Role of antioxidants in female infertility Dr. Jyoti Agarwal Lifecare IVF Center

1. Role of antioxidants in female infertility Dr. JyotiAgarwal Lifecare IVF Center New Delhi

2. 3 Concepts

3. Oxygen toxicity is an inherent challenge to aerobic life • Oxygen is essential for life. • Excess oxygen can have harmful effects. • When oxygen is metabolised in the body , it produces substances called FREE RADICALS which damage our cells.

4. Oxidative process occur in all cells and tissues throughout human body. Oxidation reactions can producefree radicals

5. Free radicals are produced by exposure to • Pollution • Smoking • Fatty food & • Stress

6. Free radicals are like robberswhich are themselves deficient in energy. They attack and snatch energy from other cells to satisfy themselves. Free radical with unpaired electron

7. Free radicals cause damage by oxidation Free radicals are highly reactive and can cause damage to molecules in the cell.

8. ROS PARADOX (delicate balance) ROS are key signal molecules in many physiological process to maintain cell homeostasis Whenever there is an excess they precipitate pathologies ROS act as double edged sword

9. Prooxidants >>> antioxidants = oxidative stress Oxidative Stress

10. Are we ready to fight the attack of oxidative stress ?

11. Counteracting free radical damage The only defence in this war against free radicals is by antioxidants.

12. What are Antioxidants ? Science behind antioxidants

13. Browning reactions are due to oxidation

14. Antioxidants in action The apple is protected when dipped in orange juice containing the antioxidant vitamin C Oxidation occurs when the apple is left exposed to air

15. Antioxidants Antioxidants “ mop-up ” the free radicals Convert ROS to water ‘scavenging molecules’

16. Antioxidants prevents • Lipid peroxidation • Oxidative damage to protein • Oxidative damage to DNA • ANTIOXIDANTS have low redox potential and will sacrifice themselves to prevent oxidative damage

17. Antioxidants are of two types • Primary / Enzymatic / natural • superoxide dismutase • catalase • glutathione peroxidase • glutathionreductase • Secondary / Non enzymatic / synthetic / dietary supplements • vitamin C • vitamin E • selenium • zinc • taurine , hypotaurine • glutathion • beta carotene , carotene

18. Antioxidants and infertility • Etiologies of unexplained infertility and recurrent pregnancy loss remains unclear and present a scientific challenge. • Oxidative stress is a piece in this puzzle.

19. Increasing evidence to show that oxidative stress affects both natural and assisted fertility.

20. Oxidative stress is associated with ROS negatively affects oocyte maturation Decreased oocyte penetration of sperm ↓ fertilization and blastocyst development Decreased implantation Increased loss of implanted embryo • Age related decline in fertility • Decline in ovarian function • Decrease steroidogenesis OS affects multiple physiological processes …..oocyte maturation to fertilization & embryo development

21. Reactive oxygen species and the endometrium • There is a cyclical variation in the expression of superoxide dismutase(SOD) in the endometrium. • In the late secretory phase SOD activity decreases and levels of ROS increases. • It is important for endometrial shedding and the onset of menstruation.

22. Reactive oxygen species and the ovary • Various biomarkers of OS has been demonstrated in normal cycling human ovaries. • A delicate balance exists between ROS and antioxidant enzymes in the ovarian tissue. • These antioxidants enzymes neutralise ROS production and protect the oocycte.

23. Role of ROS in Endometriosis • An increased generation of ROS by pelvic macrophages and increased lipid oxidation in patients with endometriosis. (Halme et al) • Antioxidant levels of superoxide dismutase (SOD) were noted to be significantly lower in peritoneal fluid in patients of endometriosis • Increased ROS levels in the tubal and the peritoneal environment negatively alter fertilization and embryonic development . (fertilsteril 2003 )

24. Role of ROS in PCOD Elevated insulin resistance and hyper homocysteinemia has been proposed to be caused by OS in patients with PCOD.(j clinendocrinometab 2006) . Antioxidant supplementation has been shown to improve insulin sensitivity. The decreased antioxidants status and the increased OS levels may contribute to the increased cardiovascular morbidity in these patients.(fertilsteril 2003)

25. Impact of OS on assisted reproduction There are multiple sources of ROS generation in patient undergoing ART… • Oocytes (4 – 5 /dish) • Cumulus cell mass • Sperms • Prolongation of sperm – occyte co incubation time of more than 20 hrs • ROS levels in day 1 culture media

26. Impact of ROS on assisted reproduction Causes a perturbed redox state and affects the development of the embryo by…. • Altering the gene expression • Impaired adenosine triphosphate production • Two cell arrest of embyro • Embyropathies ROS levels can be utilised to counsel patients before IVF. (agarwal et al fertisteri 2005 )

27. Oxidative stress influences the entire reproductive span of a women’s life and even thereafter , i.e. menopause

28. How do we measure ROS / OS levels ?

29. There are many different ways to test for antioxidants

30. Assessing levels of ROS and OS • Electron spin resonance… it identifies the molecules containing unpaired electrons. • Stable peroxidation product concentrations.. malondialdehyde , lipid peroxidase , and conjugated dienes. No standard reference values No defined Sensitivity & Specificity

31. Strategies to overcome oxidative stress in infertility and ART When OS is diagnosed treatment must focus on identifying and eliminating the source of ROS. • Smoking • Alcohol • Obesity • Stress • Poor diet Only after the treatment of primary etiology should patients be advised to take antioxidant supplementation

32. Antioxidants in Food: Mere Myth or Magic Medicine?

33. Role of Each Ingredient

34. Antioxidants Iron Magnesium Helpful in unexplained infertility since most have micronutrient deficiency with ROS In vitro study shows oocyte activation on exposure to magnesium • Important mineral for general well-being and energy • a subtle deficiency of iron Female subfertility http://conditionsinfo.mitamins.com/sexual-Health-STDs/Female-Infertility-Vitamins.html

35. Zinc essential cofactor for several metabolic reactions Zinc deficiency Impairs Synthesis & Secretion of LH Impairs Synthesis & Secretion of FSH Abnormal Ovarian development • Irregular Ovulation • Impairment of oocyte maturation Female Infertility Experientia.1994 Jul 15;50(7):626-40

36. Zinc .. Supplementation helps to: Regulate ovarian development Improves oocyte maturation & ovulation Exerts beneficial effect in female infertility

37. Vitamin C • most important antioxidants in extra cellular fluids • Vitamin C is a chain breaking antioxidant. • protects tissues against oxidative damage • It also helps recycle oxidized vitamin E and glutathion.

38. Vitamin E…strongest antioxidant present‘a free radical scavenger’ Protects the DNA in the cells from damage

39. Beta-Carotene-provitamin • Carotenoids are a group of red, orange and yellow pigments found in plant foods, particularly fruits and vegetables.

40. N-acetyl cysteine: Powerful antioxidant: NAC ↑ reduced glutathione at cellular level N-acetylcysteine. Altern Med Rev. 2000 Oct;5(5):467-71 Rizk et al. Fertil Steril. 2005 Feb;83(2):367-70 Acts directly on reactive oxidant radicals Antioxidant effect Exerts protective effects to cells Not found in diet, available as a nutritional supplement

41. Additional benefits of NAC Improve insulin sensitivity Preserve vascular integrity by vasodilator effects Antiandrogeniceffect: ↓ testosterone levels & also ↓ free androgen index (FAI) As an antioxidant and mucolytic agent it detoxify harmful substances in body and promote sperm motility & survival in female genital tract Badawy et al. ActaObstetricia et Gynecologica. 2007; 86: 218-222 Detti et al. Diabetes. 2003; 52: 499-505 Millea PJ. Am Fam Physician. 2009 Aug 1; 80(3):265-9 Pieper et al. J CardiovascPharmacol 1998; 32:101–5 Fulghesu et al. FertilSteril 2002; 77:1128 –35

42. L-Arginine • An semi-essential amino acid • Well known as a precursor to nitric oxide (NO) • Lower levels of L Arginine causes delayed conception, decline in oocyte fertilization and implantation rates ActaObstetGynecol Scand 2005; 84: 1029-1034

43. (p < 0.05) (p < 0.01) ActaObstetGynecol Scand 2005; 84: 883-886 Measure of cervical secretion: by weight & volume Nitric oxide→ increases cervical secretion → improve chances of conception

44. Takasaki A et al. 2010; 93(6): 1851-8 L-arginine improved RA-RI in eight (89%) out of nine pts & endometrial thickness (ET) in six (67%) patients which was seen in only 1 patient in control group Improves RA-RI and ET → ↑ uterine blood flow & endometrial receptivity • L-arginine supplementation was reviewed in patients with thin endometrium and high radial artery resistance index • 9 patients were given L-arginine and 10 control patients were not given any medication

45. Sources of L-arginine: • Animal sources: • Dairy products (e.g. milk, yogurt, cottage cheese etc.) • Beef, poultry food, seafood (e.g. lobster, shrimp, snails etc.) • Vegetable sources: • Wheat, oatmeal, peanuts • Nuts (coconut, walnuts, almonds etc.), soybeans

46. Grape Seed Extract

47. Grape Seed Extract In vitro study: (superoxide anion and hydroxyl radical) (Concentration: 100mg/l) In animal study: Protective effects of GS-Proanthocyanidins & other antioxidants in mice Powerful antioxidant: proanthocyanidins Bagchi D. et al. Res Commun Mol Pathol Pharmacol, 1997; 95(2): 179-89 Bagchi D. et al. Gen Pharmacol, 1998; 30(5): 771-6

48. Green tea, Vit B6, B12, • Reduces free radical damage • Improves overall reproductive health Green tea catechins are 100 times more potent than vitamin C Hot cocoa tops red wine and tea in antioxidants 2-3 times stronger than green tea, and 4-5 times stronger than black tea

49. Vitamin B12 & Vitamin B6 Deficiency of Vitamin B12 & Vitamin B6 Increase in Oxidative Stress Female Infertility Failure in Ovulation Defective Implantation Human Reproduction Update 2007; 13(3): 225–238 Asia Pac J Clin Nutr.2005;14(3):250-5 Int J Fertil.1991Jan-Feb;36(1):36-8 J Reprod Med.2001 Mar;46(3):209-12 Schweiz Med Wochenschr.1984 Feb 4;114(5):178-9

50. Vitamin B12 &Vitamin B6 Supplementation helps to : Reduce oxidative stress Improve ovulation Thus improve reproductive health J of Reproductive Medicine April 2004