redox regulation of fertility in aging male and the role of ...€¦ · redox regulation of...

Send Orders for Reprints to [email protected]

Current Pharmaceutical Design, 2017, 23, 1-13 1 REVIEW ARTICLE

1381-6128/17 $58.00+.00 © 2017 Bentham Science Publishers

Redox Regulation of Fertility in Aging Male and the Role of Antioxidants: A Savior or Stressor

Kristian Leiseganga,*, Ralf Henkelb and Ashok Agarwalc

aSchool of Natural Medicine, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa; bDepartment of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa; cAmerican Center for Reproductive Medicine, Cleveland Clinic, Cleveland, Ohio 44195, USA

A R T I C L E H I S T O R Y

Received: March 23, 2017 Accepted: April 26, 2017 DOI:

10.2174/1381612822666161019150241

Abstract: Oxidative stress (OS), an imbalance between reactive oxygen species (ROS) and antioxidant scaven-gers, is considered an important underlying mechanism associated with ageing, age-related chronic diseases and reproductive decline in males. However, OS is an important regulator of numerous molecular pathways essential for homeostasis within cells, tissues and systems. A fine balance between factors promoting OS and those that scavenge ROS is critically important. In the male reproductive tract and ejaculate, ROS are critical molecular players in essential reproductive processes, including spermatogenesis, epididymal transport, spermatozoa matu-ration and post-ejaculation processes such as motility, capacitation and the acrosome reaction. Increasing evi-dence suggests there is an overlap between age-related OS and male reproductive tract dysfunction correlating with reduced fertility potential due to semen quality decline, endocrine changes and sexual dysfunction. With increasing life expectancy in many regions of the world, together with an increasing paternal age and associated risk of for infertility, pregnancy complications and reduced health potential in the offspring, it is becoming in-creasingly important to fully understand the mechanism linked with these associations. This review considers the role of ROS and OS in fertility regulation, the ageing process, the role for OS in age-related reproductive decline, the approach to assessment in male infertility and the current use of antioxidants as a therapeutic option. Although evidence suggests exogenous antioxidant supplementation is useful in male infertility, caution to excessive or unnecessary use of these approaches is required to avoid the paradox of antioxidants inhibiting essential and beneficial ROS activities in male reproductive biology.

Keywords: Oxidative stress, reactive oxygen species, antioxidants, aging males, male infertility, redox balance.

INTRODUCTION Life expectancy is increasing worldwide, now reaching an av-erage of up to 80 years in many developed countries [1]. Ageing can be defined as a time-dependent decline in biological functions on the cellular, tissue and system level, related to a progressive decline in quality of life (QoL) alongside an increased probability of various functional and organic co-morbidities and risk of mortal-ity [2]. There is, however, much variation in how advanced co-morbidity processes are within a specific chronological age group [3]. The rate of ageing depends on genotypic inheritance, epigenetic expressions, underlying disease processes and accumulated effects of lifestyle, environmental and socioeconomic factors [4]. Some people also enter an increased risk category defined as frailty, in which advancing age is the underlying cause [3]. Although ageing is not in itself a disease, this natural biological phenomenon is fur-ther considered a major risk factor for numerous degenerative dis-eases, including various common malignancies, neurodegeneration and dementia, cardiovascular disease, osteoarthritis, osteoporosis and type-2 diabetes in the context of obesity and metabolic syn-drome [1; 2; 5]. Ageing is also associated with a shift in the balance between pro-oxidants and anti-oxidants towards oxidative stress [6]. It is therefore becoming increasingly important to understand the mechanisms of ageing associated with all clinical specialties, as well as within interdisciplinary practice, in order to develop and improve strategies associated with prevention and therapeutics, potentially increasing life span and improving quality of life. From a biological science perspective, ageing is a complex, multifactorial and relatively poorly understood phenomenon, with *Address correspondence to this author at the Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Tel: +27-21-959 2037; E-mail: [email protected]

no clear biological basis [7]. Although still an elusive area of study, numerous biomarkers of ageing have been proposed, as well as correlated to various frailty indices and pathophysiology of well-defined co-morbidities of ageing (Table 1) [8]. Within this context, oxidative stress (OS) and the broader focus of redox biology has been receiving increased scientific attention in recent decades [9]. Although the OS theory has risen to prominence in recent decades, the role of other theories of ageing, relating to immunosenescence, neuroendocrine decline, somatic mutation and error catastrophe, is less well defined [7]. The immune and neuroendocrine systems clearly deteriorate with age. However, underlying the phenotypic and clinical changes of ageing, genomic factors and OS are consid-ered important mediators that drive the cellular senescence under-pinning the ageing process [1-2]. OS is triggered by various patho-logical conditions, including inflammation, ischemia, heat-stress and ageing, as well as being an underlying cause of age-related fertility decline [10]. It is also interesting to note that more than half of all males that present with infertility and undergo relevant as-sessments are found to have excessive OS in the male reproductive tract [11]. Furthermore, evidence suggests that antioxidant defenses decline with age [12]. This provides a basis for OS being involved in the pathogenesis of male reproductive changes associated with ageing, highlighting the importance of understanding these mecha-nisms for therapeutic targets in ageing males. Although there may be a gradual decline in male reproductive potential with age, the average paternal age has been increasing in recent years for various socioeconomic reasons. Furthermore, many males want to father children at older ages, and a detailed under-standing of changes in the male reproductive system associated with ageing is of critical importance [13]. It is generally accepted that females live longer on average than males (although they have

2 Current Pharmaceutical Design, 2017, Vol. 23, No. 00 Leisegang et al.

Table 1. Proposed age associated biomarkers and direction of change [8].

Category Marker Direction of

Risk

CRP ↑

TNFα ↑

IL-6 ↑

Adiponectin ↑

TH-Lymphocytes (CD4+) (% T-Cells)

↓

TC- Lymphocytes (CD8+) (% T-Cells)

↑

CD4+: CD8+ Ratio ↓

Memory: Naïve CD4+ Ratio ↑

Memory: Naïve CD8+ Ratio ↑

Immune and Inflammatory

Markers

Memory: Naïve B-Lymphocyte Ratio

↑

Haemoglobin ↓

Platelets ↓

White Blood Cells ↑

Neutrophils ↑

Lymphocytes ↓

Monocytes ↑

Basophils ↓

Hematological Markers

Eosinophils ↑

Telomere Length ↓

DNA Repair (%) ↓

DNA Damage: DNA Repair Ratio ↑

Cellular Ageing, Genetic and

Oxidative Stress Markers

DNA methylation (%) ↑

increased rates of age related morbidities), and although there are many common co-morbidities, differences in incidence between the sexes is apparent [5]. In males specifically, cognitive decline, obe-sity and metabolic syndrome, cardiovascular disease (including hypertension as an underlying risk), depression, age-related andro-gen decline (ADAM), erectile dysfunction, testicular dysfunction with reduced sperm quantity and quality, lower urinary tract symp-toms (LUTS), benign prostatic hyperplasia (BPH) and prostate carcinomas are common co-morbidities observed [14-15]. Although testes can still produce sperm throughout the lifespan, the rate and quality of production gradually declines with age [16]. Further-more, age-related co-morbidities such as metabolic syndrome and diabetes are associated with reproductive tract inflammation and OS induced sperm damage [17-18]. The effect of ageing is also associated with increased genitourinary tract diseases in males, which directly or via their treatment options negatively influence semen quality and fertility potential [13]. In addition, an increased paternal age is also associated with increased rates of adverse birth outcomes and a negative impact on

the health of the offspring via epidemiological and animal studies. Children fathered by older males have increased risk of sporadic congenital diseases (such as achondroplasia and Alport syndrome), and increased lifetime risks of cancers (including leukemia non-Hodgkin’s lymphoma, breast and prostate cancers), neuropsy-chiatric disorders (including autism, schizophrenia and bipolar dis-order), as well as disorders with behavior and cognitive abilities [19]. These effects are postulated to be due to the accumulated DNA mutations over time, associated with reduced sperm DNA integrity as well as various epigenetic factors [19]. The accurate transmission of genotypic information is vital both prior to fertiliza-tion and following the development of the offspring during preg-nancy and through life [20].

OVERVIEW OF REDOX IN CELLULAR BIOLOGY Oxygen (O2) molecules are essential for aerobic life on earth, driving cellular metabolism and respiration, thus primarily used for ATP-generation within the mitochondria via the electron transport chain (oxidative phosphorylation) [21]. However, the paradoxical nature of this element has become apparent in recent years as the production of associated free radicals are detrimental to life. De-rived from metabolic use, O2 is transformed into a variety of mole-cules termed reactive oxygen species (ROS) as by-products of cel-lular respiration [21-23]. The broad term ROS is used to describe oxygen-derivatives that are highly reactive and contribute to a proc-ess termed oxidative stress (OS), having important cellular regula-tory roles as well as being implicated in numerous pathologies. ROS includes various sub-classes of molecules comprising of un-stable oxygen ions such as superoxide (.O2

-), free radicals that have one or more unpaired electrons in the outer molecular orbits, and peroxide molecules [24]. The terms free radicals and reactive oxy-gen species (ROS) are frequently used interchangeably, however, not all ROS are free radicals [25-26]. Hydrogen peroxide (H2O2), for example, is named among ROS, but is not a radical. An addi-tional sub-class of biologically important reactive molecules com-prises of nitrogen, termed reactive nitrogen species (RNS). These include nitrous oxide (NO), peroxynitrate and peroxynitrous acid [25]. For the purposes of this review, ROS will be used to encom-pass all endogenous highly reactive oxygen molecules. Endogenous ROS are produced intracellularly via numerous cellular mechanisms, which may vary within different tissues. Im-portantly, ROS is generated by the mitochondria during oxidative phosphorylation (mtROS). This is done via a series of reduction-oxygen (redox) reactions in which there is a transfer of electrons through five mitochondrial protein complexes. However, a small leakage of electrons from protein complex I and complex III results in the formation of superoxide (.O2

-) due to partial reductions at a rate of 0.2% - 2.0% of the oxygen consumed by the mitochondria. .O2

- is then metabolized by superoxide dismutase (SOD) 1 and SOD2 in the mitochondria into H2O2, a signaling molecule in the cell [27]. As with other endogenous ROS, H2O2 has particularly emerged as major redox metabolite operative in redox sensing, signaling and regulation in cellular function, including the male reproductive tract and sperm function [28]. H2O2 may be partially reduced further to produce a hydroxyl radical (.OH-). Moreover, other ROS can be generated directly (enzymatically) or indirectly (metal catalyzed) from .O2

- [25; 29]. Additionally, .O2- can undergo

further metabolism by NO to produce the highly detrimental oxi-dant peroxynitrite that denatures proteins and damages mitochon-drial membrane integrity [27]. In cells, ROS are used for numerous processes to regulate cellu-lar function including apoptosis for the elimination of damaged cells, immune system regulation and inflammation, response to growth factors, genomic regulation, and ion transport systems. However, due to the highly reactive nature of these molecules with half-life times in the nano-second range, ROS can not only further modify other oxygen molecules, but also damage macromolecules

Redox Regulation of Fertility in Aging Male and the Role of Antioxidants Current Pharmaceutical Design, 2017, Vol. 23, No. 00 3

such as proteins and lipids through oxidative stress (OS), defined as an imbalance between oxidants and antioxidants towards an oxida-tive status [24; 30]. Specifically, adaption to physiological concen-trations of ROS is involved as what is termed molecular redox switches (phosphorylation/dephosphorylation) as a mechanisms of cellular signaling [9]. At physiological concentrations, the cellular adaptions are important for normal cell function. However, an ex-cess in ROS production and/or reduction in savaging antioxidants is creating a state of OS in which cellular adaptions lead to patho-physiological changes in a process similar to the general adaption syndrome of cellular stressors [9; 28]. The activities of OS are me-diated through numerous intracellular pathways, including MAPK family members of ERK’s (ERK 1 and 2), c-Jun N-terminal kinases (JNK1, 2 and 3) and the p38 kinases (alpha, beta and gamma) [31-32]. Mediated by an unfavorable ROS-TAC mismatch, OS is known to contribute to the pathophysiology of many disease states in which antioxidants provide protection [24], many of which may also be mediated through the above signaling pathways in addition to direct toxic effects on molecules [31-32]. Due to the high cellular toxicity of OS, cellular defense mecha-nisms have evolved including many endogenous antioxidants that are capable of scavenging and neutralizing cellular ROS. These balances are illustrated in Fig. 1. H2O2 is quickly reduced in the mitochondria into water by catalase and glutathione peroxidase, the latter requiring glutathione as a co-factor. Three molecules of the SOD family are involved in antioxidant complexes with co-enzymes, namely copper and zinc (SOD1) or manganese (SOD2), as well as an extracellular SOD (SOD3). Other important endoge-nous antioxidant families include peroxiredoxin and thioredoxins [27]. It is therefore important that there is a balance between genera-tion of ROS and their elimination by endogenous and exogenous antioxidants within the cells. Disturbances in this balance can be due to numerous factors, both internal and environmental [26]. OS can induce accumulated cellular damage, implicated in the complex pathophysiology associated with ageing and age-related chronic diseases, including the metabolic syndrome, type-2 diabetes melli-

tus, cardiovascular disease, malignancy and neurodegeneration [24]. Scavenging of ROS is critical, and endogenous and exogenous antioxidants are used to reduce the damaging impact of excessive OS. The endogenous system consists of enzymatic antioxidants (e.g. SOD, catalase, thiol peroxidases), and non-enzymatic antioxi-dants (e.g. glutathione). The exogenous antioxidant system com-prises of various micronutrients (e.g. β-carotene, vitamins A, C and E; glutamine), phytonutrients, as well as trace elements such as zinc and selenium (involved in enzymatic functioning) [2]. Contrary to OS, a shift of the cellular redox status from normal physiologic levels into the reduced state is also highly problematic for cells since they will no longer be able to induce essential func-tions. This condition has been termed ‘reductive stress’ (RS) [33]. RS is considered as dangerous for cells as OS [34], and can be a cause for cardiac injury, neurological diseases such as Alzheimer’s disease [35-36] or dysregulations of embryogenesis [37]. This is illustrated in Figure 1.

THE FREE RADICAL THEORY OF AGEING Currently, there is no single theory that encompasses the com-plexity of the cellular and molecular mechanism that would fully explain the ageing process, which occurs on the molecular, cellular and organ level. An accumulation of damaged macromolecules and cellular components is considered a hallmark of ageing and can be characterized by an accumulation of cellular damage over time, closely associated with DNA damage and proteostasis driving cel-lular senescence [2]. Genomic factors and OS are considered two important cellular drivers for the accumulation of the cellular and molecular damage associated with ageing [1-2; 7]. Furthermore, many of the degenerative diseases associated with ageing are also closely associated with OS and chronic inflammatory processes as part of a complex mechanism of action in these pathologies [7]. Within this context, the free radical theory of ageing has achieved prominence as an underlying mediator of age-related morbidity and mortality, in which excessive endogenous and ex-ogenous ROS is detrimental to homeostasis [38]. This suggests that

Fig. (1). Oxidative interactions and metabolism in normal and abnormal cellular function.


there is an accumulation of macromolecular damage from excessive OS that is associated with age-related functional decline of organ-isms [39]. Cellular senescence is also preprogramed into the ge-nome as telomeres are unable to maintain their lengths due to the replication process. This leads to a decline in protective systems against cellular stressors, including ROS, during the ageing process [2]. Longer lived mammals, including humans, show decreased evidence of cellular oxidative damage [40]. Thus, ageing is associ-ated as a cause and effect of an increase in the ROS: total antioxi-dant capacity (TAC) ratio leading to a disruption in redox signaling [9], and this OS can act as a potent inducer of cellular senescence, particularly via H2O2, .O2

- and .OH- [2]. Cellular senescence is associated with numerous phenotypic changes within cellular structure and function. This includes in-creased cell size and protein content (accumulation of protein ag-gregates), increased oxidative protein damage, giant mitochondria (produce increasing amounts of ROS with age), enlarged nucleus, increased lipofusin accumulation and reduced proteasomal and lysosomal function. In addition, numerous secretory biomarkers are produced, including pro-inflammatory cytokines, growth factors, matrix metalloproteinases and ROS [2]. The genome is then associ-ated with a reduced capacity for DNA repair, resulting in an in-creased accumulation of genetic mutations and which is correlated with cellular senescence [1]. Mitochondria are considered a prominent cellular source of ROS where these highly reactive molecules are produced as a nor-mal metabolic process in cellular respiration during normal meta-bolic activity. These endogenous molecules oxidize and damage lipids, proteins and DNA, causing severe OS to mammalian physi-ology that drives the ageing process [41]. OS is associated as a cause of an accumulation of mitochondrial DNA (mtDNA) muta-tion associated with cellular senescence [42]. According to this theory, .O2

- production by the mitochondrial electron transport chain is readily converted to H2O2 by SOD2, the main defense against mitochondrial ROS [41]. The H2O2 can diffuse out of the mitochondria where it is converted to water via cytosolic catalase or mitochondrial glutathione peroxidase [41]. However, the H2O2 can be reduced by scavenging endogenous antioxidant molecules in-cluding mitochondrial glutathione peroxidase, mitochondrial perox-iredoxin and catalase [39]. If adequate scavenging of the reactive molecules is not effective, the imbalance leads to excessive macro-molecular damage that accumulates over time, and is driving and accelerating the degenerative and ageing process. As pointed out before, damage to mtDNA by OS is most closely associated with accelerated ageing in animal models [43]. mtDNA damage is also accumulated by replication errors made by the mtDNA polymerase [42]. Therefore, dysfunctional mitochondria are known drivers of excessive ROS production, they create an unfavorable imbalance in the redox status, and are thus considered a major cause of cellular ageing [40]. Further evidence to support this theory in mammals using transgenic mice has shown that endogenous production of ROS due to normal metabolic process is a major limiting factor of life-span, and that transgenic mammals deficient in the antioxidant SOD2 die within the first week of life. The latter phenotypic changes, with pathophysiological evidence of oxidative damage in tissues, can be reversed with SOD2 mimics injected intraperito-neally. Catalytic antioxidant defenses can favorably modulate the life span [44]. Increasing research is focused on measures to in-crease life-span by reduction of accumulated mtDNA mutations [42]. It is suggested that targeting endogenous antioxidant defenses against mitochondrial oxidative damage, particularly through over-expression of mitochondrial catalase that neutralizes the leaking superoxide from mitochondria, prevents excessive OS to DNA, lipids and proteins, and this can increase the life-span as indicated in mammalian animal models [39]. In addition to the endogenous sources of ROS, various lifestyle and environmental factors can increase exogenous sources of ROS

and add to the burden of OS, as well as accelerate the ageing proc-ess and age-related co-morbidity risk. This includes smoking, envi-ronmental pollution, poor nutritional choices, drugs, toxins (e.g. heavy metals, pesticides and xenobiotics) and ionizing radiation [45].

REDOX REGULATION OF MALE FERTILITY ROS and antioxidants are balanced in the healthy male repro-ductive tract and seminal fluid, and ROS are essential to the acqui-sition of fertilizing ability of the male partner [46]. Although ROS was initially thought to be exclusively toxic to human spermatozoa, it is now well accepted that ROS have important physiological functions in reproduction as they are essential regulators of male fertility [47-48]. As such, low ROS concentrations play a funda-mental role in various sperm processes and structure. This includes chromatin condensation, cell membrane remodeling, activation of intracellular pathways, roles in epididymal transport and matura-tion, hyperactivation of motility, triggering of capacitation and the acrosome reaction, sperm zona binding and oocyte fusion [46-48]. However, if there is an increase in OS for any reason, including age-associated increases [49], then the limited antioxidant defenses of the cell are overcome leading to excessive OS. Spermatogenesis is a highly dynamic process based on several mitotic divisions of germ cells followed by two meiotic divisions to produce spermatids, which undergo chromatin condensation and membrane remodeling before being transformed into spermatozoa. Germ cells generate a significant amount of ROS as metabolic by-products, which are important for this remodeling and chromatin condensation process in addition to other signaling and homeostatic roles [10; 49]. Yet, as compared to ROS and age associated damage to mature spermatozoa, the role of ROS in early germ cell devel-opment is still poorly studied [49]. Sperm capacitation is an essential process for fertilization and rep-resents a complex series of molecular, physiological and morpho-logical changes in the spermatozoa critical for acrosome reaction and oocyte binding. This is considered an oxidative event using low level ROS for redox reactions to coordinate a series of phosphoryla-tion reactions [50-51]. Although the ROS responsible for this are still elusive, ROS-induced ROS formation has been described in sperm capacitation [50]. Spermatozoa are known to be able to gen-erate their own ROS, and particularly H2O2 has been investigated, and this ROS activity is of physiological significance in tyrosine phosphorylation critical for capacitation [52]. This capacitation-associated ROS production is generated on the plasma membrane of the spermatozoon [50]. Ultimately, H2O2 generation, via .O2

-, exerts a role in intracellular protein kinase activation for sperm capacita-tion. Activation of the protein kinase C (PKC) pathway leads to phosphorylation of Raf to activate mitogen-activated protein kinase (MEK) proteins that are necessary to trigger late tyrosine phos-phorylation. Additionally, ROS production directly activates the protein kinase A (PKA) pathway as an early event during mammal-ian sperm capacitation, with additional phosphorylation processes occurring at later stages. The effects of MEK, extracellular-regulated kinase (ERK), phosphoinositide-3 kinase/Akt (PI3K/Akt) pathways and tyrosine phosphorylation are essential in guaranteeing activation of the spermatozoon during capacitation [50]. However, SOD and CAT both can act as inhibitors for this process [50]. Peroxiredoxins (PRDX’s) are a class of endogenous ROS scav-engers, but also act as ROS control systems, particularly involved in capacitation and oocyte binding remodeling [51]. These are well known and important antioxidant systems in seminal plasma, savag-ing ROS and modulating ROS-dependent signaling pathways [53]. PRDX’s dysfunction, and related enzymes needed for their reactivation (e.g. thioredoxin/thioredoxin reductase system and glutathione-S-transferases), is known to impair capacitation, as well as sperm motility, and promotes DNA damage in spermatozoa [50]. However, it is still to be elucidated how the spermatozoon controls


the levels of the produced ROS to keep within a normal physiologi-cal range and avoid toxicity [50].

THE EFFECTS OF AGEING IN MALE REPRODUCTION AND THE ROLE OF OS

Male Fertility and Sperm Quality Semen quality is considered an important indicator of fertility success in males, and seminal analysis guidelines and diagnostic procedures are provided by the World Health Organization (2010) [54] to routinely estimate the fertilization potential of the male partner. Age-related testicular dysfunction includes testicular atro-phy with reduced volume, reduced number of Sertoli cells (and hence spermatogenesis), reduced number of Leydig cells and Ley-dig cell function (partly responsible for hypogonadism) and thick-ening of the seminiferous tubules basement membrane [16]. In-creasing male age further correlates with poor sperm quality whereby different seminal parameters are affected and often even give conflicting results [16]. Moreover, paternal age is also associ-ated with an increased risk of reduced success rates in in vitro fer-tilization (IVF) and intracytoplasmic sperm injection (ICSI) proce-dures as it negatively influences the number of high quality em-bryos [55], spontaneous abortions [56], increased pregnancy com-plications as well as increased risk of birth defects [57]. There are two main sources of ROS in the male reproductive tract, namely leukocytes and spermatozoa. Low concentrations of leukocytes, predominantly neutrophils, are found in most normal semen samples [25], and these cells produce ROS as part of regula-tory roles during inflammation and cellular defense. ROS are also generated as a by-product of spermatozoal oxidative phosphoryla-tion, and are therefore intrinsic, as well as important, to the male reproductive tract [47-48]. H2O2 is the most prevalent ROS to be synthesized by spermatozoa from reduction of .O2

- [58]. In addition, apart from the epididymal contribution, ROS in seminal plasma as well as the exogenous antioxidants may originate from prostatic secretions or from the seminal vesicles. The seminal plasma is made up of a variety of lipids, proteins and amino acids, ions, cyto-kines and hormones, and is required to protect and nourish the ejaculated spermatozoa. Seminal plasma also contains endogenous enzymatic scavengers such as SOD, catalase and glutathione per-oxidase, as well as exogenous non-enzymatic antioxidants such as vitamins C and E, zinc, co-enzyme Q10 and ubiquinol [59]. Additional ROS in the male reproductive tract derive from poor lifestyle and other exogenous sources, such a smoking, alcohol consumption, pesticides, exogenous estrogens and heavy metal toxicity, nutritional deficiencies and advanced age. Pathological conditions such as varicocele and spinal cord injuries are also me-diated by OS as well as from the ageing process itself [47; 60]. Due to their extraordinary high plasma membrane content of polyunsaturated fatty acids (PUFA), spermatozoa are a sensitive target for OS-induced damage as they contain numerous carbon double-bonds which are sensitive to an oxidative assault. The rela-tive large surface area of membrane over the sperm head and tail, the vulnerability of DNA and the lack of cytoplasm (which is mostly confined to the midpiece and contains cellular defense mechanisms) also reduce the ability for innate antioxidant defenses such as SOD1, catalase and glutathione peroxidase [61]. As such, males with idiopathic infertility have been found to be associated with a ROS-TAC mismatch and poor semen quality [62]. A ROS-TAC mismatch in seminal fluid has numerous detrimental effects on various parameters of sperm function that parallel effects of ageing on the reproductive tract. Excessive OS has been correlated with reduced sperm concentration and motility, morphological de-rangements and damage to both cellular and mitochondrial DNA, as well as mitochondrial membrane potential (MMP) [47-48; 63-65]. Furthermore, abnormal spermatozoa are postulated to undergo a form of apoptosis associated with high concentrations of H2O2 for-

mation that is well demonstrated to initiate the spermatozoa apop-totic pathway via caspase 3 activation and annexin-V binding (which can be prevented with pre-exposure of spermatozoa to anti-oxidants such as catalase and melatonin) [61]. Thus, the state be-tween ROS and antioxidants needs to be finely balanced and main-tained by various enzymatic and non-enzymatic processes [47-48; 66]. The damage to the plasma membrane is caused by a process called lipid peroxidation (LPO) whereby ROS initiate a radical chain reaction in the membrane leading to the damage and forma-tion of toxic and mutagenic end products. LPO also leads to re-duced membrane fluidity, its inability for sperm-oocyte fusion [60; 67-68], reduced activity of calcium regulation channels which nega-tively affects motility [69-70], and a loss of intracellular ATP pro-duction resulting in further reduction of sperm motility [71]. OS also negatively affects the structure of the axonemal flagellar during epididymal maturation, causing impaired motility following ejacu-lation [11]. Studies have shown that lipid soluble antioxidants, such as vitamin A, other carotenoids and especially vitamin E, are corre-lated with increased fertility potential due to a reduction of LPO and are thereby positively correlated with improved motility. Re-duced amounts of these lipid soluble antioxidants in the reproduc-tive tract and seminal plasma negatively affect sperm concentration, motility and morphology [72]. Furthermore, due to the nature of the compaction of DNA with protamines in sperm cells, DNA repair does not occur as readily as in other mammalian cells. Therefore, DNA damage accumulates through the lifespan of spermatozoa, a process which may be associated with impaired fertilization due to apoptosis [61]. Many of the negative effects of OS, and specifically impaired motility and oocyte binding potential, can be due to the damaging effects of ROS on spermatozoa membranes. In addition to the cell membrane, DNA damage is a major char-acteristic of ROS induced defects to spermatozoa. These damages can either be due to direct oxidation of the DNA causing DNA fragmentation or due to indirect action of end products of LPO such as malondialdehyde (MDA), 4-hydroxy-2-alkenals or 2-alkenals which are mutagenic and genotoxic [73-74]. Patients with a high percentage of sperm with DNA fragmentation have an increased risk of infertility, miscarriage, and numerous disorders in the off-spring [61], as this damage can be transmitted to the progeny [12]. Mechanism of damages in spermatozoa DNA are numerous, and importantly include excessive OS, aberrant spermatozoa maturation and apoptosis [12; 48; 61; 75]. DNA fragmentation is closely asso-ciated with OS base adducts evidenced by increased 8-hydroxy-2’-deoxyguanosine (8OHdG), a known marker of DNA oxidative damage [61]. Increased spermatozoa DNA damage and decreased DNA repair is observed in experimental ageing models, and oxida-tive DNA damage is increased in young SOD1 (SOD1-/-) and Cata-lase (Cat-/-) knockout mice as well as with ageing [12]. These lines of evidence further suggest a role of OS in male ageing-related fertility outcomes. SOD1 appears particularly important in this process, as it is suggested that there are compensatory responses in wild-type and Cat-/- mice to partially alleviate OS associated with ageing [12]. In addition to DNA damage, there is extensive protein oxidation in spermatozoal and seminal plasma proteins associated with ROS. Among these are the OS markers DJ-1, PIP and lacto-transferrin and PRDX’s [59]. Mitochondria are considered to be both a source of ROS and increased OS in spermatozoa, but also a target and a critical com-ponent of OS related sperm damage as mitochondrial DNA (mtDNA) is up to 100-times more susceptible to damage and muta-tions than nuclear DNA as it is not protected against an oxidative assault [76-78]. As a result of such mitochondrial damage, more ROS could be produced leading to a vicious cycle of further mito-chondrial ROS production further mtDNA damage and loss of sperm functionality [79]. Particularly, damaged mitochondria are negatively correlated with sperm motility in a causal manner that


can be induced experimentally by ROS, and reversed with antioxi-dant treatments, such as vitamin E, in vitro [80]. High ROS concen-trations are associated with mtDNA damage and reduced mito-chondrial membrane potential (MMP) in spermatozoa, impairing ATP formation, motility and thereby fertilization capacity [78]. In a small study of oligoasthenozoospermic men, seminal MDA and ROS levels were found to be higher in infertile men than fertile men, with lower levels of antioxidants catalase and glutathione peroxidase, but no difference for SOD levels which may compen-sate for the excessive ROS. These seminal changes correlated with reduced sperm count and progressive motility [78]. Leukocytospermia is a marker of reproductive tract infection and inflammation, and the associated immune response increases ROS production alongside down regulation of endogenous antioxi-dants in order to induce OS as part of the immune response. Typi-cally, a local inflammatory response is associated with acute or chronic infections of the reproductive tract in which OS plays a significant role [60; 81-82]. Pro-inflammatory cytokines, particu-larly TNFα, IL1β and IL6, are known to unfavorably modulate both OS and antioxidant status, with increased concentrations correlating positively with ROS and OS in seminal fluid [83]. Furthermore, systemic inflammatory diseases are also associated with poor fertil-ity outcomes, possibly due to concomitant reproductive tract in-flammation. It is also suggested that low grade systemic inflamma-tion associated with metabolic syndrome and type-2 diabetes melli-tus is associated with local reproductive tract inflammation and poor fertility marker [18], which is likely to be associated with increased OS. Leukocytospermia is associated with increased .O2

-- and H2O2-positive spermatozoa associated with apoptotic markers caspase 3/7 and glutathione activation [82]. In vivo, leukocyte-induced damage can be mitigated by the addition of antioxidants to the medium, including catalase, glutathione and N-acetylcysteine [61], with further lines of evidence suggesting that leukocyte in-duced OS can also be managed with Vitamin C, Vitamin E, Glu-tathione and CoEnzyme Q10 [60], further suggesting a central role of OS in the pathogenesis of male reproductive tract inflammation.

Spermatogenesis and Germ Cells Susceptibility Although ageing has been thought to have limited effect on male fertility, as males continue to produce sperm into old age, germ cells from elderly males are of lower quality than younger males [49]. Furthermore, alongside ageing, OS-induced DNA dam-age accelerates germ cell apoptosis resulting in impaired spermato-genesis and spermatozoa [68]. Spermatogenesis occurs in the testes, primarily in association with Sertoli and germ cells [12]. In steady states, redox reactions play pivotal physiological roles in spermato-genesis [10; 49]. However, evidence shows that age-related OS compromises the development of quality germ cells. Abnormal gametes with poorly remodeled chromatin may also generate OS during spermatogenesis leading to increased abnormal spermatozoa and high percentage of DNA fragmentation. This is mediated by the increased inability to repair DNA strand breaks that occur normally during spermatogenesis. These cells are susceptible to ROS-dependent apoptotic pathway activation through caspase activation, externalization of phosphatidylserine residues, and ROS-activation from the mitochondria which further results in LPO and DNA fragmentation alongside apoptosis [61]. Age-dependent changes in spermatogenesis have previously focused on induced damage to mature spermatocytes that have un-dergone compaction but are transcriptionally inactive, with fewer studies focused on germ cells in earlier phases of spermatogenesis [49]. Ageing is associated with a decreased number of Sertoli and germ cells alongside a reduction in fertility potential [12]. Further-more, germ cells from elderly rats particularly display increased levels of ROS and associated DNA damage, when compared to younger animals [49].

In SOD1 knockout mice (SOD-/-), ageing shows a significantly increased loss of Sertoli and germ cells compared to wild type age mice, indicating a significant role of OS in detrimental spermato-genesis. This is in addition to experimental pro-oxidant exposure in these SOD-/- mice that increases ROS in spermatocytes, but this increase was not observed in Catalase-Null (Cat-/-) mice which were still able to neutralize ROS [12]. In animal models, this can be pre-vented by overexpression of the antioxidant catalase following exogenous ROS challenge that damages spermatozoa, showing increased markers of DNA repair enzymes [84]. However, it ap-pears that they respond and adapt to OS differently through the different developmental phases, and ageing results in redox dys-function as aged germ cells are less able to support appropriate defenses [49].

Leydig Cell Function and Age-Related Androgen Decline Male endocrinology, as with other systems, is complex in age-ing, with multiple mediators of the hypothalamic-pituitary-testes (HPT) axis being affected. Ageing is associated with declining androgens, affecting in particular testosterone and dehydroepian-dosterone (DHEA), with corresponding increases in luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex hor-mone-binding globulin (SHBG), manifesting as a male primary hypogonadism phenotype. Although dihydrotestosterone (DHT) remains relatively constant, there is a corresponding decline in the testosterone-DHT ratio negatively affecting the ageing process [4; 15]. Unlike women, androgen decline is a gradual age-related phe-nomenon in males, declining at an annual average rate of 1 - 2% per year (although this is highly variable, and testosterone may even increase in some men). Therefore, appropriate terms used to de-scribe this include androgen deficiency in aging men (ADAM) and late-onset hypogonadism (LOH). ADAM is driven primarily by testicular dysfunction, with or without more subtle HPT-axis dys-function and more likely secondary to the declining androgens [4; 15]. This process is closely correlated with reduced libido, sexual function and reduced fertility potential associated with hyposper-matogenesis and sperm dysfunction. However, ADAM affects be-tween 10 and 25% of the elderly, with symptomatic hypogonadism affecting 6 - 12%. Even at a very advanced age, serum testosterone levels, and associated sexual and reproductive function, may remain within normal limits for many elderly men [85]. ADAM appears to be an important mediator of age-related co-morbidities in males, and this is modulated by lifestyle factors that further affect declining testosterone levels. This includes tobacco and alcohol use, increased psychological or physiological stress, obesity, type-2 diabetes mellitus, obstructive sleep apnoea and medication use. Declining testosterone is an independent risk factor for co-morbid conditions including declining cognitive function, osteoporosis, metabolic syndrome, type-2 diabetes mellitus and cardiovascular disease, and therefore can be a cause and conse-quence in these complex pathologies. Ultimately, men with low testosterone are more likely to die prematurely. Although some studies suggest that testosterone-replacement therapy (TRT) may offer benefit in reducing the negative impact of ageing in males, long term safety and efficacy is not yet clear. These lines of evi-dence suggest a significant role for testosterone and ADAM in the ageing process in males, as well as the development of co-morbidities, although the mechanisms associated with this require further elucidation [4]. The free radical theory involves the damaging role of OS and various toxins on mitochondria [7]. It has been demonstrated that Leydig cell MMP, ATP synthesis and mitochondrial calcium con-centrations are all required for steroidogenesis [86]. These mecha-nisms may be disrupted by excessive OS which is known to inhibit both ovarian and testicular steroidogenesis, most notably the initial step of cholesterol transfer into Leydig cell mitochondria and ster-


oidogenic acute regulatory (StAR) protein transcription [86-89].The result is a reduction in cAMP production, StAR mRNA and the activity of P450 cholesterol side-chain cleavage (P450scc), and therefore the activities of Cytochrome P450 17α-hydroxylase/17, 20-lyase (CYP17) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3βHSD) [89-90]. H2O2 is derived from macrophages in close residence to Leydig cells in the testes, and these ROS are associated with reduced testosterone production and 3βHSD tran-scription alongside LPO, decreased cellular antioxidants such as SOD, catalase and glutathione transferase, and initiation of apopto-sis [91]. This is then associated with a gradual decline in testoster-one in ageing males that can be accelerated in chronic inflammatory diseases and other age-related co-morbidities [89]. Detailed under-standing of these pathways may open the possibility of novel treat-ments to improve steroidogenesis in ageing males and there is po-tential to target these pathways to modulate the consequences of ageing and improving reproductive potential and sexual function.

Prostate Ageing and OS Benign prostatic hyperplasia (BPH) and adenocarcinoma of the prostate are common co-morbidities associated with male ageing. Risk factors, in addition to ageing major risk, include genetic in-heritance and dietary factors. Prostatic hyperplasia and carcino-genesis of prostate cells, as mechanism associated with these pa-thologies, are considered to be driven by OS in ageing males. This may be initiated due to hormonal imbalances with age that modu-lates mitochondrial activity to increase ROS production and deple-tion of intracellular antioxidants such as glutathione peroxidase [6]. Other mechanisms common to both diseases include inflammatory mediators (e.g. chronic systemic inflammation or prostatitis) and additional hormone changes with ageing (including estrogen, pro-gesterone and androgens changes) [92]. Prostate OS, as with other tissues, damages DNA, cell membrane and tissue proteins that trig-ger cellular signaling disruption, impaired homeostasis and genetic mutations [6; 92]. The structural changes associated with BPH and prostate carcinoma is associated with the syndrome of LUTS that is associated as an age-related phenomenon that has increasing OS as part of the underlying mechanisms, as well as with sexual dysfunc-tion and poor semen quality [92]. This impacts on pathogenesis, response to therapeutics and prognosis in these conditions. Fur-thermore, prostatitis can increase OS in the reproductive tract and the ejaculate, impairing semen function [93].

Erectile Dysfunction Erectile dysfunction (ED) is characterized by the inability to develop and maintain a sufficient erection of the penis for satisfac-tory sexual activity. Disruption of psychological, neurological, hormonal, vascular, and cavernosal factors, individually, or in com-bination, can induce ED [94]. Major health concerns such as athe-rosclerosis, hyperlipidemia, hypertension, diabetes, and metabolic syndrome have now become well integrated into the investigation of ED [95]. Age is also an important risk factor, with higher propor-tions of ED in men over 50 years as compared to younger men (age is also a risk factor for obesity, metabolic and hypogonadism in males) [96-97]. Alongside the epidemic of obesity, ED is a com-mon complaint, with an age-standardized prevalence of more than 40% [97-98]. Age-related ED is associated with neurovascular dys-function, which is closely mediated by nitric oxide (NO) suppres-sion, is associated with vasodilation in the vascular system and increased OS in the penile tissues [99]. Antioxidants may provide some potential targets for therapeutic strategies in ED associated with OS, and in this context, various phytotherapeutic extracts have been attributed to have benefit in males with erectile dysfunction, with different levels of scientific attention, including Panax ginseng (Korean Ginseng or Chinese Ginseng), Tribulus terrestris (Devil’s Thorn or Devil’s Weed), Eurycoma longifolia (Tongkat Ali), Withania somnifera (Indian Ginseng or Poison Gooseberry) and Camellia sinensis (green tea), amongst [100-101]. Although many

herbs show benefit in traditional and pre-clinical settings, there is a need for the design and execution of well-controlled clinical studies to determine the efficacy and safety of plant aphrodisiacs, the de-sign and execution of well-controlled clinical studies to determine the efficacy and safety of plant aphrodisiacs [102].

THE ROLE OF ENDOGENOUS ANTIOXIDANT DEFENSES AND AGEING Due to the structure of the spermatozoa, these cells are signifi-cantly prone to oxidative damage particularly to the cell membrane and the DNA. The only defense is a limited system of endogenous antioxidants. These include members of the SOD family, catalase, glutathione peroxidases, PRDX’s, the nuclear factor erythroid 2-related factor 2/antioxidant response element (NRF2/ARE) path-ways and nitric oxide synthase (NOS) as critical endogenous anti-oxidants in seminal plasma and spermatozoa [49; 61; 103]. The physiological roles of some of these antioxidants have been described previously. However, in addition to endogenous antioxi-dants, there are other exogenous scavengers associated with the male reproductive tract and found in seminal fluid. This includes vitamin C, taurine, tryptophane and spermine [61]. Depletion of endogenous or exogenous antioxidants is associated with OS due to an increased ROS-TAC ratio, and therefore driving cellular and tissue dysfunction in the male reproductive tract [61]. In addition to ROS being closely associated with infertility, there is also a signifi-cant percentage of infertile males that have low levels of antioxi-dants in the ejaculate [104]. It should be considered that genotypic mutations, known as single nucleotide polymorphisms (SNP’s), result in variations of genetic expressions. Within this context, there are numerous poly-morphisms associated with the endogenous antioxidant genes in males. These include the GSTT1-nul, GSTM1-nul, GSTP1-Ile105Val, SOD2-Ala16Val, eNOS-T786C, eNOS-G894T and CAT-262T/T variants [103]. These are associated with increased potential for OS, further associated with low sperm quality, oligoasthenotera-tozoospermia, oligozoospermia and reduced fertility potential in general [103]. These SNP’s should be potentially considered in the assessment of males who may benefit from antioxidant supplemen-tation [103]. There is a loss of endogenous antioxidant production with age-ing [12]. SOD1, more than catalase, is critical for maintenance of germ cells and spermatogenesis [12]. In animal models, Selvarat-num et al. [49] showed that in younger populations, the genetic transcripts for SOD1, catalase and PRDX’s are upregulated in re-sponse to induced OS, but expressed at lower levels in elderly populations there is evidence of increased DNA damage, suggesting that age is related with a decreased ability to respond to increased ROS production [105]. SOD serves as a major antioxidant through-out the body, and SOD1 is particularly important in prevention of OS in the reproductive tract. Specifically, SOD1-/- mice show dis-rupted redox signaling, significantly advanced global ageing and age related pathologies, and reduced life-span by up to 30% [105]. PRDX’s are well known and important antioxidant systems in seminal plasma, scavanging ROS and modulating ROS-dependent signaling pathways [53]. Reduced PRDX’s in seminal plasma are associated with poor seminal quality. In a study by Gong et al. [53], reduced seminal PRDX1 and PRDX2 correlated to lower sperm concentrations, higher LPO as determined by MDA and sperm DNA damage. PRDX’s have a key role in protection of sperm from OS within the male reproductive tract. Infertile males, and ageing males, have lower levels of PRDX’s than fertile males, as these are thio-oxidized and inactive [49-51]. The dysregulation of PRDX’s and their associated reactivating systems (thioredoxin/thioredoxin reductase and glutathione‑S‑transferases) negatively modulates sperm motility, capacitation and promotes DNA damage leading to male infertility [50-51]. This provides an interesting mechanism for decreased fertilization capacity in ageing men.


FERTILITY ASSESSMENT IN THE AGEING MALE Infertility is defined by the WHO as ‘the inability of a couple to achieve conception or bring a pregnancy to term after 12 months or more of regular (at least three times per week), unprotected sexual intercourse’ [54]. It affects about 15% (approximately one in seven) of couples trying to conceive [64]. Of these cases, 25-50% can be attributed partially or solely to the male partner [106]. Male infertil-ity is dramatically increasing in recent decades, with defective sperm function considered the major contributor to this phenome-non [61]. Causes of male fertility are multifactorial, including ge-netic diseases, organic pathologies and lifestyle and environmental factors. Well defined major causes of male factor infertility include varicoceles (±25% cases), urogenital infections (±10% cases), tes-ticular failure characterized by endocrine failure leading to testos-terone deficiency with severely impaired spermatogenesis resulting in azoospermia or severe oligozoospermia (±1.1% cases), other immunogenic causes, impotence or sexual/ejaculation inadequacy, other acquired urogenital abnormalities or strictures (e.g. structural complication following mumps orchitic), and various endocrine disorders (±1.5% cases) [85; 107]. Approximately 20 - 50% of these cases have no known etiology, termed ‘idiopathic’ [106]. Lifestyle and environmental factors negatively associated with fer-tility potential include consumption of alcohol and tobacco, recrea-tional drug use (such as marijuana and cocaine), exposure to exces-sive testicular heat, prolonged sitting (related to testicular heat), exposure to endocrine disruptors (e.g. pesticides; phthalates), expo-sure to heavy metals (e.g. lead; cadmium), stress (both physiologi-cal and psychological), ionizing radiation and even exposure to cell phone radiation [85; 107-110]. Many of these risk factors are asso-ciated with OS as an important mechanisms, and are also associated with increased rates of ageing. A clinical assessment would include a complete case history relevant to andrology, a detailed clinical examination, relevant hormonal profile and a standard seminal analysis. Additional analy-sis may be warranted, including genital ultrasound imaging as re-quired, functional sperm markers (e.g. DNA fragmentation) and genetic testing [106]. The standard seminal assessment includes routine markers such as sperm concentration, motility, vitality and morphology with seminal volume, viscosity, pH and leukocyte count, and these analyses need to be done by appropriate trained clinicians and laboratory technicians within the guidelines offered [54]. A decrease in sperm quality is considered a major reflection of the decreased ability of the male partner to contribute to fertiliza-tion [106]. In must be noted, however, that although the semen analysis remains a standard test in male fertility assessments, its clinical value is limited as 5% of fertile men and 16% of infertile men display poor semen analyses [111]. In addition to the number of spermatozoa, the functional capacity of sperm is considered a more sensitive determinant of fertility potential. This includes as-sessment of percentage of sperm with DNA fragmentation and damaged MMP [63]. MMP is considered a factor which most closely represents spermatozoal mitochondrial function, critical for metabolic functioning and motility [112]. Of relevance, both DNA fragmentation and MMP (mitochondrial dysfunction) are mediated by increased OS from all underlying causes, as well as ageing, and this in turn results in dysfunctional reduced DNA integrity and MMP, which further increases ROS production [20; 47-48; 75]. Particularly, assessment of sperm DNA integrity is important in the male assessment, especially related to increasing paternal age and increased OS, to assess strand breaks, numerical abnormalities in sperm chromosome complement and alterations in epigenetic regu-lations [20].

ROS ASSESSMENT With increasing evidence on the role of OS in male infertility and reproductive system dysfunction, there are growing indications

that measurement of ROS in semen, or potentially even serum markers, should be part of standard fertility testing in males [47; 72; 113]. Accurate assessment is of importance in diagnostics, but also to guide therapeutic considerations [47; 72]. Various methods for assessment of OS exist. Currently, there is the use of both direct and indirect measure-ment of OS. The major assessments currently used include the ROS, total antioxidant capacity (TAC) and the oxidation-reduction potential (ORP), MDA, thiobarbituric acid assay (TBARS) or 4-hydroxynonenal (4-HNE) as LPO markers, caspase-3 or annexin V as apoptotic markers and proteomic analysis of oxidized protein markers (e.g. protein carbonyl is a highly sensitive marker of pro-tein oxidation in semen) that indirectly reflect increased OS [11; 20; 47; 104]. Evidence is suggesting, as with sperm DNA integrity, that diagnostic and prognostic capabilities of OS assessment may ex-ceed the capabilities of conventional semen analysis, and may more accurately differentiate fertile from infertile men [113]. However, these methods are not suited for easy clinical diagnostics, and they are generally lengthy assays that require specialized laboratory equipment and specialized techniques [104]. These systems do not evaluate the full spectrum of OS, how-ever, but rather different specific components that may prove to be incomplete as individual assays at a single point in the pathogenesis [104]. This ‘one-size-fits-all’ mentality pervades also into the ana-lytics. Measuring TAC, for example, will not give useful informa-tion on the state of the organism, and should be discouraged. Rather, individual antioxidant enzyme activities and patterns of antioxidant molecules need to be assessed [9]. Therefore, a new more comprehensive and simplified measure of OS the oxidation-reduction potential (ORP) has been suggested as diagnostic marker [104]. This novel method is a direct measurement and indicator of OS in biological samples, and can be considered a more compre-hensive marker of OS in biological systems, including seminal fluid. This involves the analysis of all known and unknown oxi-dants and antioxidants in blood, urine and seminal fluid, and re-flects damaged tissues associated with OS [104]. ORP also corre-lates with poor semen samples and infertility risks, and this can be further developed into a routine, comprehensive and sensitive diag-nostic test as it can be used as quick and cost-effective one-step screening test in clinical practice to measure OS both in semen and seminal plasma in fresh or in frozen samples [104; 114].

THERAPEUTIC CONSIDERATION IN AGE RELATED REDOX BIOLOGY AND MALE FERTILITY: The free radical theory of ageing, particularly due to endoge-nous ROS, raises the potential to target these mechanisms for thera-peutic approaches. The role of these antioxidants is to prevent ROS concentrations from elevating to non-physiological levels, and thus from derailing the finely balanced redox balance into oxidative stress [30]. Many of these antioxidants can be acquired through nutritional intake, with a study by Karayiannis et al. suggesting that deficiencies in dietary habits associated with increased antioxidant intake (Mediterranean Diet) is associated with poor semen quality [115]. Although there is indication that synthetic oral antioxidants have limited efficacy in disease prevention [23] and are not particu-larly efficient when compared to the catalytic antioxidants that mimic SOD and catalase activities [41], antioxidants have an evi-dence-base to be considered as a promising therapy option in the treatment and prevention of ageing and various co-morbidities, including cardiovascular disease, type-2 diabetes mellitus and can-cers (as observed in diets rich in phytonutrients from fruits and vegetables) [30]. In the context of rapid increases in over-the-counter dietary supplements in recent years, a large proportion of these are antioxidant-rich items such as multivitamins, including vitamin C and vitamin E [116], and the effects on male reproduc-tion potential required further investigation to further understand the benefits, risks and dosages of specific antioxidants or the com-


binations for different presentations. However, blanket use of anti-oxidants should not be encouraged without appropriate justification via demonstration of OS within each specific patient [81]. The effects of antioxidant therapy in idiopathic cases of male infertility and varicoceles has been widely studied, with conflicting data. However, antioxidants are increasingly used by urologists for sub-fertile men on the premise that OS is at least partly due to a deficiency of seminal antioxidants [104]. Although the treatment of patients with antioxidants to mitigate the effects of OS is highly debated [81], the role of OS in male factor infertility, the high inci-dences of OS diagnoses in males with fertility concerns and age associated changes in male reproductive system that can be associ-ated with OS as both a cause and a consequence suggest that there is a strong indication for a role of antioxidants for therapeutics [47; 61; 68; 113]. In this context, it is essential to evaluate the impact of ROS in the patient through appropriate clinical assessment and diagnostics in order to determine which patient sub-groups may benefit from antioxidant therapy [113]. Although there is little con-sensus still for the type and dosages of antioxidants that should be used, evidence suggests that infertile males with excessive OS would benefit from this therapeutic approach [47] [113]. However, many studies have indicated a benefit of antioxidant therapy, or freezing of semen samples from younger age, to reduce the impact of OS and age related modifications of male fertility [46; 47; 113]. Numerous antioxidants are used orally, singularly or in combi-nation, and include the following: Vitamins A, B complexes, C and E, CoEnzyme Q10, ubiquinol, glutathione, L-carnitine, β-carotene, lycopene, α-lipoic acid, N-acetyl-cysteine, selenium, zinc and or copper [47]. Ahmadi et al. [117] and Agarwal et al. [47] provide a detailed review of these approaches in clinical trials. Oral antioxi-dants need to reach high concentrations in the reproductive tract and restore OS related dysfunction to improve spermatogenesis, and should reduce levels of seminal ROS [118]. Treatment should be aimed at reducing OS by lowering ROS and/or increasing antioxi-dants to keep only a low level necessary for biological function [68]. Lipid soluble antioxidant profiles include carotenoids, vitamin A and vitamin E as antioxidants, and MDA as a non-specific marker of lipid peroxidation. Both, serum and seminal concentra-tions of these markers can reflect potential reproductive tract and spermatozoa dysfunction [72]. In experiments using an animal model (rabbit), high levels of vitamin E were suggested to be det-rimental to fatty acid composition of spermatozoa. Vitamin E may also reduce ROS production in the male reproductive tract [117]. This vitamin E (and the fish oil separately) increased sperm produc-tion in ageing animals though, and was associated with negative correlation to sperm lipid peroxidation [119]. Grecco and col-leagues [120] showed that 2 months of treatment with vitamin E and vitamin C (water soluble) significantly reduced the levels of DNA fragmentation in infertile patients. Vitamin C, a water soluble antioxidant in seminal plasma, offers protection against DNA dam-age [117]. On the other hand, any uncontrolled over-supplemen-tation with antioxidants may result in reductive stress, which is regarded as dangerous as OS [34]. Over-supplementation with vi-tamins E and β-carotene have been suggested to cause DNA dam-age by DNA adduct formation by inhibition of the glutathione-S-transferase π [121]. Glutathione levels, when reduced in seminal fluid, is associated with poor semen quality and oligozoospermia [122]. This antioxidant, which is a co-factor for glutathione peroxi-dase, react directly with ROS as a scavenger that can be recycled, and protects primarily against products associated with lipid per-oxidation [123]. Zinc (Zn) and Selenium (Se) are important factors in OS and redox balance in male genital tracts [124]. Zn is found in high con-centrations in seminal fluid and the prostate gland particularly, being important in spermatogenesis and as an element to oppose OS by occupying copper binding sites in the DNA or as a Cu/Zn cofac-

tor in SOD. Thus, seminal zinc levels are associated with sperm concentration and sperm quality [124; 125]. Although the mecha-nisms by which Se exerts protection against OS is not well estab-lished, it is hypothesized to work through glutathione peroxidase enzymes (selenoenzymes). Se deficiency is associated with poor motility and poor morphology, specifically in the sperm head. Mi-cronutrient deficiencies of these elements need to be considered in assessment and treatment of OS in male fertility as they are associ-ated with poor sperm quality [124]. An interesting area of consideration in OS may be caloric re-striction within appropriate nutritional guidelines. Caloric restric-tion is associated with increased life span, due at least in part, to reduce OS, as evidence suggests that there is significantly reduced ROS damage on mtDNA particularly [40]. This can be with a body of evidence that suggests obesity and the metabolic syndrome also impair male fertility [18; 126]. Herbal extracts that are antioxidant rich require further investi-gation for the benefits on ageing and age-related reproductive de-cline in males. Herbs with significant antioxidant activity that have been well studied for benefit in ageing and age-related OS, decreas-ing the risk of age associated co-morbidities as well as evidence of benefit for infertile males includes ECGC from green tea [127], white tea [128], curcumin [129] and resveratrol (grape seed ex-tracts) [130-131]. More specifically for the male reproductive tract, various herbs have shown indication they may be useful for repro-ductive dysfunction in ageing males with increased OS. These in-clude Astralagus membranaceus, Eurycoma longifolia, Panax gin-seng and other ginseng spp. Tribulus terrestris, Lepidium meyenii, Catha edulis, Solanum lycopersicum, Amaranthus hypochondriacus [132]. In the prostate, there is evidence that antioxidants influence the development and progression of BPH and cancer, and nutri-tional intake of vitamin E, selenium and lycopene, with evidence suggesting that micronutrient supplementation may restore ROS-TAC imbalance and improve therapeutic outcomes [133]. This problem is particularly becoming more evident as the reproductive age is increasing. However, significantly more studies are required in herbal extracts for clinical use, and research is difficult to review due to wide ranging methodologies, extracts, dosages and outcomes investigated. Antioxidant supplementation must be used with caution, and it is recommended that any oral antioxidant treatment that is initiated in patients who have demonstrated increased seminal OS should be carefully controlled. There are concerns raised on the detrimental effect of antioxidants on the physiological roles of ROS, and there has been previous particular concern with vitamins C, E and β-carotene, in the context of a very finely balanced redox system [134-135]. Uncontrolled use of antioxidants may lead to a reductive state known as the antioxidant paradox, as certain low level ROS is required for physiological function systemically and in the repro-ductive tract [81]. There are some indications that specific overcon-sumption of some antioxidants is associated with increased mortal-ity risk in some populations [30]. Further areas of concern in thera-peutic use is that, at least in adult male houseflies, endogenous anti-oxidants may also decrease endogenous antioxidant production [136]. Areas of specific concern and consideration includes lifestyle factors and (patho)physiological states associated with ROS-TAC balance. This includes smokers vs. non-smokers, dietary antioxi-dant intake, specific pharmacodynamics and pharmacokinetics of antioxidants, interaction with medication and other antioxidant combinations [30]. A complete and appropriate workup is required, including seminal OS assessment, before initiation of any specific therapy. The approach to treatment of male related infertility, following investigation for underlying disorders, further involves a multifac-torial approach that should include the identification and avoidance where possible of harmful environmental, social and occupation risk factors, as well as identification and appropriate correction of


nutritional imbalances associated with optimal spermatogenesis and seminal fluid production [60].

CONCLUSION Ageing, and age-related reproductive decline in males, is closely associated with a shift from a balanced redox state to a state of OS. Genetics, lifestyle and environmental factors modulate this balance. Although a low-level ROS is important for normal physio-logical function including fertility, increased ROS-TAC and associ-ated oxidative mechanisms have roles in all male age-related phe-nomenon affecting sexual behavior and seminal quality. Important areas of research should continue to elicit the physiological role of ROS in andrology, the impact of OS on male reproduction and age-related changes, and the standardization of OS assessment in the male reproductive tract. Of further specific interest is the role of ROS in early germ cell development and spermatozoa maturation for fertilization, the mechanisms associated with downregulation of endogenous antioxidant genetic expressions with age, identification of therapeutic targets for improving endogenous antioxidant activ-ity, and specific oral antioxidant treatment for age related OS. The OS assessment in the male reproductive tract of ageing males is important prior to any consideration of therapeutic inter-vention with oral antioxidants. However, and these must be used with caution and monitoring be done for therapeutic risks (antioxi-dant paradox and reductive stress). Evidence suggests that antioxi-dants, along with appropriate lifestyle changes and avoidance of environmental risks where possible, may offer an important strategy to improve fertility outcomes in ageing males and potentially pre-vent the impact of excessive OS on disease development in the reproductive tract as well as globally. On the other hand, the poten-tial detrimental effects of too high antioxidant levels and RS for male reproductive health must not be neglected as scientists and clinicians still do not know what the normal values for the redox potential in serum and semen is. Therefore, more research is re-quired in ageing males to fully understand the physiology, patho-logical mechanisms and best treatment options in different ageing male sub-groups. Although it is difficult to draw broader compari-sons on the efficacy of specific therapies in male infertility due to the large variability in the studies done, antioxidant treatment con-tinues to provide increased promise in clinical practice as a thera-peutic option.

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS Declared none.

REFERENCES [1] Smetana K Jr, Lacina L, Szabo P, Dvořánková B, Brož P, Šedo A.

Ageing as an important risk factor for cancer. Anticancer Res 2016; 36(10): 5009-17.

[2] Höhn A, Weber D, Jung T, et al. Happily (n)ever after: Aging in the context of oxidative stress, proteostasis loss and cellular senes-cence. Redox Biol 2017; 11: 482-501.

[3] Clegg A, Young J, Iliffe S, Rikkert MO, Rockwood K. Frailty in elderly people. Lancet 2013; 381: 752-62.

[4] Araujo AB, Wittert GA. Endocrinology of the aging male. Best Pract Res Clin Endocrinol Metab 2011; 25(2): 303-19.

[5] Gordon EH, Peel NM, Samanta M, Theou O, Howlett SE, Hubbard RE. Sex differences in frailty: A systematic review and meta-analysis. Exp Gerontol 2016; 89: 30-40.

[6] Ripple MO, Henry WF, Rago RP, Wilding G. Prooxidant-antioxidant shift induced by androgen treatment of human prostate carcinoma cells. J Natl Cancer Inst 1997; 89(1): 40-8.

[7] Knight JA. The Biochemistry of ageing. Adv Clin Chem 2000; 35: 1-62.

[8] Mitnitski A, Collerton J, Martin-Ruiz C, et al. Age-related frailty and its association with biological markers of ageing. BMC Med 2015; 13: 161.

[9] Sies H. Oxidative stress: a concept in redox biology and medicine. Redox Biol 2015; 4: 180-183.

[10] Fujii J. Imai H. Redox reactions in mammalian spermatogenesis and the potential targets of reactive oxygen species under oxidative stress. Spermatogenesis 2014; 4(2): e979108.

[11] El-Taieb MA, Herwig R, Nada EA, Greilberger J, Marberger M. Oxidative stress and epididymal sperm transport, motility and mor-phological defects. Eur J Obstet Gynecol Reprod Biol 2009; 144(1): S199-203.

[12] Selvaratnam J, Robaire B. Effects of ageing and oxidative stress on spermatozoa of superoxide dimutase 1- and catalase-null mice. Biol Reprod 2016; 95(3): 60

[13] Avellino G, Theva D, Oates RD. Common urologic diseases in older men and their treatment: how they impact fertility. Fertil Steril 2017 Jan 7. pii: S0015-0282(16)63076-5. doi: 10.1016/j.fertnstert.2016.12.008. [Epub ahead of print].

[14] Corona G, Lee DM, Forti G, et al. Age-related changes in general and sexual health in middle-aged and older men: results from the European Male Ageing Study (EMAS). J Sex Med 2010; 7: 1362-80.

[15] Golan R, Scovell JM, Ramasamy R. Age-related testosterone de-cline is due to waning of both testicular and hypothalamic-pituitary function. Aging Male 2015; 18(3): 201-4.

[16] Sharma R, Agarwal A, Rohra VK, Assidi M, Abu-Elmagd M, Turki RF. Effects of increased paternal age on sperm quality, reproductive outcome and associated epigenetic risks to offspring. Reprod Biol Endocrinol 2015; 19: 13-35.

[17] Amaral S, Oliveira PJ, Ramalho-Santos J. Diabetes and the impair-ment of reproductive function: possible role of mitochondria and reactive oxygen species. Curr Diabetes Rev 2008; 4(1): 46-54.

[18] Leisegang K, Bouic PJ, Henkel RR. Metabolic syndrome is associ-ated with increased seminal inflammatory cytokines and reproduc-tive dysfunction in a case-controlled male cohort. Am J Reprod Immunol 2016; 76(2): 155-63.

[19] Conti SL, Eisenberg ML. Paternal aging and increased risk of con-genital disease, psychiatric disorders, and cancer. Asian J Androl 2016; 18(3): 420-4.

[20] Shamsi MB, Venkatesh S, Tanwar M, et al. DNA integrity and semen quality in men with low seminal antioxidant levels. Mutat Res 2009; 665(1-2): 29-36.

[21] Greabu M, Battino M, Mohora M, Olinescu R, Totan A, Didilescu A. Oxygen, a paradoxical element? Rom J Intern Med 2008; 46(2): 125-35.

[22] Hayyan M, Hashim MA, Alnashef IM. Superoxide ion: generation and chemical implications. Chem Rev 2016; 116(5): 3029-85.

[23] Halliwell B. Free radicals and antioxidants - quo vadis? Trends Pharmacol Sci 2011; 32(3): 125-30.

[24] Morrell CN. Reactive oxygen species: Finding the right balance. Circ Res 2008; 103: 571-2.

[25] Tremellen K. Oxidative stress and male infertility - A clinical per-spective. Hum Reprod Update 2008; 14(3): 243-58.

[26] Lushchak VI. Free radicals, reactive oxygen species, oxidative stresses and their classifications. Ukr Biochem J 2015; 87(6): 11-8.

[27] Li X, Fang P, Mai J, Choi ET, Wang H, Yang X. Targeting mito-chondrial reactive oxygen species as novel therapy for inflamma-tory diseases and cancers. J Hematol Oncol 2013; 6: 19.

[28] Sies H. Hydrogen peroxide as a central redox signalling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol 2017; 11: 613-9.

[29] Turrens JF. Mitochondrial formation of reactive oxygen species. J. Physiol 2003; 552: 335-44.

[30] Seifried HE, Anderson DE, Fisher EI, Milner JA. A review of the interaction among dietary antioxidants and reactive oxygen species. J Nutr Biochem 2007; 18(9): 567-79.

[31] Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002; 82: 47-95.

[32] Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 2002; 192: 1-15.

[33] Wendel A. Measurement of in vivo lipid peroxidation and toxico-logical significance. Free Radic Biol Med 1987; 3: 355-8.


[34] Castagné V, Lefèvre K, Natero R, Clarke PG, Bedker DA. An op-timal redox status for the survival of axotomized ganglion cells in the developing retina. Neuroscience 1999; 93: 313-20.

[35] Brewer A, Banerjee MS, Murray TV, Namakkal SR, Benjamin I. Reductive stress linked to small HSPs, G6PD and NRF2 pathways in heart disease. Antioxid Redox Signal 2013; 18: 1114-27.

[36] Lloret A, Fuchsberger T, Giraldo E, Vina J. Reductive stress: A new concept in Alzheimer's Disease. Curr Alzheimer Res 2016; 13: 206-11.

[37] Ufer C, Wang CC, Borchert A, Heydeck D, Kuhn H. Redox control in mammalian embryo development. Antioxid Redox Signal 2010; 13: 833-875.

[38] Beckman KB, Ames BN. The free radical theory of ageing matures. Physiol Rev 1998; 78: 547-581.

[39] Linford NJ, Schriner SE, Rabinovitch PS. Oxidative damage and aging: Spotlight on mitochondria. Cancer Res 2000; 66(5): 2497-99.

[40] Barja G. The mitochondrial free radical theory of aging. Prog Mol Biol Transl Sci 2014; 127: 1-27.

[41] Melov S. Therapeutics against mitochondrial oxidative stress in animal models of ageing. Ann NY Acad Sci 2002; 959: 330-40.

[42] Kauppila TE, Kauppila JH, Larsson NG. Mammalian mitochondria and aging: An update. Cell Metab 2017; 10(25): 57-71.

[43] Wallace DC. A mitochondrial paradigm for degenerative diseases and ageing. Novartis Found Symp 2001; 235: 247-63.

[44] Melov S. Mitochondrial oxidative stress. Physiological conse-quences and potential for the role in ageing. Ann NY Acad Sci 2000; 908: 219-25.

[45] Lavranos G, Balla M, Tzortzopoulou A, Syriou V, Angelopoulou R. Investigating ROS sources in male infertility: a common end for numerous pathways. Reprod Toxicol 2012; 34(3): 298-307.

[46] Pons-Rejraji H, Sion B, Saez F, Brugnon F, Janny L, Grizard G. Role of reactive oxygen species (ROS) on human spermatozoa and male infertility. Gynecol Obstet Fertil. 2009; 37(6): 52935.

[47] Agarwal A, Roychoudhury S, Bjugstad KB, Cho CL. Oxidation-reduction potential of semen: what is its role in the treatment of male infertility? Ther Adv Urol 2016; 8(5): 302-18.

[48] Henkel R. The impact of oxidants on sperm function. Andrologia 2005; 37(6): 205-6.

[49] Selvaratnam J, Paul C, Robaire B. Male rat germ cells display age-dependent and cell-specific susceptibility in response to oxidative stress challenges. Biol Reprod 2015; 93(3): 72.

[50] O’Flaherty C. Redox regulation of mammalian sperm capacitation. Asian J Androl 2015; 17: 583-90.

[51] Lee D, Moawad AR, Morielli T, Fernandez MC, O'Flaherty C. Peroxiredoxins prevent oxidative stress during human sperm ca-pacitation. Mol Hum Reprod 2016; doi: 10.1093/molehr/gaw081. [Epub ahead of print].

[52] Baker MA, Aitken RJ. Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility. Reprod Biol Endocrinol 2005; 3: 67.

[53] Gong S, San Gabriel MC, Zini A, Chan P, O'Flaherty C. Low amounts and high thiol oxidation of peroxiredoxins in spermatozoa from infertile men. J Androl 2012; 33(6): 1342-51.

[54] World Health Organisation. 2010. WHO laboratory manual for the examination and processing of human semen. 5th edn. Department of Reproductive Health and Research.

[55] Wu Y, Kang X, Zheng H, Liu H, Huang Q, Liu J. Effect of paternal age on reproductive outcomes of intracytoplasmic sperm injection. PLoS One. 2016; 11(2): e0149867.

[56] Kleinhaus K, Perrin M, Friedlander Y, Paltiel O, Malaspina D, Harlap S. Paternal age and spontaneous abortion. Obstet Gynecol 2006; 108(2): 369-77.

[57] Chapuis A, Gala A, Ferrières-Hoa A, et al. Sperm quality and pa-ternal age: effect on blastocyst formation and pregnancy rates. Ba-sic Clin Androl 2017; 21: 27: 2.

[58] Holland MK, Alvarez JG, Storey BT. Production of superoxide and activity of superoxide dismutase in rabbit epididymal spermatozoa. Biol Reprod 1982; 27: 1109-18.

[59] Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M. Proteomics, oxidative stress and male infertility. Reprod Bio-med Online 2014; 29(1): 32-58.

[60] Sheweita SA, Tilmisany AM, Al-Sawaf H. Mechanisms of male infertility: role of antioxidants. Curr Drug Metab 2005; 6(5): 495501.

[61] Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid Redox Signal 2011; 14(3): 36781.

[62] Pasqualotto FF, Sharma RK, Pasqualotto EB, Agarwal A. Poor semen quality and ROS-TAC scores in patients with idiopathic in-fertility. Urol Int. 2008; 81(3): 263-70.

[63] Aitken RJ. Sperm function tests and fertility. Int J Androl 2006; 29(1): 69-75.

[64] Kefer JC, Agarwal A, Sabanegh E. Role of antioxidants in the treatment of male infertility. Int J Urol 2009; 16(5): 449-57.

[65] La Vignera S, Condorelli R, Vicari E, D'Agata R, Calogero AE. Diabetes mellitus and sperm parameters. J Androl 2012; 33(2): 145-53.

[66] Henkel R, Solomon M. Oxidative Stress. In: Zini A, Agarwal A (eds.). A clinicians guide to sperm DNA and chromatin damage. Springer Int Publishing (submitted)

[67] Aitken RJ, Harkiss D, Buckingham D. Relationship between iron catalysed lipid peroxidation potential and human sperm function. J Reprod Fertil 1993; 98: 257-65.

[68] Maneesh M, Jayalekshmi H. Role of reactive oxygen species and antioxidants on pathophysiology of male reproduction. Indian J Clin Biochem 2006; 21(2): 80-9.

[69] Hirosumi J, Ouchi Y, Watanabe M, Kusunoki J, Nakamura T, Orimo H. Effect of superoxide and lipid peroxide on cytosolic free calcium concentration in free culture pig aortic endothelial cells. Biophys Res Commun 1988; 152: 301-7.

[70] Ohta A, Mohri T, Ohyashiki T. Effect of lipid peroxidation on membrane-bound Ca2_-ATPase activity of the intestinal brush-border membrane. Biochim Biophys Acta 1989; 984: 151-7.

[71] de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an im-portant role in the inhibition of sperm motility. J Androl 1992; 13: 379-86.

[72] Benedetti S, Tagliamonte MC, Catalani S, et al. Differences in blood and semen oxidative status in fertile and infertile men, and their relationship with sperm quality. Reprod Biomed Online 2012; 25(3): 300-6.

[73] Luczaj W, Skrzydlewska E. DNA damage caused by lipid peroxida-tion products. Cell Mol Biol Lett 2003; 8: 391-413

[74] Esterbauer H. Cytotoxicity and genotoxicity of lipid-oxidation products.Am J Clin Nutr 1993; 57(5 Suppl): 779S-85S

[75] Sergerie M, Bleau G, Teulé R, Daudin M, Bujan L. Sperm DNA integrity as diagnosis and prognosis element of male fertility. Gy-necol Obstet Fertil 2005; 33(3): 89-101.

[76] Croteau DL, Stierum RH, Bohr VA. Mitochondrial DNA repair pathways. Mutat Res 1999; 434: 137-48

[77] Pesole G, Gissi C, De Chirico A, Saccone C. Nucleotide substitu-tion rate of mammalian mitochondrial genomes. J Mol Evol 1999; 48: 427-34.

[78] Kumar R , Venkatesh S, Kumar M, et al. Oxidative stress and sperm mitochondrial DNA mutation in idiopathic oligoasthenozoo-spermic men. Indian J Biochem Biophys 2009; 46(2): 1727.

[79] Henkel R. ROS and sperm DNA integrity - Implications of male accessory gland infections. In: Giwercman A, Tournaye H, Björndahl L, Weidner W (eds) Clinical Andrology, Informa healthcare (incorporating Parthenon Publishing, Marcel Dekker, and Taylor & Francis Medical (London); 2010: 481-8.

[80] Koppers AJ, De Iuliis GN, Finnie JM, McLaughlin EA, Aitken RJ. Significance of mitochondrial reactive oxygen species in the gen-eration of oxidative stress in spermatozoa. J Clin Endocrinol Metab 2008; 93(8): 3199-207.

[81] Henkel R. Leukocytes and oxidative stress: dilemma for sperm function and male fertility. Asian J Androl 2011; 13: 43-52.

[82] Mupifiga C, Fisher D, Kruger T, Henkel R. The relationship be-tween seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa. Syst Biol Reprod Med 2013; 59(6): 304-11.

[83] Sonocka D, Jedrzejczak P, Szumala-Kakol A, Fraczek M, Kurpisz M. Male Genital Tract Inflammation: The role of selected interleu-


kins in regulation of pro-oxidant and antioxidant enzymatic sub-stances in seminal plasma. J Androl 2003; 24(3): 448-55.

[84] Selvaratnam J, Robaire B. Overexpression of catalase in mice re-duces age related oxidative stress and maintains sperm production. Exp Gerontol 2016; 84: 1220.

[85] Dohle GR, Smit M, Weber RF. Androgens and male fertility. World J Urol 2003; 21(5): 341-5.

[86] Hales DB, Allen JA, Shankara T, et al. Mitochondrial function in Leydig cell steroidogenesis. Ann N Y Acad Sci 2005; 1061: 120-34.

[87] Diemer T, Allen JA, Hales KH, Hales DB. Reactive oxygen dis-rupts mitochondria in MA-10 tumor Leydig cells and inhibits ster-oidogenic acute regulatory (StAR) protein and steroidogenesis. En-docrinology 2003; 144(7): 2882-91.

[88] Midzak AS, Chen H, Aon MA, Papadopoulos V, Zirkin BR. ATP synthesis, mitochondrial function, and steroid biosynthesis in rodent primary and tumor Leydig cells. Biol Reprod 2011; 84(5): 976-85

[89] Midzak AS, Chen H, Papadopoulos V, Zirkin BR. Leydig cell aging and the mechanisms of reduced testosterone synthesis. Mol Cell Endocrinol 2009; 299(1): 23-31.

[90] Zirkin BR, Chen H. Regulation of Leydig cell steroidogenic func-tion during aging. Biol Reprod 2000; 63(4): 977-81.

[91] Gautam DK, Misro MM, Chaki SP, Sehgal N. H2O2 at physiologi-cal concentrations modulates Leydig cell function inducing oxida-tive stress and apoptosis. Apoptosis 2006; 11(1): 39-46.

[92] Minciullo PL, Inferrera A, Navarra M, Calapai G, Magno C, Gangemi S. Oxidative stress in benign prostatic hyperplasia: a sys-tematic review. Urol Int. 2015; 94(3): 249-54.

[93] Potts JM, Pasqualotto FF. Seminal oxidative stress in patients with chronic prostatitis. Andrologia 2003; 35(5): 304-8.

[94] Sáenz de Tejada I, Angulo J, Cellek S, et al. Pathophysiology of erectile dysfunction. J Sex Med 2005; 2(1): 26-39.

[95] Gratzke C, Angulo J, Chitaley K, et al. Anatomy, physiology, and pathophysiology of erectile dysfunction. J Sex Med 2010; 7: 445-75.

[96] Traish AB, Guay A, Feeley R, Saad F. The Dark Side of Testoster-one Deficiency: I. Metabolic Syndrome and Erectile Dysfunction. J Androl 2009; 30: 10-22.

[97] Rhoden EL, Telöken C, Sogari PR, Vargas Souto CA. The use of the simplified International Index of Erectile Function (IIEF-5) as a diagnostic tool to study the prevalence of erectile dysfunction. Int J Impot Res 2002; 14(4): 245-50.

[98] Schouten BWV, Bohnen AM, Groeneveld FPMJ, Dohle GR, Tho-mas S, Ruun Bosch JLH. Erectile dysfunction in the community: Trends over time in incidence, prevalence, GP consultation and medication use—the krimpen study: Trends in ED. J Sex Med 2010; 7: 2547-53.

[99] Chen D, Zhang KQ, Li B, Sun DQ, Zhang H, Fu Q. Epigallocate-chin-3-gallate ameliorates erectile function in aged rats via regula-tion of PRMT1/DDAH/ADMA/NOS metabolism pathway. 2016 doi: 10.4103/1008-682X.178486. [Epub ahead of print]

[100] George A, Henkel R. Phytoandrogenic properties of Eurycoma longifolia as natural alternative to testosterone replacement therapy. Andrologia 2014; 46: 708-21

[101] Ho CC, Tan HM. Rise of herbal and traditional medicine in erectile dysfunction management. Curr Urol Rep 2011; 12(6): 470-8.

[102] Bella AJ, Shamloul R. Traditional plant aphrodisiacs and male sexual dysfunction. Phytother Res 2014; 28(6): 831-5.

[103] Yu B, Huang Z. Variations in antioxidant genes and male infertility. Biomed Res Int 2015; 2015: 513196.

[104] Agarwal A, Sharma R, Roychoudhury S, Du Plessis S, Sabanegh E. MiOXSYS: a novel method of measuring oxidation reduction po-tential in semen and seminal plasma. Fertil Steril 2016; 106(3): 566-573.e10.

[105] Watanabe K, Shibuya S, Ozawa Y, et al. Superoxide dismutase 1 loss disturbs intracellular redox signaling, resulting in global age-related pathological changes. Biomed Res Int 2014; 2014: 140165

[106] Hamada A, Esteves SC, Nizza M, Agarwal A. Unexplained male infertility: diagnosis and management. Int Braz J Urol 2012; 38(5): 576-94.

[107] Esteves SC, Hamada A, Kondray V, Pitchika A, Agarwal A. What every gynecologist should know about male infertility: an update. Arch Gynecol Obstet 2012; 286(1): 217-29.

[108] Agarwal A, Desai NR, Ruffoli R, Carpi A. Lifestyle and testicular dysfunction: a brief update. Biomed Pharmacother 2008; 62(8): 550-3.

[109] Makker K, Varghese A, Desai NR, Mouradi R, Agarwal A. Cell phones: modern man's nemesis? Reprod Biomed Online 2009; 18(1): 148-57

[110] Mendiola J, Torres-Cantero AM, Agarwal A. Lifestyle factors and male infertility: an evidence based review. Arch Med Sci 2009; 5(1A): S3-S12.

[111] Lewis SE. Is sperm evaluation useful in predicting human fertility? Reproduction 2007; 134(1): 31-40.

[112] Paoli D, Gallo M, Rizzo F, et al. Mitochondrial membrane potential profile and its correlation with increasing sperm motility. Fertil Steril 2011; 95(7): 2315-9.

[113] Deepinder F, Cocuzza M, Agarwal A. Should seminal oxidative stress measurement be offered routinely to men presenting for infer-tility evaluation? Endocr Pract 2008; 14(4): 484-91.

[114] Agarwal A, Roychoudhury S, Sharma R, Gupta S, Majzoub A, Sabanegh E. Diagnostic application of oxidation-reduction potential assay for measurement of oxidative stress: clinical utility in male factor infertility. Reprod Biomed Online 2017; 34(1): 48-57.

[115] Karayiannis D, Kontogianni MD, Mendorou C, Douka L, Mas-trominas M, Yiannakouris N. Association between adherence to the Mediterranean diet and semen quality parameters in male partners of couples attempting fertility. Hum Reprod 2017; 32(1): 215-22.

[116] Associations between dietary patterns and semen quality in men undergoing IVF/ICSI treatment. Hum Reprod 2009; 24(6): 1304-12.

[117] Radimer KL, Bindewald B, Hughes J, Ervin B, Swanson C, Pic-ciano MF. Dietary supplement use by US adults: data from the Na-tional Health and Nutrition Examination Survey, 1999-2000. Am J Epidemiol 2004; 160: 339-49.

[118] Ahmadi S, Bashiri R, Ghadiri-Anari A, Nadjarzadeh A. Antioxidant supplements and semen parameters: An evidence based review. Int J Reprod Biomed 2016; 14(12): 729-36.

[119] Zini A, San Gabriel M, Baazeem A. Antioxidants and sperm DNA damage: a clinical perspective. J Assist Reprod Genet. 2009; 26(8): 427-32.

[120] Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl 2005; 26(3): 349-53.

[121] Vrolijk MF, Opperhuizen A, Jansen EH, et al. The shifting percep-tion on antioxidants: the case of vitamin E and β-carotene. Redox Biol 2015; 4: 272-8.

[122] Atig F, Raffa M, Habib BA, Kerkeni A, Saad A, Ajina M. Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men. BMC Urol 2012; 12: 6.

[123] Bhardwaj A, Verma A, Majumdar S, Khanduja KL. Status of vita-min E and reduced glutathione in semen of oligozoospermic and azoospermic patients. Asian J Androl 2000; 2(3): 225-8.

[124] Colagar AH, Marzony ET, Chaichi MJ. Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men. Nutr Res 2009; 29(2): 82-8.

[125] Wong WY, Merkus HMWM, Thomas CMG, Menkveld R, Zielhuis GA, Steegers-Theunissen RPM. Effects of folic acid and zinc sul-fate on male factor subfertility: a double-blind, randomized, pla-cebo-controlled trial. Fertil Steril 2002; 77: 491-8.

[126] Leisegang K, Udodong A, Bouic PJ, Henkel RR. Effect of the metabolic syndrome on male reproductive function: a case-controlled pilot study. Andrologia 2014; 46(2): 167-76.

[127] Mosbah R, Yousef MI, Mantovani A. Nicotine-induced reproduc-tive toxicity, oxidative damage, histological changes and haemato-toxicity in male rats: the protective effects of green tea extract. Exp Toxicol Pathol 2015; 67(3): 253-9.

[128] Oliveira PF, Tomás GD, Dias TR, et al. White tea consumption restores sperm quality in prediabetic rats preventing testicular oxi-dative damage. Reprod Biomed Online 2015; 31(4): 544-56.

[129] Zhang L, Diao RY, Duan YG, Yi TH, Cai ZM. 2017. In vitro anti-oxidant effect of curcumin on human sperm quality in leucocyto-spermia. Andrologia 2017: doi: 10.1111/and.12760. [Epub ahead of print]

[130] Eleawa SM, Alkhateeb MA, Alhashem FH, et al. Resveratrol re-verses cadmium chloride-induced testicular damage and subfertility


by downregulating p53 and Bax and upregulating gonadotropins and Bcl-2 gene expression. J Reprod Dev 2014; 60(2): 115-27. Epub 2014 Feb 1.

[131] Cui X, Jing X, Wu X, Yan M. Protective effect of resveratrol on spermatozoa function in male infertility induced by excess weight and obesity. Mol Med Rep 2016; 14(5): 4659-65.

[132] Ho CC, Tan HM. Rise of herbal and traditional medicine in erectile dysfunction management. Curr Urol Rep 2011; 12(6): 470-8.

[133] Udensi UK, Tchounwou PB. Oxidative stress in prostate hyperpla-sia and carcinogenesis. J Exp Clin Cancer Res 2016; 35(1): 139.

[134] Herbert V. The antioxidant supplement myth. Am J Clin Nutr 1994; 60: 157-8.

[135] Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 2002; 192(1): 1-15.

[136] Sohal RS, Allen RG, Farmer KJ, Newton RK, Toy PL. Effects of exogenous antioxidants on the levels of endogenous antioxidants, lipid-soluble fluorescent material and life span in the housefly, Musca domestica. Mech Ageing Dev 1985; 31(3): 329-36.

ahmedullah

Typewritten Text

DISCLAIMER: The above article has been published in Epub (ahead of print) on the basis of the materials provided by the author. The Editorial Department reserves the right to make minor modifications for further improvement of the manuscript.

ahmedullah

Typewritten Text

PMID: 27774895

redox regulation of fertility in aging male and the role of ...€¦ · redox regulation of...

Documents