Oxidative stress and male infertility

Abstract Background Between 30% and 80% of patients with male infertility produce excessive reactive oxygen species (ROS) in their ejaculate even though the cause of male infertility is unexplained in approximately half of cases. The strong connection between oxidative stress (OS) and male infertility has led recent investigators to propose the term “Male Oxidative Stress Infertility (MOSI)” to describe OS‐associated male infertility. Methods We searched the PubMed database for original and review articles to survey the effects of OS on male infertility, and then verified the effects and treatments. Main findings Seminal plasma contains many antioxidants that protect sperm from ROS, because low amounts of ROS are required in the physiological fertilization process. The production of excessive ROS causes OS which can lower fertility through lipid peroxidation, sperm DNA damage, and apoptosis. Several assays are available for evaluating OS, including the MiOXSYS® analyzer to measure oxidation‐reduction potential. Several measures should be considered for minimizing OS and improving clinical outcomes. Conclusion Accurately diagnosing patients with MOSI and identifying highly sensitive biomarkers through proteomics technology is vital for better clinical outcomes.

sperm-oocyte fusion. 6,7 However, lipid peroxidation (LPO) within the cellular membrane, deoxyribonucleic acid (DNA) fragmentation in nuclei and mitochondria, and apoptosis 8 can occur when the production level of ROS gets excessive. All these events negatively affect sperm parameters, [9][10][11][12][13][14][15] male fertility, 12 and pregnancy outcome of their partners. Clinically, several studies have demonstrated that OS in human semen resulted in significantly poor assisted reproductive technology (ART) outcomes, such as lower fertilization rates, arrest of embryonic development, implantation failure, recurrent pregnancy loss, and lower live birth rates. [16][17][18] Agarwal et al proposed the term and concept of "Male Oxidative Stress Infertility (MOSI)", 19 and it has been determined that there are many patients with MOSI among those who were previously classified as having idiopathic male infertility. 19 Understanding the effects of ROS on male fertile capacity, measuring OS accurately, and treating patients based on their pathophysiologies will contribute to improving male infertility outcomes. The following short review will describe the pathophysiologies of OS and discuss its treatments.
The electrons form pairs by depriving other compounds of an unpaired electron, which can cause oxidation. One of the representative free radicals, superoxide anion, reacts to form a precursor of hydroxyl radicals and hydrogen peroxide. Hydrogen peroxide is one of the non-radical species and is not very reactive. However, it generates hydroxyl radicals, 21 which are highly reactive and oxidative, when endogenous metal ions are present. It is therefore the most important factor causing oxidative damage, including DNA damage and in vivo cellular LPO, especially in human semen.
It has become apparent over the past 20 years that the physiological levels of ROS have an impact on several signaling pathways that regulate biological and physiological redox-sensitive processes. 22 These redox processes usually require ROS for interaction with the amino acid cysteine on proteins. ROS typically mediate cell proliferation and apoptotic pathways that regulate the cell cycle and programmed cell death. Likewise, ROS in semen plays a low-level role as a second messenger in the fertilization processes, including sperm maturation, hyperactivation, capacitation, acrosome reaction, egg penetration, and sperm head decondensation. 6,7 Human sperm generates ROS through several pathways which induce cyclic adenosine monophosphate in sperm, activate tyrosine kinases, and increase the tyrosine phosphorylation level. The localization of tyrosine phosphorylation in the flagellum causes spermatozoal hyperactivation within the female genital tract; furthermore, it causes binding of the sperm to the zona pellucida which is essential for acrosome reaction. 6,7 OS caused by excessive ROS production has various detrimental effects on certain components of the human body, such as the cranial nervous system, cardiovascular system, digestive system, and endocrine and metabolic systems, even though low levels of ROS have important physiological functions. [23][24][25][26] An excess of seminal ROS has been reported in 30% to 80% of infertile men. 11,15,27 It is well known that seminal OS induces LPO of the sperm cell membrane, sperm DNA fragmentation (SDF) and, consequently, apoptosis.
OS results from not only excessive ROS production but also from low antioxidant capacity. All human semen contains endogenously produced antioxidants in the seminal plasma to protect the sperm from OS via three mechanisms: prevention, interception, and repair.
Nonenzymatic antioxidants include ascorbic acid (vitamin C) and F I G U R E 1 Factors that can cause oxidative stress. Oxidative stress results from a disturbance of homeostatic balance between ROS production and antioxidant capacity in seminal plasma [Colour figure can be viewed at wileyonlinelibrary.com] alpha-tocopherol (vitamin E), coenzyme Q10, astaxanthin, myo-inositol, urate, taurine, melatonin, transferrin, L-carnitine, and lactoferrin. [28][29][30] These antioxidants function as ROS scavengers to maintain redox homeostasis. The total antioxidant capacity (TAC) assay was often used to measure the total amount of nonenzymatic antioxidants in seminal plasma. 31

| MAIN SOURCE S OF ROS
The two major sources of endogenous ROS in human semen are leukocytes (extrinsic ROS) in seminal fluid 4,32 and immature sperm (intrinsic ROS) having a morphologically abnormal head and cytoplasmic retention. [32][33][34] When leukocyte chemotaxis and activation stimulate male genital tract inflammation and infection, extrinsic ROS is produced. ROS production occurs when leukocytes break down pathogens by activating the myeloperoxidase system. 27 This excessive production of ROS by leukocytes can lead to OS in seminal fluid.
Intrinsic ROS is produced by abnormal and immature spermatozoa. Cytoplasm deposits in the mid-piece fall off to cause cell elongation and condensation during the normal spermiogenesis process.
The morphologically abnormal, immature spermatozoa retain the excess residual body containing large amounts of cytosolic glucose-6-phosphate dehydrogenase enzyme and produce intracellular nicotinamide adenine dinucleotide phosphate (NADPH). NADPH then produces ROS via NADPH oxidase called NOX5 located in the intramembrane. 35 Therefore, immature spermatozoa are characterized by the presence of cytoplasm in the residual body of the mid-piece which produces excessive ROS. It is very important to determining the source of excessive ROS production in semen because infiltrating leukocytes and immature spermatozoa have very different clinical implications.
Myeloperoxidase staining is effective for distinguishing granulocytes such as neutrophils, polymorphonuclear leukocytes from germ cells to determine the source of excessive ROS production in semen. 36,37 Leukocytes positive for peroxidase staining in semen stain brown, which reflects their capacity for producing excessive ROS through phagocytosis. 38 These activated leukocytes increase NADPH production via the hexose monophosphate shunt, allowing them to produce 100 times more ROS than non-activated leukocytes. 39 On the other hand, nitroblue tetrazolium (NBT) staining is effective for detecting ROS production by immature spermatozoa. 40 NBT is a yellow, water-soluble, nitro-substituted aromatic tetrazolium compound that reacts with cellular superoxide ions to form a blue-colored formazan derivative, which can be observed either microscopically or spectrophotometrically using an enzyme-linked immunosorbent assay (ELISA) plate reader. Oxidation within the cytoplasm helps transfer electrons from NADPH to NBT and reduces NBT to blue-colored diformazan. The intensity of staining is therefore correlated with that of intracellular ROS. 41 NBT staining is a user-friendly technique used to predict the level of ROS production and detect the source of ROS production. Table 1 shows the well-established external sources of ROS roughly classified into five factors. Many investigations have focused on the impact of these factors on seminal OS. For example, alcohol abuse reportedly causes excessive ROS production, partly due to undernutrition resulting in insufficient antioxidant intake. 42 Alcohol abuse also decreases the success rate of IVF and increases the rate of miscarriage, 43 and tobacco smokers could exhibit higher ROS production than nonsmokers. 44 However, the mechanism of smoking toxicity is complicated because tobacco contains many kinds of chemical compounds, including nicotine, tar, carbon monoxide, and heavy metals. 45 Many of these toxic compounds have an oxidizing effect and can induce in vivo chromosomal aberrations and SDF. 46 Smoking can recruit proinflammatory leukocytes which increase seminal ROS levels. A small cohort study reported that smokers had 48% higher seminal leukocyte levels and 107% higher seminal ROS production than nonsmokers. 47 The mean sperm DNA fragmentation index (DFI) of infertile smokers was also reported as being higher than in infertile nonsmokers (37.66% vs. 19.34%, P < .001). 48  Obesity is often associated with impaired spermatogenesis due to endocrinological abnormalities. Adipose fibroblasts contain aromatase which converts testosterone into estradiol.

| OTHER SOURCE S OF ROS
Moreover, cytokines are generated from adipose tissues leading to the recruitment of proinflammatory leukocytes and an increase in NADPH oxidase activity, which induces OS. 50 Previous investigations have reported a positive correlation between body mass index and DFI. 51 Environmental pollution and heavy metals can also induce OS.
For example, phthalates are a class of chemicals used as plasticizers, which are also endocrine disrupters. They are widely found in polyvinyl chloride plastics and have been implicated in OS induction.
Phthalate esters act as peroxisome proliferators and can produce H 2 O 2 and other oxidants. 52 Wearing tight-fitting underwear, using a sauna, bathing for long periods of time, using a laptop on closed legs, and cycling 53-56 may result in an elevated scrotal temperature. The location of the scrotum keeps the temperature of the testes lower than body temperature by approximately 2°C. 57 Increased scrotal temperature may inversely, but reversibly, affect spermatogenesis. Several authors have reported a correlation between scrotal heat stress and SDF. Furthermore, intermittent heat exposure reportedly impedes spermatogenesis more than continuous heat exposure. 56 Immature spermatozoa may produce excessive ROS since the process of spermiogenesis is susceptible to heat stress and heat stress may alter the normal function of the epididymis. 58 OS may contribute to spermatogenesis suppression in response to heat.
Previous investigations have shown the effect of mobile phone radiation on increasing ROS production and decreasing the activity of antioxidants, such as catalase, SOD, and glutathione peroxidase (GPX). 59 Recently, Gautam et al demonstrated that significant increase in ROS and lipid peroxidation level with simultaneously decrease in sperm count, alterations in sperm tail morphology were observed in the male Wistar rats which were exposed to 3G mobile phone for 45 days. 60 Approximately 15% of the general male population-30%-40% of men with primary infertility, and up to 80% of men with secondary infertility-are diagnosed as having clinical varicocele. 61 Recent meta-analyses have shown that varicocele repair significantly improves seminal parameters 62,63 and is considered as one of the most common causes of surgically treatable male infertility. 64 OS is one of the major contributors to male infertility in men with varicocele, and many investigations have demonstrated elevated levels of SDF in men with varicocele. 65 One of the main mechanism is considered to be a protamination and chromatin compaction disorder during the spermiogenesis process, which elevates the sensitivity of affected cells to OS causing defective spermatogenesis and SDF. 66 Most studies reported that the ROS production level of semen in men with varicocele was increased compared to the controls. 67 Furthermore, the endogenous antioxidant level in seminal plasma was decreased in patients with varicocele. 68

| IMPAC T OF OS ON S PERMATOZOA
A disturbance of the homeostatic balance between ROS and antioxidants occurs when highly reactive ROS exceed the antioxidant defense systems, and it can lead to the development of OS. This can have detrimental effects on sperm, such as LPO, SDF, and apoptosis.
We describe each effect below. for successful sperm-oocyte fusion after the capacitation and acrosome reaction biochemical cascades. 70 Byproducts of LPO bind to and disrupt mitochondrial proteins of the electron transport chain apart from disrupting the sperm cell membrane, which leads to electron leakage and consequently decreases mitochondrial membrane potential, decreases ATP production, and decreases sperm motility. 71 LPO has three phases: the first phase is "initiation," which is the extraction of hydrogen atoms from the carbon-carbon double bonds of an unsaturated fatty acid to propel free radicals. The second phase is "propagation," which is the formation of lipid radicals followed by their rapid reaction with oxygen to form peroxyl radicals. 72 When metals such as copper and iron are present, the peroxyl radicals can again abstract a hydrogen atom from an unsaturated fatty acid to produce a lipid radical and lipid hydrogen peroxide. 73 The last phase is "termination," where these formed radicals react with successive lipids and generate cytotoxic aldehydes and other end products.

| Lipid peroxidation
The main products of LPO are 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), and acrolein. One method used to quantify LPO is MDA measurement according to the spectrophotometric thiobarbituric acid reaction (TBAR) test. MDA is an essential biomarker for analyzing and monitoring PUFA peroxidation levels. 69,74

| Sperm DNA fragmentation
Excessive ROS production and decreased antioxidant levels in semen can also lead to SDF. OS can damage sperm DNA directly or indirectly through sperm caspase and endonuclease activation.
SDF is caused by DNA vulnerability due to a chromatin compaction error during the spermiogenesis process, which causes a substitution failure of chromatin structure from histone to protamine. This damage is due to ROS exposure after spermiation, during comigration of spermatozoa from the seminiferous tubules through the rete testis to the cauda epididymis. This results in the formation of 8-OH-guanine and 8-OH-2'-deoxyguanosine (8-OHdG), 75 which is an oxidized guanine adduct. Increased 8-OHdG concentration correlates significantly with DNA fragmentation and strand breaks.
DNA has a double-helix structure, and DNA fragmentation can occur in both the single-stranded (ss-) and double-stranded (ds-) forms. DNA repair can only occur during specific stages of spermiogenesis, and the repair mechanisms are no longer activated during nuclear condensation in the epididymis. The next opportunity for ss-DNA break repair is by the human oocyte, which is a critical step in embryo development, although the ability to repair SDF decreases with advanced maternal age. 76 The ds-DNA break results in genomic instability and apoptosis in the absence of repair. 77 The presence of unrepaired SDF above the critical threshold reportedly has a detrimental effect on embryo development and pregnancy outcome-also called the "late paternal effect". 78 In a cleavage-stage embryo, major activation of embryonic genome expression begins on the second day of human embryo development (the 4-cell stage), and embryogenesis switches from maternal factor dependence to the embryo's own genome dependence. 79 Therefore, a spermatozoon with SDF negatively affects blastulation, implantation, and pregnancy outcomes after fertilization. Furthermore, Kuroda et al reported that OS also had an adverse effect on cleavage embryo development, called "early paternal effect". 80

| Apoptosis
Apoptosis via multiple cell death signaling and regulatory pathways is known as physiologically programmed cell death due to DNA fragmentation. ROS-induced ds-DNA breaks can result in apoptosis. ROS also disrupts the mitochondrial membranes so that they release signaling molecule cytochrome C, which can activate the apoptotic caspases and annexin-V binding to phosphatidylserine. High cytochrome c levels in seminal plasma may suggest significant damage to mitochondria caused by high levels of ROS in infertile patients.

| E VALUATI ON OF OS IN H UMAN S EMEN
As previously reported in the literature, 19 OS has been linked to unexplained and idiopathic male infertility, and measurement of OS is essential for its subsequent management and treatment.
Currently, more than 30 different assays have been described to measure seminal OS. They can be classified as direct or indirect assays (Table 2). Direct assays quantify the levels of ROS directly.
Indirect assays quantify the adverse effects of OS, such as SDF or LPO levels. 82

| Direct measurement assays for OS
The chemiluminescence method is one of the direct assays used to quantify seminal ROS levels. Takeshima et al 11

F I G U R E 2
Measurement of ROS by chemiluminescence method. ROS production levels were calculated as the integrated chemiluminescence for 30 min after the addition of luminol (5-amino-2,3-dihydro-1,4-phtalazinedione) to unwashed semen after baseline subtraction strengths and weaknesses, which can be used according to the number of samples processed. 83 As described above, the NBT assay is also a cost-effective, user-friendly, and sensitive direct assay. Unlike chemiluminescent assays, the NBT assay can assess seminal ROS levels and microscopically or spectrophotometrically determine the source (immature sperm or leukocytes) of ROS. Diformazan crystal concentration has a positive correlation with intracellular ROS level, and the location of the crystals reveals the cellular source of ROS in semen. 40,41

| Indirect measurement assays for OS
MDA is one of the byproducts of LPO, and it can be commonly Seminal TAC can be measured as the sum of antioxidant activities available in seminal plasma using enhanced chemiluminescence or colorimetric techniques. 88 Previous studies reported that a low level of seminal TAC was associated with male infertility. 89 Mahfouz et al reported that infertile patients had lower seminal plasma TAC levels compared to the proven fertile and donor group. 90 However, the ROS-TAC score which calculates the balance between oxidation and reduction potential 82 is superior for measuring ROS and TAC alone and is more suitable for predicting MOSI. 91 The ROS-TAC score is a parameter formulated using the ratio of standardized ROS production level in washed sperm suspensions and standardized TAC in seminal plasma using a principal component analysis. A cutoff value of 30 was determined as the lower end of a normal range of ROS-TAC score, and patients with lower scores are assumed to be at risk for infertility. 92 However, TAC assays cannot be performed routinely, as they require expensive equipment and advanced technical skills while only measuring nonenzymatic antioxidants. 13 MiOXSYS ® has the potential to substitute TAC assay, as it can measure simultaneously oxidation-reduction potential (ORP) more easily and in a shorter time.

| Novel measurement assays for oxidationreduction potential
In contrast to the methods described above, ORP measurement is a di-

| MANAG EMENT AND PRE VENTI ON FOR MOS I
There are many types of precautionary measures, such as those related to lifestyle and environmental modifications and treatment options (eg, sperm selection techniques) that can be used to minimize the adverse effects of OS on the reproductive function.

| Lifestyle and environmental modification
The lifestyle and environmental factors that increase ROS production are shown in Table 1. Patients may quit smoking, 101 avoid alcohol abuse and lose weight through balanced diet and moderate exercise, 102 and decrease their exposure to phthalates 52 in order to minimize extrinsic ROS production. It is also well-established that increased scrotal temperature 55,56 and exposure to harmful substances 103

| Shorter interval of ejaculatory abstinence
Because SDF occurs after spermiation during transfer of spermatozoa from the seminiferous tubules through rete testis to the cauda epididymis due to the ROS exposure, they can be affected by a harmful seminal microenvironment of OS while stored in the epididymis and after ejaculation. Therefore, increasing ejaculation frequency and decreasing the storage interval of spermatozoa in the epididymis may reduce sperm exposure to toxic ROS, thereby increasing sperm motility and decreasing SDF. Several studies have shown that a shorter interval between ejaculatory abstinence contributed to a lower seminal ROS and sperm DFI. 105,106 A shorter interval between ejaculatory abstinence may improve sperm quality and DNA integrity by reducing sperm exposure to excessive ROS in the epididymis.

| Oral antioxidant therapy
Oral  possible toxic effect when overused. 126,127 Therefore, the efficacy is still controversial. True efficacy of these treatments should be validated by large-scale nonrandomized two-arm studies between OS-positive and -negative groups.

| Varicocele repair
As described earlier, much evidence suggests that OS and increasing SDF are the major contributors to infertility in men with a varicocele. 66 In addition, much evidence also suggests that varicocelectomy in men with clinically palpable varicocele and infertility significantly improves male fertile. 64

| Testicular sperm extraction
As mentioned above, spermatozoa in semen are affected by ROS during the ejaculation process. The testis is protected by substantial antioxidant systems, but spermatozoa are released from the Sertoli cells during the spermiation process and migrate from the seminiferous tubules through the rete testis to the epididymis; they become susceptible to OS. 131 The SDF level in testicular sperm has been reported to be one third lower than in ejaculated sperm. 132 Testicular sperm extraction (TESE) is a procedure by which sperm is surgically retrieved from the testis of patients with azoospermia, cryptozoospermia, or ejaculatory disorders. ICSI using testicular sperm has a higher implantation and clinical pregnancy rates than ICSI using ejaculated sperm. 133,134 However, testicular sperm has a significantly higher aneuploidy rate than ejaculated sperm. 132 Therefore, this method should be performed with limited indication of recurrent ART failure and severe oligozoospermia cases.

| CON CLUS I ON S AND FUTURE DIREC TIONS
Male patients with idiopathic/unexplained infertility should be screened for MOSI as mentioned above using an efficient, inexpensive, and high sensitivity/high-specificity ORP assay as a screening test. 19,94 Those who are MOSI-positive should then undergo a more extensive examination to identify the treatable factors.
In recent years, advances in proteomics technology have led to the discovery of many protein biomarkers of disease to elucidate the pathological condition and establish treatment methods. 135,136 Protein identification in spermatozoa and seminal plasma in semen samples exposed to OS will help understanding the biological pathways associated with male infertility and may lead to the discovery of new biomarkers of idiopathic male infertility. HSPA2, a member of HSP70 family, as a heat shock protein, is reportedly a key protein underexpressed in ROS-positive sperm. HSPA2 is a protein located in the sperm tail and is involved in spermatogenesis. 137 Underexpression of DJ-1 (which removes ROS such as hydrogen peroxide and inhibits apoptosis) has been determined through seminal plasma proteome analysis in ROS-positive semen. 135 These proteins could be possible biomarkers for OS in semen.
Environmental factors, such as endocrine disruptors such as BPA (bisphenol A), DBP (dibutyl phthalate), and DEHP (bis (2-ethylhexyl phthalate)) in plastics, can generate OS and cause epigenetic DNA methylation. 138 Moreover, oxidative damage can cause epigenetic changes through a variety of mechanisms, including DNA methylation, histone modifications, and chromatin remodeling. It has been reported that DNA methylation deficiency caused by deficiency of methyltransferase KMT2D is impaired by ROS and is implicated in spermatogenesis, Sertoli cell only syndrome, and the incidence of testicular cancer. 139 Exploring biomarkers and epigenetic changes may lead to additional treatment and screening options in the future.

ACK N OWLED G M ENT
This research was supported by JSPS KAKENHI Grant No. 18K16739.

Conflicts of interest:
The authors report no declarations of interest.
Human/animal rights statements and informed consent: This article does not contain any studies with human and animal subjects performed by any of the authors.