A site of Dr. Pankaj Desai

Definition:

These are highly reactive chemical entities that have a single unpaired electron in their outer most orbit.

Under certain conditions can be highly toxic to the cells.

Generally unstable and try to become stable, either by accepting or donating an electron.

Therefore if two FRs react, they neutralize each other.

However, if the FRs react with stable molecules, there is generation of more free radicals.

This characteristic enables the FRs to participate in auto catalytic chain reactions,

Molecules with which they react are themselves converted to free radicals to propagate the chain of damages.

Reactive Oxygen Species (R.O.S.): -

Normal respiration –

Transition metals present inside our body when are in free form behave as free radicals. Fe2+, Cu+

Body cells-

Ageing

Phagocytosis or biogenetics

Oxidation of foods and endogenous compounds.

Transportation of substances for energy production.

Radiation ® Breaks the water inside our body: H2O =H+ + OH-

Metabolism of drugs ® CCl3

Transition Metals ® Cu+, Fe2+

Ultraviolet rays

Emotional stress

Mechanism of Action:

These alterations in the enzymes and receptors inside the cell lead to abnormal cell behavior.

FRs cause fracturing on the cell nucleus resulting in single strand DNA damage.

This oxidative injury may be-

In the form of Medicines

Food Sources:

Oxidative stress has been implicated in the etiology of various pathological conditions.

Successful implantation and development of fertilized egg requires low superoxide environment and optimal NO concentration.

Can be studied by observing effect of O.S. on ART as there is increase in R.O.S. and lipid peroxidation product MDA (malondialdehyde) in peritoneal fluid of infertile patient, there may be defect in oocyte DNA, cumulus cell mass plus as there are multiple sources of R.O.S. generation in ART.

Embryo two cell stage is thought to be most vulnerable stage to R.O.S. during which it is protected by antioxidant in fallopian tube.

Reactive Oxygen Species (R.O.S.) – both endogenous and exogenous cause alterations in lipids, proteins, DNA, mitochondria, decrease in ATP, apoptosis and fragmentation.

In natural implantation, embryo is protected by various oxygen scavengers in fallopian tube and follicular fluid,metal binding proteins like transferrin,vitamin A, ascorbate, pyruvate, taurine, hypotaurine, cystamine are non enzymatic compounds important in embryo protection against O.S..SOD, catalase and GTX are enzymatic compounds.

Several defense mechanism against R.O.S. are present both in embryos and their surroundings.

In vivo embryos are protected against oxidative stress by oxygen scavengers present in follicular and oviductal fluids.

Oxidative stress can be avoided by preventing against R.O.S. formation, interception by antioxidants and repair.

Metal chelation is major means of controlling lipid peroxidation and DNA fragmentation, and metal binding proteins such as transferrin are important in potential radical- generating reactions.

Numerous non enzymatic compounds such as vitamin A, ascorbate and pyruvate have antioxidant function. Other sulphur compounds such as GSH, taurine, hypoturine and cysteamine are equally important in protecting embryo against R.O.S..

Enzymatic defence mechanism protecting oocytes and embryos include SOD, catalase or GPX.

Both embryos and spermatozoa possess repair mechanisms to counteract toxic effects of R.O.S..

Newly fertilized eggs are capable of repairing the damages DNA of spermatozoa.

Both in vitro and in vivo repair of damaged spermatozoa or inject DNA is known to occur in oocytes.

In addition, the pronuclei of the oxidatively damaged embryos can be rescued by transferring into normal cytoplasm.

However, the ability of mitochondrial DNA to auto repair leads to a progressive decline in mitochondrial function.

DNA damage beyond the capacity to repair results in apoptosis and fragmentation of the early embryo or morbidity in later life.

Numerous endogenous and exogenous conditions can induce oxidative stress on the embryos.

Various metabolic pathways and enzymes can produce endogenous R.O.S. including oxidative phosphorylation, B. Nicotinamide adenine dinudeotide phosphate reduced (B – NADPH) oxidase and xanthine oxidase.

Embryo 2-cell block is associated with R.O.S. at this stage.

Angiogenesis is a pathophysiological process involving formation of blood vessels.

It is necessary for follicular development, endometrial growth, embryo development, growth of embryo development growth of placenta vessel and repair.

A complex cytokine influence at the maternal fetal interface creates condition on that are necessary to implant embryo in the endometrium.

Any imbalance between cytokines and angiogenic factors, could result in implantation failure and pregnancy loss.

For a favourable pregnancy outcome, sperm DNA needs to be normal.

Sperm DNA damage can occur as a result of defective sperm chromatin packaging, apoptosis and oxidative stress.

Greater miscarriage rates in ICSI can be due to damage to DNA; while zygotes resulting from IUF have a higher blastocyst development rate over ICSI.

Also, sperm from infertile men with more DNA abnormalties tend to have abnormal blastocyst development, failed implantations and spontaneous miscarriages.

The deleterious consequences of fragmented paternal DNA become evident when embryonic genome is activated.

Sperm chromatin structure assay can detect DNA fragmentation, so that counseling of couples before ICSI & IVF is possible

DNA fragmentation, more than 30% and high DNA stainability are at a greater risk of failure to initiate ongoing pregnancy.

Umbilical cord lipid peroxide concentrations reflect the extent of cell membrane damage by R.O.S.

Persistent intrauterine asphyxia may result in ischemia to the organs, leading to permanent damage, especially to the brain.

Oxygen free radicals activity in the neonate at birth and its relationship to umbilical cord acid-base status has been investigated in singleton deliveries.

Lipoperoxide levels, cord blood pH and base excess were significantly related with singleton deliveries.

In cases of uncomplicated labor followed by spontaneous vaginal delivery, significantly higher lipid peroxide concentrations were seen compared with those delivered following elective.

Caesarean section. Especially noted was an increase in the levels of malondialdehyde (MDA), while the hydroperoxide increase of 27% and base excess increase of 78% were seen.

High levels of free oxygen radical activity in the fetus are a function of the labor process as are the changes in acid-base balance.

Several defence mechanisms against R.O.S. are present both in embryos and their surroundings.

In VIVO embryos are protected against oxidative stress by oxygen scavengers present in follicular and oviduct fluid blocks DNA damage and support cytoplasmic maturation in paracrine oocytes Vit. C deficiency characteristically with follicular atresia and premature resumption of meiosis.

Ascorbic acid can be depleted both by oxidant scavenging as well as by cellular secretion.

In the pre-ovulatory follicles, ascorbic acid is depleted by the presence of luteinizing hormone transferrin suppresses R.O.S. generation and is important for the successful development of follicles.

In the follicular fluid, transferring levels have been found for follicle maturation.

Presence of R.O.S. is detected in amniotic fluid is second and third trimester

Antioxidant capacity is reported to correlate with gestational age and estimate fetal weights.

Cystamine present in follicular fluid is a precursor of hypotaurine, maintains redox status in oocytes maintains GSH content. Increases synchronous pronuclei formation. Improves normal embryo development.

Taurine & hypotaurine present in the follicular fluid is a chelating agent, neutralization of cytotoxic aldehydes improves embryo development, neutralizes hydrotyle radicals.

In IVF patients follicular fluid R.O.S. and lipid peroxidation levels might be potential markers for predicting success in IVF patients.

The production of R.O.S., prostaglandins, proinflammatory cytokines and proteases has been implicated in the pathogenesis of term and preterm labor.

PPROM results initially from R.O.S. damage to collagen in the chorio-amnion, leading to tear/rupture in the membrane as demonstrated by epidemiological and by invitro studies.

OVER 20% of the women of reproductive age in Europe and the USA regularly smoke cigarettes, Tobacco is a major source of exogenous pro-oxidants, R.O.S. and free radical generators are present in both its gas and partyiculate phase.

Active smoking affects the pro-oxidant/antioxidant balance inside the Graffian follicle in women undergoing ovulation induction for IVF.

Cigarette smoking is associated with increased intensity of lipid peroxidation inside the mature ovarian follicle, which is accompanied by the depletion of local antioxidant scavengers. There is a depletion of both enzymatic and non-enzymatic antioxidants in the follicular fluid of smokers.

The decreased antioxidant capacity of follicular fluid in the smokers is, most likely, a secondary phenomenon caused by the utilization of antioxidants in defense reactions neutralizing R.O.S. originating from, or induced by, the tobacco smoke constituents.

A relatively high diploidy has been reported in unfertilized oocytes originating from smoking IVF-embryo transfer patients, suggesting a smoking related meiotic immaturity of the oocytes.

An increased risk of trisomy has been observed in the off-spring of mothers who smoke cigarettes.

A shift of the pro-oxidant/antioxidant balance inside the ovarian follicle towards oxidative stress may provide another possible explanation of impaired folliculogenesis in female smokers undergoing IVF-embryo transfer.

Higher levels of antioxidants are found in pregnancy as there is increase in free radicals because

Pregnancy being a stressful condition

Increased cellular activity

Increased lipid peroxidation

increased antioxidants demonstrated by -

Normal pregnancy has well balanced ratio of free radicals and antioxidants.

Increased spontaneous auto oxidation of lipids leads to increased lipid peroxides.

NO has controversial role.

It is found to be at lower levels in preterm labor patients and also, at term – the level decreased – this establishes NO as uterine relaxant. While some evidence suggest increased NO in amniotic fluid intrapartum.

Reperfusion injury occurring intrapartum also causes oxidative stress. So, there occurs decline in vitamin C level which scavenges R.O.S. in aqueous phase.

Oxidative stress in pregnancy results in –

Abortion (defective genetics and implantation)

Embryopathy

Vesicular mole (DNA damage)

Sub-clinical chorioamnionitis

Bacterial toxins

Inflammatory cells

NO2 release

Oxidative stress occurs when there is imbalance between prooxidants and antioxidants which famous oxidation. Deficient levels of Vit. E and C can lead to oxidative stress.

Both Vit. E and Vit. C are non enzymatic antioxidants.

Vitamin E:

Vitamin C:

It is also a chain breaking antioxidant.

Competitively protects the lipoproteins from peroxyl radicals and recycles vitamin E.

Reduces presence of sulfahydryls.

Supplementation reduces the risk of ovulating aneuploid and diploid oocytes.

Multivitamin supplementation during pregnancy may prevent DNA damage.

Lower dietary intake of Vit. C and E is associated with higher incidence of pre-term labors. Oxidative stress leads to focal collagen damage in the fetal membranes and results in pre-term labor.

Miscarriage (early pregnancy failure) is a pregnancy-related disease, the pathophysiology of which is still not completely understood. Lipid peroxidation and alterations in antioxidant enzyme activites may be of importance in the pathogenesis of this disorder.

Glutathione peroxidase (GSH-Px) and Catalase (CAT) activities is significantly increased while Total superoxide desmutase and non-enzymatic superoxide radical scavenger activities is decreased in patient with pregnancy failure.

Oxidative stress in placental tissues of early pregnancy failure, as the oxidative processes seem to be counteracted by the physiologic activation of antioxidant enzymes such as CAT and GSH-Px. Moreover, a compensatory mechanism might be developed against possible oxidative damage in patients with miscarriage.

Pathophysiology

R.O.S. is involved in the endometrial associated infertility and may play a role in the regulation of the expression of genes encoding immunoregulators, cytokines, and cell adhesions molecules implicated in the pathogenesis of endometriosis. Increased production of Hydroxyl radical and other free radicals in endometriosis is due to the activation of the immune system, and the various antigens in endometrial cells are excessively expressed.

Activated peritoneal macrophages promote disease by secretion of growth factors and cytokines that stimulate proliferation of ectopic endometrium and inhibit antioxidant activity.

Retrograde menstruation is likely to carry highly prooxidants factors, such as heme and iron, into the peritoneal cavity, as well as apoptotic endometrial cells, which are well known inducers of oxidative stress.

Levels of NO were demonstrated in peritoneal fluid of patents with endometriosis.

Peritoneal macrophages express higher levels of NOS, have higher NOS enzyme activity, and produce more no in response to immune-stimulation in vitro.

High levels of NO adversely affect sperm embryos, implantation and oviductal function. Thus reduction in peritoneal fluid NO production or blocking NO effects may improve fertility in women with endometriosis.

Expression of NOS is elevated in patients with endometriosis, and a common polymorphism of exon 7 at nucleotide 894 in the endothelial NOS gene may be associated with endometriosis. Hence variations in the expression of the eNOS gone may be involved in endometrial angiogenesis and thus modulate the process of endometriosis.

Phase-dependent changes of GPX expression are seen in the surface and glandular epithelia in the eutopic endometrium during the menstrual cycle in the fertile control. However, this is lost in the eutopic endometrium in endometriosis.

The aberrant expression of GPX in the eutopic endometrium throughout the cycle suggests a pathological role in endometriosis and adenomyosis.

In endometriosis, expression of SOD is pronounced in the endometrium throughout the menstrual cycle, suggesting that superoxide plays a key role in infertility in endometriosis.

There is an exaggerated expression of Cu, Zn-SOD and Mn-SOD in eutopic endometrium in endometriosis and adenomyosis.

Evidence of oxidative stress in the peritoneal fluid of women with endometriosis and presence of oxidatively modified lipids in the peritoneal fluid has been demonstrated.

Oxidation specific epitopes and macro phages are present in the endometrium and in endometriosis.

Lipid peroxides interact with proteins, resulting in oxidatively modified proteins that are antigenic .

Auto antibodies to oxidatively modified proteins have been detected in subjects of coronary artery disease, pre-eclamptic pregnancy and other vascular disease.

The detection of increased anti-oxidized modified low density lipoproteins auto-antibody titer is thought to represent a biologic marker of enhanced low-density lipoprotein oxidation in vivo.

Macrophages through scavenger receptor play an important role in the uptake and removal of modified proteins, which can be formed as a result of inflammatory processes scavenger receptors also known as oxidized low density lipoprotein receptors do not recognize native low density lipoprotein. Oxidatively modified lipid proteins constitute legends for scavenger receptor.

The presence of macrophages expressing scavenger receptor in the peritoneal cavity of women with endometriosis may indicate oxidative damage to cellular or acellular components of the peritoneum.

Staining with antibodies to oxidatively modified lipid proteins HNE-2 and MDA-2 in both endometrium and endometriosis tissues containing stromal cells, strongly indicate the occurrence of oxidatively stress in endometriotic tissue.

Nitric oxide is a potent vasodilator synthesized from L-arginine by endothelial cells.

In human, it maintains the normal low pressure vasodilated state characteristic of fetoplacental perfusion

NO is increased in women with preeclampsia as a compensatory mechanism for increased Synthesis and release of vasoconstrictors and platelet aggregating agents.

Besides causing direct inactivation of NO as a vasodilator Xanthine oxidase (XO) derived O2- reacts with NO to form a potent peroxynitrite ONOO- which has been detected both within the vasculature and in vessel walls of placenta after preeclampsia.

Increased endothelial NO synthesis, decreased SOD and increased nitro-tyrosine staining in maternal vasculature of women with preeclampsia have also been reported, indicating increased peroxynitrite formation.

Preeclampsia is a multisystem disorder and endothelial dysfunction is one of the main pathogenic features of preeclampsia.

The markers of endothelial dysfunction such as tissue plasminogen activator, von Willebrand factor, sE-selectine and fibronectin are elevated in patients with preeclampsia.

Although the exact mechanism of vascular endothelial damage in preeclampsia are unclear, increased lipid peroxidation may lead to endothelial cell dysfunction.

TNF-α, tissue factor of placental origin, endothelial nitric oxide synthase (eNOS) and excessive activity of the enzyme polymerase may contribute to endothelial dysfunction.

Compared with normotensive pregnant women, women with preeclampsia have reduced expression of endothelial mRNA and protein for eNOS. Reduced expression of constitutive NOS in the vascular system leads to reduced production of NO. NOS. inhibition lead to increased endothelial permeability and an abnormal response of the endothelial cells to the stress challenge of the preeclampsia.

endothelial dysfunction may also result from increased shedding of syncytiotrophoblast membrane micro-vesicular particles into the maternal circulation.

TNF-α, a circulating cytokine has also been implicated as causing endothelial dysfunction in preeclampsia.

Blood cultured from patient with preeclampsia showed higher TNF-α release from the leukocytes in patients with preeclampsia compared with normotensive pregnant women. Significantly higher tissue level of TNF- α where demonstrated in the placenta from with preeclampsia.

Pregnancy is characterized by increased generation of pro-oxidants from the placenta.

Poor antioxidant reserves can also tilt the balance in favour of peroxidation.

Lipid peroxidation results in primary lipid peroxidation products such as lipid hydroperoxides and secondary products such as malondialdehyde and lipid peroxides.

Lipid hydroperoxides are formed and bound to lipoproteins. They are then carried to distant sites where the hydroperoxides can cause ongoing lipid peroxidation and result in systemic oxidative stress.

Increased generation of R.O.S. leads to increased. Lipid peroxides.

Normal pregnancy is associated with physiological hyperlipidomia Pre-eclampsia is characterized by further elevation of serum triglycerides and serum free fatty acid.

Placenta may be site of generation of lipid peroxides in pre-eclamptic patient.

Both enhanced lipid peroxidation and alternation of immune responses are involved in endothelial cell dysfunction in pre-eclampsia.

Enhanced lipid peroxidation may result from excessive production of reactive oxygen species by neutrophil activation in pre-eclampsia.

The increase in antioxidants is probably of a compensatory nature responding to increased peroxide load in pre-eclampsia load in pro-eclampsia and may reflect the severity of diseases.

Increased activity of free radical promote maternal uterine vascular malformations.

FRs are promoters of maternal vasoconstriction.

O2, H2O2, & NO2 in combination. Which inactivate the NO (vasorelaxant)

Increased PG synthatase activity

Produce peroxynitrite, a potent oxidant.

Sources : In vivo anti-oxidants.

In The form of medicines

Vit. A, C & E.

Cystene, Glutathione, Methionine

Bioflavines, Se, Zn

Food sources:

Treatment

Lipid peroxide concentration 1.8 time commoner in placenta of pre-eclamptic patient.

Vitamin E level is decreased in serum of PIH patients.

Concentration of Vitamin E level has been found inversely proportional to the severity of pre-eclampsia.

Concentration of serum uric acid level is directly proportional to the severity of pre-eclampsia.

Alteration in Prostaglandin Metabolism

Oxidative stress has been suggested to contribute to increased risk of fetal malformations in poorly controlled diabetics.

There are reports of increased lipid peroxidation in cell membranes in diabetic pregnancies as LDL from pregnant diabetic mother is more susceptible to oxidation.

Periods of maternal hyperglycemia and hypoglycemia may cause marked changes in availability of glucose to the fetus.

Also increased concentration of lipids, notably the ketone bodies and branched chain amino acids in the maternal circulation contribute to altered nutrition for the embryo leading to increase FR concentration and fetal malformation in embryo.

During later part of pregnancy increased load of glucose in mitochondria may accelerate flow of electrons through respiratory chain including mitochondrial leakage of free radicals.

This leads to increased FR production in embryonic tissues to cause congenital malformations.

FRS being the hall mark of aging, are greatly increased with increased maternal age.

Embryos cultured under hyperglycemic conditions show increased formation of R.O.S. and depletion of GSH contents as well as reduced synthesis of GSH. The addition of free radical scavenging enzymes (e.g. SOD, catalase, GPX and N-acetylcysteine, butylated hydrosytolouene) to culture media reduced the incidence of embryonic malformation after hyperglycemia.

Hyperglycemia-induced embryonic malformations may be due to increased R.O.S. formation and depletion of intracellular GSH in embryonic tissues. GSH depletion and impaired responsiveness of GSH synthesizing enzyme to oxidative stress during organogenesis may have important roles in the development of embryonic malformation in diabetes.

Pregnancy complicated by poor control of diabetes is associated with a high risk of embryopathies, spontaneous abortions and perinatal mortality attributed to excessive oxidative stress. Both lipid peroxidation and scavenging enzyme activities may be valuable, sensitive indices of fetal/neonatal threat in diabetic pregnancy in humans. Adequate measurements at the time of birth would significantly contributed to clarifying the fetal/neonatal status in a medical and legal context, and may be even of value in altering therapy in newborn infants.

Dysmorphogenesis caused by maternal diabetes is correlated with R.O.S. induced inhibition of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in embryos indicating that inhibition of GAPDH plays, a causal role in diabetic embryopathy.

The offspring of women with diabetes mellitus are at increased risk for congenital defects. The incidence and severity of the defects are related to glycemia within the first weeks of pregnancy, during which organogenesis is initiated.

Maternal diabetes also inhibits the expression of PAX-3, an embryonic gene required for neural tube closure caused by increased levels of oxidative stress. Alpha-tocopherol blocks these defects.

Oxidative stress has been suggested to contribute to the increased risk of fetal malformation in poorly controlled diabetes.

Maintenance of blood glucose level to euglycemic level is always important for prevention of diabetic embryopathy.

Antioxidants such as vitamin E and butylated hydroxytoluene, Vit. C, GS4 precursor, N acetyl cystene or transgenic over expression of copper / zinc superoxide desmutase.

In vitro, embryonic dysmorphogenesis has been shown to be significantly diminished, either by restricting the influx of oxidative substrates such as pyruvates to the embryonic mitochondria or by improving the embryonic capacity to scavenge free oxygen radicals.

Elevated levels of copper zinc SOD, catalase, GPX & SOD elicit a protective effect against diabetes associated embryopathy.

O.S. is implicated in cervical cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulval cancer.

The burst of R.O.S. has carcinogenic,teratogenic and genotoxic effects and is correlated with DNA damage.

Oxidative modification of nucleic acids by R.O.S. could result in transformation of normal cells to malignant cells.

R.O.S. induced lipid peroxidation has been implicated in malignant transformation indicate the extent of lipid peroxidation in general and serve as markers of cellular damage due to FRs

Over expression of NOS is seen in chronic inflammation which leads to genotoxicity.

NO may mediate DNA damage through formation of carcinogenic nitrosamines, generation of R.O.S. and inhibition of DNA damage repair mechanism.

It can thus be implicated as tumour initiating agent.

Biochemical estimation of lipid peroxidation products,reduced Glutathione, SOD, catalase, NO can be helpful in evaluation of O.S. in gynec malignancies and their modification after therapy.