DNA Damage In Human Spermatozoa Research Articles

The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology

The purpose of this study was to evaluate the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay as a method for assessing DNA damage in human spermatozoa. The conventional assay was shown to be insensitive and unresponsive to the DNA fragmentation induced in human and mouse spermatozoa on exposure to Fenton reagents (H₂O₂ and Fe(2+) ). However, both time- and dose-dependent responses could be readily detected if the chromatin was exposed to 2 mm dithiothreitol (DTT) for 45 min prior to fixation. This modified version of the assay significantly enhanced the TUNEL signals generated by subpopulations of spermatozoa isolated on discontinuous Percoll gradients as well as the responses triggered by reagents (arachidonic acid and menadione) that are known to stimulate superoxide anion production by human spermatozoa. DTT exposure also improved the signals detected with chromomycin A₃ (CMA₃), a probe designed to determine the efficacy of chromatin protamination, and enhanced the correlation observed between this criterion of sperm quality and the TUNEL assay. Finally, the output of the TUNEL assay was found to be highly correlated with sperm vitality. The TUNEL methodology was therefore further refined to incorporate a vital stain that covalently bound to intracellular amine groups in non-viable cells. This tag remained associated with the spermatozoa during fixation and processing for the TUNEL assay so that ultimately, both DNA integrity and vitality could be simultaneously assessed in the same flow cytometry assay. The methods described in this article are simple and robust and should facilitate research into the causes of DNA damage in human spermatozoa.

Apoptosis and DNA damage in human spermatozoa.

DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria

DNA damage in human spermatozoa is known to be associated with a variety of adverse clinical outcomes affecting both reproductive efficiency and the health and wellbeing of the offspring. However, the origin of this damage, its biochemical nature and strategies for its amelioration, still await resolution. Using novel methods to simultaneously assess DNA fragmentation (modified TUNEL assay), DNA-base adduct formation (8-hydroxy-2'-deoxyguanosine [8OHdG]) and cell vitality, spermatozoa from a cohort of 50 assisted conception patients were examined and compared with a group of donors. Receiver operating characteristic (ROC) curve analysis was then used to examine the frequency distribution of the data and to determine optimized thresholds for identifying patients exhibiting abnormally high levels of DNA damage. 8OHdG formation and DNA fragmentation were highly correlated with each other and frequently associated with cell death. Percoll centrifugation improved sperm quality but, unexpectedly, increased 8OHdG formation in live cells, as did sperm fractionation using Puresperm gradients. ROC analysis indicated that the frequency distribution of 8OHdG and DNA fragmentation data were significantly different between patients and donors (P < 0.001), permitting the development of thresholds that would allow the accurate diagnosis of DNA damage in the male germ line. The aetiology of DNA damage in spermatozoa involves a cascade of changes that progress from the induction of oxidative stress and oxidized DNA base adduct formation to DNA fragmentation and cell death. Preparation of spermatozoa on discontinuous density gradients aggravates the problem by stimulating the formation of 8OHdG in live cells. However, the development of novel methods and optimized thresholds for diagnosing oxidative DNA damage in human spermatozoa should assist in the clinical management of this pathology.

In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 μM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 μM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE–DNA adducts as measured with 32P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

On the possible origins of DNA damage in human spermatozoa

DNA damage in the male germ line has been linked with a variety of adverse clinical outcomes including impaired fertility, an increased incidence of miscarriage and an enhanced risk of disease in the offspring. The origins of this DNA damage could, in principle, involve: (i) abortive apoptosis initiated post meiotically when the ability to drive this process to completion is in decline (ii) unresolved strand breaks created during spermiogenesis to relieve the torsional stresses associated with chromatin remodelling and (iii) oxidative stress. In this article, we present a two-step hypothesis for the origins of DNA damage in human spermatozoa that highlights the significance of oxidative stress acting on vulnerable, poorly protaminated cells generated as a result of defective spermiogenesis. We further propose that these defective cells are characterized by several hallmarks of 'dysmaturity' including the retention of excess residual cytoplasm, persistent nuclear histones, poor zona binding and disrupted chaperone content. The oxidative stress experienced by these cells may originate from infiltrating leukocytes or, possibly, the entry of spermatozoa into an apoptosis-like cascade characterized by the mitochondrial generation of reactive oxygen species. This oxidative stress may be exacerbated by a decline in local antioxidant protection, particularly during epididymal maturation. Finally, if oxidative stress is a major cause of sperm DNA damage then antioxidants should have an important therapeutic role to play in the clinical management of male infertility. Carefully controlled studies are now needed to critically examine this possibility.

Sperm DNA Damage: Correlation to Severity of Semen Abnormalities

To analyze the relationship between DNA damage and standard semen parameters (SSP) in patients who present for fertility evaluation. Evaluation of male fertility includes assessment of the SSP and increasingly sperm DNA damage. However, the relationship between DNA damage and SSP remains controversial. Following Institutional Research Ethics Board approval, semen samples from 2586 unselected nonazoospermic patients were subjected to computer-assisted semen analysis and flow cytometry-based sperm DNA damage assessment expressed as the DNA fragmentation index. Sperm DNA damage was significantly negatively correlated to sperm SSP (concentration, motility, and normal morphology) and positively correlated to patient's age. DNA damage increased in association with the number of abnormalities in SSP. Patients with oligoasthenoteratozoospermia had significantly higher DNA damage and more frequent DNA damage over 30% compared with normozoospermic patients and patients with abnormalities in 1 or 2 SSP. Our results indicate that DNA damage is significantly correlated to SSP as well as age. In addition, the degree of DNA damage increases with the number of abnormal parameters in a sample and is most severe in patients with oligoasthenoteratozoospermia. Complex and possibly age-related mechanisms of DNA damage in human spermatozoa may be responsible for the strong relationship between SSP and DNA fragmentation index.

Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis

Whereas studies have revealed that the cryopreservation of human semen increases sperm DNA fragmentation, the mechanisms involved in this type of cryo-injury are largely unknown. Elucidation of these mechanisms may provide insight into preventing such injury. We obtained 60 semen samples from 60 men and conducted experiments to determine the cause of cryopreservation-induced DNA fragmentation using 8-oxo-7,8-dihydro-2'deoxyguanosine (8OHdG) as a biomarker of oxidative stress, percentage caspase positive cells as an indicator of apoptosis, the potential antioxidant genistein and the caspase inhibitor Z-VAD(OMe)-FMK. Cryopreservation led to a significant increase in percentage DNA fragmentation, percentage 8OHdG and percentage caspase positive cells (P < 0.001). Percentage DNA fragmentation was positively correlated with percentage 8OHdG before (r = 0.756, P < 0.001) and after cryopreservation (r = 0.528, P = 0.017). The addition of 50 and 100 microM genistein to the cryoprotectant had a significant protective effect on sperm DNA (P < 0.001) although the caspase inhibitor demonstrated no difference to the control. Human sperm DNA fragmentation is associated with an increase in oxidative stress during cryopreservation, rather than the activation of caspases and apoptosis. The estrogenic compound genistein may be useful in reducing this effect but larger trials are needed to confirm this.

DNA Damage in Human Spermatozoa Is Highly Correlated with the Efficiency of Chromatin Remodeling and the Formation of 8-Hydroxy-2′-Deoxyguanosine, a Marker of Oxidative Stress1

DNA damage in human spermatozoa has been associated with a range of adverse clinical outcomes, including infertility, abortion, and disease in the offspring. We have advanced a two-step hypothesis to explain this damage involving impaired chromatin remodeling during spermiogenesis followed by a free radical attack to induce DNA strand breakage. The objective of the present study was to test this hypothesis by determining whether impaired chromatin protamination is correlated with oxidative base damage and DNA fragmentation in human spermatozoa. DNA fragmentation, chromatin protamination, mitochondrial membrane potential, and formation of the oxidative base adduct, 8-hydroxy-2'-deoxyguanosine (8OHdG), were monitored by flow cytometry/fluorescence microscopy. Impairment of DNA protamination during late spermatogenesis was highly correlated (P < 0.001) with DNA damage in human spermatozoa. The disruption of chromatin remodeling also was associated with a significant elevation in the levels of 8OHdG (P < 0.001), and the latter was itself highly correlated with DNA fragmentation (P < 0.001). The significance of oxidative stress in 8OHdG formation was demonstrated experimentally using H2O2/Fe2+ and by the correlation observed between this base adduct and superoxide generation (P < 0.001). That 8OHdG formation was inversely associated with mitochondrial membrane potential (P < 0.001) suggested a possible role for these organelles in the creation of oxidative stress. These results clearly highlight the importance of oxidative stress in the induction of sperm DNA damage and carry significant implications for the clinical management of this condition.

Lycopene supplementation in vitro can protect human sperm deoxyribonucleic acid from oxidative damage

To examine the effects of lycopene on human sperm motility and DNA damage. Prospective study. Andrology research laboratory. Twelve fertile donors. Preincubation of washed sperm suspensions with or without lycopene (2 or 5 micromol/L) followed by a 2-hour incubation with or without hydrogen peroxide (H2O2, 50 micromol/L). Assessments of sperm motility (percentage) and DNA fragmentation index (percent DNA fragmentation index) before and after incubation. Sperm motility (percentage) and sperm percent DNA fragmentation index. Incubation of spermatozoa with H2O2 resulted in a significant decline in mean (+/-SD) percent sperm motility (28%+/-13% vs. 73%+/-4%, respectively) and a significant increase in percent DNA fragmentation index compared with samples incubated without H2O2 (29.8%+/-39.4% vs. 11.1%+/-14.6%, respectively). Pretreatment of samples with 5 micromol/L lycopene resulted in a significantly lower percent DNA fragmentation index than samples incubated without lycopene (8.0%+/-7.9% vs. 29.8%+/-39.4%, respectively). However, lycopene did not protect spermatozoa from the decline in motility after H2O2 treatment. The data suggest that preincubation of spermatozoa with lycopene offers protection against oxidative DNA damage in vitro. These data also highlight the differential protective effects of lycopene on sperm motility and sperm DNA integrity.

Biological and clinical significance of DNA damage in the male germ line

DNA damage is a common feature of human spermatozoa with purported links to poor rates of conception, impaired embryonic development, an increased incidence of miscarriage and the appearance of various kinds of morbidity in the offspring including childhood cancer. However, difficulties in interpretation arise, because these associations are not consistently observed across all data sets. Such inconsistency reflects the inherent complexity of the reproductive process, large variations in sample size, differences in patient selection, inadequate study design as well as inter-individual differences in the type of DNA damage being detected and the effectiveness of repair mechanisms in the oocyte. This review considers the type, source and measurement of DNA damage in human spermatozoa. It also addresses the clinical utility of the information generated in such studies, and highlights areas where further research is needed to bridge the gap between an intriguing biological phenomenon and the evidence-based clinical management of male patients characterized by high levels of DNA damage in their spermatozoa.

Air pollution and mutations in the germline: are humans at risk?

Genotoxic air pollution is ubiquitous in urban and industrial areas. A variety of studies has linked human exposure to air pollution with a number of different somatic cell endpoints including cancer. However, the potential for inducing mutations in the human germline remains unclear. Sentinel animal studies of germline mutations at tandem-repeat loci (specifically minisatellites and expanded simple tandem repeats) have recently provided proof of principle that germline mutations can be induced in vertebrates (birds and mice) by air pollution under ambient conditions. Although humans may also be susceptible to induced germline mutations in polluted areas, uncertainties regarding causative agents, doses, and mutational mechanisms at repetitive DNA loci currently preclude extrapolation from animal data to the evaluation of human risk. Nevertheless, several recent studies have linked air pollution exposure to DNA damage in human sperm, indicating that our germ cells are not impervious to the genotoxic effects of air pollution. Thus, both sentinel animal and human studies have raised the possibility that ambient air pollution may increase human germline mutation rates, especially at repetitive DNA loci. Given that some human genetic conditions appear to be modulated by length mutations at tandem-repeat loci (e.g. HRAS1 cancers, type 1 diabetes, etc.), there is an urgent need for extensive study in this area. Research should be primarily focused upon: (1) the direct measurement of mutation frequencies at repetitive DNA loci in human male germ cells as a function of air pollution exposure, (2) large-scale epidemiology studies of inherited disorders and tandem-repeat associated genetic conditions and air pollution, and (3) the characterization of mutational mechanisms at hypervariable tandem-repeat loci.

Lycopene attenuates oxidative sperm DNA damage in vitro

OBJECTIVE: Oxidative stress has been implicated as a potential factor in male idiopathic infertility, and, has been shown to cause sperm DNA damage and impaired sperm motility. The objective of this study was to examine in vitro effects of the anti-oxidant lycopene on human sperm motility and DNA damage.

Human semen quality and sperm DNA damage in relation to urinary metabolites of pyrethroid insecticides

Exposure to synthetic pyrethroid insecticides is widespread, and is expected to increase among the general population due to the need to replace other common insecticides following regulatory use restrictions. On the basis of limited studies, there is animal and human evidence for altered reproductive or endocrine function following pyrethroid exposure. The present study measured urinary pyrethroid metabolites [3-phenoxybenzoic acid (3PBA) and cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (CDCCA and TDCCA)], semen quality, sperm motion parameters and sperm DNA damage with the neutral comet assay in 207 men recruited from an infertility clinic. In multivariate analysis, the highest 3PBA quartile was associated with a suggestive 20.2 million sperm/ml reduction (95% confidence interval -37.1 to + 2.6) in sperm concentration compared with men below the 3PBA median. There were significant inverse associations between TDCCA and sperm motility and sperm motion parameters when adjusting for CDCCA and other covariates. The highest TDCCA quartile was associated with a 15.5% decline (95% confidence interval -26.2 to -4.8) in sperm motility compared with men below the median. In multiple logistic analyses, there were dose-dependent increased odds for below reference sperm concentration, motility and morphology in relation to TDCCA. Among the comet assay measures, 3PBA and CDCCA were associated with increased sperm DNA damage, measured as percent DNA in the comet tail. We found evidence for reduced semen quality and increased sperm DNA damage in relation to urinary metabolites of pyrethroid insecticides. These findings may be of concern due to increased pyrethroid use and prevalent human exposure.

Estrogenic Compounds Induce Oxidative Stress in Human Spermatozoa: Implications for DNA Integrity and Fertilization Success.

Unusually high levels of DNA damage in the male germ line are, unfortunately, characteristic of our species. A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the mechanisms responsible for creating this stress have not yet been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure to estrogenic xenobiotics. We have undertaken a detailed analysis of the ability of these compounds to trigger oxidative stress and/or DNA damage in human spermatozoa. This investigation was completed using a range of techniques validated for use in these highly specialized cells. DNA integrity was assessed using the Comet and TUNEL assays, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens (reactive metabolites of estrogen), quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity in human spermatozoa while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17β-estradiol, nonylphenol, bisphenol A and 2,3- dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation and potentially preventing fertilization. These findings raise fundamental questions about the importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line.

Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (β-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17β-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.

248. Oxidative stress and DNA damage in human spermatozoa

Unusually high levels of DNA damage in the male germ line are, unfortunately, characteristic of our species. A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the mechanisms responsible for creating this stress have not yet been elucidated. We have undertaken a detailed analysis of the ability of estrogens, electromagnetic radiation and xenobiotics including metal ions to trigger reactive oxygen species (ROS) production and/or DNA damage in human spermatozoa in vitro. This investigation was completed using a range of techniques validated for use in these highly specialised cells. DNA integrity was assessed using the Comet and TUNEL assays, oxidative DNA adducts were detected by an anti-8-oxo-dG assay and cross-linking adducts were characterised by mass spectrometry. Intracellular redox activity was monitored using dihydroethidium as the probe. Of the factors evaluated, catechol estrogens, certain transition metal ions, radio frequency electromagnetic radiation and heat were all capable of stimulating ROS production in human spermatozoa. The oxidative stress created by exposure to such factors lead to the induction of significant DNA damage. Generally, redox inert compounds including non-catechol estrogens and xenobiotics such as phthalate esters did not lead to ROS production or measurable DNA damage. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin. These findings raise fundamental questions about the importance of xenobiotics, environmental factors as well endogenous compounds in creating oxidative stress and DNA damage in the male germ line.

Do heat stress and deficits in DNA repair pathways have a negative impact on male fertility?

In Europe up to one in four couples experience difficulty conceiving and in half of these cases the problem has been attributed to sub or infertility in the male partner. The development of assisted reproductive technologies (ART) such as in vitro fertilization and intra-cytoplasmic spermatozoa injection has allowed some such couples to achieve a pregnancy. Concerns have been raised over the increasing use of ART not least because of the discovery of elevated levels of DNA damage in sperm from subfertile men. The impact of damaged DNA originating in the male germ line is poorly understood, but is thought to contribute to early pregnancy loss (recurrent miscarriage), placental problems and have a long-term impact on the health of the offspring. DNA repair is essential for meiotic recombination and correction of DNA damage in germ cells and proteins involved in all the major repair pathways are expressed in the testis. In this review, we will consider evidence that the production of sperm containing damaged DNA can be the result of suboptimal DNA repair and/or a mild environmental insult, such as heat stress, and how studies in mice may give us insight into the origins and consequences of DNA damage in human sperm.

Attenuation of Oxidative Stress After Varicocelectomy in Subfertile Patients With Varicocele

We examined changes due to oxidative damage to spermatozoa and alterations in antioxidant capacity in subfertile patients with varicocele before and after varicocelectomy in a prospective study. A total of 30 young subfertile male patients with varicocele were recruited in this study. Varicocele was diagnosed by physical examination and Doppler ultrasound. Semen analysis was performed in the 30 patients before and 6 months after varicocelectomy using a computer assisted semen analyzer. The parameters for evaluating oxidative stress changes were 4977 bp deletion of mitochondrial DNA in sperm, as detected by polymerase chain reaction, the 8-OHdG (8-hydroxy-2'-deoxyguanosine) content in spermatozoa DNA, as measured by a high performance liquid chromatography electrochemical method, and seminal plasma protein thiols and ascorbic acid, as measured by spectrophotometric methods. Semen quality, including motility, morphology and sperm density, was improved in 22 patients (73.3%) after varicocelectomy. The incidence of 4977 bp deletion of mitochondrial DNA in sperm was 40% (12 of 30 patients) and 13.3% (4 of 30) before and after surgery, respectively. Mean +/- SD 8-OHdG content in sperm DNA, and seminal plasma protein thiols and ascorbic acid were 10.27 +/- 2.24/10(5) 2'-deoxyguanosine, 0.77 +/- 0.75 nmole/ml and 1.87 +/- 0.40 mg/dl before operation, and 5.95 +/- 1.46/10(5) 2'-deoxyguanosine, 3.00 +/- 1.17 nmole/ml and 3.12 +/- 0.94 mg/dl after surgery, respectively. The incidence of 4977 bp deletion of mitochondrial DNA in sperm and the level of 8-OHdG in sperm DNA were decreased, and seminal plasma protein thiols and ascorbic acid were increased significantly in all 30 patients after varicocelectomy. Also, in the 8 patients in whom semen quality did not improve after surgery a significant decrease in 8-OHdG in sperm DNA, and a significant increase in seminal plasma protein thiols and ascorbic acid were observed. Subfertile patients with varicocele had a significant decrease in oxidative damage in sperm DNA and an increase in antioxidant capacity in seminal plasma after varicocelectomy, indicating that surgery is effective treatment in such patients.

Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome.

GSTM1 genotype influences the susceptibility of men to sperm DNA damage associated with exposure to air pollution

Previous studies have provided evidence for an association between exposure to high levels of air pollution and increased DNA damage in human sperm. In these studies DNA damage was measured using the sperm chromatin structure assay (SCSA) wherein the percentage of sperm with abnormal chromatin/fragmented DNA is determined and expressed as % DNA fragmentation index (%DFI). Here we extend these observations to address the following hypothesis: men who are homozygous null for glutathione-S-transferase M1 ( GSTM1−) are less able to detoxify reactive metabolites of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) found in air pollution. Consequently they are more susceptible to the effects of air pollution on sperm chromatin. Using a longitudinal study design in which men provided semen samples during periods of both low (baseline) and episodically high air pollution, this study revealed a statistically significant association between GSTM1 null genotype and increased SCSA-defined %DFI (beta = 0.309; 95% CI: 0.129, 0.489). Furthermore, GSTM1 null men also showed higher %DFI in response to exposure to intermittent air pollution (beta = 0.487; 95% CI: 0.243, 0.731). This study thus provides novel evidence for a gene–environment interaction between GSTM1 and air pollution (presumably c-PAHs). The significance of the findings in this study with respect to fertility status is unknown. However, it is biologically plausible that increases in %DFI induced by such exposures could impact the risk of male sub/infertility, especially in men who naturally exhibit high levels of %DFI.