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Role of antioxidants in
female infertility
Dr. Jyoti Agarwal
Lifecare IVF Center
New Delhi
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.
Free radical
with unpaired electron
They attack and snatch energy from other
cells to satisfy themselves.
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 stress
Asthma
Infertility
Cancer
Arthrits
Oxidative
Stress
Diabetes
Parkinsons
CVD
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 ?
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 PCOD
Elevated 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 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
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 ferti steri 2005 )
Oxidative stress influences the entire
reproductive span of a women’s life and even
thereafter , i.e. menopause
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 values
No 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
Antioxidants
Iron
• 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
Impairs
Abnormal
Synthesis & Secretion of LH
Ovarian
development
of FSH
Irregular Ovulation
Impairment of oocyte maturation
Experientia.1994 Jul 15;50(7):626-40
Female Infertility
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
Antioxidant effect
N-acetylcysteine.
Altern Med Rev.
2000
Oct;5(5):467-71
Rizk et al. Fertil
Steril. 2005
Feb;83(2):367-70
Exerts protective effects to cells
Not found in diet, available as a nutritional supplement
Additional benefits of NAC
Improve insulin sensitivity
Preserve vascular integrity by vasodilator effects
Antiandrogenic 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
Millea PJ. Am Fam Physician. 2009 Aug 1; 80(3):265-9
Fulghesu et al. Fertil Steril 2002; 77:1128 –35
Detti et al. Diabetes. 2003; 52: 499-505
Pieper et al. J Cardiovasc Pharmacol 1998; 32:101–5
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
Measure of cervical secretion: by weight & volume
(p < 0.01)
(p < 0.05)
Nitric oxide→ increases cervical secretion → improve chances of conception
Takasaki A et al. 2010; 93(6): 1851-8
 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
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
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
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
• 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
Vitamin B12 & Vitamin B6
Deficiency of
Vitamin B12
&
Vitamin B6
Increase in
Oxidative Stress
Failure in Ovulation
Female
Infertility
Defective Implantation
Human Reproduction Update 2007; 13(3): 225–238
Asia Pac J Clin Nutr.2005;14(3):250-5
J Reprod Med.2001 Mar;46(3):209-12
Int J Fertil.1991Jan-Feb;36(1):36-8
Schweiz Med Wochenschr.1984 Feb 4;114(5):178-9
Vitamin B12 &Vitamin B6
Supplementation helps to :
Reduce oxidative stress
Improve ovulation
Thus improve reproductive health
J of Reproductive Medicine April 2004
Folate (Folic Acid)
Deficiency of folate
Homocysteine &
Reduced
folliculogenesis
Production of
inflammatory
cytokine
All leads to
Human Reproduction Update 2007; 13(3): 225–238
X
Folate
Oxidative stress
Altered
Nitric oxide
metabolism
Female Infertility
Apoptosis
Improve
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