1. ANTIOXIDANTS AND MALE INFERTILITY DR. ABHISHEK SINGH PARIHAR M.S.( OBS & GYNAE), FELLOW REPRODUCTIVE MEDICINE. CONSULTANT : LIFECARE I.V.F E), FELLOW REPRODUCTIVE MEDICINE Consultant – Life care IVF

2. In 40- 60% Of Infertile Couples A Male Related Problem Is A Cause Of Infertility. Sperm production cycle occurs in a mean 64 ± 8 days (range: 42–76 days)—(by stable isotope and gas spectrometric analysis)

3. Several reports describe reduction in male fertility over recent decades. Estimates that sperms counts in U.S males decreasing by 1.5% each year

4. 30%-80% of infertile men have elevated markers of OS Agarwal et al., Urology 2006 Reactive Oxygen Species (ROS) (Log ROS + 1; cpm) OXIDATIVE STRESS An emerging explanation for several cases of male infertility An emerging explanation for several cases of male infertility 2,5 2 1,5 1 0,5 0

5. R EACTIVE O XYGEN S PECIES (ROS) Chemical species with unpaired electron capable to oxidize lipids, proteins and nucleic acids: Superoxide anion (O-2) Hydroxyl radical (OH) Hydrogen peroxide (H2O2) Peroxyl radical (ROO) Hypochlorite ion (OCl-) Generated from aerobic metabolism in mitochondria. Sources: Leukocytes (extrinsic) Spermatozoa (intrinsic) Sperm and Seminal Plasma Antioxidants: Enzymatic (SOD, catalase, GPX): inactivate ROS (conversion into other substances) Non-enzymatic (ascorbic acid, alpha-tocopherol, carnitine, etc.): neutralize ROS Lampiao et al., Oxidative stress. In: Male Infertility. Parekattil & Agarwal (eds) 2012, pp.225-35

6. REACTIVE OXYGEN SPECIES THE NEGATIVE EFFECTS OF ROS ON SPERM QUALITY HAVE GAINED LOT OF ATTENTION

7. OXYGEN & ROS PARADOX

13. O RIGIN OF OXIDATIVE STRESS

14. S PERM DNA F RAGMENTATION AND A SSISTED C ONCEPTION Live Birth Rates by Intrauterine Insemination OR = 0.07 [95% CI: 0.01-0.48 ] Normal Elevated Pregnancy by Method in Cases of Elevated Sperm DNA Fragmentation 42% 26% P <0.05 Adapted from Bungum et al., Hum Reprod 2007 ICSI 1.5% IVF 19%

15. S PERM DNA F RAGMENTATION AND M ISCARRIAGE Population: Meta-analysis of 16 cohort studies (2,969 couples), 14 prospective. Techniques for DNA integrity: Acridine orange-based assays, TUNEL and COMET. Findings: Significant increase in miscarriage rates in patients undergoing IVF/ICSI with high sperm DNA damage compared with those with low DNA damage. Risk ratio (RR) = 2.16 (1.54, 3.03), P < 0.00001). Miscarriage rates are positively correlated with sperm DNA damage levels I Robinnson L et al. Hum Reprod. 2012; 27(10): 2908-17 Rob

16. S I GN S OF O XIDATIVE S TRESS IN S EMEN A NALYSIS Reduction of sperm parameters ( Asthenozoospermia is the best surrogate marker of OS ( Kesks et al., Ammar et al. ) DNA fragmentation ( Kao et al. 2007) Hyperviscosity– (ureaplasma infection ? ) Semen leukocytes ( round cells ) count using peroxidase or seminal elastase test ( Kopa et al. 2003) Testing Malondialdehyde level in sperm/seminal plasma Or Chemoluminescence assays using luminol/lucigenin are expensive, complicated and highly sensitive.

17. M ANAGEMENT OF OS –R ELATED I NFERTILITY In >80% of males medical treatment is ineffective. General measures 1 –Lifestyle modification ( smoking, poor diet, alcohol,obesity, stress) 2–Avoid activities heating the scrotum 3–Proper ventilation /use of personal protective equipment to reduce exposure to chemicals /metals linked with OS 4–Treatment of infections / variecocele 5–ANTIOXIDANTS,VITAMINS AND FOOD SUPPLEMENTATION.

18. R OLE OF M ICRONUTRIENTS IN F ERTILITY Nutrition plays vital role in maintaining male fertility: Involved in the successful maturation of sperm Provides nutrition for motility of sperm Improvement in sperm count and motility Helps in production of sex hormones Prevents sperm damage

19. N UTRITIONAL C ONSIDERATIONS Various micronutrients are associated with male fertility. Deficiency of these Micronutrients & ANTIOXIDANTS may result in infertility. Nutritional Factors Free radical scavengers L-Carnitine Lycopene Coenzyme Q10 Vitamin C Zinc Vitamin E Arginine Glutathione Vitamin B12 Selenium

20. I DEAL N UTRACEUTICAL FOR M ALE I NFERTILITY Coenzyme Q10 Improves sperm motility by providing energy through ATP generation in mitochondrion L-carnitine Improves sperm motility by providing energy to the sperm cell through fatty acid metabolism Lycopene Increases sperm count and improves morphology by reducing oxidative damage to sperm DNA and lipids Zinc Promotes sperm production & maturation testosterone synthesis & improves sperm morphology

21. V ITAMINS, M INERALS AND H ERBS U SED F OR I NFERTILITY T REATMENT Vitamins B complex combat the effect of oxidative stress. B5 –for hormone production Folic acid – Arole in RNA and DNA synthesis during spermatogenesis and has antioxidative properties. Vitamin C: Helps carry oxygen to sex organs Necessary for balanced hormone / sperm production Selenium is related to significantly a higher concentration of sperm per ejaculate. Herbs: Red clover / rich in every trace element, vitamins and proteins. Red raspberry – high in essential minerals. Damiana–potent aphrodisiac

23. O RAL A NTIOXIDANT T HERAPY Methodological weakness of antioxidants trials make it difficult to determine “who”,“how” and “for how long?? Patient selection and controls Associated pathology Single or combination antioxidants Dosage & formulation Outcome measures Varying duration of treatment Lack of diagnostic markers for oxidative stress Presence of molecular and genetic differences Controversies

24. O RAL A NTIOXIDANTS IN M ALE I NFERTILITY Beneficial Kodama 1997 Dawson, 1992 Kessopoulou, 1995 Vezina, 1996 Vicari, 2001; 2002 Lenzi, 2003; 2004 Cavallini, 2004 Comhaire, 2005 Grecco 2005 Menezo 2007 Tremellen 2007 Piomboni 2008 Gil Villa 2009 No effect Giovenco, 1987 Moilanen, 1993 Iwanier, 1995 Rolf, 1999 Sigman, 2006 Detrimental long-term use and high doses; increased mortality in cancer population- based studies. No Consensus Yet. Short-term use appear to be safe. Caution against indiscriminate use of high dosages for long periods.

25. Who are the candidates for Oral Antioxidant Therapy? Infertile men with OS Diagnosis

26. H OW TO USE A NTIOXIDANT T HERAPY T REATMENT S TRATEGY Once OS is diagnosed, focus on identifying and controlling source of increased ROS Varicocele Genital Infection Smoking Medication Drug abuse Systemic disease Pollution Radiation Differentiate between sperm and leukocyte source of ROS Testing for Leukocytes in Semen Select antioxidant formulation and dosage Ascorbic acid (Vit. C)  - tocopherol (Vit. E) Glutathione N-acetyl-cysteine Carnitine Coenzyme Q10 Lycopene Picnogenol Pentoxifylline Selenium Shao-Fu-Zhu-Yu-Tang Astaxanthin Lepidium meyenii  -linolenic acid and lignans Folic acid Zinc

27. O RAL A NTIOXIDANTS IN M ALE I NFERTILITY OUR FORMULA MAXOLA -L Co Enzyme Q 10-20mcg Vitamin B12 2.5 mcg Folic acid 500 mcg Zinc Sulfate 10mg Selenium 50 mcg Lycopene 2500mcg Fructose 1g L-Carnitine fumarate -1g Acetyl-L-Carnitine 0.5g Citric Acid 50mcg PLAIN MAXOLA does not contain LYCOPENE. How long: minimum 2 months From initiation of sperm production to ejaculation Old concept ~80 days New concept 60 days

28. Oral Antioxidants Cochrane Review 2011 Outcome Live birth Pregnancy rate DNA fragmentation Miscarriage, sperm count, sperm motility Adverse effects N studies 3 15 1 6-16 6 participants 214 964 64 242-700 426 Effect size (OR; 95% CI) 4.85 [1.92, 12.24] 4.18 [2.65, 6.59] -13.80 [-17.50, -10.10] No effect Improve the outcomes of live birth and pregnancy rate for subfertile couples undergoing ART cycles

29. A LTERNATIVE T REATMENT Testicular (not epididymal) sperm extraction in men with poor sperm DNA quality if conservative treatment ( AO and lifestyle modification) have failed. ( Greco2005, O ΄ Connell 2002 ). Reduce centrifugation time prior to IUI /IVF Use of non–centrifuge separation techniques (swim–up etc…) Limit the sperm culture time in media away from seminal plasma

30. A LTERNATIVE T REATMENT Culturing sperm under low oxygen tension ( 5% O2) reduces seminal leukocyte ROS. Avoid using frozen sperm (if possible ) since ROS are produced during freezing /thawing ( Watson 2000) Supplement sperm separation media with a variety of AO ( catalase—Rossi et al 2001,vit.C — Zheng and Zhang, EDTA, glutathione/hypotaurine, albumin, N– acetyl–cysteine.

31. SUMMARY Oxidative stress impairs sperm function and is a risk factor for male infertility and miscarriage. current evidence suggest that OA supplementation for subfertile males improve the chances for pregnancy and live birth for couples undergoing ART. Well-designed studies are needed to determine the best candidate for AO therapy and which formulation and dosages yield better results.

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