Reactive Oxygen Species & male infertility Jalal Ghasemzadeh Andrology lab Yazd Reproductive Sciences Institute

Jalal Ghasemzadeh Andrology lab 1387/09/17

Introduction  Male factor infertility accounts for up to 50% of all cases of infertility.  Oxidative stress is a common pathology seen in approximately half (30-80%) of all infertile men (Agarwal et al,2006).  Reactive Oxygen Species (ROS)  ROS, defined as including oxygen ions, free radicals, and peroxides are generate by sperm and seminal leukocytes within semen.

 Sperm are vulnerable to ROS attacks :  Sperm membranes contain a large amount of polyunsaturated fatty acids.  Lack a sufficient number of antioxidant systems in their cytoplasm.  ROS, produce infertility by two key mechanisms :  Damage the sperm membrane decreasing sperm motility.  ROS directly damage sperm DNA. Objective of this review is :  The mechanisms by which OS produce infertility.  Identify which men are most at risk of oxidative infertility.  The methods available for diagnosis OS and the various treatment.

 A free radical is defined as an oxygen molecule containing one or more unpaired electrons in molecular orbitals.  Superoxide anion (O 2 ) radical is the primary form of ROS.  Secondary ROS include hydroxyl radical (OH), Peroxyl radical (ROO) and hydrogen peroxide (H 2 O 2 ).  Free radicals participate in chemical reaction, resulting in the oxidation of lipids in membranes, amino acids in proteins and carbohydrates within nucleic acid (Ochsendorf, 1999). Oxidative stress biochemistry

Source of R♂S in semen  Leukocytes :  Neutrophils being the predominant leukocyte type. (Aitken et al,1995)  A positive correlation between seminal leukocyte numbers and ROS production. (Aitken et al., 1994; Sharma et al., 2001)  The rate of production of ROS by leukocytes is reported to be 1000 times higher than of spermatozoa. (Plante et al.,1994)  Spermatozoa :  The ability of sperm to produce ROS inversely correlates with their maturational state.  As a result, teratozoospermic sperm produce increased amounts of ROS compared with normal sperm.

Kinds of R ♂ S  Intrinsic ROS is seminal ROS production by sperms.  Extrinsic ROS is seminal ROS production by leukocytes. Both intrinsic and extrinsic ROS production is negatively correlated with sperm DNA integrity the relationship is significantly stronger for intrinsic ROS production. (Henkel et al., 2005) Intrinsic ROS production a more important variable in terms of fertility potential.

Antioxidant systems of human body  Enzymatic antioxidants : 1) Superoxide dismutase (SOD) : SOD is present within both sperm and seminal plasma. 2) Catalase : a link between deficient seminal catalase activity and male infertility. 3) Glutathione peroxidase (GPX 1-5 ) : Located within the testis, prostate, seminal vesicles, vas deferens, epididymis, seminal plasma and spermatozoa. Male factor infertility has been linked with a reduction in seminal plasma and spermatozoa GPX activity (Garridoetal.,2004).

 Non-enzymatic antioxidants : Ascorbic acid (Vitamin C) Glutathione flavenoids Alpha-tocopherol (Vitamin E) Carnitine Carotenoids Amino acids (taurine, hypotaurine) Albumin Urate A significant reduction in non-enzymatic antioxidants activity in seminal plasma of infertile compared with fertile men. (Fraga et al.,1996;Smith et al., 1996; Therond et al., 1996;Lewis et al., 1997; Gurbuz et al., 2003; Koca et al., 2003;Mostafa et al., 2006; Song et al., 2006)

 Low level of ROS plays a positive role in fertilization (capacitation).  H 2 O 2 stimulates the acrosome reaction and sperm hyperactivation.  Low concentrations of H 2 O 2 also cause sperm membrane binding to the ZP-3 protein and sperm–oocyte fusion (Aitken et al., 1998).  Mechanisms account for oxidative stress-mediated male infertility : 1) Impaired motility 2) Impaired fertilization 3) Oxidative DNA damage  Increased sperm oxidative DNA damage with poor blastocyst formation in vitro (Zorn et al., 2003; Meseguer et al., 2006,2007). Seminal free radicals-friend or foe?

 Iatrogenic : 1) Centrifugation 2) Cryopreservation 3) Drugs  Life style : 1) Smoking 2) Excessive alcohol consumption 3) Extremes of exercise activity 4) Obesity 5) Psychological stress 6) Advancing age 7) Dietary deficiencies :  A significant correlation between vitamin C intake and sperm concentration and between vitamin E intake and total progressively motile sperm (Eskenazi et al.,2005). Origins of oxidative stress

Laboratory identification of R ♂ S  Cost and complexity of testing and the lack of a single standardized  Direct methods : 1) assessing sperm membrane peroxidation  Measurement of MDA levels in sperm or seminal plasma 2) measurement of sperm DNA damage  TUNEL, SCSA & 8 -OHdG  Indirect methods :  Chemoluminescence assays  Nitro blue tetrazolium (NBT) assay

1. Asthenozoospermia 2. Teratozoospermia 3. High number of round cells (? Leukocytes) in semen. 4. Increased semen viscosity 5. Poor fertilization on routine IVF 6. Poor sperm motility after over night incubation with the oocyte. 7. Poor blastocyst development  NOTE : Asthenozoospermia is probably the best marker for oxidative stress in a routine semen analysis. (Kao et al., 2007) Oxidative stress & routine semen analysis “Sentinel laboratory signs”

Management of oxidative stress related infertility “Summary of treatment options in male oxidative infertility” 1. Modification ‘lifestyle’ 2. Minimize environmental exposure to heat, pollutions and toxins. 3. Direct treatment 4. Surgery. for example, ‘varicocelectomy’ 5. Vitamin and antioxidant supplements 6. Optimize laboratory procedures

Vitamin and antioxidant supplementation  The oral antioxidant carnitine or a combination of antioxidants such as acetylysteine, β-carotene, Vitamin E and essential fatty acids have reduce seminal ROS levels.  2 months treatment with 1 g of Vitamin C and Vitamin E reported a very significant reduction in sperm DNA damage (Greco et al., 2005).  A reduction in sperm DNA damage with the use of a combination of Vitamin C and E (400mg each), β-carotene (18 mg), zinc and selenium (Menezoetal.,2007).  The only parameter that appears to be possibly improved with oral antioxidant therapy is sperm motility. “ carnitine,selenium, Vitamin E, Vitamin E and selenium glutathione and Astaxanthin ”

 Reducing the time that the semen is centrifuged.  Use of non-centrifuge separation techniques such as ‘glass-wool filtration’.  Limiting the time in which sperm are cultured in media away from seminal plasma.  Avoiding use of cryopreserved sperm for fertilization.  The addition of variety antioxidants sperm preparation media. Laboratory techniques to reduce R ♂ S

C ♂ NCLUSI ♂ N  Role for oxidative stress in male infertility is now established :  Develop inexpensive assays to identify sperm oxidative stress.  Effectiveness of surgical (varicocelectomy, testicular biopsy) in the management of oxidative stress.  Determine combination and dose of antioxidant supplement.  Finally, the development of new sperm culture media.

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