Abstract
Assessment of sperm DNA damage has been suggested as a negative predictor of fertility potential. Multiple pathological factors acting at both the intra-testicular and post-testicular levels may contribute to sperm DNA damage. The relative contribution of each of these factors in an individual with high DNA damage (>30%) is unclear. The management of patients with elevated DNA damage is also challenging. The purpose of our retrospective study was to evaluate the clinical course of patients with sperm DNA damage over 30% and to assess the effect of non-specific (oral antioxidant) and cause-specific treatments on the quality of their sperm DNA. Results of our retrospective study suggest that the evaluated group with high DNA damage was diagnostically heterogeneous and comprised patients with varicoceles, bacteriospermia and idiopathic infertility. A three month course of antioxidant therapy reduced sperm DNA damage in only 30/61 (49%) patients with significant improvement between the initial and post-treatment DNA Fragmentation Index (DFI) results (46.8%±14.1 vs. 36.7%±16.6, p <. 001). The positive effect of antioxidants could be age-dependent, as patients older that 40 years of age showed no improvement after such treatment. The cause-specific treatments showed superior results compared to antioxidants alone. Improvement was observed in 7/9 (78%) of patients after surgical varicocele repair between the initial and post-treatment DFI results (44.7%±12.8 vs. 28.4%±9.5, p <. 03). The majority of the patients 13/14 (93%) with bacteriospermia had improvement in sperm DFI results after antibiotic treatment (50.4%±19.1 vs. 38.6%±18.7, p <. 001).
| Abbreviations | ||
| DNA | = | Deoxyribonucleic Acid |
| DFI | = | DNA Fragmentation Index |
| CASA | = | Computer-Assisted Semen Analysis |
| ROS | = | Reactive Oxygen Species |
| AO | = | acridine orange |
| AD | = | absolute difference |
| SD | = | standard deviation |
Introduction
Sperm DNA damage has been suggested as a negative predictor of fertility potential [Evenson et al. Citation2002; Spano et al. Citation2000]. Men with high DNA damage with greater than 30% have very low potential for in-vitro and in-vivo fertility [Benchaib et al. Citation2007; Bungum et al. Citation2007; Carrell et al. Citation2003; Tesarik et al. Citation2004a]. Multiple factors acting at both the intra-testicular and post-testicular levels may contribute to this damage. Sperm DNA damage has been found to be caused by many factors including a history of smoking and drug use, age, varicocele and urinary tract infection [Agarwal and Said Citation2003; Erenpreiss et al. 2002; Gandini et al. Citation2000; Moskovtsev et al. Citation2006; Ochsendorf Citation1999; Saleh et al. Citation2003]. However, the contribution of different clinical factors in patients with high DNA damage is not clear.
The management of patients with elevated DNA damage is also challenging and increases proportionately with the rise of such damage. Several studies showed positive results in the treatment of patients with moderate DNA damage using oral antioxidants [Agarwal et al. Citation2004; Greco et al. Citation2005; Menezo et al. Citation2007], microsurgical varicocelectomy in patients with varicocele [Zini et al. Citation2005] and antibiotics in patients with Chlamydia and Mycoplasma infection [Gallegos et al. Citation2008]. The data regarding treatment of patients with high DNA damage is lacking.
The purpose of our retrospective study was to evaluate the clinical course of patients with sperm DNA damage greater than 30% and to compare the effect of non-specific (oral antioxidant) and cause-specific treatments on the quality of their sperm DNA.
Results
Eighty-four infertile patients with initial sperm DNA damage greater than 30% as measured by DFI, who completed a course of treatment followed by successive DNA assessments, were included in this retrospective study. All patients were advised to make lifestyle modifications including cessation of smoking, avoiding recreational drugs and excessive heat and to take a three-month course of oral antioxidant treatment.
The patients were diagnostically heterogeneous and were divided into groups according to the treatment (). Group I was comprised of 24 patients (29%) with idiopathic infertility who received oral antioxidant alone. Group II contained 46 patients (55%) with clinically significant varicoceles; among them, 37 patients (Group II A) took oral antioxidants while 9 patients (Group II B) underwent standard microsurgical varicocelectomy as previously described [Goldstein et al., Citation1992] in addition to taking oral antioxidants. Bacteriospermia were present in 14 patients (16%), including 7 patients with varicocele, who received oral antioxidants and antibiotics according to the results of semen culture (Group III).
Changes in Sperm DNA Damage Following Treatment
The results of the initial semen analysis revealed that all patients had an abnormality in one or more of the standard semen parameters with no significant differences in semen volume, sperm concentration, motility, morphology or vitality following the treatment (). Patients receiving antioxidants or cause-specific treatment had similar post-treatment semen analysis results, with only one exception; significant improvement in sperm concentration was observed following surgical varicocele repair (25.6×106/mL±33.1 vs. 38.1×106/mL±39.3, p <. 03).
Changes in Semen Parameters and DNA Damage Following Treatment
Initial DFI ranged from 30.1 to 97.8% (mean 46.9%±14.8) and was negatively correlated to sperm concentration, motility and vitality (p <. 001). Post-treatment DFI ranged from 9.9 to 84.7% (mean 41.9%±18.1), and was significantly lower than that detected in the initial samples (p <. 01). However, when patients were analyzed according to diagnosis and treatment, it was revealed that DNA improvement was related to the type of treatment ().
Effect of Empirical Oral Antioxidant Treatment
In the three-month course of oral antioxidant treatment, two types of antioxidants were randomly recommended; 19 patients received Fertile One® (Coast Reproductive, Inc., San Diego, CA), and 42 were treated with daily vitamins. Post-treatment DFI showed no significant difference between the two types of antioxidants in patients with varicocele or idiopathic infertility.
When only oral antioxidants were prescribed, not every patient benefited from these therapies. Only 30/61 (49%) showed significant improvement between the initial and post-treatment DFI (46.8%±14.1 vs. 36.7%±16.6, p <. 001). When two types of antioxidants were compared, no significant differences in post-treatment DFI were observed in patients with idiopathic infertility (Group I, n=24) or varicocele (Group II A, n=37). Moreover, significant differences were observed in the response to the antioxidants based on age. The group of men younger than 40 years of age, n=35 (mean age 35.9±3.5) had modest, but significant improvement in their DFI after treatment (44.8%±14.9 vs. 41.1%±17.4, p <. 03) compared to older patients, n=26 (mean age 46.9±6.1), who showed no improvement in DFI (48.8%±13.5 vs. 49.6%±17.9).
Effect of Surgical Varicocele Repair
Nine patients (Group II B) diagnosed with clinical varicocele underwent surgical repair in addition to empirical oral antioxidant treatment. Significant improvement of DFI was observed after the surgery (p <. 03). Patients with varicocele treated with oral antioxidants alone (Group II A) did not show improvement (). We have observed the positive effect of surgical treatment of varicocele on sperm DNA damage. The majority (78%) of patients exhibited improvement in DFI after surgery (28.4%±9.5), compared to 49% of the patients showing an improvement in DFI after oral antioxidants alone (42.5%±16.7, p <. 02). In addition, significant improvement in sperm concentration was observed following the surgical varicocele repair.
Effect of Specific Antibiotic Treatment
Bacterial infection of the urinary tract was diagnosed in 14 patients (16%), including 7 patients with varicocele. The specific pathogens identified were: Enterococcus in 7 samples, including one in combination with Klebsiella; Enterobacter cloacae in 3 samples, including one in combination with Escherichia coli; E. coli as a monoculture in 2 samples; and Ureaplasma urealyticum in 2 samples.
The majority of the patients 13/14 (93%) with bacteriospermia showed an improvement in sperm DFI after antibiotic treatment. Antibiotic treatment had a positive effect on sperm DNA damage regardless of the presence of varicocele (initial DFI 53.4%±24.3 vs. post-treatment DFI 43.5%±20.1, p <. 01), or its absence (initial DFI 47.4%±13.5 vs. post-treatment DFI 33.6%±17.4, p <. 02).
Discussion
Men with greater than 30% DNA damage often exhibit reduced in vitro and in vivo fertility [Benchaib et al. Citation2007; Bungum et al. Citation2007; Spano et al. Citation2000]. High sperm DNA damage has been correlated to defective embryonic development, increased probability of implantation failure as well as the risk of recurrent miscarriages [Carrell et al. Citation2003; Tesarik et al. Citation2004a].
While potential mechanisms of sperm DNA damage are complex, defective sperm chromatin packaging, disordered apoptosis, and oxidative stress with damaging effect of ROS on sperm DNA have been suggested [Agarwal and Said Citation2003]. Human spermatozoa are susceptible to oxidative stress because of a high content of polyunsaturated fatty acids in their plasma membrane [Aitken et al. Citation1998]. Seminal plasma may combat ROS generation to some degree, but the production of excessive amounts of ROS may overcome the antioxidant protective activities of seminal plasma leading to oxidative stress [Sikka Citation2004].
It was proposed that the association of varicocele and sperm DNA damage may be due to elevated testicular temperature and increased free radicals [Saleh et al. Citation2003]. Our result confirms the negative effect of varicocele on sperm DNA. The prevalence of varicocele (63%) was increased in patients with high DFI compared to 35–40% incidence of varicoceles reported for infertile men in general [Zini Citation2007]. The reduction in levels of ROS and the improvement of sperm DNA integrity has been reported after surgical treatment of varicocele [Zini et al. Citation2005]. We have also observed the positive effect of surgical correction of varicocele on sperm DNA damage. Within this study, 78% of patients showed reduced DFI after surgery (28.4%±9.5), compared to 49% of patients after oral antioxidants alone (42.5%±16.7, p <. 02). The antioxidant treatment was not intended for treatment of varicocele but rather an empirical treatment for sperm DNA damage.
Bacteriospermia manifests as acute or chronic inflammation and can lead to an increase in the leukocyte concentration in the genital tract resulting in elevated ROS production [Ochsendorf Citation1999]. Some specific microbial pathogens have been linked to increased oxidative stress and sperm DNA damage in patients with male accessory gland infection [Vicari Citation2000]. Sperm DNA fragmentation assessed by slide-based sperm chromatin dispersion test in patients with Chlamydia and Mycoplasma infections was reported to decrease after a course of antibiotics [Gallegos et al. Citation2008; Vicari Citation2000]. Significant improvement in DNA damage following antibiotic treatment was also observed in our study, although several different bacterial pathogens were identified as the cause of the bacteriospermia.
The large proportion of patients with idiopathic infertility (29%) observed in our group, is consistent with an early report of high levels of ROS in men with unexplained infertility [Agarwal et al. Citation2004]. Elevated production of ROS by morphologically abnormal spermatozoa and/or reduction of antioxidant capacity of seminal plasma are possible causes of DNA damage in these patients [Agarwal et al. Citation2004; Moskovtsev et al. Citation2007]. Antioxidants added as dietary supplements have been shown to remove free radicals and reduce the degree of oxidative damage by improving the cellular redox equilibrium [Wong et al. Citation2000]. The benefits of antioxidant therapy in male infertility are inconclusive with both a positive effect [Greco et al. Citation2005; Menezo et al. Citation2007] and no significant effect reported [Silver et al. Citation2005]. For example, the beneficial effect of increased antioxidant consumption on sperm concentration and motility in the infertile patient population with no effect on sperm DNA integrity has been observed [Eskenazi et al. Citation2005]. Keskes-Ammar et al. [Citation2003] also demonstrated an improvement in sperm concentration and motility after a three month treatment oral antioxidants. However, the improvement in the standard semen parameters observed in these studies could not be confirmed by Greco and colleagues [Citation2005]. As reported here, we were unable to observe a positive effect of oral antioxidants on the standard semen parameters. However, most of the study patients had oligoasthenoozoospermia, known to be associated with high DNA damage [Moskovtsev et al. Citation2006].
Greco et al. [Citation2005] and colleagues observed improvement in DNA damage in 76% of patients with moderate DNA damage (≥15%) after oral treatment with vitamins. In cases with high DNA damage, reported here, improvement was seen in 49% of patients. The differences in the effects of antioxidant treatment among studies are likely to be related to differences in patient populations as well as to the type, dosage and duration of the treatment. It has been suggested, that multiple antioxidants acting through different mechanisms on diverse free radicals is a potential therapeutic approach in the treatment of male infertility [Agarwal et al. Citation2004]. However, we did not observe any difference in outcome between the two types of antioxidants (Fertile One® or the combination of vitamins and supplements) utilized in this study. Antioxidant therapy would only be of benefit to patients whose DNA damage was due primarily to oxidative stress [Tesarik et al. Citation2004b]. This might explain the reduction of DNA damage observed only in a portion of the study subjects. The variation in response to treatment may also relate to individual differences in nutrient absorption and metabolism of the antioxidants, as well as to failure of the antioxidant system or enzyme production in some patients [Aitken et al. Citation1998; Silver et al. Citation2005].
We have previously reported an age-related significant increase in sperm DNA damage [Moskovtsev et al. Citation2006]. In the present study, men younger than 40 years of age had a significant reduction in DFI after the antioxidant treatment, compared to older men who showed no such improvement. Either specific mechanisms of sperm DNA damage in older men or age-related irreversible deterioration in the spermatozoa's protective properties from this damage may be responsible for the differences in the response to the treatment [Agarwal and Said Citation2003]. This may include age-dependent accumulation of DNA damage coupled with a less efficient apoptotic cell selection system [Singh et al. Citation2003]. Alternatively, it may be attributable to age-related damage to the genes involved in the apoptotic pathway [Martin and Rademaker Citation1987] as well as an age-related cumulative birth cohort effect with altered testicular development [Sharpe and Skakkebaek Citation1993].
The results of our retrospective study suggest that the group of men with high sperm DNA damage (>30%) was diagnostically heterogeneous and comprised patients with idiopathic infertility, varicocele and bacteriospermia. A three month course of antioxidant therapy may reduce sperm DNA damage in some patients with high DFI. While the reduction in damage was modest, there was improvement in 49% of cases. The positive effect of antioxidants on sperm DNA could be age dependent, as patients older that 40 years of age showed no improvement after such treatment.
The cause-specific treatments, such as a course of antibiotics in patients with bacteriospermia or surgical repair in patients with varicocele showed superior results than antioxidants alone. This cause-specific treatment led to a significant decrease in sperm DNA damage in most of patients and could be recommended for patients with a known source of sperm DNA damage. Further study of oral antioxidant therapy alone or in combination with other treatments is warranted to independently confirm the results reported here for patients with high sperm DNA damage.
Materials and Methods
Selection of Subjects
The study was approved by the Mount Sinai Research Ethics Board and included retrospective analysis of clinical charts of 84 infertile patients with high DNA damage (>30%) with initial and post-treatment successive sperm DNA assessments. Initial infertility evaluation included collection of medical history, physical evaluation by urologist, and laboratory workup of semen analysis, semen bacterial culture and susceptibility and sperm DNA fragmentation analysis.
All patients were instructed to take a three month course of oral antioxidant treatment, either Fertile One®, a combination of vitamins (C, E, B6, B12, folic acid), herbs (ginseng root, garlic) and supplements (ferulic acid, coenzyme Q10, L-carnitine, zinc, selenium) or daily vitamins (C 500 mg, E 100 IUS, folic acid 1 mg) supplemented with 20 mg of zinc and 200 mcgs selenium. Antibiotic treatment included either a two week course of Ciprofloxacin (500 mg twice a day or extended-release (XL) 1000 mg daily) or Amoxicillin 500 mg three times a day. In case of ureaplasma infection, Zithromax (Azithromycin, Z-pack) 250 mg for 5 days was prescribed.
Assessment of Semen Sample
Semen samples were collected by masturbation on site after 2 to 5 days of sexual abstinence. The semen was allowed to liquefy and was analyzed within 60 minutes of collection. A routine Computer-Assisted Semen Analysis (CASA) was performed, which included assessment of semen volume, sperm concentration, motility, morphology and vitality as per World Health Organization [Citation1999] recommendations. An HTM-CEROS Sperm Analyzer (Hamilton Thorne, Beverly, MA) was used to analyze sperm concentration and motion parameters in fresh semen samples. Aliquots of semen samples were transferred to a microbiology laboratory for bacterial cultures. Bacteria colony counts were performed on all morphotypes of possible seminal tract pathogens; the identification and susceptibility testing was performed when ≥106 CFU/L colonies were counted [Keck et al. Citation1998].
Sperm DNA Fragmentation Analysis
All chemicals were obtained from Sigma-Aldrich Ltd. (Oakville, Ontario, Canada) unless otherwise stated. Aliquots of raw semen were frozen at −80°C for later DNA assessment performed as previously described [Evenson et al. Citation2002; Moskovtsev et al. Citation2005]. Briefly, at the time of analysis, the samples were thawed on ice and mixed with a low-pH detergent solution followed by staining with chromatographically purified AO, (Polysciences Inc., Warrington, PA). Samples were analysed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with an air-cooled argon laser. Measurements were collected in duplicate on 5000 cells per sample and two aliquots were analyzed for each semen specimen. Under these conditions, AO intercalated with double-stranded DNA emits green fluorescence and AO associated with single-stranded DNA emits red fluorescence. Reference samples were used to set the red and green photomultiplier tube voltages to avoid instrument drift and were run between every 6–10 samples. FCS Express version 2 (De Novo Software, Thornhill, Ontario, Canada) was used for off-line analysis of the flow cytometric data. DNA damage was expressed as the DNA Fragmentation Index (DFI), which is the ratio of red to total amount of red plus green fluorescing sperm in an individual sperm sample. Absolute difference in the level of DNA damage between initial and post-treatment assessments was calculated for all patients.
Statistical Analysis
Data was analyzed using SPSS 16.0 statistical computer software (SPSS Inc., Chicago, IL). Spearman's rank correlation was used to determine the relationship between age, semen parameters and DNA damage. One Way ANOVA followed by a post-hoc Tukey test was used to test DFIs and absolute differences between diagnostically different groups of patients. The treatment effect of each diagnostic group was tested using the Mann-Whitney test for related samples, comparing initial standard semen parameters and DFI with post-treatment semen results and DFI. Results are expressed as mean±SD.
Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Notes
1. Mount Sinai Hospital, Toronto, Ontario, Canada
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