Role of antioxidants in

female infertilityDr. Jyoti Agarwal

Lifecare IVF CenterNew Delhi

3 Concepts

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.

Oxidative process occur in all cells and tissues throughout human body.

Oxidation reactions can produce free radicals

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

Free radicals are like robbers which are themselves deficient in energy.

They attack and snatch energy from other cells to satisfy themselves.

Free radical with unpaired electron

Free radicals cause damage by oxidation

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

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

Prooxidants >>> antioxidants = oxidative stressAsthma

Arthrits

CVD

ParkinsonsDiabetes

Cancer

Infertility

Oxidative Stress

Are we ready to fight the attack

of oxidative stress ?

Counteracting free radical damage

The only defence in this war against free radicals is by antioxidants.

What are Antioxidants ?

Science behind

antioxidants

Browning reactions are due to oxidation

Antioxidants in action

Oxidation occurs when the apple is left exposed to air

The apple is protected when dipped in orange juice containing the antioxidant vitamin C

Antioxidants

Antioxidants “ mop-up ” the free radicals

Convert ROS to water

‘scavenging molecules’

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

Antioxidants are of two types• Primary / Enzymatic /

natural

• superoxide dismutase • catalase • glutathione

peroxidase• glutathion reductase

• Secondary / Non enzymatic / synthetic / dietary supplements• vitamin C • vitamin E • selenium • zinc • taurine , hypotaurine• glutathion • beta carotene , carotene

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.

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

Oxidative stress is associated with

• Age related decline in fertility

• Decline in ovarian function

• Decrease steroidogenesis

• ROS negatively affects oocyte maturation

• Decreased oocyte penetration of sperm

• ↓ fertilization and blastocyst development

• Decreased implantation • Increased loss of implanted

embryo

OS affects multiple physiological processes ….. oocyte maturation to fertilization & embryo

development

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.

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.

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 .

(fertil steril 2003 )

Role of ROS in PCODElevated insulin resistance and hyper homocysteinemia has been

proposed to be caused by OS in patients with PCOD.(j clin endocrino metab 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.(fertil steril 2003)

Impact of OS on assisted reproductionThere 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

Impact of ROS on assisted reproductionCauses 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 ferti steri 2005 )

Oxidative stress influences the entire reproductive span of a women’s life and even

thereafter , i.e. menopause

How do we measure ROS / OS levels ?

There are many different ways to test for antioxidants

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 valuesNo defined Sensitivity & Specificity

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

Antioxidants in Food: Mere Myth or Magic Medicine?

Role of Each Ingredient

AntioxidantsIron

• Important mineral for general well-being and energy

• a subtle deficiency of iron

Female subfertility

Magnesium• Helpful in unexplained

infertility since most have micronutrient deficiency with ROS

• In vitro study shows oocyte activation on exposure to magnesium

http://conditionsinfo.mitamins.com/sexual-Health-STDs/Female-Infertility-Vitamins.html

Zinc essential cofactor for several metabolic reactions

Zinc deficiency

Impairs Synthesis & Secretion

of FSH

Abnormal Ovarian

development

Impairs

Synthesis & Secretion of LH

Female Infertility

Irregular OvulationImpairment of oocyte maturation

Experientia.1994 Jul 15;50(7):626-40

Zinc .. Supplementation helps to:

• Regulate ovarian development• Improves oocyte maturation & ovulation

Exerts beneficial effect in female infertility

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.

Vitamin E… strongest antioxidant present‘a free radical scavenger’

Protects the DNA in the cells from damage

Beta-Carotene-provitamin • Carotenoids are a group of red, orange and yellow pigments found

in plant foods, particularly fruits and vegetables.

N-acetyl cysteine : Powerful antioxidant:

NAC

↑ reduced glutathione at cellular level

Acts directly on reactive oxidant radicals

Exerts protective effects to cells

Antioxidant effect

Not found in diet, available as a nutritional supplement

N-acetylcysteine. Altern Med Rev. 2000 Oct;5(5):467-71Rizk et al. Fertil Steril. 2005 Feb;83(2):367-70

Additional benefits of NACImprove insulin sensitivityPreserve vascular integrity by vasodilator effectsAntiandrogenic effect: ↓ 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. Acta Obstetricia et Gynecologica. 2007; 86: 218-222 Detti et al. Diabetes. 2003; 52: 499-505Millea PJ. Am Fam Physician. 2009 Aug 1; 80(3):265-9 Pieper et al. J Cardiovasc Pharmacol 1998; 32:101–5Fulghesu et al. Fertil Steril 2002; 77:1128 –35

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

Acta Obstet Gynecol Scand 2005; 84: 1029-1034

Acta Obstet Gynecol Scand 2005; 84: 883-886

(p < 0.01)(p < 0.05)

Nitric oxide→ increases cervical secretion → improve chances of conception

Measure of cervical secretion: by weight & volume

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

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

Takasaki A et al. 2010; 93(6): 1851-8

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

Grape Seed Extract

Grape Seed Extract

Bagchi D. et al. Res Commun Mol Pathol Pharmacol, 1997; 95(2): 179-89Bagchi D. et al. Gen Pharmacol, 1998; 30(5): 771-6

In vitro study: (superoxide anion and hydroxyl radical)

Antioxidants Inhibition of free radicals

GSPE 78-81 %

Vitamin C 12-19 %

Vitamin E succinate 36-44 %(Concentration: 100mg/l)

In animal study: Protective effects of GS-Proanthocyanidins & other antioxidants in mice

Antioxidants ↓ production of ROS

GSPE (20-100mg/kg) 70 %

Vitamin C (100mg/kg) 18 %

Vitamin E succinate (100mg/kg) 47 %

Beta-carotene (50mg/kg) 16 %

Powerful antioxidant: proanthocyanidins

• 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

Deficiency of Vitamin B12

&Vitamin B6

Increase in Oxidative Stress

Vitamin B12 & Vitamin B6

Failure in Ovulation

Defective Implantation

FemaleInfertility

Asia Pac J Clin Nutr.2005;14(3):250-5 Int J Fertil.1991Jan-Feb;36(1):36-8J Reprod Med.2001 Mar;46(3):209-12 Schweiz Med Wochenschr.1984 Feb 4;114(5):178-9

Human Reproduction Update 2007; 13(3): 225–238

Vitamin B12 &Vitamin B6Supplementation helps to :

Reduce oxidative stressImprove ovulation

Thus improve reproductive health

J of Reproductive Medicine April 2004

Folate (Folic Acid)Deficiency of folate

Homocysteine & Oxidative stress

Reducedfolliculogenesis

Production of inflammatory

cytokine

Altered Nitric oxidemetabolism

Apoptosis

Female InfertilityAll leads toHuman Reproduction Update 2007; 13(3): 225–238

Folate

Improve

X

Selenium .. Selenium works synergistically with vitamin A

• Sources• Nuts• Seafood• Pasta

Antioxidants in combination

Antioxidants are believed to work better in combination than supplementing alone.

• Together they protect oxidation of each other• Together they preclude the need for large doses of

single nutrient

Clin Exp Obstet Gynecol. 2006;33(4):205-8

There is lack of consenus on the type and dosage of antioxidants to be used.Validation by large randomised double blind case control trials is needed as evidence

Take Home Message…..• Oxidative stress has a significant impact on female

infertility.

• The role of oxidative stress in female reproduction cannot be underestimated.

• The best way to add antioxidants to the diet is to eat generous servings of fruits and vegetables daily.

J of Reproductive Medicine April 2004

Antioxidants optimize the environment and offers complementary role to correct and support female factor fertility and conception

“The real voyage of discovery consists of not in seeking new landscapes but in having new eyes.”

- Marcel Proust