Reactive Oxygenated Species (ROS) in Male Fertility; Source, Interaction Mechanism and Antioxidant Therapy

 

Daryoush Fatehi¹, Ardeshir Moayeri², Omid Rostamzadeh³, Ayoob Rostamzadeh^4*,

Maziar Malekzadeh Kebria⁵

¹Department of Medical Physics, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran

²Department of Anatomy, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran

³Department of Occupational Therapy, School of Rehabilitation, Iran University of Medical Sciences, Tehran, Iran

⁴Medical Plants Research Center, Basic Health Sciences Institute, Department of Anatomy and Neuroscience, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran

⁵Department of Anatomical Sciences, Faculty of Medicine, Tarbiat Modares University, Tehran, Iran

 

ABSTRACT:

Currently ROS is known as the cause of many diseases and damage to cells and tissues. Many studies have shown that ROS is related to diseases such as cancer, cardiovascular, diabetic nephropathy and even processes such as aging and infertility. The studies of the past two decades reveal that low and controlled ROS values in the physiological process of the cell are secondary; while their physiological values is essential for the normal activity of the cell. Studies show that in most cells, the physiological amounts of ROS are produced by NADPH oxidase family enzymes. These enzymes are present in the plasma membrane of the cells, where they increase the production of ROS by connecting them with calcium. ROS in sperm also plays different physiological roles from the time of its production to the time of its emergence with an oocyte, but the pathological effects of its excessive production are also evident. This includes increasing the damage to DNA and increasing sperm apoptosis, which is responsible for including infertility in an important part of interfile men. In the infertility treatment clinics, that their number of clients is increasing every day, sperms find fertilization opportunity with the oocyte in the culture medium; but prior to that, sperm should passes different washing steps. These washing processes increase ROS production. The production of ROS is increased by the method of separating the sperm by centrifuging and then allowing sperm moves to float. This is one of the common methods in infertility treatment centers for sperm washing, while the sperm in washing stages has been deprived of its antioxidant source. Therefore, the use of vitamins either orally or in a sperm medium can increase the chances of fertilization and fertility of infertile individuals by preventing the motility of sperm and preventing their increased mortality and reducing DNA damage.

 

 

 

INTRODUCTION:

Production of ROS:

Reactive oxygen species (ROS) is a component of oxygen-derived molecules that, in the case of excessive activity, is considered to be harmful oxidant. Some of these radical forms are free. Free radicals are chemically highly reactive due to their non-coupling electrons (1). ROS include: Anions superoxide 0x1 (O2°), hydrogen peroxide (H2O2), radical peroxyl (ROO°), hypochlorite ion (C1O°) and hydroxyl radical (OH°) are very reactive (2). The mechanism of activation of oxygen by metal ions is justified by the Harber-weiss, Fenton reaction. In each of these reactions, molecular oxygen and its regenerated forms (H2O2 and O2°) act as substrates (3). The first ROS produced in human spermatozoa is superoxide anion (O2°). This anion is a product of a single-electron oxidation reaction in the presence of a superoxide dismutase enzyme that produces hydrogen peroxide in the secondary reaction with oxygen. In the presence of metals such as copper and iron, as a catalyst, radical hydroxyl is produced by the reaction of fenton from hydrogen peroxide. Also, after the radical reaction of oxygen with hydrogen peroxide, radical hydroxyl is produced which is known as the Haber-Weiss reaction (4). The roles of biological ROS include: Acting as intracellular propagandists (5) and active in regulating pathways of the cell (6), also regulates the expression of the gene, in particular the gene related to the antioxidant proteins (7). Therefore, the presence of ROS is necessary for normal cell function, and any change, at its physiological level, causes oxidative stress conditions and endangers cell survival such as traumatic spinal cord injury (8, 9), ovarian torsion (10), polycystic ovary syndrome (11), and liver fibrosis (12). Sperm function is also affected by ROS. Based on their concentration, these elements can have physiological or pathologic effects on sperm (13).

 

Mechanisms of ROS production in human semen:

One of the components of the semen that can produce ROS includes abnormal spermatozoa, primordial germ cell (PGC) and leukocytes. Among these, seminal leukocytes and abnormal sperm are the main sources of ROS production in humans (14). This section explores how these ROS are produced. The level of ROS in semen in men with infertility with unknown cause is significantly higher than that of normal people and has lower antioxidant properties (1).  Research has shown that the presence of oxidative stress in men with normal sperm that is infertile can be considered as one of the causes of infertility.  Also, examination of sperm DNA damage due to ROS can indicate the hidden sperm DNA abnormalities in infertile men whose sperm count is normal (7).

 

a) ROS production by abnormal spermatozoids:

The production of ROS in abnormal sperm is due to the maintenance of additional cytoplasm in the process of spermiogenesis. If the spermatogenesis process gets impaired, the cytoplasmic deletion mechanisms will not function properly, resulting in sperm motility and inoperative performance (15). The abnormal spermatozoa produce

 

 

 

ROS in two methods:

1: The NADPH oxidase system, which is active on the surface of the sperm plasma membrane.

 

2: NADH oxide and reductase (diaphorase), which is present at the mitochondrial level (16). The mitochondrial system is considered as the main source of ROS in infertile men's spermatid (7, 16). Spermatozoa require energy due to its high mobility. Therefore, it contains massive mitochondria; that any mitochondrial defect results in excessive ROS production. This increase in ROS is due to mitochondrial membrane degradation (17).

 

b) ROS production by leukocytes:

Positive oxidase leukocytes are considered as sources of ROS in semen (18). Of the total leukocytes in the semen, 50 to 60 percent of the leukocytes are positive for peroxidase, and have the ability to produce ROS, depending on their activity in response to stimuli such as inflammation and infection (19). In activated leukocytes, the production of NADPH increases, and their activation of the myeloprosidase system; the following process increases the ROS level (20). Sperm injuries derived from ROS induced from leukocytes may be due to an abnormal increase in the concentration of seminal leukocytes, which is called leukocytospermia (15). Recent studies have shown that seminal leukocytes may also play a role in stimulating the production of ROS by spermatozoa (21).

 

ROS bioeffects:

In the process of ROS production; it can have a physiological or pathological role. In physiological conditions, spermatozoa produces a limited amount of ROS, which is necessary to regulate the sperm function, its capacity, acrosomy activity, and the ability to bind to the transparent layer of the oocyte. Superoxide anions play an important role in this process (22). Studies have shown that spermatozoa has the ability to produce ROS at different stages of puberty, that immature sperm with an abnormal head and additional cytoplasm produces the highest ROS compared to normal mature sperm (23). Background studies show that ROS plays an important role in the regulation of sperm maturation, the intensification of cAMP synthesis, and the diffusion of ejaculatory proteins at the onset of sperm motility (24). However, in the pathologic conditions, ROS increases the quality and other parameters of semen and causes oxidative stress conditions. Because of the high sensitivity of spermatozoa to oxidative stress conditions, it will face great deal of harm, including the following (figure 1):

 

 

 

a)Lipid Peroxidation:

Spermatozoa has been attacked by peroxides due to unsaturated fatty acids in the membrane, and this reaction results in the loss of unsaturated fatty acids and the production of altered oxidative products that are fatal to the cell. The presence of unsaturated fatty acids leads to the preservation of the fluidity of the spermatozoa membrane (11, 25). The reactivity of these fatty acid fluids to membrane fluid reduces the activity of membrane enzymes and ion canals; as a result the natural cellular mechanisms required for fertilization are affected. One of the methods for estimating steady-state products caused by lipid peroxidation is the measurement of malondialdehyde (12, 13).

 

b)DNA Damage:

Oxidative tissues in the sperm have a special activity; however, there is no DNA repair mechanism in the dense sperm chromatin (26), which is why sperm DNA and sphygmomanic phytoremediation are sensitive to per-oxidative damage. Thus the high level of ROS results in degradation of the breasts, transversal transmutation of proteins, and DNA degradation (27). Although, the sperm DNA damage is restored after fertilization by oocyte; its excessive damage can not be repaired. It should be noted that the degree of recovery depends on the quality of the oocyte (28). A biological marker that can determine the amount of DNA damage is "8-Hydroxy-2-Doxy-Guanosine (8-OHdG)" (28).

 

c)Apoptosis:

Extensive ROS production activates apoptotic chain reactions (22). ROS levels have a positive correlation with the level of caspases as an apoptotic protease (11, 23). Mitochondria in apoptosis play a key role, according to which, the inner mitochondrial membrane contains cytochrome C. if the mitochondrial membrane is destructed by ROS, the cytochrome from mitochondria drops, causing activation of caspases and induces the phenomenon of apoptosis. Apoptosis can also exacerbate DNA damage caused by ROS (29).

 

d)Destruction of sperm motility:

Mobility is an essential part of the spermatozoa, in order to design a pathway for the production of propagation and to fertilize itself and then penetrate the oocyte. Studies have demonstrated that ROS levels have a direct correlation with the mobility of spermatozoa. (30). in an in-vitro study, it was found that the effects of ROS on mobility may be temporary or permanent. If increased ROS causes rapid ATP discharges and reduces the phosphorylation of the axonal protein, the sperm motility will be interrupted temporarily (11, 22, 31). However, if peroxidation results from an increase in ROS to damage the sperm membrane and axonal protein, sperm motility is permanently eliminated (31). In addition, several other factors contribute to the creation of oxidative stress conditions: Examples include exposure to heavy metals and smoking. Heavy metals in humans and other mammals act as lethal metals and produce oxidative stress. These metals can increase the production of oxidants and affect the health of the membrane (32). Studies in animals have revealed that these metals produce high levels of ROS, reduce sperm motility, reduce the rate of sperm penetration into the oocyte and reduce the level of antioxidants, in a way that the level of these metals in the semen has a positive relationship with degradation of DNA bays (12, 23). The destructive effects of these metals on the reproductive pattern of men act with damage to the hypothalamus-pituitary-testicle axis. The number of sperm cells of those who are exposed to heavy metals reduces and the state of "Teratozoospermia” increases (23, 33). Cigarettes as another cause of stress contain 4,000 components, including alkaloids, nitrosamines, organic molecules and many other subunits, ROS or  reactive nitrogen species (RNS) are also radical-free (34). There is a positive correlation between the amount of smoking and DNA fragmentation; so that Frge et al. found that the level of 8-OHdG (the marker of DNA decomposition) in smokers was 50% higher than for non-smokers (35). Based on their idea, cigarettes increase the production of norepinephrine on spermatogenesis and increase the conversion of testosterone to estrogen and reduce the level of this hormone. A group of researchers also found that cigarettes increased ROS and decreased antioxidants (36).

 

Antioxidants and their Function:

There are different mechanisms for inhibiting oxidative stress and reducing the damage caused by ROS, one of which is the antioxidant system (1, 23). The presence of antioxidants creates a steady state of the ROS level in seminal plasma. They protect sperm from ROS as free radical cleansers. Antioxidants include two enzymatic and non-enzymatic groups: Types of enzymes include:  Superoxide Dismutase (SOD), Glutathione Peroxidase (GPX), Catalase and Glutathione Activated Reactor. Non-enzymatic types include: Vitamins A, C, E, Pyruvate, Glutathione, Taurine, Hypothalourein, and Coenzyme Q (37) (Table 1). The amount of oxidants and antioxidants is maintained at a specific ratio, and any changes in order to increase this ratio towards the oxidants, create oxidative stress conditions.  Antioxidants also compensate for the reduction of cytoplasmic enzymes caused by the process of spermiogenesis (23, 29).  The mechanisms of action of antioxidants against oxidative stress are as follows (figure 1): 

 

a) Prevention Mechanism:

Proteins that contain a nucleus of iron, copper or nucleus with binding capacity to these metals, including albumin, metallothionein, cerfloxacin, transferrin, myoglobin, can prevent radical hydroxyl production.

 

b) Restorative mechanism:

Biomolecules that have been damaged by oxidative stress, glutathione peroxidase in the presence of enzymes such as glutathione reductase, Methionine-sulfoxide reductase restore or vanish. 

 

c) Clearing mechanism:

Enzymes capable of clearing the excess of ROS such as Superoxide dismutase, glutathione peroxidase, catalase and other Metal Enzymes along with a series of

 

chemicals that potentially scaveng capacity, such as unsaturated fatty acids, vitamins, uric acid, bilirubin and carotenoids do the cleansing  (38).

Table 1. Some antioxidants factors in the reproductive cells.

Location in cell	Intracellular	Extracellular	Membranes
antioxidant	Superoxide dismutase (SOD), Catalase, Glutathione peroxidase	Ascorbic acid (vitamin C), Urate (ubiquinone), Sulphydryl groups (proteins, glutathione), Bilirubin, Vitamin E (tocopherols)	Tocopherols (vitamin E), Carotenoids (vitamin A), Transferrin, ceruloplasmin, Haptoglobin, hemopexin

 

Figure 1. main risk factor and key mediators between oxidative stress, ROS and germ cells in inducing fertility failure in male reproductive system.

 

 

Non-enzymatic antioxidants:

Vitamins C and E are non-enzymatic antioxidants, which, depending on the dose, show their protective effects (39).  The intake of vitamins C and E leads to decreased concentrations of lipid peroxidation markers and DNA degradation (40).  Glutathione is the most abundant non-thyroid protein in mammals (41).  It protects the plasmid membrane from lipid peroxidation and prevents radical formation of peroxide and superoxide (42).

 

Enzymatic antioxidants:

The SOD cleanses the anion and extra and intracellular superoxide (22, 31). It also prevents over-activation before sperm maturation and its capacity to pre-ejaculate, which occurs in the presence of radical superoxide (28).  Catalase purifies hydrogen peroxide and plays an important role in reducing peroxidation of lipids and sperm proteins (22, 28). Glutathione peroxidase, together with glutathione, as an electron donor, eliminates various peroxides (44). Another system that is important in the process of clearing oxidative stress is called redox. This system of components includes the following:

 

a) Glutathione:

which is a peptide molecule is present in the presence of glutathione reductase enzymes in two forms of oxidation (GSSG) and resuscitation (GSH) (45). 

 

b) Thioredoxin (Trx):

which is a small Redox protein. Reconstruction of this protein is accomplished by the presence of the thyrudoxine reductase enzyme (45).

 

Finally, Oxidative condition is incomplete as a result of spermatogenesis and is associated with a cytoplasmic appendage (Residual cytoplasm), which is one of the characteristics of this group. ROS can directly damage sperm by induction of peroxidation of the plasma membrane of the sperm. Therefore, high levels of oxidative stress play an important role in reducing sperm motility and the infertility. Apoptosis and DNA damage can prevent sperm maturation, that imbalance in these pathways creates the condition of low count of sperm and slow motility that’s some most key factors for fertility and embryo formation.  In physiological conditions, apoptosis preserves a number of germ cells in sertoli cells. If this pathway is disturbed, spermatogenesis stops and the level of apoptosis increases, which the frequency of DNA damage in the cells that are not mature is elevated.

 

CONCLUSION:

All aerobic living cells are naturally exposed to ROS. If the ROS level increases, oxidants are formed and this leads to increased cell damage. Oxidative stress has been identified as one of the main causes of male infertility. In most infertile men, the level of ROS seminal increases and causes various DNA damage, including: Cross-linking chromatin, chromosomal removal, DNA strand destruction, and base oxidation.  On the other hand, ROS also plays an important role in the induction of cytochrome C and type III caspases in the process of apoptosis. Therefore, there is a relationship between male infertility oxidative seminal stress, sperm DNA damage and apoptosis. Oxidative stress is caused by an imbalance between oxidants and antioxidants in semen plasma. Determining the sources of ROS production can affect the treatment method in oxidative stress infertility. Factors involved in abnormal leukocyte penetration in semen i.e. inflammation, infection and smoking should be minimized. In order to remove leukocytes and cysts containing cytoplasmic sperms that are the main sources of ROS production a sperm separation method can be used. It should be kept in mind that the process of centrifugation in these methods can itself be ROS-inducing in the sperm. Antioxidant protective agents against ROS are supposed to be useful therapeutic agents for male infertility. However, some of these studies suggest effective antioxidant supplements and some without effect. On the other hand, some researchers believe that adding supplements to sperm can improve its function and can be considered as a useful option in treating infertile patients.

 

REFERENCES:

1.       Halliwell B, Gutterridge J. M. C. Free radicals in biology and medicine. New York: Oxford University press; 1996:617-783

2.       Halliwell B. Tell me about free radicals, doctor: a review. J R Soc Med. 1989; 82(12):747-52.

3.       Sohal R. S, Dubey A. Mitochondrial oxidative damage, hydrogen peroxide release, and ageing, free radicalBiol. Med. 1994; 16: 621-626.

4.       Agarwal A, Ikemoto I, Loughlin KR. Effect of sperm washing on levels of reactive oxygen species in semen. Arch Andorl. 1994; 33(3):157-62.

5.       Cooper CE, Patel RP, Brookes PS, Darley-Usmar VM. Nanotransducers in cellular redox signaling: modification of thiols by reactive oxygen and nitrogen species. Trends Biochem sci. 2002; 27(10):489-92.

6.       Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol. 1998; 10(2):248-53. Review.

7.       Jones RD, Hancock JT, Morice AH. NADPH oxidase: a universal oxygen sensor? Free Radic Biol Med. 2000; 29(5):416-24. Review.

8.       Fatehi D, Naleini F, Salehi MG, Afshari D, Mirfendereski S, Farzizadeh M. Traumatic spinal cord injury; theranostic applications of advanced MRI techniques. Biomedical and Pharmacology Journal. 2015 Dec 28;8(2):891-903.

9.       Fatehi D, Dayani MA, Rostamzadeh A. Role of CT scan in theranostic and management of traumatic spinal cord injury. Saudi Journal of Biological Sciences. 2016 Aug 26.

10.     Rostamzadeh A, Mirfendereski S, Rezaie MJ, Rezaei S. Diagnostic efficacy of sonography for diagnosis of ovarian torsion. Pakistan journal of medical sciences. 2014 Mar; 30(2):413.

11.     Ahmadi M, Rostamzadeh A, Fathi F, Mohammadi M, Rezaie MJ. The effect of Melatonin on histological changes of ovary in induced polycystic ovary syndrome model in mice. Middle East Fertility Society Journal. 2017 Apr 12.

12.     Mohammadi M, Rezaie MJ, Rostamzadeh A, Allahveisi A, Mohammadi HR, Mohammadi F. Signaling Pathways of Melatonin in Prevention of Liver Disorders via Suppressing of Oxidative Stress in Cellular Level. Biomedical and Pharmacology Journal. 2016 Aug 21;9(2):555-61.

13.     Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology. 1996; 48(6):835-50. Review.

14.     Kessopoulou E, Tomlinson MJ, Barratt CL, Bolton AE, Cooke ID. Origin of reactive oxygen species in human semen: spermatozoa or leucocytes? J Reprod Fertil. 1992; 94(2):463-70.

15.     Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta  1,4-galactosyltransferase,  cytoplasmic  creatine  phosphokinase, and creatine phosphokinase isoform ratios. Biol Reprod. 1997; 56(4):1020-4.

16.     Aitken RJ. Buckingham DW, West KM. Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminal- and lucigenin- dependent chemiluminescence. J Cell Physiol. 1992; 151(3):466-77.

17.     Ochsendrof FR. Infections in the male genital tract and Reactive oxygen species. Hum Reprod Update. 1999; 5(5): 399-420. Review.

18.     Shekarriz M, Sharma RK, Thomas AJ, Agarwal A. Positive myeloperoxidase staining (Endtz test) as an indicator of excessive reactive reactive oxygen species formation in semen. J Assist Reprod Genet. 1995; 12(2): 70-4.

19.     Blake DR, Allen RE, Lunec J. Free radicals in biological systems—a review orientated to inflammatory processes. Br Med Bull. 1987; 43(2): 371-85. Reviw.

20.     Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod. 1989; 41(1): 183-97.

21.     Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Feertil Steril. 2003; 79(4): 429-43. Review.

22.     Ollero M, Gil- Guzman E, Lopez MC, Sharma PK, Agarwal A, Larson K, Evenson D, Thomas AJ Jr, Alvarez JG. Characterizayion of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod. 2001 Sep; 16(9): 1912-21.

23.     Aitken RJ, Vernet P. Maturation of redox regulatory mechanisms in the epididymis. J Reprod Fertil Suppl. 1998; 53: 109-18.

24.     Pogany GC, Corzett M, Weston S, Balhorn R. DNA and protein content of mouse sperm. Exp Cell Res. 1981; 136(1): 127-36.

25.     Kemal Duru N, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa. Fertil Steril. 2000; 74(6): 1200-7.

26.     Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update. 2003; 9(4): 331-45. Review.

27.     Sikka SC. Role of oxidative stress and antioxidants in andrology and assisted reproductive technology. J Androl. 2004; 25(1): 5-18. Review.

28.     Wang X, Sharma PK, Sikka SC. Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril. 2003; 80(3): 531-5.

29.     Armstrong JS, Rajasekaran M, Chamulitrat W, Gatti P, Hellstrom WJ, Sikka SC. Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radic Biol Med. 1999; 26(7-8): 896-80.

30.     De Lamirrande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J Androl. 1992; 13(5): 379-86.

31.     Xu SX, Shen HM, Zhu QX, Chua L, Wang QN, Chia SE, Ong CN. The associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma. Mutat Res. 2003; 534(1-2): 155-63.

32.     Osborn BH, Haney AF, Misukonis MA, Weinberg JB: Inducible nitric oxide synthase expression by peritoneal macrophages in endometriosis-associated infertility. Fertil Steril 2002; 77: 46-51.

33.     Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res. 1996; 351(2): 199-203.

34.     Pasqualotto FF, Lucon AM, Sobreiro BP, Pasqualotto EB, Arap S. Effects of medical therapy, alcohol, smoking, and endocrine disruptors on male infertility. Rev Hosp Clin Fac Med Sao Paulo. 2004; 59(6): 375-85. Review.

35.     Agarwal A, Saleh RA. Role of oxidants in male infertility: rationale, Significance, and treatment. Urol Clin North Am. 2002; 29(4): 417-27. Review.

36.     Martinez Sanchez, G.; Delgado Hernandez, R.; Garrido, G.; Guevara Garcia, M.; Garcia Rivera, D.; Paez Betancourt, E.; Nunez Selles, A. J.; Mitos y Realidades de la Terapia Antioxidante. Vimang: Nuevo Producto Natural Antioxidante, Center of Pharmaceutical Chemistry: Havana, 2003.

37.     Hull MG, North K, Taylor H, Farrow A, Ford WC. Delayed conception and active and passive smoking. The Avon Longitudinal Study of Pregnancy and Childhood Study Team. Fertill Steril. 2000; 74(4): 725-33.

38.     Suleiman SA, Ali ME, Zaki ZM, el-Malik EM, Nasr MA. Lipid peroxidation and human sperm motility: protective role of vitamin E. J Androl. 1996; 17(5): 530-7.

39.     Keskes-Ammar L, Feki-Chakroun N, Rebai T, Sahnoun Z, Ghozzi H, Hammami D, Zghal K, Fki H, Damak J, Bahloul A. Sperm oxidative stress and the effect of an oral vitamin E and selenium supp;ement on semen quality in infertile men. Arch Androl. 2003; 49(2): 83-94.

40.     De Lamirande E, Gagnon C, Capacitation-associated production of superoxide anion by human spermatozoa. Free Radic Biol Med. 1995; 18(3): 487-95.

41.     Lenzi A, Culasso F, Gandini L, Lombardo F, Dondero F. Placebo-controlled, double-blind, cross-over trial of glutathione therapy in male ingertility. Hum Reprod. 1993; 8(10): 1657-62.

42.     Jeulin C, Soufir JC, Weber P, Laval- Martin D, Calvayrac R. Catalase activity in human spermatozoa and seminal. Gamete Res. 1989; 24(2): 185-96.

43.     Baker HW, Brindle J, Irvine DS, Aitken RJ. Protective effect of antioxidants on the impairment of sperm motility by activated ploymorphonuclear leukocytes. Fertil Steril. 1996; 65(2): 411-9.

44.     Meister A. Glutathione biosynthesis and its inhibition. Methods Enzymol. 1995; 252: 26-30.

45.     Said TM, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Hum Reprod Updete. 2004; 10(1): 39-51. Review.

 

 

 

 

Accepted on 24.12.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2): 791-796.