Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.

The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2-), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 +/- 20.9 U/ml (mean +/- SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 +/- 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehyde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (IVF) or artificial insemination.     

A Human Sperm Coating Antigen Isolated From Sperm Cell Membrane

ABSTRACT: A human sperm cell membrane antigen that is highly specific to sperm and seminal plasma was isolated from plasma membrane fraction of spermatozoa using rabbit antiserum against human seminal plasma. In addition to the high specificity to sperm and seminal plasma, the isolated antigen showed the following characteristics: (1) It is a glycoprotein of approximately 12,000 daltons that has an affinity to lentil lectin; (2) it is distributed in human milk other than in sperm and seminal plasma, but is not found in any other organs and tissues including testis; (3) seminal plasma contains the largest amount of the antigen activity, 60-fold greater than spermatozoa and 900-fold greater than milk, suggesting that this antigen could be a sperm-coating seminal plasma antigen.     

In-vitro effects of anti-sperm antibodies on human sperm movement

The present study was designed to investigate whether autoantibodlesto external domains of the sperm plasma membrane affect themovement of normal motile spermatozoa. Eight sera and 20 semInalplasma samples containing high levels of anti-sperm antibodiesas well as antibodies eluted from the sperm fraction of 19 autounmuneejaculates were incubated with donor's motile spermatozoa, obtainedby swim-up migration in Tyrode's solution. Sperm movement wasanalysed using is exposure microphotography when >70% ofthe spermatozoa were coated with antibodies (after 30–90mi of incubation). At least 50 tracks of progressively motilespermatozoa were analysed in order to obtain the mean valuesof the amplitude of lateral head displacement (ALH) and thevelocity of progression (VSL). Serum antibodies and sperm elutedantibodies had quite consistent but opposite effects on spermmovement; serum antibodies Increased ALH and decreased VSL whereaseluted antibodies decreased ALH and increased VSL. Seminal antibodiesdid not affect these two parameters significantly. Furthermore,seminal antibodies and sperm eluted antibodies obtained fromthe same ejaculates had distinct effects on ALH and/or VSL.This diversity was apparently not linked to antibody isotypeor localization on the sperm membrane; it might be due to differencesin the composition of the extracellular media. These resultssuggest a dynamic effect of anti-sperm antibodies on sperm movement,a possibility that merits further investigation.     

Reproductive Immunology and Microbiology News*

ABSTRACT: A fertile man had sperm-agglutinating activity in his serum (titers 1:16-1:128) and in his seminal plasma (titers 1:128-1:2048). The antibodies in the seminal plasma could be absorbed with anti-IgA antiserum but not with anti-IgG antiserum. A fresh ejaculate showed strong auto-agglutination of the spermatozoa. With mixed antiglobulin reaction tests (MART) and/or immunobead tests (IBT), IgA and IgG were detected on almost all motile spermatozoa; the erythrocytes, in the MART, and the latex spheres, in the IBT, adhered mainly to the tip of the tail. After mixing the fresh semen with cervical mucus, only 40% of the spermatozoa were locally shaking. The spermatozoa showed excellent penetration of cervical mucus in vitro. This case shows that IgA coating of the tails of the spermatozoa does not necessarily lead to adherence of these spermatozoa to the micelles of the cervical mucus and that the sperm cervical mucus contact test has a better predictive value than the sperm agglutination titer in the seminal plasma.     

Identification and Characterization of a Human Sperm Antigen Corresponding to Sperm-Immobilizing Antibodies

ABSTRACT: A sperm antigen has been isolated front radiolabeled human sperm cell membrane by detergent solubilization, lectin affinity chromatography, gel filtration, and indirect immune precipitation using sperm-immobilizing antisera from patients with unexplained infertility. Isolated material was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Among 20 infertile women's sera with sperm-immobilizing antibodies, two were found to react predominantly with a sperm membrane polypeptide having the approximate molecular weight of 15,000 daltons. No significant binding to this molecule was observed in any sera from pregnant women, unmarried women, and normal men. By the absorption with spermatozoa, the antisera lost their binding activity to the molecule, while the sera absorbed with seminal plasma did not lose the activity. The results indicated that the molecule is a genuine sperm antigen and not a sperm-coating seminal plasma antigen. By the indirect immunofluorescence of washed ejaculated spermatozoa with the antisera, strong fluorescence was localized only in an equatorial segment of the acrosome, while no specific staining was observed in the controls. The antigen is relatively unstable against acid, alkali, and heat treatment. Treatment with proteolytic enzymes such as pronase and trypsin inactivated the antigen activity, indicating that the antigen epitope could be a peptide portion of the glycoprotein.     

Calcium fractions in seminal plasma and functional properties of human spermatozoa

The concentration of ionized calcium [Ca⁺⁺] was reproducibly determined in human seminal plasma with a calcium sensitive electrode (Orion Space Stat 20). Dilution of the seminal plasma gave a linear decrease in free calcium concentration. Freezing and thawning or storage of the seminal plasma under anaerobic conditions did not influence the level of ionized calcium. Storage under aerobic conditions gave a temperature dependent decrease in CA⁺⁺ which parallelled a spontaneous increase in pH. The mean concentration of ionized calcium (measured after a standardized air exposition) was 0.17 mM±0.05 (SD) (range 0.09–0.29 mM). The Ca⁺⁺ level was not correlated to the total calcium concentration or to markers for prostatic (zinc) or seminal vesicular (fructose) secretions. There were, however, more motile spermatozoa in semen samples with an ionized calcium level less than the average (0.17 mM) than in semen samples with a higher Ca⁺⁺ level (53.8%±8.4, N=17 vs 45.0%±12.8, N=15, respectively, p<0.05). The percentage live spermatozoa was also higher (62.4%± 10.4, N=17 vs 50.7%± 14.2, N=15, p<0.01) in the semen samples with a low [Ca⁺⁺]. Spermatozoa in the low [Ca⁺⁺] group did also exhibit a better progressive motility than spermatozoa in the high [Ca⁺⁺] group (p<0.05). It is suggested that the low level of ionized calcium in seminal plasma is of importance for motility of human spermatozoa and that the transfere of spermatozoa from a high Ca⁺⁺ in the epididymis to the low levels in accessory sex glands secretion might be of significans for activation of spermatozoa upon ejaculation.     

Effects of chamber depth on the motion pattern of human spermatozoa in semen or in capacitating medium.

The computer-aided sperm analysis system (CASA) permits precise calculation of the trajectory characteristics of human spermatozoa. Comparison between different chamber depths (10, 20 and 100 microns) revealed variations in the results, which were more evident as the magnitude of the spermatozoon flagellar beat increased. In seminal spermatozoa, the reduced amplitude of movement, linked to the relatively short flagellum and high viscosity of seminal plasma, indicates that the 10 microns-deep chamber can be used for motion analysis without involving extensive modifications. On the other hand, analysis of quicker movement, such as in capacitated spermatozoa, revealed large variations; in particular the proportion of non-progressive hyperactivated spermatozoa was higher in the 20 microns than in the 10 microns chamber (17.9 +/- 14% and 6.9 +/- 4.5% respectively, P < 0.01). In fact the distribution of non-progressive and progressive hyperactivated spermatozoa is depth-dependent. It is therefore necessary to use a chamber of at least 20 microns in depth for sperm analysis in capacitated medium.     

Phagocytosis of human post-capacitated spermatozoa by macrophages

BACKGROUND: Earlier studies demonstrated that macrophages phagocytize spermatozoa in the female genital tract of mammals. In spite of this phagocytosis, fecundity is not affected, raising questions of how the resulting decrease in the number of spermatozoa does not reduce the fertilization rate and of the role of this phagocytosis. We hypothesized that its role is to rid the female genital tract of spermatozoa past their fertilizing stage (post-capacitated spermatozoa). Here we examined whether, indeed, phagocytosis is restricted to post-capacitated spermatozoa. METHODS: Spermatozoa were incubated for 22 h either in a medium that allows them to become capacitated and then post-capacitated, or in a medium that prevents them from acquiring these states. These sperm populations were compared for their susceptibilities to macrophage phagocytosis. RESULTS: Phagocytosis was significantly higher (P < 0.001) in the sperm population containing post-capacitated spermatozoa. Vitality, motility, the acrosomal status and the proportion of capacitated cells did not affect phagocytosis. CONCLUSION: Post-capacitated spermatozoa are, probably, preferentially phagocytized by macrophages.     

Monoclonal Sperm Antibodies: Their Potential for Investigation of Sperms as Target of Immunological Contraception

ABSTRACT: We generated 149 hybridoma cell lines secreting antibodies against human spermatozoal antigens of which antibodies from 136 hybridoma lines also reacted with seminal plasma constituents. The occurrence of common antigeneic determinants on spermatozoa and seminal plasma was further demonstrated by competitive inhibition ELISA tests. We found that seven hybridoma clones secreted antibodies reactive with spermatozoa but nonreactive with seminal plasma. Antibodies from 5 clones were sperm-agglutinating and from 15 clones sperm-immobilizing. Localization of sperm antigens reactive with the monoclonal antibodies was demonstrated by indirect immunoperoxidase staining. A synthetic decapeptide, earlier shown to be reactive with naturally occurring human iso- and autosperm antibodies, was shown to be reactive with the monoclonal antibody VII-5 in ELISA tests.     

Motion Characteristics of Spermatozoa From Men With Cytotoxic Sperm Antibodies

ABSTRACT: Semen samples from 55 fertile nonautoimmune and 44 infertile sperm autoimmune men were evaluated by computerized sperm cell motion analysis. Sperm counts (mean ± SEM, 59.6 ± 10.3 ± 10⁶ per ml), motility (39.0 + 4.6%), mean swimming speed (μm/sec, 26.5 ± 0.9), mean linearity (straight line distance of the cell track divided by the actual track length and multiplied by 10, 6.5 + 0.2), and motility index (% motility ± mean speed, 10.7 ± 1.4) in 23 men with significant titers of cytotoxic sperm antibodies in their serum and seminal plasma were less (p<0.0001) than those in the fertile controls. However, these parameters were comparable in 18 men with sperm antibodies in their seminal plasma but not in their serum, and the control group. Infertile men with serum cytotoxic sperm antibodies had more sperm cells swimming at 11–30 μm/sec, and fewer moving at 31 μm or higher; this was in contrast to results obtained from fertile men (p < 0.05). The percentages of sperm cells moving at 21–30 μm/sec were increased, while those moving at 51–60 μm/sec were decreased in men with seminal plasma sperm antibodies, versus controls. Spermatozoa with low linearities (≤6) were higher (p<0.05) in men with serum and seminal plasma cytotoxic sperm antibodies than in the fertile group. The numbers of sperm cells moving at a linearity of ten, on the other hand, were lower in the infertile men with sperm antibodies in the serum and seminal plasma (21.8 ± 2.7 versus 36.4 ± 1.3; p< 0.00001) or seminal plasma only (28.0 ± 1.5; p< 0.0001). We suggest that the spermatozoa of men with cytotoxic sperm antibodies in their serum and seminal plasma have poorer sperm motion characteristics than those of fertile men.     

Low seminal zinc bound to high molecular weight proteins in asthenozoospermic patients: evidence of increased sperm zinc content in oligoasthenozoospermic patients [published erratum appears in Hum Reprod 1998 Jun;13(6):1750]

Total seminal zinc concentration, seminal zinc fraction bound to high molecular weight proteins (HMW-Zn%) and zinc content in spermatozoa were assayed in the ejaculates of 90 asthenozoospermic patients subdivided into two study groups: normoasthenozoospermics (group I: n = 50) and oligoasthenozoospermics (group II: n = 40). The zinc concentrations of patients were compared with those of a control group of donors showing normal semen parameters. All samples were also investigated for their sperm membrane functional integrity by the hypo- osmotic swelling test (HOS). The results showed normal total zinc concentrations but very low HMW-Zn% values (P < 0.001) in seminal plasma of the two groups of asthenozoospermic patients compared to the controls. Furthermore higher zinc amounts (P < 0.001) were measured in spermatozoa of oligoasthenozoospermic patients compared to group I and to the control group. Oligoasthenozoospermics also displayed a lower HOS score (P < 0.001) compared to the other two groups. These data suggest that the increased unbound seminal zinc could contribute to the decrease of sperm motility in normoasthenozoospermic and oligoasthenozoospermic patients. A further impairment in sperm motility could occur in the oligoasthenozoospermic patients where the increase of seminal free zinc was followed by a major zinc uptake by spermatozoa. The higher intrasperm zinc content in these patients could be a reflection of their low sperm membrane functionality.      

Further Characterization of a Human Sperm Coating Antigen (gp12)

ABSTRACT: In our previous paper, we identified a novel sperm-coating antigen with molecular weight of approximately 12,000 daltons that is highly specific to sperm, seminal plasma, and milk from the plasma membrane fraction of human spermatozoa by using rabbit antiseminal plasma antiserum. In the present study, this 12,000-daltons component, termed gp12, has been investigated for its tissue distribution and antigenic stability. The largest amounts of the antigen are found in seminal plasma, although individual variation is rather high. In seminal plasma, the gp12 molecule presents in a large form with a molecular weight of approximately 50,000 daltons. Its antigenicity is stable when treated with acid, alkali, heat, and various protein denaturants.     

Anti-Müllerian hormone as a seminal marker for spermatogenesis in non-obstructive azoospermia.

Anti-Müllerian hormone (AMH) also known as Müllerian inhibiting substance or factor, is a Sertoli cell-secreted glycoprotein responsible in male embryos for Müllerian duct regression. However, its role in adults remains unknown. AMH seminal concentrations have been evaluated using an enzyme-linked immunoassay in three groups of young men: group 1, fertile donors (n = 18); group 2, obstructive azoospermia (n = 9) after vasectomy or associated with deferent duct agenesia; and group 3, non-obstructive azoospermia with spermatogenesis deficiency and normal karyotype (n = 23). AMH was present in seminal plasma of most fertile donors at concentrations ranging from undetectable (<3.5 pmol/l) up to 543 pmol/l (geometric mean: 153 pmol/l), higher than the serum level (range <3.5 up to 67 pmol/l, geometric mean: 10.7 pmol/l, n = 13). Seminal AMH concentrations were undetectable in all obstructive azoospermic patients, confirming its testicular origin. In non-obstructive azoospermia (group 3), seminal AMH concentration was lower (range <3. 5-68.5 pmol/l, geometric mean: 17 pmol/l) than in fertile donors (P < 0.003) without correlation with plasma follicle stimulating hormone values. In group 3, comparison of seminal AMH concentration and the results of histological analysis of testicular biopsies revealed that undetectable AMH found in 14 cases was associated in 11 of them with lack of spermatozoa, while detectable concentrations of AMH (10-68.5 pmol/l) found in nine cases were associated in seven of them with persistent spermatogenesis. In the adult, AMH is secreted preferentially towards the seminiferous lumen. Although its relationship with spermatogenesis requires further investigation, our results suggest that seminal AMH may represent a non-invasive marker of persistent hypospermatogenesis in cases of non-obstructive azoospermia which may indicate the likely success of testicular spermatozoa recovery before intracytoplasmic sperm injection.     

Glutathione and free sulphydryl content of seminal plasma in healthy medical students during and after exam stress

BACKGROUND: It has been reported that there is a relationship between stress and infertility. The mechanisms of stress-related semen quality alterations have not been fully elucidated. In the present study, we investigated the effect of examination stress on seminal glutathione and free sulphydryl content and sperm quality. METHODS: Semen samples were collected from 34 healthy volunteers who were students of medical school in the fourth semester just before (stress period) and 3 months after (non-stress period) their final examinations. Their psychological examination stress was measured by the State Trait Anxiety Inventory (STAI) questionnaire. After standard semen analysis, semen samples were centrifuged at 10 000g for 15 min. Glutathione and free sulphydryl concentration of seminal plasma were measured. RESULTS: During the period of examination stress, the glutathione and free sulphydryl content of seminal plasma and the motility index of spermatozoa were significantly lower, whereas the percentage of morphologically abnormal spermatozoa was higher, than during the non-stress period (P < 0.001, for all). An association between seminal plasma glutathione and motility index was observed at both periods (P < 0.05 and P < 0.01, respectively). CONCLUSIONS: This study demonstrated that glutathione and free sulphydryl levels in seminal plasma decreased in subjects undergoing examination stress. Furthermore, poor sperm quality may be due to loss of glutathione and free sulphydryl content of seminal plasma.     

Studies on Sperm Survival and Motility in the Presence of Cytotoxic Sperm Antibodies

ABSTRACT: The effect of cytotoxic sperm antibodies and native complement in the serum and secretions from 40 fertile and 93 infertile couples on in vitro sperm survival and motion characteristics was studied. Sperm survival in vitro was unaffected by sera from fertile and infertile subjects without cytotoxic sperm antibodies and from infertile men with antibodies to control but not to autologous sperm. Sperm survival was reduced (P < .001) by sera from infertile men with antibodies to autologous sperm or to antologous and control sperm and from women with cytotoxic antibodies to sperm from both. Sera from fertile couples without sperm antibodies enhanced sperm swimming speed and motility index (P < .0001). Sera from infertile women with or without cytotoxic sperm antibodies did not affect sperm motility. Sperm survival and motility were reduced by seminal plasma from infertile men with cytotoxic antibodies to autologous and/or control sperm. Seminal plasma from fertile men enhanced sperm survival. Cervical mucus from infertile women with antibodies to autoimmune husbands' sperm or to husbands' and control sperm inhibited sperm motion, whereas cervical mucus from infertile women without sperm antibodies and women with antibodies to control sperm failed to have any effect. It is concluded that cytotoxic sperm antibodies developed through exposure to sperm antigens in autoimmune infertile men decrease in vitro sperm survival and/or motility.     

Frequent structural chromosome aberrations in immotile human sperm exposed to culture media

BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.     

Identification of evolutionary conserved mouse sperm surface antigens by human antisperm antibodies (ASA) from infertile patients

PROBLEM: The presence of antisperm antibodies (ASA) in semen may impair sperm function leading to immunological infertility. The aim of the study was to identify the evolutionary conserved antigens on mouse sperm surface that react with human ASA in order to study the mechanism of autoimmune infertility. METHODS OF STUDY: The binding of human ASA to mouse sperm was investigated by means of indirect immunofluorescence. 2D-electrophoresis was applied to separate the biotin-labelled mouse membrane proteins using isoelectric focusing followed by polyacrylamide gel electrophoresis. Cognate antigens of ASA from seminal plasma of infertile patients were analysed by Western blotting. Performing avidin-blots it was detected which of the proteins recognized were sperm surface proteins. The spots of interest were analysed by means of mass spectrometry. RESULTS: ASA bound most frequently (36%) to the post-acrosomal region and to the midpiece of mouse spermatozoa. About 30% of ASA recognized apo lactate dehydrogenase (LDHC4) as a cognate antigen, 30% voltage-dependent anion channel (VDAC2). ASA of 20% bound to outer dense fibre protein and 20% of samples recognized glutathione S-transferase mu5. CONCLUSIONS: Human ASA bound to specific cognate antigens of mouse spermatozoa, offering the possibility to study their functional relevance in the mouse model.     

Effect of sperm-antibodies on acrosome reaction of human sperm used for the hamster egg penetration assay

The effect of anti-sperm antibodies (ASA) on the rate of acrosome reactions (AR) during "in vitro" capacitation of human sperm used for the hamster egg penetration assay (HEPA) was assessed. Motile sperm suspensions from donors were exposed to several sera and seminal plasma with sperm head-directed ASA, then they were washed and capacitated "in vitro." After capacitation, the proportion of acrosome-reacted viable sperm was assessed by staining with Fluoresceinated Pisum Sativum Agglutinin and supravital stain Hoechst 33258. ASA of any immunoglobulin class did not significantly affect either the AR rate, or the hamster egg penetration rate. In conclusion, interference of ASA on spontaneous AR rate during "in vitro" capacitation can not be advocated as an explanation of the impairment of the interaction of human sperm with egg or its vestments, which have been reported in several studies.     

Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia

Testicular or epididymal spermatozoa were obtained for in-vitrofertilization and intracytoplasmic sperm injection ICSI) in27 cycles out of 33 (in six men the azoospermia proved to havetesticular causes). Testicular needle biopsy carried out inaddition to surgical open biopsy proved to be an effective methodto obtain spermatozoa for ICSI from patients with obstructiveazoospermia. Thus it might be possible to replace scrotal operationsby simple needle biopsies. Embryos resulting from ICSI withtesticular spermatozoa were used in 19 transfers that resultedin six pregnancies. One pregnancy resulted from six embryo transfersfrom ICSI after microsurgical-epididymal sperm aspiration (MESA).The normal fertilization rates with testicular (37.3%) and MESAspermatozoa (53.7%) did not differ significantly from each other,but with testicular spermatozoa the rate was significantly lowerthan that obtained with ejaculated spermatozoa and ICSI (59.7%)in the matched couples. The abnormal fertilization of oocyteswith one pronucleus was significantly higher with testicularspermatozoa than with ejaculated spermatozoa in the controlcouples.     

Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men

Male factor infertility patients can have anomalies in their sperm nuclei, displaying high levels of loosely packaged chromatin and damaged DNA. The primary objectives of this study were to compare the extent of DNA fragmentation in the spermatozoa of healthy light and heavy smokers versus non-smokers, and to investigate its correlation with concentrations of the smoking markers cotinine and cadmium. A secondary objective was to compare the concentrations of blood cadmium and serum cotinine with corresponding concentrations in seminal plasma. Ninety-seven healthy male volunteers were divided into three groups: non-smokers, light and heavy smokers. There was no difference between the three groups with respect to age, number of ejaculations per week, serum testosterone concentration, and parameters of semen analysis. The percentages of DNA fragmentation in spermatozoa were not statistically different in the heavy smokers (12.11%), light smokers (11.66%) and non-smokers (20.41%). Serum and seminal plasma concentrations of cotinine were significantly higher in heavy smokers compared with the other groups (P < 0.0001). Median values for blood cadmium concentration were higher in heavy smokers (4.50 microg/l) than in light smokers (0.20 microg/l) and non-smokers (0.20 microg/l) (P < 0.001). Cadmium concentration in seminal plasma was significantly higher in heavy smokers (0.20 microg/l) than in light smokers (0.10 microg/l) and non-smokers (0. 10 microg/l) (P < 0.05). In summary, our results indicate no association between smoking and DNA fragmentation in the spermatozoa of healthy men.