Role of Oxidative Stress and Antioxidants in Male Infertility: An Interventional Study.

AIMS: The study aims, in infertile men, (i) to assess oxidative stress parameters in semen plasma and (ii) to study the effect of antioxidants in those with abnormal semen parameters. SETTINGS AND
DESIGN: This was an interventional study. POPULATION: Ninety men attending and infertility clinic in a tertiary center were enrolled in the study.
MATERIALS AND METHODS: The present study was conducted in the departments of obstetrics and gynecology and biochemistry in a tertiary center. Ethical approval was obtained from the institute ethics subcommittee, and the study was conducted between July 2014 and July 2016. The study was conducted on two groups of 45 men with normal semen parameters in group 1 and 45 men with abnormal semen parameters in group 2.
RESULTS: Malondialdehyde (MDA) value was higher in men with abnormal semen parameters, which was statistically significant. The total antioxidant assay was higher in men with abnormal semen parameters, which was not statistically significant. Oxidative stress index (OSI) value was higher in men with normal semen parameters, which was not statistically significant. After 90 days of antioxidants therapy to men with abnormal semen parameters, MDA value decreased, total antioxidant assay increased, and OSI value decreased, which were statistically significant. Semen parameters such as sperm concentration, motility, and normal morphology improved after 90 days of antioxidant therapy, which were statistically significant.
CONCLUSIONS: Oxidative stress is increased in men with abnormal semen parameters. Antioxidant therapy improves sperm concentration and motility and decreases oxidative stress in the semen plasma. Copyright:

INTRODUCTION

In modern era, there are multiple, cost-effective medical treatment options available for female infertility; however, in cases of male infertility, the option of medical management is limited. In this regard, antioxidants seem to be promising options for the management of male infertility. Among young married couples, approximately 85%–90% conceive within 1 year that too mostly within 6 months.[1] Infertility affects 10%–15% of married couples. Male factor infertility contributes to 20%–30% of cases, both male and female factors together contribute to around 40%, female factor alone contributes 40%–55%, and remaining 10% of the factors are unexplained.

Superoxide anion, hydroxyl radicals, and hydrogen peroxides are the major reactive oxygen species (ROS) in the seminal plasma. In physiological amount, ROS help in sperm capacitation, acrosomal reaction, and sperm oocyte fusion. However, excessive amount of ROS causes infertility in two ways; first, by damaging sperm plasma membrane that leads to loss of sperm motility and ability of sperm to fuse with oocytes,[2] and second, ROS can alter the sperm DNA, resulting in passage of defective paternal DNA to the conceptus that leads to implantation failure. To counter excessive amount of ROS, human body has natural antioxidants such as glutathione peroxidase and superoxide dismutase. In healthy male, delicate balance is maintained between ROS and antioxidant mechanism in the reproductive system.[3]

Seminal oxidative stress results from imbalance between ROS production and antioxidant defense in the seminal plasma. Seminal oxidative stress is one of the main factors in pathogenesis of male infertility. Oxidative stress can be quantified by measuring individual biomarkers such as malondialdehyde (MDA) and total antioxidant assay of the semen plasma. MDA is a stable lipid peroxidation product, which reflects total oxidative status of semen plasma. Oxidative stress index (OSI) is calculated as (total oxidative stress/total antioxidant status) ×100.

Several methods have been proposed for the management of infertility caused by oxidative stress. Positive lifestyle changes such as maintaining normal weight, reducing smoking or alcohol intake, and taking diet rich in fruits and vegetables would have beneficial effect on sperm health. Antioxidants are most commonly used as defense against oxidative stress-induced infertility.[4] Several clinical studies demonstrated beneficial effect of oral antioxidant in male infertility. Antioxidants augment scavenging capacity of seminal plasma by reducing ROS within semen plasma. The low cost and minimal toxicity of antioxidants are main contributing factors for its use in male infertility.

There are few studies on the role of oxidative stress and antioxidants in male infertility. In our study, we have assessed oxidative stress parameters in infertile men with both normal and abnormal semen parameters at the start of study, and then, we have assessed oxidative stress parameters and semen parameters after giving antioxidants to infertile men with abnormal semen parameters, which is one of the first types of study in male infertility.

MATERIALS AND METHODS

The present study was conducted in the departments of obstetrics and gynecology and biochemistry in a tertiary center. Ethical approval was obtained from the institute ethics subcommittee, and the study was conducted between July 2014 and July 2016. The sample size was calculated by nMASTER software (CMC, Vellore, Tamil Nadu, India), with α error = 5%, power 80%, and dropout 20%. The sample size was 90 that were divided into 45 each in two groups. Men with normal semen parameters were included in group 1 and men with abnormal semen parameters were included in group 2.

Inclusion criteria for our study were infertile men with oligozoospermia (<15 million/ml) or asthenozoospermia (<40% motile spermatozoa) or teratozoospermia (<4% of normal shape and form spermatozoa) or combination of these and men with normal semen parameters. Exclusion criteria in our study were infertile men with testicular atrophy and other congenital testicular diseases such as cryptorchidism as antioxidants have no role in management of these conditions.

Outcome parameters we studied were baseline characteristics such as age, body mass index (BMI), and duration of infertility and laboratory parameters such as MDA, total antioxidant status, OSI, sperm count, sperm motility, and sperm morphology.

Brief procedure

Men who attended infertility clinic were recruited after fulfilling the inclusion criteria. Detailed history was taken and physical examination was done. Semen analysis was performed. Based on semen analysis, these men were divided into two groups – men with normal semen parameters and men with abnormal semen parameters. Baseline characteristics such as age, height, weight, and BMI were documented. The semen samples were collected from the subjects, centrifuged for separation of plasma, and stored at −80°C until further analysis was done for MDA and total antioxidant capacity. From this sample, semen plasma MDA level was measured by colorimetric method and expressed as micromole (μM) while total antioxidant capacity was measured by colorimetric method and expressed as millimole (mM). At start of the study, semen plasma MDA and total antioxidant capacity tests were done on both men with normal and abnormal semen parameters. Then, infertile men with abnormal semen parameters were treated with antioxidants for 90 days. Repeat semen analysis and oxidative stress test were done after 90 days on men with abnormal semen parameters only.

Estimation of malondialdehyde and total antioxidant assay

Semen plasma MDA was estimated by colorimetric method using QUANTIchrom thiobarbituric acid reactive substances (TBARS) assay kit, and the values were expressed as μM

Semen plasma total antioxidant assay was estimated by colorimetric method using Caymanʼs antioxidant assay kit (Product Number: 709001), and the values were expressed as mM.

Principle of the assay for malondialdehyde

TBARS are low-molecular weight end-products (mainly MDA) that are formed during the decomposition of lipid peroxidation. TBRAS assay is based on the reaction of TBRAS with TBA to form a pink-colored product. The color intensity at 535 nm is directly proportional to TBRAS concentration in the sample.[56]

Principle of the assay for total antioxidant capacity

Total antioxidant capacity assay depends on the ability of antioxidants in the sample to inhibit the oxidation of 2,2-azino-di-3-ethylbenzthiazoline sulfonate (ABTS®) to ABTS®·+ by metmyoglobin. The amount of ABTS produced can be monitored by reading the absorbance at 750 or 405 nm. Under the reactive conditions used, the antioxidants in the sample cause suppression of the absorbance at 750 or 405 nm to a degree which is proportional to their concentration. The capacity of the antioxidants in the sample to prevent ABTS®·+ oxidation is compared with that of Trolox, a water-soluble tocopherol analog and is quantified as millimolar Trolox equivalents.[78]

Total antioxidant capacity assay depends on the ability of antioxidants in the sample to inhibit the oxidation of ABTS® to ABTS®·+ by metmyoglobin. The amount of ABTS produced can be monitored by reading the absorbance at 750 or 405 nm. Under the reactive conditions used, the antioxidants in the sample cause suppression of the absorbance at 750 or 405 nm to a degree which is proportional to their concentration. The capacity of the antioxidants in the sample to prevent ABTS®·+ oxidation is compared with that of Trolox, a water-soluble tocopherol analog and is quantified as millimolar Trolox equivalents.[78]

Medications used in study

Antioxidants containing per tablet:

L- Carnitine - 500 mg

Lycopene - 2500 mcg

L-Glutathione - 10 mg

Coenzyme Q10-50 mg

Selenium - 75 mcg

Zinc - 60 mg

Twice daily doses - for 3 months.

Statistical analysis

Data were analyzed using software SPSS for Windows 20.0 (IBM Company, Illinois, USA).

Following parameters between men with normal and abnormal semen were compared using appropriate statistical tests. Age, BMI, and duration of infertility were tested using unpaired t-test. Semen plasma MDA and total antioxidant capacity were compared using independent t-test. OSI expressed as median using Mann–Whitney U test.

Before and after values of MDA and total antioxidant assay were compared using paired t-test. Before and after values of OSI values were compared using Wilcoxon signed-rank test. Before and after values of semen parameters were compared using paired t-test. All statistical analyses were carried out at 5% level of significance (P < 0.05).

RESULTS

Group 1: Men with normal semen parameters

Group 2: Men with abnormal semen parameters.

Semen plasma MDA, total antioxidant capacity, and OSI were done in both two groups. Forty-five men with abnormal semen parameters were given antioxidant for 3 months. After which semen analysis, semen plasma MDA, total antioxidant assay, and OSI were repeated. Only two men dropped out from our study.

In our study, there was no significant difference in demographic variables such as age, BMI, and duration of infertility between the two groups [Table 1].

Table 1

Demographic factor of both the groups (n=45)

Factors	Mean±SD		P (<0.05 is significant)
	Group 1	Group 2
Age (years)	29.40±4.95	28.51±3.29	0.31
BMI (kg/m2)	20.84±1.33	21.18±3.19	0.51
Duration of infertility (years)	4.42±1.43	4.31±1.98	0.76

SD=Standard deviation, BMI=Body mass index

MDA value was higher in men with abnormal semen parameters, which was statistically significant. The total antioxidant assay was higher in men with abnormal semen parameters, which was not statistically significant. OSI value was lower in men with normal semen parameters, which was not statistically significant [Table 2].

Table 2

Values of malondialdehyde, total antioxidant assay, and oxidative stress index of two groups (n=45)

Factors	Mean±SD		P (<0.05 is significant)
	Group 1	Group 2
MDA (µM)	4.98±2.84	6.45±1.90	0.006
Total antioxidant capacity (mM)	2.78±1.90	3.52±1.77	0.060
Oxidative stress index*	0.16 (0.13-0.34)	0.22 (0.1-0.36)	0.900

*Median (IQR). MDA=Malondialdehyde, SD=Standard deviation, IQR=Interquartile range

We found reduction in the seminal plasma MDA values in men with abnormal semen parameters after giving antioxidants with values of 6.46 ± 1.93 μM before and 5.14 ± 2.29 μM after treatment, which was statistically significant. Improvement of total antioxidant capacity was also statistically significant with values 3.52 ± 1.81 mM before and 5.14 ± 2.29 mM after treatment. In our study, reduction in OSI was observed with median values of 0.16 before and 0.09 after treatment [Table 3].

Table 3

Comparison of malondialdehyde, total antioxidant capacity, and oxidative stress index before and after treatment in Group 2 (n=43)

Factors	Mean±SD		P (<0.05 is significant)
	Before	After
MDA (µM)	6.46±1.93	5.32±2.01	<0.001
Total antioxidant capacity (mM)	3.52±1.81	5.14±2.29	<0.001
Oxidative stress index*	0.16 (0.13-0.34)	0.09 (0.6-0.13)	0.05

*Median (IQR). MDA=Malondialdehyde, SD=Standard deviation, IQR=Interquartile range

There was significant difference in the semen volume in men with abnormal semen parameters after giving antioxidant with values before 3.28 ± 1.84 ml and after 2.81 ± 1.91 ml. However, the semen volume was decreased after antioxidant therapy. There was significant improvement in the sperm concentration after giving antioxidant with values 29.10 ± 23.58 million and 43.33 ± 30.34 million, respectively. After antioxidant therapy, there was statistically significant improvement in total motility (27.90% ± 14.21% to 48.48% ± 23.73%). There was significant improvement in sperms with normal morphology after giving antioxidant with values of 75.71% ± 17.09% before and 82.32% ± 17.33% after the treatment. In addition, there was decline in sperm with abnormal morphology with values 24.29% ± 17.09% and 15% ± 11.23%, respectively, before and after treatment [Table 4].

Table 4

Comparison of semen parameters before and after treatment in Group 2 (n=43)

Factors	Mean±SD		P (<0.05 is significant)
	Before	After
Volume (ml)	3.28±1.84	2.81±1.91	0.050
Concentration (×106/ml)	29.10±23.58	43.33±30.34	0.003
Total motility (%)	27.90±14.21	48.48±23.73	<0.001
Normal morphology (%)	75.71±17.09	82.32±17.33	0.020
Abnormal morphology (%)	24.29±17.09	15±11.23	<0.001

DISCUSSION

Baseline characteristics such as age and BMI were comparable between two groups and did not have significant difference. The mean age was similar between two groups with 28.51 ± 3.29 years in Group 1 and 29.40 ± 4.95 years in Group 2. Our study result was comparable to other studies done by Kumar et al., Colagar et al., and Sheikh et al.[91011] The mean BMI was also comparable between the groups with 20.84 ± 1.33 kg/m2 in Group 1 and 21.18 ± 3.19 kg/m2 in Group 2. Our study result was similar to studies done by MacDonald et al. and Nadjarzadeh et al.[1213] The duration of infertility was also similar in both the groups, 4.42 ± 1.43 years in Group 1 and 4.31 ± 1.98 years in Group 2, and it was similar to the study done by Nadjarzadeh et al. and Verit et al.[1314]

In the present study, seminal plasma MDA level was higher in men (6.42 ± 1.90 μM) with abnormal semen parameters than that in normal semen parameters (4.98 ± 2.84 μM), which was statistically significant. Our study result was similar to the studies done by Colagar et al., Fazeli and Salimi, and Hosen et al., and Asbagh et al.[10151617] Excessive oxidative stress in the semen of men with abnormal semen parameters was the reason for higher MDA value.

The total antioxidant capacity was higher in men with abnormal semen parameters than that in men with normal semen parameters with values 3.52 ± 1.77 and 2.78 ± 1.90 mM, respectively. Our study result was not similar to the studies done by Fazeli and Salimi and Hosen et al.[1516] A case–control study was done by Fazeli and Salimi[15] on 35 infertile and 34 fertile men where total antioxidant capacity was measured by ferric reducing ability of plasma method. They found total antioxidant capacity more in the fertile men than that in infertile men. Hosen et al.[16] did a study on 25 fertile men and 41 infertile men using ABTS method to assay total antioxidant assay. They found total antioxidant assay value higher in the fertile men than infertile men. We got total antioxidant capacity higher in men with abnormal semen parameters as we have compared infertile men in both the groups unlike other studies, and also, our result was not statistically significant.

In our study, we found OSI value (0.16) was lower in men with normal semen parameters than in men with abnormal semen parameters (0.22), which was not statistically significant. In a study done by Verit et al.,[14] (prospective study) on 32 infertile men and 30 fertile men, the OSI value was higher in the infertile men than fertile men.

We had studied oxidative stress parameters after giving 90 days of antioxidants to men with abnormal semen parameters. We found significant reduction of the semen plasma MDA level after treatment with antioxidant with values 4.053 μm before and 3.073 μm after treatments, which was statistically significant. This was similar to other studies done by Suleiman et al. and Singh et al.[1819] In a study done by Suleiman et al.,[18] on 52 infertile patients giving antioxidants, they found significant reduction of MDA level and improvement of sperm motility. Singh et al.[19] did a study on 40 infertile men who received antioxidants for 3 months. They found significant reduction of MDA levels after 3 months of antioxidant therapy. The level of MDA which is marker of oxidative stress was decreased after antioxidant therapy in the present study.

The present study showed significant improvement of total antioxidant capacity after treatment with antioxidant, with mean levels being 3.52 ± 1.81 and 5.14 ± 2.29 mM before and after, respectively. This was similar to studies done by Singh et al. and Yadav et al.;[1920] however, in a study done by Kumar et al.,[9] on 21 infertile men, antioxidant for 3 months found no significant improvement in total antioxidant capacity. This difference may be due to less sample size of 21 infertile men. Total antioxidant capacity which reflects total antioxidant power of the semen plasma was improved after antioxidant therapy. Antioxidant therapy improves infertility outcomes by supplying synthetic antioxidants to seminal plasma and by reducing MDA level in semen plasma.

In our study, we found significant reduction in OSI before and after antioxidant treatment, with mean levels of 0.16 before and 0.09 after treatments. We did not find any study comparing OSI before and after antioxidant therapy.

We had studied semen parameters after giving antioxidants to men with abnormal semen parameters for 90 days. There was a significant difference in semen volume (3.28 ± 1.84 vs. 2.81 ± 1.91 ml) after 3 months of antioxidant therapy. This decrease in semen volume may be due to incomplete collection or a short abstinence interval. Our study result was not similar to studies done by Kacem et al. and Kobori et al.[2122] In the study done by Kacem et al.,[21] on 48 infertile couples after giving antioxidant therapy for a variable period of 3–5 months, they found no significant difference in semen volume. Kobori et al.[22] conducted a study on 169 infertile men after giving antioxidants; they observed semen parameters after 3 and 6 months and found no improvement of semen volume after antioxidant therapy.

Sperm concentration is an important parameter in the management of male infertility. With antioxidant therapy, we observed a significant improvement in sperm concentration which can help in change in the management plan from in vitro fertilization to intrauterine insemination. In our study, there was a significant improvement in sperm concentration (29.10 ± 23.58 × 106/ml vs. 43.33 ± 30.34 × 106/ml). Our study result was similar to the studies done by Singh et al., Khani et al., and Muneshwar et al.[192324] Kacem et al.[21] did another study on 48 infertile men after giving antioxidant for 2-month duration; they found no increase in sperm volume, concentration, and motility. The length of treatment for 2 months is less than the life cycle of sperm to show any effect of antioxidant in their study.

Antioxidants improve sperm motility which is one of the important parameters in the management of male infertility. In our study, there was a significant improvement in motility after antioxidants in men with abnormal semen parameters (27.90% ± 14.21% vs. 48.48% ± 23.73%). Our study was similar to the studies done by Suleiman et al., Singh et al., Khani et al., Muneshwar et al., Comhaire et al., and Piomboni et al.[181923242526] All the above studies used either single or combination of antioxidants for 3 months and observed improvement in sperm motility, except Suleiman et al.,[18] who observed improvement in sperm motility after giving Vitamin E alone for 6 months.

In our study, we found significant improvement in sperm morphology after antioxidant therapy. Our result was similar to the studies done by Piomboni et al. and Heidary et al.[2627] as both studies observed improvement in the sperm morphology after 3 months of antioxidant therapy; however, Khani et al.[23] performed a study on 25 infertile men to determine the effect of antioxidant therapy on sperm morphology, and they found no significant improvement in sperm morphology. This difference may be due to less sample size in their study.

In our study, we got significant improvement in semen parameters such as sperm count and motility after 3 months of antioxidant therapy, and also, biochemical parameter such as MDA value was significantly decreased and total antioxidant capacity was increased significantly after treatment. As spermatogenic cycle completes in 3 months, our idea was to give antioxidants for 3 months to cover sperm damage at any level of spermatogenesis and to analyze the results. Our study of antioxidants use in male infertility is supported by many studies.

CONCLUSIONS

From the present study, it may be concluded that oxidative stress is increased in men with abnormal semen parameters. Antioxidant therapy improves sperm concentration and motility and decreases oxidative stress in the semen plasma.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.