Agilent plasma ICP-MS spectrophotometer from Agilent Technologies Inc. (Palo Alto, CA, USA). We used the stove of Czylok firm and aluminum mineralizer of Tusnovics firm. Test tubes, from boron and silicon glass each of 25 ml volume, used in the procedure were an advantage. An aliquot of whole available volume, i.e., 1-1.5 ml, was mineralized before measuring. Semen sample was evaporated in a mineralizer at the temperature of 105??C. Evaporated semen sample was burned at the temperature of 450??C within 14 h of order (4 h of access to the temperature of burning). After self-cooling, the sample was poured with 3 ml of 69.0-70.0% nitric acid (Baker Instra analyzed). Then, sample was mixed and located in aluminum mineralizer (heated electrically block up to 400??C supplied with the adjuster and the measure of the temperature). First, mixture was heated at the temperature of 100??C (1 h, 15 min of access to the temperature of heating); then, temperature was raised to 150??C (1 h). After self-cooling to room temperature and addition of 1 ml of H2O2 (35%), sample was again heated at the temperature of 100??C (1 h). After self-cooling, solution was made up of 6 ml of bi-distilled water (0.5 ??S*cm-1), mixed up, and then poured to polyethylene tightly closed containers and determined by ICP-MS method. The concentration of elements was given in terms of milligrams per kilogram of dry weight (ppm dw).
Catalase assay in seminal plasma
CAT activity was measured in 149 of 168 samples (group I: n=37; group II: n=112). The fresh semen was centrifuged at 2000 rpm for 15 min. The supernatant (seminal plasma) was collected and stored at ’80??C until assayed. CAT activity in the seminal plasma (samples were diluted 1:10) was measured by enzyme analysis reactions using a Catalase Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA).
Statistical analysis
The obtained data was analyzed by using Statistica StatSoft data analysis software system, version 8.0., StatSoft, Inc. (2008) computer program. The results are summarized as arithmetic mean values and standard deviation (SD). The differences between the mean values of Co, Cr and Pb concentration in semen and CAT activity in seminal plasma were analyzed for statistical significance by U-Mann-Whitney test (Z) and by Kruskal-Wallis test. Probability level values at P<0.05 were regarded as significant. The relationships between CAT activity and Co, Cr and Pb concentration and semen parameters were examined by Spearman’s rank correlation coefficients.
This study was undertaken following to the Guidelines of the European Union Council and the current laws in Poland, according to the Ethical Commission (05/2005). The work required a permit from the Local Committee for Bioethical Research of Nicolaus Copernicus University Collegium Medicum in Bydgoszcz. This was obtained and had the following number: KB/538/2007.
Results
Statistical analyses were performed in the three groups of males, i.e., group of all individuals, group of normozoospermic males (group I), and group of males with pathological spermiogram (group II).
Co concentration in semen of men from group I was (0.0007 ?? 0.0021) mg*kg-1 dry weight and it was lower than in semen of men from group II (0.0013 ?? 0.0026 mg*kg-1 dry weight), but the difference was not significant. No significant difference was also detected between Cr concentration in semen of men from group I (0.016 ?? 0.0291 mg*kg-1 dry weight) and of men from group II (0.0333 ?? 0.1092 mg*kg-1 dry weight). The mean Pb concentration in semen of men from group I was 0.2569 (?? 0.4295) mg*kg-1 dry weight and it was higher than Pb concentration in semen of men from the group II (0.1898 ?? 0.27 mg*kg-1 dry weight), but the difference was not significant. No significant difference was also detected between CAT activity in seminal plasma of semen from men from group I (130.78 ?? 217.80 nmol*min.-1*ml-1) and of men from group II (64.88 ?? 91.62 nmol*min.-1*ml-1; Tables 1, 2, 3).
In the group of all individuals (group I+II) and group of males with pathological spermiogram (group II), the concentration of Co was significantly lower in males with correct sperm motility (group I+II: n=60; 0.0005??0.0017 mg*kg-1 dry weight; group II: n=22; 0.0002??0.0009 mg*kg-1 dry weight) than in asthenozoospermic males (group I+II: n=101; 0.0015??0.0028 mg*kg-1 dry weight; group II: n=101; 0.0015??0.0028 mg*kg-1 dry weight). Significant difference was also detected in the group of males with pathological spermiogram (group II) between CAT activity in seminal plasma of semen from normozoospermic men (n=19; 43.235 ?? 28.064 nmol*min.-1*ml-1) and of asthenozoospermic men (n=92; 69.828 ?? 99.698 nmol*min.-1*ml-1). The concentration of Co in semen of men from group of all individuals (group I+II) and group of males with pathological spermiogram (group II) was also significantly lower in males with normal morphology of spermatozoa (group I+II: n=105; 0.0005??0.0017 mg*kg-1 dry weight; group II: n=67; 0.0005??0.0015 mg*kg-1 dry weight) than in teratozoospermic males (group I+II: n=56; 0.0023??0.0032 mg*kg-1 dry weight; group II: n=56; 0.0023??0.0032 mg*kg-1 dry weight). Significant difference was also detected in the group of all individuals (group I+II) and group of males with pathological spermiogram (group II) between Pb concentration in semen from normozoospermic men (group I+II: n=105; 0.2341??0.3541 mg*kg-1 dry weight; group II: n=67; 0.2212??0.3115 mg*kg-1 dry weight) and of teratozoospermic men (group I+II: n=56; 0.1531??0.2097 mg*kg-1 dry weight; group II: n=56; 0.1531??0.2097 mg*kg-1 dry weight). The concentration of Pb in semen of men from group of males with pathological spermiogram (group II) was significantly higher in humans with consumption of alcohol in their life history (n=90; 0.18166??0.26272 mg*kg-1 dry weight) than individuals without consumption of alcohol (n=24; 0.16269??0.26514 mg*kg-1 dry weight).
Statistical analyses of relationships between examined variables performed in the group of all individuals showed significant negative correlation (P<0.05) between Co and Pb concentration in semen. The concentration of Co in semen was positively associated with immotility (category D) and negatively associated with slow progressive motility (category B), progressive motility (category A+B) and normal morphology (percentage of normal spermatozoa). There was significant positive correlation between Pb concentration in semen and progressive motility (category A+B) and normal morphology (percentage of normal spermatozoa), and negative correlation between Pb concentration and immotility (category D). We also stated significant negative correlation between Cr concentration in semen and slow progressive motility (category B), and between CAT activity in seminal plasma and volume of ejaculate (ml); Table 4.
In the group of normozoospermic males (group I), significant negative correlation (P<0.05) was demonstrated between Co and Pb concentration in semen. CAT activity was negatively associated with volume of ejaculate (ml). There were significant correlations between both Co (positive) and Pb (negative) concentration in semen and rapid progressive motility (category A). Pb concentration in semen was also positively associated with slow progressive motility (category B; Table 4).
In the group of males with pathological spermiogram (group II), significant negative correlation (P<0.05) was demonstrated between Co and Pb concentration in semen. CAT activity was negatively associated with volume of ejaculate (ml). The concentration of Co in semen was positively associated with immotility (category D) and negatively associated with slow progressive motility (category B), progressive motility (category A+B) and normal morphology (percentage of normal spermatozoa). There was significant positive correlation between Pb concentration in semen and rapid progressive motility (category A) and normal morphology (percentage of normal spermatozoa), and negative correlation between Pb concentration and immotility (category D). We also stated significant negative correlation between Cr concentration in semen and slow progressive motility (category B; Table 4).
Discussion
In the present study we examined the concentration of Co, Cr and Pb in semen and CAT activity in seminal plasma in samples obtained from normozoospermic males and males with pathological spermiogram. We have stated lower, but no significant, concentration of Co in normozoospermic males than in males with pathological spermiogram, but we did not find any information for and against these findings. We observed significantly lower concentration of Co in males with correct sperm motility and normal morphology of spermatozoa than in asthenozoospermic and teratozoospermic males. There was also significant correlation between Co concentration in semen and sperm motility, and normal morphology of spermatozoa. These findings suggest that cobalt concentration might have influence on formation of abnormality in these sperm parameters. Kumar et al. (1990) noticed the loss of motility of human spermatozoa in vitro as a consequence of cobalt treatment which could be explain by observed the loss of sperm surface thiol groups and the augmented production of superoxide anion radicals. A diet containing cobalt caused loss of sperm tail filaments and degeneration of sperm mitochondria in adult male rats (Mollenhauer et al. 1985). The correlations were also found between Co and Pb concentration in semen, but we did not find any information for and against these findings.
We cannot establish the differences in the concentrations of Cr in semen from man in this study between normozoospermic males and males with pathological spermiogram. However, we did not find any information for and against these findings. We also found a significant negative correlation between Cr concentration in semen and slow progressive motility (category B) which indicate adverse influence of chromium on this sperm parameter. The investigations by Li et al. (2001) confirm that male workers occupationally exposed to hexavalent chromium (VI) have decreased sperm motility compared to the control group. At the same time it must be emphasized that in Danadevi et al. (2003) research the sperm concentration showed a negative correlation with blood chromium content in workers from a welding plant. On the other hand, Kumar et al. (2005) found no significant alterations in sperm motility between chromium exposed and unexposed workers.
Our results concerning cobalt concentration in seminal plasma and sperm parameters in semen from man (progressive motility ‘ A, B, and normal morphology) may be useful for determinations in artificial reproduction technics. We can summarize that analyses of cobalt level in semen plasma may be useful for wide diagnostics especially of men infertility.
We have also stated no significant differences between normozoospermic males and males with pathological spermiogram in the concentrations of Pb in semen from man in this study. Concentrations of lead in semen samples of subjects with abnormal semen parameters as compared to normozoospermic patients also did not differ in study of Kasperczyk et al. (2002). Xu et al. (2003) also observed no statistically significant correlation between Pb and semen quality as a whole. We observed significantly higher concentration of Pb in males with normal morphology of spermatozoa than in teratozoospermic males.We also found significant correlation between Pb in semen and sperm motility, and normal morphology of spermatozoa. Studies by Eibensteiner et al. (2005), which examined lead exposure and semen quality among traffic police officers in Arequipa (Peru), showed decreased in sperm motility when blood lead level increased. Huang et al. (2001), in experiment with incubation of spermatozoa with metal ions, showed that incubation with Pb2+ significantly inhibited sperm motility. A negative correlation between Pb concentration in seminal plasma and percentage of motile spermatozoa was found by both Benoff et al. (2003) and Kasperczyk et al. (2008). Hovatta et al. (1998), Noack-Fuller et al. (1993) and Stachel et al. (1989) showed no significant correlations between both sperm motility and morphology of spermatozoa, and concentration of Pb in semen. Also in the research of Xu et al. (1993) the concentrations of lead in blood or seminal plasma did not appear to have any correlation with the sperm parameters studied. On the other hand, Benoff et al. (2003) found a significant positive correlation of Pb concentration in seminal plasma with normal morphology of spermatozoa. The examinations by Telisman et al. (2007) indicated a lead-related increase in immature sperm concentration, in percentages of pathologic sperm, wide sperm, round sperm and short sperm. An increase in lead levels in the infertile men and a significant negative correlation of cadmium semen concentration with sperm motility and sperm concentration was observed in oligoasthenospermic men by Pant et al. (2003). The analysis performed by Slivkova et al. (2009) showed negative correlation between lead and flagellum ball. These findings suggest still unclear influence of lead concentration on formation of abnormality in these sperm parameters. We also observed significantly higher Pb concentration in the individuals with consumption of alcohol in their life history than those without consumption of alcohol. We did not find any information for and against these findings, however toxic effect of alcohol might manifest, among other things, by disturbance of chemical element equilibrium in human body (Ford et al. 1995, Rylander et al. 2001).
We cannot establish the differences in the activity of CAT in seminal plasma from man in this study between normozoospermic males and males with pathological spermiogram. Similarly, mean seminal catalase-like activity in the fertile group was not significantly different from that of the infertile group in research of Zini et al. (2000, 2002). These findings suggest that the high semen ROS levels in some infertile men are likely due to excessive generation of ROS rather than deficient ROS scavenging activity in semen. We observed significantly lower activity of CAT in males with correct sperm motility than in asthenozoospermic males. Although these findings did not confirm previous reports (Ben Abdallah et al. 2009, Tavilani et al. 2008), Khosrowbeygi, Zarghami (2004, 2007) found positive correlations between sperm motility and CAT activity. We also found significant negative correlation between CAT activity in seminal plasma and volume of ejaculate, while Zini et al. (2000, 2002) did not show correlation between them. These findings indicate that, though antioxidants activity in semen is connected with volume of ejaculate, it rather not influence on occurrence or not occurrence of volume disorders of ejaculate.
Summarizing our results, we suggest cobalt, chromium and lead concentration in semen and CAT activity in seminal plasma are related to sperm characteristics (volume of ejaculate, motility and morphology of spermatozoa) and determinations of their levels in semen and seminal plasma might provide new diagnostic tools to improve evaluation of male fertility. Simultaneously, the relationships of Co, Cr and Pb content with sperm parameters are still not clear. Therefore, further investigations of Co, Cr and Pb concentration impact on spermatozoa, and relationships with the parameters of human semen are needed.
Conclusions
1. Co, Cr and Pb concentration and CAT activity are related to sperm characteristics and probably human fertility and their survey could improve the diagnosis of infertility.
2. Effect of alcohol might manifest by disturbance of Pb equilibrium in organism.
3. Co and Pb in semen plasma may be useful for wide diagnostics especially of men infertility as essential factors influencing progressive motility and normal morphology.