Clinical Application of Antioxidants to Improve Human Oocyte Mitochondrial Function: A Review

Mitochondria produce adenosine triphosphate (ATP) while also generating high amounts of reactive oxygen species (ROS) derived from oxygen metabolism. ROS are small but highly reactive molecules that can be detrimental if unregulated. While normally functioning mitochondria produce molecules that counteract ROS production, an imbalance between the amount of ROS produced in the mitochondria and the capacity of the cell to counteract them leads to oxidative stress and ultimately to mitochondrial dysfunction. This dysfunction impairs cellular functions through reduced ATP output and/or increased oxidative stress. Mitochondrial dysfunction may also lead to poor oocyte quality and embryo development, ultimately affecting pregnancy outcomes. Improving mitochondrial function through antioxidant supplementation may enhance reproductive performance. Recent studies suggest that antioxidants may treat infertility by restoring mitochondrial function and promoting mitochondrial biogenesis. However, further randomized, controlled trials are needed to determine their clinical efficacy. In this review, we discuss the use of resveratrol, coenzyme-Q10, melatonin, folic acid, and several vitamins as antioxidant treatments to improve human oocyte and embryo quality, focusing on the mitochondria as their main hypothetical target. However, this mechanism of action has not yet been demonstrated in the human oocyte, which highlights the need for further studies in this field.


Introduction
Mitochondria produce the energy required by cells to carry out all cellular processes. Energy is generated in the form of adenosine triphosphate (ATP) through oxidative phosphorylation, a process that takes place in the inner mitochondrial membrane under aerobic conditions. Along this membrane, electrons from the controlled oxidation of nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH 2 ), both products of the citric acid cycle, travel through several enzymatic complexes forming the electron transport chain (ETC). The movement of electrons throughout the ETC is coupled with the transfer of protons across the membrane into the intermembrane space, generating an electrochemical proton gradient over the inner mitochondrial membrane that is harnessed by F1-F0 ATPase to phosphorylate adenosine diphosphate (ADP) into ATP [1] (Figure 1).
Mitochondrial respiration is a form of aerobic metabolism and uses oxygen to produce energy, with oxygen as the ultimate electron acceptor of the electron flow system of the mitochondrial ETC. However, mitochondrial electron flow may become uncoupled at several sites along the chain, resulting in unpaired single electrons that react with oxygen or other electron acceptors and generate free radicals. When these electrons react with oxygen, the resulting free radicals are referred to as Coenzyme-Q10 (CoQ10) carries electrons from complexes I and II to complex III in the mitochondrial respiratory chain and participates in the synthesis of ATP [36] (Figure 1). CoQ10 is a source of superoxide anion radical, though it also acts as an antioxidant, making it both a prooxidant and an antioxidant. The reduced form of CoQ10, ubiquinol, protects biological membranes from lipid peroxidation by recycling vitamin E and is also an antioxidant [37].
The dual role of CoQ10 in controlling mitochondrial function makes it an essential molecule for cellular performance. CoQ10 plasma levels decrease with advancing age, and this decline coincides with a decline in fertility and an increase in embryo aneuploidies [38]. Supplementation with CoQ10 may improve the reproductive outcome in infertile patients by improving mitochondrial function. Indeed, preovulatory CoQ10 supplementation improves mitochondrial function in aged mice [39] and improves non-aging related MD in mice [40] and other species [41]. In addition, CoQ10 treatment prevented mitochondrial ovarian aging in a rat model [42]. CoQ10 supplementation in culture media also restored the age-induced deterioration of oocyte quality in mice [43] and porcine models [44]. Finally, CoQ10 injected into aged mice increased mitochondrial respiratory activity and glucose uptake in cumulus cells, as well as the number of cumulus cells per oocyte, leading to improved reproductive capacity. These findings suggest that CoQ10 supplementation can benefit not only oocytes but also cumulus cells; this can be translated to humans because the human aging process also leads to reduced CoQ10-synthesis gene expression in cumulus cells [45]. In humans, higher CoQ10 levels in follicular fluid are related to improved embryo quality and higher pregnancy rates [46]. However, although CoQ10 supplementation increases CoQ10 levels in

Antioxidant Supplementation in Reproduction
Antioxidant treatment in the reproductive field can be carried out either by oral supplementation before infertility treatment or by culture media supplementation during ART. Oral supplementation attempts to improve gamete quality in vivo, while culture media supplementation attempts to do so in vitro. The latter approach can also be used to improve the in vitro maturation (IVM) process and to counteract high ROS production within the IVF setting.
Antioxidant supplementation is generally described in the literature as being applied to the male [31]. In this review, we discuss the use of antioxidants to improve oocyte and embryo quality, both in vivo and in vitro. We focus on mitochondrial function because its enhancement may be the main mechanism by which antioxidants manage to improve gamete quality. However, although mitochondrial function has been restored by different antioxidant molecules in many other tissues [32][33][34][35], this direct association has not yet been demonstrated in the human oocyte. A summary of the main evidence regarding the current utility of each of the antioxidants described is presented in Table 1, while a more extensive summary of the results of the discussed human studies is presented in Table 2. Table 1. Brief summary of evidence from published studies on the utility of the antioxidants reviewed. Only human studies are summarized. A green tick means that at least one study found beneficial effects on oocyte/embryo quality; a red cross means that the studies reviewed did not find any beneficial effects; a question mark means that the antioxidant effect has not been studied in that scenario.
Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 30 Another method of inducing the antioxidant effect is caloric restriction (CR). CR consists of limiting the daily diet to 25-50% of the normal diet. CR extended the lifespan and delayed aging in rodents [168]. In addition, CR can improve fertility and prolong reproductive life by delaying the process of ovarian aging [169]. However, its implementation is not easy in practice, and there are alternative substances that can induce the same effects [170]. For instance, metformin decreases the production of hepatic glucose by inhibiting the mitochondrial ETC complex I; thus, it mimics CR effects while reducing ROS production [171].
Other compounds enhance reproductive performance, though their potential antioxidant role has not been evaluated. For example, vitamin D [172] and myo-inositol [173] administration to infertile patients undergoing IVF treatments improved clinical outcomes. The addition of myoinositol to other molecules with proven antioxidant properties also showed promising results in PCOS [174] and IVF patients [175].
In sum, infertility therapy based on antioxidant supplementation is continuously evolving, and new applications of molecules may be adopted to keep oxidative stress in balance. This review covers antioxidants already described in the literature but constitutes only a subset of all molecules with antioxidant properties available. Therefore, continued research in this area is critical to the development of antioxidant therapies and applications.

Conclusions
Antioxidants are molecules that are easily obtained from natural sources. Their mechanisms of action are diverse, but they typically enhance mitochondrial function or directly scavenge free

Coenzyme-Q10
Coenzyme-Q10 (CoQ10) carries electrons from complexes I and II to complex III in the mitochondrial respiratory chain and participates in the synthesis of ATP [36] (Figure 1). CoQ10 is a source of superoxide anion radical, though it also acts as an antioxidant, making it both a prooxidant and an antioxidant. The reduced form of CoQ10, ubiquinol, protects biological membranes from lipid peroxidation by recycling vitamin E and is also an antioxidant [37]. The dual role of CoQ10 in controlling mitochondrial function makes it an essential molecule for cellular performance. CoQ10 plasma levels decrease with advancing age, and this decline coincides with a decline in fertility and an increase in embryo aneuploidies [38]. Supplementation with CoQ10 may improve the reproductive outcome in infertile patients by improving mitochondrial function. Indeed, preovulatory CoQ10 supplementation improves mitochondrial function in aged mice [39] and improves non-aging related MD in mice [40] and other species [41]. In addition, CoQ10 treatment prevented mitochondrial ovarian aging in a rat model [42]. CoQ10 supplementation in culture media also restored the age-induced deterioration of oocyte quality in mice [43] and porcine models [44]. Finally, CoQ10 injected into aged mice increased mitochondrial respiratory activity and glucose uptake in cumulus cells, as well as the number of cumulus cells per oocyte, leading to improved reproductive capacity. These findings suggest that CoQ10 supplementation can benefit not only oocytes but also cumulus cells; this can be translated to humans because the human aging process also leads to reduced CoQ10-synthesis gene expression in cumulus cells [45].

Use of Coenzyme-Q10 in Infertility
In humans, higher CoQ10 levels in follicular fluid are related to improved embryo quality and higher pregnancy rates [46]. However, although CoQ10 supplementation increases CoQ10 levels in follicular fluid in infertile patients [47], clinical outcome results are inconclusive. CoQ10 supplementation (600 mg/day for 2 months and up to the day of oocyte retrieval) in women with infertility of advanced age (between 35 and 43 years old) was insufficient to prevent ROS prolonged exposure effects on the meiotic apparatus because there was no significant difference in aneuploidy rates between the treated and the control groups. However, the study ended prematurely due to concerns related to the possible deleterious effects of polar body biopsy on embryo quality and implantation. The study did not recruit the number of participants initially proposed, which could underlie the non-significant difference in aneuploidy rate (46.5% CoQ10 group vs. 62.8% control group) [26]. Moreover, the dose and duration of CoQ10 treatment were based on mice studies, so humans may require longer exposure or higher doses to achieve remarkable benefits [26].
Additionally, CoQ10 supplementation benefited PCOS patients who exhibited ovulatory disorders and a higher proportion of immature follicles within the ovary [19]. Indeed, an RCT showed a significant increase in the number of follicles ≥14 and 18 mm of clomiphene citrate (CC)-resistant PCOS patients after the addition of CoQ10 as an adjuvant to CC treatment [50].
CoQ10 supplementation is proposed as an adjuvant during IVM, which may promote this technique by improving mitochondrial function in immature oocytes. Despite the initial discrepancies between animal studies [51,52], a 2020 report by Ma et al. described increased maturation rates (82.6% vs. 63.0%; p = 0.035) and reduced post-meiotic aneuploidies (36.8% vs. 65.5%; p = 0.02) after IVM in oocytes from women of advanced age supplemented with CoQ10. They did not find significant differences in young women [53]. However, mitoquinol (a CoQ10 analog) addition to the culture media from fertilization until the blastocyst stage did not improve embryo quality in women of advanced age. There were no significant differences in day 5 (18% vs. 20%) or total (48% vs. 45%) good quality blastocyst development per zygote, total blastocyst development (63% vs. 62%), and euploidy rates (33% vs. 30%) between the control and the treatment groups, respectively [54].
In sum, although CoQ10 is a promising therapy and is non-pharmaceutical, inexpensive, and safe, there is a need for well-designed clinical trials involving a greater number of patients to fully assess the effects of CoQ10 treatment on human fertility.

Resveratrol
Resveratrol is a natural polyphenol synthetized by several plants in response to pathogens. It is found in grapes, red wine, peanuts, and several medicinal plants [55]. Over the past decade, resveratrol has emerged as a therapeutic treatment for many diseases due to its anti-aging, antioxidant, anti-inflammatory, insulin-sensitizing, cardioprotective, vasodilating, and anti-neoplastic properties [56]. In the reproductive field, resveratrol may benefit women with diminished ovarian function, PCOS, endometriosis, and uterine fibroids [55,[57][58][59]. Resveratrol may improve age-related decline in ovarian function through the activation of sirtuin 1 (SIRT1) [60], a molecule that protects mitochondrial function from oxidative stress and whose levels are undetectable in aged oocytes [61]. In rats, resveratrol supplementation inhibited the process of follicular atresia, increased ovarian follicular reserve, and prolonged ovarian lifespan [62]. Moreover, mice treated with resveratrol for 12 months exhibited a higher number of follicles than controls and improved the number and quality of oocytes, evidenced by proper spindle morphology and chromosome alignment. In addition, telomerase activity, telomerase length, and age-related gene expression in the ovaries of mice supplemented with resveratrol resembled those of younger mice [63]. Resveratrol may also improve ovarian dysfunction caused by POI through the inhibition of the PI3K/AKT [64] and the NF-kB signaling pathways [65]. Resveratrol inhibited oxidative stress and inflammatory events in a rat POI model [66], and its antiapoptotic effect prevented oogonial stem cell loss in a mouse model of POI [67]. Furthermore, the addition of resveratrol to culture media improved oocyte maturation and embryo developmental potential in different animal studies, both in IVM [68,69] and conventional in vitro culture [70], by decreasing ROS production and increasing ATP content. Resveratrol may enhance mitochondrial homeostasis in both oocytes and granulosa cells via SIRT1 activation and regulation of the balance between mitochondrial biogenesis and autophagy [69]. Resveratrol may also have an antiapoptotic effect on granulosa cells through the inhibition of NF-kB signaling [65]. Nevertheless, the effects of resveratrol depend largely on cell type [55]. Contrary to granulosa cells, resveratrol exerts a proapoptotic effect on theca cells and counteracts insulin's stimulatory effect on cell proliferation [71]. In addition, resveratrol inhibits theca-interstitial cell androgen production, primarily by inhibiting the rate-limiting enzyme in the androgen biosynthesis pathway [72]. Therefore, it is useful in PCOS treatment, a pathology related to insulin resistance/hyperinsulinemia, theca-interstitial cell hyperplasia, and hyperandrogenism [55]. In vivo studies have demonstrated the antioxidant effect of resveratrol and its ability to return ovarian morphology to normal limits in a PCOS rat model [73].
In the endometrium, resveratrol's antiapoptotic and anti-proliferative effects inhibited the progression of ectopic endometrium, countering endometriosis [58]. In addition, resveratrol reduced vascular endothelial growth factor (VEGF) expression [74], improving the treatment of ovarian hyperstimulation syndrome (OHSS) and endometriosis, both gynecologic disorders associated with excessive VEGF activity [55]. However, its anti-inflammatory properties may inhibit the inflammatory-related process of decidualization [75], leading to decreased endometrial receptivity.
Therefore, resveratrol has the potential to benefit women with diminished ovarian reserve and function through its antioxidant effects and the stimulation of mitochondrial biogenesis, though it also has adverse effects on implantation and endometrial decidualization [76] (Figure 3). Human studies involving resveratrol are needed to validate the success observed in animal models. excessive VEGF activity [55]. However, its anti-inflammatory properties may inhibit the inflammatory-related process of decidualization [75], leading to decreased endometrial receptivity.
Therefore, resveratrol has the potential to benefit women with diminished ovarian reserve and function through its antioxidant effects and the stimulation of mitochondrial biogenesis, though it also has adverse effects on implantation and endometrial decidualization [76] (Figure 3). Human studies involving resveratrol are needed to validate the success observed in animal models.

Use of Resveratrol in Infertility
In humans, resveratrol supplementation in the IVM medium of aged immature oocytes demonstrated improved oocyte maturation rates after 24 h (55.3% vs. 37.84%; p < 0.05) and 36 h of culture (71.1% vs. 51.35% in the study and control group, respectively; p < 0.05). Resveratrol also improved oocyte quality, as evidenced by improved mitochondrial immunofluorescence intensity (53.0% vs. 31.1%, p < 0.05) and a reduced proportion of oocytes with abnormal spindle morphology and irregular chromosomal disposition (p < 0.05) [68]. In a double-blind RCT, oral resveratrol (1500

Use of Resveratrol in Infertility
In humans, resveratrol supplementation in the IVM medium of aged immature oocytes demonstrated improved oocyte maturation rates after 24 h (55.3% vs. 37.84%; p < 0.05) and 36 h of culture (71.1% vs. 51.35% in the study and control group, respectively; p < 0.05). Resveratrol also improved oocyte quality, as evidenced by improved mitochondrial immunofluorescence intensity (53.0% vs. 31.1%, p < 0.05) and a reduced proportion of oocytes with abnormal spindle morphology and irregular chromosomal disposition (p < 0.05) [68]. In a double-blind RCT, oral resveratrol (1500 mg/day) supplementation in women with PCOS showed reduced ovarian and adrenal androgen levels [77]. A triple-blinded RCT comparing 800 mg/day of resveratrol to a placebo in patients with PCOS found an increased high-quality oocyte rate (81.9% vs. 69.1%; p = 0.002), increased high-quality embryo rate (89.8% vs. 78.8%; p = 0.024), and reduced expression of the VEGF gene in granulosa cells (p = 0.0001) [78]. Finally, a retrospective study evaluated the impact of resveratrol (200 mg/day) on human pregnancy outcomes during fresh or frozen embryo transfer cycles compared to no treatment. The study group showed significantly lower clinical pregnancy rates (10.8% vs. 21.5%; p = 0.0005), as well as higher miscarriage rates (52.4% vs. 21.8%; p = 0.0022), even after a multivariate logistic regression analysis (CPR: OR 0.539, 95% CI 0.341-0.853; MR: OR 2.602, 95% CI 1.070-6.325). Patients with resveratrol treatment had poor pregnancy outcomes, even though good quality embryos had been transferred [27], which may be related to the suppressor effect of resveratrol on decidualization [75]. This study focused on pregnancy outcomes after embryo transfer and did not evaluate ovarian function before or after resveratrol treatment [76].
Following these results, an alternative resveratrol administration protocol was proposed to avoid negative effects on the endometrium. Because the half-life of resveratrol is between 3 and 9 h [79], it may not affect implantation or pregnancy if its endometrial tissue levels drop before decidualization. Thus, discontinuation of resveratrol intake on the day of ovulation, or the freeze-all policy with a deferred frozen embryo transfer without supplementation, may help overcome these adverse effects [76].
Resveratrol may have adverse effects depending on the dose administered. Some side effects were reported after its administration, including headaches, nausea, diarrhea, dizziness, and liver dysfunction [79]. High-dose resveratrol should not be administered, and its supplementation should be discontinued during pregnancy due to its negative effect on the endometrial decidualization process Antioxidants 2020, 9, 1197 8 of 29 and scarce data about adverse effects and embryo-fetal toxicity. Randomized controlled trials are needed along with human studies to define a standard dose and duration of treatment since there is a high heterogeneity between all the studies described [55].

Melatonin
Melatonin is synthesized from the amino acid tryptophan, almost exclusively by the pineal gland. It is released into the blood in a circadian manner, and its production is restricted to nighttime [80]. Melatonin may be produced in high amounts in mitochondria [34], and it has an important role in reducing oxidative stress. Its antioxidant properties come from its excellent free radical scavenging, as well as its capacity to upregulate the expression of antioxidant enzymes and a spectrum of stress-responsive genes. In addition, melatonin indirectly accelerates electron transport through the ETC, decreasing electron leakage and reducing ROS formation, a process called radical avoidance [81]. Moreover, this molecule has antiapoptotic [82], anti-inflammatory [83], and antiandrogenic properties [84].
Melatonin modulates the hypothalamic-pituitary-gonadal axis at different levels, thus modulating reproductive system activity [85]. Melatonin may reduce intrafollicular oxidative stress, and melatonin levels in follicular fluid are suggested as a biomarker to predict IVF outcomes and ovarian reserve [86]. Melatonin production decreases with age [87], and in the ovary, this age-related decline is related to the increase in follicle-stimulating hormone levels associated with menopause [88]. Therefore, melatonin may delay ovarian aging. Interestingly, Song et al. found reduced aging ovary parameters, increased oocyte quality and quantity, and increased litter size in mice treated with melatonin for 12 months [89].

Use of Melatonin in Infertility
In humans, melatonin treatment improves fertility outcomes in infertile women [28,[90][91][92], patients with PCOS [84], patients with endometriosis, and many other gynecologic pathologies. In addition, melatonin may help normalize menstrual disorders, such as dysmenorrhea [93]. Further, melatonin supplementation during pregnancy may protect the embryo from oxidative stress since this hormone crosses the placental barrier during pregnancy [94].
Melatonin supplementation is proposed as a treatment for poor oocyte quality due to its antioxidant effect in oocytes and granulosa cells. In 2003, Takasaki et al. found that melatonin treatment (3 mg/day from the previous cycle until the day of triggering) improved oocyte quality in women with a previous IVF failure due to poor oocyte quality. However, this improvement was evidenced only in the number of degenerated oocytes retrieved and not in the number of oocytes or mature oocytes obtained. In the treatment group, melatonin levels were significantly higher in the follicular fluid, while levels of lipid peroxide were reduced [90]. Lipid peroxide is implicated in free radical reactions [5]. Furthermore, melatonin accelerates the action of the maturation-inducing hormone, a molecule important to final oocyte maturation [95]. Thus, melatonin may improve oocyte quality by protecting oocytes from oxidative stress and enhancing the oocyte maturation processes.
Clinical trials found that melatonin supplementation during an IVF cycle significantly increased the number of retrieved (11.5 vs. 6.9 in the control group; p = 0.0001) and mature oocytes (9.0 vs. 4.4 in the control group; p = 0.0001) [28], or at least the proportion of metaphase II (MII)/retrieved oocytes (81.9% vs. 75.8% in the control group; p = 0.034) [92]. In addition, the fertilization success of women with low fertilization rates (≤50%) in the previous IVF cycle was improved by melatonin treatment compared to the previous cycle (29.8% increment in the melatonin group vs. 1.9% increment in the non-melatonin group; p < 0.01) [91]. However, an RCT conducted in 2018 found no difference in the number of oocytes retrieved, number of MII, fertilization rate, embryo quality, clinical pregnancy rate, or live birth rate between the treatment and control groups [96].
In 2019, Espino et al. found reduced concentrations of melatonin at the systemic level and in the follicular fluid of women with unexplained infertility. Interestingly, melatonin levels were correlated with different parameters of oxidative balance in follicular fluid. In comparison with a control group of fertile women, patients with unexplained infertility who underwent melatonin supplementation during ovarian stimulation until the day of oocyte retrieval (one group 3 mg/day and the other group 6 mg/day) had better outcomes than patients with no melatonin treatment. This supplementation restored concentrations of diverse oxidative stress markers, improved oocyte quality, and, consequently, increased the number of transferable embryos and the success of ART [97]. Lastly, melatonin supplementation of culture media also improved outcomes in animal studies in IVM [98] and conventional in vitro culture [99]. IVM of immature oocytes from PCOS patients cultured with melatonin achieved elevated but not significantly higher maturation rates [100].
The main advantage of melatonin antioxidant therapy is its relatively well-documented safety due to its frequent use as a sleep aid [101]. However, studies conducted so far have evaluated melatonin only in the short-term, so long-term clinical trials are needed [102].

Vitamins
Despite their proven antioxidant properties, the use of vitamins as supplements to improve mitochondrial oocyte function has not been extensively addressed. No human clinical trials have assessed the role of vitamins as antioxidants in improving oocyte and embryo quality in IVF patients.

Vitamin A
Vitamin A is a lipid-soluble vitamin obtained from the diet in the form of vitamin A esters or provitamin A carotenoids. Dietary sources of vitamin A include green and yellow vegetables, dairy products, fish, eggs, and organ meats. There are at least a dozen forms of vitamin A esters and approximately 600 types of carotenoids, although only about 50 forms of the latter have provitamin A activity. Once within the body, physiologically active forms of vitamin A are retinol, retinal, and retinoic acid [103]. Antioxidant activity has been described for retinol, the main physiological form of vitamin A, as well as for α and β carotenes, two examples of provitamin A carotenoids. Vitamin A forms are potent antioxidants that act by scavenging peroxyl radicals, thus reducing ROS levels [103]. In the reproductive field, vitamin A and its metabolites have important roles in follicular growth [104], steroidogenesis [105], oocyte maturation [106,107], and embryo development [108]. Its supplementation has been tested in both in vivo [109,110] and in vitro studies. In vitro studies found that vitamin A addition to IVM medium increased maturation rates by enhancing mitochondrial membrane potential activity, lowering ROS levels, and decreasing apoptosis [111]. Indeed, retinoic acid supplementation to the IVM medium has obtained beneficial results in several species, including cows [112] and mice [113].

Use of Vitamin A in Infertility
In humans, high follicular fluid levels of all-trans retinoic acid (ATRA), the active metabolite of retinol, at the time of oocyte retrieval, were related to oocytes giving rise to embryos of the highest quality. In addition, follicular fluid ATRA concentrations were positively correlated with patient serum ATRA [114]. A later study from the same group confirmed that this higher oocyte competence was the result of higher ATRA synthesis of cumulus-oocyte complexes, which in turn was positively correlated with higher fertilization rates [115].
Therefore, minimum levels of vitamin A or its metabolites are essential for proper oocyte maturation and acquisition of competence. However, due to the essential role of vitamin A in the signaling pathways that control ovarian function, including oocyte maturation and development [116], it is unknown if its antioxidant properties also play a role in these processes. In any case, human clinical trials assessing the effect of vitamin A supplementation in oocyte quality have not been performed yet, neither in vitro nor in vivo.

Folic Acid
Folate, or vitamin B9, is a B vitamin found in green leafy vegetables, dark green vegetables, beans, and other legumes. The synthetic form of folate, folic acid, is common in dietary supplements in fortified foods due to its high stability and low cost [117]. At the cellular level, folate acts as a methyl donor to support the methylation of homocysteine to become methionine [118], which is a critical intermediary in the production of glutathione, the primary intracellular antioxidant. Therefore, folic acid may protect from oxidative stress by increasing intrinsic antioxidant levels within the cell. In addition, different molecules derived from folate metabolism are also essential to DNA, RNA, protein synthesis, proper epigenetic activity, and chromosome structure maintenance, making folate indispensable during periods of rapid cell growth and proliferation, such as germ cell maturation, pregnancy, and fertilization [119].
In Europe, the recommended folate intake in adult women ranges from 170 to 300 µg/day, and 400 µg/day of supplemental folic acid is recommended for pregnant women [120]. Folate deficiency can occur due to poor dietary intake, either of folate itself or of the micronutrients necessary for its synthesis, and/or malabsorption, mainly by defects in folate-metabolizing genes. Folate deficiency leads to homocysteine accumulation [121] or hyperhomocysteinemia, which is associated with several pathophysiological mechanisms during pregnancy, including oxidative stress [122] and decreased cellular antioxidant potential [123].
Preconception folate deficiency hampered female fertility, as well as embryo and fetal viability, in several animal models. This condition partially inhibits ovulation in rats [124] and increases the number of degenerated follicles in rhesus monkeys [125], suggesting the essential role of folate in folliculogenesis [119]. In humans, women with folate deficiency undergoing ovarian stimulation often have impaired ovarian function, lower oocyte quality, and lower pregnancy rates [126,127]. However, no human studies have hypothesized a relationship between folate deficiency-related mitochondrial impairment and reduced fertility.
Folic acid supplementation, therefore, may be useful in the reproductive field. Its use during pregnancy is widespread, as it seems to prevent fetal neural tube defects [128] as well as heart defects [129], Down syndrome [130], intrauterine growth restriction, and pre-term birth [131]. However, its preconception use is less studied. Recently, the impact of folic acid supplementation was investigated regarding the earlier stages of female reproductive physiology, in particular its role in folliculogenesis [119]. During the preovulatory stage, folic acid decreased the proportion of developmentally delayed embryos in a mouse model [132] and increased glutathione synthesis at the oocyte level in a rat model, although it was unable to revert the altered expression pattern of genes related to mitochondrial function and dynamics [133].

Use of Folic Acid in Infertility
In humans, folate and homocysteine are present in the follicular fluid [134], and these levels correlate with their blood concentrations [135]. Moreover, folic acid supplementation increases serum folate concentration and reduces serum hyperhomocysteinemia [135], exerting the same effect at the follicular level. A negative correlation was found between homocysteine levels in the follicular fluid and oocyte maturity [127], as well as in vitro day-3 embryo quality [136]. Similar results were obtained in a recent study conducted in PCOS patients, where negative correlations between homocysteine concentrations and fertilization rates, as well as oocyte and embryo quality, were observed [137]. Furthermore, higher folate intake was related to higher implantation, clinical pregnancy, and live birth rates [138].
Supplemental folic acid may improve ART outcomes and is favored over food-based sources due to lower amounts of bioavailable folate in food [139]. However, literature evaluating the impact of folate levels on reproduction analyze the effects of different concentrations within the normal dietary intake range, and folic acid supplementation studies are scarce. A prospective study conducted in women with unexplained infertility found no difference in clinical pregnancy rates (32.8% users vs. 35.7% non-users) and live birth rates (24.1% users vs. 31.0% non-users) after folic acid supplementation, even though folate blood levels were increased [140].
Therefore, a diet rich in folate is essential to achieve good pregnancy outcomes. Its antioxidant properties may have an important impact in folliculogenesis and early embryo development, although folate has multiple other functions that may also assist pregnancy. However, there are no clinical trials confirming its therapeutic advantages as an antioxidant supplement to benefit infertility treatment or its potential to enhance oocyte mitochondrial function.

Ascorbic Acid
Ascorbic acid, also known as vitamin C, is a powerful antioxidant that scavenges free radicals [141]. Its addition to culture and vitrification/warming media significantly improves the quality and survival rates of porcine cryopreserved embryos by regulating some crucial genes implicated in mitochondrial redox status [142]. Despite this, ascorbic acid supplementation to fertilization and conventional culture media did not exert any significant beneficial effect on maturation, fertilization, or embryo development parameters [143].

Use of Ascorbic Acid in Infertility
In humans, ascorbic acid supplementation significantly increased serum and follicular fluid ascorbic acid levels [144][145][146]. However, no differences in implantation or clinical pregnancy rates were found after ascorbic acid supplementation in women undergoing an IVF procedure, either during hormonal ovarian stimulation [144] or during the luteal phase [145]. In addition, this therapy was incapable of reducing oxidative stress markers in women with endometriosis after two months of treatment [146]. Therefore, although promising, vitamin C supplementation in infertile patients has yet to show a beneficial effect on fertility. Further clinical trials are needed to confirm preliminary results.

Vitamin D
Vitamin D plays a crucial role in dietary calcium absorption. In the reproductive field, vitamin D deficiency has been suggested to impact reproductive performance [147], but the evidence is not conclusive. Its potential antioxidant effect in the human female gamete has not been investigated. Currently, an RCT is being conducted in which follicular fluid and cumulus cells samples will be processed in order to evaluate the effect of vitamin D supplementation on oocyte quality, although the main primary endpoint is the clinical pregnancy rate [148]. Because vitamin D shows antioxidant properties [149] and the ability to improve mitochondrial function in other tissues [150], it could become an antioxidant treatment in the future, although its implications as an antioxidant at the oocyte molecular level need to be elucidated.

Vitamin E
Vitamin E is an essential antioxidant mainly found in high-fat vegetable products, and α-tocopherol is its most common form [151]. Its main role is the protection of cell membranes from oxidative damage by reaction with lipid radicals produced in the lipid peroxidation chain reaction [5].
Culture media supplementation with vitamin E increased the blastocyst development rate in a bovine model, presumably by protecting from ROS [152]. Similar results were found in a mouse model, although with less benefit compared to vitamin C supplementation [153]. Interestingly, combined oral supplementation with vitamins C and E successfully prevented ovarian aging in a mouse model [154].

Use of Vitamin E in Infertility
Bahadori et al. described several vitamin E concentration intervals related to higher human oocyte and embryo quality. Follicular fluid vitamin E levels in the ranges 0.35-1 mg/dL and 1.5-2 mg/dL were related to higher oocyte maturation rates (89.2% and 84.9%, respectively, vs. 69.6% in 1-1.5 mg/dL and 76.7% in 2-7.4 mg/dL; p = 0.002) while serum vitamin E levels in the range 10-15 mg/dL were related to a higher proportion of high-quality embryos (87.5% vs. 46.2 in 1-5 mg/dL, 54.9% in 5-10 mg/dL, 42.9% in 15-20 mg/dL; p = 0.007). However, no significant relationship between serum vitamin E levels and oocyte maturation was found, nor was there a correlation between follicular fluid vitamin E levels and embryo quality [155].
A recent RCT showed that the concomitant administration of vitamin E and vitamin D to women with PCOS was associated with higher implantation (35.1% vs. 8.6%; p < 0.001), pregnancy (69.0% vs. 25.8%; p < 0.001), and clinical pregnancy rates (62.1% vs. 22.6%; p = 0.002) compared to a control group [156]. However, these outcomes were not associated with an antioxidant mechanism. Therefore, the findings of this trial do not support the use of vitamins D and E as a dual antioxidant treatment in IVF, and further research is needed to determine whether the antioxidant mechanism of vitamin E can improve mitochondrial oocyte function.

Antioxidants in Combination
Antioxidants can be supplied alone or in combination, and different cocktails of these molecules were evaluated for their potential to improve oocyte quality both in animal [157,158] and human studies [159,160]. Providing antioxidants in combination may more closely resemble in vivo conditions, where the molecules participate in a complex system with multi-faced interactions and feedback mechanisms [161]. It is important that antioxidants be evaluated both alone and in combination to distinguish which specific antioxidant is responsible for certain benefits.

Other Antioxidant Mechanisms
The molecules described in this review have been evaluated for their potential to improve mitochondrial function in reproduction, though many additional molecules may also serve this purpose. Many molecules have antioxidant properties, including growth hormone [162], progesterone [163], and curcumin [164], which successfully reduce oxidative stress in the animal model; only the mechanism of action for growth hormone is suggested to be related to mitochondrial activity improvement [162]. In addition, putrescine supplementation improves oocyte quality and reproductive performance in aged mice [165] and is related to improved mitochondrial activity [166]. Because human granulosa cells from aged follicles present periovulatory putrescine deficiency [167], putrescine supplementation is suggested as a novel therapy to restore human ovarian function.
Another method of inducing the antioxidant effect is caloric restriction (CR). CR consists of limiting the daily diet to 25-50% of the normal diet. CR extended the lifespan and delayed aging in rodents [168]. In addition, CR can improve fertility and prolong reproductive life by delaying the process of ovarian aging [169]. However, its implementation is not easy in practice, and there are alternative substances that can induce the same effects [170]. For instance, metformin decreases the production of hepatic glucose by inhibiting the mitochondrial ETC complex I; thus, it mimics CR effects while reducing ROS production [171].
Other compounds enhance reproductive performance, though their potential antioxidant role has not been evaluated. For example, vitamin D [172] and myo-inositol [173] administration to infertile patients undergoing IVF treatments improved clinical outcomes. The addition of myo-inositol to other molecules with proven antioxidant properties also showed promising results in PCOS [174] and IVF patients [175].
In sum, infertility therapy based on antioxidant supplementation is continuously evolving, and new applications of molecules may be adopted to keep oxidative stress in balance. This review covers antioxidants already described in the literature but constitutes only a subset of all molecules with antioxidant properties available. Therefore, continued research in this area is critical to the development of antioxidant therapies and applications.        No difference in the mean number of MII oocytes between the treated (12.7) and the control group (13.2); p = 0.7.

Conclusions
Antioxidants are molecules that are easily obtained from natural sources. Their mechanisms of action are diverse, but they typically enhance mitochondrial function or directly scavenge free radicals, which in turn protects mitochondria and other cellular components from oxidative stress. Given the crucial role of mitochondrial activity in oocyte maturation, fertilization, and embryo development, antioxidants may improve ART outcomes by improving oocyte quality.
In ART, antioxidant supplementation can be prescribed as an oral pre-treatment or as an adjuvant in the media during in vitro culture, although the extent of its effects have not been fully elucidated. Indeed, the majority of studies described throughout this review evaluate the indirect consequences of antioxidant supplementation on oocyte quality, evidenced by endpoints such as oocyte maturation, aneuploidy, and pregnancy rates, which may or may not be related to improved mitochondrial function. Although the direct relationship between antioxidant support and improved mitochondrial function is likely, further studies are needed to fully evaluate the consequence of antioxidant treatment on specific mitochondrial parameters, such as mitochondrial membrane potential, morphology, and distribution, as well as oxidative stress markers. In addition, there is no consensus on the optimal dose and duration of treatment, so further evaluation of these parameters is necessary before clinical application of antioxidant strategies.
Although antioxidant therapy is a promising and safe therapy, well-designed human clinical trials are needed before it is incorporated into routine clinical practice. The population that can benefit from their use must also be clearly defined, and their short-and long-term safety must be evaluated. Further, the mechanisms of each antioxidant's action at the molecular level and the administration protocol must be clearly defined.