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Home , Reactive oxygen species

Male infertility testing: reactive species and capacity

Edmund Y. Ko, M.D.,a Edmund S. Sabanegh Jr., M.D.,b and Ashok Agarwal, Ph.D.c a Department of Urology, Loma Linda University, School of Medicine, Loma Linda, California; and b Department of Urology and c Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic Foundation, Cleveland, Ohio

Reactive oxygen species (ROS) are an integral component of sperm developmental , capacitation, and function. Elevated ROS levels, from processes such as infection or inflammation, can be associated with aberrations of sperm development, function, and fertil- izing capacity. We review the impact of ROS on sperm physiology, its place in infertility evaluation, the implications for reproductive outcomes, and antioxidant therapy. Our systematic review of PubMed literature from the last 3 decades focuses on the physiology and etiology of ROS and (OS), evaluation of ROS, and . ROS is normally produced physiologically and is used to maintain cellular processes such as sperm maturation, capacitation, and sperm-oocyte interaction. When ROS production exceeds the buffering capacity of antioxidants, OS occurs and can have a negative impact on sperm and fertility. ROS and antioxidant capacity testing can potentially add additional prognostic information to standard laboratory testing for the infertile male, although its role as standard part of an evaluation has yet to be determined. Elevated ROS levels have been implicated with abnormal semen parameters and , but the impact of ROS on fertilization rates and pregnancy is controversial. This is partly because of the lack of consensus on what type of patients may be suitable for ROS testing and assay standardization. Routine ROS testing for the infertile male is not currently recommended. (Fertil SterilÒ Use your smartphone 2014;102:1518–27. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Antioxidant capacity, antioxidants, male infertility, reactive oxygen species and connect to the discussion forum for Discuss: You can discuss this article with its authors and with other ASRM members at http:// this article now.* fertstertforum.com/koe-reactive-oxygen-species-antioxidant-capacity/ * Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.

nfertility, defined as the inability to described by MacLeod in 1943 (6). His- Reactive oxygen species have a sig- I achieve pregnancy after 12 months torical studies have associated elevated nificant impact on spermatogenesis as of regular intercourse, affects 15% ROS concentrations with infertility in well as on sperm function. Supraphy- of reproductive-aged couples (1).A up to 40% men (7, 8). Contemporary siologic levels of ROS can affect all as- quarter of these cases can be explained studies have also found elevated pects of the semen analysis. Multiple by male-only factors, and up to 50% ROS levels in 30% to 80% of infertile studies have demonstrated the detri- can be explained by combined male men (9–11). Oxidative stress (OS) mental effects of ROS on sperm con- and female factors (2). Many infertile in the genitourinary system can centration (13, 15, 16), motility (15, men have abnormal semen analyses, damage sperm, resulting in decreased 16), morphology (15, 17), and ROS are of which the etiology is often poorly motility, , increased implicated in sperm DNA damage (18) explained (3). These etiologies can deoxyribonucleic acid (DNA) damage, and (19). Conversely, other include environmental, dietary, medi- and decreased oocyte-sperm fusion studies have demonstrated no cal, genetic, and physiologic factors (12). An imbalance between ROS levels correlation between ROS and sperm (4, 5). and physiologic antioxidants can result motility (20, 21). Recent studies have Reactive oxygen species (ROS) in OS, with subsequent adverse effects demonstrated that ROS values have a have long been implicated as a poten- on the body's normal physiologic pro- sensitivity of 68.8% and specificity of tial cause of male infertility, as first cesses (13, 14). 93.8% in diagnosing male factor infertility (infertility due to abnormal Received July 30, 2014; revised and accepted October 14, 2014. E.Y.K. has nothing to disclose. E.S.S. has nothing to disclose. A.A. has nothing to disclose. semen parameters, not female-related Reprint requests: Edmund Y. Ko, M.D., Assistant Professor, Department of Urology, Loma Linda Uni- factors) (15). versity School of Medicine, 11234 Anderson Street, A560, Loma Linda, California 92354 (E-mail: [email protected]). Not all OS is harmful to sperm function, however. Excessive levels of Fertility and Sterility® Vol. 102, No. 6, December 2014 0015-0282/$36.00 ROS can have detrimental effects on Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.10.020 sperm quality, but lower physiologic

1518 VOL. 102 NO. 6 / DECEMBER 2014 Fertility and Sterility® levels are necessary for sperm capacitation, hyperactivation, FIGURE 1 and sperm-oocyte fusion, along with other critical cellular processes (22–24). We review the impact of ROS on sperm physiology and antioxidant capacity, the role of ROS testing in the male fertility evaluation, the implications for reproduction outcomes, and the potential treatments to improve antioxidant capacity.

PHYSIOLOGY OF REACTIVE OXYGEN SPECIES AND ANTIOXIDANT CAPACITY Reactive oxygen species are byproducts of normal physio- logic processes and cellular . Cellular aerobic metabolism within the mitochondria occurs via oxidative phosphorylation. Molecular oxygen (O2) normally carries two unpaired , thereby making oxygen susceptible to formation. The primary form of ROS occurs with Mechanism of oxidative stress in human semen. From: Agarwal A, the addition of an extra to O2, resulting in a super- Sekhon LH. Oxidative stress and antioxidants for idiopathic oxide anion radical (.O2 ) (25). The anion can oligoasthenoteratospermia: is it justified? Indian J Urol 2011;27:74–85. then be converted to other ROS, including hydrogen Used with permission. Ko. Male infertility and ROS and TAC. Fertil Steril 2014. peroxide (H2O2), hydroxyl (.OH ), and peroxyl (HO2) radicals (23, 24, 26). Free radicals are formed as a byproduct of the enzymatic reduction of oxygen during energy production (9). Free radicals are oxygen containing one or more unpaired electrons, making them unstable and very tem via enzymatic and nonenzymatic antioxidant pathways reactive in the presence of amino acids, lipids, and nucleic (11, 13, 29). This finely balanced oxidant-antioxidant system acids (23, 24, 27). allows the formation of beneficial oxidants for normal Reactive oxygen species are important intermediaries cellular functions and concurrently prevents the damaging involved in vascular tone and gene regulation within the effects of excess oxidative stress. testes. Normal physiologic ROS levels are required for sperm A high concentration of antioxidants and scavengers are maturation, hyperactivation, capacitation, chemotaxis, zona contained within seminal plasma. These antioxidants act to pellucida binding, acrosome reaction, and sperm-oocyte protect sperm from the deleterious effects of ROS. Enzymatic fusion (28, 29). Within normal physiologic conditions, antioxidants include , , gluta- antioxidants help maintain a low level of oxidative stress in thione peroxidase, transferase, and cerulo- the semen, thereby allowing normal signaling processes plasmin. Nonenzymatic antioxidants include albumin, and spermatic function and avoiding ROS-induced cell dam- b-carotenes, L-carnitine, glutathione, pyruvate, taurine, age (14). hypotaurine, ubiquinol, vitamins C (ascorbic acid) and E (a- The main ROS producers in semen are leukocytes and ), and zinc (3, 38–40). immature spermatozoa (Fig. 1). Leukocytes can produce up Glutathione provides the most important intracellular to 1,000 times more ROS compared with spermatozoa under protection against ROS. It contains a sulfhydryl group that physiologic conditions (30). The sperm membrane remodeling directly scavenges free radicals. The cycle is completed process during spermatogenesis may be the common origin when oxidized glutathione is reduced/regenerated by gluta- for both abnormal spermatozoa and ROS. Failures in the pro- thione reductase and nicotinamide adenine dinucleotide cess, such as head-tail attachment abnormalities, incomplete phosphate (NADPH) (41). Superoxide dismutase (SOD) is a acrosome development, or sperm cytoskeleton alterations, metal-containing that reduces two into can lead to the creation of ROS and abnormal sperm O2 and less toxic H2O2 (9). Catalase is an enzyme found within morphology (23, 24, 31, 32). that completes the reduction process by breaking Sperm membranes contain a high concentration of poly- down H2O2 to and O2. Other that also reduce unsaturated lipids, making them sensitive to ROS and oxida- H2O2 include glutathione transferase and ceruloplasmin, or tive damage (24, 33). Lipid peroxidation can result in loss of heme oxygenase (42). membrane fluidity and integrity, leading to increased Not all OS is harmful to sperm function. Within normal permeability. This can lead to impaired motility, abnormal physiologic conditions, antioxidants help maintain a low morphology, reduced sperm-oocyte fusion, DNA damage, level of oxidative stress in the semen, thereby allowing reduced acrosomal reaction, and even sperm cell death (34– normal processes and spermatic function and 36). More recent studies have demonstrated that the avoiding ROS-induced cell damage (3, 14). Normal incidence of teratozoospermia may be directly correlated to physiologic ROS levels are required for sperm maturation, excess ROS (37). hyperactivation, chemotaxis, zona pellucida binding, Although ROS are formed during normal enzymatic reac- acrosome reaction, and sperm-oocyte fusion (22–24, 29, tions, cellular damage is prevented through a scavenging sys- 43–45).

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ETIOLOGIES OF OXIDATIVE STRESS coal, has an oxidative effect in the testis (67). Exposure to Oxidative stress can arise intrinsically from sperm itself and high levels of certain metals such as cadmium (68) or lead from a variety of conditions that are known to have detri- (69) can raise testicular OS and epididymal ROS production mental effects on male fertility. These conditions can range and result in decreased sperm production, antioxidant capac- from intrinsic abnormalities to environmental exposures. ity, and motility and increase lipid peroxidation (70). Toxins Some etiologies are reviewed here. from cigarette smoking have been noted to increase ROS pro- Intrinsic ROS can originate from damaged or deficient duction in many tissues, including the testes, and have been spermatozoa (46). Impaired spermatogenesis resulting in associated with infertility (71, 72). abnormal spermatozoa can yield excess ROS (47). Other Men with cancer who have been treated with chemo- intrinsic etiologies include , cryptorchidism, testic- therapy and/or radiation therapy are at risk of infertility. ular torsion, infection/inflammation, and aging. Rapidly dividing spermatogonia are more sensitive to damage Varicocele, a condition that is caused by dilation of the compared with later stage cells. Commonly used chemo- spermatic veins, has been found to be associated with infer- therapy agents that have gonadotoxic properties include tility in approximately 30% of men (48). The increased retro- (73), cyclophosphamide (74), and doxyrubicin (75). grade flow leads to an elevation of scrotal and testicular Heat shock may facilitate the testicular OS response temperatures (49). This results in OS from rising ROS concen- (76). trations in the testes and semen of varicocele patients (50, 51). Oxidative stress is induced in the testis from ionizing ra- Varicocele is also associated with a decrease in antioxidant diation (77). This leads to germ cell apoptosis (78). Despite the capacity (50). developing spermatogonia being sensitive, Sertoli and Leydig Cryptorchidism also results in increased testicular cells are relatively resistant to the effects of radiation (79). temperature and has been associated with increased ROS This may be, in part, due to the rise of total antioxidant capac- production and OS (52, 53). The elevation of ROS levels can ity noted in these cells after irradiation. lead to germ cell apoptosis as well as alterations in genes responsible for energy and lipid metabolism, stress IMPACT OF ELEVATED ROS ON SPERM AND response, and redox reactions (54). Testicular torsion and repair can induce significant OS re- FERTILITY CONSEQUENCES sulting from an -reperfusion (55). Torsion can Reactive oxygen species have a significant impact on sper- result in disruption of the seminiferous tubules and germ cell matogenesis as well as on sperm function. Supraphysiologic loss (56). Increased OS has been found within the testis after levels of ROS have been found to potentially impact all as- testicular torsion repair (57). pects of the semen analysis (15). Pathologic damage from Localized genitourinary infections and inflammation can oxidative stress occurs with an imbalance in the oxidant- affect male fertility by causing increased OS. These conditions antioxidant system, and ROS levels increase beyond the anti- increase the leukocyte concentration in the local environment oxidant buffering capacity of the system (41). Oxidative stress (58). The inflammatory response results in the rise of testicular can occur when there is an increase in ROS production, a inducible nitric oxide synthase, interleukin-1b, and decrease in antioxidant scavenging capacity, or both (25). cyclooxygenase-2 with concomitant decline in antioxidant Infertile men are more likely to have pathologic levels of enzymes and germ cells leading to an overabundance of seminal ROS as a result of increased ROS production rather ROS (59). Testicular inflammation has also been demonstrated than low antioxidant concentrations when compared with to result in a reduction in mitochondrial membrane potential, fertile controls (80). Up to 80% of men with infertility have steroidogenesis, and ultimately germ cell apoptosis (60). elevated seminal ROS levels (40). A recent study compared Aging has been linked to elevations in testicular OS (61, the semen of infertile men to proven donors (15). The semen 62). This has been suggested to be linked to the age related of infertile men had significantly higher levels of ROS declines in steroidogenesis (63). Aging Leydig cells have compared with those of proven donors. This study found been demonstrated to have reduced expression of enzymatic that elevated ROS values had a sensitivity of 68.8% and spec- and nonenzymatic antioxidants decreasing the buffering ificity of 93.8% in correlating with poor semen parameters capacity within the testes thereby increasing the risk of such as concentration, motility, and morphology, and could damage from ROS (62, 64). result in infertility (15). Environmental or extrinsic factors can also impact testic- Multiple studies have demonstrated the association of ular ROS levels thereby raising OS levels, leading to abnor- elevated ROS levels with abnormal sperm concentration (15, malities in spermatogenesis (13). These include exposure to 16, 81), motility (16), and morphology (17), with sperm toxins, chemotherapy, and . DNA damage (18, 82, 83), and with apoptosis (19). Elevated There are many environmental and industrial toxins that ROS levels can also decrease sperm-oocyte fusion (41). cause elevated ROS levels in the testes and impact spermato- Conversely, other studies have demonstrated no correlation genesis and sperm function. Briefly, methoxyethanol, found between ROS and sperm motility (20, 21). in paints, brake fluid, and other industrial chemicals, can in- Sperm motility can be negatively impacted by elevated crease ROS levels (65). Toluene by products found in solvents ROS levels. Reactive oxygen species may result in decreased can also cause oxidative DNA damage (66). Sulfur dioxide, a axonemal phosphorylation and reduced membrane combustion byproduct of burning petroleum products and fluidity (83, 84). Certain ROS, like H2O2, can diffuse across

1520 VOL. 102 NO. 6 / DECEMBER 2014 Fertility and Sterility® cellular membranes and disrupt enzyme activity, such as with analysis, Agarwal et al. (105) reviewed nine studies that spe- G6PD (85). Reduction in the availability of NADPH results in cifically addressed the association of ROS and IVF outcomes. increased levels of oxidized glutathione, reduced levels of Only three of nine studies were included in the final meta- glutathione, decreased antioxidant capacity, and increased analysis, with selection criteria including use of density peroxidation of membrane phospholipids (45). Reactive gradient for sperm preparation and N-formylmethionyl- oxygen species have also been implicated in the underlying leucyl-phenylalanine-stimulated ROS levels with correlation mechanism of oligozoospermia; patients with values to IVF outcomes, for a total study population of n ¼ oligoasthenoteratospermia and oligoasthenospermia show 122. The estimated overall correlation for this analysis significantly higher levels of ROS compared with normal was 0.374 (95% confidence interval [CI], 0.205 to healthy donors (86). 0.520) suggesting that ROS levels negatively impact fertil- Increased OS can increase DNA damage to mitochondrial ization rates for IVF. The meta-analysis, however, was not and nuclear material of spermatozoa (87). This can occur without weaknesses, including a lack of consistent ROS on an amino acid or molecular level in the form of base testing methods, unknown male or female factor infertility, modification, production of base-free sites, deletions, DNA incomplete statistical information in the original studies, cross-links, frame shifts, or even on a higher level with rear- and inclusion of normal semen parameters into the analysis. rangement of chromosomes (13). High ROS levels are associ- The investigators proposed that if all nine studies had had ated with an increased frequency of sperm DNA strand breaks similar ROS tests with complete statistical analyses, the out- in infertile men (88, 89). Previous studies have not established comes of the meta-analysis might have been different. a cutoff value for ROS or sperm DNA fragmentation at which pregnancy would not be possible (90). ROS AND ANTIOXIDANT CAPACITY TESTING Damage to sperm DNA ultimately can be clinically man- Testing for ROS can be achieved with direct or indirect assays ifested with decreased fertilization ability, impaired embry- (Table 1). Direct assays measure the net oxidative imbalance onic development, pregnancy loss, and potentially birth between ROS production and the antioxidant concentration defects (91, 92). Mouse models have suggested that sperm in semen by measuring the amount of oxidation within the with high levels of DNA damage used for conception can sperm cell membrane (37). These tests include chemilumines- result in aberrant growth and behavior, premature cence, nitro blue tetrazolium (NBT) test, cytochrome C reduc- aging, and an increased incidence of mesenchymal tumors tion test, flow cytometry, electron spin resonance, and in offspring (93). Increased rates of aneuploidy have also xylenol orange-based assay (106). Direct ROS assays have been found to be associated with elevated sperm DNA limited widespread clinical application at this time due to fragmentation rates, potentially resulting in genetic cost and practicality issues, rendering them more suitable abnormalities (94). for research purposes (107). Elevated ROS levels have been directly correlated with Indirect assays evaluate the downstream effects of oxida- increased apoptosis in mature spermatozoa, resulting in oli- tive stress, such as DNA damage or lipid peroxidation levels gozoospermia (40, 95). Oxidative stress has also been (37). Although indirect assays do not give a direct measure implicated in abnormal apoptosis via disrupting of the level of ROS within the system, they can provide impor- mitochondrial membranes and altering the caspase activity tant information regarding the negative effects that ROS has that induces apoptosis. Although sperm apoptosis may upon the spermatozoa as well as the components within the appropriately control overproduction of male gametes, spermatozoa. Indirect testing includes the myleperoxidase abnormal apoptosis may allow the persistence of abnormal or Endtz test, redox potential, lipid peroxidation levels via thi- spermatozoa that are marked for elimination, thereby obarbituric acid-reactive substances, and the isoprostane increasing teratozoospermia (96, 97). method, chemochine, and DNA damage testing. DNA damage Elevated ROS levels can affect many aspects of the sperm levels are not solely a measure of OS or ROS, but are used as a quality, including structural integrity, motility, morphology, surrogate measurement of ROS-related damage (103). count, viability, and DNA integrity, thereby making it one Currently, there are over 30 different assays that can be of the potential etiologies of male factor infertility (13, 15– used to measure ROS and OS in semen. Most ROS testing 18, 40, 98, 99). Despite the negative effect of ROS on sperm, controversy continues on the effect of elevated ROS levels on assisted TABLE 1 reproductive technology (ART), including in vitro fertilization (IVF) and pregnancy outcomes. There have been studies that Various direct and indirect semen assays of reactive oxygen species. have demonstrated that despite the negative impact on sperm quality and function, elevated ROS levels had no negative ef- Direct assays Indirect assays fect on IVF or intracytoplasmic sperm injection (ICSI) rates Chemiluminescence Antioxidants (100, 101). Yeung et al. (100) proposed that ROS might have Cytochrome C reduction test Chemokines Electron spin resonance DNA damage a positive effect on fertilization. Flow cytometry Lipid peroxidation levels Other studies have demonstrated negative effects of ROS Nitroblue tetrazolium test Measurement of redox potential on ART outcomes (102–104), including a meta-analysis Thiobarbituric acid assay Myeloperoxidase or Endtz test demonstrating a negative correlation between ROS levels Xylenol orange-based assay and fertilization rates using IVF (105). In their meta- Ko. Male infertility and ROS and TAC. Fertil Steril 2014.

VOL. 102 NO. 6 / DECEMBER 2014 1521 VIEWS AND REVIEWS does not identify the source of ROS (leukocytes versus imma- considered in idiopathic male infertility with normal bulk ture or abnormal spermatozoa) (106). semen parameters. Unexplained idiopathic oligoasthenotera- Seminal antioxidant capacity can be tested for each indi- tospermia or abnormalities in other specialized semen testing, vidual antioxidant in the seminal system or as a cumulative such as DNA fragmentation, acrosome reaction, or sperm- sum of the antioxidants (Table 2). The total antioxidant ca- oocyte binding, may also indicate ROS testing (3, 40).As pacity (TAC) is measured by evaluating the reducing ability with any test, ROS evaluation should be considered if it of the antioxidants within the semen against an oxidative provides additional information that would potentially reagent such as , and measuring the effect change or improve the management of male infertility and on the substrate (29). Depending on the substrate, the reaction ultimately pregnancy outcomes. However, the currently can be measured with a calorimeter, spectrophotometer, available studies correlating ROS levels and pregnancy or visually. Testing of individual antioxidants within semen outcomes are limited, and some are even contradictory. is costly and time consuming (29). In addition, measurement Oxidative stress and TAC testing may provide couples of individual antioxidants does not provide the entire with additional diagnostic data as to whether the male factor antioxidant picture. Individual antioxidant capacity can be is the primary source of their infertility, what the likelihood of determined by testing for catalase, , achieving pregnancy is if given time, and whether treatment and reductase, and superoxide dismutase activity (108). with antioxidant therapy is warranted if elevated ROS levels Total antioxidant capacity assesses the cumulative effect are the only abnormality. A 4-year prospective analysis of all antioxidants present within the semen (29). Studies have comparing the diagnostic significance among conventional demonstrated that normospermia is directly correlated with semen analysis, hamster-oocyte fusion, and ROS generation higher TAC levels compared with those of patients with in 139 couples demonstrated that ROS generation had a nega- asthenoteratospermia or oligoasthenoteratospermia (109). tive impact on sperm-oocyte fusion and fertility status despite Conversely, decreased seminal TAC levels are correlated normal semen analyses (114). with impaired semen parameters (110). However, TAC levels Testing for ROS has been used in combination with other are not routinely measured as a standard part of an infertility tests, including conventional semen analysis, acrosome reac- evaluation because of the lack of a proven benefit and the cost tion, and acridine orange staining to predict IVF outcomes, and time-consuming nature of this diagnostic testing when with an overall accuracy of 83.6% (115). The utility of ROS compared with a routine semen analysis (37). testing has also been demonstrated with sperm donors and A ROS-TAC score is a parameter that is derived from the proven donors, who have considerably better semen parame- ratio between ROS concentrations and seminal TAC (111). The ters and significantly lower ROS levels compared with infer- ROS-TAC score may be more predictive of subfertility than tile men (15). ROS levels or TAC alone (112). Infertile men have lower scores Despite their potential for providing additional prognostic when compared with their fertile counterparts, with a information, ROS and TAC testing are not routinely used during score <30 predicting risk for prolonged inability to conceive the initial male fertility workup because of their high cost, (13, 33). inconvenience, and lack of efficiency. In addition, there is an Elevated seminal OS on testing is abnormal and may reflect absence of standard protocols to assess seminal oxidants an oxidative imbalance (113). The OS level is not measured despite the ample data implicating ROS in male infertility directly but is a reflection of excess ROS, or lack of adequate (37). There has been no consensus on ROS and TAC testing be- antioxidant capacity to buffer the ROS, which can create a ing included as a routine part of an initial male infertility eval- detrimental environment for sperm production as well as func- uation owing to the lack of proper studies, the poor tion. The etiologies of OS were discussed earlier in this article. understanding of the ROS measurement in multiple assays, and the confusion as to whether neat or washed seminal ROS INDICATIONS FOR ROS AND TAC TESTING should be measured and the impact on the results (113, 116). In addition, smaller laboratories may find the testing Testing of ROS or TAC can be considered when an abnormal costly as considerable capital expenditure is required to semen analysis is found in an infertile male. It can also be obtain the necessary equipment and assays. However, the economies of scale make ROS and/or TAC testing efficient and cost effective for some laboratories (such as high- TABLE 2 volume academic centers), and they potentially can provide additional clinically relevant information to the treating cli- Tests to determine antioxidant capacity. nicians and their patients (15, 29). Individual Total Currently, no literature recommends against ROS testing, Catalase Colorimetric assay but the American Urologic Association Best Practices State- Dismutase Ferric reducing ability of plasma (FRAP) ment on evaluating the infertile male does not advocate the Glutathione peroxidase Luminol routine use of ROS testing as part of managing couples (117). Oxygen radical absorbance capacity (ORAC) This is because ROS testing lacks the ability to predict preg- Lipids Phycoerythrin fluorescence-based assay nancy outside of the semen analysis, and there are no proven Proteins therapies to treat elevated ROS levels if they are found. In their Vitamins opinion statement, the American Society for Reproductive Ko. Male infertility and ROS and TAC. Fertil Steril 2014. Medicine Practice Committee supports this view as well (118).

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ANTIOXIDANT THERAPY supplements (e.g., supplemental vitamins, minerals, or herbs) Antioxidants can be obtained through two main sources: and are thought to increase the physiologic TAC (Table 3). A physiologic or dietary. Physiologic antioxidants are present higher intake of antioxidant-rich foods can potentially in the seminal plasma and the spermatozoa (9, 24). Intrinsic improve sperm DNA integrity and overall protection against ROS cellular damage is provided by (123). This is contrasted by studies demonstrating that a lower intracellular enzymatic and nonenzymatic antioxidants. intake and subsequent lower antioxidant concentration may Sperm contain minimal intracellular antioxidants due to be associated with poor semen quality (124, 125). their lack of significant cytoplasm (24, 119, 120). The Antioxidant supplement is not without risk, however. majority of TAC is thus contained in the seminal plasma. Adverse events, although uncommon at doses at or below the The enzymatic antioxidants include catalase, glutathione recommended daily allowance, usually occur with excess peroxidase, and superoxide dismutase. The nonenzymatic intake of supplements (126). Supplement-related adverse antioxidants include carnitine, , glutathione, events can be of similar magnitude to those experienced with urate, and vitamins C and E (29, 41, 120). conventional pharmaceuticals. A link has been established be- Antioxidant supplements can further be divided into two tween plasma L-carnitine levels and atherosclerosis promotion categories: natural and synthetic antioxidants. Natural anti- resulting in an increased risk of and ma- oxidants are those that are synthesized physiologically and jor adverse cardiac events (127). Vitamin A supplementation can be isolated from food sources. Synthetic antioxidants has been linked with an increased incidence of mortality from are those that are chemically synthesized as an isolate com- lung cancer and cardiovascular disease (128, 129). pound and packaged into pill form. Several studies have sug- supplementation has been shown to increase the risk of gested that synthetic compounds have suboptimal cardiac complications, breast cancers in women, and prostate – antioxidant properties due to their chemical composition cancer men (130 132) and has been found to increase all- and having been isolated from other synergistic compounds cause mortality at doses greater than 400 IU/day (128). present in natural food sources (121, 122). Combination supplement therapy may also have addi- Dietary antioxidants are obtained through oral intake tional side effects attributed to the synergistic effects of the in- of food (e.g., fruits, vegetables, nuts, dairy, and meat) or dividual agents (133). Concurrent selenium and high-dose

TABLE 3

Mechanism of action of various antioxidants and supplements. Antioxidant/supplement Mechanism of action Arginine (139) Potent immunomodulator Role in cellular inflammatory response and protection against oxidative damage Carnitines (140) Central role in cellular energy metabolism Transporter of activated long-chain fatty acids into mitochondria through b-oxidation and buffering of acetyl-coenzyme A (CoA) to CoA ratio Coenzyme Q10 (141) Crucial component of mitochondrial oxidative phosphorylation Electron transporter in mitochondrial respiratory chain Oxidation-reduction (redox) link between flavoproteins and cytochromes Redox control of cellular signaling origin and transmission Folic acid (142) Provides carbon groups for purine and pyrimidine production Important role in DNA synthesis Important role in proper cell function Glutathione (143, 144) Maintains exogenous antioxidants in reduced states Detoxification of carcinogens and foreign compounds Lycopene (145) Protection of lipid peroxidation Gap junction communication Cell growth regulation Gene expression modulation Immune response N-acetylcysteine (146) Free radical scavenger Promotes cellular glutathione production Selenium (147) Cofactor in reduction of antioxidant enzymes as a component of glutathione peroxidase Vitamin A (carotenoids, retinoids) (148) Mechanism unknown (27, 149, 150) Important cofactor for hydroxylation and amidation reactions Assists in recycling oxidized vitamin E Neutralizes ROS in concentration-dependent manner Vitamin E () (151, 152) Prevents oxidant-induced lipid peroxidation Inhibits free-radical-induced damage to cell membranes Prevents lipid peroxidation Improves activity of other antioxidants Zinc (142, 153) Cofactor for metalloenzymes involved in DNA transcription and protein synthesis Antiapoptotic and antioxidant properties Ko. Male infertility and ROS and TAC. Fertil Steril 2014.

VOL. 102 NO. 6 / DECEMBER 2014 1523 VIEWS AND REVIEWS vitamin E has also been associated with an increased incidence What impact will treatment with antioxidants have on of prostate cancer and high-grade prostate cancer risk (134). pregnancy rates and ultimately live births? Several extensive reviews have investigated the effectiveness of single or combination antioxidant therapies. Some supple- ments, such as b-carotene, vitamins A, C, and E, and selenium, CONCLUSION fi have not been proven to have distinct bene ts and may even be Elevated ROS levels have been found to involve 30%–80% of harmful and increase all-cause mortality (135, 136). infertile males and may potentially be the cause of their infer- A recent Cochrane Collaboration that included a pooled tility. Reactive oxygen species can cause abnormalities of analysis of 34 randomized controlled trials comprising 2,876 sperm morphology, motility, concentration, and DNA integ- fi couples extensively reviewed antioxidant studies speci cally rity, resulting in difficulties achieving pregnancy. The impact for male infertility in couples undergoing ART (137).No of ROS on fertilization and pregnancy is controversial. This is conclusion could be drawn on the effects of antioxidants on partly because of the lack of consensus on what type of pa- fi sperm parameters. There was a statistically signi cant increase tients may be suitable for ROS testing and also what kind of in pregnancy and live-birth rates in the couples who had un- assay should be used. Therefore, routine ROS and TAC testing dergone treatment with antioxidant supplements. In looking is not currently recommended as a part of a standard male at the actual results, there were 96 pregnancies in 964 couples infertility evaluation. combined from 15 trials (pooled odds ratio [OR] 4.18; 95% CI, – P< 2 ¼ 2.65 6.59; .0001; I 0%). There were only 20 live births in REFERENCES the 214 antioxidant couples, obtained from three of the studies included in this review (pooled OR 4.85; 95% CI, 1.92–12.24; 1. Practice Committee of American Society for Reproductive Medicine. Defi- P¼.0008; I2 ¼ 0%) compared with a control group. That review nitions of infertility and recurrent pregnancy loss: a committee opinion. Fer- suggests that there are negative effects of elevated ROS on til Steril 2013;99:63. 2. Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, et al. fertilization and that antioxidant therapy may be useful in Best practice policies for male infertility. Fertil Steril 2002;77:873–82. treating men with elevated levels. 3. Agarwal A, Sekhon LH. 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