8-hydroxy-2--deoxyguanosine and Infertility--Male

The expanding research interest in the last two decades on reactive oxygen species (ROS), oxidative stress, and male infertility has led to the development of various techniques for evaluating oxidative DNA damage in human spermatozoa. Measurement of 8-hydroxydeoxyguanosine (8-OHdG) offers a specific and quantitative biomarker on the extent of oxidative DNA damage caused by ROS in human sperm. The close correlations of 8-OHdG level with male fertility, sperm function and routine seminal parameters indicate the potential diagnostic value of this technique in clinical applications. On the other hand, single cell gel electrophoresis (SCGE or comet assay) and terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay have also been demonstrated to be sensitive, and reliable methods for measuring DNA strand breaks in human spermatozoa. As certain technical limitations were inherent in each of these tests, it is believed that a combination of these assays will offer more comprehensive information for a better understanding of oxidative DNA damage and its biological significance in sperm function and male infertility.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Biomarkers; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA Damage; Electrochemistry; Fertility; Humans; Infertility, Male; Male; Oxidation-Reduction; Oxidative Stress; Spermatozoa

SARS-CoV-2 negatively affects semen characteristics, impairs various biochemical processes in seminal fluid and within spermatogenic cells ultimately leading to male fertility decline. However, the distinct mechanisms, in particular, the role of oxidative stress on the consequences of coronavirus infection, have not been well investigated, which is the purpose of the present study. The standard semen parameters, its pro- and antioxidant system state, as well as the level of sperm DNA fragmentation, were assessed in 17 semen samples of men five months after the coronavirus infection and in 22 age-matched control patients. We determined that the DNA fragmentation rate negatively correlated with the period after coronavirus recovery, as well as seminal fluid superoxide dismutase activity and uric acid level. It was demonstrated that COVID-19 is not always associated with increased DNA fragmentation, allowing them to be considered as two independent factors. Thus, the most significant changes were noted in the samples of men after COVID-19 and abnormal TUNEL results: increased round cell number, decreased seminal fluid's nitrotyrosine level, and total antioxidant capacity and Zn, as well as an increased 8-hydroxy-2'-deoxyguanosine level within spermatozoa. The data obtained indicate that increased DNA fragmentation and diminished semen quality in men can be the result of an imbalance in semen pro- and antioxidant components after COVID-19.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Antioxidants; Biomarkers; COVID-19; DNA Fragmentation; Humans; Infertility, Male; Male; Oxidative Stress; SARS-CoV-2; Semen; Semen Analysis; Sperm Motility; Spermatozoa

The aim of study was to evaluate reactive oxygen species (ROS), total antioxidant capacity (TAC) andROS-TAC score as indicator for oxidative stress status as well as 8-hydrodeoxyguanosine (8-OHdG) levels as amarker for DNA damage in the seminal plasma of asthenozoospermia patients compared to normozoospermiasamples.. The semen samples of 28 fertile normozoospermic donors and 25 infertile men withasthenozoospermia were analyzed according to World Health Organization (WHO) criteria. ROS production wasmeasured in neat semen samples by the chemiluminescent assay. Plasma levels of TAC was measured by commercially available colorimetric assays. The levels of DNA oxidative damage were measured by seminal plasma levelsof 8-OHdG using ELISA method. ROS-TAC score was measured using principal component analysis.. Asthenozoospermic men had a higher ROS levels compared to the normozoospermic men (P = .01).However, no significant difference was observed in TAC levels between the groups. ROS-TAC score in asthenozoospermicmen was lower than normozoospermic men (P = .02). The levels of 8-OHdG in the asthenozoospermicmen were higher than normozoospermic men (P = .01).. The present study demonstrated a decrease in ROS-TAC score and, a high DNA damage in asthenozoospermiacompared to normozoospermia. ROS-TAC score can predict the oxidative damage of semen samplesof astenozoospermic infertile males.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Asthenozoospermia; Biomarkers; Deoxyguanosine; DNA Damage; Fertility; Humans; Infertility, Male; Male; Oxidative Stress; Reactive Oxygen Species; Semen

Varicocele is commonly associated with male infertility because it impairs normal sperm morphology and activity. Polyunsaturated fatty acids (PUFA) are important determinants of sperm cell structure and function, but their relationship with varicocele remains unclear. The aim of the present study was to investigate the PUFA composition in spermatozoa of infertile men with varicocele and to evaluate the potential relationship between PUFA and varicocele. This case control study recruited 92 infertile men with varicocele, 99 infertile men without varicocele and 95 fertile male control subjects. Semen morphology and activity parameters were assessed and seminal plasma 8-hydroxy-2-deoxyguanosine (8-OHdG) content was determined by ELISA. Sperm concentrations of omega-3 and omega-6 fatty acids were measured by gas chromatography. Infertile men with varicocele had lower concentrations of omega-3 PUFA, higher omega-6:omega-3 PUFA ratios and greater oxidative DNA damage in spermatozoa compared with infertile men without varicocele and normal subjects. The degree of varicocele and DNA damage was associated with decreased omega-3 PUFA concentrations and semen quality in infertile men with varicocele. The findings suggest that omega-3 PUFA deficiency could be implicated in varicocele-associated infertility, and highlight the need for intervention trials to test the usefulness of omega-3 supplementation in reducing sperm abnormalities in infertile men with varicocele.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Case-Control Studies; Chromatography, Gas; Deoxyguanosine; DNA Damage; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Fertility; Humans; Infertility, Male; Male; Oxidative Stress; Semen; Sperm Count; Sperm Motility; Spermatozoa; Varicocele

Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress?. Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress.. Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive.. The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n = 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2-8 weeks and compared with control groups for sperm DNA damage and pregnancy rates.. In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded.. Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P < 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P < 0.05 for increase in fertility in the scrotal heat stress model).. It was not possible to determine the exact level of antioxidant consumption for each mouse during the treatment period.. Recent clinical studies confirm moderate to severe sperm DNA damage in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility. Our results, if confirmed in humans, will impact clinical fertility practice because they support the concept of using an efficacious antioxidant supplementation as a preconception therapy, in order to optimize fertilization rates, help to maintain a healthy pregnancy and limit the mutational load carried by children.. The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Biomarkers; Deoxyguanosine; DNA Damage; Female; Glutathione Peroxidase; Infertility, Male; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Oxidative Stress; Pregnancy; Pregnancy Outcome; Pregnancy Rate; Semen Analysis; Spermatozoa

Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6(-/-) mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6(-/-) males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6(-/-) males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6(-/-) males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aging; Animals; Chromatin; Deoxyguanosine; DNA; DNA Fragmentation; Female; Immunohistochemistry; Infertility, Male; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Peroxiredoxin VI; Sperm Motility; Spermatozoa

In the present work, the effect of exposure to cigarette smoke on male fertility in rats, as characterized by changes in the relative weight of sex organs, epididymal sperm count, activity of marker enzymes and DNA damage was evaluated. Exposure of rats to cigarette smoke caused a gradual decrease in total body weight gain and relative weight of the epididymis and seminal vesicles by 30 and 40% respectively. Epididymal sperm count was reduced significantly by 25% (P 0.05) after 2 weeks and by 41% (P 0.001) after 4 weeks of exposure. Exposure to cigarette smoke had reduced the activity of sorbitol dehydogenase by 18% (P < or = 0.05) and increased the activity of lactate dehydrogenase by 28% (P < or = 0.05). The changes in both key enzymes were significant, which reflected the inhibitory effect of cigarette smoke on spermatogenesis and sperm maturation. The toxic effect of exposure could be explained partially due to induction of DNA damage and oxidative stress as shown by the significant increase in serum 8-hydroxy-2'-deoxyguanosine from 22.83 to 37.33 ng ml(-1) blood.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Deoxyguanosine; DNA Damage; Infertility, Male; L-Iditol 2-Dehydrogenase; L-Lactate Dehydrogenase; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Smoking; Sperm Count; Sperm Maturation; Spermatogenesis

Oxidative stress (OS) is involved in many disoders including male infertility. Human spermatozoa are very sensitive targets of reactive oxygen species (ROS) and most sperm functions are impaired in the case of OS. In addition unbalanced production of ROS is considered one of the most important causes of sperm DNA fragmentation, a semen trait of infertile men. The relationship between oxidative damage and semen quality is partially controversial, probably due to the different methods and/or targets used to reveal the OS. In this study, by fluorescence microscopy and flow cytometry, we compared two methods to reveal 8-hydroxy,2-deoxyguanosine (8-OHdG), the hallmark of oxidative DNA damage: an immunofluorescence method and the commercial OxyDNA kit. We found that although both methods localized the labelling in sperm nuclei they yielded different measures, and only with the immunofluorescence method was the labelling specific for sperm 8-OHdG. The immunofluorescence method, coupled to flow cytometry, was thus selected to analyse the 8-OHdG content in semen samples from 94 subfertile patients and to investigate the relationship with semen quality. We found that the percentages of spermatozoa with 8-OHdG (mean±s.d., 11.4±6.9%) were related to sperm count (Pearson's correlation coefficient (r)=-0.27, P=0.04 (ANOVA and student's t-test)), motility (progressive: r=-0.22, P=0.04; non-progressive: r=0.25, P=0.01), and normal morphology (r=-0.27, P=0.01). In conclusion, we demonstrate that immunofluorescence/flow cytometry is a reliable and specific method to detect 8-OHdG at single-cell level and show that oxidative damage only partially overlaps poor semen quality, suggesting that it could provide additional information on male fertility with respect to routine semen analysis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Analysis of Variance; Biomarkers; Cell Nucleus; Cohort Studies; Deoxyguanosine; DNA Damage; Flow Cytometry; Humans; Infertility, Male; Male; Microscopy, Fluorescence; Oxidative Stress; Predictive Value of Tests; Reagent Kits, Diagnostic; Reproducibility of Results; Semen Analysis; Sperm Count; Sperm Motility; Spermatozoa

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods. The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10(6) dG were respectively 7.85 and 5.87 (p=0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Ascorbic Acid; Deoxyguanosine; DNA; DNA Damage; Fertility; Guanine; Humans; Infertility, Male; Leukocytes; Male; Oxidative Stress; Semen; Smoking; Spermatozoa

8-Oxoguanine DNA glycosylase 1 (OGG1) plays an important role in repairing oxidative DNA damage induced by chemical agents, such as tobacco. This study examined the effects of OGG1 Ser326Cys polymorphism and cigarette smoking, alone or combined, on sperm oxidative DNA damage and the risk of male infertility. A total of 620 idiopathic infertile subjects and 480 fertile controls were recruited in this study. Sperm 8-hydroxydeoxyguanine (8-OHdG) was measured by immunofluorescent assay using flow cytometry and genotypes were determined by OpenArray platform with a chip-based Taq-Man genotyping technology. Our results demonstrated that both cigarette smoking and OGG1 polymorphism can affect the sperm 8-OHdG levels. Individuals with variant Cys/Cys homozygote showed higher levels of sperm 8-OHdG than wide-type homozygote carriers (Ser/Ser). Stratified analysis found that the association between OGG1 polymorphism and sperm 8-OHdG levels was only observed among smokers with pack-years ≥5 but not among those subjects with pack-years<5 (pack-years=packs smoked per day×years as a smoker). Further analysis based on the case-control study revealed that variant allele (Cys) of OGG1 was significantly associated with male infertility risk in a dominant model (OR=1.35, 95% CI: 1.01-1.82; trend P<0.001). Furthermore, we found a significant gene-environment interaction between OGG1 Ser326Cys polymorphism and cigarette smoking in relation to male infertility risk (Pinteration=0.0003). These findings provided the first evidence about potential interactive effects of OGG1 polymorphism and cigarette smoking on male infertility risk.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Case-Control Studies; DNA Damage; DNA Glycosylases; Gene-Environment Interaction; Genotype; Guanine; Humans; Infertility, Male; Male; Oxidative Stress; Polymorphism, Single Nucleotide; Risk Factors; Smoking; Spermatozoa

To test the hypothesis that polymorphisms in antioxidant genes are more susceptible to sperm DNA damage and male infertility, we examined 11 single-nucleotide polymorphisms from six antioxidant genes (GPX1, CAT, PON1, NQO1, SOD2/MnSOD, and SOD3) in 580 infertility cases and 580 controls from a Chinese population-based case-control study (NJMU Infertility Study). Genotypes were determined using the OpenArray platform. Sperm DNA fragmentation was detected using the Tdt-mediated dUTP nick-end labeling assay, and the level of 8-hydroxydeoxyguanosine (8-OHdG) in sperm DNA was measured using immunofluorescence. The adjusted odds ratio and 95% confidence interval (CI) were estimated using unconditional logistic regression. The results indicated that the PON1 Arg192Glu (rs662) and SOD2 Val16Ala (rs4880) variant genotypes were associated with a significantly higher risk of male infertility. In addition, subjects carrying variant genotypes of both loci had a twofold (95% CI, 1.42-2.90) increase in the risk of male infertility, indicating a significant gene-gene interaction between these two loci (P for multiplicative interaction=0.045). Moreover, linear regression analysis showed that individuals carrying the PON1 Arg192Glu (rs662) or SOD2 Val16Ala (rs4880) variants have significantly higher levels of sperm DNA fragmentation and 8-OHdG. These data suggest that genetic variations in antioxidant genes may contribute to oxidative sperm DNA damage and male infertility.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aryldialkylphosphatase; Case-Control Studies; Catalase; China; Deoxyguanosine; DNA Fragmentation; Epistasis, Genetic; Genetic Association Studies; Genotype; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; Infertility, Male; Male; Oxidative Stress; Polymorphism, Single Nucleotide; Risk Factors; Sequence Analysis, DNA; Smoking; Spermatozoa; Superoxide Dismutase

To discuss seminal plasma oxidative stress and sperm DNA oxidative damage in patients with idiopathic asthenozoospermia.. Infertile couples were selected from the clinic outpatients of the Reproductive Center of Xiangya Hospital, Central-South University from December 2010 to March 2011. Fresh semen of 28 men with idiopathic asthenozoospermia was collected as an experiment group, and 24 fertile men with normal semen and normal reproductive history served as a control group. Level of reactive oxygen species (ROS) in the seminal plasma was assessed with luminer chemiluminescence method. Density of sperm DNA oxidation product 8-hydroxy-2'-deoxyguanosine (8-OHdG) was assessed with enzyme linked immunosorbent assay.. 1) ROS level in the experiment group was higher than that in the control group (P<0.01). There was negative correlation between the percentage of progressive motility spermatozoa and the ROS level in the seminal plasma in the 2 groups (r=-0.72, P<0.01). 2) Density of sperm 8-OHdG in the experiment group was higher than that in the control group (P<0.01). There was negative correlation between the percentage of progressive motility spermatozoa and the density of sperm 8-OHdG (r=-0.73, P<0.01). 3) There was positive correlation between the ROS level in the seminal plasma and the density of sperm 8-OHdG (r=0.77, P<0.01).. There is sperm DNA oxidative damage in patients with idiopathic asthenozoospermia, which may be related with the oxidative stress. Excessive generation of reactive oxygen species may be a cause of low sperm motility in patients with idiopathic asthenozoospermia.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Asthenozoospermia; Case-Control Studies; Deoxyguanosine; DNA Damage; Humans; Infertility, Male; Male; Oxidative Stress; Spermatozoa; Young Adult

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.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Biomarkers; Chromatin; Chromatin Assembly and Disassembly; Deoxyguanosine; DNA Damage; Efficiency; Humans; Infertility, Male; Male; Membrane Potential, Mitochondrial; Oxidative Stress; Protamines; Spermatozoa; Superoxides; Young Adult

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Ascorbic Acid; Deoxyguanosine; DNA, Mitochondrial; Humans; Infertility, Male; Male; Oxidative Stress; Semen; Spermatozoa; Sulfhydryl Compounds; Varicocele

To investigate the causal role of oxidative-stress status on human sperm motility.. To demonstrate that sperm with higher oxidative damage have a lower antioxidant capacity.. University hospital infertility center.. Seventy-eight semen samples were obtained from 35 healthy donors who had normal semen characteristics and from 43 infertile or subfertile males.. The levels of oxidative damage (8-hydroxy-2'-deoxyguanosine [8-OHdG] and lipid peroxides) and antioxidants (retinol, alpha-tocopherol, ascorbate, and protein thiols) in the spermatozoa and/or seminal plasma were measured.. We analyzed the specific content of 8-OHdG and lipid peroxides by using high-performance liquid chromatography (HPLC)-electrochemical detection and HPLC-fluorescence analysis, respectively. Retinol and alpha-tocopherol were analyzed by using an HPLC system, whereas ascorbate and protein thiols were determined by using spectrophotometry. 8-Hydroxy-2'-deoxyguanosine was visualized by immunofluorescent staining with an anti-8-OHdG antibody that was conjugated with fluorescein isothiocyanate conjugate. Lipid peroxides in spermatozoa were stained with a fluorescent dye, C11-BODIPY(581/591).. Statistically significant negative correlations were revealed between sperm motility and 8-OHdG and between motility and lipid peroxides. Statistically significant positive correlations were found between sperm motility and the levels of retinol, alpha-tocopherol, ascorbate, and protein thiols of seminal plasma. 8-Hydroxy-2'-deoxyguanosine and lipid peroxides in spermatozoa were found to be present mostly in mitochondria.. Oxidative stress and oxidative damage were increased significantly in spermatozoa with declined motility, and the antioxidant capacities in the spermatozoa and seminal plasma were lower in males who had infertility or subfertility.

Topics: 8-Hydroxy-2'-Deoxyguanosine; alpha-Tocopherol; Antioxidants; Ascorbic Acid; Deoxyguanosine; DNA Damage; Humans; Infertility, Male; Lipid Peroxides; Male; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Semen; Sperm Motility; Spermatozoa; Sulfhydryl Compounds; Vitamin A

One byproduct resulting from free radical damage is the DNA hydroxylation also known as DNA oxidation. Our aim with this work was to determine the relevance of sperm DNA oxidation on embryo quality in oocyte donation cycles.. We prospectively studied pairs of oocyte donation cycles, i.e., the same oocyte donors, donating to two recipients, where the only difference between the two treatments was the use of a different sperm sample.. University-affiliated private IVF setting.. Infertile male partners from couples undergoing oocyte donation cycles (n = 38): 76 semen aliquots analyzed before and after semen processing by swim up.. None.. We measured sperm DNA oxidation by flow cytometry using the OxiDNA assay and correlated it with embryo quality parameters, implantation, and pregnancy outcome.. A positive correlation was seen between embryo fragmentation and DNA oxidation of capacitated samples at 48 hours and 72 hours after fertilization. However, when we analyzed the differences in the IVF outcome parameters of the couples who shared the oocyte cohort (same donor) with the differences in the OxiDNA values, we observed increased and further relationships with cell embryo division 48 hours after fertilization. A negative association with blastocyst formation was also detected.. Oxidative damage in the DNA is clearly increased in samples with lower sperm motility. An association between early and late embryo quality and sperm DNA oxidation supports the relevance of the hydroxylation of 8-oxoguanine as a biomarker of sperm quality reflecting the free radical damage in human sperm.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Biomarkers; Deoxyguanosine; DNA; Embryonic Development; Female; Fertilization in Vitro; Humans; Infertility, Male; Linear Models; Male; Middle Aged; Oocyte Donation; Oxidation-Reduction; Pregnancy; Pregnancy Outcome; Prognosis; Prospective Studies; Sperm Capacitation; Spermatozoa

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.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Ascorbic Acid; Deoxyguanosine; DNA, Mitochondrial; Follow-Up Studies; Humans; Infertility, Male; Male; Oxidative Stress; Prospective Studies; Semen; Spermatozoa; Sulfhydryl Compounds; Varicocele

Methylenetetrahydrofolate (MTHFR) and DNMT3b play imperative roles in DNA synthesis and de novo methylation. GSTM1 is involved in detoxification of carcinogens. Mitochondrial DNA deletion has been associated with lower motility in human sperm. We analysed if polymorphisms in MTHFR (C677T and A1298C) and DNMT3b (C46359T) are associated with non-obstructive male infertility. We also analysed if folate, vitamin B(12), homocysteine (Hcy), 8'-hydroxy-2'-deoxygnanosine (8-OHdG) levels, dietary folate intake and mtDNA deletion (4977 bp) affects fertility, such interactions are modified by deletion and methylation of GSTM1. In this case-control study, we included 179 oligoasthenoteratozoospermia patients and 200 fertile men. Single-nucleotide polymorphism analysis was performed by PCR-restriction fragment length polymorphism. The MTHFR (C677T and A1298C) and DNMT3b (C46359T) frequencies did not differ significantly in two groups. GSTM1 in association with mtDNA 4977 deletion is significantly associated with infertility. Plasma folate and vitamin B(12) levels are decreased and total Hcy is elevated in infertile men. GSTM1 methylation status was investigated by methylation-specific PCR. Methylation is significantly correlated with GSTM1 reduced/loss of expression in infertile men. Infertile men have significantly higher 8-OHdG levels. Dietary folate intake is not linked with GSTM1 methylation. Low folate intake in association with CT + TT genotypes (C677T) has significant protective effect on GSTM1 methylation. Results indicate that micronutrients, 8-OHdG levels, mtDNA deletion and GSTM1 promoter methylation are frequent alterations in infertility.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Base Sequence; Case-Control Studies; CpG Islands; Deoxyguanosine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; DNA, Mitochondrial; Folic Acid; Gene Frequency; Genetic Predisposition to Disease; Glutathione Transferase; Homocysteine; Humans; India; Infertility, Male; Male; Methylenetetrahydrofolate Reductase (NADPH2); Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Sequence Deletion; Vitamin B 12; White People

We examined oxidative damage to leukocyte DNA in the spermatic vein and sperm DNA of patients with varicocele.. A total of 32 young male patients with varicocele (group 1), 20 young male patients with subclinical varicocele (group 2) and 15 normal young males without varicocele (group 3) were recruited in this study. Varicocele and subclinical varicocele were confirmed by physical examination and Doppler ultrasonography. Blood samples were drawn from peripheral and spermatic veins of controls and patients before varicocelectomy. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) in leukocyte DNA and sperm DNA were measured by high performance liquid chromatography. The 4977 bp deletion of mitochondrial DNA (mtDNA) in spermatozoa was detected by polymerase chain reaction.. The mean 8-OHdG level +/- SD in leukocyte DNA of spermatic veins was significantly higher than that of corresponding peripheral veins in groups 1 and 2 (12.39 +/- 2.90 vs 7.11 +/- 0.75/10 deoxyguanosine for group 1 and 10.28 +/- 2.43 vs 6.82 +/- 0.62/10 deoxyguanosine for group 2, p <0.001). The 8-OHdG level in leukocyte DNA of the spermatic vein and 8-OHdG in sperm DNA were highest in group 1 followed by those in groups 2 and 3, and correlated inversely with motility, morphology and density of spermatozoa. The incidence of 4977 bp deletion of mtDNA in sperm was 40.6%, 20% and 0% in groups 1, 2 and 3, respectively. These results indicate that oxidative stress in patients with varicocele or subclinical varicocele was greater than in healthy subjects.. 8-OHdG in leukocyte DNA of the spermatic vein and in sperm DNA, and 4977 bp deletion of mtDNA in sperm might be useful markers for the assessment of oxidative stress in patients with varicocele and subclinical varicocele.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Biomarkers; Chromosome Deletion; Deoxyguanosine; DNA; DNA, Mitochondrial; Humans; Infertility, Male; Leukocytes; Male; Oxidative Stress; Palpation; Predictive Value of Tests; Reference Values; Sperm Count; Sperm Motility; Spermatic Cord; Spermatozoa; Ultrasonography, Doppler; Varicocele; Veins

Oxidative stress in the reproductive system is thought to affect the fertilizing ability of sperm. Since 8-hydroxydeoxyguanosine (8-OHdG) and lipid peroxides are widely used as markers to quantify oxidative stress, we compared 8-OHdG and lipid peroxide concentrations in seminal plasma and spermatozoa from subfertile and fertile men.. Semen obtained from 37 men of subfertile couples (21 men with normozoospermia and 16 with asthenozoospermia) and from eight fertile volunteers were examined. Seminal plasma and spermatozoa were fractionated by four-step discontinuous Percoll gradient centrifugation. 8-OHdG in seminal plasma was measured by ELISA, and lipid peroxides in seminal plasma and spermatozoa were determined using a thiobarbituric acid (TBA) assay.. The concentrations of 8-OHdG and lipid peroxides in the seminal plasma of the subfertile group were significantly higher than those of the fertile group. There were no significant differences in these values between patients with normozoospermia and asthenozoospermia. In all four fractions obtained by Percoll gradient fractionation, the lipid peroxide levels in spermatozoa recovered from subfertile males were significantly higher than those of fertile controls.. Seminal plasma and spermatozoa from subfertile males showed elevated levels of oxidative stress that were detectable in ejaculated semen specimens by ELISA or TBA assay. Even the spermatozoa fraction considered to be mature and normal showed elevated oxidative stress in the subfertile group. Our results confirm the importance of oxidative stress in male reproductive function, and could be applied for the selection of patients for antioxidant therapy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Centrifugation, Density Gradient; Deoxyguanosine; Enzyme-Linked Immunosorbent Assay; Humans; Infertility, Male; Lipid Peroxides; Male; Malondialdehyde; Oxidative Stress; Semen; Spermatozoa; Thiobarbituric Acid Reactive Substances

To examine glutathione S-transferase M1 (GST M1) gene polymorphism and male infertility in Taiwanese patients with varicocele, 80 young male patients with varicocele (group 1), 62 young male patients with subclinical varicocele (group 2) and 60 normal young males (group 3) were recruited in this study.. GST M1 null homozygous genotype [GST M1-] and the occurrence of a 4977 bp deletion of sperm mitochondrial DNA (mtDNA) were determined by polymerase chain reaction. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) content of sperm DNA was measured by high-performance liquid chromatography.. The frequencies of GST M1- genotype were 43.8, 41.9 and 45% for patients in groups 1, 2 and 3 respectively. In group 1 patients with GST M1- genotype, the frequency of the presence of the 4977 bp deletion in sperm mtDNA (54.3%) was significantly higher than that of the patients without the 4977 bp deletion in sperm mtDNA (45.7%, OR: 2.63, P = 0.04). Patients of groups 1 and 2 with GST M1- genotype had significantly higher 8-OHdG content in sperm DNA and lower protein thiols and ascorbic acid in seminal plasma than those with GST M1+ genotype.. GST M1- genotype predisposes to increased oxidative damage to sperm of patients with varicocele.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Ascorbic Acid; Deoxyguanosine; DNA, Mitochondrial; Gene Deletion; Gene Frequency; Genotype; Glutathione Transferase; Homozygote; Humans; Infertility, Male; Male; Polymorphism, Genetic; Reference Values; Semen; Sulfhydryl Compounds; Varicocele

To determine the effects of oxidative stress on the quality of oocytes and embryos, 8-hydroxy-2'-deoxyguanosine (8-OHdG) in granulosa cells was quantitatively studied during an in vitro fertilization and embryo transfer (IVF-ET) program.. Immunocytochemical staining of 8-OHdG in granulosa cells was quantitatively estimated using a charge-coupled device camera and analyzed using the National Institute of Health Image (NIH Image) freeware on a computer .. Obstetrics and gynecology department in a university hospital.. Ninety-six infertile couples undergoing IVF-ET treatment and intracytoplasmic sperm injection (IVF, n = 72; intracytoplasmic sperm injection, n = 24).. Oocytes, granulosa cells, and follicular fluids were collected 35-36 hours after the administration of hCG.. 8-OHdG indices were obtained for mural [8-OHdG index (m)] and cumulus [8-OHdG index (c)] granulosa cells.. A negative correlation between the fertilization rate and both 8-OHdG indices (c and m) was found. The rate of production of good embryos also showed a negative correlation with the 8-OHdG index (m) and the 8-OHdG index (c). Negative correlations between the 8-OHdG index (c) and E2 levels in follicular fluid were observed. Endometriosis patients showed a higher 8-OHdG index (c) than did patients with other infertility causes, such as tubal, male factor, and unknown.. Oxidative stress in granulosa cells lowered fertilization rates and subsequently led to a decrease in the quality of embryos. The quality of oocytes for endometriosis patients was impaired by the presence of 8-OHdG. This might be one causative factor in infertility in endometriosis patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Deoxyguanosine; Embryo Transfer; Embryo, Mammalian; Endometriosis; Estradiol; Female; Fertilization; Fertilization in Vitro; Follicular Fluid; Granulosa Cells; Humans; Infertility, Female; Infertility, Male; Male; Oocytes; Sperm Injections, Intracytoplasmic

To investigate whether a high level of oxidative DNA damage in spermatozoa occurs in infertile male patients and to examine the influence of antioxidant treatments on the levels of this damage.. Controlled clinical study and uncontrolled pilot study.. Department of Obstetrics and Gynecology, Akita University School of Medicine.. Nineteen infertile male and 17 control patients.. The levels of oxidative DNA damage in spermatozoa of infertile male and control patients were compared. In addition, 14 infertile males were given antioxidants for 2 months.. The levels of 8-hydroxy-2'-deoxyguanosine, a form of oxidative damage, in the spermatozoa were determined using high-performance liquid chromatography with electrochemical detection.. The levels of 8-hydroxy-2'-deoxyguanosine in sperm DNA were significantly higher in male infertile patients than in the control patients (1.5 +/- 0.2 versus 1.0 +/- 0.1 per 10(5) deoxyguanosine) and were correlated with sperm concentrations in ejaculates. Antioxidant treatment resulted in significant positive effects on sperm concentrations, with a significant reduction in sperm 8-hydroxy-2'-deoxyguanosine levels (from 1.5 +/- 0.2 to 1.1 +/- 0.1 per 10(5) deoxyguanosine).. Our present data demonstrate an association between the level of oxidative DNA damage in spermatozoa and male infertility and point to the possible use of antioxidants to reduce this damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Antioxidants; Deoxyguanosine; DNA; DNA Damage; Humans; Infertility, Male; Male; Oxidation-Reduction; Spermatozoa