Abstract
Purpose
Superoxide dismutase (SOD) is an important component of antioxidative defense systems and plays an important role in protecting spermatozoa from oxidative damage. In this study, we assessed seminal SOD activity, its association with semen parameters, and also genetic and non-genetic factors contributing to the determination of SOD activity in infertile men.
Methods
Semen samples were obtained from 435 male infertility patients. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL) assay. Four single nucleotide polymorphisms (SNPs) in SOD2 and SOD3 genes were genotyped using OpenArray platform.
Results
We found that seminal SOD activity was positively associated with sperm concentration and overall motility, whereas inversely with sperm DNA fragmentation. In addition, infertile men with SOD2 rs4880 CC variants showed a low level of SOD activity when compared with TT carriers (Mean ± SD: 268.3 ± 102.3 and 342.8 ± 98.2, respectively, P = 0.005). Those who consumed vitamin C/E (≥3 times per week) had a significantly higher SOD activity level than those who did not (mean ± SD: 379.8 ± 93.3 and 332.2 ± 94.9, respectively, P = 0.001).
Conclusions
Seminal SOD activity and other factors influencing SOD activity play a role in determining sperm fertilization potential and male infertility.
Keyword: SOD activity, Semen quality, Polymorphism, Vitamin C, Vitamin E, Male infertility
Introduction
Oxidative stress has been implicated in the pathophysiology of damage to human spermatozoa [1, 2]. It is a consequence of an imbalance between Reactive Oxygen Species (ROS) production and degradation. Germ cells produce physiological amounts of ROS that are required for maturation, capacitation, the acrosome reaction, and oocyte fusion under normal physiological conditions [3]. However, excessive generation of ROS can impair normal sperm function by attacking the fluidity of the sperm plasma membrane and the integrity of sperm DNA, and ROS-induced DNA damage could accelerate the process of germ cell apoptosis, resulting in the decline in sperm counts associated with male infertility [2].
To protect spermatozoa from oxidative damage, seminal plasma has a highly specialized ROS scavenger system, containing superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT). Among them, SOD is known to be the most important antioxidant enzyme. Leydig and Sertoli cells have been reported to produce SOD in the testis [4, 5]. A significantly lower semen SOD activity was found in oligoasthenozoospermic cases, when compared to normospermic men [6]. Previous studies have detected a positive correlation between seminal SOD with abnormal sperm morphology, percentage of dead sperms, sperm concentration and overall motility in male infertility patients [7, 6]. However, other researchers reported a nonsignificant correlation between SOD activity in seminal plasma and semen quality [8].
Thus, the purpose of this work was to evaluate SOD activity in seminal plasma of male infertility cases in a Chinese population, and its relationship with selected human semen parameters and sperm DNA fragmentation. As is known to all, genetic factors and life style factors might contribute to the determination of SOD activity. This study includes also these influence factors of SOD activity. Therefore, knowledge of activity of SOD in seminal plasma, its association with sperm parameters and influencing factors could be a useful tool for determining sperm fertilization potential and could improve the diagnosis and the prevention of male infertility.
Materials and methods
Subjects and sample collection
The study was approved by the Ethics Review Board of Nanjing Medical University. All the studies involving human subjects were conducted in full compliance with government policies and the Declaration of Helsinki. A total of 709 infertile patients diagnosed with unexplained male factor infertility were drawn from the Center of Clinical Reproductive Medicine between April 2005 and March 2009 (NJMU Infertility Study). All patients underwent at least two semen analyses, a series of physical examinations and serum determination, which helped us to exclude 274 individuals: 210 azoospermia (6 obstructive azoospermia), 7 with cryptorchidism,11 with abnormal karyotype, 12 with Y-chromosome microdeletions and 34 secondary sterility cases . In the final analysis, 435 idiopathic infertility patients aged 24 to 42 years were included. All participants were ethnic Han Chinese and completed an informed consent and a questionnaire including detailed information, such as age, cigarette smoking, alcohol intake, tea and vitamin C/E consumption, and abstinence time. Each subject donated 5 ml of blood for genomic DNA extraction. In addition, the semen samples were were obtained in private by masturbation into a sterile widemouth and metal-free glass container after a recommended at least 3-day sexual abstinence. The semen samples, frozen at −70 °C, were used for routine semen analysis, CASA motion analysis, the assessment of sperm DNA fragmentation, and the SOD activity.
Semen quality analysis
After liquefaction at 37 °C for 30 min, conventional semen analysis was conducted according to WHO criteria [9] by using Micro-cell slide and the computer-aided semen analysis (CASA, WLJY 9000, Weili New Century Science and Tech Dev.). Recorded semen parameters were sperm concentration, sperm motility, and CASA motion parameters, including straight-line velocity (VSL), curvilinear velocity (VCL), and linearity (LIN).
Determination of seminal SOD activity
SOD activity in seminal plasma was measured by the inhibition of nitrite (NIT) reduction due to the superoxide anion generated by the combination xanthine and xanthine oxidase. SOD assay kit (NO.A001-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to assess the SOD activity. One unit of SOD was the amount that caused a 50 % inhibition in the rate of NIT reduction. The SOD activity was expressed as U/mL seminal plasma.
DNA fragmentation analysis
As described previously [10], the Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay was performed to detect DNA fragmentation. We used the APO-DIRECT kit (BD Biosciences Pharmingen, SanDiego, CA, USA) according to the manufacturer’s protocol. Briefly, semen samples were thawed in a 37 °C water bath and immediately diluted with buffer (0.15 mol/l NaCl, 0.01 mol/l Tris, 0.001 mol/l EDTA, pH 7.4) to obtain a sperm concentration of 1–2 × 106/ml. Washed sperm were resuspended in 2 % paraformaldehyde for 30 min at room temperature. After being rinsed with PBS, samples were resuspended in permeabilisation solution (0.2 % Triton X-100, 0.1 % sodium citrate) for 10 min on ice. Fifty millilitres of TUNEL reagent were then added to the sample. For each batch, samples that were not treated with the Tdt enzyme were used as negative controls, and samples treated with DNase I were included as positive controls. After incubation for 1 h at 37 °C, samples were analysed immediately by flow cytometry (FACSCalibur; BD Biosciences Pharmingen).
Genotyping
Given that the SOD2 and SOD3 genes are all active in the ROS detoxification pathway, we selected four potential functional polymorphisms in SOD2 and SOD3 for genotyping. All of these single nucleotide polymorphisms (SNPs) have a reported minor allele frequency of ≥0.05 in the general Han Chinese population.
Briefly, DNA was extracted from leukocyte pellets of the venous blood by phenol-chloroform extraction with proteinase K digestion, and frozen until use. Genotype analysis was done using the OpenArray platform (Applied Biosystems, Foster City, CA, USA), which employs a chip-based Taq-Man genotyping technology. Genotyping was conducted according to the manufacturer’s standard protocols, and genotype calls were made by OpenArray SNP Genotyping Analysis Software version 1.0.3. For quality control, 10 % of the samples were randomly genotyped twice by different individuals, and the reproducibility was 100 %. To confirm the genotyping results, selected PCR-amplified DNA samples (n = 2, for each genotype) were examined by DNA sequencing and the results were also consistent.
Statistical analyses
All the statistical analyses were performed using the STATA 11.0 (STATA Corp, College Station, Texas). Sperm DNA fragmentation and sperm concentration were normalized by natural logarithm (ln) transformation. Linear regression models were used to estimate the correlations of seminal SOD activity with ln-transformed sperm fragmentation, ln-transformed sperm concentration values and other semen parameters after adjusted for age, smoking, drinking status, tea consumption and vitamin C/E using. The effects of selected individual characteristics on seminal SOD activity were analyzed by oneway ANOVA. Multivariate logistic regression analysis was employed to examine the effects of SOD polymorphisms on seminal SOD activity after adjusted for age, smoking, drinking status, tea consumption and vitamin using. The data were expressed as the mean ± SD. All P-values were two-sided and the significance level was set at P < 0.05.
Results
Characteristics of the study population
Subjects’ demographic information and semen quality in 435 infertile men were presented in Table 1. All these participants were Han Chinese with an average age of 28.7 years, mean Body Mass Index (BMI) of 23.6 kg/m2, an average abstinence period of 5.8 days. One hundred and ninety-four (44.6 %) participants were never smoker, 207 (47.6 %) were current smoker, and 34 (7.81 %) were former smoker. Of the 435 semen samples evaluated, the mean sperm concentration (106/ml) and sperm motility (% motile) were 69.6 and 57.9, respectively. Eighty-one (18.6 %) participants having sperm concentration values less than 20 million sperm/ml and 220 (50.6 %) having sperm motility values less than 50 % motile sperm. In addition, the sperm DNA damage was also detected, and the average sperm DNA fragmentation was 18.7 %, with 161 (37.0 %) having sperm DNA damage higher than 20 % TUNEL.
Table 1.
Subjects’ demographic information and semen quality in 435 infertility men
| Characteristic | Mean ± SD | No (%) |
|---|---|---|
| Age (years) | 28.7 ± 3.35 | |
| Smoking status | ||
| Never smoker | 194 (44.6) | |
| Current smoker | 207 (47.6) | |
| Former smoker | 34 (7.81) | |
| Body mass index (kg/m2) | 23.6 ± 3.23 | |
| Abstinence time (days) | 5.8 ± 4.47 | |
| 3 | 62 (14.2) | |
| 4–5 | 205 (47.1) | |
| 6–7 | 117 (26.9) | |
| ≥8 | 51 (11.7) | |
| Semen parameters | ||
| Sperm concentration (106/ml) | 69.6 ± 59.4 | |
| Subjects <20 million sperm/mla | 81 (18.6) | |
| Sperm motility (% motile) | 57.9 ± 25.9 | |
| Subjects <50 % motile sperma | 220 (50.6) | |
| Sperm DNA damage (%) | 18.7 ± 15.6 | |
| Subjects >20 % TUNEL | 161 (37.0) | |
aAccording to WHO (1999)
Associations between individual characteristics and seminal SOD activity in infertile subjects
The individual characteristics and the effects on seminal SOD activity levels in 435 infertile men were shown in Table 2. We found that individuals who consumed vitamin C/E (≥3 times per week) had significantly higher SOD activity levels than those who did not (mean ± SD: 379.8 ± 93.3 versus 332.2 ± 94.9, respectively, P = 0.001). However, other characteristics, such as age, alcohol intake, smoking status, and tea consumption appeared to have no obvious effects on seminal SOD activity.
Table 2.
Distribution of SOD activity results, exposure variables, and potential confounders in 435 infertility men
| Variables | Subjects N (%) | SOD activity (Mean ± S.D. U/ml) | P a |
|---|---|---|---|
| Age (years) | |||
| <30 | 205 (47.1) | 339.8 ± 102.5 | 0.432 |
| ≥30 | 230 (52.9) | 332.5 ± 91.3 | |
| Smoking status | |||
| Never | 194 (44.6) | 332.1 ± 104.4 | 0.2537 |
| Current | 207 (47.6) | 335.7 ± 90.8 | |
| Former | 34 (7.81) | 361.9 ± 86.7 | |
| Drinking status | |||
| Yes | 65 (14.9) | 323.5 ± 94.5 | 0.2805 |
| No | 370 (85.1) | 338.2 ± 97.3 | |
| Tea consumption | |||
| Yes | 130 (29.9) | 343.6 ± 86.78 | 0.311 |
| No | 305 (70.1) | 333.1 ± 100.8 | |
| Vitamin C/E using (≥3 times per week) | |||
| Yes | 59 (13.6) | 379.8 ± 93.3 | 0.0013 |
| No | 376 (86.4) | 332.2 ± 94.9 | |
aOneway ANOVA
Data in boldface represent P < 0.05
The correlation between seminal SOD activity and semen quality
As shown in Table 3, there was a positive association between sperm concentration and seminal SOD activity. The ln-transformed sperm concentration (106/ml) increased, on average, by 0.002 (95 % confidence interval (CI): 0.001, 0.003, P < 0.001) per 1-unit increase in the seminal SOD activity. Seminal SOD activity was also significantly associated with the percentage of sperm motility (coefficient: 0.039, 95 % CI: 0.010, 0.068, P = 0.008). Additionally, a negative relationship was found between ln-transformed sperm DNA fragmentation (%) and seminal SOD activity (coefficient: −0.001, 95 % CI: −0.002, −0.0003, P = 0.014). However, no associations were found between the seminal SOD activity and CASA motion parameters.
Table 3.
Correlations of seminal SOD activity with semen parameters and sperm DNA fragmentation after adjusted for age, smoking, drinking status, tea consumption and vitamin using in 435 infertility men
| Variables | Coef.a | Std. Err | 95 % Conf. Interval | P-values |
|---|---|---|---|---|
| Semen quality parameters | ||||
| Sperm concentration (106/ml) b | 0.002 | 0.0005 | 0.001, 0.003 | <0.001 |
| Sperm motility (%) | 0.039 | 0.0147 | 0.010, 0.068 | 0.008 |
| CASA motion parameters | ||||
| VSL (μm/s) | 0.840 | 0.478 | −0.100, 1.78 | 0.080 |
| VCL (μm/s) | 0.313 | 0.372 | −0.418, 1.04 | 0.401 |
| LIN (%) | 0.913 | 0.465 | −0.043, 1.87 | 0.092 |
| Sperm DNA fragmentation (%)b | −0.001 | 0.0005 | −0.002, −0.0003 | 0.014 |
aAdjusted linear regression coefficients
bln-transformed
Data in boldface represent P < 0.05
Association between SOD2, SOD3 polymorphisms and seminal SOD activity in infertile subjects
The associations between the four defined functional polymorphisms and seminal SOD activity were evaluated by multiple linear regressions in Table 4. We found that individuals with SOD2 rs4880 CC variants showed a low level of SOD activity when compared with common TT genotype carriers (Mean ± SD: 268.3 ± 102.3 and 342.8 ± 98.2, for CC and TT, respectively, P = 0.005). However, we did not find any significantly association between other studied SNPs (SOD2 rs5746136, SOD3 rs2536512, and SOD3 rs2695232) and seminal SOD activity in our population.
Table 4.
Effects of SOD polymorphisms on seminal SOD activity in 435 infertility men
| Genotype | Subjects N (%) | SOD activilty | P a | |
|---|---|---|---|---|
| Mean ± S.D.(U/ml) | Range (U/ml) | |||
| SOD2 rs4880 | ||||
| TT | 292 (67.1) | 342.8 ± 98.2 | 83.5–740.2 | Reference |
| TC | 124 (28.5) | 329.6 ± 88.7 | 101.2–621.3 | 0.437 |
| CC | 19 (4.4) | 268.3 ± 102.3 | 79.8–421.5 | 0.005 |
| SOD2 rs5746136 | ||||
| GG | 116 (26.7) | 344.5 ± 97.3 | 108.1–740.2 | Reference |
| GA | 214 (49.2) | 332.6 ± 99.3 | 79.8–621.3 | 0.572 |
| AA | 105 (24.1) | 332.7 ± 91.4 | 83.5–556.0 | 0.666 |
| SOD3 rs2536512 | ||||
| GG | 344 (79.1) | 337.0 ± 96.9 | 79.8–740.2 | Reference |
| GA | 83 (19.1) | 332.9 ± 98.3 | 83.5–522.0 | 0.944 |
| AA | 8 (1.8) | 315.0 ± 85.4 | 211.4–434.8 | 0.818 |
| SOD3 rs2695232 | ||||
| CC | 169 (38.9) | 348.6 ± 86.4 | 86.3–556.0 | Reference |
| CT | 183 (42.1) | 326.0 ± 100.1 | 79.8–621.3 | 0.091 |
| TT | 83 (19.1) | 331.5 ± 107.2 | 108.1–740.2 | 0.419 |
aAdjusted for age, smoking, drinking status, tea consumption and vitamin using
Data in boldface represent P < 0.05
Discussion
In the present study, we evaluated SOD activity in seminal plasma of 435 male infertility patients, and their relationship with selected human semen parameters and sperm DNA fragmentation, and investigated genetic factors that might contribute to the determination of SOD activity as well. Our analysis showed that SOD activity in seminal plasma was positively associated with sperm concentration and overall motility, whereas inversely with sperm DNA fragmentation. In addition, we found that SOD2 rs4880 variant genotype was associated with a low level of SOD activity, and those who consumed vitamin had significantly higher SOD activity levels comparing to those who did not.
ROS affect sperm membrane fluidity by oxidising the polyunsaturated fatty acids (PUFAs), and sperm motility is closely correlated with sperm membrane integrity. As SOD is an important element of ROS scavenger system that protects sperm from oxidative damage, the positive relationship between seminal SOD activity and sperm motility found in this study seems biologically reasonable. This result also confirms to the previous observations of Murawski et al. [6]. Additionally, a positive correlation between seminal SOD activity and sperm concentration, and a negative relationship between sperm DNA fragmentation and seminal SOD activity were also found in this study, suggesting a critical role of SOD in protecting sperm form ROS-induced DNA damage which could accelerate the process of germ cell apoptosis, resulting in the decline in sperm counts associated with male infertility [2].
Of the four defined functional polymorphisms in SOD2 and SOD3 studied here, we found a strong association between SOD2 rs4880 (nsSNP/Val16Ala) and lower SOD activity (Mean ± SD: 268.3 ± 102.3 and 342.8 ± 98.2, for CC and TT, respectively, P = 0.005). SOD2 (MnSOD) plays a critical role in protecting cells from free radicals and oxidative damage, and localization of SOD2 into the mitochondrial matrix is essential for it to protect sperm from oxidation [11]. It has been demonstrated that the SOD2 Ala variant at a mitochondrial targeting sequence allows for efficient MnSOD import into the mitochondrial matrix, while the Val variant causes partial arrest of the precursor within the inner membrane and decreased formation of the active MnSOD tetramer in the mitochondrial matrix [12]. Since the mitochondria are protected from H2O2 by MnSOD enzyme, cells could become susceptible to H2O2-related damages when the activity of MnSOD in the mitochondria is reduced. Thus, we could speculate that lower SOD activity in seminal plasma caused by the Val16Ala polymorphism may result in male infertility. To test the hypothesis, our previous study examined this SNP with male infertility risk in a case–control study, and found that SOD2 Val16Ala (rs4880) variant genotypes were associated with a significantly higher risk of male infertility [13].
With respect to vitamin supplementation, we observed individuals who consumed vitamin C/E (≥3 times per week) had significantly higher SOD activity levels than those who did not (mean ± SD: 379.8 ± 93.3 versus 332.2 ± 94.9, respectively, P = 0.001). The role of ascorbic acid (vitamin C) and tocopherol (vitamin E) in the prevention of the damage caused by free radicals and lipid peroxidation was well established [14]. Vitamin E, a lipid-soluble antioxidant, is able to repair oxidizing radicals directly, preventing the chain propagation step during lipid peroxidation, thus playing a vital role in protecting cell membranes [15]. Vitamin C is a water-soluble antioxidant that exerts its antioxidant effect through scavenging ROS by very rapid aqueous phase electron transfer, and preventing initiation of lipid peroxidation [16]. Previously, a systematic review of randomized studies was conducted to evaluate the effects of oral antioxidants (vitamins C and E, zinc, selenium, folate, carnitine and carotenoids) on sperm quality and pregnancy rate in infertile men, and showed an improvement in either sperm quality or pregnancy rate after antioxidant therapy [17]. Thus, vitamin C/E using might have a unique effect on SOD activity and a preventive role in clinical condition caused by oxidative stress-derived male infertility.
It’s worth mentioning that cigarette smoking, one of the main exogenous sources of oxidants, is correlated with alterations in sperm concentration, motility, morphology, and sperm function [18]. It has been long reported that ROS are found in high concentrations in semen of infertile smoking men [19]. However, in this study, we found no change of SOD activity in patient smokers. It might be possible that the use of vitamins combated the negative effect of smoking on SOD activity. Thus, further studies are needed to validate it.
In summary, SOD activity in seminal plasma was positively associated with sperm concentration and overall motility, whereas inversely with sperm DNA fragmentation. Additionally, SOD2 rs4880 was associated with low levels of SOD activity, and those who consumed vitamin had significantly higher SOD activity levels, when compared to those who did not. Thus, knowledge of activity of SOD in seminal plasma, its relationship with sperm parameters and influencing factors could be a useful tool for determining sperm fertilization potential and could improve the diagnosis and the prevention of male infertility.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (Grant No.81202243, Grant No.81172694 and Grant No.30901210); the Natural Science Foundation of Jiangsu Province (Grant No. BK2012087). A project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We thank Dr. C. Chen for statistical analysis.
Conflict of interest
No competing financial interests exist.
Footnotes
Capsule
SOD activity was found to be associated with semen quality and in individuals consuming vitamins C and E, a significantly higher expression level of SOD was observed.
Contributor Information
Guixiang Ji, Phone: +86-25-85287201, Email: jgx@nies.org.
Aihua Gu, Phone: +86-25-86862834, Email: aihuagu@njmu.edu.cn.
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