The aim of this study was to measure reactive oxygen species (ROS) production and total antioxidant capacity (TAC) in the seminal fluid of the male partners in couples undergoing intrauterine insemination and to evaluate correlations between these values and their semen parameters.
The study was conducted at Vamsam Fertility Center, Coimbatore, India and enrolled 110 male patients from whom semen samples were collected. ROS production was measured by a thiobarbituric acid reactive species assay, and TAC was measured by a 2,2-diphenyl-2-picrylhydrazyl free radical assay. The differences in the TAC and malondialdehyde (MDA) levels between the subfertile and fertile groups were analysed. Correlations between sperm parameters and TAC and MDA levels were statistically analysed, and cutoff values with respect to the controls were determined. All hypothesis tests used were two-tailed, with statistical significance assessed at the level of
A total of 87 subfertile and 23 fertile men were included in the study. The mean MDA level was significantly higher in the subfertile subjects than in the fertile subjects, and the mean antioxidant level was significantly lower in the subfertile subjects than in the fertile subjects. Seminal MDA levels were negatively associated with sperm concentration, motility, and morphology, whereas the opposite was seen with TAC levels.
Measurements of seminal TAC and ROS are valuable for predicting semen quality, and hence predicting the outcomes of fertility treatment.
Infertility has become a widespread problem in recent years. For the normal fertilization process to occur, a healthy, undamaged sperm cell is vital. Approximately one-third of infertility problems are attributed to the male partner. In order to improve the fertility rate, potential causes of damage leading to abnormal semen parameters should be evaluated. Semen analysis constitutes the initial step in screening for the male partner's contribution in an infertile couple. Extensive debate exists concerning the diagnostic and prognostic significance of semen parameters in the evaluation of male infertility, except in patients with azoospermia or severe oligozoospermia [
Oxidative stress has been found to be one of the most prevalent causes of sperm damage, subsequently affecting fertilization [
The purpose of this study was to quantify malondialdehyde (MDA), one of the by-products of lipid peroxidation and antioxidants in the seminal plasma. Measuring the level of free radicals and total antioxidants present in the seminal plasma enables an assessment of the correlations of these values with semen parameters such as motility, concentration, and morphology, thereby helping to improve the success rate of fertility treatment. Supplementing patients with necessary antioxidants for a prescribed duration after performing the antioxidant assay may help them to improve their antioxidant capacity and maintain an appropriate balance between ROS and antioxidants. This, in turn, may help maintain the integrity of DNA, leading to an improved fertility rate in patients with male factor infertility. Hence, standardizing these assays for measuring the level of total antioxidants present in the seminal plasma and establishing cutoff values for easy reference will be useful for treating patients by targeting oxidative stress.
The study was approved by the Institutional Human Ethics Committee (No. 15/278). Written informed consent was obtained from all subjects whose samples were analysed in this study. A questionnaire about the participants' lifestyle and medical history was also administered. Semen samples from 87 subfertile men attending a private fertility center in Coimbatore from May 2015 to April 2016 were obtained for this analysis. Twenty-three fertile men were enrolled as controls. The mean age of the fertile subjects and subfertile subjects was 33.73± 4.64 and 33.74±5.21 years, respectively.
The study group included 26 normozoospermic male partners, 51 teratozoospermic male partners, 10 oligoteratozoospermic male partners, and 23 fertile healthy volunteers as a control group. The semen parameters were used to classify the patients into these groups following the 2010 World Health Organization (WHO) guidelines [
Subjects whose partners did not conceive after a period of unprotected intercourse for more than a year were included in the study, regardless of their semen parameters. Exclusion criteria are: (1) subjects who smoked and/or consumed alcohol, (2) subjects with leukocytospermia or pyospermia, (3) subjects suffering from diabetes mellitus or tuberculosis, or who had a previous history of mumps, and (4) subjects with a known male factor anomaly, such as varicocele.
ROS levels were measured using a thiobarbituric acid reactive species assay [
TAC was measured using a 2,2-diphenyl-2-picrylhydrazyl free radical (DPPH) assay [
Data were expressed as mean±standard deviation. The Mann-Whitney
Semen samples from 110 individuals, including 87 subfertile participants (26 normozoospermic, 51 teratozoospermic, and 10 oligoteratozoospermic) and 23 fertile donors, were studied. The mean age of the fertile subjects and subfertile subjects was 33.73±4.64 years and 33.74±5.21 years, respectively. The semen parameters of both groups, including sperm concentration, motility, morphology, MDA levels, and TAC are presented in
The correlations between semen parameters and seminal plasma ROS and TAC levels were examined using Spearman correlation analysis, as shown in
In order to analyze the effectiveness of oxidative stress in discriminating between the fertile and subfertile patients, an ROC curve was generated with a 95% confidence interval for TAC levels, comparing the fertile controls with the subfertile participants. The results are presented in
Cutoff values for TAC were derived through a comparison of the control group with the individual subgroups of subfertile participants, as well as through a comparison of the control group with the subfertile participants as a whole. No effective cutoff value of TAC was derived when individual subgroups were compared with the fertile controls, while a TAC value of 77.4% was determined to be an efficient discriminating value when comparing the fertile participants with the subfertile participants as a whole (
Imbalances between ROS and TAC in the male reproductive tract have a major influence on the fertilization potential of sperm. Measuring and standardizing these parameters would be helpful for treating patients with antioxidant therapy in order to increase the natural conception rate. The subfertile men included in this study had lower seminal TAC levels than the fertile men. Antioxidant levels showed positive associations with sperm concentration, motility, and morphology. The lower TAC levels in subfertile men were accompanied by higher levels of free radicals. The mean MDA level was significantly higher in the subfertile subjects than in the fertile subjects; this was due to the overproduction of ROS in semen, which is associated with reduced sperm fertilizing potential [
Based on the ROC curve, the most suitable cutoff value for TAC levels, along with sensitivity, specificity, and the area under the curve (AUC), was obtained by comparing the control group with the subgroups of subfertile men, as well as by comparing the control group with the subfertile participants as a single group. The cutoff value that was found is important because it discriminates between fertile and subfertile patients and provides a threshold value for various semen parameters based on clinical data. Previous studies have shown sperm morphology to have a high predictive power of various semen parameters, with AUCs of 0.78 and 0.697, and the 4% cutoff value of sperm morphology showed good predictive value, as shown by an AUC of 0.782 [
In addition to normal sperm concentration and other semen parameters measured in routine semen analysis, other markers are also needed to assess the fertilization potential [
At high levels of lipid peroxidation, spermatozoa are rendered dysfunctional and their membrane function is altered. Lipid peroxidation triggers the loss of membrane integrity, causing increased cell permeability, enzyme inactivation, structural damage to DNA, and cell death [
Initially, antioxidants are present in spermatozoa in a quantity capable of counteracting the free radicals that are produced such that a balance is maintained between antioxidants and ROS. On prolonged exposure, the antioxidant mechanism breaks down and an imbalance is created, due to which DNA fragmentation occurs [
In accordance with previous studies in which morphologically abnormal spermatozoa have been suggested to be a potential source of ROS, in our study, participants with teratozoospermia and oligoteratozoospermia showed elevated MDA levels. Of the three subfertile groups included in the study, the patients with oligoteratozoospermia seem to have had significantly low TAC levels and very high MDA levels. In this condition, morphologically immature or abnormal sperm cells, which are a potential source of ROS, lead to increased lipid peroxidation. The decline in sperm concentration in these patients might have been due to extended exposure to high levels of ROS produced by immature or abnormal spermatozoa. This likely resulted in damage to tubules, causing testicular atrophy, reduction in motility, and DNA damage to mature sperm cells, all of which affect fertility potential.
Exogenous antioxidant supplementation should be considered as a therapeutic measure in patients with abnormal TAC and MDA levels. Several
In conclusion, our study demonstrated that there were significant differences in the levels of ROS and TAC in the seminal plasma of subfertile and fertile participants. A significant threshold level for the TAC of semen (77.4%) was established. Supplementation with necessary antioxidants for a certain period of time after performing an antioxidant assay may help patients to improve their antioxidant capacity and maintain a balance between ROS and antioxidants. This, in turn, may help maintain the integrity of DNA, thereby improving the fertility rate in patients with male factor infertility. Hence, standardizing these assays for measuring the total level of antioxidants present in the seminal plasma and establishing a cutoff value for easy reference will facilitate the treatment of patients for oxidative stress.
The authors are grateful to the PSG Institution, Coimbatore, India for funding this work.
This work was supported by PSG Institution, Coimbatore, India.
Values are presented as mean±standard deviation.
ROS, reactive oxygen species; TAC, total antioxidant capacity; MDA, malondialdehyde.
a)Indicates statistical significance at
MDA, malondialdehyde; TAC, total antioxidant capacity.
a)Indicates statistical significance at
ROC, receiving operator characteristic; TAC, total antioxidant capacity.
a)Indicates statistical significance at