Moreover, GPx4 activity is deficient in the spermatozoa of infertile men Imai et al. This multipurpose enzyme not only protects spermatozoa from oxidative stress but is also important in facilitating the cross-linking of nuclear protamines during epididymal transit, thereby contributing to sperm chromatin condensation and stability Conrad et al.

Another extremely important enzyme involved in peroxide removal within the male reproductive system is peroxiredoxin PRDX. This class of enzyme traditionally uses thioredoxin to recycle the oxidized enzyme. Thioredoxin itself then becomes oxidized and has to be reduced by thioredoxin reductase using NAD P H as a source of electrons in a similar manner to glutathione reductase.

This enzyme is versatile; in addition to its peroxidase activity, PRDX6 also removes and replaces damaged fatty acids via its inherent PLA2 and lysophospholipid acyltransferase activities Fisher PRDX6 therefore possesses the full range of enzymatic activities needed to effect phospholipid repair in damaged sperm plasma membranes.

To illustrate this point, PRDX6 has been observed to translocate from a cytoplasmic position to the plasma membrane in response to the oxidative damage associated with cryopreservation and asthenozoospermia Xin et al.

The pKa for this protonation reaction is 4. Nevertheless, it has been tentatively suggested that there may be microenvironments within the cell where the pH may be low enough to support HO 2 · formation Storey , Salvador et al. Since there are no enzymes capable of detoxifying OH·, it is an extremely dangerous radical species.

The pernicious nature of this radical explains the lengths that biological systems go to, in order to control the availability of catalytic metals, such as iron and copper, through the complex interaction of metal binding proteins such as ferritin, lactoferrin, ceruloplasmin, and metallothionein Collins et al.

Along similar lines, spermatozoa are active generators of the reactive nitrogen radical species, nitric oxide ·NO via enzymatic nitic oxide synthase and non-enzymatic means Herrero et al.

This oxidant is thought to play an important role in the redox regulation of sperm capacitation Herrero et al.

Indeed, the induction of capacitation and the emergence of sperm senescence have been portrayed as a continuum mediated by ROS such as ONOO- Aitken et al. The reactivity of ONOO- is enhanced in biological systems through its reaction with carbon dioxide to form nitrosoperoxycarbonate, which can then undergo homolytic fission to generate nitrogen dioxide and carbonate radicals, both of which are reactive and can cause damage to biomolecules.

For example, they can react with tyrosine residues or tyrosyl radicals on proteins, resulting in 3-nitrotyrosine adducts which cause a broad range of pathologies including poor sperm motility in infertile males Cassina et al. Thus overall, ROS appear to be important for normal cellular function when generated in small physiological amounts.

One of the most intensely studied aspects of oxidative stress is lipid peroxidation. In the non-enzymatic version of this process, ROS attack the unsaturated fatty acids at position 2 of membrane glycerophospholipids, initiating a lipid peroxidation chain reaction by abstracting hydrogen from an unsaturated fatty acid to create a carbon centred lipid radical L·.

The latter then combines with molecular oxygen the universal electron acceptor to generate an unstable lipid peroxyl radical LOO· which then abstracts a hydrogen atom from another unsaturated fatty acid to stabilize as the lipid hydroperoxide LOOH.

This process invariably creates another lipid radical that will again combine with oxygen thereby propagating the chain reaction Fig. Lipid peroxides can also be created enzymatically by lipoxygenases generating hydroperoxyeicosatetraenoic acids, lipoxins, leukotrienes, or hepoxilins , cyclooxygenases generating prostaglandins , and cytochrome P generating epoxyeicosatrienoic acids, leukotoxins, thromboxane, or prostacyclin.

In general, these products of lipid peroxidation are produced in a tightly controlled manner and serve as important biological signalling molecules that use receptor activation and second messenger generation to influence a wide variety of cellular processes.

The lipid peroxidation process. Lipid peroxidation cascades are initiated by the abstraction of a hydrogen atom from an unsaturated fatty acid generating a lipid radical L· which then reacts with oxygen to form a lipid peroxyl radical LOO·.

In order to stabilize, this radical then abstracts another hydrogen atom from an unsaturated fatty acid in the immediate vicinity, to generate the corresponding lipid hydroperoxide LOOH , thereby propagating the lipid peroxidation chain reaction. In the presence of transition metals such as iron, these lipid hydroperoxides can be induced to breakdown, generating a series of highly electrophilic toxic lipid aldehydes such as acrolein, 4-hydroxynonenal, 4-oxononenal, and malondiadehyde, which by binding to the nucleophilic centres of proteins and DNA can cause significant cellular damage.

However, it is also clear that the generation of lipid peroxides via these mechanisms can become dysregulated and cause oxidative stress. In the case of male fertility, particular attention has focused on the lipoxygenase, arachidonate lipoxygenase ALOX This enzyme is located in the cytoplasmic droplet of mammalian spermatozoa Fischer et al.

Human and rodent ALOX15 enzymes are thought to act on linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid when presented not only as free acids but also when incorporated as esters in phospholipids, glycerides, or cholesteryl esters.

A role for this enzyme in the induction of oxidative stress was first suggested by Brütsch et al. This result suggested that the antioxidant properties of GPx4 and the prooxidant properties of ALOX15 exist in a state of dynamic tension and, as a result, excessive lipoxygenase activity might well be associated with oxidative stress and male subfertility.

In keeping with this suggestion, the pharmacological suppression of ALOX15 activity has been found to reduce ROS generation and inhibit the generation of 4-hydroxynonenal 4HNE; a marker of lipid peroxidation in precursor male germ cells and the GC-2 cell line Bromfield et al.

In addition, inhibition of ALOX15 suppressed ROS generation and 4-HNE generation in human spermatozoa and, in the process, preserved the functional competence of these cells in terms of both acrosomal exocytosis and zona binding Walters et al. The clinical significance of this work is suggested by the observation that the spermatozoa of infertile patients possess significantly elevated levels of ALOX15 protein, in concert with an increase in the appearance of proteins adducted by 4HNE Walters et al.

In light of the above, it is clear that in the male germ line, lipid peroxidation is a key factor in the aetiology of defective sperm function and that both enzymatic and non-enzymatic pathways are probably involved in the mediation of this process.

In the presence of transition metals such as iron and copper, lipid hydroperoxides can be induced to break down, creating a series of highly cytotoxic lipid aldehydes such as acrolein, 4-HNE, and malondialdehyde Aitken et al.

These aldehydes are powerful electrophiles and will bind to the nucleophilic centres of proteins, phospholipids, and nucleic acids in the immediate vicinity, causing significant damage and, particularly in the case of acrolein and 4-HNE, damaging cell function and promoting inflammation Guo et al.

The lipid aldehydes generated as a consequence of lipid peroxidation may bind directly to proteins involved in the regulation of sperm function such as the dynein heavy chain component of the axoneme that drives motility Baker et al.

In addition, the ability of this aldehyde to bind heat shock protein A2 HSPA2 is known to disrupt the ability of spermatozoa to bind to the zona pellucida Bromfield et al. Importantly, several of the proteins adducted by 4-HNE are components of the mitochondrial electron transport chain such as ATP synthase subunit β ATP5B , succinate dehydrogenase [ubiquinone] flavoprotein subunit SDHA , and NADH dehydrogenase [ubiquinone] iron—sulphur protein 2 NDUFS2 Aitken et al.

Mitochondria therefore play a key role in the perpetuation of oxidative stress and the propagation of damage throughout affected cells Aitken et al.

Given the particular importance of oxidative phosphorylation in the metabolism of oocytes and the spermatozoa of ungulate species, damage to the mitochondria will have a major impact on energy generation in these cells, compromising their viability and function Lord et al.

Given the importance of lipid peroxidation as a pathological mechanism, sophisticated mechanisms have evolved to limit the initiation and propagation of peroxidative damage in biological systems. The enzymatic defences against peroxidative damage have been discussed earlier and include the superoxide dismutatases, catalase, peroxidases, and peroxiredoxins.

In addition, there are a range of small molecular mass antioxidant molecules that are capable of suppressing the peroxidative process. This is achieved by interfering with the initiation or the propagation of the lipid peroxidation chain reaction.

Various radical-scavenging antioxidants are known that may be either hydrophilic or lipophilic. Ascorbic acid vitamin C , uric acid, bilirubin, albumin, carotenoids, and thiols such as glutathione are examples of hydrophilic, radical-scavenging antioxidants.

Another major mechanism by which ROS can influence cell function is via the modification of proteins. We have already discussed how aldehydes, e.

These electrophilic aldehydes preferentially bind to cysteine, lysine, and histidine residues in proteins and via this mechanism are known to have a major impact on male and female gametes, influencing a variety of functions from the completion of meiosis to the induction of sperm capacitation Windsor et al.

Protein carbonyl expression in human semen is negatively correlated with sperm motility, fertility rate, and subsequent embryo quality in human intracytoplasmic sperm injection ICSI cycles Al Smadi et al.

On the female side, protein oxidative stress markers are increased in peritoneal fluids from women with deep infiltrating endometriosis, when compared with endometriosis-free controls Santulli et al. In addition, reproductive ageing appears to be accompanied by an increase in maternal uterine carbonylated albumin that, in turn, disrupts extravillous trophoblast function and placentation Mendes et al.

Another form of oxidative protein damage occurs when reactive nitrogen species including ONOO- attack proteins leading to their nitration.

As with oxidative stress, protein nitration is probably a two-edged sword as far as sperm function is concerned. At low doses, nitration may well be helpful, reflecting the impact of powerful oxidants such as ONOO- on sperm capacitation Herrero et al.

This positive association between protein nitration and sperm function is also supported by the observation that seminal 3-NT levels are significantly reduced in cases of male infertility Kalezic et al.

However, if the nitroxidative stress is sustained, it ultimately overwhelms the antioxidative defence capacity of these cells and precipitates a loss of sperm function. It may be for this reason that we see negative correlations between ONOO- and sperm movement and why 3-NT expression is elevated in the spermatozoa of asthenozoospermic patients Vignini et al.

Similarly in the ovary, repeated induction of ovulation leads to significantly decreased ovarian function and oocyte quality and increased oxidative stress reflected by the increased expression of both 4-HNE and 3-NT Nie et al.

Increased protein nitration has also been observed in the granulosa cells recovered from women exhibiting poor oocyte quality in association with endometriosis Goud et al. DNA is notoriously vulnerable to oxidative attack. The guanine residues are particularly vulnerable because they possess the lowest redox potential of the four DNA bases.

The adverse developmental consequences of oxidative DNA damage in the male germ line have been demonstrated using the glutathione peroxidase 5 Gpx5 knockout mouse. Gpx5 is the major antioxidant enzyme in the epididymis; as a result, knocking out this gene leads to the generation of oxidatively stressed male mice with high levels of 8OHdG in their epididymal spermatozoa.

Following from this research, a high incidence of 8OHdG lesions in the spermatozoa of male infertility patients Vorilhon et al.

However, the fact that the DNA damage precipitated by GPx5 inactivation can be ameliorated using antioxidant therapy suggests a possible means of resolving this problem in a clinical context Gharagozloo et al.

The damage to lipids, proteins, and nucleic acids described earlier occurs because the exposure to ROS has overwhelmed the defensive capacity of antioxidant systems that have evolved to maintain redox homeostasis in reproductive cells and tissues.

Oxidative stress may occur when these antioxidant systems are diminished due to such factors as a lack of dietary micronutrients e. vitamin C or selenium , the impaired synthesis of effective antioxidant enzymes, or the excessive consumption of antioxidants in the wake of an oxidative attack.

Stress may also arise because of exposure to excessive levels of ROS. In vitro , excessive ROS production may be induced by high oxygen tensions affecting, for example, the developmental potential of embryos in an in vitro fertilization setting Arias et al.

In vivo , the cellular systems responsible for ROS generation may have become excessively active for a variety of reasons, key examples of which are outlined below. The mechanisms responsible for the elevation of ROS generation in human spermatozoa have recently been reviewed in depth Aitken et al.

One of the key mechanisms by which oxidative stress is created in the human ejaculate is via the infiltration of free radical-generating leukocytes, particularly macrophages and neutrophils, and the consequent development of leukocytospermia Aitken et al. Whether the presence of leukocytes in the ejaculate will create a state of oxidative stress that will suppress sperm function depends on the number and type of leukocytes involved, the site of infiltration epididymis, prostate, seminal vesicles, or urethra , whether the leukocytes are activated, how they were activated, and when they were activated.

Although these questions are often difficult to answer, overall, there seems to be good evidence to suggest a causative chain of events involving leukocytic infiltration, oxidative stress, sperm DNA damage, and impaired sperm function Aitken et al.

Similarly in the ovary, resident and infiltrating leukocytes represent a source of oxidative stress that can seriously impact ovarian function and oocyte quality. ROS are generated by infiltrating leukocytes during luteolysis, and this response is associated with reversible depletion of ascorbic acid, uncoupling of the luteinizing hormone receptor from adenylate cyclase, and the inhibition of steroidogenesis Behrman et al.

ROS are also produced within the follicle at ovulation, with both leukocytes and granulosa cells being major sources of these damaging metabolites. In much the same manner as we observe in the male, ROS serve important physiologic roles in biological processes surrounding female fertility, such as the resumption of oocyte maturation and ovulation, but the sustained production of these damaging metabolites by infiltrating leukocytes may lead to an increased cumulative risk of ovarian pathology Behrman et al.

A major site of ROS generation in all cells, including germ cells, are the mitochondria. In order to generate ATP, the mitochondria have to split hydrogen atoms into electrons and protons.

The former are passaged along a carefully orchestrated series of redox centres culminating in the four electron reduction of oxygen to water by cytochrome oxidase. This process is not fool-proof, and there is a tendency for electrons to leak from the mitochondrial electron transport chain.

In some cell types, such as equine spermatozoa, the intense metabolic activity required to maintain rapid sperm velocity means that there is a constant leakage of electrons from the electron transport chain, with the downstream effect being that in this cell type, mitochondrial ROS generation is, somewhat paradoxically, positively correlated with fertility Gibb et al.

Of course, even equine spermatozoa are not completely immune to oxidative stress and with the passage of time even these cells become overwhelmed, if ROS generation is sustained Aitken et al. Mitochondrial ROS generation can be promoted by a variety of factors that encourage electron leakage from the mitochondria including the above-mentioned lipid aldehydes such as 4-HNE, the male contraceptive agent, gossypol, the vitamin K mimetic, menadione, and the homocysteine cyclic congener, homocysteine thiolactone Aitken et al.

A variety of synthetic, environmental, and natural oestrogens will also trigger mitochondrial ROS generation in spermatozoa. These include compounds such as parabens, catechol oestrogen, bisphenol A, genistein, and pyrogallol, all of which contain the common biochemical feature of a hydroxylated aromatic ring Aitken et al.

Another group of compounds that are involved in the stimulation of mitochondrial ROS generation by mammalian spermatozoa are PUFA. Exposure of these cells to free, unesterified, unsaturated fatty acids elicits a powerful mitochondrial ROS response in human spermatozoa. The magnitude of this response is correlated with the degree of unsaturation, such that the major naturally occurring PUFAs in spermatozoa arachidonic and docosahexaenoic acids are, in their free unesterified form, powerful instigators of mitochondrial ROS generation Aitken et al.

The observation that defective human spermatozoa contain an abnormally high content of free PUFA and that these levels positively correlate with ROS generation by the mitochondria suggests that this association is causative Koppers et al.

There may be significant implications here for the impact of fat-rich, Westernized diets on the biochemical composition and function of human spermatozoa.

Putting energy into mitochondria in the form of radiofrequency electromagnetic radiation De Iuliis et al. These observations are important because they suggest a plausible mechanism by which electromagnetic radiation, in all of its various forms, can influence cellular function.

Finally, many different disruptive pathways converge with induction of the intrinsic apoptotic cascade in both spermatozoa and oocytes, and this process is also associated with the activation of mitochondrial ROS generation, thereby accelerating the rate at which affected cells lose their viability Koppers et al.

This enzyme only requires the presence of substrate to generate both hydrogen peroxide and ammonia:. While this scenario has been validated for bovine, equine, and ovine spermatozoa, it does not play out in the case of human spermatozoa where this oxidase is lost from non-viable cells.

In this cell type, the L-amino acid oxidase has been shown to play a physiological role in the redox regulation of sperm capacitation and acrosomal exocytosis Houston et al.

Similarly in the boar, the L-amino acid gene has been positively associated with spermatozoa capable of generating high letter sizes Kwon et al. A second group of oxidases in mammalian spermatozoa that are thought to be involved in the regulation of sperm function are the NADPH oxidases NOX.

In this case, particular attention has focused on NOX5, an isoform of this enzyme that possesses EF-hand motifs rendering it is sensitive to calcium Bánfi et al. This enzyme has been demonstrated in man Musset et al.

NOX5 is thought to play a key role in the redox regulation of sperm capacitation, where it controls key elements of this process including intracellular pH, cAMP generation, tyrosine phosphorylation, and cholesterol efflux from the plasma membrane Aitken et al. Interestingly, NOX5 expression is also elevated in cases of teratozoospermia Ghani et al.

These morphologically abnormal cells retain a larger volume of residual cytoplasm following spermiogenesis and therefore contain more cytoplasmic glucosephosphate dehydrogenase to generate the NADPH needed to fuel NOX5 activity Gomez et al.

Surprisingly, the NOX5 gene is absent from the genomes of rodent species such as mouse, rat, and hamster despite the fact that sperm capacitation in these species is known to be redox regulated Bize et al. In these species, the redox regulation of sperm capacitation must be controlled by other members of the NOX family such as NOX2 Shukla et al.

In the ovary, it is also clear that enzymatic ROS generation via NOX enzymes plays a critical role in the processes of ovulation and oocyte maturation Chen et al. Granulosa cells, in particular, are known to contain a rich mixture of NOX-like enzymes NOX4, NOX5, DUOX1, and DUOX2 that are thought to regulate the redox status of the ovarian follicle, orchestrating both physiological ROS generation Buck et al.

The existence of NOX activity in the endometrium also signals the potential importance of these enzymes in the regulation of fundamental processes such as decidualization and implantation that have yet to be extensively explored Yu et al.

If oxidative stress is such an important factor in the promotion and dysregulation of reproductive function, then it is imperative that we develop biomarkers by which such stress can be recognized.

The classical markers of oxidative stress focus on assessment of oxidative changes to lipids, proteins, and DNA. Of all these potential assays, the thiobarbituric acid assay for the detection of malondialdehyde or, more accurately, thiobarbituric acid reactive substances, is the most widely used marker of oxidative stress in biological systems.

The assay is simple and sensitive and gives results that parallel the outcome of more sophisticated gas chromatography-mass spectrometric assays Liu et al. Providing its lack of specificity is recognized, it provides a robust measure of oxidative stress for diagnostic purposes and has been widely used in a reproductive context to measure the stress associated with such conditions as varicocele Barradas et al.

For greater specificity, assays of lipid aldehydes can be conducted using antibodies against specific metabolites such as malondialdhyde, 4HNE, and acrolein in flow cytometry protocols Moazamian et al.

Oxidative damage to proteins is generally assessed using the dinitrophenylhydrazine method to detect carbonyl groups Dalle-Donne et al. This method of monitoring oxidative stress has been successfully used to demonstrate the importance of protein carbonyl formation in several reproductive situations including impaired semen quality in elephants Satitmanwiwat et al.

The use of Western blot techniques to reveal the adduction of proteins by lipid aldehydes such as 4HNE has also been used to monitor oxidative stress in a reproductive context.

For example, 4HNE adduction of AKAP4 has been linked with failures of sperm capacitation Nixon et al. Similarly, the binding of 4HNE to proteins in the mitochondrial electron transport chain has been associated with the enhanced generation of mitochondrial ROS Aitken et al.

The development of probes to detect DNA oxidation in the form of 8OHdG has also been used as an oxidative stress biomarker in a reproductive context. For example, the ability of xenobiotics to induce oxidative stress during pregnancy Lan et al.

The measurement of antioxidant levels in biological fluids such as semen, blood, and follicular fluid is another approach to monitoring oxidative stress that has found application in a wide range of reproductive contexts including the potential role of antioxidant deficiency in the aetiology of male infertility Sharma et al.

Redox homeostasis is critical for the entire reproductive process. Both male and female germ lines rely on oxidative processes to achieve a wide range of biological functions from capacitation and chromatin cross linking in the spermatozoa to the maturation and ovulation of oocytes, preimplantation embryo development, implantation, fetal development to term and, if conception does not occur, menstruation and luteolysis Manfredi Romanini et al.

However, if ROS generation becomes dysregulated and exceeds the capacity of the reproductive system to defend itself against these potentially toxic oxygen metabolites then a state of oxidative stress is induced that can compromise the reproductive process at many different points Fig.

To date much attention has focused on the role of oxidative stress in male infertility and placental function during pregnancy Aitken , Cindrova-Davies et al.

However, in reality, an appropriate redox balance is critical for all aspects of the reproductive process from conception to parturition. A disruption of redox homeostasis is one of the major mechanisms by which age, lifestyle and environmental factors impact the reproductive system Shahin et al.

Mechanisms regulating oxidative stress in both male and female reproductive systems. Dysregulation of NADPH oxidase acid and high levels of leucocytic infiltration can cause oxidative stress in both the ovary and the testes. In addition, the mitochondria can make a significant contribution to oxidative stress in the reproductive tract particularly when the free flow of electrons along the electron transport chain in compromised by the ability of electrophilic aldehydes to bind to mitochondrial redox carriers.

These pernicious electrophiles may be generated as a result of lipid peroxidation e. the glutamate semialdehyde generated during proline metabolism. Specifically in spermatozoa, an L-amino acid oxidase can generate sufficient ROS to compromise sperm function particularly in the presence of eye yolk-based cryopreservation media that contain high quantities of the aromatic amino acids, such as phenylalanine, needed to stimulate oxidase activity.

All of these various pathways converge at the generation of excessive ROS, particularly H 2 O 2 , which can have a devastating impact at all levels of the reproductive process from gametogenesis to parturition as well as impacting the health and wellbeing of any subsequent progeny.

Tiboni et al. Beta-carotene has previously been reported to be one of the micronutrients whose concentration is most strongly influenced by smoking Alberg, Bolumar et al.

Analysis of non-smokers in the cohort showed no association between BMI and time-to-pregnancy, however women who did not become pregnant, regardless of smoking status, were not studied. This report indicates that even moderate alcohol consumption can affect OS in certain female populations.

No relationship with alcohol intake was observed among male partners Jensen et al. Self-reported alcohol intake among a cohort of 39 pregnant women showed no marked reduction in waiting time to pregnancy with alcohol consumption; however, women who failed to conceive were not available for analysis since women entered the cohort once they were pregnant Juhl et al.

A positive dose—response relationship between alcohol consumption and ovulatory factor infertility has also been reported in a case—control study in the USA Grodstein et al.

Compared with non-drinkers, the OR for ovulatory infertility was 1. In addition to alcohol and tobacco use, other exogenous agents such as environmental pollutants acting as endocrine disruptors or in an otherwise antagonistic manner may interfere with ovarian development, folliculogenesis and steroidogenesis Uzumcu and Zachow, Although many of these contaminants have pro-oxidant capabilities, oxidation is not the likely mechanism of action causing the endocrine disruption.

Pesticides e. dichlorodiphenyltrichloroethane, DDT; methoxychlor, MXC; vinclozolin; and atrazine , detergents and surfactants e.

octyphenol, nonylphenol and bisphenol-A plastics e. phthalates , and industrial compounds e. Moreover, fetal exposure to certain environmental pollutants may alter DNA methylation Li et al. In vitro animal work suggests that the exposure to mercury and cadmium decreases the quantity of ATP in the ovary and uterus via a reduction in the ATP-hydrolyzing enzyme and may affect mammalian fertility Milosevic et al.

Liu et al. Following hormonal influence at puberty, a number of primary oocytes begin to grow each month. One primary oocyte outgrows the others and resumes meiosis I MI. Interestingly, resumption of MI is induced by an increase in ROS and inhibited by antioxidants Takami et al.

However, it has been suggested that cyclical ROS production may, over time, contribute to oophoritis associated with autoimmune premature ovarian failure Behrman et al. Oocyte maturation occurs with the second meiotic division MII , which arises in response to an increase in pre-ovulatory luteinizing hormone LH Thibault et al.

The process is suspended in metaphase and does not resume unless fertilization occurs following ovulation of the mature oocyte. In both the human and rat, granulosa and luteal cells respond negatively to ROS and adversely affect MII progression, leading to diminished gonadotrophin and anti-steroidogenic actions, DNA damage, and inhibited protein ATP production Behrman et al.

Glutathione GSH , a non-protein sulphydryl tripeptide and key cellular antioxidant, has also been identified as critical for oocyte maturation, particularly in the cytoplasmic maturation required for pre-implantation development and formation of the male sperm pronucleus Yoshida et al.

In bovine models, beta-carotene has been recognized for its ability to enhance cytoplasmic maturation, further supporting reports in other species Ikeda et al. The contrasting relationship of antioxidants, detrimental for the progression of MI, but beneficial for MII, suggests a complex role for antioxidants and ROS in the ovarian environment.

Such findings, along with others discussed below suggesting a threshold for ROS beneficence where embryo formation is compromised by ROS concentration during IVF treatment, requires an appreciation of ROS as multifunctional agents in which their effects may vary over the continuum of concentration and developmental stages.

Attendant to the increase in steroid hormone production of developing follicles is an increase in the activity of cytochrome P, which in turn generates ROS such as hydrogen peroxide H 2 O 2 Ortega-Camarillo et al. Behl and Pandey sought to investigate whether changes in the antioxidant enzyme catalase which converts H 2 O 2 to H 2 O and O 2 and estradiol E 2 activity of ovarian follicular cells in various stages of development fluctuated with follicle-stimulating hormone FSH.

Concurrent catalase and E 2 fluctuation may signal a developmental role of catalase in folliculogenesis. Since the dominant follicle will be the follicle with the highest estrogen concentration, the concomitant increases in catalase and E 2 in response to FSH suggest a role for catalase in follicular selection and prevention of apoptosis Behl and Pandey, Although the exact role of transferrin in folliculogenesis has not been elucidated, Briggs et al.

Reverse transcription—polymerase chain reaction indicated transferrin mRNA in the ovary but not in the oocyte; suggesting that local production of transferrin by the ovary is likely. FF concentrations of transferrin were found to be similar to serum concentrations.

Hypoxia of the granulosa cells is a normal event during the growth of ovarian follicles Tropea et al. Oxygen limitation is known to stimulate follicular angiogenesis, which is important for follicular growth and development.

Impairment of angiogenesis within ovarian follicles contributes to follicular atresia Greenwald and Terranova, ROS may act as signal transducers Schroedl et al. Basini et al.

Cells were held in normoxic, hypoxic and anoxic environments followed by measurement of ROS O 2 and H 2 O 2 and scavenging enzymes superoxide dismutase SOD , catalase, peroxidase.

Catalase activity was unaffected by hypoxic or anoxic conditions. Lack of change in catalase may be due to localization of catalase in the peroxisomes Kinnula et al. Mitochondria are the major consumers of cellular oxygen, thereby providing support to the hypothesis that ROS are involved in intracellular signaling between tissue hypoxia and angiogenic response Basini et al.

OS and apoptosis are the consequences of folliculogenesis, follicular atresia and luteal regression. However, the ROS increase can be countered be it desirable or undesirable by antioxidant status.

Antioxidant properties of E 2 were investigated in pig luteal and follicular tissue exposed to in vitro H 2 O 2. Follicular ROS initiate apoptosis whereas follicular GSH, in addition to FSH, protect against apoptosis in cultured pre-ovulatory rat follicles Tsai-Turton and Luderer, Oocyte GSH synthesis is believed to be stimulated by low-molecular weight thiol compounds including cysteine, cysteamine and β-mercaptoethanol de Matos and Furnus, ; Luberda, However, both cysteamine and β-mercaptoethanol supplementation were found to decrease intracellular peroxidase content, most likely via increased GSH synthesis de Matos et al.

Interestingly, increased serum GSH reductase GSHR was significantly associated with decreased time-to-pregnancy in 83 female participants in a prospective pregnancy study with preconception enrollment recruited from the New York Angler Cohort.

No statistically significant associations were found with GSHPx, SOD, catalase or thiobarbituric acid Jackson et al. Das and Chowdhury investigated the effect of a vitamin E deficient diet on uterine estrogen inducing enzymes and gonadal—pituitary axis and ovarian histological changes among pre-pubertal female rats.

Animals, which were days old at initiation of the experiment, were assigned to one of four diet regimens for a day experiment period: Group 1 received normal control chow diet for the entire period, Group 2 received vitamin E deficient chow for the entire period, Group 3 received normal chow for the first 45 days and deficient chow for the next 25 days and Group 4 received deficient chow for the first 45 days and normal diet for the last 25 days.

Interestingly, the mean body weight of animals in Group 4 did not significantly differ from that of control animals at the end of the day experiment. In contrast, animals fed days deficient Group 2 or days normal chow followed by days vitamin E deficient chow Group 3 weighed significantly less than control animals at the study's conclusion.

Uterine peroxidase and uterine alkaline phosphatase are estrogen-inducible enzymes associated with uterine growth Manning et al.

Activity of uterine peroxidase was significantly lower in the three groups that experienced deficiency compared with the control group, indicating that although having the last 25 days as control diet was effective in restoring body weight, it was not effective in restoring peroxidase concentration to control levels.

These findings suggest that vitamin E deficiency inhibits uterine growth. Among measured hormone levels, plasma LH was significantly lower than the control group in Groups 2 and 4.

FSH was not significantly different from the control in any of the groups, but serum estrogen was significantly lower in Groups 2 and 3, but not Group 4, again indicating that 25 days of a vitamin E adequate diet has restorative properties. Histological analysis indicated that the ovaries of animals on the day vitamin E deficient diet showed degenerated follicles, follicles with increased diameter and hypertrophy of the granulosa cells; whereas the control animals exhibited healthy, large follicles.

In addition to vitamin E, other antioxidants such as manganese a cofactor for SOD are known to influence LH secretion in female rats Pine et al. Duleba et al. T-I cells develop in the secondary follicle stage and control follicle growth and atresia, regulate ovarian steroidogenesis, and may provide mechanical support for ovarian follicles Erickson et al.

However, caution is always warranted in extrapolating the results of in vitro studies to the in vivo milieu. Chao et al. An increase in degenerative oocytes and ovulated immature oocytes was seen with repeated stimulation, indicating a decrease in oocyte quality.

In addition, an increase in mtDNA large scale deletions was noted with increased ovarian stimulation. Related work by Tarin et al. Mice were given a mixture of vitamins C and E, either after weaning early administration or beginning at 32 weeks of age late administration and continuing through sacrifice at 40—42, 50—52 or 57—62 weeks after exogenous stimulation Tarin et al.

To evaluate the overall quality of ovulated oocytes, the number of retrieved oocytes from both antioxidant supplementation groups at all three times points of sacrifice and total percentage of ovaries exhibiting morphological traits indicative of apoptosis were summed and compared with the control.

Animals receiving antioxidant supplements showed an increased number of normal MII oocytes compared with the control group In general, when supplemental vitamins C and E were given to older mice, the age-associated reduction in ovulation was partially prevented, but the preventive effects of supplementation were greatest when supplementation began after weaning and continued to time of sacrifice.

GSH in mature oocytes is thought to be a highly relevant biochemical marker for the viability of mammalian oocytes Zuelke et al. Samples collected during hamster IVM indicate ovulated oocytes suspended in metaphase of MII have approximately twice the concentration of GSH as immature germinal vesicle stage oocytes Zuelke et al.

GSH was found through the pre-implantation stage in bovine oocytes Furnus et al. The corpus luteum CL has a high concentration of antioxidants, particularly beta-carotene, which gives the CL its bright yellow color Rodgers et al.

Other carotenoids and vitamins C and E are also present in relatively high concentrations in the CL where they may play an important role in scavenging ROS Aten et al.

In addition to its antioxidant function, ascorbic acid is a required cofactor in the synthesis of collagen in the luteal extra-cellular matrix Luck and Zhao, ROS are produced during luteal regression Behrman et al.

Over-exposure of the ovary to H 2 O 2 causes the LH receptor to uncouple from adenylate cyclase, thereby impairing protein synthesis and cholesterol utilization by mitochondrial P side-chain cleavage P scc , most likely through impaired production of steroidogenic acute regulatory protein StAR Behrman et al.

StAR is responsible for moving cholesterol to the inner mitochondrial membrane where P scc converts cholesterol to pregnenolone Behrman and Aten, ; Stocco et al.

Lecithin—cholesterol acyltransferase LCAT plays an important role in reverse cholesterol transport and follicular synthesis of estrogen. Cigliano et al. High FF LCAT activity was positively associated with ascorbate and α-tocopherol accumulation, and lower LCAT activity was associated with their consumption, so the mature follicle appears to accumulate these vitamins in the FF to protect LCAT from oxidative damage and promote steroidogenesis.

As noted above, GSH concentrations in mature, metaphase hamster and mouse oocytes are higher than those found in most other tissues, and mature oocytes have a higher concentration than immature or fertilized oocytes Zuelke et al.

High GSH concentrations may also aid in meiotic spindle formation Oliver et al. Zuelke et al. High-performance liquid chromatography analyses were conducted to measure oxidation of GSH to GSH disulfide GSSG , recovery of GSH from GSSG after the removal of diamide, and the overall effect of diamide on MII completion and zygote formation following IVF.

Diamide oxidized GSH in a time- and concentration-dependent manner. Diamide exposure resulted in disruption of spindle morphology, chromosome clumping and altered oocyte cortex microtubules. Recovery from these aberrations was possible if the diamide exposure was followed by either a 1.

Diamide exposure did not affect fertilization or development of the male pronuclei, but oocytes exposed to 50 µm but not 25 µm before IVF exhibited abnormal female pronuclei. Thus, exposure to OS before fertilization appears to disrupt the meiotic spindle and increase risk of abnormal zygote formation.

The activity of ROS generated during gamete fusion is inhibited, due to increased production of antioxidants, particularly SOD. Nitric oxide NO , a free radical produced by NO synthases NOS , functions as an important vasodilator, neurotransmitter, regulator of embryonic development and implantation Guerin et al.

Schmidt et al. NOS type I NOS-I and NADPH-diaphorase NADPH-d were found to be highly concentrated in endometrial epithelial cells. The function of NO in endometrial epithelial cells is not established, but may include regulation of cyclic GMP, which may mediate the estrogen-stimulated rapid uterine secretory response at the implantation site.

Reports on the origin of these enzymes in rat endometrium are inconsistent Shew et al. SOD in human endometrial stromal cells increases with decidualization and is thought to be an important component of implantation Sugino et al.

In addition, both ROS and SOD may act as second messengers to regulate endometrial function Sugino, Furthermore, uterine expression of the gene for α-tocopherol transfer protein, a major determinant of serum α-tocopherol status, increases after implantation, suggesting a protective action of α-tocopherol during embryogenesis Jishage et al.

A prospective study by Oyawoye et al. Of the samples, A total of Baseline TAC was significantly higher in FF samples of oocytes that achieved successful fertilization, suggesting that higher TAC may predict increased fertilization potential. However, significantly lower baseline TAC was observed in the FF where the resultant embryo survived to the day of transfer.

The observation that higher FF TAC is associated with successful fertilization is consistent with the findings of Paszkowski et al. However, the latter finding of lower baseline TAC among embryos surviving to transfer conflicts with those of Paszkowski and Clarke , Paszkowski et al.

Oyawoye et al. suggest that the discrepancy may be due to the effects of ROS being dependent on the stage of embryo development. The percent of TAC loss h post-harvest did not differ significantly between follicles containing oocytes and those that did not, nor did it differ by fertilization status or embryo survival to time of transfer.

These results suggest that antioxidant consumption in FF may have little value in predicting successful fertilization and embryo viability up to the time of transfer.

However, the variation in outcomes may also reflect differential impact on OS by the various causes of infertility and confounding by indication for IVF. ROS and TAC were measured by chemiluminescence in the FF of 53 women undergoing IVF by Attaran et al. Individuals who became pregnant had significantly higher FF ROS levels than those who did not, although TAC did not differ by pregnancy status.

However, lack of a reference value for healthy women with unstimulated cycles precludes the comparison of the study population with a healthy fertile population. Nonetheless, this study suggests FF ROS, at physiologic concentrations, may be indicative of a metabolically active system and a potential marker of IVF success.

Wiener-Megnazi et al. All pregnancies occurred when the FF TCL amplitude at 50 sec was within the range of — cps. These results suggest a beneficial threshold level for OS.

The existence of an acceptable threshold level was also suggested in the evaluation of FF samples from 78 women undergoing controlled ovarian stimulation Das et al. Similar to the findings of Pasqualotto et al. However, the Das et al. The unique finding of the Das et al.

Pasqualotto et al. However, the categorical nature of the correlation precludes the ability to detect differences over the continuum of values. Many thiols are capable of scavenging free radicals.

Plasma Hcy is negatively associated with fruit and vegetable consumption and endurance exercise and positively associated with alcohol intake, caffeine intake and tobacco use Chrysohoou et al.

Elevated Hcy induces endothelial dysfunction and promotes disease of the vasculature, in part by reducing the availability of NO and activation of protease activated receptors PARs to generate ROS Tyagi et al.

Ebisch et al. No difference in the FF concentration of Hcy was detected among etiologic subfertility classifications or among the other substances under investigation.

Free radical activity of human FF increases with age Wiener-Megnazi et al. Takahashi et al. Both aged oocytes and H 2 O 2 treated fresh oocytes exhibited a lower fertilization rate and decreased blastocyst development compared with fresh untreated oocytes.

Follicular fluid aspirates from 12 young women aged 27—32 years and 12 older women aged 39—45 years undergoing IVF treatment were analysed for the activity and protein expression of catalase, SOD, GSHPx, GSH transferase GST and GSHR Carbone et al. No difference in activity between the two groups was observed for GSHPx or GSHR.

No difference in protein expression of catalase or SOD was observed between the two groups, indicating that the age-associated change is due to a post-translational process. A potential weakness of the study was the failure to describe the reason the women were seeking IVF treatment, because the etiology of the infertility may have influenced FF antioxidant and ROS activity.

Whole ovaries were removed and homogenized from reproductive aging rats aged 8—9 months and control animals day old following CL induction and prostaglandin F2α PGF2α administration in the mid-luteal phase to induce luteal regression Yeh et al.

Analysis of homogenized ovaries at baseline from animals given a 0. It is hypothesized that diminished antioxidant status may induce apoptosis during luteal regression and lead to decreased progesterone synthesis. No significant differences in GSHPx, catalase or thiobarbituric acid-reacting substances TBARS , an index of lipid peroxidation, were detected.

Thus, a shift toward a higher concentration of vitamin E may occur to help protect the aging ovary during luteolysis and compensate for the decline in the luteal cell ability to quench ROS, as evidenced by lower GSHR.

Hydrosalpinx is caused by blockage of the Fallopian tube with serous fluid, typically secondary to Fallopian viral infection, which is known to augment OS.

Recent evidence suggests a role for ROS in HSF as a contributor to embryotoxicity. Bedaiwy et al. Several possible mechanisms for the embryotoxic properties of the HSF exist, including presence of microorganisms, endotoxins, cytokines, OS and lack of nutrients Strandell and Lindhard, Alternatively, HSF without ROS may indicate extensive endosalpingeal damage or, as seen in the positive correlation of successful IVF pregnancy with ROS Attaran et al.

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Advanced Search Help. Oxidative stress and reproductive function in Reproduction. Author: Robert John Aitken Robert John Aitken Priority Research Centre for Reproductive Science , Discipline of Biological Sciences, School of Environmental and Life Sciences, College of Engineering Science and Environment, University of Newcastle, Callaghan, NSW, Australia Hunter Medical Research Institute , New Lambton Heights, NSW, Australia Search for other papers by Robert John Aitken in Current site Google Scholar PubMed Close.

Correspondence should be addressed to John Aitken; Email: john. aitken newcastle. Article Type: Editorial Online Publication Date: 18 Nov Copyright: © Society for Reproduction and Fertility Free access. Download PDF. Check for updates. Full Text PDF Article Impact.

Declaration of interest RJA is funded by Memphasys Ltd and CellOxess. Funding No funding was used in the preparation of this article. Author contribution statement RJA was solely responsible for the preparation of this editorial. pub5 PubMed de Ligny W Smits RM Mackenzie-Proctor R Jordan V Fleischer K de Bruin JP Showell MG Antioxidants for male subfertility.

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