Send Orders of Reprints at [email protected] Current Women’s Health Reviews, 2012, 8, 183-207 183 Female Infertility and Assisted Reproduction: Impact of Oxidative Stress-- An Update

Beena J. Premkumar and Ashok Agarwal*

Center for Reproductive Medicine, Cleveland Clinic, Cleveland, USA

Abstract: Augmented levels of reactive oxygen species (ROS) that overpower the body’s antioxidant defenses result in oxidative stress (OS). Physiologically balanced levels of ROS and antioxidants maintain homeostasis in the body and allow for normal physiological processes to proceed. Physiological processes that involve oxygen consumption inevitably produce ROS. However, an overabundance of ROS leads to widespread injury to cells, and can damage DNA, lipid membranes, and proteins. An unfavorable reproductive environment hinders normal physiology secondary to this disruption of homeostasis. Infertility may be attributed to reproductive pathologies, leading to OS. Infertile couples often turn to assisted reproductive techniques (ART) to improve their chances for a successful pregnancy. In vitro techniques create an unfavorable environment for gametes and embryos by exposing them to a surplus of ROS in the absence of enzymatic antioxidant protection that normally exists in vivo. This article will review the currently available literature on the effects of ROS and OS on ART outcomes. The role of antioxidant supplementation of ART culture media continues to be a subject of interest to increase the likelihood for ART success. Keywords: Antioxidants, assisted reproduction, female infertility, oxidative stress.

BACKGROUND INFORMATION the role of OS in female infertility and its impact on gametes and embryos in the ART setting. Close to 10% of reproductive aged couples fail to conceive through natural means. Associated pathologies and Reactive Oxygen Species and Oxidative Stress causative factors of infertility may be identified in either partner, however, the cause of subfertility remains unknown Oxidative stress occurs in the midst of an unbalanced in about 30% of these cases [1]. Female factors include ratio of pro-oxidants to antioxidants in favor of the former endometriosis, polycystic ovary syndrome (PCOS) [2], tubal [10]. This ratio can be disrupted by an abundance of reactive factors, premature ovarian failure, oocyte aging, and oxygen species (ROS) and/or by weakened antioxidant unexplained infertility (Fig. 1). In males, conditions affecting defenses [11, 12]. Normal physiological functions including the sperm, including azoospermia, asthenospermia, damaged processes of cell signaling, proliferation, and differentiation, sperm DNA, and varicocele, may contribute to infertility, as depend on the presence of a certain amount of ROS well as unexplained and idiopathic causes [3]. Women with [13, 14]. In the reproductive environment, physiological high body mass index (BMI) [4] and those who smoke levels of ROS are necessary not only for ovulation [15, 16], cigarettes [5] have been documented to be at an increased but also for sperm-oocyte interactions, fertilization [17], risk for infertility. Irrespective of the etiology of infertility, implantation, and early embryo development. However, the infertile couples often turn to assisted reproductive overproduction of ROS can overwhelm the body’s natural techniques (ART) to improve their chances of conception. antioxidant defenses, disturbing the female reproductive However, even those who seek the help of ART to conceive environment and disrupting normal female physiological have still been shown to have lower pregnancy success [6-8] reactions [10]. Growing literature suggests that OS may rates than normal weight and non-smoking women. be paramount to determining successful outcomes with Over the years, oxidative stress (OS) has been ART. The methods used in ART are known to over-expose increasingly thought to exert adverse effects on the sperm, oocytes, and embryos to unnecessary levels of ROS, reproductive abilities of both men and women [3]. The which likely contributes to the reportedly low fertility contributions of OS to the pathogenesis of infertility have outcomes. been more extensively studied in the realm of male infertility Reactive oxygen species have been implicated as an [9], specifically in terms of its ability to affect various important contributor to embryonic arrest and apoptosis in in aspects of sperm quality. This review however, will focus on vitro culture, as they can inflict serious damage to proteins,

lipids, DNA, enzyme functions, and mitochondria [9]. Both radical and non-radical derivatives of O2 are known as *Address correspondence to this author at the Center for Reproductive Medicine, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, ROS. Unpaired electron(s) in the atom’s outer shell account Ohio 44195, USA; Tel: (216) 444-9485; Fax: (216) 445-6049; for the high reactivity of free radicals [18]. During oxidative E-mail: [email protected] phosphorylation and ATP production, electron leakage from

1875-6581/12 $58.00+.00 © 2012 Bentham Science Publishers 184 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

Hydrosalpinx

Endometriosis

Polycystic Ovary Syndrome

Oxidative Stress Ovarian aging

High BMI

Cigarette smoking

Cell Membrane Fig. (1). Factors influencing OS in female infertility. the mitochondrial electron transport chain (ETC) forms the associated with the production of superoxide from NADPH majority of ROS [19, 20]. Redox reactions occurring in [27], which likely leads to the induction of LH. In addition biological systems mainly involve the superoxide anion, to regulating steroid production within cells, ROS may also hydrogen peroxide (H2O2), and nitric oxide (NO) [21]. aid in stimulating corpus luteum release of progesterone. Reactions involving oxygenase and electron transfers use O2 Controlled amounts of oxidation are also important for the as a substrate to produce ROS, of which the superoxide formation of disulfide bonds during ovulation in females and anion is the most common [22]. In the ovary and other in sperm nuclei in males. Carbonyl production is stimulated steroidogenic tissues, ROS are also generated by cytochrome by ROS; levels of carbonyl compounds increase during cell P450 enzymes [23]. proliferation, steroid hormone generation, and ovulation [20]. Thus, the utilization of ROS by the reproductive system The Haber-Weiss reaction, catalyzed by transition is essential for certain vital reproductive processes. metals, produces the hydroxyl radical, which is considered the most toxic inflictor of oxidative damage due to its ability Throughout various developmental stages, oocytes and to modify purines and pyrimidines, thereby causing DNA embryos are exposed to fluctuating levels of the superoxide damage [24]. The highly reactive hydroxyl radical and the anion and H2O2; however, in high amounts, these ROS can reactive nitrogen species (RNS), peroxynitrite, are formed cause apoptosis of oocytes and embryos by damaging from exogenous non-detoxified H O [25]. Technically, mitochondria [28], DNA, lipids, and proteins [29, 30]. The 2 2 H2O2 is not a free radical, but is often considered in the process of ovulation is stimulated by NO, an RNS produced same category because of its involvement in free radical from nitric oxide synthase (NOS) III. Similarly, in vitro production and breakdown [24]. In the presence of metal embryo development heavily relies on a critical concentration ions such as iron, accumulated H2O2 stimulates the production of NO, in the culture medium. When NO is present in of H2O and O2 through the Fenton and Haber-Weiss reactions excess, however, embryos have been shown to exhibit [26]. developmental arrest as well as apoptosis [31]. Reactions between ROS or RNS with lipids, proteins, and nucleic acids A conservative elevation of ROS can stimulate the can damage cells [29, 30]. Intracellular lipid peroxidation growth and proliferation of cells. In fact, a baseline level of secondary to ROS results from increased production of fatty ROS is necessary for cell signaling processes. In endocrine acid peroxides [32], and weakens cell membranes [33]. organs including the ovary and testes, ROS produced by the When levels of ROS are high enough to disturb processes P450-mediated monooxygenase reaction plays an important mediated by cellular redox status, shifting of the oxidant/ role in the synthesis of steroid hormones from cholesterol. antioxidant balance occurs, and OS results [34, 35]. Fig. (2) During regression of the corpus luteum, levels of ROS summarizes ROS/RNS and the main antioxidants and increase. In murine ovaries, the pre-ovulatory phase in antioxidant enzymes. cycling females and the luteal phase in pregnant mice are Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 185

2 H2O

2 H20 + O2

Xanthine oxidase Catalase NADPH oxidase Fe2+ Fe3+ Mitochondria Superoxide dismutase O2 O2∙ˉ H2O2 ∙OH

R‐SH Myeloperoxidase RNOS Clˉ ∙NO GPx ONOOˉ Nitric oxide synthase HOCl Tyrosine Nitrotyrosine 2 GSH GSSG L‐arginine

Peroxiredoxin GR

2 Trx‐SH Thioredoxin disulfide

Thioredoxin reductase

Fig. (2). ROS/RNS and the major antioxidant enzymes and antioxidants.

Inflammation Antioxidants Inflammatory processes have generally been associated A delicate balance between levels of ROS and with increased generation of ROS. However, recently, some antioxidant defenses exists to regulate physiological research groups have proposed a protective mechanism for functions of cells [42]. Several antioxidants in enzymatic ROS in inflammatory situations. In autoimmune diseases, and non-enzymatic forms exist within cells to protect the production of ROS such as superoxide anion induces them against oxidative damage by neutralizing excess ROS formation of H2O2 and hydroxyl radical, all by the NOX2 [12, 43]. Endogenous antioxidant enzymes include catalase, complex, which has been suggested to actually halt, rather superoxide dismutase (SOD), and glutathione peroxidase than induce autoimmune responses [36, 37]. For example, (GPx) [22]. mice lacking NOX2 have been shown to have hyper- inflammatory responses in mice models such as cigarette Dismutation of the superoxide anion to H2O2 and O2 is smoke-induced lung inflammation [38], influenza infection catalyzed by the SOD enzyme, which in humans, exists in of the lung [39], and UV-light induced sunburn [40]. three isoforms: Cu,Zn-SOD in the cytosol, Mn-SOD in the Phagocytes exert protection to the host by generating mitochondria, and extracellular SOD [44]. Subsequently, superoxide to eradicate tissue-invading bacteria in order H2O2 is degraded to H2O by catalase and GPx. Glutathione to prevent the development of pathogenic infection [41]. peroxidase exists in selenium-dependent (Se-Gpx) and In patients with chronic granulomatous disease, aseptic selenium-independent forms. It catalyzes the reduction of H2O2 to oxidized glutathione (GSSG) and H2O, and degrades inflammation results from recurrent infections with bacteria + and fungi in the setting of deficient ROS. It has thus been peroxides, using glutathione (GSH) as a H ion donor [24, suggested that therapy with ROS-inducers may be of benefit 45]. As a reductant, NADPH is utilized by glutathione to patients with chronic granulomatous disease and other reductase (GR) to catalyze the regeneration of GSH [45]. In autoimmune or inflammatory conditions [36]. the cell, GSH is chiefly responsible for maintaining the cell in a reduced state [20]. The idea of ROS as merely pro-inflammatory has recently been challenged by several research groups, who In female reproduction, the Thioredoxin (Trx) system propose that ROS may actually play a role in limiting regulates cell growth and differentiation, as well as inflammation, as shown by the results of studies on apoptosis. It also regulates gene and enzyme functions [46]. autoimmune and inflammatory conditions. However, this The release of H+ ion from cysteine’s thiol group can novel concept warrants further and more thorough investigation cause proteins to fold improperly, stimulating formation for the potential of NOX2-induced ROS as a target for of disulfide bonds; as a result, protein functions can therapy of autoimmune diseases. be altered and apoptosis can occur [18]. Peroxiredoxins, 186 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal such as GPx, work to detoxify and reduce H2O2, generating Endometriosis H O in the process, and are vital for the removal of peroxides 2 The presence of endometrial tissue in extra-uterine [47]. locations is characteristic of endometriosis, a benign, but Studies have demonstrated the dual role of estrogen as chronic, estrogen-dependent gynecological disorder. Although both an antioxidant [48,49] and a pro-oxidant [50, 51]. benign, the disease leaves many affected females infertile Interestingly, estrogen can stimulate production of enzymatic [68]. Endometriosis is considered a multifactorial disease. antioxidants, including SOD [52, 53]. The phenol structure Although its etiology remains unclear, it may be explained of estrogen makes it capable of scavenging free radicals. by one or more proposed theories including retrograde Estrogen has been found to limit damage from oxidation of menstruation, altered immunologic responses, genetic DNA and LDL [52, 53] and markedly reduce lipid oxidation. predisposition, and inflammatory mechanisms [69], which Non-enzymatic antioxidants consist of GSH, ascorbate have all been suggested to involve OS. The subfertility often (vitamin C), and tocopherols (vitamin E), amongst others. experienced by patients with endometriosis may also result Ascorbic acid and tocopherols are direct scavengers of ROS. from excessive ROS in the peritoneal fluid environment, These compounds may be endogenous, or obtained from diet which could be toxic to sperm and impair oocyte and embryo [54, 55]. The antioxidant activities of vitamin E neutralize quality [70]. Unfortunately, however, studies evaluating OS products of lipid peroxidation [56] and produce the markers in patients with endometriosis have produced tocopheroxyl radical. Vitamin C is water-soluble and largely conflicting results. functions in concert with the lipid-soluble vitamin E to Recent data suggest that estrogen may act as a pro- regenerate -tocopherol from the tocopheroxyl radical. In oxidant. Results from a study by Laloraya et al. (1996) humans, ascorbate is present in the ovary and follicular fluid revealed that the addition of estrogen to progesterone (FF) [57, 58]. injections given to female mice led to a dramatic increase in Glutathione is a tripeptide that contains cysteine. It generation of oxyradicals along with a significant decline in SOD, suggesting a regulatory role for estrogen in the can scavenge free radicals directly through chemical reactions or by acting as a co-factor for GPx to reduce production of superoxide radical via SOD suppression [71]. peroxides [21]. Thus, GSH can alternate between reduced In this way, the Laloraya’s group has proposed that estrogen (GSH) or oxidized (GSSG) forms. Furthermore, vitamins C may actually stimulate production of superoxide radical and E can be regenerated to their active forms by GSH. during implantation by inhibiting or suppressing uterine Glutathione is strong predictor of oocyte quality [59, 60]. secretion of SOD. Measurements of ROS [72] and In oocytes, intracellular GSH content can be increased antioxidant levels could potentially assist in the monitoring by cysteine and cysteamine. Besides performing as an of disease recurrence. Given the estrogen-dependent nature antioxidant, cysteamine also has scavenging properties that of endometriosis, estrogen may act as a pro-oxidant in the assist in the maintenance of high GSH levels within cells disease by enhancing superoxide radical production and [9, 61]. suppressing SOD, thus acting as a key mediator in disease progression. Melatonin, a hormone and well-known antioxidant, is produced by the body and may be obtained through the diet. Several research groups have measured higher levels of When melatonin is oxidized, however, stable end-products OS markers such as the lipid peroxidation marker, malonaldehyde (MDA) [36], pro-inflammatory cytokines are produced, preventing it from returning to its reduced state [16]. [73, 74], and oxidized LDL (ox-LDL) [75] in the peritoneal fluid of subjects with endometriosis [73, 76-79], whereas REPRODUCTIVE PATHOLOGIES ASSOCIATED other groups have failed to report similar findings [80, 81]. WITH INFERTILITY: A ROLE FOR OXIDATIVE Another biomarker of lipid peroxidation known as 8-iso- STRESS? prostaglandin F2 (8-iso-PGF2) has been used to measure OS [82-84]. In comparison to controls, high amounts of 8- Hydrosalpinx iso-PGF2 have been detected in the peritoneal fluid and Hydrosalpinx is a condition characterized by unilateral or plasma of patients with endometriosis [79]. Measurements of bilateral tubal buildup of serous fluid [62]. Consequently, the pro-inflammatory [72] cytokines and oxidative biomarkers fallopian tube(s) become occluded and distended with such as 8-iso-PGF2 may be useful to isolate the cause of hydrosalpinx fluid (HSF) [62], usually as a result of chronic infertility associated with diseases such as endometriosis, tubal infection [63, 64]. Hydrosalpinx fluid contains bacteria while establishing a link with OS. and cytokines [65]. Because HSF lacks antioxidants, OS may The buildup of iron within endometriotic lesions is a ensue [65], and has been suggested to play a role in the possible contributor to the OS implicated in the development embryotoxicity [66] and infertility [62] associated with of endometriosis, although this occurrence of OS may be hydrosalpinx. Additionally, products of lipid peroxidation localized to the lesions and has been suggested to be have been detected in human HSF by Bedaiwy et al. (2002), transient [85]. In the Fenton reaction, iron acts as a catalyst although the group failed to find a significant relationship in the generation of ROS [86]. Bound iron can be released between lipid peroxidation and rate of blastocyst develop- from ferritin by superoxide, and in the presence of abundant ment [67]. Importantly, they did report that the presence of free iron, hydroxyl radicals are produced via the Fenton ROS in HSF was positively associated with blastocyst reaction when superoxide reacts with H2O2. The -cleavage development, suggesting that low levels of ROS are vital to of alkoxyl radicals that are formed from lipid hydroperoxides endosalpinx health. yields aldehydes such as MDA [87]. Plasma iron levels have Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 187 been shown to positively correlate with plasma MDA, underlie the association with obesity [101]. Moreover, demonstrating that free iron can damage cell membranes by significant negative correlations between levels of MDA inducing lipid peroxidation [88]. To this effect, further and insulin sensitivity and between levels of MDA and GSH studies should evaluate the possible association of iron have been made [102]. Significantly higher MDA levels with other established biomarkers of lipid peroxidation have also been measured in PCOS patients compared to by quantifying other products of lipid peroxides. Examples healthy controls, independent of both obesity and insulin of such include isoprostanes, which are formed at locations resistance [103]. It should also be noted that although PCOS of free radical production on phospholipids [89], and and obesity are often co-morbid conditions, they have prostaglandin-F2-like compounds called F2-isoprostanes, that been demonstrated to have independent effects on in-vitro result from free radical-mediated non-enzymatic peroxidation fertilization (IVF) outcomes [104, 105]. of arachidonic acid [87], namely 8-iso-PGF2. These Recently, a study examining murine oocytes concluded products should also be measured and their presence in that the poor oocyte quality of insulin-resistant mice was a the setting of abundant free iron should be investigated result of oxidative damage to mitochondrial functions [106]. further. Moreover, both iron and heme are considered highly The mitochondrial dysfunction in PCOS patients [94, 107] active free radicals, given their ability to destroy DNA [90]. occurs secondary to decreased consumption of O and Continual exposure to these free radicals from iron overload 2 increased generation of ROS, with a decline in antioxidant may account, at least in part, for the role of OS in levels [95, 108]. Accordingly, it has been suggested that endometriosis. insulin resistance suppresses antioxidant levels, while Women with endometriosis have also been found with increasing lipid hydroperoxide [109], and thus, OS. decreased levels of antioxidants [74, 91]. The consumption of antioxidant defenses in response to overwhelming ROS Results from a study by one research group on IVF led the authors to propose that ROS and lipid peroxide levels in might explain the low levels of antioxidants observed in the peritoneal fluid of patients with endometriosis [91]. the follicular fluid (FF) of developing oocytes could predict embryo quality [110]. In a later study, the same group Polycystic Ovary Syndrome demonstrated that meiotic spindle formation, in relation to low OS levels, was linked to successful intracytoplasmic Polycystic Ovary Syndrome (PCOS) is the most common sperm injection (ICSI) outcomes in PCOS patients. In this endocrine abnormality affecting women of reproductive age study, Chattopadhayay et al. (2010) reported that compared

[92]. It is characterized by hyperandrogenism, ovulatory with controls, the oocytes of PCOS women exhibited less dysfunction, and multiple cysts on one or both ovaries [2]. meiotic spindles, possibly due to excessive ROS production Between 4% and 18% of reproductive-aged women are along with low FF content of antioxidants. A positive affected by PCOS [93]. Patients often experience menstrual correlation between the presence of meiotic spindles in disturbances and the vast majority fails to conceive. oocytes and low OS was shown; likewise, the absence of Oxidative stress has emerged as a possible link between meiotic spindles was found to correlate with high FF levels PCOS and its associated infertility. of OS [111]. Oyawoye et al. (2009) examined the effects of Women with PCOS commonly exhibit signs of insulin different factors on IVF outcome, and could not find a resistance, including hypertension and central obesity, which significant association between polycystic ovaries and places them at serious risk to develop conditions such as reproductive outcomes, when taking into account FF cardiovascular disease and diabetes [94]. In response to measurements of total antioxidant capacity (TAC) [112]. hyperglycemia, the mononuclear cells of women with PCOS produces increased amounts of ROS. The presence of OS Unexplained Infertility triggers inflammation, which could contribute to the insulin Couples who fail to conceive despite having practiced resistance and hyperandrogenism associated with PCOS 12 months of unprotected sexual intercourse and in [95]. Increased concentrations of C-reactive protein have which known causes of infertility have been excluded, are been measured in PCOS patients, supporting the relationship considered to have unexplained infertility, a condition between inflammation and the disease [96]. In addition to affecting up to 15% of couples in the United States. In these increased C-reactive protein levels, the inflammatory cases, the pathophysiology of infertility has yet to be markers IL-6, and TNF-, have also been measured in obese elucidated, but may involve high levels of ROS along with women with PCOS when compared with BMI-matched low levels of antioxidants in the peritoneal fluid of female controls and a normal weight group [97]. Other circulating patients. Two studies by Polak et al. collectively reported markers of inflammation have been measured in PCOS high peritoneal levels of MDA in affected females [113, patients in correlation with insulin resistance and obesity 114]. Additionally, antioxidant levels have been found to be [98]. Coupled with an altered hormonal milieu, the decreased in the peritoneal cavity [115]. Taken together, the inflammatory reproductive environments of PCOS patients share similarities to those of women with high BMIs, and peritoneal environment of these patients is likely dominated could adversely affect oocyte maturation and quality [99]. by ROS, resulting in diminished antioxidant defenses [116]. Larger measurements of waist circumference have been ASSISTED REPRODUCTIVE TECHNIQUES found to account for increased levels of ox-LDL detected in PCOS patients [100]. Increased lipolysis leading to Assisted reproductive techniques (ART) are the treatment increased fatty acid levels is lipotoxic to lean muscle and has of choice in many cases of female and male infertility been observed in both insulin resistance and PCOS, and may as an alternative to overcome some of the known and 188 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal unknown causative factors of infertility [117, 118]. Amongst shown that the success of IVM largely depends on the these techniques are in-vitro maturation (IVM), in-vitro seamless maturation of nuclei and cytoplasm [128, 129], fertilization (IVF), intracytoplasmic sperm injection (ICSI), which is in keeping with the notion that close simulation of and cryopreservation, amongst other up and coming the in vivo environment must be achieved in vitro [125] to procedures. These techniques utilize advanced technologies obtain positive outcomes from IVM. to achieve reproductive success in the in vitro setting. However, despite the considerable advancement of ART Cryopreservation over the last few decades, only 25% of cycles produce Cryopreservation utilizes low temperatures to essentially favorable pregnancy outcomes [119]. freeze and maintain reproductive cells and tissues, and IVF & ICSI embryos [130] until their use is desired, prior to which they must be thawed. To stimulate ovarian production of mature oocytes in women undergoing IVF, many ART programs utilize FACTORS ASSOCIATED WITH OS AND DECREASED hormone therapy. Oocytes are then retrieved from the ovary ART SUCCESS and sperm-oocyte interaction is allowed to take place in the laboratory setting within culture media [120] before transfer Ovarian Aging and Age-related Fertility Decline of the produced embryo into the uterus. As a woman gets older, her ability to reproduce declines, In general, causes of female factor infertility such as and as she approaches menopause, her chances of conception tubal factors, endometriosis, and abnormal cervical mucus are almost nil [131]. However, some women experience are the usual indications for IVF. The need for women to depletion of ovarian reserve at a faster rate than would undergo ovarian hyperstimulation protocols with hCG or normally be expected by a woman’s chronological age and progesterone can cause unfavorable side effects, the most the oocytes that remain are decreased in quality [132]; this common of which is ovarian hyperstimulation syndrome may lead to premature ovarian failure which cannot be [120]. reversed [133]. The factors that influence the process of ovarian aging have not yet been fully determined, and the In cases of male factor infertility or when IVF cycles fail reasons why some women age faster than others, have yet to ICSI may be used. Unlike IVF, ICSI allows sperm-oocyte be elucidated. interaction to occur directly, as a single sperm is injected into the oocyte’s cytoplasm [120]. Although fertilization can The chances of a successful pregnancy with ART cycles occur in a direct manner by ICSI, the methods of natural also declines significantly with age [134, 135]; for a 29-year- selection present in the in vivo environment are lacking, thus old female, they are approximately 40%, which decreases to permitting the injection of damaged spermatozoon into an 32% by the age of 36, and is almost diminished by age 46, as oocyte. As such, a crucial set of processes that rely on stable she nears menopause [136]. In vitro fertilization studies have sperm DNA (e.g. acrosome reaction, sperm capacitation, established that the most important determinant of ART sperm-oocyte fusion) can take place both naturally and outcome is the woman’s age [134]. through IVF, but are circumvented during ICSI [121]. Oocyte quality is a well-founded predictor of the embryo The in vitro environment of ICSI subjects ova and sperm development [137, 138]. Both endogenous cell functions and to OS during gamete manipulation techniques, particularly exogenous sources in the ovary make it a generous producer causing damage to DNA [122]. This aspect could explain the of free radicals. It is plausible that the accelerated follicular associated low take-home-baby rate per cycle of ICSI [123]. atresia and reduced ovarian reserve [139, 140], along Recently, Lee et al. (2012) determined that high ROS levels with the loss of oocyte quality and consequent infertility in FF were associated with poor embryo quality on day 3 of encountered with aging [141, 142] could be mediated by ICSI cycles, while increased ROS in culture media positively oxidative pathways [56, 143, 144], given the well-documented correlated with embryo fragmentation, and negatively role of OS in physiological processes of other age-related correlated with embryo implantation potential [124]. diseases [145]. The aging process seems to be accelerated in fresh In Vitro Maturation of Oocytes oocytes upon exposure to SO anion, H2O2, and hypochlorous Women with and without polycystic ovaries who desire acid (another type of ROS) separately, and to a greater both fertility preservation while avoiding ovarian hyper- extent in previously aged oocytes [146]. Fresh oocytes have stimulation protocol increasingly considering IVM as a more also been found to have higher levels of glutathione- affordable alternative to conventional ART methods [62, S-transferase and thiols than aged oocytes [147], which 125]. As a developing technique, IVM yields a disappointing have been found to have decreased GSH content [148], clinical pregnancy rate of 18-30% [126]. The IVM process indicating lower oocyte quality [59, 60]. Higher levels of begins with the retrieval of pre-ovulatory or anovulatory ROS observed in aged oocytes verses fresh young oocytes immature oocytes from antral follicles. After oocytes are have been observed by several research groups [56, 149, matured in vitro, conventional IVF or ICSI and embryo 150]. These results may explain weakened antioxidant transfer are performed [127]. Through this process, the defenses recorded in the reproductive environment of older number of immature oocytes for retrieval is preserved. females, as demonstrated by lower granulosa cell content of Moreover, it allows for substantial growth of amount of antioxidant enzymes in older women compared with younger matured oocytes available for routine IVF. Studies have women [151, 152]. Diminished antioxidant defenses could Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 189 possibly account for the greater sensitivity of older oocytes of fat noted in central adiposity allows for overflow of fatty to ROS. Supporting data from Wiener-Megnazi et al. (2004) acids and leak of electrons from the mitochondrial ETC, revealed increased FF free radical activity during the creating a lipotoxic atmosphere throughout the female aging process [153]. Furthermore, low FF levels of TAC in reproductive milieu [166, 167] and triggering insulin women > 37 years old has been associated with unsuccessful resistance [168]. fertilization [112]. Overabundant production of ROS in the reproductive The course of aging consistently subjects a woman’s environment of women with high BMI can lead to OS, and body to repeated ovulation. Since OS plays a key role in low antioxidant status [169, 170], both of which have been ovulation [151], its involvement has also been proposed in linked to obesity [171, 172]. Obesity and OS are also repeated ovulation which may lead to damage to the ovary considered inflammatory conditions [167]. The state of and oocyte secondary to OS buildup [154]. obesity has been described to involve increased fatty acid Cumulus cells (CC) are vital for the processes of oocyte breakdown with decreased uptake, intrusion of inflammatory maturation, ovulation, and fertilization to take place [155]. In cells, along with adipokine secretion [173, 175]. Levels of mice, however, CCs have been found to hasten the aging of oxidized lipoproroteins such as ox-LDL, a known mediator oocytes [156]. In conjunction with FF, CCs surrounding of inflammatory processes, have been measured in the FF of oocytes may confer protection against ROS-induced damage, obese patients [176], demonstrating an association between as was observed in porcine oocytes [157]. an inflammatory reproductive environment and OS in obesity [172]. Furthermore, upregulated antioxidant defenses Female aging is invariably associated with a decline in such as catalase and GPx have been measured in both the estrogen levels, most notably post-menopause. As mentioned circulation and FF of obese women compared to normal earlier, estrogen has been proposed to act as a pro-oxidant, weight women [176]; the recruitment of antioxidants and the lack of estrogen in post-menopausal women may suggests an attempt for to overcome excessive ROS. As actually be a protective mechanism against the development inflammatory cytokine production increases, factors that rely of OS. Recently, Victorino et al. (2012) have reported heavily on mitochondrial functions and ATP for energy that post-menopausal women have markedly low OS levels [177, 178] such as oocyte function and embryo development and higher antioxidant status compared to pre-menopausal may be compromised by OS [179]. In addition to modulating women [158]. Specifically, lipid peroxidation, which was follicular development, the cytokine TNF- has been measured by lipid hydroperoxide formation, was found to be suggested to contribute to oocyte atresia [180]. significantly lower in post-menopausal women than in pre- menopausal women, implying a positive association between The effects of female obesity on ART outcomes remain estrogen and lipid peroxidation [158]. In mammary epithelial controversial. It is generally acknowledged, however, that cells, aromatic hydroxylation of either endogenous or obese women usually need higher doses of gonadotropins synthetic estrogens leads to the production of catechol than non-obese females for ovarian hyperstimulation protocols [172], possibly due to deranged metabolic processes metabolites [51], which have been shown to yield H2O2 and hydroxyl radicals in the setting of endogenous estrogen causing impaired drug absorption and which may also [159]. The concept of estrogen as a pro-oxidant may actually account for lower oocyte numbers [181]. Furthermore, shield post-menopausal women from the perils of OS. successful outcomes with ART are reportedly lower for However, infertility rates remain high in the post-menopausal women with high BMIs than those of normal weight, and population, suggesting that either that 1) the seemingly they are at increased risk for miscarriage [7, 161, 164, 173, protective role of estrogen may be inadequate to counter the 182, 183], although two studies have failed to relate OS associated with infertility, or 2) that the pro-oxidant high BMI with adverse IVF outcome parameters, such as effects of estrogen have no effect on fertility and that other decreased oocyte number and quality, implantation, and mechanism(s) are responsible for high rates of infertility in pregnancy rates [184, 185]. post-menopausal women. Indeed, future studies, particularly Oocyte quality is considered a crucial regulator of early those with large sample sizes are needed to clarify the embryo development [186], thus, unfavorable oocyte quality potential role of estrogen as a pro-oxidant. may well contribute to the decreased pregnancy rates of obese females [173]. Maternal obesity has also been linked High Body Mass Index to reduced antral follicle count, ovarian reserve [187], and It is estimated that around 34% of women of embryo quality [174] in those undergoing IVF. Moreover, a reproductive-age in the United States are obese [160]. In significantly low number of mature oocytes have been addition to being at an increased risk for miscarriage and retrieved from obese women [188]. Reduced odds of recurrent miscarriage [161], overweight and obese women successfully cryopreserved embryos have also been reported have also been found to have longer times to conception in obese women [174]. [162] than normal weight women do. Hormonal and menstrual Deficiencies in oocyte and embryo quality may partially irregularities are well-established cause of subfertility in stem from insulin resistance, as seen in PCOS [189, 190]. obese females [163], and decreased endometrial receptivity Increased levels of inflammatory markers have been found in has more recently been considered a contributing factor [164, the FF of obese women who are infertile [191], including 165]. With an increased tendency to store fat centrally, obese inappropriately high levels of C-reactive protein [192, 193], women often have increased waist circumferences, or a which could also contribute to the decline in oocyte quality. waist to hip ratio > 0.85 [7], which reduces their chances for This altered ovarian follicular environment could impact natural conception by half [7, 166]. The intracellular buildup fertilization, implantation, and rates of live births from IVF 190 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal cycles [194, 195]. Obese women undergoing ICSI have also pregnancy [213] similar to active smokers, while taking been observed to have poor clinical pregnancy rates, possibly longer to conceive [7, 192, 214, 215] than non-smokers not due to decreased oocyte quality [196]. Nevertheless, the exposed to cigarette smoke. Women exposed to second-hand association between high BMI and low oocyte quality likely smoke were found with high levels of cotinine, a metabolite stems from multiple factors. of nicotine, in their FF, which was associated with increased odds for implantation failure and decreased odds for live Results from many studies confirm that obesity can birth compared to non-exposed women [216]. negatively affect IVF outcomes [172, 174, 197, 198], with the potential for pregnancy loss [186, 199]. Although Depalo The detrimental effects of cigarette smoking on et al. (2011) found no significant difference in fertilization reproductive outcomes are most likely multifactorial. Early and embryo quality after ICSI between women of normal placental development is disrupted by smoking, and has been and high BMI, they did report lower rates of implantation in shown to induce trophoblast apoptosis [217]. Cigarette smoke the high BMI group [200], which was in agreement with also adversely affects placental development by altering O2 Kahraman et al. [201]. Conversely, Metwally et al. (2007) tension-sensing mechanisms [218]. In addition, smoking has observed a negative effect of obesity on embryo quality, been shown to impair tissue oxygenation while exposing the rather than oocyte quality in women undergoing IVF/ICSI. reproductive environment to the myriad of toxins contained These findings may point instead to a negative effect of the in cigarette smoke. It is only recently, however, that OS has uterine environment. been implicated as an important contributor to smoking- induced infertility. Studies exploring the link between obesity and fertility outcomes after IVF reported that obese women had a Many of the constituents of tobacco smoke are free significantly lower live birth rate than normal weight women radicals or ROS [219, 220] that can lead trigger OS and [173, 190, 198], which positively correlated with increasing apoptosis through various mechanisms [221]. Two of the BMI [190]. On the other hand, some groups failed to report major toxic components implicated in impaired embryo differing rates of clinical pregnancy between obese and development secondary to OS are nicotine and benzo[] normal weight women [202-206] and one study determined pyrene [214, 222]. A grouping of tobacco smoke metabolites that embryo quality was not affected in obese women in FF was discovered to correlate with cigarette smoke undergoing IVF [190]. Interestingly, the transfer of high exposure [223, 224]. Levels of metabolites were further quality embryos has been shown to surmount the adverse linked to a substantial increase in lipid peroxidation along effects of obesity on rates of implantation and pregnancy in with suppressed antioxidant defenses [223]. Cotinine levels females undergoing IVF [207]. in the FF of smokers who failed to achieve pregnancy through IVF also correlated to an increase of blood and FF Studies thus far fail to agree with regard to the possible DNA adducts [225], possibly originating from ROS-induced impairment of oocyte and embryo quality in obese females DNA damage [226]. Thus, the oocyte and surrounding undergoing ART, although a bulk of evidence suggests an association in between obesity and miscarriage both in granulosa cells are left susceptible to damage by toxins that reach the FF environment [222]. women conceiving naturally and in those undergoing ART. These discrepancies are likely due to differences in study Similar to aging, smoking has been strongly linked to design and clinical classifications such as varied BMI cutoff diminished ovarian reserve in humans [227-229] and in mice points. More prospective studies will be needed to confirm [229, 230] through enhanced apoptotic cascades [229], whether increased BMI is associated with impaired oocyte which could be induced by OS. In mice, cigarette smoke and/or embryo quality in women conceiving naturally as exposure has been found to decrease oocyte quality. well as those undergoing ART. Embryos exposed maternally to cigarette smoke displayed defective development secondary to OS and cell death [231]. Cigarette Smoking and Secondhand Exposure Nicotine and carbon monoxide contained in tobacco Controllable lifestyle habits such as cigarette smoking [5] smoke can cross the placenta, resulting in placental hypoxia, have also been linked to oxidative disturbances that may and thus utero-placental insufficiency. In this way, the contribute to subfertility. In general, women who smoke developing fetus becomes deficient in O2 and nutrients [214]. undergo double the number of IVF cycles than non-smokers Although actively smoking mothers may lower their chances in order to conceive [7]. Smokers also have significantly of fertility, their fertility may be restored after they quit lower odds for pregnancy and live delivery per cycle than smoking [231]. those of non-smokers, as reported by a 2009 meta-analysis of Furthermore, women who smoke have been observed 21 studies [114]. By smoking, women decrease their odds of with hampered follicular growth [232, 233], follicular loss having a live birth by close to 50%, and increase their [228, 234, 235], and lower numbers of oocytes retrieved chances of miscarriage 2.65-fold [196]. Smokers also are during IVF [236]. Increased production of ROS and decreased thought to have almost four times [208] the risk for ectopic antioxidants [237] in various cells of mice have been pregnancy than non-smoking women [208, 209] and have reported in association with chronic cigarette smoke been found to be 3-4 times more likely [210] to take longer exposure [238], suggesting a role for oxidative-induced than one year to conceive than their non-smoking damage to fertility via tobacco smoke exposure. The counterparts [210-212]. heightened expression of Hsp25, a heat shock protein known Even non-smokers who are passively exposed to smoke to be induced by OS [239], has also been measured in have been reported with lower rates of implantation and women exposed to cigarette smoke [229]. The expression of Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 191

SOD protein was also found to be decreased in mice exposed The membrane of spermatozoa is largely composed of to cigarette smoke [229]. Moreover, increased TAC has been polyunsaturated fatty acids, which facilitates fusion with measured in the FF of smokers compared to non-smokers oocyte(s) for fertilization. Aerobic conditions promote the [112]. The aforementioned data supports the up-regulation of production of ROS by spermatozoa. When ROS is produced antioxidants in settings of OS to buffer an overproduction of in excess, OS can ensue and cause damage to spermatozoa. free radicals and ROS. Infertility has been thought to result from OS-induced sperm damage. A study by Yumura et al. (2009) compared pregnancy Even smokers who are able to produce high-quality rates from semen samples containing ROS with those embryos still maintain risks for lower pregnancy outcomes lacking ROS. Although pregnancy rates did not seem to with IVF. In these cases, decreased endometrial receptivity differ between the use of ROS-containing and ROS-lacking may account for failed IVF attempts. The adverse effects of semen samples, they observed a significant difference in the cigarette smoke on IVF success may be reversible for those levels of ROS in pregnant and non-pregnant groups [248]. An only passively exposed, as similar pregnancy rates have been inverse correlation between sperm motility and ROS levels reported for non-smokers and passively exposed women in the semen samples was reported, similar to the findings [240]. of prior studies [249, 250]. Thus, the observed negative impact of seminal ROS on pregnancy rates suggests that SOURCES OF ROS IN THE ART SETTING identification of ROS in semen might be an important In vitro studies have long implicated OS as a contributor prognostic factor in assessing idiopathic male infertility [8]. to defective embryo development [69], however, a specific threshold level OS may be beneficial in vitro, just as it is in Exogenous Sources of ROS vivo . Although oocyte quality is often used as a predictor of The production of ROS can be stimulated by several embryo development [241, 242], various other factors in the factors in the in vitro setting. In vivo, fertilization and embryo’s environment may also influence embryo growth pregnancy normally take place in a protected environment of and viability through ROS production. Reactive oxygen enzymatic antioxidants. In contrast, the in vitro setting is species can originate from two types of sources: endogenous deprived of the antioxidant defenses usually present in vivo, (intrinsic) or exogenous (extrinsic) [9]. Overabundant ROS and thus, an overabundant amount of ROS could easily be originating from either type of source can create OS that may generated in the absence of antioxidants to counter them. adversely affect gametes and embryos. Furthermore, the techniques of ART itself necessitate the manipulation of sperm, oocytes, and embryos, during which Endogenous Sources of ROS ROS is produced [9]. Endogenous sources of ROS include spermatozoa, Male/Sperm Contributions oocytes, and the embryo itself [117], as they too depend on ATP as their energy source for their own metabolic processes. In IVF, sperm-oocyte interaction takes place within the The human body relies heavily on the mitochondrial culture media [115]. Factors within the culture media can processes of oxidative phosphorylation and glycolysis for inhibit the fertilization of an oocyte by spermatozoa energy in the form of ATP. The processes of folliculogenesis containing damaged DNA [251]. In their study, Bakos et al. and oocyte maturation consume O2 during the conversion of (2008) observed that the use of sperm samples containing ADP to ATP via oxidative phosphorylation, producing ROS high levels of DNA damage in ICSI resulted in a significant in the process. During development, the preimplantation reduction of pregnancy rates [252]. These results indicate embryo switches energy utilization from oxidative that DNA damage in spermatozoa could impair its ability to phosphorylation to glycolysis in order to generate more ATP fertilize an oocyte. During ICSI, the utilization of a single [234]. spermatozoon actually minimizes gamete production of OS compared with IVF. Nonetheless, the quantitative assessment In addition to oxidative phosphorylation, the enzymes of sperm DNA damage by terminal deoxynucleotidyl NADPH oxidase and xanthine oxidase comprise the main transferase dUTP nick end labeling, or TUNEL, has been sources of embryo-generated intrinsic ROS. In either proposed as a useful tool to predict poor ART outcomes in the presence or absence of O2, physiological amounts patients with high amounts of DNA damaged sperm [1]. of ROS are produced through the reactions involved in Damaged DNA in sperm may be repaired by oocytes and these metabolic processes. Moreover, inadequate antioxidant embryos, although the mechanisms through which this defenses in combination with the surrounding FF environment occurs are unclear. Evidence exists, however, that if DNA of the oocyte and embryo can further generate ROS [244]. damage in sperm is present in excess of a threshold amount, Some reproductive disorders are associated excessive ROS oocytes and embryos may be unable to reverse this damage in the tubal and uterine environments, which could affect [253]. embryo development and reproductive outcomes in general [245, 246]. Oxidant-induced alterations in sperm motility not only reduce its fertilization capability, but may also induce age- In ART, successful fertilization depends on spermatozoa related changes in the oocyte and decreased developmental of high quality [247]. The normal functions of sperm competence with lengthened fertilization times. Inseminated including oocyte fusion, capacitation, and acrosome reaction, bovine oocytes matured over 22 hours were more likely to which all require a certain amount of ROS, however, produce cleaved embryos that progressed to the blastocyst oxidative damage to the oocyte may occur by spermatozoa- stage than inseminated oocytes that were matured over 28 or generated OS, lowering the chance for fertilization [62]. 34 hours [254]. 192 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

Female Contributions rates of fertilization have been linked to enhanced activity of the antioxidant enzyme Se-GPx [261]. Within the FF Successful fertilization likely requires a physiological microenvironment, a low TAC may predict diminished level of OS. Thus, a delicate balance between ROS and oocyte fertilization potential, but has also been found to protective antioxidants must exist both in vivo and in vitro to signify higher chances of embryo survival till transfer [244]. achieve successful reproductive outcomes in either case To assess the degree of OS present in a given environment, [255, 256]. measurements of TAC and biomarkers of lipid peroxidation Follicular fluid composition is a key factor for predicting may be useful [118]. successful ART outcomes [104]. The oocyte, granulosa cells, Reactive oxygen species can also be generated through and surrounding cells such as endothelial and thecal cells higher numbers of oocytes per dish and cumulus cell mass, make up the FF environment. The FF also contains cytokines, with exception to the beginning of culture, during which neutrophils, and macrophages, which can trigger free radical cumulus cells contain higher antioxidant activity than generation. Reactive oxygen species are also generated as denuded oocytes [268]. As a result, the presence of abundant by-products of the P450-regulated monooxygenase reaction ROS can damage mitochondria, deplete ATP energy stores, that mediates steroidogenic processes [20]. Since oocyte promote apoptosis, and cause embryo fragmentation [9]. development occurs within the FF environment, factors such Specifically, high amounts of the adapter OS protein, PC66 as oocyte quality, sperm-oocyte interaction, implantation, SHC, a stimulator of embryo apoptosis, have been measured and early development of the embryo, are directly affected in arrested embryos [9, 269, 270]. by its composition [257] and redox status [85]. Murine mitochondrial activity and basal ATP synthesis Ovulation is a normal physiological process of the ovary is regulated by sirt3, which protects pre-implantation that invariably generates OS, as evidenced by the development from OS-induced disturbances in vitro. Pre- identification of oxidative biomarkers in FF. In response to implanted embryos from mice deficient in sirt3 showed ovulation, fluctuating levels of OS biomarkers have been arrested development secondary to ROS-activated p53 [271]. measured in the FF environment and altered levels of these On the contrary, results from Wiener-Megnazi et al. (2011) biomarkers could affect ART outcomes [258-261]. An designated early embryo oxidative status as an accurate imbalanced ratio of oxidants to antioxidants can give rise to predictor of successful embryo implantation, and suggested OS, which can be detrimental to developing oocytes and that it could be of value to improve the selection of high embryos, consequently affecting fertilization and pregnancy quality embryos prior to transfer [272]. outcomes. ROS from Gamete Manipulation Methods in ART Oxidative stress and antioxidant production are modulated by low-molecular weight metabolites, which are During ART, gamete manipulation in vitro triggers their in abundant in FF, and have been used to assess oocyte production of ROS, while they remain susceptible to damage competence [262] and predict oocyte and embryo viability by exposure to overabundant amounts of ROS [273]. [263, 264]. Jana et al. (2010) found a significant negative It has been proposed that the gamete manipulation correlation between ROS levels of < 107 cps (counted technique of ICSI could exert strain and pressure of the photons per second) in FF with respect to IVF outcome oocyte’s plasma membrane, which could dictate effects of [257]. follicular ROS on oocytes, and lead to decreased fertilization, Oxidative stress can induce damage to oocyte DNA, as cleavage, and implantation rates [9, 69, 124]. Results measured by 8-hydroxydeoxyguanosine (8-OHdG). Low presented by Bedaiwy et al. (2004) associated ROS levels fertilization rates and poor embryo quality have been linked in day 1 culture media with day 3 embryo quality [274], to elevated intrafollicular levels of 8-OHdG [256] in women indicating that OS-induced damage on fertilization and with a prior failed IVF cycle secondary to low oocyte quality embryo development could be affected by the ICSI [105]. procedure itself [124]. Interestingly, there was no relation Conversely, results from a study by Attaran et al. (2000) between ROS in day 3 culture media and levels of ROS in FF [274]. indicated that successful IVF pregnancies were associated with markedly higher levels of ROS in the FF than failed According to standard insemination protocols, sperm and pregnancies [258]. On the other hand, Fujimoto et al. (2011) oocytes are usually co-incubated overnight [275], for as long failed to demonstrate a significant association between OS in as 15-20 hours. Co-incubation times can influence the the FF and morphological parameters of early embryo production of ROS by human spermatozoa [276]. Higher development [266]. Other studies have also reported that levels of ROS in fertilization medium of mice have been oocyte development is unaffected by variations in ROS [108, measured after co-incubation for 18 hours compared to ROS 267], and in fact, associated processes such as lipid levels after 4 hours [275]. Supra-physiological levels of ROS peroxidation may be required to achieve pregnancy through in vitro can cause harm to oocytes and zygotes [274, 275, ART [110]. Overall, it appears that some threshold level of 277], which may decrease rates of fertilization, embryo OS exists for optimal oocyte and embryo development; this quality, and pregnancy outcomes [278, 279]. level, however, remains to be determined. Through the freeze and thaw techniques of Antioxidants are another component of FF, which serve cryopreservation, ROS are increasingly generated. To defend to buffer overabundant ROS, although studies investigating against abundant ROS, cells develop mechanisms that antioxidant status in FF are largely conflicting. Increased produce large amounts of energy, and consequently induce Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 193

OS [280, 281]. Cryopreservation can generate OS through can also become elevated in a hyperoxic environment. In different mechanisms. Osmotic stress, which may occur IVF specifically, increased exposure to O2 lengthens the during cryopreservation, may lead to the production of incubation time [9]. superoxide anion [162]. The post-warming rescue process On the other hand, low O tension may mimic the can also generate OS, demonstrated by damage to 2 reproductive tract in vivo [11] and suppress ROS formation membranes and mitochondria in addition to altered oxidative in culture during early embryo development [293]. Studies metabolism [282-284]. Thus, after thawing, cells need the have demonstrated that the reduction of O tension from protection of antioxidants to shield them against ROS- 2 20% (atmospheric) to 5% (low) has resulted in enhanced induced damage [162, 285] from glucose and re-exposure embryo development in vitro [273, 294, 295]. More recently, of preserved tissues to O [285, 286]. Furthermore, increased 2 Waldenström et al. (2009) reported improved human ROS induced by vitrification has also been shown in in vivo blastocyst quality and a significant increase in birth rate oocytes of mice [162]. These results may suggest a similar effect of heightened ROS production during the for blastocysts cultured under low (5%) compared to high cryopreservation of human oocytes. (19%) O2 concentration. It was suggested that free radical production increases in conjunction with both prolonged ROS in Culture Systems and Media duration of culture (5 days) and increasing O2 tension [296]. et al. The use of culture media in ART provides a means for Similarly, Bermejo-Alverez (2010) reported enhanced sperm-oocyte interaction with subsequent fertilization, and is bovine embryo development, despite failing to observe another source of ROS. Extrinsic factors in culture media increased blastocyst quality [297]. Thus, it has been advised that culturing embryos under O2 tension above 5%-10%, such as O2 concentration, metallic cations, visible light, and spermatozoa, amongst others, are used to enhance embryonic such as atmospheric O2 concentration (~20%), should be avoided [298], while others feel making this recommendation production of ROS at physiologically beneficial amounts. However, the combination of these factors can trigger a is too premature in the absence of results of further series of harmful chemical reactions [287], which expose randomized controlled trials [299]. gametes and embryos to higher than ideal levels of ROS, and The effects of varied O2 concentration, however, may be thereby, increase their susceptibility to detrimental effects dependent on the stage of embryo development upon in vitro from OS. The chief factors of culture systems that exposure. Bain et al. (2011) found that H2O2 exerted a dose- could be toxic to embryos are ROS in conjunction with dependent decrease in blastocyst formation that related to the heavy metal ions, but may vary with different culture stage when they were treated. Furthermore, the frequencies systems (See Table 1). of embryo cleavage and blastocyst formation did not change significantly with the addition of catalase for embryos cultured under low (5%) O tension; however, for embryos Table 1. Factors that Contribute to OS within the ART 2 cultured under high (20%) O tension, there was significant Culture Media 2 improvement in blastocyst development. Catalase was confirmed to exert antioxidant effects when markedly Factors contributing to OS in ART culture media reduced intracellular ROS and significantly increased developmental potential was observed in catalase-treated O2 concentration: or O2 tension embryos further subjected to H2O2 [300]. Previously, the Metallic cations: iron, copper same group had reported markedly increased H2O2 levels in permanently arrested embryos [301]. Taken together, data Visible light: UV light from Bain et al. (2011) indicate that some level of H2O2 or Composition: presence or absence of antioxidants ROS signaling is vital to proper embryo development; beyond this level, however, developmental failure could Female contributions: constituents of follicular fluid, including result [127]. oocytes, cytokines, antioxidant content Both animal and human studies have demonstrated Male contributions: spermatozoa, especially those with high levels of substantial benefits of low O2 concentration on embryo DNA damage development, implantation, and pregnancy rates, as opposed to atomospheric O2 concentrations [293, 302-305]. On the contrary, other studies have failed to report differences in

O2 Concentration implantation and pregnancy rates with low and atmospheric O concentrations [303, 306, 307]. Thus far, results have in vitro 2 The production of ROS within culture media can been inconclusive and the possible benefits of culturing produce an environment of high O2 tension which has been human oocytes and embryos under low O2 concentrations proved to be deleterious to embryo development and result regarding ART outcomes need to be investigated further. in apoptosis [288, 289], damage to DNA [9], and altered gene expression [290, 291]. Likewise, a setting of high O2 Metallic Cations tension can promote the generation of ROS [292]. In two studies, high O2 tension was found to up-regulate the gene The formation of ROS in ART culture media can be expressions of the antioxidant enzymes SOD2 and GPx1, exogenously promoted by the presence of traces of metallic although catalase was unaffected [286, 287]. Through cations [9] through the Fenton and Haber-Weiss reactions heightened oxidase enzyme activity, levels of the SO radical discussed earlier. 194 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

Visible Light Handling of Gametes and Embryos In contrast to the dark setting of the reproductive tract, Reactive oxygen species can be generated through oocytes and embryos are intermittently exposed to light sperm-oocyte interactions and by embryos themselves during during ART prior to transfer, another factor that can ART. Mechanical removal of ROS has been investigated stimulate ROS production and damage to DNA [308, 309]. in patients with endometriosis undergoing IVF-embryo Exposure to light has demonstrated an overall negative effect transfer. The granulosa cells of these patients contain high on embryo development [310-313]. Takenaka et al. (2007) levels of OS biomarkers. Furthermore, endometriotic exposed mouse and hamster zygotes to fluorescent light, peritoneal fluid has been found to contain inflammatory and noted increased H2O2 in both species, more so in cytokines and high levels of TNF-, which has been the latter [308]. Another study by Oh et al. (2007) reported implicated in oocyte atresia [180]. In those with ovarian that embryos cultured under high intensity lighting of endometriomas, enhanced oocyte and embryo quality has short wavelength exhibited enhanced expression of the resulted from cumulus oophorus rinsing [329]. It could be HSP70 gene (a mediator of apoptosis) and increased ROS that through this rinsing procedure, the effects of toxic production, indicating increased apoptotic blastomeres, constituents contained in the endometriotic environment reduced blastocyst formation, and decreased embryo survival surrounding retrieved oocytes are eliminated. and quality [314]. Duration of Sperm-oocyte Co-incubation Composition As aforementioned, standard sperm-oocyte incubation Rates of ROS production by the culture media can be times have been reported to increase ROS production [276] influenced by its composition [257], which can inflict compared to short co-incubation [275]. Hence, reducing the serious damage to preimplantation embryos [315, 316]. The duration co-incubation may limit the amount of ROS impact of OS triggered by culture media can deplete oocyte produced. Improved embryo quality as well as significantly GSH content, enhancing the effect of sustained OS and thus, increased rates of implantation and pregnancy have been risking oocyte fertilization and viability [317]. observed with sperm-oocyte incubation of 1 or 2 hours compared with co-incubation overnight in prospective Within the IVM medium, cumulus-oocyte complexes randomized controlled studies [330, 331]. In patients with (COC) reportedly release the antioxidant enzymes SOD, complete IVF failure, short sperm-oocyte co-incubation of 3 catalase [318], and GSH [319, 320], all of which scavenge ROS, and provide the COCs some degree of protection hours in conjunction with early rescue ICSI significantly improved clinical pregnancy outcomes, despite non- against oxidative damage [321] and apoptosis [320]. Until significant differences detected in fertilization rates and the blastocyst stage of development is reached, GSH has embryo quality [332]. Conversely, studies have also reported been suggested to exert a protective effect on the embryo no apparent benefit of shortened co-incubation times over against harmful effects of ROS [322]. the standard overnight times [333]. Interestingly, Barraud- Heightened activity of ROS has been reported in vitrified Lange et al. (2008) found that co-incubation for 1 hour porcine oocytes matured in vitro [323] and has been actually resulted in significantly lower fertilization rates evidenced by decreased GSH and increased H2O2 [324]. compared to the standard length of 18 hours. Additionally, Peroxynitrite and NO2 have been suggested to be cytotoxic no significant improvement in embryo quality was observed to mouse oocytes by decreasing their survival and between the short (1 hour) and standard (18 hour) groups fertilization potential [325]. [334]. Whether short gamete co-incubation times definitively Maintaining a physiological and beneficial level of ROS improve IVF outcomes by limiting ROS-induced damage, balanced with antioxidants in the in vitro setting would while still maintaining fertilization rates remains controversial. closely replicate the reproductive environment in vivo. One Minimizing the Influence of Factors Affecting the major obstacle encountered during ART is the inability to Culture Media sufficiently and adequately emulate the in vivo reproductive environment. To this effect, the functions of antioxidants Culture media can generate ROS at different rates, within the FF [326] environment need to be explored in depending on its composition. Oocyte viability and greater depth. fertilization can compromise GSH content of oocytes and can be altered by OS generated within the culture media OVERCOMING DAMAGING EFFECTS OF ROS [317]. Embryos of poor quality were found to be cultured in Despite the apparent advancement of ART methods, less media with low TAC, which suggests that OS may have had than 40% of IVF cycles are successful. Failed fertilization a negative impact on embryo quality [335]. in ART is mainly attributed to poor quality of oocytes O2 Concentration [327]. Today, the goals of assisted reproduction have shifted away from obtaining a large number of oocytes; Meitotic maturation and ATP generation of bovine COCs scientists now aim to acquire oocytes of high quality [328]. were found to progress under conditions of low O2 tension As highlighted in our previous review [326], obtaining (<5%), as encountered in the vivo environment. Atmospheric levels of ROS in females seeking treatment for infertility O2 tension (~20%) has been shown to impair bovine oocytes could prove valuable in finding potential sources of the secondary to abundant generation of ROS [149]. Improved infertility. embryo development has also been demonstrated by

Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 195 decreasing the culture’s O2 concentration from 20% to 5% by supplementing the maturation medium with antioxidant [9, 270]. More recently, however, a meta-analysis reported compounds that could maintain cell viability by shielding that fertilization, implantation, and pregnancy rates did not against OS-induced damage. differ significantly between embryos cultured under low and Co-culture refers to the use of live human and non- 20% O2 concentrations [299]. human cells in the culture media surrounding the embryo. The atmospheric O2 concentration of 21% is significantly Co-culturing cells is one method of improving the higher than the 1.5%-6% O2 concentrations of the mammalian environment in which the embryo is growing. Co-culture oviduct and uterus. Although studies continue to produce with tubal epithelial cells enhances human, bovine, and conflicting results, it seems that in general, mammalian porcine IVF. Rates of implantation [341] and clinical embryo culture systems should employ low O2 concentrations pregnancy have shown improvement with cumulus co- [336, 337] similar to those in vivo. Further decreases of culture or cumulus-aided embryo transfer. A meta-analysis in vitro O2 concentration, however, should be avoided, as of 17 prospective randomized controlled studies revealed deleterious effects may result [315]. higher blastomere numbers, implantation, and improved rates of clinical and ongoing pregnancy with the use of co- Metallic Cations cultures for IVF [342]. Metal chelators such as EDTA and transferrin can Bovine IVM/IVF embryos showed enhanced development decrease ROS production by binding metallic cations like with bovine oviduct epithelial cell (BOEC) co-culture. The co- iron [9] and should be included in the culture medium [315, culture of bovine IVM/IVF embryos in BOEC + astaxanthin 338]. Transferrin, an iron-chelating agent [339], can suppress increased development of blastocysts significantly. The lipid peroxidation, shown by decreasing MDA levels, as well group concluded that the antioxidant effect of the carotenoid, as inhibit the formation of hydroxyl radicals [26, 340]. astaxanthin, on cell viability and lipid peroxidation of BOEC, was demonstrated through the observed increased Visible Light expression of the genes for the antioxidant enzymes Cu,Zn- SOD, Mn-SOD, and catalase, and decreased expression A decrease in the amount of time an embryo is exposed of apoptotic genes [343]. Results from Jeon et al. (2008) to visible light has been found to suppress culture media were in agreement and suggested that bovine embryo generation of ROS, resulting in higher quality embryos [9, 270, 317]. The exposure of oocytes and embryos to low developmental competence was improved by co-culture with BOEC + astaxanthin, evidenced by increased blastocyst intensity visible light was reported to minimize OS development, presumably as a result of astaxanthin-induced compared to exposure to cool white light [308]. Limited scavenging of NO radicals [344]. exposure of oocytes and embryos to visible light would likely benefit their development and thus, ART outcomes, by As suggested by Tatone et al. (2011), media used for more closely mimicking in vivo conditions. freezing and thawing may benefit from supplementation with antioxidants to maintain or even improve the quality Antioxidant Supplementation of ART Culture Media of aged oocytes [162]. Assessing FF levels of ROS through direct measurements and through TAC of retrieved Supplementing ART culture media with antioxidants oocytes will identify patients who would likely benefit continues to be investigated for its potential to counteract from having their oocytes cryopreserved and cultured in high levels of ROS, and perhaps improve ART success rates. media supplemented with antioxidants [170]. A greater High levels of ROS appear to suppress in vitro oocyte understanding of the role of OS in cryopreservation is clearly maturation and embryo development, although some level of needed before we can confidently establish optimal freezing ROS is likely beneficial to oocytes and embryos that are rates that will enhance pregnancy outcomes. matured, fertilized, and developed in vitro. In addition to its L-carnitine damaging effects on oocytes, sustained OS depletes the oocyte’s pool of antioxidants. Interestingly, adding anti- L-carnitine mediates the movement of long-chain fatty oxidants with lipophilic and hydrosoluble properties to acids from the cellular cytosol to the mitochondrial matrix oocyte’s culture media has been found to suppress OS, for energy production via oxidation [345] in addition to while improving embryo quality [317]. The presence of fueling the complexes of the mitochondrial ETC [346]. L- antioxidants in the culture media could curb the negative carnitine has been observed suppress high levels of H2O2, effects of ROS, although the mechanisms through which this while reducing apoptosis and DNA damage when added to occurs within in vitro culture is unclear. Further studies are the culture medium of mouse embryos. These results were needed to expand our understanding of these mechanisms. attributed to its potent antioxidant effects [347] and ability to As IVM is in its developing stages, scientists are counteract high levels of ROS, thus decreasing OS in the currently focusing on optimizing the maturation media. This IVF setting. remains a daunting task, which requires avoiding exogenous The addition of L-carnitine to the IVM medium of ROS while maintaining a delicate balance between pro- porcine oocytes was observed to improve developmental oxidants and antioxidants in an effort to closely replicate in competence, demonstrated by accelerated nuclear and vivo conditions. Increased survival of oocytes in vitro has cytoplasmic maturation and enhanced post-parthenogenetic been observed in association with the addition of ROS blastocyst development. Importantly, the addition of scavenging antioxidant enzymes to the maturation medium 0.5mg/mL of L-carnitine to IVM medium reduced oocyte [86]. Simulation of in vivo defense systems may be achieved levels of ROS and increased oocyte GSH levels significantly 196 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal compared to the control. Similarly, ROS levels in embryos and oocyte quality [359], thereby increasing pregnancy rates decreased when the same dose of L-carnitine was added of older women who desire children [131]. to the in vitro culture medium, although GSH content did Nitric Oxide not significantly differ with that of the control. In porcine embryos, the role of L-carnitine on cytoplasmic maturation Nitric oxide is abundantly present in the oocyte’s was demonstrated by the observed increase in GSH microenvironment and is an important regulator of concentration, while nuclear maturation enhanced by L- development through the early stages of the embryo [360]. carnitine was indicated by augmented nuclear progression The ability of NO to prevent oocyte aging has been shown in that resulted in better parthenogenetic embryo development both fresh and aged oocytes, and may be an attest to its [348]. known scavenging properties of ROS and lipid peroxyl Repeated ovulation occurs with aging and may involve radicals [361]. The ability to control the aging of oocytes in vitro mechanisms of OS. Accumulated OS in the ovarian matured could be a big step forward to improve environment could affect a woman’s ability to conceive as success rates when used for IVF or ICSI. her age increases [132]. Similar to results demonstrated Antioxidant Enzymes in IVM medium [306, 349], Miyamoto et al. (2010) reported that the consequences of ovarian aging via repeated The addition of the antioxidant enzyme catalase to the ovulation, such as decreased numbers of ovulated oocytes, culture medium of bovine embryos was found to enhance abnormal mitochondrial distribution in cells, and hampered their development when cultured under a high concentration progression to the blastocyst stage, could be curbed by orally of O2 (>20%), a setting known to fuel production of ROS [127]. supplementing L-carnitine [132]. Freeze-thaw media supplemented with SOD and Cytoskeleton damage in oocytes and apoptosis of hemoglobin separately or together increased survival and fertilization of mouse oocytes [362]. When hemoglobin is embryos incubated in peritoneal fluid of patients with - endometriosis was also decreased through incubation with autoxidized, O2 is produced and is detoxified by SOD L-carnitine [350]. In the future, the use of L-carnitine may throughout O2 metabolism in erythrocytes. However, in be useful to improve IVF outcomes in patients with the presence of excessive H2O2 production, H2O2 takes endometriosis. over the oxidization of oxyhemoglobin; SOD is then either useless, or may actually aid in degradation of hemoglobin The antioxidant-like properties of L-carnitine have been [363]. shown to shield OS-induced damage and apoptosis in an array of cell types [351-354]. L-carnitine may possess the Anthocyanin ability to scavenge free radicals, combat ROS, and chelate metal ions [355], and it may be through these mechanisms A reduction in ROS accompanied by increased GSH and that it decreases damage to oocytes and embryos. enhanced embryo development has been observed with supplementation of the antioxidant anthocyanin to the Coenzyme Q10 culture media of cloned porcine embryos [364]. Coenzyme Q10 (CoQ10), a natural lipid-soluble antioxidant involved in cell metabolism and energy Melatonin processes, is thought to mediate cellular redox potential by Poor oocyte quality is considered a major cause of regulating oxidation in mitochondrial DNA [356, 357]. infertility. Importantly, oocyte quality declines with Bentov et al. (2010) reported that supplementation of CoQ10 increasing age, but may also result from reproductive in bovine embryos cultured in vitro significantly enhanced pathologies such as PCOS. As an oxidizing agent and marker their development and increased ATP levels, indicating of OS, 8-OHdG measured in high levels in FF indicates poor improved embryo quality [358]. oocyte quality. In women with high FF levels of 8-OHdG, A preliminary study by Fujiwara et al. (2007) melatonin supplementation caused FF melatonin levels to investigated the role of coenzyme Q10, which is well known increase, as 8-OHdG levels decreased. It was concluded that for its antioxidant properties on aging murine ovaries. In OS was reduced by the antioxidant effects of melatonin. The mice administered CoQ10, significantly high levels of administration of melatonin to mice also resulted in CoQ10 were recorded in both venous blood and ovarian decreased oocyte damage secondary to ROS [105]. tissue. Importantly, systemic OS was reduced in mice treated The same group subsequently conducted a trial with with CoQ10 compared to control mice, although anti- women who had a prior IVF failure due to poor quality of oxidative stress did not differ significantly between the two oocytes, and presented their results at The World Congress groups. Furthermore, the ovaries of mice treated with CoQ10 on Fertility and Sterility [365]. Compared to untreated were found to weigh 30% more than those of control mice. controls, women supplemented with 3mg melatonin prior to Taken together, these results indicate that OS contributes to their next IVF cycle had substantially higher intrafollicular age-related decline of ovarian function, and that this levels of the hormone in conjunction with considerably oxidative damage could be prevented by administration of lower levels of 8-OHdG. Moreover, a fertilization rate CoQ10, as evidenced by improved ovarian function in of 50% of oocytes was reported for women supplemented treated aged mice. These results could potentially extend to with melatonin compared to the relatively unchanged humans and CoQ10 supplementation may prove promising 22.8% of oocytes for women in the control group. Successful for the prevention of age-related decline of ovarian function pregnancy occurred in 19% of supplemented women, Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 197 while only 10.2% in the control group achieved pregnancy In vitrified embryos, ascorbate was also shown to considerably following embryo transfer. According to Tamura (2010), reduce the production of H2O2, but was unable to do so on this trial was the first to implement the clinical use of slow-frozen embryos [376]. The slow-freezing method may melatonin to treat infertility [304]. The results of the study, be a more potent producer of H2O2 than vitrification. however, must be replicated by several future trials before Vitamins C and E have also demonstrated preventative melatonin can be considered as a treatment option for effects on age-related decline of ovarian function in mice infertile women with poor oocyte quality to improve both the [377, 378]. Successful IVF is thought to depend heavily on quality of their oocytes and fertilization rates. Further studies the presence of antioxidant compounds. Higher serum must also determine the mechanisms by which ROS causes concentrations of GSH as well as vitamins C and E, have damage to oocytes and the ways in which melatonin been reported with the use of oral vitamins and minerals ameliorates this damage through the reduction of OS in the [379]. oocyte. Melatonin is a well-known for its antioxidant properties Cysteine, Cysteamine, -mercaptoethanol as a scavenger of free O2 radicals and initiator of antioxidant Cysteamine and -mercaptoethanol are low molecular defense activities within cells [366]. During IVM of porcine weight thiols that promote the uptake of cystine. oocytes, melatonin has been suggested to enhance nuclear Supplementation of the maturation medium of denuded and cytoplasmic maturation [367]. The maturation of oocytes bovine oocytes with cysteine, cysteamine, and - has been shown to depend on varied levels of enzymes mercaptoethanol has been reported to advance embryo et al. such as Cu,Zn-SOD, Mn-SOD, and GPx [368]. Cetica development to the blastocyst stage [322], and measurements (1999) reported substantially decreased activity of SOD, of cysteine in particular can be used to gauge intracellular GPx, and catalase in denuded oocytes compared to cumulus- levels of GSH [380]. As such, studies have observed intact oocytes [369]. In melatonin-supplemented culture amplified production of GSH by oocytes in cysteine- et al. medium, Kim (2011) observed the expressions of supplemented media [381-384]. Gasparrini et al. (2003) and et al. Cu,Zn-SOD, Mn-SOD, and catalase [366]. El-Raey Beheshti et al. (2011) further reported that bovine oocytes (2011) measured markedly lower levels of ROS in bovine matured faster and were less likely to degenerate than were oocytes treated with melatonin compared with untreated controls, when matured in media supplemented with cysteine controls [370]. Taken together, the results from these studies [385, 386]. demonstrate the benefits of the ROS-scavenging properties of melatonin during the IVM process. Alpha-lipoic Acid Myo-inisitol +/- Melatonin In mice, improved follicular maturation and develop- High myo-inisitol content in FF has been associated with mental competence in vitro was recently achieved with IVM enhanced oocyte quality and maturation [371]. Treatment medium supplementation with alpha-lipoic acid versus non- with myo-inisitol has also been shown to decrease the supplemented controls [387]. The antioxidant properties of number of germinal vesicles and degenerated oocytes alpha-lipoic acid have been well established in biological without negatively affecting the number of oocytes retrieved systems [388]. It is also a direct ROS scavenger, a metal [372]. Given the seemingly positive effect of melatonin on chelator, and partakes in reutilizing other intracellular oocyte quality, Unfer et al. (2011) assessed the effect of antioxidants [389-391]. The group suggested that improved daily administration of combined myo-inisitol + melatonin developmental competence of pre-antral follicles in vitro was for 3 months on pregnancy outcomes in women with a a result of the observed reduced follicular ROS levels and previously failed IVF attempt due to low oocyte quality. The elevated TAC during culture [397]. combined treatment led to a significant increase in the number of mature oocytes, despite retrieval of the same N-acetyl-L-cyteine (NAC) number of oocytes as the previous cycle, in addition to Recently, oocyte aging in mice was reportedly delayed improved embryo quality. As a result, previously infertile with oral administration of NAC [392]. Liu et al. (2012) women with poor oocyte quality achieved better implantation administered NAC to two groups of mice in their drinking and pregnancy rates [373]. water- one group was administered NAC over a period of 2 months (short term) and the other group was given NAC Ascorbic Acid, Alpha Tocopherol over a period of 1 year (long term). At the end of both In mice, enhanced development of denuded oocytes intervals, both groups of mice exhibited improved the quality and COCs was reported in the presence of ascorbic acid. of fertilized oocytes and embryos. Additionally, mice treated It was also determined that a 250μM/ml dose of ascorbic with NAC long term produced larger litter sizes when they acid conferred protection against degeneration of cumulus were older, compared to age-matched controls. The group cells [374]. Results from an earlier study by Dalvit concluded that NAC’s antioxidant properties were effective et al. 2005, however, did not demonstrate developmental in delaying the process of aging in murine oocytes by changes in bovine oocytes cultured in maturation medium reducing ROS. supplemented with ascorbic acid, alpha tocopherol, or a The amide form of NAC, glutathione N-acetylcysteine combination of the two [375]. amide (NAC amide), is both a low molecular weight 2+ During cryopreservation, the presence of ascorbate seems thiol antioxidant and a Cu chelator. As a free radical to have positive effects on subsequent embryo development. scavenger, it can protect cells from oxidative damage [393]. 198 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

Supplementation of culture medium with NAC amide ABBREVIATIONS significantly increased intracellular GSH concentration of 8-iso-PGF2 = 8-iso-prostaglandin F2 oocytes, which correlated with a significantly higher percentage of embryos that progressed to the 2-cell and blastocyst stages 8-OHdG = 8-hydroxydeoxyguanosine than those in non-supplemented medium [394]. The increase in intracellular GSH concentrations may have suppressed ART = assisted reproductive techniques OS, thus enhancing oocyte quality and improving fertilization BMI = body mass index and early embryonic development in vitro. BOEC = bovine oviduct epithelial cell Weight Loss CC = cumulus cells Through diet and exercise regimens [186], overweight and obese women with or without comorbid PCOS may be COC = cumulus-oocyte complexes able to normalize their fertilization abilities [173] through CoQ10 = coenzyme Q10 resumption of ovulation [395], decreased insulin resistance, and improved oocyte quality. With weight loss, levels of the ETC = electron transport chain inflammatory markers CRP, IL-6, and TNF- that correlate FF = follicular fluid with BMI can be reduced [348, 396]. Even a minimal reduction in weight has been found to significantly improve GPx = glutathione peroxidase reproductive outcomes in women with high BMI [63, 397]. GSH = glutathione The underlying mechanisms through which BMI affects oocyte quality remain to be determined and warrant future GSSG = oxidized GSH investigation. Subfertile women with higher than normal H O = hydrogen peroxide BMI should be advised to lose weight, ideally prior to 2 2 attempting conception. Since obesity is a modifiable risk HSF = hydrosalpinx fluid factor for infertility, pre-conceptional counseling should highlight the importance of weight maintenance through ICSI = intracytoplasmic sperm injection proper diet and physical activity. IVF = in-vitro fertilization Smoking Cessation IVM = in-vitro maturation Women who actively smoke should be strongly advised MDA = malonaldehyde to quit prior to pregnancy to minimize the risk for experiencing negative effects of smoking on fecundity, NO = nitric oxide oocyte and embryo development, and ART success [192]. NOS = nitric oxide synthase CONCLUDING REMARKS OS = oxidative stress When ROS are overproduced and overwhelm the body’s Ox-LDL = oxidized LDL capacity of antioxidant defenses, a state of OS results. Several pathologies and factors such as aging, BMI, and PCOS = polycystic ovary syndrome smoking status have been associated with decreased female RNS = reactive nitrogen species fertility through mechanisms involving OS. The adverse effects of excess ROS production and the resulting OS on ROS = reactive oxygen species gametes and embryos are considered largely responsible for SOD = superoxide dismutase the poor reproductive outcomes documented with ART. Without the protection from antioxidant enzymes present TAC = total antioxidant capacity in vivo, sperm, oocytes, and embryos are left open to Trx = thioredoxin deleterious effects of surplus ROS production of the in vitro environment. Successful ART outcomes are contingent REFERENCES on the replication of reproductive conditions in vivo and in vitro [1] Snick HK, Snick TS, Evers JL, Collins JA. The spontaneous their use in the setting of ART. Some studies pregnancy prognosis in untreated subfertile couples: the Walcheren have demonstrated the effectiveness of antioxidant primary care study. Hum Reprod 1997; 12(7): 1582-8. supplementation both orally and in culture media to improve [2] Group REA-SPCW. Revised 2003 consensus on diagnostic criteria pregnancy outcomes. Overall, there is still insufficient data and long-term health risks related to polycystic ovary syndrome. to recommend their use, and whether they can benefit female Fertil Steril 2004; 81(1): 19-25. [3] Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen reproductive abilities is still controversial. species in the pathophysiology of human reproduction. Fertil Steril 2003; 79(4): 829-43. CONFLICT OF INTEREST [4] Metwally M, Cutting R, Tipton A, Skull J, Ledger WL, Li TC. Effect of increased body mass index on oocyte and embryo quality The authors declare that they have no competing in IVF patients. Reprod Biomed Online 2007; 15(5): 532-8. interests. [5] Kovacic P. Unifying mechanism for addiction and toxicity of abused drugs with application to dopamine and glutamate ACKNOWLEDGEMENTS mediators: electron transfer and reactive oxygen species. Med Hypotheses 2005; 65(1): 90-6. Declared none. Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 199

[6] Waylen AL, Metwally M, Jones GL, Wilkinson AJ, Ledger WL. [33] Halliwell B. Lipid peroxidation, antioxidants and cardiovascular Effects of cigarette smoking upon clinical outcomes of assisted disease: how should we move forward? Cardiovasc Res 2000; reproduction: a meta-analysis. Hum Reprod Update 2009; 15(1): 47(3): 410-8. 31-44. [34] Jones DP. Redefining oxidative stress. Antioxid Redox Signal [7] Dondorp W, de Wert G, Pennings G, et al. Lifestyle-related factors 2006; 8(9-10): 1865-79. and access to medically assisted reproduction. Hum Reprod 2010; [35] Jones DP. Radical-free biology of oxidative stress. Am J Physiol 25(3): 578-83. Cell Physiol 2008; 295(4): C849-68. [8] Penzias AS. Recurrent IVF failure: other factors. Fertil Steril 2012; [36] Hultqvist M, Olsson LM, Gelderman KA, Holmdahl R. The 97(5): 1033-8. protective role of ROS in autoimmune disease. Trends Immunol [9] Guerin P. OS and protection against ROS in the preimplantation 2009; 30(5): 201-8. embryo and its surroundings. HumReprodUpdate 2001; 7: 175-89. [37] Deng J, Wang X, Qian F, et al. Protective role of reactive oxygen [10] Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive species in endotoxin-induced lung inflammation through oxygen species, oxidative stress and antioxidants in pregnancy modulation of IL-10 expression. J Immunol 2012; 188(11): 5734- outcomes. Int J Biochem Cell Biol 2010; 42(10): 1634-50. 40. [11] Burton GJ, E J. Oxidative stress. Best Pract Res Clin Obstet [38] Yao H, Edirisinghe I, Yang SR, et al. Genetic ablation of NADPH Gynaecol 2010; 25(3): 287-99. oxidase enhances susceptibility to cigarette smoke-induced lung [12] Ruder EH, Hartman TJ, Goldman MB. Impact of oxidative stress inflammation and emphysema in mice. Am J Pathol 2008; 172(5): on female fertility. Curr Opin Obstet Gynecol 2009; 21(3): 219-22. 1222-37. [13] Chandra ASN, Kesavan SAA. Significance of oxidative stress in [39] Snelgrove RJ, Edwards L, Rae AJ, Hussell T. An absence of human reproduction. Arch Med 2009; 5(1A): 528-42. reactive oxygen species improves the resolution of lung influenza [14] Rhee SG. Cell signaling. H2O2, a necessary evil for cell signaling. infection. Eur J Immunol 2006; 36(6): 1364-73. Science 2006; 312(5782): 1882-3. [40] Komatsu J, Koyama H, Maeda N, Aratani Y. Earlier onset of [15] Imai M, Qin J, Tamakawa N, Miyado K, Umezawa A, Takahashi neutrophil-mediated inflammation in the ultraviolet-exposed skin Y. Molecular alterations during female reproductive aging: Can of mice deficient in myeloperoxidase and NADPH oxidase. aged oocytes remind youth? In: Pereira LV, editor. Embryology - Inflamm Res 2006; 55(5): 200-6. Updates and Highlights on Classic Topics: InTech; 2012; pp. 3-22. [41] Rada B, Leto TL. Oxidative innate immune defenses by Nox/Duox [16] Shkolnik K, Tadmor A, Ben-Dor S, Nevo N, Galiani D, Dekel N. family NADPH oxidases. Contrib Microbiol 2008; 15: 164-87. Reactive oxygen species are indispensable in ovulation. Proc Natl [42] Harvey AJ. The role of oxygen in ruminant preimplantation Acad Sci U S A 2011; 108(4): 1462-7. embryo development and metabolism. Anim Reprod Sci 2007; [17] Lopes AS, Lane M, Thompson JG. Oxygen consumption and ROS 98(1-2): 113-28. production are increased at the time of fertilization and cell [43] Zimniak P. Detoxification reactions: relevance to aging. Ageing cleavage in bovine zygotes. Hum Reprod 2010; 25(11): 2762-73. Res Rev 2008; 7(4): 281-300. [18] Burton GJ, Jauniaux E. Oxidative stress. Best Pract Res Clin Obstet [44] Landis GN, Tower J. Superoxide dismutase evolution and life span Gynaecol 2011; 25(3): 287-99. regulation. Mech Ageing Dev 2005; 126(3): 365-79. [19] Halliwell B. Oxidative stress and neurodegeneration: where are we [45] Perkins AV. Endogenous anti-oxidants in pregnancy and now? J Neurochem 2006; 97(6): 1634-58. preeclampsia. Aust N Z J Obstet Gynaecol 2006; 46(2): 77-83. [20] Fujii J, Iuchi Y, Okada F. Fundamental roles of reactive oxygen [46] Borchert A, Savaskan NE, Kuhn H. Regulation of expression of the species and protective mechanisms in the female reproductive phospholipid hydroperoxide/sperm nucleus glutathione peroxidase system. Reprod Biol Endocrinol 2005; 3: 43. gene. Tissue-specific expression pattern and identification of [21] Hancock JT, Desikan R, Neill SJ. Does the redox status of functional cis- and trans-regulatory elements. J Biol Chem 2003; cytochrome C act as a fail-safe mechanism in the regulation of 278(4): 2571-80. programmed cell death? Free Radic Biol Med 2001; 31(5): 697- [47] Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview 703. and speculative preview of novel mechanisms and emerging [22] Chandra A, Surti N, Kesavan S, Agarwal A. Significance of concepts in cell signaling. Free Radic Biol Med 2005; 38(12): oxidative stress in human reproduction. ArchMed 2009; 5(1A): 1543-52. S28-S42. [48] Borras C, Sastre J, Garcia-Sala D, Lloret A, Pallardo FV, Vina J. [23] Hanukoglu I. Antioxidant protective mechanisms against reactive Mitochondria from females exhibit higher antioxidant gene oxygen species (ROS) generated by mitochondrial P450 systems in expression and lower oxidative damage than males. Free Radic steroidogenic cells. Drug Metab Rev 2006; 38(1-2): 171-96. Biol Med 2003; 34(5): 546-52. [24] Agarwal AAS. Oxidants and antioxidants in human fertility. [49] Borras C, Gambini J, Lopez-Grueso R, Pallardo FV, Vina J. Direct Middle East Society Fertility J. 2004; 9(3): 187-97. antioxidant and protective effect of estradiol on isolated [25] Klaunig JE, Kamendulis LM. The role of oxidative stress in mitochondria. Biochim Biophys Acta 2010; 1802(1): 205-11. carcinogenesis. Annu Rev Pharmacol Toxicol 2004; 44: 239-67. [50] Russo J, Hasan Lareef M, Balogh G, Guo S, Russo IH. Estrogen [26] Emerit J, Beaumont C, Trivin F. Iron metabolism, free radicals, and and its metabolites are carcinogenic agents in human breast oxidative injury. Biomed Pharmacother 2001; 55(6): 333-9. epithelial cells. J Steroid Biochem Mol Biol 2003; 87(1): 1-25. [27] Jain S, Saxena D, Kumar GP, Laloraya M. NADPH dependent [51] Okoh V, Deoraj A, Roy D. Estrogen-induced reactive oxygen superoxide generation in the ovary and uterus of mice during species-mediated signalings contribute to breast cancer. Biochim estrous cycle and early pregnancy. Life Sci 2000; 66(12): 1139-46. Biophys Acta 2011; 1815(1): 115-33. [28] Velez-Pardo C, Morales AT, Del Rio MJ, Olivera-Angel M. [52] Ayres S, Abplanalp W, Liu JH, Subbiah MT. Mechanisms involved Endogenously generated hydrogen peroxide induces apoptosis via in the protective effect of estradiol-17beta on lipid peroxidation and mitochondrial damage independent of NF-kappaB and p53 DNA damage. Am J Physiol 1998; 274(6 Pt 1): E1002-8. activation in bovine embryos. Theriogenology 2007; 67(7): 1285- [53] Subbiah MT, Kessel B, Agrawal M, Rajan R, Abplanalp W, 96. Rymaszewski Z. Antioxidant potential of specific estrogens on [29] Roberts RA, Laskin DL, Smith CV, et al. Nitrative and oxidative lipid peroxidation. J Clin Endocrinol Metab 1993; 77(4): 1095-7. stress in toxicology and disease. Toxicol Sci 2009; 112(1): 4-16. [54] Sies H, Stahl W, Sevanian A. Nutritional, dietary and postprandial [30] Roede JR, Jones DP. Reactive species and mitochondrial oxidative stress. J Nutr 2005; 135(5): 969-72. dysfunction: mechanistic significance of 4-hydroxynonenal. [55] Lenaz G. Mitochondria and reactive oxygen species. Which role in Environ Mol Mutagen 2010; 51(5): 380-90. physiology and pathology? Adv Exp Med Biol 2012; 942: 93-136. [31] Tranguch S, Steuerwald N, Huet-Hudson YM. Nitric oxide [56] Behrman HR, Kodaman PH, Preston SL, Gao S. Oxidative stress synthase production and nitric oxide regulation of preimplantation and the ovary. J Soc Gynecol Investig 2001; 8(1 Suppl embryo development. Biol Reprod 2003; 68(5): 1538-44. Proceedings): S40-2. [32] Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. [57] Luck MR, Jeyaseelan I, Scholes RA. Ascorbic acid and fertility. Reactive oxygen species, lipid peroxidation and enzymatic defence Biol Reprod 1995; 52(2): 262-6. systems in human spermatozoa. J Reprod Fertil 1995; 103(1): [58] Zreik TG, Kodaman PH, Jones EE, Olive DL, Behrman H. 17-26. Identification and characterization of an ascorbic acid transporter in 200 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

human granulosa-lutein cells. Mol Hum Reprod 1999; 5(4): 299- throughout normal pregnancy. Am J Obstet Gynecol 1991; 165(6 302. Pt 1): 1690-4. [59] Zuelke KA, Jeffay SC, Zucker RM, Perreault SD. Glutathione [82] Montuschi P, Barnes PJ, Roberts LJ, 2nd. Isoprostanes: markers (GSH) concentrations vary with the cell cycle in maturing hamster and mediators of oxidative stress. FASEB J 2004; 18(15): 1791- oocytes, zygotes, and pre-implantation stage embryos. Mol Reprod 800. Dev 2003; 64(1): 106-12. [83] Morrow JD, Awad JA, Boss HJ, Blair IA, Roberts LJ, 2nd. Non- [60] Luberda Z. The role of glutathione in mammalian gametes. Reprod cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed Biol 2005; 5(1): 5-17. in situ on phospholipids. Proc Natl Acad Sci U S A 1992; 89(22): [61] Orsi NM, Gopichandran N, Leese HJ, Picton HM, Harris SE. 10721-5. Fluctuations in bovine ovarian follicular fluid composition [84] Pavlovic D, Dordevic V, G K. A ‘‘cross-talk’’ between oxidative throughout the oestrous cycle. Reproduction 2005; 129(2): 219-28. stress and redox cell signaling. Med Biol 2002; 19: 131-7. [62] Agarwal A, Gupta S, Sekhon L, Shah R. Redox considerations in [85] Sekhon L, Gupta S, Kim Y, Agarwal A. Female Infertility and female reproductive function and assisted reproduction: from Antioxidants. Curr Women’s Health Rev 2010; 6(2): 84-95. molecular mechanisms to health implications. Antioxid Redox [86] Yamaguchi K, Mandai M, Toyokuni S, et al. Contents of Signal 2008; 10(8): 1375-403. endometriotic cysts, especially the high concentration of free iron, [63] Ruder EH, Hartman TJ, Blumberg J, Goldman MB. Oxidative are a possible cause of carcinogenesis in the cysts through the iron- stress and antioxidants: exposure and impact on female fertility. induced persistent oxidative stress. Clin Cancer Res 2008; 14(1): Hum Reprod Update 2008; 14(4): 345-57. 32-40. [64] Su H, Messer R, Whitmire W, Fischer E, Portis JC, Caldwell HD. [87] Moore K, Roberts LJ, 2nd. Measurement of lipid peroxidation. Vaccination against chlamydial genital tract infection after Free Radic Res 1998; 28(6): 659-71. immunization with dendritic cells pulsed ex vivo with nonviable [88] Amirkhizi F, Siassi F, Minale S, et al. Plasma iron is associated Chlamydiae. J Exp Med 1998; 188(5): 809-18. with lipid peroxidation in women. ARYA Atherosclerosis J. 2006; [65] Strandell A, Lindhard A. Why does hydrosalpinx reduce fertility? 2(3): 134-7. The importance of hydrosalpinx fluid. Hum Reprod 2002; 17(5): [89] Cracowski JL, Durand T, Bessard G. Isoprostanes as a biomarker 1141-5. of lipid peroxidation in humans: physiology, pharmacology and [66] Chanr LY, Chiu PY, Lau TK. Hydrosalpinx fluid induced clinical implications. Trends Pharmacol Sci 2002; 23(8): 360-6. embryotoxicity and lipid peroxidation. Reprod Toxicol 2004; [90] Kobayashi H, Yamada Y, Kanayama S, et al. The role of iron in 19(1): 147-8. the pathogenesis of endometriosis. Gynecol Endocrinol 2009; [67] Bedaiwy MA, Goldberg JM, Falcone T, et al. Relationship between 25(1): 39-52. oxidative stress and embryotoxicity of hydrosalpingeal fluid. Hum [91] Mier-Cabrera J, Jimenez-Zamudio L, Garcia-Latorre E, Cruz- Reprod 2002; 17(3): 601-4. Orozco O, Hernandez-Guerrero C. Quantitative and qualitative [68] Giudice LC, Kao LC. Endometriosis. Lancet 2004; 364(9447): peritoneal immune profiles, T-cell apoptosis and oxidative stress- 1789-99. associated characteristics in women with minimal and mild [69] Carvalho L, Podgaec S, Bellodi-Privato M, Falcone T, Abrao MS. endometriosis. BJOG 2011; 118(1): 6-16. Role of eutopic endometrium in pelvic endometriosis. J Minim [92] Hart R, Hickey M, Franks S. Definitions, prevalence and symptoms Invasive Gynecol 2011; 18(4): 419-27. of polycystic ovaries and polycystic ovary syndrome. Best Pract [70] Augoulea A, Mastorakos G, Lambrinoudaki I, Christodoulakos G, Res Clin Obstet Gynaecol 2004; 18(5): 671-83. Creatsas G. The role of the oxidative-stress in the endometriosis- [93] March WA, Moore VM, Willson KJ, Phillips DI, Norman RJ, related infertility. Gynecol Endocrinol 2009; 25(2): 75-81. Davies MJ. The prevalence of polycystic ovary syndrome in a [71] Laloraya M, Jain S, Thomas M, Kopergaonkar S, Pradeep Kumar community sample assessed under contrasting diagnostic criteria. G. Estrogen surge: a regulatory switch for superoxide radical Hum Reprod 2010; 25(2): 544-51. generation at implantation. Biochem Mol Biol Int 1996; 39(5): [94] Hardiman P, Pillay OC, Atiomo W. Polycystic ovary syndrome and 933-40. endometrial carcinoma. Lancet 2003; 361(9371): 1810-2. [72] Catenacci M, Falcone T. Surgical Management Options for Patients [95] Gonzalez F, Rote NS, Minium J, Kirwan JP. Reactive oxygen with Infertility and Endometriosis. Curr Women’s Health Rev species-induced oxidative stress in the development of insulin 2010; 6(2): 161-6. resistance and hyperandrogenism in polycystic ovary syndrome. J [73] Mier-Cabrera M J-ZL, Garcia-Latorre E, Cruz-Orozco O, and Clin Endocrinol Metab 2006; 91(1): 336-40. Hernandez-Guerrero C. Quantitative and qualitative peritoneal [96] Kelly CC, Lyall H, Petrie JR, Gould GW, Connell JM, Sattar N. immune profiles, T-cell apoptosis and oxidative stress-associated Low grade chronic inflammation in women with polycystic ovarian characteristics in women with minimal and mild endometriosis. syndrome. J Clin Endocrinol Metab 2001; 86(6): 2453-5. BJOG 2010; 118: 6-16. [97] Samy N, Hashim M, Sayed M, Said M. Clinical significance of [74] Khan KN, Kitajima M, Imamura T, et al. Toll-like receptor 4- inflammatory markers in polycystic ovary syndrome: their mediated growth of endometriosis by human heat-shock protein 70. relationship to insulin resistance and body mass index. Dis Markers Hum Reprod 2008; 23(10): 2210-9. 2009; 26(4): 163-70. [75] Rong R, Ramachandran S, Santanam N, Murphy AA, Parthasarathy [98] Festa A, D'Agostino R, Howard G, Mykkanen L, Tracy RP, S. Induction of monocyte chemotactic protein-1 in peritoneal Haffner SM. Chronic subclinical inflammation as part of the insulin mesothelial and endometrial cells by oxidized low-density resistance syndrome: the Insulin Resistance Atherosclerosis Study lipoprotein and peritoneal fluid from women with endometriosis. (IRAS). Circulation 2004; 102: 42-7. Fertil Steril 2002; 78(4): 843-8. [99] Qiao J, Feng HL. Extra- and intra-ovarian factors in polycystic ovary [76] Jackson LW, Schisterman EF, Dey-Rao R, Browne R, Armstrong syndrome: impact on oocyte maturation and embryo developmental D. Oxidative stress and endometriosis. Hum Reprod 2005; 20(7): competence. Hum Reprod Update 2011; 17(1): 17-33. 2014-20. [100] Crist BL, Alekel DL, Ritland LM, Hanson LN, Genschel U, Reddy [77] Murphy AA, Santanam N, Parthasarathy S. Endometriosis: a MB. Association of oxidative stress, iron, and centralized fat mass disease of oxidative stress? Semin Reprod Endocrinol 1998; 16(4): in healthy postmenopausal women. J Womens Health (Larchmt) 263-73. 2009; 18(6): 795-801. [78] Szczepanska M, Kozlik J, Skrzypczak J, Mikolajczyk M. Oxidative [101] Ek I, Arner P, Ryden M, et al. A unique defect in the regulation of stress may be a piece in the endometriosis puzzle. Fertil Steril visceral fat cell lipolysis in the polycystic ovary syndrome as an 2003; 79(6): 1288-93. early link to insulin resistance. Diabetes 2002; 51(2): 484-92. [79] Sharma I, Dhaliwal LK, Saha SC, Sangwan S, Dhawan V. Role of [102] Sabuncu T, Vural H, Harma M. Oxidative stress in polycystic 8-iso-prostaglandin F2alpha and 25-hydroxycholesterol in the ovary syndrome and its contribution to the risk of cardiovascular pathophysiology of endometriosis. Fertil Steril 2010; 94(1): 63-70. disease. Clin Biochem 2001; 34(5): 407-13. [80] Polak G, Koziol-Montewka M, Gogacz M, Blaszkowska I, [103] Kuscu NK, Var A. Oxidative stress but not endothelial dysfunction Kotarski J. Total antioxidant status of peritoneal fluid in infertile exists in non-obese, young group of patients with polycystic ovary women. Eur J Obstet Gynecol Reprod Biol 2001; 94(2): 261-3. syndrome. Acta Obstet Gynecol Scand 2009; 88(5): 612-7. [81] Wang YP, Walsh SW, Guo JD, Zhang JY. Maternal levels of prostacyclin, thromboxane, vitamin E, and lipid peroxides Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 201

[104] Fedorcsak P, Storeng R, Dale PO, Tanbo T, Abyholm T. Obesity is [126] Suikkari AM. In-vitro maturation: its role in fertility treatment. a risk factor for early pregnancy loss after IVF or ICSI. Acta Obstet Curr Opin Obstet Gynecol 2008; 20(3): 242-8. Gynecol Scand 2000; 79(1): 43-8. [127] McKenzie LJ. Granulosa cells and their impact on human ART. In: [105] Dokras A, Baredziak L, Blaine J, Syrop C, VanVoorhis BJ, Sparks Gardner DK, Rizk B, Falcone T, Eds. Human Assisted A. Obstetric outcomes after in vitro fertilization in obese and Reproductive Technology: Future Trends for Laboratory and morbidly obese women. Obstet Gynecol 2006; 108(1): 61-9. Clinical Practice. New York: Cambridge University Press 2011; pp. [106] Ou XH, Li S, Wang ZB, et al. Maternal insulin resistance causes 209-22. oxidative stress and mitochondrial dysfunction in mouse oocytes. [128] Kim DH, Ko DS, Lee HC, et al. Comparison of maturation, Hum Reprod 2012; 27(7): 2130-45. fertilization, development, and gene expression of mouse oocytes [107] Victor VM, Rocha M, Banuls C, et al. Induction of oxidative stress grown in vitro and in vivo. J Assist Reprod Genet 2004; 21(7): 233- and human leukocyte/endothelial cell interactions in polycystic 40. ovary syndrome patients with insulin resistance. J Clin Endocrinol [129] Moon JH, Jee BC, Ku SY, et al. Spindle positions and their Metab 2011; 96(10): 3115-22. distributions in in vivo and in vitro matured mouse oocytes. Hum [108] Palacio JR, Iborra A, Ulcova-Gallova Z, Badia R, Martinez P. The Reprod 2005; 20(8): 2207-10. presence of antibodies to oxidative modified proteins in serum [130] Tatone C, Di Emidio G, Vento M, Ciriminna R, Artini PG. from polycystic ovary syndrome patients. Clin Exp Immunol 2006; Cryopreservation and oxidative stress in reproductive cells. 144(2): 217-22. Gynecol Endocrinol 2010; 26(8): 563-7. [109] Lee JY, Baw CK, Gupta S, Aziz N, Agarwal A. Role of Oxidative [131] Baird DT, Collins J, Egozcue J, et al. Fertility and ageing. Hum Stress in Polycystic Ovary Syndrome. Curr Women’s Health Rev Reprod Update 2005; 11(3): 261-76. 2010; 6(2): 96-107. [132] te Velde ER, Pearson PL. The variability of female reproductive [110] Das S, Chattopadhyay R, Ghosh S, Goswami SK, Chakravarty ageing. Hum Reprod Update 2002; 8(2): 141-54. BN, Chaudhury K. Reactive oxygen species level in follicular [133] Krysko DV, Diez-Fraile A, Criel G, Svistunov AA, Vandenabeele fluid--embryo quality marker in IVF? Hum Reprod 2006; 21(9): P, D'Herde K. Life and death of female gametes during oogenesis 2403-7. and folliculogenesis. Apoptosis 2008; 13(9): 1065-87. [111] Chattopadhayay R, Ganesh A, Samanta J, Jana SK, Chakravarty [134] Wright VC, Chang J, Jeng G, Macaluso M. Assisted reproductive BN, Chaudhury K. Effect of follicular fluid oxidative stress on technology surveillance--United States, 2003. MMWR Surveill meiotic spindle formation in infertile women with polycystic Summ 2006; 55(4): 1-22. ovarian syndrome. Gynecol Obstet Invest 2010; 69(3): 197-202. [135] Alviggi C, Humaidan P, Howles CM, Tredway D, Hillier SG. [112] Oyawoye OA, Abdel-Gadir A, Garner A, Leonard AJ, Perrett C, Biological versus chronological ovarian age: implications for Hardiman P. The interaction between follicular fluid total assisted reproductive technology. Reprod Biol Endocrinol 2009; 7: antioxidant capacity, infertility and early reproductive outcomes 101. during in vitro fertilization. Redox Rep 2009; 14(5): 205-13. [136] Leridon H. Can assisted reproduction technology compensate for [113] Polak G, Koziol-Montewka M, Tarkowski R, Kotarski J. Peritoneal the natural decline in fertility with age? A model assessment. Hum fluid and plasma 4-hydroxynonenal and malonyldialdehyde Reprod 2004; 19(7): 1548-53. concentrations in infertile women. Ginekol Pol 2001; 72(12A): [137] Wang Q, Sun QY. Evaluation of oocyte quality: morphological, 1316-20. cellular and molecular predictors. Reprod Fertil Dev 2007; 19(1): [114] Polak G, Rola R, Gogacz M, Koziol-Montewka M, Kotarski J. 1-12. Malonyldialdehyde and total antioxidant status in the peritoneal [138] Tatone C, Amicarelli F, Carbone MC, et al. Cellular and molecular fluid of infertile women. Ginekol Pol 1999; 70(3): 135-40. aspects of ovarian follicle ageing. Hum Reprod Update 2008; [115] Gupta S, Surti N, Metterle L, Chandra A, Agarwal A. Antioxidants 14(2): 131-42. and female reproductive pathologies. Arch Med 2009; 5: 151-73. [139] Tatone C. Oocyte senescence: a firm link to age-related female [116] Wang Y, Sharma RK, Falcone T, Goldberg J, Agarwal A. subfertility. Gynecol Endocrinol 2008; 24(2): 59-63. Importance of reactive oxygen species in the peritoneal fluid of [140] Coccia ME, Rizzello F. Ovarian reserve. Ann N Y Acad Sci 2008; women with endometriosis or idiopathic infertility. Fertil Steril 1127: 27-30. 1997; 68(5): 826-30. [141] Ottolenghi C, Uda M, Hamatani T, et al. Aging of oocyte, ovary, [117] Gupta S, Sekhon L, Agarwal A. The Role of Oxidative Stress and and human reproduction. Ann N Y Acad Sci 2004; 1034: 117-31. Antioxidants in Assisted Reproduction. Curr Women’s Health Rev [142] Broekmans FJ, Soules MR, Fauser BC. Ovarian aging: mechanisms 2010; 6: 227-38. and clinical consequences. Endocr Rev 2009; 30(5): 465-93. [118] Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG. [143] Tarin JJ. Aetiology of age-associated aneuploidy: a mechanism Oxidative stress in an assisted reproductive techniques setting. based on the 'free radical theory of ageing'. Hum Reprod 1995; Fertil Steril 2006; 86(3): 503-12. 10(6): 1563-5. [119] Andersen AN, Goossens V, Gianaroli L, Felberbaum R, de [144] Lim J, Luderer U. Oxidative damage increases and antioxidant Mouzon J, Nygren KG. Assisted reproductive technology in gene expression decreases with aging in the mouse ovary. Biol Europe, 2003. Results generated from European registers by Reprod 2011; 84(4): 775-82. ESHRE. Hum Reprod 2007; 22(6): 1513-25. [145] Salmon AB, Richardson A, Perez VI. Update on the oxidative [120] DeUgarte CM, DeCherney AH, Penzias AS. Assisted Reproductive stress theory of aging: does oxidative stress play a role in aging or Technologies: In Vitro Fertilzation and Related Techniques. In: healthy aging? Free Radic Biol Med 2010; 48(5): 642-55. Sydor AM, Edmonson K, Eds. Current Diagnosis & Treatment [146] Goud AP, Goud PT, Diamond MP, Gonik B, Abu-Soud HM. Obstetrics & Gynecology, Tenth Edition. 10th ed: The McGraw- Reactive oxygen species and oocyte aging: role of superoxide, Hill Companies 2007; pp. 948-53. hydrogen peroxide, and hypochlorous acid. Free Radic Biol Med [121] Lewis SE, Aitken RJ. DNA damage to spermatozoa has impacts on 2008; 44(7): 1295-304. fertilization and pregnancy. Cell Tissue Res 2005; 322(1): 33-41. [147] Rausell F, Pertusa JF, Gomez-Piquer V, et al. Beneficial effects of [122] Li JP, Zhong Y, Wu D, et al. Relationships among human follicular dithiothreitol on relative levels of glutathione S-transferase activity fluid-induced acrosome reaction, sperm morphology and in vitro and thiols in oocytes, and cell number, DNA fragmentation and fertilization rates. Zhonghua Nan Ke Xue 2006; 12(7): 605-7. allocation at the blastocyst stage in the mouse. Mol Reprod Dev [123] Patrizio P, Bianchi V, Lalioti MD, Gerasimova T, Sakkas D. High 2007; 74(7): 860-9. rate of biological loss in assisted reproduction: it is in the seed, not [148] Tarin JJ, Gomez-Piquer V, Pertusa JF, Hermenegildo C, Cano A. in the soil. Reprod Biomed Online 2007; 14(1): 92-5. Association of female aging with decreased parthenogenetic [124] Lee TH, Lee MS, Liu CH, Tsao HM, Huang CC, Yang YS. The activation, raised MPF, and MAPKs activities and reduced levels association between microenvironmental reactive oxygen species of glutathione S-transferases activity and thiols in mouse oocytes. and embryo development in assisted reproduction technology Mol Reprod Dev 2004; 69(4): 402-10. cycles. Reprod Sci 2012; 19(7): 725-32. [149] Tatone C, Di Emidio G, Barbaro R, Vento M, Ciriminna R, Artini [125] Demirtas E, Holzer H, Gidoni Y, Chian RC, Lin Tan S. In Vitro PG. Effects of reproductive aging and postovulatory aging on the Maturation of Human Oocytes. In: Rizk B, editor. Infertility and maintenance of biological competence after oocyte vitrification: Assisted Reproduction. New York: Cambridge University Press insights from the mouse model. Theriogenology 2011; 76(5): 2008; p p. 448-55. 864-73. 202 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

[150] Takahashi T, Igarashi H, Kawagoe J, Amita M, Hara S, Kurachi H. [173] Jarvie E, Hauguel-de-Mouzon S, Nelson SM, Sattar N, Catalano Poor embryo development in mouse oocytes aged in vitro is PM, Freeman DJ. Lipotoxicity in obese pregnancy and its potential associated with impaired calcium homeostasis. Biol Reprod 2009; role in adverse pregnancy outcome and obesity in the offspring. 80(3): 493-502. Clin Sci (Lond) 2010; 119(3): 123-9. [151] Carbone MC, Tatone C, Delle Monache S, et al. Antioxidant [174] Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, enzymatic defences in human follicular fluid: characterization and Ferrante AW, Jr. Obesity is associated with macrophage age-dependent changes. Mol Hum Reprod 2003; 9(11): 639-43. accumulation in adipose tissue. J Clin Invest 2003; 112(12): 1796- [152] Tatone C, Carbone MC, Gallo R, et al. Age-associated changes in 808. mouse oocytes during postovulatory in vitro culture: possible role [175] Vincent HK, Powers SK, Dirks AJ, Scarpace PJ. Mechanism for for meiotic kinases and survival factor BCL2. Biol Reprod 2006; obesity-induced increase in myocardial lipid peroxidation. Int J 74(2): 395-402. Obes Relat Metab Disord 2001; 25(3): 378-88. [153] Wiener-Megnazi Z, Vardi L, Lissak A, et al. Oxidative stress indices [176] Bausenwein J, Serke H, Eberle K, et al. Elevated levels of oxidized in follicular fluid as measured by the thermochemiluminescence low-density lipoprotein and of catalase activity in follicular fluid of assay correlate with outcome parameters in in vitro fertilization. obese women. Mol Hum Reprod 2010; 16(2): 117-24. Fertil Steril 2004; 82 (Suppl 3): 1171-6. [177] Comhaire F, Decleer W. Comparing the effectiveness of infertility [154] Miyamoto K, Sato EF, Kasahara E, et al. Effect of oxidative stress treatments by numbers needed to treat (NNT). Andrologia 2012; during repeated ovulation on the structure and functions of the doi: 10.1111/j.1439-0272.2012.01295.x. [Epub ahead of print]. ovary, oocytes, and their mitochondria. Free Radic Biol Med 2010; [178] Harper ME, Green K, Brand MD. The efficiency of cellular energy 49(4): 674-81. transduction and its implications for obesity. Annu Rev Nutr 2008; [155] Tanghe S, Van Soom A, Nauwynck H, Coryn M, de Kruif A. 28: 13-33. Minireview: Functions of the cumulus oophorus during oocyte [179] Cui H, Kong Y, Zhang H. Oxidative stress, mitochondrial maturation, ovulation, and fertilization. Mol Reprod Dev 2002; dysfunction, and aging. J Signal Transduct 2012; 2012: 646354. 61(3): 414-24. [180] Vujisic S, Zidovec S. Follicular Immunology Environment and the [156] Miao YL, Liu XY, Qiao TW, Miao DQ, Luo MJ, Tan JH. Cumulus Influence on In Vitro Fertilization Outcome. Curr Women’s Health cells accelerate aging of mouse oocytes. Biol Reprod 2005; 73(5): Rev 2005; 1(1): 49-60. 1025-31. [181] Robker RL. Evidence that obesity alters the quality of oocytes and [157] Tatemoto H, Muto N, Sunagawa I, Shinjo A, Nakada T. Protection embryos. Pathophysiology 2008; 15(2): 115-21. of porcine oocytes against cell damage caused by oxidative stress [182] Bellver J, Ayllon Y, Ferrando M, et al. Female obesity impairs in during in vitro maturation: role of superoxide dismutase activity in vitro fertilization outcome without affecting embryo quality. Fertil porcine follicular fluid. Biol Reprod 2004; 71(4): 1150-7. Steril 2010; 93(2): 447-54. [158] Victorino VJ, Panis C, Campos FC, et al. Decreased oxidant profile [183] Luke B, Brown MB, Stern JE, Missmer SA, Fujimoto VY, Leach and increased antioxidant capacity in naturally postmenopausal R. Female obesity adversely affects assisted reproductive women. Age (Dordr) 2012; [Epub ahead of print]. technology (ART) pregnancy and live birth rates. Hum Reprod [159] Fussell KC, Udasin RG, Smith PJ, Gallo MA, Laskin JD. Catechol 2011; 26(1): 245-52. metabolites of endogenous estrogens induce redox cycling and [184] Sathya A, Balasubramanyam S, Gupta S, Verma T. Effect of body generate reactive oxygen species in breast epithelial cells. mass index on in vitro fertilization outcomes in women. J Hum Carcinogenesis 2011; 32(8): 1285-93. Reprod Sci 2010; 3(3): 135-8. [160] Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and [185] Vilarino FL, Christofolini DM, Rodrigues D, et al. Body mass trends in obesity among US adults, 1999-2008. JAMA 2010; index and fertility: is there a correlation with human reproduction 303(3): 235-41. outcomes? Gynecol Endocrinol 2011; 27(4): 232-6. [161] Metwally M, Ong KJ, Ledger WL, Li TC. Does high body mass [186] van der Steeg JW, Steures P, Eijkemans MJ, et al. Obesity affects index increase the risk of miscarriage after spontaneous and spontaneous pregnancy chances in subfertile, ovulatory women. assisted conception? A meta-analysis of the evidence. Fertil Steril Hum Reprod 2008; 23(2): 324-8. 2008; 90(3): 714-26. [187] Freeman EW, Gracia CR, Sammel MD, Lin H, Lim LC, Strauss JF, [162] Metwally M, Li TC, Ledger WL. The impact of obesity on female 3rd. Association of anti-mullerian hormone levels with obesity in reproductive function. Obes Rev 2007; 8(6): 515-23. late reproductive-age women. Fertil Steril 2007; 87(1): 101-6. [163] Maheshwari A, Stofberg L, Bhattacharya S. Effect of overweight [188] Esinler I, Bozdag G, Yarali H. Impact of isolated obesity on ICSI and obesity on assisted reproductive technology--a systematic outcome. Reprod Biomed Online 2008; 17(4): 583-7. review. Hum Reprod Update 2007; 13(5): 433-44. [189] Abenhaim HA, Kinch RA, Morin L, Benjamin A, Usher R. Effect [164] Bellver J, Rossal LP, Bosch E, et al. Obesity and the risk of of prepregnancy body mass index categories on obstetrical and spontaneous abortion after oocyte donation. Fertil Steril 2003; neonatal outcomes. Arch Gynecol Obstet 2007; 275(1): 39-43. 79(5): 1136-40. [190] Minge CE, Bennett BD, Norman RJ, Robker RL. Peroxisome [165] Bellver J, Melo MA, Bosch E, Serra V, Remohi J, Pellicer A. proliferator-activated receptor-gamma agonist rosiglitazone Obesity and poor reproductive outcome: the potential role of the reverses the adverse effects of diet-induced obesity on oocyte endometrium. Fertil Steril 2007; 88(2): 446-51. quality. Endocrinology 2008; 149(5): 2646-56. [166] Igosheva N, Abramov AY, Poston L, et al. Maternal diet-induced [191] La Vignera S, Condorelli R, Bellanca S, et al. Obesity is associated obesity alters mitochondrial activity and redox status in mouse with a higher level of pro-inflammatory cytokines in follicular fluid oocytes and zygotes. PLoS One 2010; 5(4): e10074. of women undergoing medically assisted procreation (PMA) [167] Robker RL, Wu LL, Yang X. Inflammatory pathways linking obesity programs. Eur Rev Med Pharmacol Sci 2011; 15(3): 267-73. and ovarian dysfunction. J Reprod Immunol 2011; 88(2): 142-8. [192] Anderson K, Nisenblat V, Norman R. Lifestyle factors in people [168] Rains JL, Jain SK. Oxidative stress, insulin signaling, and diabetes. seeking infertility treatment - A review. Aust N Z J Obstet Free Radic Biol Med 2011; 50(5): 567-75. Gynaecol 2010; 50(1): 8-20. [169] Brown LA, Kerr CJ, Whiting P, Finer N, McEneny J, Ashton T. [193] Robker RL, Akison LK, Bennett BD, et al. Obese women exhibit Oxidant stress in healthy normal-weight, overweight, and obese differences in ovarian metabolites, hormones, and gene expression individuals. Obesity (Silver Spring) 2009; 17(3): 460-6. compared with moderate-weight women. J Clin Endocrinol Metab [170] Crescenzo R, Bianco F, Falcone I, Coppola P, Liverini G, Iossa S. 2009; 94(5): 1533-40. Increased hepatic de novo lipogenesis and mitochondrial efficiency [194] Sneed ML, Uhler ML, Grotjan HE, Rapisarda JJ, Lederer KJ, in a model of obesity induced by diets rich in fructose. Eur J Nutr Beltsos AN. Body mass index: impact on IVF success appears age- 2012 [Epub ahead of print]. related. Hum Reprod 2008; 23(8): 1835-9. [171] Bloomer RJ, Fisher-Wellman KH. Systemic oxidative stress is [195] Luke B, Brown MB, Missmer SA, Bukulmez O, Leach R, Stern JE. increased to a greater degree in young, obese women following The effect of increasing obesity on the response to and outcome of consumption of a high fat meal. Oxid Med Cell Longev 2009; 2(1): assisted reproductive technology: a national study. Fertil Steril 19-25. 2011; 96(4): 820-5. [172] Mistry HD, Williams PJ. The importance of antioxidant [196] Ferreira RC, Halpern G, Figueira Rde C, Braga DP, Iaconelli A, Jr., micronutrients in pregnancy. Oxid Med Cell Longev 2011; 2011: Borges E, Jr. Physical activity, obesity and eating habits can 841749. Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 203

influence assisted reproduction outcomes. Womens Health (Lond effects with other respirable particles. Int J Environ Res Public Engl) 2010; 6(4): 517-24. Health 2009; 6(2): 445-62. [197] Pasquali R, Gambineri A. Metabolic effects of obesity on [220] Northrop-Clewes CA, Thurnham DI. Monitoring micronutrients in reproduction. Reprod Biomed Online 2006; 12(5): 542-51. cigarette smokers. Clin Chim Acta 2007; 377(1-2): 14-38. [198] Shah DK, Missmer SA, Berry KF, Racowsky C, Ginsburg ES. [221] Tagawa Y, Hiramatsu N, Kasai A, et al. Induction of apoptosis by Effect of obesity on oocyte and embryo quality in women cigarette smoke via ROS-dependent endoplasmic reticulum stress undergoing in vitro fertilization. Obstet Gynecol 2011; 118(1): and CCAAT/enhancer-binding protein-homologous protein 63-70. (CHOP). Free Radic Biol Med 2008; 45(1): 50-9. [199] Rittenberg V, Sobaleva S, Ahmad A, et al. Influence of BMI on [222] Neal MS, Foster WG, Younglai EV. The Detrimental Effect of risk of miscarriage after single blastocyst transfer. Hum Reprod Smoking on Female Fertility and IVF Success. Curr Women’s 2011; 26(10): 2642-50. Health Rev 2008; 4(1): 16-24. [200] Depalo R, Garruti G, Totaro I, et al. Oocyte morphological [223] Tiboni GM, Bucciarelli T, Giampietro F, Sulpizio M, Di Ilio C. abnormalities in overweight women undergoing in vitro Influence of cigarette smoking on vitamin E, vitamin A, beta- fertilization cycles. Gynecol Endocrinol 2011; 27(11): 880-4. carotene and lycopene concentrations in human pre-ovulatory [201] Kahraman S, Yakin K, Donmez E, et al. Relationship between follicular fluid. Int J Immunopathol Pharmacol 2004; 17(3): granular cytoplasm of oocytes and pregnancy outcome following 389-93. intracytoplasmic sperm injection. Hum Reprod 2000; 15(11): [224] Neal MS, Zhu J, Foster WG. Quantification of benzo[a]pyrene and 2390-3. other PAHs in the serum and follicular fluid of smokers versus [202] Wittemer C, Ohl J, Bailly M, Bettahar-Lebugle K, Nisand I. Does non-smokers. Reprod Toxicol 2008; 25(1): 100-6. body mass index of infertile women have an impact on IVF [225] Al-Saleh I, El-Doush I, Arif J, et al. Levels of DNA adducts in the procedure and outcome? J Assist Reprod Genet 2000; 17(10): 547- blood and follicular fluid of women undergoing in vitro 52. fertilization treatment and its correlation with the pregnancy [203] Frattarelli JL, Kodama CL. Impact of body mass index on in vitro outcome. Bull Environ Contam Toxicol 2010; 84(1): 23-8. fertilization outcomes. J Assist Reprod Genet 2004; 21(6): 211-5. [226] Wang Y. Bulky DNA lesions induced by reactive oxygen species. [204] Lashen H, Ledger W, Bernal AL, Barlow D. Extremes of body Chem Res Toxicol 2008; 21(2): 276-81. mass do not adversely affect the outcome of superovulation and in- [227] Soares SR, Simon C, Remohi J, Pellicer A. Cigarette smoking vitro fertilization. Hum Reprod 1999; 14(3): 712-5. affects uterine receptiveness. Hum Reprod 2007; 22(2): 543-7. [205] Nichols JE, Crane MM, Higdon HL, Miller PB, Boone WR. [228] Soares SR, Melo MA. Cigarette smoking and reproductive Extremes of body mass index reduce in vitro fertilization function. Curr Opin Obstet Gynecol 2008; 20(3): 281-91. pregnancy rates. Fertil Steril 2003; 79(3): 645-7. [229] Freour T, Masson D, Mirallie S, et al. Active smoking [206] Wang JX, Davies M, Norman RJ. Body mass and probability of compromises IVF outcome and affects ovarian reserve. Reprod pregnancy during assisted reproduction treatment: retrospective Biomed Online 2008; 16(1): 96-102. study. BMJ 2000; 321(7272): 1320-1. [230] Tuttle AM, Stampfli M, Foster WG. Cigarette smoke causes [207] Farhi J, Ben-Haroush A, Sapir O, Fisch B, Ashkenazi J. High- follicle loss in mice ovaries at concentrations representative of quality embryos retain their implantation capability in overweight human exposure. Hum Reprod 2009; 24(6): 1452-9. women. Reprod Biomed Online 2010; 21(5): 706-11. [231] Huang J, Okuka M, McLean M, Keefe DL, Liu L. Effects of [208] Bouyer J, Coste J, Shojaei T, et al. Risk factors for ectopic cigarette smoke on fertilization and embryo development in vivo. pregnancy: a comprehensive analysis based on a large case-control, Fertil Steril 2009; 92(4): 1456-65. population-based study in France. Am J Epidemiol 2003; 157(3): [232] Bordel R, Laschke MW, Menger MD, Vollmar B. Nicotine does 185-94. not affect vascularization but inhibits growth of freely transplanted [209] Shaw JL, Oliver E, Lee KF, et al. Cotinine exposure increases ovarian follicles by inducing granulosa cell apoptosis. Hum Reprod Fallopian tube PROKR1 expression via nicotinic AChRalpha-7: a 2006; 21(3): 610-7. potential mechanism explaining the link between smoking and [233] Neal MS, Zhu J, Holloway AC, Foster WG. Follicle growth is tubal ectopic pregnancy. Am J Pathol 2010; 177(5): 2509-15. inhibited by benzo-[a]-pyrene, at concentrations representative of [210] Baird DD, Wilcox AJ. Cigarette smoking associated with delayed human exposure, in an isolated rat follicle culture assay. Hum conception. JAMA 1985; 253(20): 2979-83. Reprod 2007; 22(4): 961-7. [211] Alderete E, Eskenazi B, Sholtz R. Effect of cigarette smoking and [234] Barbieri RL, Sluss PM, Powers RD, et al. Association of body coffee drinking on time to conception. Epidemiology 1995; 6(4): mass index, age, and cigarette smoking with serum testosterone 403-8. levels in cycling women undergoing in vitro fertilization. Fertil [212] Hull MG, North K, Taylor H, Farrow A, Ford WC. Delayed Steril 2005; 83(2): 302-8. conception and active and passive smoking. The Avon [235] Sobinoff AP, Pye V, Nixon B, Roman SD, McLaughlin EA. Longitudinal Study of Pregnancy and Childhood Study Team. Jumping the gun: smoking constituent BaP causes premature Fertil Steril 2000; 74(4): 725-33. primordial follicle activation and impairs oocyte fusibility through [213] Neal MS, Hughes EG, Holloway AC, Foster WG. Sidestream oxidative stress. Toxicol Appl Pharmacol 2012; 260(1): 70-80. smoking is equally as damaging as mainstream smoking on IVF [236] Fuentes A, Munoz A, Barnhart K, Arguello B, Diaz M, Pommer R. outcomes. Hum Reprod 2005; 20(9): 2531-5. Recent cigarette smoking and assisted reproductive technologies [214] Ornoy A. Embryonic oxidative stress as a mechanism of outcome. Fertil Steril 2010; 93(1): 89-95. teratogenesis with special emphasis on diabetic embryopathy. [237] Chelchowska M, Ambroszkiewicz J, Gajewska J, Laskowska-Klita Reprod Toxicol 2007; 24(1): 31-41. T, Leibschang J. The effect of tobacco smoking during pregnancy [215] Dechanet C, Anahory T, Mathieu Daude JC, et al. Effects of on plasma oxidant and antioxidant status in mother and newborn. cigarette smoking on reproduction. Hum Reprod Update 2011; Eur J Obstet Gynecol Reprod Biol 2011; 155(2): 132-6. 17(1): 76-95. [238] Talukder MA, Johnson WM, Varadharaj S, et al. Chronic [216] Benedict MD, Missmer SA, Vahratian A, et al. Secondhand cigarette smoking causes hypertension, increased oxidative stress, tobacco smoke exposure is associated with increased risk of failed impaired NO bioavailability, endothelial dysfunction, and cardiac implantation and reduced IVF success. Hum Reprod 2011; 26(9): remodeling in mice. Am J Physiol Heart Circ Physiol 2011; 300(1): 2525-31. H388-96. [217] Gruslin A, Qiu Q, Tsang BK. Influence of maternal smoking on [239] Stacchiotti A, Morandini F, Bettoni F, et al. Stress proteins and trophoblast apoptosis throughout development: possible oxidative damage in a renal derived cell line exposed to inorganic involvement of Xiap regulation. Biol Reprod 2001; 65(4): 1164-9. mercury and lead. Toxicology 2009; 264(3): 215-24. [218] Genbacev O, McMaster MT, Zdravkovic T, Fisher SJ. Disruption [240] Ben-Haroush A, Ashkenazi J, Sapir O, Pinkas H, Fisch B, Farhi J. of oxygen-regulated responses underlies pathological changes in High-quality embryos maintain high pregnancy rates in passive the placentas of women who smoke or who are passively exposed smokers but not in active smokers. Reprod Biomed Online 2011; to smoke during pregnancy. Reprod Toxicol 2003; 17(5): 509-18. 22(1): 44-9. [219] Valavanidis A, Vlachogianni T, Fiotakis K. Tobacco smoke: [241] Lonergan P, Rizos D, Kanka J, et al. Temporal sensitivity of involvement of reactive oxygen species and stable free radicals in bovine embryos to culture environment after fertilization and the mechanisms of oxidative damage, carcinogenesis and synergistic 204 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

implications for blastocyst quality. Reproduction 2003; 126(3): fertilization-embryo transfer program. Fertil Steril 2002; 77(6): 337-46. 1184-90. [242] Rodriguez KF, Farin CE. Gene transcription and regulation of [266] Fujimoto VY, Bloom MS, Huddleston HG, Shelley WB, Ocque AJ, oocyte maturation. Reprod Fertil Dev 2004; 16(1-2): 55-67. Browne RW. Correlations of follicular fluid oxidative stress [243] Harvey AJ, Kind KL, Thompson JG. REDOX regulation of early biomarkers and enzyme activities with embryo morphology embryo development. Reproduction 2002; 123(4): 479-86. parameters during in vitro fertilization. Fertil Steril 2011; 96(6): [244] Oyawoye O, Abdel Gadir A, Garner A, Constantinovici N, Perrett 1357-61. C, Hardiman P. Antioxidants and reactive oxygen species in [267] Pasqualotto EB, Agarwal A, Sharma RK, et al. Effect of oxidative follicular fluid of women undergoing IVF: relationship to outcome. stress in follicular fluid on the outcome of assisted reproductive Hum Reprod 2003; 18(11): 2270-4. procedures. Fertil Steril 2004; 81(4): 973-6. [245] Leese HJ, Hugentobler SA, Gray SM, et al. Female reproductive [268] Combelles CM, Gupta S, Agarwal A. Could oxidative stress tract fluids: composition, mechanism of formation and potential influence the in-vitro maturation of oocytes? Reprod Biomed role in the developmental origins of health and disease. Reprod Online 2009; 18(6): 864-80. Fertil Dev 2008; 20(1): 1-8. [269] Dennery PA. Oxidative stress in development: nature or nurture? [246] Leese HJ. Quiet please, do not disturb: a hypothesis of embryo Free Radic Biol Med 2010; 49(7): 1147-51. metabolism and viability. Bioessays 2002; 24(9): 845-9. [270] Agarwal A, Gupta S, Sharma R. Oxidative stress and its [247] Tarozzi N, Bizzaro D, Flamigni C, Borini A. Clinical relevance of implications in female infertility - a clinician's perspective. Reprod sperm DNA damage in assisted reproduction. Reprod Biomed Biomed Online 2005; 11(5): 641-50. Online 2007; 14(6): 746-57. [271] Kawamura Y, Uchijima Y, Horike N, et al. Sirt3 protects in vitro- [248] Yumura Y, Iwasaki A, Saito K, Ogawa T, Hirokawa M. Effect of fertilized mouse preimplantation embryos against oxidative stress- reactive oxygen species in semen on the pregnancy of infertile induced p53-mediated developmental arrest. J Clin Invest 2010; couples. Int J Urol 2009; 16(2): 202-7. 120(8): 2817-28. [249] Iwasaki A, Gagnon C. Formation of reactive oxygen species in [272] Wiener-Megnazi Z, Shiloh H, Avraham L, et al. Oxidative spermatozoa of infertile patients. Fertil Steril 1992; 57(2): 409-16. parameters of embryo culture media may predict treatment [250] Agarwal A, Ikemoto I, Loughlin KR. Relationship of sperm outcome in in vitro fertilization: a novel applicable tool for parameters with levels of reactive oxygen species in semen improving embryo selection. Fertil Steril 2011; 95(3): 979-84. specimens. J Urol 1994; 152(1): 107-10. [273] du Plessis SS, Makker K, Desai NR, Agarwal A. Impact of [251] Sikka SC. Role of oxidative stress response elements and oxidative stress on IVF. Expert Rev Obstet Gynecol 2008; 3(4): antioxidants in prostate cancer pathobiology and chemoprevention- 539=54. -a mechanistic approach. Curr Med Chem 2003; 10(24): 2679-92. [274] Bedaiwy MA, Falcone T, Mohamed MS, et al. Differential growth [252] Bakos HW, Thompson JG, Feil D, Lane M. Sperm DNA damage is of human embryos in vitro: role of reactive oxygen species. Fertil associated with assisted reproductive technology pregnancy. Int J Steril 2004; 82(3): 593-600. Androl 2008; 31(5): 518-26. [275] Enkhmaa D, Kasai T, Hoshi K. Long-time exposure of mouse [253] Shimura T, Inoue M, Taga M, et al. p53-dependent S-phase embryos to the sperm produces high levels of reactive oxygen damage checkpoint and pronuclear cross talk in mouse zygotes species in culture medium and relates to poor embryo development. with X-irradiated sperm. Mol Cell Biol 2002; 22(7): 2220-8. Reprod Domest Anim 2009; 44(4): 634-7. [254] Agung B, Otoi T, Wongsrikeao P, et al. Effect of maturation [276] Huang Z, Li J, Wang L, Yan J, Shi Y, Li S. Brief co-incubation of culture period of oocytes on the sex ratio of in vitro fertilized sperm and oocytes for in vitro fertilization techniques. Cochrane bovine embryos. J Reprod Dev 2006; 52(1): 123-7. Database of Systematic Rev 2011; (10): 1-11. [255] Suzuki T, Sugino N, Fukaya T, et al. Superoxide dismutase in [277] Agarwal A, Allamaneni SS, Nallella KP, George AT, Mascha E. normal cycling human ovaries: immunohistochemical localization Correlation of reactive oxygen species levels with the fertilization and characterization. Fertil Steril 1999; 72(4): 720-6. rate after in vitro fertilization: a qualified meta-analysis. Fertil [256] Shiotani M, Noda Y, Narimoto K, et al. Immunohistochemical Steril 2005; 84(1): 228-31. localization of superoxide dismutase in the human ovary. Hum [278] Simon L, Lutton D, McManus J, Lewis SE. Sperm DNA damage Reprod 1991; 6(10): 1349-53. measured by the alkaline Comet assay as an independent predictor [257] Jana SK, K NB, Chattopadhyay R, Chakravarty B, Chaudhury K. of male infertility and in vitro fertilization success. Fertil Steril Upper control limit of reactive oxygen species in follicular fluid 2011; 95(2): 652-7. beyond which viable embryo formation is not favorable. Reprod [279] Henkel R, Kierspel E, Hajimohammad M, et al. DNA fragmentation Toxicol 2010; 29(4): 447-51. of spermatozoa and assisted reproduction technology. Reprod [258] Attaran M, Pasqualotto E, Falcone T, et al. The effect of follicular Biomed Online 2003; 7(4): 477-84. fluid reactive oxygen species on the outcome of in vitro [280] Fuller B, Paynter S. Fundamentals of cryobiology in reproductive fertilization. Int J Fertil Womens Med 2000; 45(5): 314-20. medicine. Reprod Biomed Online 2004; 9(6): 680-91. [259] Baka S, Malamitsi-Puchner A. Novel follicular fluid factors [281] Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of influencing oocyte developmental potential in IVF: a review. human embryos by vitrification or slow freezing: which one is Reprod Biomed Online 2006; 12(4): 500-6. better? Curr Opin Obstet Gynecol 2009; 21(3): 270-4. [260] Hammadeh ME, Fischer-Hammadeh C, Amer AS, Rosenbaum [282] Whiteley GS, Fuller BJ, Hobbs KE. Deterioration of cold-stored P, Schmidt W. Relationship between cytokine concentration in tissue specimens due to lipid peroxidation: modulation by serum and preovulatory follicular fluid and in vitro fertilization/ antioxidants at high subzero temperatures. Cryobiology 1992; intracytoplasmic sperm injection outcome. Chem Immunol Allergy 29(6): 668-73. 2005; 88: 80-97. [283] Odani M, Komatsu Y, Oka S, Iwahashi H. Screening of genes that [261] Paszkowski T, Traub AI, Robinson SY, McMaster D. Selenium respond to cryopreservation stress using yeast DNA microarray. dependent glutathione peroxidase activity in human follicular fluid. Cryobiology 2003; 47(2): 155-64. Clin Chim Acta 1995; 236(2): 173-80. [284] Storey KB. Strategies for exploration of freeze responsive gene [262] Tamura H, Takasaki A, Miwa I, et al. Oxidative stress impairs expression: advances in vertebrate freeze tolerance. Cryobiology oocyte quality and melatonin protects oocytes from free radical 2004; 48(2): 134-45. damage and improves fertilization rate. J Pineal Res 4(3): 280-7. [285] Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe M, Larman MG. [263] Botros L, Sakkas D, Seli E. Metabolomics and its application for Analysis of oocyte physiology to improve cryopreservation non-invasive embryo assessment in IVF. Mol Hum Reprod 2008; procedures. Theriogenology 2007; 67(1): 64-72. 14(12): 679-90. [286] Varghese AC, Nagy ZP, Agarwal A. Current trends, biological [264] Singh R, Sinclair KD. Metabolomics: approaches to assessing foundations and future prospects of oocyte and embryo oocyte and embryo quality. Theriogenology 2007; 68(Suppl 1): cryopreservation. Reprod Biomed Online 2009; 19(1): 126-40. S56-62. [287] Mermillod P, Locatelli Y, Dalbies-Tran R, et al. In vitro Production [265] Seino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, of Ruminant Embryos: Results, Limits and Perspectives. 2006 Kurachi H. Eight-hydroxy-2'-deoxyguanosine in granulosa cells is symposium Coa/Inra Scientific Cooperation in Agriculture; correlated with the quality of oocytes and embryos in an in vitro November 7-10; Tainan (Taiwan, R.O.C.) 2006; pp. 59-78. Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 205

[288] Yuan YQ, Van Soom A, Coopman FO, et al. Influence of oxygen [310] Takahashi M, Saka N, Takahashi H, Kanai Y, Schultz RM, Okano tension on apoptosis and hatching in bovine embryos cultured in A. Assessment of DNA damage in individual hamster embryos by vitro. Theriogenology 2003; 59(7): 1585-96. comet assay. Mol Reprod Dev 1999; 54(1): 1-7. [289] Arias ME, Sanchez R, Felmer R. Evaluation of different culture [311] Squirrell JM, Wokosin DL, White JG, Bavister BD. Long-term systems with low oxygen tension on the development, quality and two-photon fluorescence imaging of mammalian embryos without oxidative stress-related genes of bovine embryos produced in vitro. compromising viability. Nat Biotechnol 1999; 17(8): 763-7. Zygote 2011: 1-9. [312] Umaoka Y, Noda Y, Nakayama T, Narimoto K, Mori T, Iritani A. [290] Harvey AJ, Kind KL, Pantaleon M, Armstrong DT, Thompson JG. Effect of visual light on in vitro embryonic development in the Oxygen-regulated gene expression in bovine blastocysts. Biol hamster. Theriogenology 1992; 38(6): 1043-54. Reprod 2004; 71(4): 1108-19. [313] Barlow P, Puissant F, Van der Zwalmen P, Vandromme J, Trigaux [291] Correa GA, Rumpf R, Mundim TC, Franco MM, Dode MA. P, Leroy F. In vitro fertilization, development, and implantation Oxygen tension during in vitro culture of bovine embryos: effect in after exposure of mature mouse oocytes to visible light. Mol production and expression of genes related to oxidative stress. Reprod Dev 1992; 33(3): 297-302. Anim Reprod Sci 2008; 104(2-4): 132-42. [314] Oh SJ, Gong SP, Lee ST, Lee EJ, Lim JM. Light intensity and [292] Vanroose G, Van Soom A, de Kruif A. From co-culture to defined wavelength during embryo manipulation are important factors for medium: state of the art and practical considerations. Reprod maintaining viability of preimplantation embryos in vitro. Fertil Domest Anim 2001; 36(1): 25-8. Steril 2007; 88(4 Suppl): 1150-7. [293] Leese HJ. Metabolism of the preimplantation embryo: 40 years on. [315] Lonergan P, Fair T, Corcoran D, Evans AC. Effect of culture Reproduction. 2012; 143(4): 417-27. environment on gene expression and developmental characteristics [294] Adam AA, Takahashi Y, Katagiri S, Nagano M. Effects of oxygen in IVF-derived embryos. Theriogenology 2006; 65(1): 137-52. tension in the gas atmosphere during in vitro maturation, in vitro [316] Duranthon V, Watson AJ, Lonergan P. Preimplantation embryo fertilization and in vitro culture on the efficiency of in vitro programming: transcription, epigenetics, and culture environment. production of mouse embryos. Jpn J Vet Res 2004; 52(2): 77-84. Reproduction 2008; 135(2): 141-50. [295] Kitagawa Y, Suzuki K, Yoneda A, Watanabe T. Effects of oxygen [317] Martin-Romero FJ, Miguel-Lasobras EM, Dominguez-Arroyo JA, concentration and antioxidants on the in vitro developmental Gonzalez-Carrera E, Alvarez IS. Contribution of culture media to ability, production of reactive oxygen species (ROS), and DNA oxidative stress and its effect on human oocytes. Reprod Biomed fragmentation in porcine embryos. Theriogenology 2004; 62(7): Online 2008; 17(5): 652-61. 1186-97. [318] Cetica PD, Pintos LN, Dalvit GC, Beconi MT. Antioxidant enzyme [296] Waldenstrom U, Engstrom AB, Hellberg D, Nilsson S. Low- activity and oxidative stress in bovine oocyte in vitro maturation. oxygen compared with high-oxygen atmosphere in blastocyst IUBMB Life 2001; 51(1): 57-64. culture, a prospective randomized study. Fertil Steril 2009; 91(6): [319] de Matos DG, Furnus CC. The importance of having high 2461-5. glutathione (GSH) level after bovine in vitro maturation on embryo [297] Bermejo-Alvarez P, Lonergan P, Rizos D, Gutierrez-Adan A. Low development effect of beta-mercaptoethanol, cysteine and cystine. oxygen tension during IVM improves bovine oocyte competence Theriogenology 2000; 53(3): 761-71. and enhances anaerobic glycolysis. Reprod Biomed Online 2010; [320] Tatemoto H, Sakurai N, Muto N. Protection of porcine oocytes 20(3): 341-9. against apoptotic cell death caused by oxidative stress during In [298] Gardner DK. Dissection of culture media for embryos: the most vitro maturation: role of cumulus cells. Biol Reprod 2000; 63(3): important and less important components and characteristics. 805-10. Reprod Fertil Dev 2008; 20(1): 9-18. [321] Ali AA, Bilodeau JF, Sirard MA. Antioxidant requirements for [299] Gomes Sobrinho DB, Oliveira JB, Petersen CG, et al. IVF/ICSI bovine oocytes varies during in vitro maturation, fertilization and outcomes after culture of human embryos at low oxygen tension: a development. Theriogenology 2003; 59(3-4): 939-49. meta-analysis. Reprod Biol Endocrinol 2011; 9: 143. [322] de Matos DG, Furnus CC, Moses DF. Glutathione synthesis during [300] Bain NT, Madan P, Betts DH. The early embryo response to in vitro maturation of bovine oocytes: role of cumulus cells. Biol intracellular reactive oxygen species is developmentally regulated. Reprod 1997; 57(6): 1420-5. Reprod Fertil Dev 2011; 23(4): 561-75. [323] Gupta MK, Uhm SJ, Lee HT. Effect of vitrification and beta- [301] Favetta LA, St John EJ, King WA, Betts DH. High levels of p66shc mercaptoethanol on reactive oxygen species activity and in vitro and intracellular ROS in permanently arrested early embryos. Free development of oocytes vitrified before or after in vitro Radic Biol Med 2007; 42(8): 1201-10. fertilization. Fertil Steril 2010; 93(8): 2602-7. [302] Kovacic B, Vlaisavljevic V. Influence of atmospheric versus [324] Somfai T, Ozawa M, Noguchi J, et al. Developmental competence reduced oxygen concentration on development of human of in vitro-fertilized porcine oocytes after in vitro maturation and blastocysts in vitro: a prospective study on sibling oocytes. Reprod solid surface vitrification: effect of cryopreservation on oocyte Biomed Online 2008; 17(2): 229-36. antioxidative system and cell cycle stage. Cryobiology 2007; 55(2): [303] Kovacic B, Sajko MC, Vlaisavljevic V. A prospective, randomized 115-26. trial on the effect of atmospheric versus reduced oxygen [325] Huang J, Li Q, Zhao R, et al. Effect of sugars on maturation rate of concentration on the outcome of intracytoplasmic sperm injection vitrified-thawed immature porcine oocytes. Anim Reprod Sci 2008; cycles. Fertil Steril 2010; 94(2): 511-9. 106(1-2): 25-35. [304] Ciray HN, Aksoy T, Yaramanci K, Karayaka I, Bahceci M. In vitro [326] Krajcir N, Chowdary H, Gupta S, Agarwal A. Female Infertility culture under physiologic oxygen concentration improves and Assisted Reproduction: Impact of Oxidative Stress. Curr blastocyst yield and quality: a prospective randomized survey on Women’s Health Rev 2008; 4(1): 9-15. sibling oocytes. Fertil Steril 2009; 91(4 Suppl): 1459-61. [327] Serhal PF, Ranieri DM, Kinis A, Marchant S, Davies M, Khadum [305] Harvey AJ, Navarrete Santos A, Kirstein M, Kind KL, Fischer B, IM. Oocyte morphology predicts outcome of intracytoplasmic Thompson JG. Differential expression of oxygen-regulated genes sperm injection. Hum Reprod 1997; 12(6): 1267-70. in bovine blastocysts. Mol Reprod Dev 2007; 74(3): 290-9. [328] Macklon NS, Fauser BC. Mild stimulation in in vitro fertilization. [306] Kea B, Gebhardt J, Watt J, et al. Effect of reduced oxygen Ann N Y Acad Sci 2003; 997: 105-11. concentrations on the outcome of in vitro fertilization. Fertil Steril [329] Lornage J. [Biological aspects of endometriosis in vitro 2007; 87(1): 213-6. fertilization]. J Gynecol Obstet Biol Reprod (Paris) 2003; 32(8 Pt [307] Bahceci M, Ciray HN, Karagenc L, Ulug U, Bener F. Effect of 2): S48-50. oxygen concentration during the incubation of embryos of women [330] Dirnfeld M, Shiloh H, Bider D, et al. A prospective randomized undergoing ICSI and embryo transfer: a prospective randomized controlled study of the effect of short coincubation of gametes study. Reprod Biomed Online 2005; 11(4): 438-43. during insemination on zona pellucida thickness. Gynecol [308] Takenaka M, Horiuchi T, Yanagimachi R. Effects of light on Endocrinol 2003; 17(5): 397-403. development of mammalian zygotes. Proc Natl Acad Sci U S A [331] Kattera S, Chen C. Short coincubation of gametes in in vitro 2007; 104(36): 14289-93. fertilization improves implantation and pregnancy rates: a [309] Schultz RM. Of light and mouse embryos: less is more. Proc Natl prospective, randomized, controlled study. Fertil Steril 2003; 80(4): Acad Sci U S A 2007; 104(37): 14547-8. 1017-21. 206 Current Women’s Health Reviews, 2012, Vol. 8, No. 3 Premkumar and Agarwal

[332] Zhang NY, Sun HX, Hu YL, Wang B, Xu ZP. [Combination of [354] Hino K, Nishikawa M, Sato E, Inoue M. L-carnitine inhibits short-period sperm-oocyte coincubation and early rescue hypoglycemia-induced brain damage in the rat. Brain Res 2005; intracytoplasmic sperm injection after total failure of in vitro 1053(1-2): 77-87. fertilization]. Zhonghua Nan Ke Xue 2009; 15(6): 538-41. [355] Gulcin I. Antioxidant and antiradical activities of L-carnitine. Life [333] Swenson K, Check JH, Summers-Chase D, Choe JK, Check ML. A Sci 2006; 78(8): 803-11. randomized study comparing the effect of standard versus short [356] Molyneux SL, Young JM, Florkowski CM, Lever M, George incubation of sperm and oocyte on subsequent pregnancy and PM. Coenzyme Q10: is there a clinical role and a case for implantation rates following in vitro fertilization embryo transfer. measurement? Clin Biochem Rev 2008; 29(2): 71-82. Arch Androl 2000; 45(1): 73-6. [357] Pravst I, Zmitek K, Zmitek J. Coenzyme Q10 contents in foods [334] Barraud-Lange V, Sifer C, Pocate K, et al. Short gamete co- and fortification strategies. Crit Rev Food Sci Nutr 2010; 50(4): incubation during in vitro fertilization decreases the fertilization 269-80. rate and does not improve embryo quality: a prospective auto [358] Bentov Y, Esfandiari N, Burstein E, Casper RF. The use of controlled study. J Assist Reprod Genet 2008; 25(7): 305-10. mitochondrial nutrients to improve the outcome of infertility [335] Paszkowski T, Clarke RN. Antioxidative capacity of preimplantation treatment in older patients. Fertil Steril 2010; 93(1): 272-5. embryo culture medium declines following the incubation of poor [359] Fujiwara T, Nakabayashi M, Goto M, et al. Preliminary study on quality embryos. Hum Reprod 1996; 11(11): 2493-5. action of coenzyme Q10 in female reproductive system. 5th [336] Vajta G, Rienzi L, Cobo A, Yovich J. Embryo culture: can we Conference of International Coenzyme Q10 Association November perform better than nature? Reprod Biomed Online 2010; 20(4): 9-12, 2007 Kobe Gakuin University at Port Island, Kobe, Japan 453-69. 2007. [337] Meintjes M, Chantilis SJ, Ward DC, et al. A randomized controlled [360] Jablonka-Shariff A, Olson LM. The role of nitric oxide in oocyte study of human serum albumin and serum substitute supplement as meiotic maturation and ovulation: meiotic abnormalities of protein supplements for IVF culture and the effect on live birth endothelial nitric oxide synthase knock-out mouse oocytes. rates. Hum Reprod 2009; 24(4): 782-9. Endocrinology 1998; 139(6): 2944-54. [338] Quinn P. The development and impact of culture media for assisted [361] Goud AP, Goud PT, Diamond MP, Abu-Soud HM. Nitric oxide reproductive technologies. Fertil Steril 2004; 81(1): 27-9. delays oocyte aging. Biochemistry 2005; 44(34): 11361-8. [339] Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative [362] Dinara S, Sengoku K, Tamate K, Horikawa M, Ishikawa M. Effects stress. Curr Med Chem 2005; 12(10): 1161-208. of supplementation with free radical scavengers on the survival and [340] van Campenhout A, van Campenhout CM, Lagrou AR, Manuel-y- fertilization rates of mouse cryopreserved oocytes. Hum Reprod Keenoy B. Transferrin modifications and lipid peroxidation: 2001; 16(9): 1976-81. implications in diabetes mellitus. Free Radic Res 2003; 37(10): [363] D'Alessandro A, Zolla L. The SODyssey: superoxide dismutases 1069-77. from biochemistry, through proteomics, to oxidative stress, aging [341] Parikh FR, Nadkarni SG, Naik NJ, Naik DJ, Uttamchandani SA. and nutraceuticals. Expert Rev Proteomics 2011; 8(3): 405-21. Cumulus coculture and cumulus-aided embryo transfer increases [364] You J, Kim J, Lim J, Lee E. Anthocyanin stimulates in vitro pregnancy rates in patients undergoing in vitro fertilization. Fertil development of cloned pig embryos by increasing the intracellular Steril 2006; 86(4): 839-47. glutathione level and inhibiting reactive oxygen species. [342] Kattal N, Cohen J, Barmat LI. Role of coculture in human in vitro Theriogenology 2010; 74(5): 777-85. fertilization: a meta-analysis. Fertil Steril 2008; 90(4): 1069-76. [365] Tamura H. Influence of oxidative stress on oocyte quality [343] Jang HY, Ji SJ, Kim YH, et al. Antioxidative effects of astaxanthin and protective role of melatonin as an antioxidant. In: IFFS, editor. against nitric oxide-induced oxidative stress on cell viability and The 20th World Congress on Fertility and Sterility. Munich, gene expression in bovine oviduct epithelial cell and the Germany2010. developmental competence of bovine IVM/IVF embryos. Reprod [366] Kim JT, Jang HY, Park CK, Cheong HTP, Yang BK. Melatonin Domest Anim 2010; 45(6): 967-74. attenuates nitric oxide induced oxidative stress on viability and [344] Jeon K, Kim EY, Tae JC, et al. Survivin protein expression in gene expression in bovine oviduct epithelial cells, and subsequently bovine follicular oocytes and their in vitro developmental increases development of bovine IVM/IVF embryos. Asian-Aust J competence. Anim Reprod Sci 2008; 108(3-4): 319-33. Anim Sci 2011; 24(2): 190-7. [345] Wildman REC, Medeiros DM. Advanced Human Nutrition. [367] Kang JT, Koo OJ, Kwon DK, et al. Effects of melatonin on London: CRC Press; 2000. in vitro maturation of porcine oocyte and expression of melatonin [346] Ruiz-Pesini E, Alvarez E, Enriquez JA, Lopez-Perez MJ. Association receptor RNA in cumulus and granulosa cells. J Pineal Res 2009; between seminal plasma carnitine and sperm mitochondrial 46(1): 22-8. enzymatic activities. Int J Androl 2001; 24(6): 335-40. [368] El Mouatassim S, Guerin P, Menezo Y. Expression of genes [347] Abdelrazik H, Sharma R, Mahfouz R, Agarwal A. L-carnitine encoding antioxidant enzymes in human and mouse oocytes during decreases DNA damage and improves the in vitro blastocyst the final stages of maturation. Mol Hum Reprod 1999; 5(8): 720-5. development rate in mouse embryos. Fertil Steril 2009; 91(2): [369] Cetica PD, Dalvit GC, Beconi MT. Study of evaluation criteria 589-96. used for in vitro bovine oocyte selection and maturation. Biocell [348] Wu GQ, Jia BY, Li JJ, et al. L-carnitine enhances oocyte 1999; 23(2): 125-33. maturation and development of parthenogenetic embryos in pigs. [370] El-Raey M, Geshi M, Somfai T, et al. Evidence of melatonin Theriogenology 2011; 76(5): 785-93. synthesis in the cumulus oocyte complexes and its role in [349] Hashimoto S, Miyata Y, Yamanaka M, Morimoto Y. L-Carnitine enhancing oocyte maturation in vitro in cattle. Mol Reprod Dev decreased the apoptosis of granulose cells and improved meiotic 2011; 78(4): 250-62. competence of porcine growing oocytes. Reprod Domest Anim [371] Chiu TT, Rogers MS, Law EL, Briton-Jones CM, Cheung LP, 2008; 43(Suppl): 190-1. Haines CJ. Follicular fluid and serum concentrations of myo- [350] Mansour G, Abdelrazik H, Sharma RK, Radwan E, Falcone inositol in patients undergoing IVF: relationship with oocyte T, Agarwal A. L-carnitine supplementation reduces oocyte quality. Hum Reprod 2002; 17(6): 1591-6. cytoskeleton damage and embryo apoptosis induced by incubation [372] Papaleo E, Unfer V, Baillargeon JP, Fusi F, Occhi F, De Santis L. in peritoneal fluid from patients with endometriosis. Fertil Steril Myo-inositol may improve oocyte quality in intracytoplasmic 2009; 91(5 Suppl): 2079-86. sperm injection cycles. A prospective, controlled, randomized trial. [351] Chang B, Nishikawa M, Sato E, Utsumi K, Inoue M. L-Carnitine Fertil Steril 2009; 91(5): 1750-4. inhibits cisplatin-induced injury of the kidney and small intestine. [373] Unfer V, Raffone E, Rizzo P, Buffo S. Effect of a supplementation Arch Biochem Biophys 2002; 405(1): 55-64. with myo-inositol plus melatonin on oocyte quality in women who [352] Chang B, Nishikawa M, Nishiguchi S, Inoue M. L-carnitine failed to conceive in previous in vitro fertilization cycles for poor inhibits hepatocarcinogenesis via protection of mitochondria. Int J oocyte quality: a prospective, longitudinal, cohort study. Gynecol Cancer 2005; 113(5): 719-29. Endocrinol 2011; 27(11): 857-61. [353] Furuno T, Kanno T, Arita K, et al. Roles of long chain fatty acids [374] Nadri B, Zeinoaldini S, Kohram H. Ascorbic acid effects on in and carnitine in mitochondrial membrane permeability transition. vitro maturation of mouse oocyte with or without cumulus cell. Biochem Pharmacol 2001; 62(8): 1037-46. African Journal of Biotechnology 2009; 8(20): 5627-31. Effects of Oxidative Stress in Female Infertility and Artificial Reproduction Current Women’s Health Reviews, 2012, Vol. 8, No. 3 207

[375] Dalvit G, Llanes SP, Descalzo A, Insani M, Beconi M, Cetica P. [386] Beheshti R, Mohammadi-Roshandeh A, Ghaemmaghami S, Effect of alpha-tocopherol and ascorbic acid on bovine oocyte in Houshangi AF. Effect of Antioxidant Supplements on in Vitro vitro maturation. Reprod Domest Anim. 2005; 40(2): 93-7. Maturation of Bovine Oocyte. Advances in Environmental Biology [376] Lane M, Maybach JM, Gardner DK. Addition of ascorbate during 2011; 5(7): 805-10. cryopreservation stimulates subsequent embryo development. Hum [387] Talebi A, Zavareh S, Kashani MH, Lashgarbluki T, Karimi I. The Reprod 2002; 17(10): 2686-93. effect of alpha lipoic acid on the developmental competence of [377] Tarin J, Ten J, Vendrell FJ, de Oliveira MN, Cano A. Effects of mouse isolated preantral follicles. J Assist Reprod Genet 2012; maternal ageing and dietary antioxidant supplementation on 29(2): 175-83. ovulation, fertilisation and embryo development in vitro in the [388] Yamasaki M, Kawabe A, Nishimoto K, et al. Dihydro-alpha-lipoic mouse. Reprod Nutr Dev 1998; 38(5): 499-508. acid has more potent cytotoxicity than alpha-lipoic acid. In Vitro [378] Tarin JJ, Perez-Albala S, Cano A. Oral antioxidants counteract the Cell Dev Biol Anim 2009; 45(5-6): 275-80. negative effects of female aging on oocyte quantity and quality in [389] Packer L, Witt EH, Tritschler HJ. alpha-Lipoic acid as a biological the mouse. Mol Reprod Dev 2002; 61(3): 385-97. antioxidant. Free Radic Biol Med 1995; 19(2): 227-50. [379] Ozkaya MO, Naziroglu M. Multivitamin and mineral supplementation [390] Moini H, Packer L, Saris NE. Antioxidant and prooxidant activities modulates oxidative stress and antioxidant vitamin levels in serum of alpha-lipoic acid and dihydrolipoic acid. Toxicol Appl and follicular fluid of women undergoing in vitro fertilization. Pharmacol 2002; 182(1): 84-90. Fertil Steril 2010; 94(6): 2465-6. [391] Freitas RM. The evaluation of effects of lipoic acid on the lipid [380] Zenger F, Russmann S, Junker E, Wuthrich C, Bui MH, Lauterburg peroxidation, nitrite formation and antioxidant enzymes in the BH. Decreased glutathione in patients with anorexia nervosa. Risk hippocampus of rats after pilocarpine-induced seizures. Neurosci factor for toxic liver injury? Eur J Clin Nutr 2004; 58(2): 238-43. Lett 2009; 455(2): 140-4. [381] Gasparrini B, Neglia G, Palo RD, Campanile G, Zicarelli L. Effect [392] Liu J, Liu M, Ye X, et al. Delay in oocyte aging in mice by the of cysteamine during in vitro maturation on buffalo embryo antioxidant N-acetyl-L-cysteine (NAC). Hum Reprod 2012; 27(5): development. Theriogenology 2000; 54(9): 1537-42. 1411-20. [382] Bai J, Hou J, Guan H, et al. Effect of 2-mercaptoethanol and [393] Grinberg L, Fibach E, Amer J, Atlas D. N-acetylcysteine amide, a cysteine supplementation during in vitro maturation on the novel cell-permeating thiol, restores cellular glutathione and developmental competence of oocytes from hormone-stimulated protects human red blood cells from oxidative stress. Free Radic lambs. Theriogenology 2008; 70(5): 758-64. Biol Med 2005; 38(1): 136-45. [383] de Matos DG, Furnus CC, Moses DF, Martinez AG, Matkovic M. [394] Golstein GA, inventor 20100137676, assignee. N-acetylcysteine Stimulation of glutathione synthesis of in vitro matured bovine amide (NAC AMIDE) for treatment of oxidative stress associated oocytes and its effect on embryo development and freezability. Mol with infertility. New York, NY, United States 2010; 06/03/2010. Reprod Dev 1996; 45(4): 451-7. [395] Rich-Edwards JW, Spiegelman D, Garland M, et al. Physical [384] Gasparrini B, Boccia L, Marchandise J, et al. Enrichment of in activity, body mass index, and ovulatory disorder infertility. vitro maturation medium for buffalo (Bubalus bubalis) oocytes Epidemiology 2002; 13(2): 184-90. with thiol compounds: effects of cystine on glutathione synthesis [396] Brunn JM, Pederson SB, Kristensen K, Richelsen B. Opposite and embryo development. Theriogenology 2006; 65(2): 275-87. regulation of interleukin 8 and tumor necrosis factor-alpha by [385] Gasparrini B, Sayoud H, Neglia G, Matos DG, Donnay I, Zicarelli weight loss. Obesity Research 2002; 10: 499-506. L. Glutathione synthesis during in vitro maturation of buffalo [397] Clark AM, Ledger W, Galletly C, et al. Weight loss results in (Bubalus bubalis) oocytes: effects of cysteamine on embryo significant improvement in pregnancy and ovulation rates in development. Theriogenology 2003; 60(5): 943-52. anovulatory obese women. Hum Reprod 1995; 10(10): 2705-12.

Received: May 16, 2012 Revised: September 28, 2012 Accepted: October 03, 2012