Introduction

Infertility has been a major medical problem as well as social stigma. Despite of enormous progress in research, most of the blame for infertility is placed on the female. Advances in understanding of gonadal/sperm physiology have increased our knowledge of male infertility. Defective sperm function is the most prevalent cause of male infertility. ^[1]. Many environmental, physiological, and genetic factors have been implicated in infertility. Hence, it is very important to identify factors, which affect normal sperm function. Free radical-induced oxidative damage to spermatozoa is a condition, which is gaining considerable attention for its role in inducing poor sperm function ^[2]. Understanding of such conditions will help in designing new and effective treatment strategies.

Until recently, Reactive oxygen species (ROS), were considered toxic to the human spermatozoa. However a strong evidence suggests that small amounts of ROS are necessary for spermatozoa to acquire fertilizing capabilities ^{[3], [4],[5]}.

Theoretically, cellular damage in the semen is due to improper balance between ROS generation and scavenging activities which causes ‘Oxidative stress’.

The increased ROS formation leads to lipid peroxidation. This is correlated with reduced sperm motility ^{[6], [7]}, due to decrease in axonemal protein phosphorylation and sperm immobilization, both of which are associated with a reduction in membrane fluidity ^[8]. Another hypothesis is that H₂O₂ can diffuse across the cell membranes and inhibit the activity of enzyme, glucose-6-phosphate dehydrogenase (G6PD). Inhibition of G6PD decreases the availability of NADPH and results in accumulation of oxidized glutathione, which reduces the antioxidant defense of the spermatozoa ^[9].

The seminal plasma is well endowed with an array of antioxidants such as Superoxide dismutase (SOD), Catalase. These antioxidants protect spermatozoa against oxidative stress ^{[10],[11],[12]}. SOD scavenges superoxide anion and converts it into less potent hydrogen peroxide. Catalase detoxifies H₂O₂ to water and oxygen ^[13]

This study was undertaken to correlate detrimental effect of free radicals on sperm physiology in infertile males. The prime aim of the study was to estimate the lipid peroxidation product; MDA & some antioxidants in men with oligoasthenozoospermia compared to normospermia & their correlation with seminal parameters.

Results

Figure 1

: Showing sperm count, motility, MDA, SOD and Catalase in control and study group

Note: The catalase activity is inversely proportional to the amount of perchromic acid formed by reaction between H₂O₂ and potassium dichromate due to lack of activity of catalase.

Figure 2

Showing comparison of sperm count, motility and levels of seminal MDA in control and study group.

In the above graph sperm count is expressed in millions/ml, motility in percent and MDA in nmols/ml, SOD in units/ml, catalase in U/mgs of protein

Figure 3

Showing correlation of sperm count and seminal MDA concentration

Figure 4

: Showing correlation of seminal SOD activity with Sperm Count

Figure 5

: Showing correlation of seminal Catalase activitiy with Sperm Count

The catalase activity is inversely proportional to the amount of perchromic acid formed by reaction between H₂O₂ and potassium dichromate due to lack of activity of catalase

Figure 6

: Showing correlation of seminal SOD activity with seminal MDA concentration

Figure 7

: Showing correlation of seminal Catalase activitiy with seminal MDA

Result

The concentration of seminal MDA in both groups were significantly different. Individuals with oligoasthenozoospermia are related with the elevated MDA concentrations. The levels of MDA in Group I & Group II were 12.65 ± 1.68 and 21.58 ± 1.54 nmols/ml respectively ( p< 0.0001). The activities of seminal plasma SOD in Group I & Group II were 27.99 ± 4.36 & 12.96 ± 3.35 unit/ml respectively ( p< 0.0001). The values of catalase activities in Group I & Group II were 1.360 ± 0.44 & 5.12 ± 1.03 U/mg of protein respectively ( p< 0.0001). The Sperm concentration is negatively correlated seminal MDA concentration (Graph 2) ( r = -0.0086) whereas seminal SOD & CAT activities ( measured in terms of perchromic acid which is inversely proportional ) positively correlated with sperm concentration (Graph 3 & 4) ( r = 0.083, r = -0.58) resp. We also correlated seminal MDA concentration with levels of seminal SOD & CAT where we noted negative correlation of these antioxidant with seminal MDA (Graph 5 & 6) resp.

Discussion

The most relevant findings of this study are

i) There is significant increase in concentration of MDA in oligoasthenozoospermia on comparison with normospermia and negative correlation was observed between the MDA concentration and sperm count. MDA production, which reflects the peroxidation of

polyunsaturated phospholipids, the major components of sperm membrane^[18]. MDA

measurements are physiological and relevant because major loss of sperm function may occur with minimal damage to the membranes that envelop the sperm and/or divide key intracellular sperm compartments Thus, increased MDA activity represents lipid peroxidation of sperm membrane and inhibits sperm motility and viability.

Our results of MDA are concurrent with Kobayashi et al. and Hiesh et al. Kobayashi et al. ^[19] Kobayashi et al ^{demonstrated elevated seminal MDA concentration in patients with oligoasthenoteratozoospermia. While our results are in contrast with Suleiman et al. and Heidar et al. Suleiman et al. (2001) [20] demonstrated that MDA concentration in the seminal plasma was not related with the sperm concentration and motility.}

ii) We also measured antioxidant enzymes superoxide dismutase and Catalase. The SOD & CAT activities showed positive correlation with sperm concentration. We found negative correlation between SOD, CAT and MDA in seminal plasma. Immature spermatozoa generate primary superoxide anion. This anion is dismutated to hydrogen peroxide by SOD. Detoxification of hydrogen peroxide is carried out by Catalase. Hydrogen peroxide is the primary toxic ROS for human spermatozoa, as its high concentration induces lipid peroxidation and results in cell death.

Therefore, the balance of the SOD and Catalase activities in semen is important for maintaining sperm motility. Our studies of SOD are at par with Marek et al. and Siciliano et al. Marek et al.(2007)^[21] reported significantly lower semen SOD activity in oligoasthenozoospermic patients, comparing to the normospermic men. Their study showed a positive correlation between seminal SOD activity and semen parameters - concentration and overall motility, where as Siciliano et al. (2001)^[22] observed decline in SOD activity in oligoasthenozoospermia. Our findings of Catalase are in accordance with Khosrowbeygi et al.and Siciliano et al. Khosrowbeygi et al (2007)^[23] observed decrease in seminal Catalase in infertile group compared to fertile group. Siciliano et al observed significant decrease in catalase activity and total antioxidant capacity (TAC) in men with asthenozoospermia compared to normozoospermia. In contrast Heidar et al.(2008) ^[24] found a positive correlation between total activity of CAT with total content of MDA in seminal plasma in normozoospermic samples.

Human spermatozoa exhibit a capacity to generate ROS. Production of very low amounts of ROS in semen appears to play a physiological role in regulating normal sperm functions, whereas high levels of ROS endanger sperm function and viability. Oxidative stress due to excessive production of ROS, impairs the antioxidant defense mechanisms, or it precipitates a range of pathologies that are believed to affect the male reproductive function ^[25]. Abnormal ROS production is associated with defective sperm function ^[26],[27]. A fine balance between ROS production and recycling is essential for spermatogenesis. Excessive generation of seminal ROS, mainly by neutrophils but also by immobile sperm, morphologically abnormal sperm, or morphologically normal but functionally abnormal sperm, can be a cause of male infertility ^[28]. Exposure of spermatozoa to ROS has been associated with cellular injury, which includes DNA damage and lipid peroxidation^[29]. High lipid peroxidation may reduce the capacity of the sperm to undergo acrosomal reaction and fertilization ^[30] along with it’s capability for oocyte fusion.

Seminal Antioxidant enzymes, such as SOD, Catalase could scavenge and detoxify the free radicals. A balance is maintained between the amount of ROS and antioxidants. Cellular damage arises on disturbed equilibrium, especially when the cellular scavenging systems cannot eliminate the increased ROS. Lipid peroxidation damages the cell plasma membrane, which leads to loss of cytosolic components and cell death.

Materials and methods

Thirty normal & thirty infertile males between age group 20-45 years were included in the study on voluntary basis. Exclusion criteria included positive HIV status, history of tuberculosis, endocrinological disorders. Semen was obtained by masturbation after at least 72 hours of sexual abstinence. Semen samples were collected into sterile containers. After liquefaction, samples were centrifuged to separate seminal plasma which was analyzed for volume and sperm count, sperm motility and morphology microscopically according to WHO guidelines ^[14]. All semen samples were divided into two groups: Group I - Normal healthy controls i.e. Normospermia (n=30) with sperm count > 20 x 10⁶ per ml & motility > 50%; Group II – Infertile males i.e. Oligoasthenozoospermia (n=30) with sperm count < 20x10⁶ per ml & motility <50%.

Then the semen samples were analyzed for MDA concentration and SOD, Catalase activities. MDA levels were analyzed according to methods described by Rao et al ^[15] MDA was assessed using the thiobarbituric acid method. Briefly, 800 μl of seminal plasma was added into glass tube. To each tube, 1.2 ml of thiobarbituric acid was added and then heated for ten minutes in a boiling water bath. After cooling, to each tube 2 ml of freshly prepared 1 N NaOH was added and absorbance was read on a spectrophotometer at 534 nm. The activity of the superoxide dismutase was estimated by method described by Marklund & Marklund ^[16] bases on autoxidation of pyrogallol. Catalase activity has been determined in human seminal plasma of normal and infertile groups by potassium dichromate method described by Sinha ^[17] based on reduction of dichromate acid to perchromic acid in presence of hydrogen peroxide. The concentration of perchromic acid is inversely proportional to activity of catalase. To calculate the activity of catalase the values of seminal proteins required are calculated by lowry’ method.

Conclusion

There has been a phenomenal growth in our knowledge of male reproduction, sperm function and development of diagnostic tools and treatment modalities. Evaluation of oxidative stress status and use of antioxidants is not routine in clinical practice.

MDA measurements are physiological and relevant because major loss of sperm function may occur with minimal damage to the membranes that envelop the sperm and/or divide key intracellular sperm compartments. It has been studied that the reduction of MDA by using antioxidant therapy was correlated with the improvement of fertilization rates⁴. Increased MDA concentrations has shown a significant increase in the number of immotile spermatozoa whereas the reduction in MDA level by using antioxidants therapy correlates with improvement in fertilization rate^{[22], [23]}.

Our study shows, high ROS levels in study group due to lipid peroxidation represented by MDA in semen, should be used as an additional marker in diagnosis of male infertility. It is also important to determine the antioxidant status of semen as it is a major defense mechanism.

These tests have significant bearing on the fertility of semen sample. Our study found that these tests are highly reliable, reproducible & simple to perform.

Thus to conclude - these tests should be used as an adjunct to routine analysis of semen which are carried out in the laboratories in order to find out root cause of male infertility.

They are an established index for the analysis of sperm quality.