1. بسم الله الرحمن الرحيم

2. Antioxidants, Vitamins, Trace Elements and Food Supplements in Male Infertility Dr. Akram Shalabi Consultant OBGYN Director IVF Unit/ Istiklal Hospital President /Fertility Group JSOG Secretary General /AFFS

3. Baby– Making In the old days baby–making was a pretty easy and natural activity. Husband and wife bedded down and few weeks later a baby was on the way. Now fertility and pregnancy are multi–billion dollar industry with high–tech language,expensive lab procedures and a battalion of white–coated lab technicians. Lisa Turner Supplements for childbirth and beyond

4. Some Facts Sperm production cycle occurs in a mean 64 ± 8 days (range: 42–76 days)(by stable isotope and gas spectrometric analysis) Infertility affects 15–20% of couples Male factor accounts for 40–50% (5% of general men population) In another 20% both male and female are involved Thus in 60% of infertile couples, a male related problem is a cause of infertility

5. Radical Oxygen Species (ROS) ROS : Oˉ,OHˉ, peroxyl radical, H2O2 etc… ROS –during oxidative cell respiration ROS :Intrinsic: by sperm have a stronger –ve relationship with sperm DNA integrity. ( Henkel et al.2005) Extrinsic by seminal leukocytes Abnormal–midpiece/immature sperm produce more ROS (Gomez,Said 2005) ROS cause infertility by : 1– Damaging sperm membrane leading to decreased motility and ability to fuse with the oocyte. 2– Altering sperm DNA, resulting in the passage of defective paternal DNA to the conceptus ending in miscarriage.

6. Oxidative Stress ( OS) (OS) –when ROS production exceeds natural AO defense –in 50% of infertile men Sperm are vulnerable to oxidative damage during epididymal transit, especially with epididymitis. The 3 inter–related mechanisms accounting for OS–mediated male infertility are: 1– Impaired motility 2– Impaired fertilization 3– Oxidative DNA damage.

7. Origins of Oxidative Stress 1–Infection (prostatitis,epididymitis,systemic ) 2–Hb–pathies 3– Diabetes 4–Alcohol ( Maneesh e al 2006) 5– Teratospermia, testicular torsion, vasectomy, variecocele (increases oxidative DNA damage in germ cells/spermatocytes– Ishikawa et al. 2007) 6 ART( Potts et al 2000) 7 Cyclophosphamide, Aspirin, Paracetamol ( Agarwal and Said 2005 ) 8Smoking ( 48% in seminal leukocytes and 107% in seminal ROS (Fraga 1996, Saleh 2002) 9–Dietary deficiencies ( Vit C,E folate, Zinc ) Metthews 2002 10–Sperm cryopreservation ( Watson 2000)

8. Origin of OS cont… 11–Obesity :fats release heat /cytokines, that leukocyte ROS (Singer /Graner 2007, Banks et al 2005) 12–Stress : seminal plasma ROS /reduction of natural AO ( Eskiocak et al 2006) 13–Advancing age: DNA damage by systemic OS.( Zubkova et al 2005) 14– Plastic softeners exposure through diet, dermal contact or inhalation DNA damage 15–Pesticides, herbicides, sulfur dioxide ( preservative ), diesel ( leukocyte ROS). 16–Heavy metals –Cadmium, lead------- DNA oxidation 17– Idiopathic.

9. Sings of OS in Semen Analysis Reduction of sperm parameters ( asthenospermia 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.

10. Management of OS –Related Infertility 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.

11. Antioxidants The best–studied fertility supplements, notably vitamins E and C, acetyl–cysteine,L–carnitine and glutathione. The most protective antioxidative mechanism uses thiol– or glutathione–dependent enzymes. Currently, AOs are used empirically in cases of asthenospermia and to reduce levels of sperm DNA fragmentation.

12. Antioxidants in the Management of OS AOs improving sperm motility : 1. Carnitine (Lenzi et al.2004,Balercia et al 2005 ) 2. Selenium (Scott et al 1998), 3. Vitamin E (Suleiman et al.,1996), 4.Viatmin E and selenium (Keskes–Ammaret al.,2003), 5. Glutathione (Lenzi et al.,1993) 6. Astaxanthin (Comhaire et al.,2005 ) 7. Vitamin C 8. B group vitamins( homocysteine clearance) (Matthews 2002) Antioxidants dont boost sperm count or morphology (Agarwal 2004 )

13. Natural Antioxidants Enzymati c: Super–oxide dismulase/catalase, glutathione peroxidase (GPX 1–5) GPX is present in male genital organs and sperm (Vernit et al. 2004) Male infertility is linked with a reduction in seminal plasma /sperm GPX activity. Non–enzymatic: Vit.C, E, Glutathione,aminoacids (taurine,hypotaurine),albumin, carnitine, carotinoids,flavenoids etc.. All are reduced in infertile men ( Song et al.2006, Koca et al.2003 ) Herbs : Bilberry, curcumin, grape seed, flaxseed, pine bark extracts gingko biloba and others.

14. AOs and Evidence Based Medicine Two prospective RCTs comparing vit. C and E with placebo have found no ability to improve sperm motility or PR when given for 2 months (Rolf et al.1999, Greco et al.2005) Vit. E increased PR in 6 months ( 21% vs 0% in placebo group)Suleiman 1999 A recent RCT comparing menevit ( AO formulation ) with placebo reported a significant increase in PR if taken for 3 months prior to IVF/ICSI trial.

15. cont… Menevit improves sperm quality by 3 possible mechanisms: 1–Sperm protection from ROS already produced vit C,E,selenium and lycopene effect. 2 –Reduction of semen leukocyte ROS production garlic –anti– inflammatory effect (Hodge et al 2002, Chang et al. 2005) 3–Augmenting protamine packaging of sperm DNA to protect sperm from ROS attack Zinc, selenium and folate effect (Pfiefer et al. 2001 ) It appears logical to use several AOs with different modes of action together with an agent to reduce leukocyte ROS production (Vicari et al.2002, Gambera et al 2007, Tremellen et al 2007

16. Cont… Prospective trials measuring OS end points (sperm peroxidation and DNA damage ) AOs: Directly reducing seminal ROS levels Astaxanthin( Comhaire et al. 2005) L–carnitine ( Vicari and Calogero2001) Acetyl–cysteine, β– carotene, vit.E and essential fatty acids (EFA) ( Comhaire 2000) Vit C 600mg /day( Kessopolou 1995) Vit E 400mg + 225µg Selenium OR 300mg Vit E for 3 months reduce sperm malondialdehyde levels( Keskes–Ammar 2003 )

17. Cont… AOs Reducing sperm DNA damage 1g vit C and vit.E for 2 months ( Greco et al 2005) 400mg Vit C and Vit E, β– carotene ( 18mg), Zinc and Selenium(Meneso et al 2007) Acetyl – cysteine 180mg, Vit.E 30mg, β –carotene and EFA (Comhaire et al. 2000)

18. L–Carnitine Highly polar, water soluble,vitamin –like amine. Fundamental role in intracellular energy metabolism Highly concentrated in epididymis and important for sperm metabolism and maturation. 75% of body stores ----- diet 25% synthesized ------- lysine / methionine.

19. L–Carnitine cont… Epididymal /serum concentration 2000:1 (10–50 µ mol/L) Caput epididymal fluid and sperm contain very low or undetectable concentrations of L–carnitine, increasing dramatically during transit towards the cauda. Infertile men have reduced seminal L– carnitine levels.

20. Role of Carnitine in Eukaryotic Cells 1– Transport of major FA into mitochondria for β oxidation 2– Buffering medium in maintaining sperm acetyl–CoA: CoA ratio. 3–Improves sperm motility in vitro ( Tanphaichitr N) Seminal plasma L –carnitine content has been correlated with sperm count and motility ( Menchini– Fabris GF, Bornman MS)

21. L–Carnitine in Research RCTs Lenzi et al.2003 n=86 2g L–carnitine/Day for 2 months Results:Motility by 11% vs 8.8% in placebo group ( 5 outliers excluded )P=.02 Lenzi et al.2004 n=56 2g l–carnitine +1g acetyl–L–carnitine for 6 months Results : No change in motility or sperm count P>.05 Sigman et al. Fertil Steril 2006 vol 85 :1409–1414

22. L–Carnitine In nonrandomized noncontrolled studies Costa et al. 1994 n=100 3g L–carnitine /d for 90days. Motility to 37.7% from27%, progressive mot. to 18% from 11%(P<.001). Total sperm count to 163 mil from 142(<.001) Vitali et al 1995 n=47 3g L–carnitine /d for 120days Motility to 53.5% from 27%, 7 pts worsened. Oral carnitine improves sperm motility Campaniello et al 1989, Moncada et al. 1992, Arnaud A. et al 1994, Vitali G et al. 1995 Sigman et al. Fertil Steril 2006 vol 85 :1409–1414

23. Vitamins, Minerals and Herbs Used For Infertility Treatment B vitamins combat the effect of stress. B5 –for hormone production B6 – synthesis of RNA and DNA Vitamin C: Helps carry oxygen to sex organs Necessary for balanced hormone / sperm production Coenzyme Q10 a fat–soluble, vitamin–like compound serving as important intracellular AO,DNA protector and immuno–modulator Selenium is related to significantly a higher concentration of sperm per ejaculate. Zinc essential for normal function of reproductive organs and sperm morphology Herbs: Red clover / rich in every trace element,vitamins and proteins. Red raspberry – high in essential minerals. Damiana–potent aphrodisiac

24. Hawthorn berry – stimulates pelvic blood flow Astragalus increases sperm motility ? Curcumin : Strong AO Anti–inflammatory Anti–platelet Tribulus : Tribestolic -------used to treat hormone insufficiency and impotence -------to increase libido by preventing testosterone from changing to estrogen. Gensing – elevates sperm count and motility –Increases pelvic blood flow Saw palmetto helps nourish prostate and regulates spermatogenesis Flaxseed : Very rich in n–3 FAs (for depression related impotence)

25. Lycopene : AO, found in high concentration in the testes and seminal plasma Decreased levels have been demonstrated in infertile men. 200 µg of Lycopene, twice a day for 3 months. Results : 66 % (n=20) showed an improvement in sperm concentration, 53% (n=16) had improved motility and 46% (n=14) showed improved sperm morphology. All improvements were statistically significant (P< 0.05) Base line sperm concentration < 5 mill/ml was associated with no significant improvement. Gupta NP, Kumar R. Int Urol Nephrol. 2002; 34(3): 369 –72 Lycopene in Idiopathic Male Infertility

26. Folic Acid A role in RNA and DNA synthesis during spermatogenesis and has antioxidative properties. Older studies have shown no benefit on the semen quality of infertile men. Newer studies suggest that there may be a benefit, especially for tobacco users. Most recently, when combined with zinc supplementation (5mg/day) it increases sperm count.

27. Zinc Important role in testicular development, spermatogenesis and sperm motility Important for testosterone conversion into its active form. Predominantly secreted by the prostate and found in maturing sperm 80% of subjects experienced oligospermia after Zn restriction for 24–40 weeks, which subsequently resolved after Zn supplementation. Large RCTs of Zn supplementation in infertile men are lacking.

28. Combined Supplements Proceptin : non–herbal nutritional supplement used to support sperm function Contains : Vit A,C,E,B6,B12,folic acid,L–carnitine, Acetyl L– carnitine, Ferrulic acid, Zinc, Seleniun, Co Q10,pycnogenol Side effects : Metabolic changes as a result of a cumulative burden on the liver. Advice Herbal blends should be taken for 6 months Nonherbal 4 months

29. AOs and Essential FAs 27 patients given AO ( N–acetylcysteine plus EFAs) vs ( Vit A,E plus EFAs) Evaluation included : sperm characteristics, seminal ROS,FA of sperm membrane, sperm oxidized DNA and induced acrosome reaction (AR). Result s : No improvement in sperm motility or morphology No decrease in semen WBCs Sperm count in oligospermic men ROS significantly reduced AR PUFA and fluidity of sperm membrane Overall PR 4.5% in 5months treatment PR tended to be higher in partners of (ex)–smokers 7.2 % vs 1.6% Comhaire et al. 2000

30. AOs Interaction L– Carnitine enhances AO properties of vitamin C /E. Interactions may occur between fish oil supplements and aspirin, NSAID and some herbs ( ginger )in form of bleeding tendency. Fish oil should be preferably stopped before surgery Flaxseed ALA component may be associated with macular degeneration and prostate cancer Curcumin is extremely safe. However, persons with symptoms of gallstones should avoid turmeric Zn inhibits Cu absorption, and competes for absorption with Fe,Ca, and Mg. In smokers, synthetic beta–carotene appears to increase lung cancer risk.

31. AOs Interaction cont… Excessive methionine intake with inadequate intake of folic acid, B6 and B12 can increase the conversion of methionine to homocysteine. B6 the amount of Mg entering cells, which augments FAs formation and B vitamins activation Zn maintains proper vit. E level. Beta–carotene should be supplemented with vitamin E if taken for a long period of time. B2 and B3 are important enzyme cofactors for the replenishment of antioxidants.

32. Cost–Effectiveness of Medical Treatment of Male Infertility There is virtually no literature that specifically addresses this issue. The dearth of literature in this area is due to: 1–The empirical nature of many medical treatments for infertility 2–The widespread belief that male infertility is not truly a disease that needs specific treatment 3–The increasing effectiveness of ART to help couples overcome infertility

33. Alternative Treatment 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 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.

34. Conclusions Evidence now supports a role for OS as a significant cause of male infertility, commonly ignored by infertility practitioners. It looks prudent to consider using AO in all infertile men exhibiting OS. Patient acceptance of AO supplementation in combination with ART is increasing as they may boost PR. Offer AOs in combination with changes in life style. A single cheap assay to identify sperm OS is needed in every andrology lab. A new sperm culture media providing better sperm protection from the ravages of ROS is required. Further research is required to determine what combination and dose of AOs provide sperm with maximal protection against OS Keep in mind the interactions of various AOs

35. Conclusions Evidence now supports a role for OS as a significant cause of male infertility, commonly ignored by many infertility practitioners. Patient acceptance of AO supplementation in combination with traditional ART is increasing. Offer AOs with different modes of action with an agent to reduce leukocyte ROS production in addition to changes in lifestyle to all infertile men exhibiting OS, particularly before ART procedures. Further research is required to determine combinations and doses of AOs providing maximal sperm protection against OS. Development of new sperm culture media protecting sperm from the ravages of ROS damage is clearly required. Thank You For Attention