Sperm Nuclear DNA Damage Research Articles

Low human sperm motility coexists with sperm nuclear DNA damage and oxidative stress in semen.

Low sperm motility, one of the common causes of male infertility, is associated with abnormal sperm quality. Currently, important sperm/semen biomarkers are sperm chromatin status and oxidation‒reduction potential (ORP) in semen. Because the association between sperm motility and these biomarkers is still not fully clarified, our study was designed to verify the distribution and risk of sperm DNA fragmentation (SDF) and oxidative stress in semen in asthenozoospermic men. This study was carried out on discharged sperm cells of asthenozoospermic men (isolated asthenozoospermia or coexisted with reduced sperm number and/or morphology), nonasthenozoospermic men (reduced total sperm count and/or sperm morphology) (experimental groups) and normozoospermic men (proven and presumed fertility) (control group). Basic semen analysis was evaluated according to the 6th edition of the World Health Organization manual guidelines. SDF was assessed using the sperm chromatin dispersion test, while static(s) ORP in semen was measured by means of a MiOXSYS analyser. The men from the asthenozoospermic group had lower basic semen parameters than those from the control and nonasthenozoospermic groups. In men with poor sperm motility SDF and sORP, prevalence and risk for>20% SDF (high level of DNA damage) and for>1.37 sORP (oxidative stress) were significantly higher than those of control and nonasthenozoospermic subjects. The risk for sperm DNA damage and oxidative stress in asthenozoospermic men was over 10-fold higher and almost 6-fold higher than those in control subjects and almost or over 3-fold higher than those in nonasthenozoospermic men. Poor human sperm motility coexisted with low basic sperm quality. Sperm DNA damage and oxidative stress in semen were much more frequent in asthenozoospermia. These abnormalities can decrease the sperm fertilizing capability under both natural and medically assisted reproduction conditions. Thus, in asthenozoospermia, the evaluation of sperm chromatin status and oxidation-reduction potential in semen is justified and inevitable, and the appropriate antioxidant therapy can be suggested.

Sperm telomere length is associated with sperm nuclear DNA integrity and mitochondrial DNA abnormalities among healthy male college students in Chongqing, China.

Is sperm telomere length (STL) associated with sperm nuclear DNA damage and mitochondrial DNA abnormalities? Sperm telomere length is related to sperm nuclear DNA integrity and mitochondrial DNA abnormalities in healthy young college students. Many studies have revealed the correlations between sperm genetic alterations in both the nucleus and mitochondria and sperm functionality, however, the possible associations between the telomere, an important component of chromosome, and conventional indicators of mitochondrial DNA and nuclear DNA changes have not been investigated. A prospective cohort study, Male Reproductive Health in Chongqing College Students (MARHCS), was conducted from June 2013 to June 2015. We pooled data collected from the follow-up study in 2014 and a total of 444 participants were included. STL was measured by quantitative (Q)-PCR. Sperm nuclear DNA integrity was determined using sperm chromatin structure assay (SCSA) and comet assay. Mitochondrial DNA damage was assessed by mitochondrial DNA copy number (mtDNAcn) evaluated with Q-PCR, and mtDNA integrity was determined with long PCR. The univariable-linear regression analysis revealed that STL was significantly positively correlated with markers of sperm nuclear DNA damage including the DNA fragmentation index (DFI) and comet parameters (the percentage of DNA in the tail, tail length, comet length, and tail moment). Additionally, STL was also significantly positively correlated with mtDNAcn and significantly negatively correlated with mtDNA integrity. After adjustment for potential confounders, these relationships remained appreciable. Moreover, we investigated potential effects of biometric factors, including age, parental age at conception, and BMI on STL and found that STL was increased with paternal age at conception. A mechanistic explanation of the correlation between STL, sperm nuclear DNA integrity, and mtDNA abnormalities cannot be provided with a cross-sectional study design, so well-designed longitudinal studies are still necessary. In addition, a single semen samples were provided and were not all obtained at the same time point, which may increase the intraindividual bias in this study. The findings extend the literature including assessment of mitochondrial dysfunction, sperm nuclear DNA damage, and telomere length and provide new insights into the relevance of STL in male reproduction. This work was supported by the National Natural Science Foundation of China (No. 82073590), the National Natural Science Foundation of China (No. 81903363), the National Natural Science Foundation of China (No. 82130097), and the National Key R&D Program of China (2022YFC2702900). The authors declare no conflicts of interest. N/A.

Evaluation of selected semen parameters and biomarkers of male infertility - preliminary study.

Background: Because the etiopathogenesis of male infertility is multifactorial our study was designed to clarify the relationship between standard semen parameters, testicular volume, levels of reproductive hormones and the fragmentation of sperm nuclear DNA (SDF). Methods: Patients (n = 130) were clustered as subjects: 1) with an abnormal volume (utrasonography) of at least one testis (<12 mL) or with a normal volume of testes and 2) with abnormal levels of at least one of the reproductive hormones (FSH, LH, PRL, TSH, total T - electrochemiluminescence method) or with normal hormonal profiles and 3) with high level of SDF (>30%), moderate (>15-30%) or low (≤15%) (sperm chromatin dispersion test). Results: In subjects with a decreased testicular volume and in subjects with abnormal levels of reproductive hormones, decreased basic semen parameters were found. Participants with abnormal testicular volume had a higher percentage of SDF and a higher level of FSH (Mann-Whitney U test). In turn, men with a high level of SDF had lower testicular volume and conventional sperm parameters than men with a low level of SDF (Kruskal-Wallis test). Conclusions: We showed that spermatogenesis disorders coexisted with decreased testicular volume and increased FSH levels. The disorders of spermatogenesis were manifested by reduced basic sperm characteristics and a high level of sperm nuclear DNA damage.

Protective Effects of Sesamin on Cytoxan-Induced Spermatogenesis Dysfunction by Regulating RNF8-ubH2A/ubH2B Pathways in Male Mice.

Most of the clinically infertile patients show spermatogenesis dysfunction. Cyclophosphamide, as an anticancer drug, can induce spermatogenesis dysfunction. Sesamin is the main bioactive component of natural lignans in sesame. It is abundant in sesame oil and has strong biological activities such as antioxidant, antibacterial, and hypoglycemic properties. By establishing the model of spermatogenic dysfunction induced by cyclophosphamide in male mice and then feeding sesamin (50, 100, and 200 mg/kg) for 2 weeks, we proved that sesamin can improve the reproductive organ damage induced by cyclophosphamide and increase the number and activity of sperms. Sesamin can resist cyclophosphamide-induced sperm nuclear maturity and DNA damage by increasing the expression levels of histones H2A and H2B in the testis. In addition, sesamin can improve the ubiquitination of histones regulated by RNF8 to protect the testis. In conclusion, these results suggest that sesamin can improve spermatogenic dysfunction induced by cyclophosphamide, which may be mediated by ubiquitination of histones.

Sperm DNA status in infertile patients with clinical varicocele

To evaluate if a potential relationship exists between sperm nuclear DNA damage and clinical varicocele and to determine the impact of clinical varicocele on standard semen parameters. A prospective study involving 30 infertile patients with clinical varicocele and 15 controls patients referred to our laboratory for routine spermiological exploration. Spermograms were performed and analyzed according to World Health Organisation (WHO) guidelines 2010. The DNA fragmentation was detected by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay. The DNA fragmentation index (DFI) was significantly higher in patients with clinical varicocele compared to controls (13.3±3.4% versus 6.1±2.5%, P=0.0001). In addition, the DFI was positively and significantly correlated with the degree of severity of varicocele thus the DFI was 15.24±1.9% in patients with grade 3 versus 12.92±3.5% in those with grade 2(P<0.0001). However, an abnormality of at least one of the spermatic parameters was found in 90% of varicocele patients, and all semen characteristics such as sperm count, vitality, mobility and typical forms were decreased compared to the controls. Furthermore, statistically significant negative correlations were noted between sperm DNA fragmentation index and sperm concentration (P=0.0001), motility (P=0.03), and normal sperm morphology (P=0.03). Clinical varicocele generates a significant increase of sperm abnormalities and DNA damage, and these changes are positively correlated with varicocele grade. Sperm DNA damage independent of its cause, may affect the quality of the ejaculated sperm and may have implications on patient's fertility potential. 3.

Signs of ROS-Associated Autophagy in Testis and Sperm in a Rat Model of Varicocele.

Since autophagy was suspected to occur in the pathological situation of varicocele (VCL), we have attempted to confirm it here using a surgical model of varicocele-induced rats. Thirty Wistar rats were divided into three groups (varicocele/sham/control) and analyzed two months after the induction of varicocele. Testicular tissue sections and epididymal mature sperm were then monitored for classic features of varicocele, including disturbance of spermatogenesis, impaired testicular carbohydrate and lipid homeostasis, decreased sperm count, increased sperm nuclear immaturity and DNA damage, oxidative stress, and lipid peroxidation. At the same time, we evaluated the Atg7 protein content and LC3-II/LC3-1 protein ratio in testis and mature sperm cells, two typical markers of early and late cellular autophagy, respectively. We report here that testis and mature sperm show higher signs of autophagy in the varicocele group than in the control and sham groups, probably to try to mitigate the consequences of VCL on the testis and germ cells.

Relationship between sperm DNA damage with sperm parameters, oxidative markers in teratozoospermic men

ObjectiveThis study was aimed at determining the extent of sperm nuclear DNA damage in patients with isolated teratozoospermia and examining its relationship with oxidative stress. Study designSemen samples from 60 patients with isolated teratozoospermia and 30 normozoospermic donors were examined. DNA damage was evaluated by the COMET assay. Seminal antioxidant activities (Superoxide dismutase; Glutathione peroxidase; Catalase), iron and malondialdehyde concentrations were measured spectrophotometrically. ResultsSperm DNA damage; malondialdehyde and iron levels were more elevated in studied groups than controls. Nevertheless, the antioxidant enzyme activity obtained was significantly lower in the group of patients with teratozoospermia compared to the controls. Sperm DNA damage was positively correlated to malondialdehyde and seminal iron level while reduced seminal antioxidant status was negatively associated with sperm DNA breaks. Interestingly, we noted that sperm DNA damage; lipid peroxidation, iron level, and impaired antioxidant status were negatively correlated to normal sperm morphology. ConclusionThese findings may explain the complex biological relationship between teratozoospermia, oxidative stress, and DNA damage. In fact, an impaired seminal antioxidant status and an increased seminal level of both lipid peroxidation and iron can affect sperm nuclear integrity resulting in DNA breaks and can be associated with poor sperm morphology.

Diabetes and Sperm DNA Damage: Efficacy of Antioxidants

Diabetes mellitus (DM) represents one of the major threats to human health all over the world, affecting almost every system of the body. Its prevalence is escalating globally and is associated with reproductive impairment in both males and females. Reports on male infertility due to DM is lacking since most affected individuals are unaware of their infertility condition due to the late onset of DM. Glucose metabolism is an imperative occasion in spermatogenesis, causing adverse effects on male fertility, principally on sperm DNA quality, motility, and ingredients of seminal plasma. DM is coupled with an increased oxidative stress (OS), causing sperm nuclear and mitochondrial DNA damage. Reactive oxygen species (ROS) hassles the fluidity of sperm plasma membrane, decreases sperm motility and ability to fuse with oocyte as well as altering the sperm DNA integrity. Lamentably, spermatozoa cannot repair the damage initiated by excessive ROS as they do not have the cytoplasmic enzymes required to achieve the repair. Diabetes may impact the epigenetic change during spermatogenesis which may be inherited through male gamete to more than one generation increasing the risk of diabetes in offspring. Administration of antioxidants in male infertility has started to pull in significant intrigue. Many studies have shown that antioxidants amazingly reduce the oxidative stress markers and boost the antioxidant enzymes. Currently treatment strategies are aimed at lowering ROS levels to maintain normal cell function. This review highlights the potential impact of DM on sperm DNA integrity, epigenetic dysregulation and efficacy of antioxidant therapy.

Role of nuclear and mitochondrial genes in human male infertility: a review

Infertility affects very high percentage (15%) of couples and nearly 50% of these are accountable to the male partners. Reproduction encompassed two important biological processes are successful gametogenesis and fertility. Mainly, male infertility is influenced by the sperm quantity and quality. Male infertility is genetically heterogeneous. Numerical and structural chromosomal abnormalities are associated with male infertility. Genetic studies on male infertility suggest that mutations in some Y chromosomal genes lead to male infertility. The major spermatogenic candidate genes are present in the Azoospermia factor loci (AZFa, AZFb and AZFc) of Y chromosome that largely control the successful spermatogenesis. Deletions or mutations in these genes results in abnormal spermatogenesis or abnormal semen profile affecting the fertility. The gr/gr deletion, a partial 1.6 Mb deletions in AZFc region of Y chromosome, has also been described specifically in infertile men with varying degrees of spermatogenic failure. A number of X-chromosomal and autosomal genes mutations have also been implicated for male infertility. It is suggested that impaired sperm chromatin integrity or sperm nuclear DNA damage has been associated with male infertility. It is not only genetic but genomic imprinting, an epigenetic phenomena, play a crucial role in male infertility. PLOG (DNA polymerase gamma) gene mapped to the long arm of chromosome 15 (15q25) includes CAG repeats and its mutations may affect the adenosine triphosphate production and fertility. Studies on mitochondrial genes revealed that mitochondrial genes are involved in normal growth, development and differentiation of sperms, and are also responsible for flagellar movement of sperms after ejaculation. Mutations in these mitochondrial genes result in abnormal semen parameters and impaired sperm flagellar movements which result in problem in fertilization and ultimately in infertility.

Protective effect of alpha glucosyl hesperidin (G-hesperidin) on chronic vanadium induced testicular toxicity and sperm nuclear DNA damage in male Sprague Dawley rats.

The study was conducted to evaluate the vanadium-induced testicular toxicity and its effect on sperm parameters, sperm nuclear DNA damage and histological alterations in Sprague Dawley rats and to assess the protective effect of G-hesperidin against this damage. Treatment of rats with vanadium at a dose of 1mgkgbw(-1) for 90days resulted in significant reduction in serum testosterone levels, sperm count and motility. Further, a parallel increase in abnormal sperm morphology and adverse histopathological changes in testis was also associated with vanadium administration when compared to normal control. Moreover, sperm chromatin dispersion assay revealed that vanadium induces sperm nuclear DNA fragmentation. A marked increase in testicular malondialdehyde levels and decreased activity of antioxidant enzymes such as superoxide dismutase and catalase indicates vanadium-induced oxidative stress. Co-administration of G-hesperidin at a dose of 25 and 50mgkgbw(-1) significantly attenuated the sperm parameters and histological changes by restoring the antioxidant levels in rat testis. These results suggested that vanadium exposure caused reduced bioavailability of androgens to the tissue and increased free radical formation, thereby causing structural and functional changes in spermatozoa. G-hesperidin exhibited antioxidant effect by protecting the rat testis against vanadium-induced oxidative damage, further ensures antioxidant potential of bioflavonoids.

Raman microspectroscopy: shining a new light on reproductive medicine

BACKGROUND The last 20 years have seen an enormous upsurge in the number of publications reporting findings obtained by Raman spectroscopy, a non-invasive, non-destructive method which uses the inelastic scattering of light to provide a 'fingerprint' of the sample's chemical composition and constituents. Long neglected because of practical difficulties, the technique has been transformed by recent technological advances into a powerful analytical tool capable of opening avenues of investigation that were previously out of the reach of biomedical scientists. Beyond introducing the approach and describing its relative merits and weaknesses, the aim of this review is to provide a spur for discussion of what may become an invaluable tool for biomedical investigations. METHODS A comprehensive review of the literature was conducted searching PubMed and Ovid databases using numerous MeSH terms associated with reproductive medicine. Furthermore, the reference lists of all reported literature were explored. The searches were restricted to English language articles published in the last 50 years. RESULTS Beginning with simple characterizations of biologically and medically important substances, aided by increasing technological sophistication, the use of Raman spectroscopy in biomedicine has quickly expanded to the investigation of complex biochemical interactions, the assessment of organelles and now the evaluation of living cells and tissue. The first Raman investigations of reproductive organs were primarily oncological in nature; however, the past few years have seen an increase in the application of the technique for the assessment and evaluation of both male and female gametes. In particular, progress has been made in the characterization, identification and localization of sperm nuclear DNA damage. CONCLUSIONS The use of Raman spectroscopy has already provided many tantalizing glimpses into the potential that the technique has to answer many of the unresolved issues in investigative and therapeutic reproductive medicine. However, without stringent assessment and the clear representation of the methods' findings, their true meaning cannot be revealed nor should any conclusions be hastily derived. For the potential of Raman microspectroscopy to be truly realized, the dependability and reliability of the technique and its results can only be ascertained by multidisciplinary collaborations that undertake carefully conducted, controlled and analysed studies.

The study of spermatic DNA fragmentation and sperm motility in infertile subjects

Although the pathophysiology of the testicular damage associated with varicocele remains unclear, sperm DNA damage has been identified as a potential explanation for this cause of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele, and to examine its relationship with parameters of seminal motility. Semen samples from 60 patients with clinical varicocele and 90 infertile men without varicocele were examined. Varicocele sperm samples were classified as normal or pathological according to the 1999 World Health Organizzation guidelines. Sperm DNA damage was evalutated using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. The DNA fragmentation index (DFI: percentage of sperm with denatured nuclei) values was significantly higher in patients with varicocele, either with normal or abnormal (DFI 25.8 ± 3.2 vs 17.4 ± 2.8 - P < 0,01) semen profiles. In addition, an inverse correlation was found between spermatic motility and the degree of spermatic DNA fragmentation in patients with clinical varicocele. Varicocele is associated with high levels of DNA-damage in spermatozoa. In addition, in subjects with varicocele, abnormal spermatozoa motility is associated with higher levels of sperm DNA fragmentation. DNA fragmentation may therefore be an essential additional diagnostic test that should be recommended for patients with clinical varicocele.

Light exposure and sperm nuclear DNA damage in ICSI procedures

OBJECTIVE: IVF and ICSI procedures imply that gametes and embryos are exposed to light during microscopic observation. The possible detrimental effect of light on oocytes and embryos has been subject for several different studies. However a lack of information is related to the effect of light exposure on spermatozoa functionality. The aim of this study was to evaluate the effect of the light exposure during microscopic observation on the spermatozoa DNA fragmentation measured by TUNEL.

Reducing oxidative DNA damage by adding antioxidants in human semen samples undergoing cryopreservation procedure

To evaluate the protective effects of oxidative DNA damage by adding antioxidants: ascorbate, catalase (CAT), and superoxide dismutase (SOD) in human semen samples undergoing cryopreservation procedure. Semen sample form 30 fertile men were mixed with modified cryoprotectant and divided into six groups according to the category and concentration of antioxidants: ascorbate 300 micromol/L, ascorbate 600 micromol/L, CAT 200 U/ml, CAT 400 U/ml, SOD 200 U/ml, and SOD 400 U/ml. Comet assay was conducted to measure the percentage of comet cells, and the nuclear DNA damaged parameters: tail DNA percentage (TD%) and Olive tail moment (OTM). Flow cytometry was used to detect the reactive oxidative species (ROS). The motility (a + b grade), viable recovery rate, nuclear DNA integrity and reactive oxidative species (ROS) of all groups were analyzed before and/or after freeze-thawing. (After cryopreservation, compared with the control group, the a + b grade sperm rates of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were all higher than that of the control group (all P < 0.05), however, the levels of reactive oxygen species (ROS) of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 30 +/- 13, 30 +/- 11, and 30 +/- 11 respectively, all significantly lower than that of the control group (37 +/- 17 , all P < 0.05). The viable recovery rates of the ascorbate 300 micromol/L , CAT 200 U, and CAT 400 U groups were 67% +/- 14%, 68% +/- 14%, and 69% -/+ 15% respectively, all significantly higher than that of the control group (59% +/- 10%, all P < 0.05). (2) The TD% levels of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 41% +/- 4%, 40% +/- 7%, 40% +/- 6%, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (46% +/- 6%, all P < 0.01). The OTM levels of the ascorbate 300 micromol/ L, CAT 200 U, and CAT 400 U groups were 7.7 +/- 1.2, 7.5 +/- 1.6, and 7.8 +/- 1.9, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (10.1 +/- 3.1, all P < 0.01) too. The TD% and OTM levels of the other groups were all significantly higher than that of the raw semen (all P < 0.01), but not significantly different from those of the control group (all P > 0.05). (3) ROS was significantly negatively correlated with the motility in all groups (P < 0.05 or P < 0.01). Apart from the ascorbate 600 micromol/L group, the TD% and OTM of the other groups were all significantly positively correlated with the ROS (P < 0.05 or P < 0.01). Supplementation of ascorbate or CAT reduces the level of ROS that induces sperm nuclear DNA damage, and improves the human sperm quality in the process of freeze-thawing.

Aumento del daño en el ADN y estrés oxidativo en espermatozoides de pacientes con oligozoospermia idiopática y antecedentes de criptorquidismo

Cryptorchidism and oligozoospermia are clinical conditions closely associated with impaired fertility. Oxidative stress and related sperm DNA damage have been identified as significant causes of male infertility. To determine the extent of sperm nuclear DNA damage in patients affected with idiopathic oligozoospermia or undescended testes and to examine its relationship with oxidative stress. We studied 20 patients with idiopathic oligozoospermia and 18 with undescended testes (who previously underwent orchiopexy) and 25 normozoospermic healthy controls. All subjects underwent semen analysis. Sperm DNA damage was evaluated by the sperm chromatin structure assay/flow cytometry (SCSA-FCM) and by the dUTP-biotin nick end labeling (TUNEL) assay. Levels of reactive oxygen species (ROS) and total antioxidant capacity (TAC) were assessed by a chemiluminescence assay. DFI (percentage of sperm with denatured DNA) values and percentage of TUNEL positive cells were significantly greater in patients with oligozoospermia (DFI: 28.8+/-5.6; TUNEL+: 26.9+/-3.0) or cryptorchidism (DFI: 26.4+/-10.1; TUNEL+: 29.1+/-3.9), compared with controls (DFI: 7.1+/-0.9; TUNEL+: 14.2+/-1.2). Similarly, both groups of patients had significantly higher (p<0.01) levels of ROS. TAC levels did not differ between control and patient groups, suggesting that the DNA damage occurs before spermiation. Sperm DNA damage is significantly increased in men with idiopathic oligozoospermia and in cryptorchid subjects. The finding of increased ROS levels may indicate that seminal oxidative stress may be involved in the pathogenesis of sperm DNA damage in these patients.

Treatment of severe male infertility by micromanipulation-assisted fertilization: an update

In the past 5-10 years the evolution of micromanipulation-assisted fertilization for the treatment of severe male infertility was marked by the introduction of new technical support, refinement of diagnostic methods for the evaluation of sperm developmental potential, and development of new treatment regimens for the newly discovered abnormalities. The new technical support involves the use of non-contact laser technology to assist micromanipulation for fertilization, the evolution of polarized microscopy-based optical systems to non-invasively detect the position of the meiotic spindle in living human oocytes, and the development of high-magnification optical systems for a better morphological selection of spermatozoa to be used for fertilization. Diagnostic approaches were enriched by commercial availability of kits for the analysis of sperm DNA integrity, leading to the definition of sperm nuclear DNA damage as a distinct cause of male infertility, and by the development of tests, based on heterologous ICSI, for detection of sperm failure to activate oocytes. Several treatment options for these conditions have been proposed and are currently being tested in larger-scale trials. Some technical improvement was also achieved in the field of in vitro maturation of germ cells from men with in vivo maturation arrest, but only a modest clinical improvement resulted from their application. As to the risk for the offspring, recent data are rather reassuring. Except for the risk of transmission of genetically based infertility, no straightforward evidence for a health risk derived from these techniques has been provided. Nevertheless, caution is necessary, particularly concerning the eventual increase in genomic-imprinting abnormalities.

Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment

Previous studies have shown that repeated intracytoplasmic sperm injection failures can be associated with sperm DNA damage. This paper reviews the current understanding of the mechanism of sperm DNA damage, discusses different diagnostic methods and their threshold values to discriminate between good- and poor-prognosis patients, and outlines the currently available treatment options. A rational approach to the interpretation of sperm DNA fragmentation data and to the choice of the optimal treatment method is suggested.

Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress

The pathophysiology of the testicular damage in varicocele has not been completely understood. Oxidative stress and related sperm DNA damage have been identified as significant causes of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele and to examine its relationship with oxidative stress. Semen samples from 55 patients with clinical varicocele and 25 normozoospermic donors were examined. Varicocele sperm samples were classified as normal or abnormal according to World Health Organization guidelines. Sperm DNA damage was evaluated by the sperm chromatin structure assay/flow cytometry and by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Levels of reactive oxygen species (ROS) and total antioxidant capacity were assessed by a chemiluminescence assay. DNA fragmentation index (DFI) (percentage of sperm with denatured DNA) values and the percentage of TUNEL-positive cells were significantly greater in patients with varicocele, either with normal (DFI, 20.7 +/- 4.0; TUNEL positive, 26.1 +/- 3.2) or with abnormal (DFI, 35.5 +/- 9.0; TUNEL positive, 32.2 +/- 4.1) semen profile, compared with controls (DFI, 7.1 +/- 0.9; TUNEL positive, 14.2 +/- 1.2). Similarly, ROS levels were significantly higher (P < 0.01) in both groups of patients with varicocele. The presence of a varicocele is associated with high levels of DNA-damage spermatozoa even in the presence of normal semen profile. The results also indicate that oxidative damage is associated with sperm DNA damage in these patients.

1516: Relationship between Cigarette Smoking and the Levels of Antioxidants and Leukocytes in Infertile Men: A Prospective Study

Introduction and Objective: Cigarette smoke is a known somatic cell mutagen and carcinogen and it is known to adversely affect male reproductive health. Even though studies have shown the harmful effets of smoking on sperm quality, a handful of studies have found no association between smoking and sperm function or sperm nuclear DNA damage. Superoxide dismutase (SOD) and catalase are important antioxidant enzymes that can quench excess free radicals such as: superoxide anion and hydrogen peroxide respectively. The objective of our study was to evaluate and correlate the seminal antioxidant enzymatic activity (SOD and catalase levels) among fertile and infertile men who are cigarette smokers. Methods: The Institutional Review Board approved this study. Ten fertile donors and 112 infertile patients were included in the study. Semen analysis was performed according to the World Health Organization guidelines and sperm morphology according to Kruger strict criteria. Superoxide dismutase and catalase levels were determined with a spectrophotometer. Results: A significant difference was noted in the number of cigarettes smoked/day between infertile (4.8 ± 0.9) and fertile men (1.5 ± 0.5; p = 0.03). Significantly lower levels of SOD (14.67 ± 12.27 and 38.03 ±21.65) and catalase (14.87 ± 16.95 and 34.03 ± 20.65) were seen in infertile patients compared to fertile donors (P <0.0001). A significant correlation between catalase and SOD was observed (r = 0.461, P = 0.0001). Superoxide dismutase was significantly correlated with sperm concentration (r = 0.204, P = 0.034) and negatively correlated with leukocytospermia (r = -0.228, P = 0.021). Cigarette smoking was inversely correlated with SOD levels (r = -0.251, P = 0.01), but not with catalase levels (r = -0.147, P = 0.130). Conclusions: Decreased antioxidant enzyme levels are associated with male infertility. The detrimental effects of cigarette smoking on sperm motility and antioxidant levels (negative correlation with SOD) may be a possible reason for infertility in men who smoke cigarettes. As low antioxidant levels are linked to decreased fertility, physicians should advise infertile patients who smoke cigarettes to quit smoking. Author Disclosure Block: E.B. Pasqualotto, None; F.M. Umezu, None; A. Agarwal, None; M. Salvador, None; F.F. Pasqualotto, None.

Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa

Objective To investigate the role of increased seminal leukocytes in enhancing reactive oxygen species (ROS) production by human spermatozoa. Design A prospective study. Setting Male infertility clinic. Patient(s) Forty-eight infertile men. Intervention(s) Standard semen analysis. Assessment of sperm nuclear DNA damage by sperm chromatin structure assay. Incubation of spermatozoa from nonleukocytospermic samples with blood neutrophils. Main outcome measure(s) Spontaneous and phorbol 12-myristate 13-acetate (PMA)-induced ROS production in pure-sperm suspensions (after removal of leukocytes) as measured by a chemiluminescence assay. Result(s) Levels of spontaneous and PMA-induced ROS production in pure-sperm suspensions from the infertile men with a diagnosis of leukocytospermia (n = 16) were significantly higher compared with the case of infertile men without leukocytospermia (n = 32) and with the case of a control group of healthy volunteers (n = 13). A similar pattern of increased ROS was observed when spermatozoa were incubated with blood neutrophils. Leukocytospermia was associated with a significant decrease in sperm motility and increase in DNA damage. Conclusion(s) Increased seminal leukocytes may play a role in stimulating ROS production by human spermatozoa. Such stimulation may be mediated via direct cell–cell contact or by soluble products released by leukocytes. Poor sperm quality in leukocytospermic samples may be due to leukocyte-mediated oxidative stress.