Human Reproduction Update Advance Access originally published online on February 14, 2008
Human Reproduction Update 2008 14(3):243-258; doi:10.1093/humupd/dmn004
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Oxidative stress and male infertility—a clinical perspective
1 Repromed, 180 Fullarton Road, Dulwich, 5065 Adelaide, South Australia, Australia 2 Discipline of Obstetrics and Gynaecology, University of Adelaide, South Australia, Australia
3 Correspondence address. Tel: +618-83338111; Fax: +618-83338188; E-mail: kelton.tremellen{at}adelaide.edu.au
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Abstract |
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Oxidative stress occurs when the production of potentially destructive reactive oxygen species (ROS) exceeds the bodies own natural antioxidant defenses, resulting in cellular damage. Oxidative stress is a common pathology seen in approximately half of all infertile men. ROS, defined as including oxygen ions, free radicals and peroxides are generated by sperm and seminal leukocytes within semen and produce infertility by two key mechanisms. First, they damage the sperm membrane, decreasing sperm motility and its ability to fuse with the oocyte. Second, ROS can alter the sperm DNA, resulting in the passage of defective paternal DNA on to the conceptus. This review will provide an overview of oxidative biochemistry related to sperm health and will identify which men are most at risk of oxidative infertility. Finally, the review will outline methods available for diagnosing oxidative stress and the various treatments available.
Key words: oxidative stress / sperm / male infertility / antioxidant / treatment options
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Introduction |
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Male factor infertility accounts for up to half of all cases of infertility and affects one man in 20 in the general population (McLachlan and de Kretser, 2001). Evidence now suggests that reactive oxygen species (ROS)-mediated damage to sperm is a significant contributing pathology in 30–80% of cases (Iwasaki and Gagnon, 1992
; Zini et al., 1993; Ochsendorf, 1994; Shekarriz et al., 1995a, b; Agarwal et al., 2006a). ROS, defined as including oxygen ions, free radicals and peroxides, cause infertility by two principal mechanisms. First, ROS damage the sperm membrane which in turn reduces the sperm's motility and ability to fuse with the oocyte. Secondly, ROS directly damage sperm DNA, compromising the paternal genomic contribution to the embryo. Despite the common association between compromised sperm quality and oxidative damage, men are rarely screened for oxidative stress nor treated for this condition. Instead they are usually offered ‘mechanical’ treatments such as intracytoplasmic sperm injection (IVF-ICSI) or intrauterine insemination (IUI). This is less than optimal as oxidative damage to sperm DNA is not directly ameliorated by either IVF-ICSI or IUI treatment. In addition, direct treatment of oxidative stress may allow for natural conception, thereby conserving scarce medical resources. This review will provide an overview of who is at risk of oxidative stress, the mechanisms by which oxidative stress produces infertility and the methods available for its diagnosis and treatment. |
Overview of oxidative stress biochemistry |
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ROS are products of normal cellular metabolism. Most of the body's energy is produced by the enzymatically controlled reaction of oxygen with hydrogen in oxidative phosphorylation occurring within the mitochondria during oxidative metabolism. During this enzymatic reduction of oxygen to produce energy, free radicals are formed (Valko et al., 2007
). A free radical is defined as an oxygen molecule containing one or more unpaired electrons in atomic or molecular orbitals. The addition of one electron to dioxygen (O2) forms the superoxide anion radical (O2•–), the primary form of ROS. This superoxide anion can then be directly or indirectly (enzymatic, metal catalyzed) converted to secondary ROS such as the hydroxyl radical (•OH), peroxyl radical (ROO•) or hydrogen peroxide (H2O2). The terms free radical and ROS are commonly used in an interchangeable manner, despite the fact that not all ROS are free radicals (Cheeseman and Slater, 1993). For example, hydrogen peroxide (H2O2) is considered a ROS but it is not a free radical since it does not contain unpaired electrons. In addition, there is a sub-class of free radicals derived from nitrogen which includes nitrous oxide, peroxynitrite, nitroxyl anion and peroxynitrous acid. Free radicals seek to participate in chemical reactions that relieve them of their unpaired electron, resulting in the oxidation of lipids in membranes, amino acids in proteins and carbohydrates within nucleic acids (Ochsendorf, 1999).
Within semen there are two principal sources of production of free radicals; leukocytes and sperm. The vast majority of semen specimens contain leukocytes, with neutrophils being the predominant leukocyte type (Aitken et al., 1994; Aitken and Baker, 1995). As the production of ROS is one of the principal mechanisms by which neutrophils destroy pathogens, it is not surprising that seminal leukocytes have the potential to cause oxidative stress. However, a link between the presence of leukocytes in semen and male oxidative infertility is still under debate (Wolff, 1995). Several researchers have reported a positive correlation between seminal leukocyte numbers and ROS production (Aitken et al., 1994; Whittington et al., 1999; Sharma et al., 2001). However, other studies have failed to find a significant difference in seminal leukocyte concentration between fertile and infertile men (Christiansen et al., 1991; Tomlinson et al., 1993; Aitken and Baker, 1995; Rodin et al., 2003), and the activation state of leukocytes must also play an important role in determining final ROS output. This is supported by the observation of a positive correlation between seminal ROS production and pro-inflammatory seminal plasma cytokines such as interleukin IL-6 (Camejo et al., 2001; Nandipati et al., 2005), IL-8 (Rajasekaran et al., 1995; Martinez et al., 2007) and tumour necrosis factor TNF
(Sanocka et al., 2003; Martinez et al., 2007).
Every human ejaculate contains leukocytes which make the quantification of spermatozoa-specific ROS production more complex. However, sperm isolation techniques have been used to confirm that spermatozoa themselves are responsible for some ROS generation, not just contaminating seminal leukocytes (Baker et al., 2003). Separation of sperm from seminal leukocytes using density-gradient centrifugation has shown the ‘sperm fraction’ to produce significant ROS. As this fraction may still contain a very low number of leukocytes, experiments have been conducted where leukocytes are further depleted using magnetic beads coated with leukocyte-specific CD45 antibodies (Aitken et al., 1996). After removing all detectable leukocyte contamination, ROS production can still be recorded, confirming the ability of sperm to generate ROS. The relative importance of sperm and leukocyte production of ROS varies between individuals but can be estimated using the leukocyte specific activator, N-formyl-methionine-leucine-phenylalanine (FMLP).
The ability of sperm to produce ROS inversely correlates with their maturational state. During spermatogenesis there is a loss of cytoplasm to allow the sperm to form its condensed, elongated form. Immature teratozoospermic sperm are often characterized by the presence of excess cytoplasmic residues in the mid-piece. These residues are rich in the enzyme glucose-6-phosphate dehydrogenase, an enzyme which controls the rate of glucose flux and intracellular production of β-nicotinamide adenine dinucleotide phosphate (NADPH) through the hexose monophosphate shunt. NADPH is used to fuel the generation of ROS via NADPH oxidase located within the sperm membrane (Gomez et al., 1996; Fisher and Aitken, 1997; Said et al., 2005). As a result, teratozoospermic sperm produce increased amounts of ROS compared with morphologically normal sperm.
The existence of NADPH oxidase activity within sperm was questioned when addition of NADPH was unable to elicit any production of the superoxide anion measured by electron paramagnetic resonance spectroscopy, a very sensitive and specific assay for the superoxide anion (Richer and Ford, 2001). However, since then the presence of a calcium-dependant NADPH oxidase called NOX 5 has been confirmed within sperm (Banfi et al., 2001; Armstrong et al., 2002; Sabeur and Ball, 2007). This sperm-specific NADPH oxidase (NOX 5) is reported to be quite distinct from leukocyte NADPH oxidase, with NOX 5 activity not being controlled by protein kinase C as occurs in the leukocyte (Armstrong et al., 2002). Whether NOX 5 is over expressed in spermatozoa of patients exhibiting infertility associated with oxidative stress is presently unknown.
The relative importance of leukocytes and sperm in the aetiology of oxidative stress is currently under debate. The rate of production of ROS by leukocytes is reported to be 1000 times higher than that of spermatozoa at capacitation (Plante et al., 1994), making leukocytes the likely dominant producer of seminal ROS. When seminal ROS production is divided into that produced by the sperm themselves (intrinsic ROS) and that made by the leukocytes (extrinsic), an interesting observation is seen (Henkel et al., 2005). While both intrinsic and extrinsic ROS production is negatively correlated with sperm DNA integrity, the relationship is significantly stronger for intrinsic ROS production. This suggests that while leukocytes produce more ROS than sperm on a per cell basis, the close proximity between intrinsic ROS production and sperm DNA makes intrinsic ROS production a more important variable in terms of fertility potential.
The human body has developed several antioxidant strategies to protect itself from ROS damage. This allows for normal oxidative metabolism to occur without damaging the cells, while still allowing for normal ROS-mediated cellular responses such as destruction of infectious pathogens and intracellular signalling (Valko et al., 2007). Oxidative stress occurs when the production of ROS overwhelms the antioxidant defense mechanisms leading to cellular damage. Seminal plasma and sperm themselves are well endowed with an array of protective antioxidants (Fujii et al., 2003; Garrido et al., 2004a). Superoxide dismutase (SOD) and catalase are enzymatic antioxidants which inactivate the superoxide anion (O•2–) and peroxide (H2O2) radicals by converting them into water and oxygen. SOD is present within both sperm and seminal plasma (Mennella and Jones, 1980; Zini et al., 1993). The addition of SOD to sperm in culture has been confirmed to protect them from oxidative attack (Kobayashi et al., 1991). While some investigators have reported minor reductions in seminal plasma SOD activity in infertile men (Alkan et al., 1997; Sanocka et al., 1997), many have not (Miesel et al., 1997; Zini et al., 2000; Hsieh et al., 2002). However, the majority of evidence does support a link between deficient seminal catalase activity and male infertility (Jeulin et al., 1989; Alkan et al., 1997; Miesel et al., 1997; Sanocka et al., 1997; Zini et al., 2000). Glutathione peroxidase (GPX) is the final member of the seminal enzymatic antioxidant triad. GPX consists of a family of antioxidants (GPX1-5) that are involved in the reduction of hydroperoxides using glutathione as an electron donor. The GPXs are located within the testis, prostate, seminal vesicles, vas deferens, epididymis, seminal plasma and spermatozoa themselves (reviewed by Vernet et al., 2004). GPX must play an important protective role against oxidative attack since its specific inhibition in vitro using mercaptosuccinate leads to a large increase in sperm lipid peroxidation (Twigg et al., 1998). Male factor infertility has been linked with a reduction in seminal plasma (Giannattasio et al., 2002) and spermatozoa (Garrido et al., 2004b) GPX activity, further supporting an important role for this enzyme in male fertility. In addition, men exhibiting leukospermia-associated oxidative stress have been reported to have significantly reduced GPX activity within their spermatozoa (Therond et al., 1996). Finally, the continued activity of GPX depends on the regeneration of reduced glutathione by glutathione reductase (GTR). Selective inhibition of GTR reduces the availability of reduced glutathione for maintaining GPX activity, thereby exposing sperm to oxidative stress (Williams and Ford, 2004). The coordinated activity of GPX, GTR and glutathione clearly play a pivotal role in protecting sperm from oxidative attack.
The non-enzymatic antioxidants present within semen include ascorbic acid (Vitamin C), -tocopherol (Vitamin E), glutathione, amino acids (taurine, hypotaurine), albumin, carnitine, carotenoids, flavenoids, urate and prostasomes. These agents principally act by directly neutralizing free radical activity chemically. However, they also provide protection against free radical attack by two other mechanisms. Albumin can intercept free radicals by becoming oxidized itself, thereby sparing sperm from attack (Twigg et al., 1998). Alternatively, extracellular organelles (prostasomes) secreted by the prostate have been shown to fuse with leukocytes within semen and reduce their production of free radicals (Skibinski et al., 1992; Saez et al., 1998). A substantial number of researchers have reported a significant reduction in non-enzymatic antioxidant activity in seminal plasma of infertile compared with fertile men (Fraga et al., 1991; Fraga et al., 1996; Smith et al., 1996; Therond et al., 1996; Lewis et al., 1997; Gurbuz et al., 2003; Koca et al., 2003; Mostafa et al., 2006; Song et al., 2006).
Antioxidants contained within seminal plasma are obviously helpful for preventing sperm oxidative attack following ejaculation. However, during spermatogenesis and epididymal storage, the sperm are not in contact with seminal plasma antioxidants and must rely on epididymal/testicular antioxidants and their own intrinsic antioxidant capacity for protection. Sperm are therefore vulnerable to oxidative damage during epididymal transit, especially when there is epididymal inflammation such as male genital tract infection. In addition, testicular biopsies from men with varicocele-associated oxidative stress have shown an increase in oxidative DNA damage within spermatogonia and spermatocytes (Ishikawa et al., 2007). Therefore, while seminal plasma antioxidants may help minimize ejaculated sperm oxidative stress, they have no capacity to prevent oxidative damage initiated ‘up stream’ at the level of the testis and epididymis.
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Seminal free radicals—friend or foe? |
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 TOPAbstractIntroductionOverview of oxidative stress... Seminal free radicals--friend or... Origins of oxidative stressLaboratory identification of...Management of oxidative stress...OverviewFundingReferences |
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Sperm were the first type of cell reported to produce free radicals. In this pioneering report, MacLeod (1943)
noted that incubation of sperm under conditions of high oxygen tension lead to a rapid loss of their motility. The addition of the antioxidant catalase to the medium preserved sperm motility, prompting MacLeod to suggest that sperm must produce hydrogen peroxide during normal oxidative metabolism. Since this publication, it has evolved that three inter-related mechanisms account for oxidative stress-mediated male infertility—impaired motility, impaired fertilization and oxidative DNA damage.
The underlying pathology behind free radicals ability to reduce sperm motility was first reported by Jones et al. (1979). They reported that ROS-induced peroxidation of the sperm membrane decreasing its flexibility and therefore tail motion. Sperm membranes are vulnerable to this type of damage as they contain large amounts of unsaturated fatty acids. Direct ROS damage to mitochondria, decreasing energy availability, may also impede sperm motility (de Lamirande and Gagnon, 1992; de Lamirande et al., 1997, 1998). By either mechanism, oxidative stress impairs sperm motility and will result in less sperm reaching the oocyte for fertilization (Whittington et al., 1999; Kao et al., 2007).
Low level production of free radicals by sperm plays a positive role in preparation for fertilization (capacitation). Hydrogen peroxide stimulates the acrosome reaction and sperm hyperactivation (de Lamirande and Gagnon, 1993), thereby assisting the sperm's transit through the cumulus and zona pellucida. Low concentrations of hydrogen peroxide also cause tyrosine phosphorylation, which augments sperm membrane binding to the zona pellucida ZP-3 protein (Aitken et al., 1995b), ultimately boosting sperm–oocyte fusion (Aitken et al., 1998). However, high levels of ROS production lead to peroxidation of the sperm acrosomal membrane and diminished acrosin activity (Zalata et al., 2004), and impaired sperm–oocyte fusion (Aitken et al., 1989; Ichikawa et al., 1999; Saleh et al., 2003a, b; Zorn et al., 2003a; Jedrzejczak et al., 2005).
Free radicals have the ability to directly damage sperm DNA by attacking the purine and pyrimidine bases and the deoxyribose backbone. Normally, sperm DNA is tightly packaged by protamines protecting it from free radical attack. However, infertile men often exhibit deficient protamination, leaving the sperm DNA particularly vulnerable to ROS attack (Oliva, 2006). Alternatively, free radicals can initiate apoptosis within the sperm, leading to caspase-mediated enzymatic degradation of the DNA (Kemal Duru et al., 2000; Wang et al., 2003; Moustafa et al., 2004; Villegas et al., 2005). Several investigators (Kodama et al., 1997; Aitken et al., 1998; Saleh et al., 2002b; Oger et al., 2003; Wang et al., 2003; Henkel et al., 2005; Kao et al., 2007) have now confirmed the link between oxidative stress and sperm DNA damage using various techniques such as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), sperm chromatin structure assay (SCSA) and measurement of the byproduct of DNA oxidation, 8-hydroxydeoxyguanosine (8-OHdG). Furthermore, two groups have now correlated increased sperm oxidative DNA damage with poor blastocyst formation in vitro (Zorn et al., 2003a; Meseguer et al., 2006, 2007). Damaged paternal DNA is recognized to be a significant cause for poor blastocyst development (Seli et al., 2004). Finally, a large prospective study of 225 couples planning their first pregnancy found a strong inverse relationship between seminal 8-OHdG concentration and monthly natural fecundity (Loft et al., 2003).
During natural conception or routine IVF, oxidative damage to the sperm membrane will normally block fertilization, preventing the damaged paternal DNA from creating an embryo. However, during IVF-ICSI this natural barrier to fertilization is lost and sperm containing significantly damaged DNA can still achieve fertilization following microinjection (Zorn et al., 2003a). While many of these embryos will ultimately fail at the blastocyst or early fetal stage, there is the potential for a child to be born with damaged paternal derived DNA. The consequences of this are as yet unknown but it has been suggested to include the initiation of genetic defects and childhood cancer (Aitken and Krausz, 2001; Aitken et al., 2003).
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Origins of oxidative stress |
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The origins of sperm oxidative stress are summarized in Fig. 1. While pathologies such as genitourinary tract infection and varicocele are well established causes of oxidative stress, others such as hyper-homocysteinaemia and diabetes are only now just becoming recognized as possible causes. It is hoped that this review will stimulate further research in these less well established potential causes of male oxidative infertility.
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Idiopathic
Idiopathic male factor infertility has been linked with oxidative stress by several research groups. One of the principal causes of this association is the observation that morphologically abnormal sperm have an increased capacity to generate ROS, but also a reduced antioxidant capacity (Gomez et al., 1996; Garrido et al., 2004b; Said et al., 2004; Said et al., 2005). As approximately one-third of infertile men exhibit teratozoospermia (Thonneau et al., 1991), it is not surprising that sperm oxidative stress is commonly identified in the idiopathic infertile male population. Even men with normozoospermic idiopathic infertility exhibit significantly higher seminal ROS production and lower antioxidant capacity than fertile men (Pasqualotto et al., 2001; Agarwal et al., 2006b), for as yet unknown reasons.
The use of assisted reproductive technologies (ART) has the potential to exacerbate sperm oxidative stress. During IVF and IUI treatment semen is centrifuged to separate sperm from seminal plasma. This exacerbates oxidative stress as centrifugation increases sperm ROS production many fold (Iwasaki and Gagnon, 1992; Shekarriz et al., 1995a, b), while removing sperm from protective antioxidants within seminal plasma (Potts et al., 2000a, b). In addition cryopreservation of sperm, another commonly used technique in ART, is associated with an increase in sperm oxidative stress (Watson, 2000).
Drugs such as the chemotherapy agent cyclophosphamide have been linked with sperm oxidative stress. Administration of cyclophosphamide to animals is reported to increase testicular malondialdehyde (MDA) levels and produce a fall in testicular catalase, implying the presence of oxidative stress (Das et al., 2002; Ghosh et al., 2002). Drugs such as aspirin and paracetamol (acetaminophen) can also produce oxidative stress by increasing cytochrome P450 activity, thereby boosting ROS generation (Agarwal and Said, 2005).
Smoking results in a 48% increase in seminal leukocyte concentrations and a 107% increase in seminal ROS levels (Saleh et al., 2002a). Smokers have decreased levels of seminal plasma antioxidants such as Vitamin E (Fraga et al., 1996) and Vitamin C (Mostafa et al., 2006), placing their sperm at additional risk of oxidative damage. This has been confirmed by the finding of a significant increase in levels of 8-OHdG within smoker's seminal plasma (Fraga et al., 1996).
Dietary deficiencies have been linked with sperm oxidative damage by several research groups. The Age and Genetic Effects in Sperm (AGES) study examined the self-reported dietary intake of various antioxidants and nutrients (vitamins C and E, β-carotene, folate and zinc) in a group of 97 healthy non-smokers and correlated this with sperm quality (Eskenazi et al., 2005). This study did observe a significant correlation between vitamin C intake and sperm concentration and between vitamin E intake and total progressively motile sperm. This is also consistent with earlier reports of a significant link between seminal plasma vitamin E levels and an increase in percentage of motile sperm (Therond et al., 1996). However, the AGES study was unable to confirm a link between low intake of antioxidants and sperm DNA damage (Silver et al., 2005). This was surprising given that other researchers had linked low seminal plasma vitamin C levels with increased sperm DNA damage (Fraga et al., 1991; Song et al., 2006). It is possible that levels of individual antioxidants within seminal fluids may more accurately reflect biological effect than self-reported dietary intake as different food sources and preparation techniques can vastly modify antioxidant intake. Alternatively, differences in the populations studied may explain the discrepant results. Song et al. (2006) correlated sperm DNA damage with dietary antioxidant intake in infertile men, while Silver et al. (2005) and Fraga et al. (1991) examined this relationship in healthy presumed fertile patients. Fertile men with low levels of oxidative attack may not be as dependant on seminal antioxidants for protection of their sperm DNA integrity. Therefore, a dietary deficiency in antioxidants may not lead to sperm oxidative DNA damage in this fertile cohort.
Excessive alcohol consumption causes an increase in systemic oxidative stress as ethanol stimulates the production of ROS, while many alcohol abusers have diets deficient in protective antioxidants (Wu and Cederbaum, 2003; Koch et al., 2004). A study of 46 alcoholic men of reproductive age has suggested the presence of oxidative stress within the testicle by reporting a significant reduction in plasma testosterone, increase in serum lipid peroxidation byproducts and a drop in antioxidants (Maneesh et al., 2006). However, no study to date has directly examined the link between alcohol intake and sperm oxidative damage.
Extremes of exercise activity, at both ends of the spectrum, have been linked with oxidative stress. It is not surprising that high impact exercise is linked with oxidative stress since muscle aerobic metabolism creates a large amount of ROS (Peake et al., 2007). In a rodent model, increasing levels of exercise are linked with a reduction in sperm count and motility and a corresponding increase in biochemical signs of testicular oxidative stress (Manna et al., 2004). Conversely, obesity produces oxidative stress as adipose tissue releases pro-inflammatory cytokines that increase leukocyte production of ROS (Singer and Granger, 2007). Furthermore, accumulation of adipose tissue within the groin region results in heating of the testicle which has been linked with oxidative stress and reduced sperm quality (Banks et al., 2005; Ishii et al., 2005; Perez-Crespo et al., 2007).
Psychological stress produces a reduction in semen quality; with the underlying mechanism previously felt to be related to a central impairment of gonadotrophin drive (Fenster et al., 1997). However, recent prospective studies have linked a period of psychological stress with a reduction in sperm quality mediated by an increase in seminal plasma ROS generation and a reduction in antioxidant protection (Eskiocak et al., 2005, 2006).
Several studies have reported that sperm DNA damage increases with advancing age in both fertile (Wyrobek et al., 2006) and infertile men (Singh et al., 2003; Moskovtsev et al., 2006). It is possible that an increase in oxidative sperm DNA damage is the underlying pathology. A large observational study has confirmed that systemic oxidative stress increases with age (Junqueira et al., 2004). Animal studies using the Brown Norway rat, an established model of male reproductive aging, confirm that sperm from older animals produce more free radicals than from young animals and have a reduced enzymatic antioxidant activity, resulting in an increase in ROS-mediated sperm DNA damage (Zubkova et al., 2005; Weir and Robaire, 2007).
Phthalates are chemicals used as a plastics softener and are contained in a wide range of food packaging and personal care products. Exposure to phthalates can occur via dietary consumption, dermal absorption or inhalation and has been linked with impaired spermatogenesis and increased sperm DNA damage (Agarwal et al., 1985; Srivastava et al., 1990; Kasahara et al., 2002; Hauser et al., 2007). Oral administration of phalate esters to rats is reported to increase the generation of ROS within the testis and a concomitant decrease in antioxidant levels, culminating in impaired spermatogenesis (Lee et al., 2007).
Several environmental pollutants have been linked with testicular oxidative stress. Pesticides such as lindane (Chitra et al., 2001), methoxychlor (Latchoumycandane et al., 2002) and the herbicide dioxin-TCDD (Latchoumycandane et al., 2003) have all been linked with testicular oxidative stress in rodent models. The commonly used preservative sulfur dioxide has also been shown to produce testicular oxidative stress in laboratory animals (Meng and Bai, 2004). Air pollutants such as diesel particulate matter act as potent stimuli for leukocyte ROS generation (Gonzalez-Flecha, 2004; Alaghmand and Blough, 2007). While no study has directly linked airborne pollutants with testicular oxidative stress, it is possible that this oxidative insult is responsible for the increase in sperm DNA damage seen following periods of airborne pollution (Rubes et al., 2005).
Heavy metal exposure has been conclusively linked with sperm oxidative damage. Both cadmium and lead are linked with an increase in testicular oxidative stress (Hsu and Guo, 2002; Acharya et al., 2003) and a resultant increase in sperm DNA oxidation (Xu et al., 2003; Naha and Chowdhury, 2006). The increase in infertility and miscarriage observed in the partners of welders and battery/paint factory workers (Gennart et al., 1992; Bonde, 1993) may be due to oxidative damage to sperm DNA initiated by the inhalation of metal fumes.
Genitourinary tract infection
Up to 50% of men will experience prostatitis at some point in their lives, with prostatitis becoming chronic in 10% of men (Schaeffer, 2003). Bacteria responsible for prostate infection may originate from the urinary tract or can be sexually transmitted (Fraczek and Kurpisz, 2007; Fraczek et al., 2008). Typical non-sexually-transmitted pathogens include Streptococci (S. viridans and S. pyogens), coagulase-negative Staphylococci (S. epidermidis, S. haemolyticus), gram-negative bacteria (E. coli, Proteus mirabilis) and atypical mycoplasma strains (Ureaplasma urealyticum, Mycoplasma hominis). All of these pathogens will create an acute inflammatory response with an influx of leukocytes into the genital tract and a resulting increase in ROS production (Mazzilli et al., 1994; Depuydt et al., 1996; Ochsendorf, 1999; Potts et al., 2000a, b). Men prone to recurrent genitourinary tract infections, such as paraplegics, have been confirmed to have high degrees of sperm oxidative pathology (Padron et al., 1997; Brackett et al., 2008). Current or past Chlamydia infection has also been linked with an increase in oxidative damage to sperm (Segnini et al., 2003).
Viral infections may also initiate oxidative damage to sperm. The link between common viral pathogens such as cytomegalovirus, herpes simplex virus (HSV), Epstein-Barr virus and oxidative infertility has been examined by several groups. Only HSV appears to have a possible role in the initiation of oxidative damage to sperm. Herpes simplex DNA is found in 4–50% of infertile men's semen (Kapranos et al., 2003, Bezold et al., 2007), with IgM antibodies towards HSV being associated with a 10-fold increase in the rate of leukospermia (Krause et al., 2002, 2003). Given the well recognized link between leukospermia and seminal ROS levels, together with the observation of a reduction in sperm motility in men positive for seminal HSV DNA (Kapranos et al., 2003), it is likely that HSV is a viral pathogen involved in oxidative stress.
Systemic infection
Several chronic systemic infections have been linked with increased oxidative stress throughout the body. Human immunodeficiency virus (HIV) infection is associated with an increase in leukocyte number and activation within semen (Umapathy et al., 2001). Hepatitis B and C infection has also been correlated with significant hepatic oxidative stress (Chen and Siddiqui, 2007; Seronello et al., 2007). At present it is unknown if this oxidative stress extends to the semen, but impaired sperm motility seen in hepatitis B and C patients (Durazzo et al., 2006; Vicari et al., 2006), makes this possible. Finally, chronic infections such as tuberculosis (Srinivasan et al., 2004), leprosy (Vijayaraghavan et al., 2005), malaria (Guha et al., 2006) and Chagas disease (Macao et al., 2007) have all been linked with elevated degrees of systemic oxidative stress. While no study has directly linked these chronic infectious diseases with sperm oxidative stress, it is unlikely that the male reproductive tract would be spared from this systemic oxidative insult.
Chronic non-bacterial prostatitis (NIH Category III) is a chronic inflammation of the prostate in the absence of infection and has been reported by several groups to be associated with considerably elevated oxidative stress within semen (Pasqualotto et al., 2000; Shahed and Shoskes, 2000; Potts and Pasqualitis, 2003). Chronic non-bacterial prostatitis accounts for in excess of 90% of all cases and effects 10% of men (Schaeffer, 2003). In the majority of cases of chronic non-bacterial prostatitis it is reported that an adverse autoimmune response to seminal or prostate antigens is responsible for the pathology, leading to an increase in pro-inflammatory cytokines and activated ROS producing leukocytes within the semen (Batstone et al., 2002; Motrich et al., 2005; Motrich et al., 2007). While the exact trigger for this response is unknown, one report has linked a polymorphism of the TH-2 cytokine IL-10 with chronic non-bacteria prostatitis (Shoskes et al., 2002). A lack of this Th-2 cytokine may tip the immune balance towards the Th-1 direction leading to the generation of T lymphocytes reactive against prostate antigens. These T cells will liberate cytokines such as IFN-
, TNF- and IL-1β that stimulate chemotaxis and activation of leukocytes, leading to increased seminal oxidative stress (Motrich et al., 2005). It is therefore not surprising to see the majority of studies linking chronic non-bacterial prostatitis with a significant reduction in sperm density, motility, morphology and membrane integrity (Christiansen et al., 1991; Leib et al., 1994; Krieger et al., 1996; Engeler et al., 2003; Motrich et al., 2005; Henkel et al., 2006); although this is refuted by some groups (Pasqualotto et al., 2000; Ludwig et al., 2003).
Oxidative stress has been proposed as a significant cause for infertility after vasectomy reversal. It is believed that vasectomy disrupts the normal blood-testis barrier, leading to a loss of immune privilege and activation of immune responses against sperm (Filippini et al., 2001). Several studies have documented an increase in seminal leukocytes, pro-inflammatory cytokines and free radical production within semen following vasectomy reversal (Shapiro et al., 1998; Kolettis et al., 1999; Sharma et al., 1999; Nandipati et al., 2005).
Oxidative stress is now widely believed to be the principal underlying pathology linking varicocele with male infertility (Hendin et al., 1999; Barbieri et al., 1999; Saleh et al., 2003b; Nallella et al., 2004; Smith et al., 2006; Agarwal et al., 2006c; Ishikawa et al., 2007; Smith et al., 2007). The increase in varicocele-related ROS production is strongly correlated with a reduction in sperm DNA integrity when assessed by either TUNEL (Smith et al., 2006) or 8-hydroxy-2'-deoxyguanosine DNA oxidative metabolite levels (Chen et al., 2004).
Cryptorchidism is a common cause for male factor infertility in which the primary pathology is hypo-spermatogenesis due to deficient maturation of gonocytes to type A spermatogonia (Huff et al., 1991). However, recently it has been reported that men with cryptorchidism surgically treated with orchidoplexy early in life still have markedly elevated sperm ROS production and DNA fragmentation compared with fertile controls (Smith et al., 2007).
Torsion of the spermatic cord has long been recognized as a cause of male infertility, even when this torsion is unilateral. It is now generally accepted that oxidative stress related to ischemia-reperfusion injury is the underlying cause of damage to both the torted and contra-lateral testis. A prolonged period of ischemia followed by surgical or spontaneous restoration of blood flow leads to an influx of activated leukocytes into both testis (Turner et al., 2004) and a consequent increase in generation of free radicals (Filho et al., 2004). Oxidative stress then leads to necrosis of the germinal cells with resulting subfertility or infertility.
Diabetes has long been recognized to impair male fertility by interfering with both spermatogenesis and erectile function. Recently it has been reported that diabetic men have significantly higher levels of sperm DNA fragmentation than normal controls (Agbaje et al., 2007). While this study did not directly measure oxidative stress, the authors proposed that the most likely mechanism for the observed increase in sperm DNA damage was an increase in oxidative stress as this is now recognized as a key pathology underlying many chronic complications of diabetes. In support, studies using the Streptozotocin-induced diabetic rat model have found a significant increase in testicular oxidative stress within 6 weeks of initiation of the diabetic state (Shrilatha and Muralidhara, 2007).
Chronic inflammation and oxidative stress are highly prevalent in patients with chronic kidney disease and end-stage renal disease (Oberg et al., 2004). Surprisingly, even when uraemia is reversed by haemodialysis, a persisting state of chronic inflammation and oxidative stress persists (Danielski et al., 2003; Pupim et al., 2004). Furthermore, renal transplant patients with stable renal function and no obvious signs of immune rejection of their graft also have elevated levels of oxidative stress (Moreno et al., 2005).
Patients with haemaglobinopathies such as beta-thalassemia major have high degrees of systemic oxidative stress (Livrea et al., 1996), with this oxidative damage confirmed to involve sperm (Carpino et al., 2004). The likely cause of oxidative stress is iron overload from multiple blood transfusions. Iron is a potent pro-oxidant capable of redox cycling when not safely bound to transferrin in the blood or stored as ferritin in tissue.
The toxic accumulation of homocysteine may cause reproductive dysfunction and oxidative stress within the testis (Forges et al., 2007; Sonmez et al., 2007). Hyper-homocysteinaemia usually occurs due to suboptimal re-methylation of homocysteine to methionine by the enzyme methyl tetrahydrofolate reductatse (MTHFR) caused by a dietary deficiency of folate or a single-nucleotide polymorphism (SNP) in the MTHFR gene (Selhub, 1999; Matthews, 2002). Several investigators have reported that SNPs (C677T and others) in the MTHFR gene are more commonly found in the infertile men (Bezold et al., 2001; Park et al., 2005; Lee et al., 2006; Zhou-Cun et al., 2007), placing these men at increased risk of homocysteine-induced oxidative stress.
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Laboratory identification of oxidative stress-related male infertility |
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One of the main reasons why screening for oxidative stress is not routine in andrology laboratories is the cost and complexity of testing and the lack of a single standardized measure of oxidative stress. At present there are over 30 assays of oxidative stress (Ochsendorf, 1999
), broadly divided into three different types. This review will focus on the most popular and clinically useful assays currently being performed. These assays measure damage created by excess free radicals against the sperm lipid membrane or DNA. As oxidative stress is the result of an in balance between ROS production and total antioxidant capacity (TAC), direct tests reflect the net biological effect between these two opposing forces.
The most widely used method of assessing sperm membrane peroxidation is the measurement of MDA levels in sperm or seminal plasma with the thiobarbituric acid assay. MDA levels in sperm are quite low and therefore require the use of sensitive high-pressure liquid chromatography (HPLC) equipment (Li et al., 2004; Shang et al., 2004) or the use of iron-based promoters and spectrofluometry measurement (Aitken et al., 1993). Seminal plasma levels of MDA are 5–10-fold higher than sperm, making measurement on standard spectrophotometers possible (Sanocka et al., 1997; Nakamura et al., 2002; Tavilani et al., 2005). Measurement of MDA appears to be of some clinical relevance since its concentration within both seminal plasma and sperm is elevated in infertile men with excess ROS production, compared with fertile controls or normozoospermic individuals (Sanocka et al., 1997; Nakamura et al., 2002; Tavilani et al., 2005; Hsieh et al., 2006). Furthermore, in vitro impairment of motility, sperm DNA integrity and sperm–oocyte fusion capacity by ROS is accompanied by an increase in MDA concentration (Aitken et al., 1989, 1993). Other direct tests of sperm membrane lipid peroxidation such as measurement of the isoprostane 8-Iso-PGF2 (Khosrowbeygi and Zarghami, 2007) and the c11-BODIPY assay (Aitken et al., 2007; Kao et al., 2007) are showing promise but are not yet in common usage.
It is well recognized that oxidative stress is one of the major causes of sperm DNA damage (Aitken et al., 1998; Oger et al., 2003; Saleh et al., 2003a, b). However, measurement of sperm DNA damage by TUNEL or SCSA is an imperfect assessment of oxidative stress as sperm DNA can be damaged by non-oxidative mechanisms such as aberrant apoptosis and incomplete sperm protamination (Ozmen et al., 2007). The best direct assessment of sperm DNA oxidative damage is the measurement of the oxidized deoxynucleoside, 8-oxo-7,8-dihydro 2' deoxyguanosine (8-OHdG). This can be measured in sperm or seminal plasma by HPLC (Fraga et al., 1991; Loft et al., 2003), enzyme-linked immunoabsorbent assay (Nakamura et al., 2002) or directly within sperm using immunoflurorescence (Kao et al., 2007). Since a large prospective study has reported that chances of natural conception is inversely correlated with sperm 8-OHdG levels (Loft et al., 2003), measurement of this direct marker of sperm oxidative stress appears to have some clinical utility.
Chemoluminescence assays using either Luminol or Lucigenin are the most commonly described technique to detect ROS production within semen. These probes are very sensitive and have the advantage of relatively well established reported ranges for both the fertile and infertile population (Ochsendorf et al., 1994; Williams and Ford, 2005; Athayde et al., 2007). However, general uptake by clinical andrology laboratories has been hampered by expensive equipment (luminometer) and difficulties with quality control created by assay confounders such as incubation time, leukocyte contamination and presence of seminal plasma contamination (Kobayashi et al., 2001; Aitken et al., 2004). Furthermore, Lucigenin has been shown to undergo auto-oxidization which itself leads to the production of superoxide anions (Liochev and Fridovich, 1997). This makes chemoluminescent probes such as Lucigenin less than ideal reagents for measurement of sperm superoxide anion production. A simpler alternative may be light microscopy quantification of nitroblue tetrazolium (NBT) activity. NBT is a yellow water soluble compound that reacts with superoxide anions within cells to produce a blue pigment diformazan. The amount of diformazan crystals seen within a leukocyte or sperm reflects its superoxide anion production. The NBT assay has been shown to correlate well with traditional chemoluminescence techniques (Esfandiari et al., 2003) but has two distinct advantages. First, the NBT assay is inexpensive to set up as it only requires a light microscope. Secondly, the NBT assay can discriminate between production of ROS by sperm and leukocytes without the need for addition of activating peptides (FMLP) used in chemoluminescence assays (WHO manual, 1999).
Measurement of TAC within semen can be conducted in a variety of ways. The ability of seminal plasma to inhibit chemoluminescence elicited by a constant source of ROS (horse-radish peroxidase) is a commonly used technique. The TAC is usually quantified against a Vitamin E analogue (Trolox) and expressed as a ROS-TAC score (Sharma et al., 1999). However, colourimetry techniques based on the colour change of ABTS (2,2'-azinobis3-ethylbenzo-thiazoline-6-sulphate) are now becoming more popular as they are cheaper and easier to perform (Said et al., 2003; Erel, 2004). The reduced ABTS molecule is oxidized to ABTS+ using hydrogen peroxide and a peroxidase to form a relatively stable blue-green colour measured at 600 nm with a standard spectrophotometer. Antioxidants present within seminal plasma suppress this colour change to a degree that is proportional to their concentrations. Again the antioxidant activity is quantified using Trolox.
Oxidative stress implied from routine semen analysis
A summary of the routine laboratory test ‘sentinel signs’ suggesting the possible presence of sperm oxidative stress is contained in Table I. While a reduction in any of the sperm parameters (count, motility, morphology) is more commonly seen in men with oxidative stress, asthenozoospermia is probably the best surrogate marker for oxidative stress in a routine semen analysis (Aitken and Baker, 1995; Aitken et al., 1995a, b; Whittington et al., 1999; Keskes-Ammar et al., 2003; Kao et al., 2007). A link between impaired sperm motility and oxidative stress also extends to the sperm DNA as a recent study has identified a highly significant correlation between oxidation of sperm DNA and reduced motility (Kao et al., 2007).
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Hyperviscosity of seminal plasma is associated with increased levels of seminal plasma MDA (Aydemir et al., 2008
) and reduced seminal plasma antioxidant status (Siciliano et al., 2001), making impaired viscosity a reasonable surrogate marker of oxidative stress. Infection of the semen with Ureaplasma urealyticum is associated with increased seminal plasma viscosity (Wang et al., 2006) and an increase in ROS production (Potts et al., 2000a, b). It is possible that these infections may damage the prostate and seminal vesicle, altering the substrates required for creation of normal semen viscosity.
A large number of round cells within semen may suggest the presence of oxidative stress as they may represent seminal leukocytes (Sharma et al., 2001). However, round cells may also be immature sperm rather than leukocytes, so formal identification of leukocytes requires ancillary tests such as the peroxidase test, CD45 staining or measurement of seminal elastase (WHO manual, 1999; Zorn et al., 2003b; Kopa et al., 2005). Finally, poor sperm membrane integrity assessed by the hypo-osmolar swelling test has been linked with the presence of sperm oxidative stress (Dandekar et al., 2002).
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Management of oxidative stress related infertility |
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TOPAbstractIntroductionOverview of oxidative stress...Seminal free radicals--friend or...Origins of oxidative stressLaboratory identification of...Management of oxidative stress...OverviewFundingReferences |
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Once an individual has been identified as having oxidative stress related infertility, treatment should be aimed at identification and amelioration of the underlying cause before considering antioxidant treatment. The following paragraphs are the author's suggestions for investigation and management based on the underlying causes of oxidative stress outlined in previous paragraphs. These recommendations are summarized in Table II.
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Lifestyle modification
Lifestyle behaviours such as smoking, poor diet, alcohol abuse, obesity or psychological stress have all been linked with oxidative stress. While the effectiveness of elimination of these lifestyle triggers for oxidative stress has not been formally tested, it is likely that making positive lifestyle changes such as a diet high in fruit/vegetables, maintenance of normal weight and a reduction in smoking/alcohol intake would have at least some beneficial effect on sperm health.
Exposure to heat, pollution and toxins (heavy metals and plasticizers) have all been linked with oxidative stress. Men should be advised to avoid activities which may heat the scrotum such as long baths and saunas. Proper ventilation and use of personal protective equipment at work will hopefully reduce men's exposure to chemical and metal vapours linked with oxidative stress.
Treatment of infection/inflammation
Infection of the semen and male accessory sex glands with Chlamydia and Ureaplasma has been conclusively linked with an increase in oxidative stress. As both of these infections are treatable with antibiotics, it makes sense to screen all men with known oxidative stress for these bacterial pathogens. Two studies have now confirmed the ability of antibiotic treatment to reduce sperm oxidative stress and subsequently improve sperm quality (Omu et al., 1998; Vicari, 2000). One relatively large and well-conducted study randomized men with Chlamydia or Ureaplasma infection to either 3 months of antibiotics or no treatment (Vicari, 2000). Compared with the controls, the antibiotic treated group exhibited a significant fall in seminal leukocytes and ROS production at 3 months, an improvement in sperm motility and a significant improvement in natural conception (28.2 versus 5.4%, P = 0.009). A smaller study using only 10 days of antibiotic treatment did not produce any significant decline in seminal leukocyte count or improvement in motility (Krause et al., 2003). While this study did not measure ROS production in semen, it is likely that prolonged courses of antibiotics (3 months) are required to completely irradiate difficult-to-treat male accessory gland infections and reverse oxidative pathology.
In addition to antibiotic treatment, non-steroidal anti-inflammatory (NSAID) drugs may also reduce seminal leukocytes production of free radicals. In one study men with antibiotic treated Chlamydia or Ureaplasma infection were randomized to either a NSAID or carnitine antioxidant and monitored for improvements in sperm quality over the next 4 months (Vicari et al., 2002). Those men treated with 2 months of NSAID followed by 2 months of carnitine had the most significant reduction in seminal ROS production and improvement in sperm motility/viability. In addition, a one month course of a COX-2 anti-inflammatory has been shown to significantly reduce sperm leukocyte count, while improving sperm motility, morphology and viability (Gambera et al., 2007). It would therefore appear that a combination of antibiotics followed by a course of anti-inflammatory medication is the preferred treatment path in infection related oxidative stress.
Direct treatment of oxidative pathology
Several investigators have reported that surgical treatment of a varicocoele can reduce seminal ROS levels and improve sperm DNA integrity (Mostafa et al., 2001; Zini et al., 2005; Hurtado de Catalfo et al., 2007; Werthman et al., 2007). While the most recent meta-analysis examining the effect of varicocelectomy on spontaneous conception shows a significant benefit (Marmar et al., 2007), the Cochrane Database suggests that there is no benefit (Evers and Collins, 2004). Well-conducted randomized studies measuring oxidative end-points (sperm lipid peroxidation and oxidative DNA damage) and pregnancy rates need to be performed before routine use of varicocelectomy can be advocated in men with oxidative stress. Until these studies become available, selective ligation of grade II/III varicoceles in men with poor reproductive outcomes despite oral antioxidant therapy is probably reasonable practice.
Vitamin and antioxidant supplementation
Elevated homocysteine has been linked with oxidative stress. The B group vitamins folate, Vitamin B6 and Vitamin B12 are known to increase the enzymatic efficiency of the MTHFR and cystathionine β-synthase enzymes responsible for removing homocysteine from the circulation (Matthews, 2002). While yet to be proven to enhance sperm quality, the use of a B group vitamin supplement (5 mg folate, 100 mg Vitamin B6