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Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility

Published:March 06, 2014DOI:https://doi.org/10.1016/j.rbmo.2014.02.004

      Abstract

      DNA fragmentation is an important factor in the aetiology of male infertility. However, it is still underevaluated and its inclusion in routine semen analysis is debated. DNA fragmentation has been shown to be a robust indicator of fertility potential, more so than conventional semen parameters. Men with high DNA fragmentation levels have significantly lower odds of conceiving, naturally or through procedures such as intrauterine insemination and IVF. Couples may be counselled to proceed directly to intracytoplasmic sperm injection as it is more successful in this group, avoiding costly procedures, recurrent failures or pregnancy losses; however, this treatment is not without limitations or risks. Ideally DNA fragmentation should be minimized where possible. Oxidative stress is the major cause of DNA fragmentation in spermatozoa. Endogenous and exogenous factors that contribute to oxidative stress are discussed, and in many cases are shown to be easily modifiable. Antioxidants play a protective role, although a delicate balance of reduction and oxidation is required for essential functions, including fertilization. Reducing oxidative stress may improve a couple’s chances of conception either naturally or via assisted reproduction. Sources of oxidative stress therefore should be thoroughly examined in men with high levels of DNA fragmentation and modified where possible.
      DNA fragmentation is an important factor in the aetiology of male infertility. However it is still underevaluated and its inclusion in routine semen analysis is still debated. DNA fragmentation has been shown to be a robust indicator of fertility potential, more so than conventional semen parameters. Men with high levels of DNA fragmentation will have significantly lower odds of conceiving naturally or through procedures such as intrauterine insemination and IVF. Intracytoplasmic sperm injection (ICSI) may be much more successful in this group, and couples may be counselled to proceed directly to ICSI, avoiding costly procedures, recurrent failures or pregnancy losses. However, ICSI is not without its limitations or risks. Ideally, DNA fragmentation should be investigated and minimized where possible in men trying to conceive naturally or through assisted reproduction technology. Oxidative stress is the major cause of DNA fragmentation in spermatozoa. Endogenous and exogenous factors that contribute to oxidative stress are discussed and in many cases are easily modifiable. Antioxidants play a protective role, although a delicate balance of reduction and oxidation is required for essential sperm function, including fertilization. Reducing oxidative stress may improve a couple’s chances of conception either naturally or via assisted reproduction treatment. Sources of oxidative stress therefore should be thoroughly examined in men with high levels of DNA fragmentation and modified where possible.

      Keywords

      Introduction

      DNA fragmentation is now considered an important factor in the aetiology of male infertility (
      • Erenpreiss J.
      • Elzanaty S.
      • Giwercman A.
      Sperm DNA damage in men from infertile couples.
      ,
      • Venkatesh S.
      • Singh A.
      • Shamsi M.B.
      • Thilagavathi J.
      • Kumar R.
      • Mitra D.K.
      • Dada R.
      Clinical significance of sperm DNA damage threshold value in the assessment of male infertility.
      ,
      • Zhang Y.
      • Wang H.
      • Wang L.
      • Zhou Z.
      • Sha J.
      • Mao Y.
      • Cai L.
      • Feng T.
      • Yan Z.
      • Ma L.
      • Liu J.
      The clinical significance of sperm DNA damage detection combined with routine semen testing in assisted reproduction.
      ). However, DNA fragmentation is not routinely assessed in semen analysis according to World Health Organization (WHO) guidelines (
      WHO
      WHO Laboratory Manual for the Examination and Processing of Human Semen.
      ) and approximately 15–30% of couples are ‘diagnosed’ with unexplained infertility after a routine analysis (
      Practice Committee of the American Society for Reproductive Medicine
      Effectiveness and treatment for unexplained infertility.
      ,
      • Ray A.
      • Shah A.
      • Gudi A.
      • Homburg R.
      Unexplained infertility: an update and review of practice.
      ). DNA damage may be present in men with both abnormal and normal semen parameters (
      • Erenpreiss J.
      • Elzanaty S.
      • Giwercman A.
      Sperm DNA damage in men from infertile couples.
      ,
      • Oleszczuk K.
      • Augustinsson L.
      • Bayat N.
      • Giwercman A.
      • Bungum M.
      Prevalence of high DNA fragmentation index in male partners of unexplained infertile couples.
      ) and routine semen parameters are not robustly predictive of infertility or outcome of assisted reproduction treatment (
      • Guzick D.S.
      • Overstreet J.W.
      • Factor-Litvak P.
      • Brazil C.K.
      • Nakajima S.T.
      • Coutifaris C.
      • Carson S.A.
      • Cisneros P.
      • Steinkampf M.P.
      • Hill J.A.
      • Xu D.
      • Vogel D.L.
      Sperm morphology, motility, and concentration in fertile and infertile men.
      ,
      • Virro M.R.
      • Larson-Cook K.L.
      • Evenson D.P.
      Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles.
      ). It is still debated whether DNA fragmentation should become part of the routine analysis in fertility investigation (
      Practice Committee of the American Society for Reproductive Medicine
      The clinical utility of sperm DNA integrity testing.
      ,
      Practice Committee of the American Society for Reproductive Medicine
      The clinical utility of sperm DNA integrity testing.
      ,
      Practice Committee of the American Society for Reproductive Medicine
      The clinical uitility of sperm DNA integrtiy testing: a guideline.
      ), although many research groups are greatly in favour of promoting this test (
      • Abu D.A.
      • Franken D.R.
      • Hoffman B.
      • Henkel R.
      Sequential analysis of sperm functional aspects involved in fertilisation: a pilot study.
      ,
      • Bungum M.
      Sperm DNA integrity assessment: a new tool in diagnosis and treatment of fertility.
      ,
      • Giwercman A.
      • Lindstedt L.
      • Larsson M.
      • Bungum M.
      • Spano M.
      • Levine R.J.
      • Rylander L.
      Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case-control study.
      ,
      • Lewis S.E.
      • John Aitken R.
      • Conner S.J.
      • Iuliis G.D.
      • Evenson D.P.
      • Henkel R.
      • Giwercman A.
      • Gharagozloo P.
      The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment.
      ,
      • Omran H.M.
      • Bakhiet M.
      • Dashti M.G.
      DNA integrity is a critical molecular indicator for the assessment of male infertility.
      ).
      The level of DNA fragmentation correlates negatively with pregnancy and delivery in both natural and assisted conceptions, although not after intracytoplasmic sperm injection (ICSI), as will be discussed (
      • Evenson D.
      • Wixon R.
      Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay.
      ,
      • Zhang Y.
      • Wang H.
      • Wang L.
      • Zhou Z.
      • Sha J.
      • Mao Y.
      • Cai L.
      • Feng T.
      • Yan Z.
      • Ma L.
      • Liu J.
      The clinical significance of sperm DNA damage detection combined with routine semen testing in assisted reproduction.
      ,
      • Zini A.
      Are sperm chromatin and DNA defects relevant in the clinic?.
      ). It is also strongly associated with recurrent spontaneous abortion (
      • Gil-Villa A.M.
      • Cardona-Maya W.
      • Agarwal A.
      • Sharma R.
      • Cadavid A.
      Role of male factor in early recurrent embryo loss: do antioxidants have any effect?.
      ,
      • Gil-Villa A.M.
      • Cardona-Maya W.
      • Agarwal A.
      • Sharma R.
      • Cadavid A.
      Assessment of sperm factors possibly involved in early recurrent pregnancy loss.
      ,
      • Kennedy C.
      • Ahlering P.
      • Rodriguez H.
      • Levy S.
      • Sutovsky P.
      Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction.
      ,
      • Zini A.
      Are sperm chromatin and DNA defects relevant in the clinic?.
      ,
      • Kumar K.
      • Deka D.
      • Singh A.
      • Mitra D.K.
      • Vanitha B.R.
      • Dada R.
      Predictive value of DNA integrity analysis in idiopathic recurrent pregnancy loss following spontaneous conception.
      ). Using DNA fragmentation analysis routinely may allow couples to avoid costly assisted reproduction treatments, repeated failures or recurrent pregnancy losses by proceeding directly to ICSI. In addition, the source of DNA damage may be assessed and relevant treatment may increase the likelihood of spontaneous conception or successful pregnancy using assisted reproduction technology and/or ICSI. The most common cause of DNA fragmentation in spermatozoa is reactive oxygen species (ROS) and oxidative stress (
      • Aitken R.J.
      • De Iuliis G.N.
      On the possible origins of DNA damage in human spermatozoa.
      ). This may be an area where treatment is warranted in subfertility or ahead of proceeding to assisted reproduction treatment. This area is the focus of this review.

      Functions of ROS in spermatozoa

      ‘Reactive oxygen species’ is a collective common term that includes highly oxidative radicals such as hydroxyl radicals (OH·) and nonradical species such as the superoxide anion (O2) or hydrogen peroxide (H2O2). The term can also include reactive nitrogen species (
      • Doshi S.B.
      • Khullar K.
      • Sharma R.K.
      • Agarwal A.
      Role of reactive nitrogen species in male infertility.
      ), and both species are normal byproducts of metabolism (
      • Valko M.
      • Leibfritz D.
      • Moncol J.
      • Cronin M.T.
      • Mazur M.
      • Telser J.
      Free radicals and antioxidants in normal physiological functions and human disease.
      ). Low concentrations of ROS are required for many cellular processes while overproduction is controlled and/or ameliorated by antioxidants. In spermatozoa, ROS are required for a number of specific and essential functions, which explains why they produce ROS themselves. The principal type of ROS produced in spermatozoa is O2, which spontaneously generates H2O2 (
      • Aitken R.J.
      • Clarkson J.S.
      Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.
      ,
      • Alvarez J.G.
      • Touchstone J.C.
      • Blasco L.
      • Storey B.T.
      Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
      ). Due to their short half-life, these ROS are relatively harmless in spermatozoa under normal circumstances and antioxidant mechanisms help to maintain the key balance that is required for ROS-related functions (
      • Sharma R.K.
      • Agarwal A.
      Role of reactive oxygen species in male infertility.
      ).
      ROS, including H2O2 (
      • Rivlin J.
      • Mendel J.
      • Rubinstein S.
      • Etkovitz N.
      • Breitbart H.
      Role of hydrogen peroxide in sperm capacitation and acrosome reaction.
      ), O2 and nitrogen oxide (NO), play an important role in capacitation (
      • de Lamirande E.
      • Lamothe G.
      Reactive oxygen-induced reactive oxygen formation during human sperm capacitation.
      ,
      • Doshi S.B.
      • Khullar K.
      • Sharma R.K.
      • Agarwal A.
      Role of reactive nitrogen species in male infertility.
      ). Capacitation is a term that describes a complex series of events that occurs post ejaculation in the female genital tract to allow spermatozoa to reach the oocyte, bind to the zona pellucida and fertilize the egg (
      • de Lamirande E.
      • Leclerc P.
      • Gagnon C.
      Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization.
      ). ROS induce cyclic adenosine monophosphate (cAMP) in spermatozoa, inhibit tyrosine phosphatase and drive essential tyrosine phosphorylation (
      • Leclerc P.
      • de Lamirande E.
      • Gagnon C.
      Cyclic adenosine 3′,5′monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility.
      ,
      • Leclerc P.
      • de Lamirande E.
      • Gagnon C.
      Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives.
      ,
      • O’Flaherty C.
      • de Lamirande E.
      • Gagnon C.
      Reactive oxygen species modulate independent protein phosphorylation pathways during human sperm capacitation.
      ). The localization of tyrosine phosphorylation to the flagellum is linked with hyperactivation (
      • Leclerc P.
      • de Lamirande E.
      • Gagnon C.
      Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives.
      ,
      • Nassar A.
      • Mahony M.
      • Morshedi M.
      • Lin M.H.
      • Srisombut C.
      • Oehninger S.
      Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility.
      ). Hyperactivation describes the change in motility of spermatozoa once in the female genital tract that allows chemotaxis towards, and interaction with, the oocyte (
      • de Lamirande E.
      • Leclerc P.
      • Gagnon C.
      Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization.
      ). Tyrosine phosphorylation also correlates with binding of the spermatozoon to the zona pellucida (
      • Liu D.Y.
      • Clarke G.N.
      • Baker H.W.
      Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm-zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction.
      ,
      • Urner F.
      • Leppens-Luisier G.
      • Sakkas D.
      Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose.
      ) and is necessary for the acrosome reaction (
      • Dona G.
      • Fiore C.
      • Tibaldi E.
      • Frezzato F.
      • Andrisani A.
      • Ambrosini G.
      • Fiorentin D.
      • Armanini D.
      • Bordin L.
      • Clari G.
      Endogenous reactive oxygen species content and modulation of tyrosine phosphorylation during sperm capacitation.
      ,
      • Varano G.
      • Lombardi A.
      • Cantini G.
      • Forti G.
      • Baldi E.
      • Luconi M.
      Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa.
      ).
      In vitro experiments have demonstrated the fine balance of ROS and antioxidants that are required for capacitation and ultimately fertilization. H2O2 can stimulate capacitation and tyrosine phosphorylation in vitro, while the antioxidant enzyme catalase (CAT) prevents this (
      • Aitken R.J.
      • Paterson M.
      • Fisher H.
      • Buckingham D.W.
      • van Duin M.
      Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function.
      ). Another study showed more specifically that, while low concentrations of H2O2 promoted capacitation, elevated concentrations acted to reduce hyperactivation, zona pellucida binding and the acrosome reaction (
      • Oehninger S.
      • Blackmore P.
      • Mahony M.
      • Hodgen G.
      Effects of hydrogen peroxide on human spermatozoa.
      ). Specific physiological concentrations of NO have also been shown to drive hyperactivation, capacitation and zona pellucida binding, while excess concentrations have inhibitory effects (
      • Doshi S.B.
      • Khullar K.
      • Sharma R.K.
      • Agarwal A.
      Role of reactive nitrogen species in male infertility.
      ). ROS are also required for chemotaxis, but with prolonged exposure chemotaxis is inhibited (
      • Sanchez R.
      • Sepulveda C.
      • Risopatron J.
      • Villegas J.
      • Giojalas L.C.
      Human sperm chemotaxis depends on critical levels of reactive oxygen species.
      ). Antioxidants are generally used to inhibit capacitation reactions in vitro: addition of superoxide dismutase (SOD) can inhibit capacitation (
      • de Lamirande E.
      • Harakat A.
      • Gagnon C.
      Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion.
      ) and reverse hyperactivation (
      • de Lamirande E.
      • Gagnon C.
      Human sperm hyperactivation and capacitation as parts of an oxidative process.
      ), while addition of ascorbic acid can prevent tyrosine phosphorylation and the acrosome reaction (
      • Dona G.
      • Fiore C.
      • Tibaldi E.
      • Frezzato F.
      • Andrisani A.
      • Ambrosini G.
      • Fiorentin D.
      • Armanini D.
      • Bordin L.
      • Clari G.
      Endogenous reactive oxygen species content and modulation of tyrosine phosphorylation during sperm capacitation.
      ).
      In addition, the correct balance of ROS and antioxidants is required for chromatin compaction in maturing spermatozoa during epididymal transit. During spermiogenesis (the final stage of spermatogenesis), histones are replaced by transitional proteins and then protamines (P1 and P2), which tightly compact chromatin in toroid structures (
      • Gonzalez-Marin C.
      • Gosalvez J.
      • Roy R.
      Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells.
      ). Further compaction occurs in the epididymis during maturation when disulphide bonds are created by the oxidation of thiol groups. Sperm-specific nuclear glutathione peroxidise (nGPX4) utilizes thiol donors for the reduction of ROS (
      • Conrad M.
      • Moreno S.G.
      • Sinowatz F.
      • Ursini F.
      • Kolle S.
      • Roveri A.
      • Brielmeier M.
      • Wurst W.
      • Maiorino M.
      • Bornkamm G.W.
      The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability.
      ,
      • Pfeifer H.
      • Conrad M.
      • Roethlein D.
      • Kyriakopoulos A.
      • Brielmeier M.
      • Bornkamm G.W.
      • Behne D.
      Identification of a specific sperm nuclei selenoenzyme necessary for protamine thiol cross-linking during sperm maturation.
      ). GPX5, bound near the acrosomal membrane, may also play an oxidative role in forming disulphide bonds, whereas free or otherwise membrane-bound GPX5 has an antioxidant role, protecting spermatozoa from oxidative damage in this environment by tightly controlling the concentration of H2O2 (
      • Drevet J.R.
      The antioxidant glutathione peroxidase family and spermatozoa: a complex story.
      ). Concentrations of these GPX proteins then decrease as spermatozoa travel from the caput to the cauda region of the epididymis during maturation (
      • Seligman J.
      • Newton G.L.
      • Fahey R.C.
      • Shalgi R.
      • Kosower N.S.
      Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of gamma-glutamyl-transpeptidase in sperm maturation.
      ,
      • Weir C.P.
      • Robaire B.
      Spermatozoa have decreased antioxidant enzymatic capacity and increased reactive oxygen species production during aging in the Brown Norway rat.
      ). Incorrect compaction of chromatin by the time spermatozoa reach the cauda region is associated with abnormal morphology and infertility (
      • Conrad M.
      • Moreno S.G.
      • Sinowatz F.
      • Ursini F.
      • Kolle S.
      • Roveri A.
      • Brielmeier M.
      • Wurst W.
      • Maiorino M.
      • Bornkamm G.W.
      The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability.
      ,
      • Hammadeh M.E.
      • Zeginiadov T.
      • Rosenbaum P.
      • Georg T.
      • Schmidt W.
      • Strehler E.
      Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility.
      ,
      • Molina J.
      • Castilla J.A.
      • Castano J.L.
      • Fontes J.
      • Mendoza N.
      • Martinez L.
      Chromatin status in human ejaculated spermatozoa from infertile patients and relationship to seminal parameters.
      ).

      Oxidative stress causes DNA damage in spermatozoa

      Oxidative stress occurs when the concentration of ROS becomes too high and/or antioxidant defences become overwhelmed and is linked with many disease states (
      • Valko M.
      • Leibfritz D.
      • Moncol J.
      • Cronin M.T.
      • Mazur M.
      • Telser J.
      Free radicals and antioxidants in normal physiological functions and human disease.
      ). Increased concentrations of seminal ROS have been detected in infertile men (
      • Aktan G.
      • Dogru-Abbasoglu S.
      • Kucukgergin C.
      • Kadioglu A.
      • Ozdemirler-Erata G.
      • Kocak-Toker N.
      Mystery of idiopathic male infertility: is oxidative stress an actual risk?.
      ,
      • Chen H.
      • Zhao H.X.
      • Huang X.F.
      • Chen G.W.
      • Yang Z.X.
      • Sun W.J.
      • Tao M.H.
      • Yuan Y.
      • Wu J.Q.
      • Sun F.
      • Dai Q.
      • Shi H.J.
      Does high load of oxidants in human semen contribute to male factor infertility?.
      ,
      • Moustafa M.H.
      • Sharma R.K.
      • Thornton J.
      • Mascha E.
      • Abdel-Hafez M.A.
      • Thomas Jr., A.J.
      • Agarwal A.
      Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility.
      ). Highly oxidative ROS cause damage to cell components, particularly lipids, proteins and DNA (
      • Valko M.
      • Rhodes C.J.
      • Moncol J.
      • Izakovic M.
      • Mazur M.
      Free radicals, metals and antioxidants in oxidative stress-induced cancer.
      ). Two major effects of oxidative stress impacting fertility are lipid peroxidation and DNA damage.
      Docosahexapentanoic acid is the predominant fatty acid in immature spermatozoa (
      • Lenzi A.
      • Gandini L.
      • Maresca V.
      • Rago R.
      • Sgro P.
      • Dondero F.
      • Picardo M.
      Fatty acid composition of spermatozoa and immature germ cells.
      ) although the percentage is reduced during epididymal maturation (
      • Ollero M.
      • Powers R.D.
      • Alvarez J.G.
      Variation of docosahexaenoic acid content in subsets of human spermatozoa at different stages of maturation: implications for sperm lipoperoxidative damage.
      ,
      • Ollero M.
      • Gil-Guzman E.
      • Lopez M.C.
      • Sharma R.K.
      • Agarwal A.
      • Larson K.
      • Evenson D.
      • Thomas Jr., A.J.
      • Alvarez J.G.
      Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.
      ). Due to the high proportion of this polyunsaturated acid, maturing sperm membranes are particularly vulnerable to lipid peroxidation. Lipid peroxidation occurs when the double bonds of an unsaturated fatty acid (docosahexapentanoic acid has six) are attacked by a free radical to create a lipid peroxide radical. This reaction is self-propagating by reacting with neighbouring fatty acid molecules, causing marked damage to lipid membranes and affecting membrane fluidity (
      • Mylonas C.
      • Kouretas D.
      Lipid peroxidation and tissue damage.
      ). Lipid peroxidation is commonly measured by quantification of the peroxidation product malondialdehyde. As shown in numerous studies, increased concentrations of malondialdehyde correlate with maturation arrest and decreased spermatozoa concentration and morphology and, most notably, motility, due to alterations in the membrane (
      • Atig F.
      • Raffa M.
      • Ali H.B.
      • Abdelhamid K.
      • Saad A.
      • Ajina M.
      Altered antioxidant status and increased lipid per-oxidation in seminal plasma of tunisian infertile men.
      ,
      • Benedetti S.
      • Tagliamonte M.C.
      • Catalani S.
      • Primiterra M.
      • Canestrari F.
      • De Stefani S.
      • Palini S.
      • Bulletti C.
      Differences in blood and semen oxidative status in fertile and infertile men, and their relationship with sperm quality.
      ,
      • Hsieh Y.Y.
      • Chang C.C.
      • Lin C.S.
      Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility.
      ,
      • Kao S.H.
      • Chao H.T.
      • Chen H.W.
      • Hwang T.I.
      • Liao T.L.
      • Wei Y.H.
      Increase of oxidative stress in human sperm with lower motility.
      ,
      • Koksal I.T.
      • Usta M.
      • Orhan I.
      • Abbasoglu S.
      • Kadioglu A.
      Potential role of reactive oxygen species on testicular pathology associated with infertility.
      ). Measurements of malondialdehyde also positively correlate with DNA damage in spermatozoa, indicating that oxidative stress may link these two (
      • Aktan G.
      • Dogru-Abbasoglu S.
      • Kucukgergin C.
      • Kadioglu A.
      • Ozdemirler-Erata G.
      • Kocak-Toker N.
      Mystery of idiopathic male infertility: is oxidative stress an actual risk?.
      ,
      • Atig F.
      • Kerkeni A.
      • Saad A.
      • Ajina M.
      Effects of reduced seminal enzymatic antioxidants on sperm DNA fragmentation and semen quality of Tunisian infertile men.
      ,
      • Shamsi M.B.
      • Venkatesh S.
      • Tanwar M.
      • Talwar P.
      • Sharma R.K.
      • Dhawan A.
      • Kumar R.
      • Gupta N.P.
      • Malhotra N.
      • Singh N.
      • Mittal S.
      • Dada R.
      DNA integrity and semen quality in men with low seminal antioxidant levels.
      ). Products of lipid peroxidation are both mutagenic and genotoxic to DNA (
      • Luczaj W.
      • Skrzydlewska E.
      DNA damage caused by lipid peroxidation products.
      ).
      Oxidative stress is considered to be the major cause of DNA damage in spermatozoa (
      • Aitken R.J.
      • De Iuliis G.N.
      On the possible origins of DNA damage in human spermatozoa.
      ). Decreased levels of individual and total antioxidant capacity and high concentrations of seminal ROS have been detected in men with elevated DNA damage in numerous studies (
      • Aktan G.
      • Dogru-Abbasoglu S.
      • Kucukgergin C.
      • Kadioglu A.
      • Ozdemirler-Erata G.
      • Kocak-Toker N.
      Mystery of idiopathic male infertility: is oxidative stress an actual risk?.
      ,
      • Atig F.
      • Raffa M.
      • Ali H.B.
      • Abdelhamid K.
      • Saad A.
      • Ajina M.
      Altered antioxidant status and increased lipid per-oxidation in seminal plasma of tunisian infertile men.
      ,
      • Khosravi F.
      • Valojerdi M.R.
      • Amanlou M.
      • Karimian L.
      • Abolhassani F.
      Relationship of seminal reactive nitrogen and oxygen species and total antioxidant capacity with sperm DNA fragmentation in infertile couples with normal and abnormal sperm parameters.
      ,
      • Mahfouz R.
      • Sharma R.
      • Thiyagarajan A.
      • Kale V.
      • Gupta S.
      • Sabanegh E.
      • Agarwal A.
      Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species.
      ,
      • Shamsi M.B.
      • Kumar R.
      • Malhotra N.
      • Singh N.
      • Mittal S.
      • Upadhyay A.D.
      • Dada R.
      Chromosomal aberrations, Yq microdeletion, and sperm DNA fragmentation in infertile men opting for assisted reproduction.
      ). 8-Hydroxy-2-deoxyguanosine (8-OHdG), an oxidized guanine base adduct formed when DNA is damaged by the OH· radical, can be used to measure DNA oxidative damage. Increased 8-OHdG concentration correlates markedly and significantly (r = 0.756, P < 0.001) with DNA fragmentation and strand breaks (
      • Aitken R.J.
      • De Iuliis G.N.
      • Finnie J.M.
      • Hedges A.
      • McLachlan R.I.
      Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria.
      ,
      • De Iuliis G.N.
      • Thomson L.K.
      • Mitchell L.A.
      • Finnie J.M.
      • Koppers A.J.
      • Hedges A.
      • Nixon B.
      • Aitken R.J.
      DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress.
      ).
      ROS can damage DNA directly by generation of oxidized DNA adducts (e.g. 8-OHdG, 1,N6-ethenoadenosine, 1,N6-ethenoguanosine), leading to abasic sites that destabilize the DNA structure and cause subsequent single-strand breaks (
      • Badouard C.
      • Menezo Y.
      • Panteix G.
      • Ravanat J.L.
      • Douki T.
      • Cadet J.
      • Favier A.
      Determination of new types of DNA lesions in human sperm.
      ). NO can induce abasic sites also through the processes of nitration or deamination (
      • Doshi S.B.
      • Khullar K.
      • Sharma R.K.
      • Agarwal A.
      Role of reactive nitrogen species in male infertility.
      ). It was suggested that activation of caspases by ROS also caused indirect DNA damage via endonuclease activation (
      • Sakkas D.
      • Alvarez J.G.
      Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis.
      ). However, it has also been proposed that endonucleases are physically separate from activated caspases in spermatozoa (
      • Aitken R.J.
      • Koppers A.J.
      Apoptosis and DNA damage in human spermatozoa.
      ). ROS stimulation of caspases may induce further free radical release from mitochondria during the initiation of apoptosis, further propagating damage (
      • Aitken R.J.
      • Koppers A.J.
      Apoptosis and DNA damage in human spermatozoa.
      ,
      • Sakkas D.
      • Alvarez J.G.
      Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis.
      ,
      • Wang X.
      • Sharma R.K.
      • Sikka S.C.
      • Thomas Jr., A.J.
      • Falcone T.
      • Agarwal A.
      Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility.
      ).
      DNA repair is limited in spermatozoa and only occurs during specific stages of spermiogenesis. Repair mechanisms are necessary then to restore induced nicks and strand breaks that allow chromatin remodelling at this stage in haploid spermatozoa. Repair mechanisms are no longer activated during nuclear condensation in the epididymis (
      • Leduc F.
      • Nkoma G.B.
      • Boissonneault G.
      Spermiogenesis and DNA repair: a possible etiology of human infertility and genetic disorders.
      ,
      • Marcon L.
      • Boissonneault G.
      Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling.
      ). Spermatozoa, however, are exposed to oxidative damage in the epididymis and during transport in seminal fluid. The next opportunity for DNA repair is by the oocyte, which is a critical step in embryo development. However, whereas DNA adducts induced by oxidative stress may be repairable by the oocyte, single- or double-stranded DNA breaks may not and they can have significant impact on fertilization and pregnancy outcome (
      • Gonzalez-Marin C.
      • Gosalvez J.
      • Roy R.
      Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells.
      ,
      • Menezo Jr., Y.
      • Russo G.
      • Tosti E.
      • El Mouatassim S.
      • Benkhalifa M.
      Expression profile of genes coding for DNA repair in human oocytes using pangenomic microarrays, with a special focus on ROS linked decays.
      ).

      Measuring DNA damage

      Several methods are employed by routine diagnostic laboratories in the investigation of DNA fragmentation, most common among which are the sperm chromatin structure assay (SCSA), the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) and Comet assays and the sperm chromatin dispersion test (SCD) (
      • Lewis S.E.
      • John Aitken R.
      • Conner S.J.
      • Iuliis G.D.
      • Evenson D.P.
      • Henkel R.
      • Giwercman A.
      • Gharagozloo P.
      The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment.
      ) (Table 1).
      Table 1Methods for detection of DNA damage in spermatozoa.
      MethodBasisAdvantagesDisadvantages
      SCSAAcridine orange staining and analysis using flow cytometryStatistically robust, accepted thresholds, predictor of infertility, low variability, can detect chromatin decondensationFlow cytometer is expensive, manual stain has high variability and may produce erroneous results
      TUNELdUTP labelling of strand breaksReproducible when using flow cytometry, working thresholds, detects both single- and double-strand breaksFlow cytometer is expensive, manual stain has high variability
      COMETElectrophoretic techniqueHighly sensitiveSeparate conditions required for detection of double-and single-strand breaks, difficult to optimize and standardize
      SCDStaining of DNA ‘loop’ in agaroseSimple and inexpensive, allows for analysis of sperm populationsLow numbers of spermatozoa analysed, some technician variability
      SCD = sperm chromatin dispersion test; SCSA = sperm chromatin structure assay; TUNEL = TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling.
      According to the literature, the SCSA, giving a DNA fragmentation index (DFI) value, is perceived as the most statistically robust and reproducible test and is a valuable predictor of fertility (
      • Bungum M.
      Sperm DNA integrity assessment: a new tool in diagnosis and treatment of fertility.
      ,
      • Giwercman A.
      • Lindstedt L.
      • Larsson M.
      • Bungum M.
      • Spano M.
      • Levine R.J.
      • Rylander L.
      Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case-control study.
      ,
      • Oleszczuk K.
      • Giwercman A.
      • Bungum M.
      Intra-individual variation of the sperm chromatin structure assay DNA fragmentation index in men from infertile couples.
      ,
      • Smit M.
      • Dohle G.R.
      • Hop W.C.
      • Wildhagen M.F.
      • Weber R.F.
      • Romijn J.C.
      Clinical correlates of the biological variation of sperm DNA fragmentation in infertile men attending an andrology outpatient clinic.
      ,
      • Venkatesh S.
      • Singh A.
      • Shamsi M.B.
      • Thilagavathi J.
      • Kumar R.
      • Mitra D.K.
      • Dada R.
      Clinical significance of sperm DNA damage threshold value in the assessment of male infertility.
      ). A valuable threshold for DFI was set by
      • Evenson D.P.
      • Jost L.K.
      • Marshall D.
      • Zinaman M.J.
      • Clegg E.
      • Purvis K.
      • de Angelis P.
      • Claussen O.P.
      Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic.
      at 30%, which is robustly indicative of fertility potential. This method also yields information about chromatin decondensation via measurement of high-density staining (i.e. staining of DNA over an intensity threshold indicates excessive access of the stain to the DNA via chromatin decondensation). The SCSA is based on acridine orange staining of DNA, whereby intact DNA fluoresces green and fragmented DNA fluoresces red (
      • Chohan K.R.
      • Griffin J.T.
      • Lafromboise M.
      • De Jonge C.J.
      • Carrell D.T.
      Comparison of chromatin assays for DNA fragmentation evaluation in human sperm.
      ). The SCSA employs a flow cytometer, on which the samples are analysed immediately post incubation with acridine orange and up to 10,000 cells are counted. While the disadvantage here is the expense of a flow cytometer, there is little intratechnician variability (
      • Shamsi M.B.
      • Imam S.N.
      • Dada R.
      Sperm DNA integrity assays: diagnostic and prognostic challenges and implications in management of infertility.
      ). On the other hand, performing an acridine orange stain manually and counting using a fluorescent microscope is subject to high variation and prolonged incubation time can cause erroneous results.
      The TUNEL assay is a commonly used technique that incorporates biotinylated dUTP at 3′ ends of DNA strand breaks using template-independent TdT (
      • Shamsi M.B.
      • Kumar R.
      • Dada R.
      Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction.
      ). TUNEL can detect both single- and double-strand breaks and the labelled bases can be quantified using a bright field microscope, fluorescent microscope or flow cytometer. Again the flow cytometer is an expense but one that reduces intratechnician variability and issues with background staining. Intra-assay variability using the flow cytometer was shown to be <7% but the correlation with SCSA only gave a Pearson coefficient of 0.63 (
      • Erenpreiss J.
      • Jepson K.
      • Giwercman A.
      • Tsarev I.
      • Erenpreisa J.
      • Spano M.
      Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays.
      ). Reliable thresholds for TUNEL require further confirmation, although a working threshold of 15–20% is considered high, correlating with reduced fertility potential (
      • Benchaib M.
      • Braun V.
      • Lornage J.
      • Hadj S.
      • Salle B.
      • Lejeune H.
      • Guerin J.F.
      Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique.
      ,
      • Benchaib M.
      • Lornage J.
      • Mazoyer C.
      • Lejeune H.
      • Salle B.
      • Francois Guerin J.
      Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome.
      ,
      • Greco E.
      • Scarselli F.
      • Iacobelli M.
      • Rienzi L.
      • Ubaldi F.
      • Ferrero S.
      • Franco G.
      • Anniballo N.
      • Mendoza C.
      • Tesarik J.
      Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa.
      ,
      • Sharma R.K.
      • Sabanegh E.
      • Mahfouz R.
      • Gupta S.
      • Thiyagarajan A.
      • Agarwal A.
      TUNEL as a test for sperm DNA damage in the evaluation of male infertility.
      ).
      The Comet assay is an electrophoretic technique based on the principle that smaller fragmented DNA will migrate faster towards the anode than intact DNA (
      • Shamsi M.B.
      • Kumar R.
      • Dada R.
      Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction.
      ). The Comet assay is an extremely sensitive technique that can detect both single- and double-stranded DNA breaks but under different conditions, alkali versus neutral respectively, which is a disadvantage. Double-strand breaks may be considered more vital to pregnancy outcome and thus the neutral Comet assay is most often used in relation to infertility. The alkali Comet assay can overestimate DNA damage due to alkali-labile sites, which decrease the sensitivity of this assay. However, there is vast interlaboratory variation and many modifications that need to be optimized to standardize this technique (
      • Forchhammer L.
      • Ersson C.
      • Loft S.
      • Moller L.
      • Godschalk R.W.
      • van Schooten F.J.
      • Jones G.D.
      • Higgins J.A.
      • Cooke M.
      • Mistry V.
      • Karbaschi M.
      • Collins A.R.
      • Azqueta A.
      • Phillips D.H.
      • Sozeri O.
      • Routledge M.N.
      • Nelson-Smith K.
      • Riso P.
      • Porrini M.
      • Matullo G.
      • Allione A.
      • Stepnik M.
      • Komorowska M.
      • Teixeira J.P.
      • Costa S.
      • Corcuera L.A.
      • Lopez de Cerain A.
      • Laffon B.
      • Valdiglesias V.
      • Moller P.
      Inter-laboratory variation in DNA damage using a standard comet assay protocol.
      ).
      The SCD test utilizes the ability of intact DNA to loop around the nucleus once embedded in agarose, giving a characteristic ‘halo’ appearance (
      • Shamsi M.B.
      • Imam S.N.
      • Dada R.
      Sperm DNA integrity assays: diagnostic and prognostic challenges and implications in management of infertility.
      ). The presence of single-strand breaks in the DNA prevents this halo from forming. Agarose gels are stained with eosin and azure B and the halos are analysed by eye. This is a simple and inexpensive technique but intra-individual variation in halo assessment and background staining are drawbacks. In one large study of men undergoing IVF/ICSI, the SCD was valuable in predicting fertilization rates with a threshold of 18%, but not of clinical pregnancy (
      • Velez de la Calle J.F.
      • Muller A.
      • Walschaerts M.
      • Clavere J.L.
      • Jimenez C.
      • Wittemer C.
      • Thonneau P.
      Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study.
      ). The SCD test was, however, not valuable in predicting outcome of intrauterine insemination (IUI;
      • Muriel L.
      • Meseguer M.
      • Fernandez J.L.
      • Alvarez J.
      • Remohi J.
      • Pellicer A.
      • Garrido N.
      Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study.
      ). While the numbers of spermatozoa analysed are much lower, this technique does allow for quantification of subpopulations such as highly DNA-degraded spermatozoa, the results of which may be valuable (
      • Abad C.
      • Amengual M.J.
      • Gosalvez J.
      • Coward K.
      • Hannaoui N.
      • Benet J.
      • Garcia-Peiro A.
      • Prats J.
      Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.
      ,
      • Garcia-Peiro A.
      • Oliver-Bonet M.
      • Navarro J.
      • Abad C.
      • Amengual M.J.
      • Lopez-Fernandez C.
      • Gosalvez J.
      • Benet J.
      Differential clustering of sperm subpopulations in infertile males with clinical varicocele and carriers of rearranged genomes.
      ).

      Impact on fertility

      High levels of DNA fragmentation have been shown to be a robust indicator of male infertility (
      • Evenson D.P.
      • Jost L.K.
      • Marshall D.
      • Zinaman M.J.
      • Clegg E.
      • Purvis K.
      • de Angelis P.
      • Claussen O.P.
      Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic.
      ,
      • Giwercman A.
      • Lindstedt L.
      • Larsson M.
      • Bungum M.
      • Spano M.
      • Levine R.J.
      • Rylander L.
      Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case-control study.
      ,
      • Nicopoullos J.D.
      • Gilling-Smith C.
      • Almeida P.A.
      • Homa S.
      • Norman-Taylor J.Q.
      • Ramsay J.W.
      Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables.
      ). As significant DNA fragmentation can be present in normozoospermic subfertile men, it may be an important factor in unexplained infertility (
      • Erenpreiss J.
      • Elzanaty S.
      • Giwercman A.
      Sperm DNA damage in men from infertile couples.
      ,
      • Oleszczuk K.
      • Augustinsson L.
      • Bayat N.
      • Giwercman A.
      • Bungum M.
      Prevalence of high DNA fragmentation index in male partners of unexplained infertile couples.
      ). However, it also coexists with reduced count, increased abnormal forms (
      • Chi H.J.
      • Chung D.Y.
      • Choi S.Y.
      • Kim J.H.
      • Kim G.Y.
      • Lee J.S.
      • Lee H.S.
      • Kim M.H.
      • Roh S.I.
      Integrity of human sperm DNA assessed by the neutral comet assay and its relationship to semen parameters and clinical outcomes for the IVF-ET program.
      ,
      • Mehdi M.
      • Khantouche L.
      • Ajina M.
      • Saad A.
      Detection of DNA fragmentation in human spermatozoa: correlation with semen parameters.
      ,
      • Moskovtsev S.I.
      • Willis J.
      • White J.
      • Mullen J.B.
      Sperm DNA damage: correlation to severity of semen abnormalities.
      ,
      • Virro M.R.
      • Larson-Cook K.L.
      • Evenson D.P.
      Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles.
      ) and notably with reduced motility (
      • Appasamy M.
      • Muttukrishna S.
      • Pizzey A.R.
      • Ozturk O.
      • Groome N.P.
      • Serhal P.
      • Jauniaux E.
      Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility.
      ,
      • Erenpreiss J.
      • Elzanaty S.
      • Giwercman A.
      Sperm DNA damage in men from infertile couples.
      ,
      • Giwercman A.
      • Richthoff J.
      • Hjollund H.
      • Bonde J.P.
      • Jepson K.
      • Frohm B.
      • Spano M.
      Correlation between sperm motility and sperm chromatin structure assay parameters.
      ,
      • Lin M.H.
      • Kuo-Kuang Lee R.
      • Li S.H.
      • Lu C.H.
      • Sun F.J.
      • Hwu Y.M.
      Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates.
      ). High DNA fragmentation has also been implicated in the aetiology of age-related infertility in the male (
      • Das M.
      • Al-Hathal N.
      • San-Gabriel M.
      • Phillips S.
      • Kadoch I.J.
      • Bissonnette F.
      • Holzer H.
      • Zini A.
      High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age.
      ,
      • Smit M.
      • Romijn J.C.
      • Wildhagen M.F.
      • Weber R.F.
      • Dohle G.R.
      Sperm chromatin structure is associated with the quality of spermatogenesis in infertile patients.
      ,
      • Vagnini L.
      • Baruffi R.L.
      • Mauri A.L.
      • Petersen C.G.
      • Massaro F.C.
      • Pontes A.
      • Oliveira J.B.
      • Franco Jr., J.G.
      The effects of male age on sperm DNA damage in an infertile population.
      ).
      In the comprehensive study by
      • Evenson D.P.
      • Jost L.K.
      • Marshall D.
      • Zinaman M.J.
      • Clegg E.
      • Purvis K.
      • de Angelis P.
      • Claussen O.P.
      Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic.
      , the threshold of DFI >30% indicated a marked and significant reduction in fertility potential (natural conception) including time to pregnancy. In a more recent analysis,
      • Evenson D.P.
      • Wixon R.
      Data analysis of two in vivo fertility studies using Sperm Chromatin Structure Assay-derived DNA fragmentation index vs. pregnancy outcome.
      confirmed that the likelihood of a natural pregnancy with DFI >30% is very low. Success in IUI has also been shown in numerous studies to be very limited when DFI >30%. In a meta-analysis,
      • Evenson D.
      • Wixon R.
      Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay.
      reported that couples were 7.3-times more likely to achieve pregnancy/delivery after IUI with DFI <30%. A large analysis of 998 cycles (387 IUI) from 637 couples demonstrated very limited success (1.5%) of IUI with DFI >30% (
      • Bungum M.
      • Humaidan P.
      • Axmon A.
      • Spano M.
      • Bungum L.
      • Erenpreiss J.
      • Giwercman A.
      Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.
      ).
      For IVF, high DFI (>30%) has been associated with a reduced number of quality embryos in some studies (
      • Jiang H.
      • He R.B.
      • Wang C.L.
      • Zhu J.
      The relationship of sperm DNA fragmentation index with the outcomes of in-vitro fertilisation-embryo transfer and intracytoplasmic sperm injection.
      ,
      • Niu Z.H.
      • Shi H.J.
      • Zhang H.Q.
      • Zhang A.J.
      • Sun Y.J.
      • Feng Y.
      Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF.
      ,
      • Zhang Y.
      • Wang H.
      • Wang L.
      • Zhou Z.
      • Sha J.
      • Mao Y.
      • Cai L.
      • Feng T.
      • Yan Z.
      • Ma L.
      • Liu J.
      The clinical significance of sperm DNA damage detection combined with routine semen testing in assisted reproduction.
      ) although this may not have a significant effect on resulting pregnancy rates (
      • Bungum M.
      • Humaidan P.
      • Axmon A.
      • Spano M.
      • Bungum L.
      • Erenpreiss J.
      • Giwercman A.
      Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.
      ,
      • Niu Z.H.
      • Shi H.J.
      • Zhang H.Q.
      • Zhang A.J.
      • Sun Y.J.
      • Feng Y.
      Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF.
      ). However, it has been repeatedly shown that ICSI outcome is independent of DFI and that clinical pregnancy rates are actually higher than with IVF (
      • Bungum M.
      • Humaidan P.
      • Spano M.
      • Jepson K.
      • Bungum L.
      • Giwercman A.
      The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI.
      ,
      • Bungum M.
      • Humaidan P.
      • Axmon A.
      • Spano M.
      • Bungum L.
      • Erenpreiss J.
      • Giwercman A.
      Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.
      ,
      • Chi H.J.
      • Chung D.Y.
      • Choi S.Y.
      • Kim J.H.
      • Kim G.Y.
      • Lee J.S.
      • Lee H.S.
      • Kim M.H.
      • Roh S.I.
      Integrity of human sperm DNA assessed by the neutral comet assay and its relationship to semen parameters and clinical outcomes for the IVF-ET program.
      ,
      • Evenson D.
      • Wixon R.
      Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay.
      ). Selecting for normal morphology and movement in ICSI can decrease the likelihood of fragmented DNA in fertilizing spermatozoa (
      • Maettner R.
      • Sterzik K.
      • Isachenko V.
      • Strehler E.
      • Rahimi G.
      • Alabart J.L.
      • Sanchez R.
      • Mallmann P.
      • Isachenko E.
      Quality of human spermatozoa: relationship between high-magnification sperm morphology and DNA integrity.
      ,
      • Sivanarayana T.
      • Krishna Ch.R.
      • Prakash G.J.
      • Krishna K.M.
      • Madan K.
      • Rani B.S.
      • Sudhakar G.
      • Raju G.A.
      CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation.
      ). Using this selection process, a recent study has shown that success rates with ICSI with semen showing very high DFI (>50%) are similar to those with low DFI (<15%;
      • Dar S.
      • Grover S.A.
      • Moskovtsev S.I.
      • Swanson S.
      • Baratz A.
      • Librach C.L.
      In vitro fertilization-intracytoplasmic sperm injection outcome in patients with a markedly high DNA fragmentation index (>50%).
      ). It is therefore advised that couples contemplating assisted reproduction treatment with DFI >30% go directly to ICSI to avoid costly IUI and IVF failures.
      However, ICSI with high DFI is not without its limitations. There is a significantly increased risk of miscarriage, implantation failure or failure to progress to delivery within this group (
      • Benchaib M.
      • Lornage J.
      • Mazoyer C.
      • Lejeune H.
      • Salle B.
      • Francois Guerin J.
      Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome.
      ,
      • Bungum M.
      • Humaidan P.
      • Axmon A.
      • Spano M.
      • Bungum L.
      • Erenpreiss J.
      • Giwercman A.
      Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.
      ,
      • Kennedy C.
      • Ahlering P.
      • Rodriguez H.
      • Levy S.
      • Sutovsky P.
      Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction.
      ). Even with spontaneous conception, DNA damage is positively correlated with recurrent pregnancy loss (
      • Brahem S.
      • Mehdi M.
      • Landolsi H.
      • Mougou S.
      • Elghezal H.
      • Saad A.
      Semen parameters and sperm DNA fragmentation as causes of recurrent pregnancy loss.
      ,
      • Carrell D.T.
      • Liu L.
      • Peterson C.M.
      • Jones K.P.
      • Hatasaka H.H.
      • Erickson L.
      • Campbell B.
      Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss.
      ,
      • Gil-Villa A.M.
      • Cardona-Maya W.
      • Agarwal A.
      • Sharma R.
      • Cadavid A.
      Role of male factor in early recurrent embryo loss: do antioxidants have any effect?.
      ,
      • Kumar K.
      • Deka D.
      • Singh A.
      • Mitra D.K.
      • Vanitha B.R.
      • Dada R.
      Predictive value of DNA integrity analysis in idiopathic recurrent pregnancy loss following spontaneous conception.
      ). Using ICSI bypasses many of the natural hurdles that spermatozoa undergo in order to fertilize an oocyte in vivo. The oocyte has limited capacity to repair such high levels of DNA fragmentation after fertilization during early embryo development and thus pregnancy is less likely to progress. Couples should be counselled as to the increased risk for biochemical pregnancy or early pregnancy loss under these circumstances. An additional concern is the future health of offspring. Paternal gametes are the major source of de-novo structural chromosome reorganizations (
      • Thomas N.S.
      • Morris J.K.
      • Baptista J.
      • Ng B.L.
      • Crolla J.A.
      • Jacobs P.A.
      De novo apparently balanced translocations in man are predominantly paternal in origin and associated with a significant increase in paternal age.
      ). Chromosomal abnormalities are higher in men selected for ICSI and can be passed down to offspring using this technique (
      • Bonduelle M.
      • Van Assche E.
      • Joris H.
      • Keymolen K.
      • Devroey P.
      • Van Steirteghem A.
      • Liebaers I.
      Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters.
      ,
      • Mau U.A.
      • Backert I.T.
      • Kaiser P.
      • Kiesel L.
      Chromosomal findings in 150 couples referred for genetic counselling prior to intracytoplasmic sperm injection.
      ,
      • Meschede D.
      • Lemcke B.
      • Exeler J.R.
      • De Geyter C.
      • Behre H.M.
      • Nieschlag E.
      • Horst J.
      Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection – prevalence, types, sex distribution and reproductive relevance.
      ). Balanced structural reorganizations that allow successful pregnancy may result in phenotypically normal children but may cause infertility in these children if the reorganization is present in the gamete of the child (
      • Egozcue S.
      • Blanco J.
      • Vendrell J.M.
      • Garcia F.
      • Veiga A.
      • Aran B.
      • Barri P.N.
      • Vidal F.
      • Egozcue J.
      Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion.
      ). An often-cited example of the relationship between DNA damage and the health of the offspring can be found with paternal smoking. Paternal smoking can increase DNA fragmentation by 50% (
      • Fraga C.G.
      • Motchnik P.A.
      • Wyrobek A.J.
      • Rempel D.M.
      • Ames B.N.
      Smoking and low antioxidant levels increase oxidative damage to sperm DNA.
      ) and is linked with increased incidence of childhood cancers (
      • Ji B.T.
      • Shu X.O.
      • Linet M.S.
      • Zheng W.
      • Wacholder S.
      • Gao Y.T.
      • Ying D.M.
      • Jin F.
      Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers.
      ,
      • Shu X.O.
      • Ross J.A.
      • Pendergrass T.W.
      • Reaman G.H.
      • Lampkin B.
      • Robison L.L.
      Parental alcohol consumption, cigarette smoking, and risk of infant leukemia: a Childrens Cancer Group study.
      ). Thus men undergoing assisted reproduction treatment should aim to reduce DNA fragmentation regardless of the potential success rates offered by ICSI.

      ROS generation in spermatozoa

      Like somatic cells, spermatozoa produce small amounts of ROS as a byproduct of the electron transfer chain in mitochondria (
      • Aitken R.J.
      • Clarkson J.S.
      Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.
      ,
      • Koppers A.J.
      • De Iuliis G.N.
      • Finnie J.M.
      • McLaughlin E.A.
      • Aitken R.J.
      Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa.
      ). The major ROS produced are O2 and H2O2, and antioxidants such as GPX, CAT and SOD, that neutralize these, are present both within mitochondria and in the secretions of the reproductive tract (
      • Starkov A.A.
      The role of mitochondria in reactive oxygen species metabolism and signaling.
      ,
      • Vernet P.
      • Aitken R.J.
      • Drevet J.R.
      Antioxidant strategies in the epididymis.
      ). Where there is excess ROS production, or in the absence of adequate antioxidants, concentrations of ROS may increase. H2O2 can also form the highly reactive and damaging OH· radical in the presence of copper or iron (Haber–Weiss and Fenton reactions;
      • Kehrer J.P.
      The Haber-Weiss reaction and mechanisms of toxicity.
      ). Elevated concentrations of both copper and iron have been detected in seminal plasma of subfertile men (
      • Aydemir B.
      • Kiziler A.R.
      • Onaran I.
      • Alici B.
      • Ozkara H.
      • Akyolcu M.C.
      Impact of Cu and Fe concentrations on oxidative damage in male infertility.
      ). The OH· radical is particularly damaging to DNA and lipids (
      • Kehrer J.P.
      The Haber-Weiss reaction and mechanisms of toxicity.
      ,
      • Kwenang A.
      • Kroos M.J.
      • Koster J.F.
      • van Eijk H.G.
      Iron, ferritin and copper in seminal plasma.
      ,
      • Lloyd D.R.
      • Phillips D.H.
      Oxidative DNA damage mediated by copper(II), iron(II) and nickel(II) fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks, 8-hydroxydeoxyguanosine and putative intrastrand cross-links.
      ). Mitochondrial DNA is also damaged by ROS which can limit ATP production and energy provision for motility in spermatozoa, reducing fertility (
      • Shamsi M.B.
      • Kumar R.
      • Bhatt A.
      • Bamezai R.N.
      • Kumar R.
      • Gupta N.P.
      • Das T.K.
      • Dada R.
      Mitochondrial DNA Mutations in etiopathogenesis of male infertility.
      ). Mitochondrial DNA mutations do not, however, impact the health of the offspring, as male mitochondrial DNA is degraded by the oocyte, leaving only maternal inheritance (
      • Sutovsky P.
      • Van Leyen K.
      • McCauley T.
      • Day B.N.
      • Sutovsky M.
      Degradation of paternal mitochondria after fertilization: implications for heteroplasmy, assisted reproductive technologies and mtDNA inheritance.
      ).
      Cytoplasmic generation of ROS in spermatic cells has also been suggested, although the exact mechanism is yet to be elucidated. During normal spermiogenesis, the majority of cytoplasm is extruded from the maturing spermatozoa by the action of Sertoli cells (
      • Rengan A.K.
      • Agarwal A.
      • van der Linde M.
      • du Plessis S.S.
      An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet.
      ). The remaining cytoplasmic droplet in the midpiece contains enzymes required for energy production such as creatinine kinase and glucose-6-phosphate dehydrogenase and it is here that the essential ROS production for capacitation occurs (
      • Rengan A.K.
      • Agarwal A.
      • van der Linde M.
      • du Plessis S.S.
      An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet.
      ). Glucose-6-phosphate dehydrogenase is required for the reduction of NADP + to NADPH, and it is proposed that this fuels ROS production by NADPH oxidase (
      • Aitken R.J.
      • Fisher H.M.
      • Fulton N.
      • Gomez E.
      • Knox W.
      • Lewis B.
      • Irvine S.
      Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine.
      ,
      • Dona G.
      • Fiore C.
      • Andrisani A.
      • Ambrosini G.
      • Brunati A.
      • Ragazzi E.
      • Armanini D.
      • Bordin L.
      • Clari G.
      Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system.
      ). It is thought that excess residual cytoplasm remaining from defective spermiogenesis increases the amount of glucose-6-phosphate dehydrogenase, leading to increased ROS production (
      • Gomez E.
      • Buckingham D.W.
      • Brindle J.
      • Lanzafame F.
      • Irvine D.S.
      • Aitken R.J.
      Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function.
      ). For this reason, immature spermatozoa, or ‘incorrectly matured’ spermatozoa, represent a major source of ROS in oxidative stress and may cause DNA damage in mature spermatozoa during cotransit in the epididymis (
      • Gil-Guzman E.
      • Ollero M.
      • Lopez M.C.
      • Sharma R.K.
      • Alvarez J.G.
      • Thomas Jr., A.J.
      • Agarwal A.
      Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation.
      ,
      • Gomez E.
      • Buckingham D.W.
      • Brindle J.
      • Lanzafame F.
      • Irvine D.S.
      • Aitken R.J.
      Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function.
      ,
      • Ollero M.
      • Gil-Guzman E.
      • Lopez M.C.
      • Sharma R.K.
      • Agarwal A.
      • Larson K.
      • Evenson D.
      • Thomas Jr., A.J.
      • Alvarez J.G.
      Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.
      ). In a study where spermatozoal subsets were isolated from the ejaculate, the number of immature spermatozoa directly correlated with levels of DNA damage in mature spermatozoa (
      • Ollero M.
      • Gil-Guzman E.
      • Lopez M.C.
      • Sharma R.K.
      • Agarwal A.
      • Larson K.
      • Evenson D.
      • Thomas Jr., A.J.
      • Alvarez J.G.
      Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.
      ).

      Other sources of ROS and modifiable factors

      Leukocytes

      In addition to immature/abnormal spermatozoa, leukocytes are the major source of ROS and oxidative stress (
      • Aitken R.J.
      • West K.M.
      Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients.
      ,
      • Whittington K.
      • Ford W.C.
      Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions.
      ). Low numbers of leukocytes are present in the majority of normal ejaculates, and the WHO has defined leukocytospermia as >1 × 106 spermatozoa/ml (
      WHO
      WHO Laboratory Manual for the Examination and Processing of Human Semen.
      ). However, the significance of this threshold is still debated. While studies have demonstrated a negative correlation of leukocyte concentration with sperm parameters (
      • Aziz N.
      • Agarwal A.
      • Lewis-Jones I.
      • Sharma R.K.
      • Thomas Jr., A.J.
      Novel associations between specific sperm morphological defects and leukocytospermia.
      ,
      • Moskovtsev S.I.
      • Willis J.
      • White J.
      • Mullen J.B.
      Leukocytospermia: relationship to sperm deoxyribonucleic acid integrity in patients evaluated for male factor infertility.
      ,
      • Omu A.E.
      • Al-Qattan F.
      • Al-Abdul-Hadi F.M.
      • Fatinikun M.T.
      • Fernandes S.
      Seminal immune response in infertile men with leukocytospermia: effect on antioxidant activity.
      ), others have shown that this may not be clinically relevant unless >2 × 106 spermatozoa/ml (
      • Yanushpolsky E.H.
      • Politch J.A.
      • Hill J.A.
      • Anderson D.J.
      Is leukocytospermia clinically relevant?.
      ). In one study, leukocytospermia as defined by the WHO (1–3 × 106 spermatozoa/ml) was shown to have a positive effect and to correlate with initiation of the acrosome reaction and hypo-osmotic swelling test (HOST) scores (
      • Kaleli S.
      • Ocer F.
      • Irez T.
      • Budak E.
      • Aksu M.F.
      Does leukocytospermia associate with poor semen parameters and sperm functions in male infertility? The role of different seminal leukocyte concentrations.
      ). Importantly though, in the context of this review, several studies have shown that leukocytospermia commonly coexists with elevated DNA damage in spermatozoa (
      • Alvarez J.G.
      • Sharma R.K.
      • Ollero M.
      • Saleh R.A.
      • Lopez M.C.
      • Thomas Jr., A.J.
      • Evenson D.P.
      • Agarwal A.
      Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay.
      ,
      • Erenpreiss J.
      • Hlevicka S.
      • Zalkalns J.
      • Erenpreisa J.
      Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples.
      ,
      • Fariello R.M.
      • Del Giudice P.T.
      • Spaine D.M.
      • Fraietta R.
      • Bertolla R.P.
      • Cedenho A.P.
      Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity.
      ,
      • Saleh R.A.
      • Agarwal A.
      • Kandirali E.
      • Sharma R.K.
      • Thomas A.J.
      • Nada E.A.
      • Evenson D.P.
      • Alvarez J.G.
      Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa.
      ). Increased seminal ROS associated with leukocytospermia coexists with reduced concentrations of antioxidants (
      • Omu A.E.
      • Al-Qattan F.
      • Al-Abdul-Hadi F.M.
      • Fatinikun M.T.
      • Fernandes S.
      Seminal immune response in infertile men with leukocytospermia: effect on antioxidant activity.
      ,
      • Yadav S.B.
      • Suryakar A.N.
      • Huddedar A.D.
      • Shukla P.S.
      Effect of antioxidants and antibiotics on levels of seminal oxidative stress in leukocytospermic infertile men.
      ). Thus, interindividual differences in the total seminal antioxidant capacity may explain variations in susceptibility to ROS damage from leukocytes and related dysfunction.
      With infection or chronic inflammation, activated leukocytes greatly increase the concentrations of ROS and can release 1000-times more ROS than spermatozoa (
      • Plante M.
      • de Lamirande E.
      • Gagnon C.
      Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility.
      ). These concentrations of ROS may overwhelm seminal fluid antioxidant defences. Not discounting this, the time spent in the ejaculate is relatively small and therefore infection of the epididymis or testes have the greatest significance (
      • Ochsendorf F.R.
      Infections in the male genital tract and reactive oxygen species.
      ). Thus it is clear that infection or inflammation should be treated accordingly in cases of male infertility. Notably, antibiotic treatment of low-level leukocytospermia (0.2–1 × 106 spermatozoa/ml) resulted in a significant increase in spontaneous pregnancy and thus the threshold for treatment may need to be re-evaluated (
      • Hamada A.
      • Agarwal A.
      • Sharma R.
      • French D.B.
      • Ragheb A.
      • Sabanegh Jr., E.S.
      Empirical treatment of low-level leukocytospermia with doxycycline in male infertility patients.
      ).

      Varicocele

      Varicocele is the leading cause of male factor infertility, with an incidence of 15% in the whole male population and 40% among infertile men (
      Practice Committee of the American Society for Reproductive Medicine
      Report on varicocele and infertility.
      ). Oxidative stress is a major factor in the pathophysiology (
      • Agarwal A.
      • Hamada A.
      • Esteves S.C.
      Insight into oxidative stress in varicocele-associated male infertility: part 1.
      ) and elevated levels of DNA fragmentation have been demonstrated in numerous studies (
      • Blumer C.G.
      • Restelli A.E.
      • Giudice P.T.
      • Soler T.B.
      • Fraietta R.
      • Nichi M.
      • Bertolla R.P.
      • Cedenho A.P.
      Effect of varicocele on sperm function and semen oxidative stress.
      ,
      • Saleh R.A.
      • Agarwal A.
      • Sharma R.K.
      • Said T.M.
      • Sikka S.C.
      • Thomas Jr., A.J.
      Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele.
      ,
      • Smit M.
      • Romijn J.C.
      • Wildhagen M.F.
      • Veldhoven J.L.
      • Weber R.F.
      • Dohle G.R.
      Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate.
      ,
      • Smith R.
      • Kaune H.
      • Parodi D.
      • Madariaga M.
      • Rios R.
      • Morales I.
      • Castro A.
      Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress.
      ,
      • Talebi A.R.
      • Moein M.R.
      • Tabibnejad N.
      • Ghasemzadeh J.
      Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests.
      ,
      • Zini A.
      • Dohle G.
      Are varicoceles associated with increased deoxyribonucleic acid fragmentation?.
      ). Increased concentrations of ROS and decreased antioxidant capacity coexist in the majority of subjects (
      • Abd-Elmoaty M.A.
      • Saleh R.
      • Sharma R.
      • Agarwal A.
      Increased levels of oxidants and reduced antioxidants in semen of infertile men with varicocele.
      ,
      • Mostafa T.
      • Anis T.
      • El Nashar A.
      • Imam H.
      • Osman I.
      Seminal plasma reactive oxygen species-antioxidants relationship with varicocele grade.
      ,
      • Pasqualotto F.F.
      • Sundaram A.
      • Sharma R.K.
      • Borges Jr., E.
      • Pasqualotto E.B.
      • Agarwal A.
      Semen quality and oxidative stress scores in fertile and infertile patients with varicocele.
      ,
      • Saleh R.A.
      • Agarwal A.
      • Sharma R.K.
      • Said T.M.
      • Sikka S.C.
      • Thomas Jr., A.J.
      Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele.
      ,
      • Smith R.
      • Kaune H.
      • Parodi D.
      • Madariaga M.
      • Rios R.
      • Morales I.
      • Castro A.
      Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress.
      ). Interestingly some studies have shown elevated concentrations of antioxidants such as SOD and CAT (
      • Altunoluk B.
      • Efe E.
      • Kurutas E.B.
      • Gul A.B.
      • Atalay F.
      • Eren M.
      Elevation of both reactive oxygen species and antioxidant enzymes in vein tissue of infertile men with varicocele.
      ,
      • Hurtado de Catalfo G.E.
      • Ranieri-Casilla A.
      • Marra F.A.
      • de Alaniz M.J.
      • Marra C.A.
      Oxidative stress biomarkers and hormonal profile in human patients undergoing varicocelectomy.
      ,
      • Ozbek E.
      • Cekmen M.
      • Simsek A.
      • Turkoz Y.
      • Soylu A.
      • Ilbey Y.O.
      • Balbay M.D.
      Comparison of antioxidant enzyme activity in the internal spermatic vein and brachial veins of patients with infertile varicocele.
      ), which may account for an early increase in defences before becoming overwhelmed.
      Although previously debated, it is now the consensus that varicocele, either symptomatic or as a cause of infertility, is best treated surgically and the gold standard is microsurgical repair (
      • Ficarra V.
      • Crestani A.
      • Novara G.
      • Mirone V.
      Varicocele repair for infertility: what is the evidence?.
      ,
      • Goldstein M.
      • Tanrikut C.
      Microsurgical management of male infertility.
      ,
      • Mehta A.
      • Goldstein M.
      Microsurgical varicocelectomy: a review.
      ). Surgical repair is associated with improved semen parameters, including DNA fragmentation, decreased ROS and increased antioxidants and with improved success with assisted reproduction, but above all with increased incidence of spontaneous conception in previously infertile men (
      • Baker K.
      • McGill J.
      • Sharma R.
      • Agarwal A.
      • Sabanegh Jr., E.
      Pregnancy after varicocelectomy: impact of postoperative motility and DFI.
      ,
      • Chen S.S.
      • Huang W.J.
      • Chang L.S.
      • Wei Y.H.
      Attenuation of oxidative stress after varicocelectomy in subfertile patients with varicocele.
      ,
      • Hurtado de Catalfo G.E.
      • Ranieri-Casilla A.
      • Marra F.A.
      • de Alaniz M.J.
      • Marra C.A.
      Oxidative stress biomarkers and hormonal profile in human patients undergoing varicocelectomy.
      ,
      • Leung L.
      • Ho K.L.
      • Tam P.C.
      • Yiu M.K.
      Subinguinal microsurgical varicocelectomy for male factor subfertility: a ten-year experience.
      ,
      • Mostafa T.
      • Anis T.H.
      • El-Nashar A.
      • Imam H.
      • Othman I.A.
      Varicocelectomy reduces reactive oxygen species levels and increases antioxidant activity of seminal plasma from infertile men with varicocele.
      ,
      • Smit M.
      • Romijn J.C.
      • Wildhagen M.F.
      • Veldhoven J.L.
      • Weber R.F.
      • Dohle G.R.
      Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate.
      ).

      Smoking

      Tobacco smoke contains high concentrations of ROS including O2 and OH·, shown to participate in Fenton reactions to produce H2O2 and cause DNA damage (
      • Valavanidis A.
      • Vlachogianni T.
      • Fiotakis K.
      Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles.
      ). Cadmium and lead derived from cigarette smoke also cause DNA strand breaks (
      • Hengstler J.G.
      • Bolm-Audorff U.
      • Faldum A.
      • Janssen K.
      • Reifenrath M.
      • Gotte W.
      • Jung D.
      • Mayer-Popken O.
      • Fuchs J.
      • Gebhard S.
      • Bienfait H.G.
      • Schlink K.
      • Dietrich C.
      • Faust D.
      • Epe B.
      • Oesch F.
      Occupational exposure to heavy metals: DNA damage induction and DNA repair inhibition prove co-exposures to cadmium, cobalt and lead as more dangerous than hitherto expected.
      ) and are present in seminal fluid associated with indices of oxidative stress (
      • Kiziler A.R.
      • Aydemir B.
      • Onaran I.
      • Alici B.
      • Ozkara H.
      • Gulyasar T.
      • Akyolcu M.C.
      High levels of cadmium and lead in seminal fluid and blood of smoking men are associated with high oxidative stress and damage in infertile subjects.
      ). Nicotine is oxidative and can induce double-stranded DNA breaks in sperm DNA in vitro (
      • Arabi M.
      Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa.
      ). Cotinine, its major metabolite, is detected in the seminal plasma of smokers (
      • Wong W.Y.
      • Thomas C.M.
      • Merkus H.M.
      • Zielhuis G.A.
      • Doesburg W.H.
      • Steegers-Theunissen R.P.
      Cigarette smoking and the risk of male factor subfertility: minor association between cotinine in seminal plasma and semen morphology.
      ).
      In a small study (20 smokers excluding controls), smoking was associated with dramatic increases in seminal ROS concentrations (by 107%) and leukocyte numbers (by 48%) compared with nonsmokers (
      • Saleh R.A.
      • Agarwal A.
      • Sharma R.K.
      • Nelson D.R.
      • Thomas Jr., A.J.
      Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study.
      ). Smoking decreases overall concentrations of antioxidants in the body, indicating reduced protection against oxidative stress (
      • Lesgards J.F.
      • Durand P.
      • Lassarre M.
      • Stocker P.
      • Lesgards G.
      • Lanteaume A.
      • Prost M.
      • Lehucher-Michel M.P.
      Assessment of lifestyle effects on the overall antioxidant capacity of healthy subjects.
      ). Direct correlations with DNA damage indices have been demonstrated in a number of studies in fertile men (
      • Linschooten J.O.
      • Laubenthal J.
      • Cemeli E.
      • Baumgartner A.
      • Anderson D.
      • Sipinen V.E.
      • Brunborg G.
      • Haenen G.R.
      • Fthenou E.
      • Briede J.J.
      • van Schooten F.J.
      • Godschalk R.W.
      Incomplete protection of genetic integrity of mature spermatozoa against oxidative stress.
      ,
      • Taha E.A.
      • Ez-Aldin A.M.
      • Sayed S.K.
      • Ghandour N.M.
      • Mostafa T.
      Effect of smoking on sperm vitality, DNA integrity, seminal oxidative stress, zinc in fertile men.
      ), men with varicoceles (
      • Fariello R.M.
      • Pariz J.R.
      • Spaine D.M.
      • Gozzo F.C.
      • Pilau E.J.
      • Fraietta R.
      • Bertolla R.P.
      • Andreoni C.
      • Cedenho A.P.
      Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele.
      ) and men with idiopathic infertility (
      • Elshal M.F.
      • El-Sayed I.H.
      • Elsaied M.A.
      • El-Masry S.A.
      • Kumosani T.A.
      Sperm head defects and disturbances in spermatozoal chromatin and DNA integrities in idiopathic infertile subjects: association with cigarette smoking.
      ). The greatest difference was shown in the notable study of men with idiopathic infertility (n = 70): the mean DFI of infertile smokers was 37.66% compared with 19.34% in infertile nonsmokers (P < 0.001) or 14.51% in fertile controls (not significantly different from infertile nonsmokers). The correlation of DFI with smoking gave a Pearson coefficient of 0.796. This study carefully excluded confounding factors such as age, leukocytospermia, varicocele and other potential sources of DNA damage. This demonstrates the importance of smoking in the aetiology of idiopathic infertility.
      As aforementioned, smoking has been linked with heritable DNA damage and incidence of childhood cancer. Cessation of smoking is therefore advised in the treatment of male factor infertility (Table 2).
      Table 2Lifestyle factors modifiable without risk.
      Lifestyle factorResultsRecommendationsReferences
      SmokingStrong correlation with % DFI, DFI markedly higher in infertile smokersCessation of smoking
      • Elshal M.F.
      • El-Sayed I.H.
      • Elsaied M.A.
      • El-Masry S.A.
      • Kumosani T.A.
      Sperm head defects and disturbances in spermatozoal chromatin and DNA integrities in idiopathic infertile subjects: association with cigarette smoking.
      POP/PCBPositive correlation between exposure and % DFIPCB accumulate in food chain: avoid fatty fish, particularly farmed
      • Rignell-Hydbom A.
      • Rylander L.
      • Giwercman A.
      • Jonsson B.A.
      • Lindh C.
      • Eleuteri P.
      • Rescia M.
      • Leter G.
      • Cordelli E.
      • Spano M.
      • Hagmar L.
      Exposure to PCBs and p,p′-DDE and human sperm chromatin integrity.
      ,
      • Spano M.
      • Toft G.
      • Hagmar L.
      • Eleuteri P.
      • Rescia M.
      • Rignell-Hydbom A.
      • Tyrkiel E.
      • Zvyezday V.
      • Bonde J.P.
      Exposure to PCB and p,p′-DDE in European and Inuit populations: impact on human sperm chromatin integrity.
      ,
      • Stronati A.
      • Manicardi G.C.
      • Cecati M.
      • Bordicchia M.
      • Ferrante L.
      • Spano M.
      • Toft G.
      • Bonde J.P.
      • Jonsson B.A.
      • Rignell-Hydbom A.
      • Rylander L.
      • Giwercman A.
      • Pedersen H.S.
      • Bonefeld-Jorgensen E.C.
      • Ludwicki J.K.
      • Lesovoy V.
      • Sakkas D.
      • Bizzaro D.
      Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p, p’-DDE in European and Inuit populations.
      OrganophosphorusMarked increase in % DFI (>30%) in exposed workersAvoid pesticide exposure
      • Sanchez-Pena L.C.
      • Reyes B.E.
      • Lopez-Carrillo L.
      • Recio R.
      • Moran-Martinez J.
      • Cebrian M.E.
      • Quintanilla-Vega B.
      Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers.
      LeadIncrease in percentage of spermatozoa with DNA fragmentationAvoid occupational exposure and smoking or exposure to cigarette smoke
      • Hsu P.C.
      • Chang H.Y.
      • Guo Y.L.
      • Liu Y.C.
      • Shih T.S.
      Effect of smoking on blood lead levels in workers and role of reactive oxygen species in lead-induced sperm chromatin DNA damage.
      ,
      • Vani K.
      • Kurakula M.
      • Syed R.
      • Alharbi K.
      Clinical relevance of vitamin C among lead-exposed infertile men.
      Bisphenol ASignificant trend of increased DNA damage with increased urinary bisphenol A concentrationsAvoid plastic packaging, tinned foods, heating or storing foods in plastic
      • Meeker J.D.
      • Ehrlich S.
      • Toth T.L.
      • Wright D.L.
      • Calafat A.M.
      • Trisini A.T.
      • Ye X.
      • Hauser R.
      Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from an infertility clinic.
      Testicular heatIncrease in DNA fragmentation with 2–3°C temperature increaseAvoid cycling with tight pants, avoid sauna use, avoid using laptop on closed legs
      • Southorn T.
      Great balls of fire and the vicious cycle: a study of the effects of cycling on male fertility.
      ,
      • Ahmad G.
      • Moinard N.
      • Esquerre-Lamare C.
      • Mieusset R.
      • Bujan L.
      Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men.
      ,
      • Sheynkin Y.
      • Welliver R.
      • Winer A.
      • Hajimirzaee F.
      • Ahn H.
      • Lee K.
      Protection from scrotal hyperthermia in laptop computer users.
      ,
      • Garolla A.
      • Torino M.
      • Sartini B.
      • Cosci I.
      • Patassini C.
      • Carraro U.
      • Foresta C.
      Seminal and molecular evidence that sauna exposure affects human spermatogenesis.
      Mobile phone radiationNo specific studies on DNA fragmentation, increased ROS and decreased antioxidantsDo not store mobile phone in trouser pocket
      • Desai N.R.
      • Kesari K.K.
      • Agarwal A.
      Pathophysiology of cell phone radiation: oxidative stress and carcinogenesis with focus on male reproductive system.
      ObesityPositive correlation of body mass index and DNA fragmentation, higher incidence in obese malesWeight loss through diet and moderate exercise
      • Kort H.I.
      • Massey J.B.
      • Elsner C.W.
      • Mitchell-Leef D.
      • Shapiro D.B.
      • Witt M.A.
      • Roudebush W.E.
      Impact of body mass index values on sperm quantity and quality.
      ,
      • Chavarro J.E.
      • Toth T.L.
      • Wright D.L.
      • Meeker J.D.
      • Hauser R.
      Body mass index in relation to semen quality, sperm DNA integrity, and serum reproductive hormone levels among men attending an infertility clinic.
      ,
      • Fariello R.M.
      • Pariz J.R.
      • Spaine D.M.
      • Cedenho A.P.
      • Bertolla R.P.
      • Fraietta R.
      Association between obesity and alteration of sperm DNA integrity and mitochondrial activity.
      ,
      • La Vignera S.
      • Condorelli R.A.
      • Vicari E.
      • Calogero A.E.
      Negative effect of increased body weight on sperm conventional and nonconventional flow cytometric sperm parameters.
      ,
      • Dupont C.
      • Faure C.
      • Sermondade N.
      • Boubaya M.
      • Eustache F.
      • Clement P.
      • Briot P.
      • Berthaut I.
      • Levy V.
      • Cedrin-Durnerin I.
      • Benzacken B.
      • Chavatte-Palmer P.
      • Levy R.
      Obesity leads to higher risk of sperm DNA damage in infertile patients.
      DFI = DNA fragmentation index; PCB = polychlorinated biphenyls; ROS = reactive oxygen species.

      Xenobiotics and toxic metals

      Xenobiotics represent a diverse source of potential DNA damage. Infertile men under oxidative stress may have increased susceptibility, and a combined or additive effect may occur between different xenobiotics and other sources of ROS. It is for this reason that a thorough history and questionnaire is advised in treating male infertility so that exposure to such damaging chemicals can be reduced or avoided as part of a holistic approach.
      Exposure to persistent organochlorine pollutants such as polychlorinated biphenyls and metabolites of dichlorodiphenyltrichloroethane may cause DNA fragmentation in spermatozoa. In studies of European men, exposure levels correlated with higher levels of DFI, while this was not observed in Inuit populations in Greenland where exposure would be higher (contaminated fatty fish) (
      • Rignell-Hydbom A.
      • Rylander L.
      • Giwercman A.
      • Jonsson B.A.
      • Lindh C.
      • Eleuteri P.
      • Rescia M.
      • Leter G.
      • Cordelli E.
      • Spano M.
      • Hagmar L.
      Exposure to PCBs and p,p′-DDE and human sperm chromatin integrity.
      ,
      • Spano M.
      • Toft G.
      • Hagmar L.
      • Eleuteri P.
      • Rescia M.
      • Rignell-Hydbom A.
      • Tyrkiel E.
      • Zvyezday V.
      • Bonde J.P.
      Exposure to PCB and p,p′-DDE in European and Inuit populations: impact on human sperm chromatin integrity.
      ,
      • Stronati A.
      • Manicardi G.C.
      • Cecati M.
      • Bordicchia M.
      • Ferrante L.
      • Spano M.
      • Toft G.
      • Bonde J.P.
      • Jonsson B.A.
      • Rignell-Hydbom A.
      • Rylander L.
      • Giwercman A.
      • Pedersen H.S.
      • Bonefeld-Jorgensen E.C.
      • Ludwicki J.K.
      • Lesovoy V.
      • Sakkas D.
      • Bizzaro D.
      Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p, p’-DDE in European and Inuit populations.
      ). However, genetic variation might be expected to confer protection in highly exposed populations. DFI has been shown to be markedly higher in exposed men with modifications of the androgen receptor gene (
      • Giwercman A.
      • Rylander L.
      • Rignell-Hydbom A.
      • Jonsson B.A.
      • Pedersen H.S.
      • Ludwicki J.K.
      • Lesovoy V.
      • Zvyezday V.
      • Spano M.
      • Manicardi G.C.
      • Bizzaro D.
      • Bonefeld-Jorgensen E.C.
      • Toft G.
      • Bonde J.P.
      • Giwercman C.
      • Tiido T.
      • Giwercman Y.L.
      Androgen receptor gene CAG repeat length as a modifier of the association between persistent organohalogen pollutant exposure markers and semen characteristics.
      ).
      • Sanchez-Pena L.C.
      • Reyes B.E.
      • Lopez-Carrillo L.
      • Recio R.
      • Moran-Martinez J.
      • Cebrian M.E.
      • Quintanilla-Vega B.
      Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers.
      showed that 75% of Mexican workers exposed to organophosphorus pesticides had DFI >30% whereas unexposed controls had mean DFI 9.9%. Mean DFI of Venezuelan farmers exposed to organophosphorus and carbamate pesticides showed similar elevations to 34.8% (P < 0.0001) although unexposed controls had a higher DFI than the Mexican cohort (24.6%) (
      • Miranda-Contreras L.
      • Gomez-Perez R.
      • Rojas G.
      • Cruz I.
      • Berrueta L.
      • Salmen S.
      • Colmenares M.
      • Barreto S.
      • Balza A.
      • Zavala L.
      • Morales Y.
      • Molina Y.
      • Valeri L.
      • Contreras C.A.
      • Osuna J.A.
      Occupational exposure to organophosphate and carbamate pesticides affects sperm chromatin integrity and reproductive hormone levels among Venezuelan farm workers.
      ). Occupational exposure to lead also correlates with DNA fragmentation in spermatozoa, even when adjusting for smoking (
      • Hsu P.C.
      • Chang H.Y.
      • Guo Y.L.
      • Liu Y.C.
      • Shih T.S.
      Effect of smoking on blood lead levels in workers and role of reactive oxygen species in lead-induced sperm chromatin DNA damage.
      ,
      • Vani K.
      • Kurakula M.
      • Syed R.
      • Alharbi K.
      Clinical relevance of vitamin C among lead-exposed infertile men.
      ). Seasonally increased air pollution (sulphur dioxide, nitric oxide and particulate matter) in the Czech Republic was associated with significantly increased DNA fragmentation during months of highest exposure, adjusting for smoking and other variables (
      • Rubes J.
      • Selevan S.G.
      • Evenson D.P.
      • Zudova D.
      • Vozdova M.
      • Zudova Z.
      • Robbins W.A.
      • Perreault S.D.
      Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality.
      ).
      Bisphenol A is an important endocrine disruptor and human exposure is widespread from food and drink containers and the environment (
      • Rubin B.S.
      Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects.
      ). Animal studies have demonstrated mutagenic effects, DNA damage induced in spermatozoa and epigenetic modifications in offspring (
      • Dobrzynska M.M.
      • Radzikowska J.
      Genotoxicity and reproductive toxicity of bisphenol A and X-ray/bisphenol A combination in male mice.
      ,
      • Manikkam M.
      • Tracey R.
      • Guerrero-Bosagna C.
      • Skinner M.K.
      Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations.
      ,
      • Tiwari D.
      • Vanage G.
      Mutagenic effect of Bisphenol A on adult rat male germ cells and their fertility.
      ). Limited studies have been carried out in humans. One study by
      • Meeker J.D.
      • Ehrlich S.
      • Toth T.L.
      • Wright D.L.
      • Calafat A.M.
      • Trisini A.T.
      • Ye X.
      • Hauser R.
      Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from an infertility clinic.
      measured urinary bisphenol A and DNA damage by Comet assay in 132 men from subfertile couples. Adjusting for variables and other sources of DNA damage, a significant trend was seen for increasing Tail% DNA, which the authors attributed to single-strand breaks.
      Some medications and drugs are associated with DNA damage in spermatozoa such as selective serotonin reuptake inhibitors (
      • Safarinejad M.R.
      Sperm DNA damage and semen quality impairment after treatment with selective serotonin reuptake inhibitors detected using semen analysis and sperm chromatin structure assay.
      ,
      • Tanrikut C.
      • Feldman A.S.
      • Altemus M.
      • Paduch D.A.
      • Schlegel P.N.
      Adverse effect of paroxetine on sperm.
      ) and opiates (
      • Safarinejad M.R.
      • Asgari S.A.
      • Farshi A.
      • Ghaedi G.
      • Kolahi A.A.
      • Iravani S.
      • Khoshdel A.R.
      The effects of opiate consumption on serum reproductive hormone levels, sperm parameters, seminal plasma antioxidant capacity and sperm DNA integrity.
      ). This reiterates the importance of taking an adequate history due to the high intake of these drugs, including over-the-counter drugs such as codeine. Avoidance of these xenobiotic sources would be advised in cases where exposure is evident (Table 2).

      Heat

      The position of the scrotum acts to maintain the temperature of the testes lower than that of the body. Elevation in scrotal temperature, or hyperthermia, is associated with male infertility and impaired spermatogenesis (
      • Mieusset R.
      • Bujan L.
      • Mondinat C.
      • Mansat A.
      • Pontonnier F.
      • Grandjean H.
      Association of scrotal hyperthermia with impaired spermatogenesis in infertile men.
      ). Spermatogenic arrest increases the number of immature or ‘incorrectly matured’ spermatozoa in the epididymis and ejaculate (
      • Dada R.
      • Gupta N.P.
      • Kucheria K.
      Spermatogenic arrest in men with testicular hyperthermia.
      ), and these are a major source of ROS (
      • Gil-Guzman E.
      • Ollero M.
      • Lopez M.C.
      • Sharma R.K.
      • Alvarez J.G.
      • Thomas Jr., A.J.
      • Agarwal A.
      Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation.
      ,
      • Ollero M.
      • Gil-Guzman E.
      • Lopez M.C.
      • Sharma R.K.
      • Agarwal A.
      • Larson K.
      • Evenson D.
      • Thomas Jr., A.J.
      • Alvarez J.G.
      Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.
      ). Scrotal hyperthermia may result from close-fitting underwear although the degree of hyperthermia caused by briefs rather than boxer shorts is debated as somewhat of a myth (
      • Southorn T.
      Great balls of fire and the vicious cycle: a study of the effects of cycling on male fertility.
      ). Specially designed tight-fitting underwear that maintained the testes at inguinal position did elevate scrotal temperature by 2°C in one study (
      • Ahmad G.
      • Moinard N.
      • Esquerre-Lamare C.
      • Mieusset R.
      • Bujan L.
      Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men.
      ). This study demonstrated a significant increase in both DNA fragmentation and chromatin decondensation if sustained for 20 days or more at this temperature increase. Cycling has also been proposed as a source of hyperthermia. Moderate cycling under tightly controlled laboratory conditions was shown not to cause scrotal hyperthermia (
      • Jung A.
      • Strauss P.
      • Lindner H.J.
      • Schuppe H.C.
      Influence of moderate cycling on scrotal temperature.
      ). However, the subjects of this study were wearing loosely fitted cotton trousers whereas most cyclists wear tight-fitting lycra, which is proposed to be the source of hyperthermia (
      • Southorn T.
      Great balls of fire and the vicious cycle: a study of the effects of cycling on male fertility.
      ). Regular use of a sauna, Jacuzzi or hot bath is also contraindicated. In a small study of 10 normozoospermic healthy volunteers, there was a temperature increase from 34.5°C to 37.5°C with regular sauna use (
      • Garolla A.
      • Torino M.
      • Sartini B.
      • Cosci I.
      • Patassini C.
      • Carraro U.
      • Foresta C.
      Seminal and molecular evidence that sauna exposure affects human spermatogenesis.
      ). This usage resulted in nonsignificant increases in DNA fragmentation (by both TUNEL and acridine orange assay) and a significant increase in chromatin decondensation. Incidence of acute febrile illness is also associated with impaired semen parameters and excludes men from routine tests, trials and donation for assisted reproduction. Fever has been shown to significantly increase DNA fragmentation for a period lasting at least 79 days, peaking at approximately 1-month post illness (
      • Sergerie M.
      • Mieusset R.
      • Croute F.
      • Daudin M.
      • Bujan L.
      High risk of temporary alteration of semen parameters after recent acute febrile illness.
      ). Finally, positioning a laptop on the lap of closed legs markedly increases scrotal temperature (
      • Sheynkin Y.
      • Welliver R.
      • Winer A.
      • Hajimirzaee F.
      • Ahn H.
      • Lee K.
      Protection from scrotal hyperthermia in laptop computer users.
      ). This and other sources of scrotal hyperthermia should be avoided in men that are trying to conceive, particularly men diagnosed with elevated DNA fragmentation (Table 2).

      Mobile phone radiation

      Increased usage of mobile phones and storage of phones in trouser pockets has been suggested as a source of damaging radiation to the male reproductive system (Table 2). A recent study showed that DNA fragmentation was the only parameter altered in mobile phone users, in a group of high usage (>4 h daily) that stored their phone in the trouser pocket (
      • Rago R.
      • Salacone P.
      • Caponecchia L.
      • Sebastianelli L.
      • Marcucci I.
      • Calogero A.E.
      • Condorelli R.
      • Vicari E.
      • Morgia G.
      • Favilla V.
      • Cimino N.
      • Arcoria A.F.
      • La Vignera S.
      The semen quality of the mobile phone users.
      ). Previous studies have shown alterations in motility, morphology and hormonal disturbances (
      • Agarwal A.
      • Deepinder F.
      • Sharma R.K.
      • Ranga G.
      • Li J.
      Effect of cell phone usage on semen analysis in men attending infertility clinic: an observational study.
      ,
      • Fejes I.
      • Zavaczki Z.
      • Szollosi J.
      • Koloszar S.
      • Daru J.
      • Kovacs L.
      • Pal A.
      Is there a relationship between cell phone use and semen quality?.
      ,
      • Gutschi T.
      • Mohamad Al-Ali B.
      • Shamloul R.
      • Pummer K.
      • Trummer H.
      Impact of cell phone use on men’s semen parameters.
      ). Mobile phone radiation can increase ROS production and decrease activity of CAT, SOD and GPX (
      • Desai N.R.
      • Kesari K.K.
      • Agarwal A.
      Pathophysiology of cell phone radiation: oxidative stress and carcinogenesis with focus on male reproductive system.
      ). DNA damage may result from this, as has been clearly demonstrated in vitro (
      • De Iuliis G.N.
      • Newey R.J.
      • King B.V.
      • Aitken R.J.
      Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro.
      ).

      Age

      Age-related infertility has been linked with DNA damage where age correlates positively with DNA fragmentation (
      • Das M.
      • Al-Hathal N.
      • San-Gabriel M.
      • Phillips S.
      • Kadoch I.J.
      • Bissonnette F.
      • Holzer H.
      • Zini A.
      High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age.
      ,
      • Hammiche F.
      • Laven J.S.
      • Boxmeer J.C.
      • Dohle G.R.
      • Steegers E.A.
      • Steegers-Theunissen R.P.
      Sperm quality decline among men below 60 years of age undergoing IVF or ICSI treatment.
      ,
      • Rybar R.
      • Kopecka V.
      • Prinosilova P.
      • Markova P.
      • Rubes J.
      Male obesity and age in relationship to semen parameters and sperm chromatin integrity.
      ). Because age is a nonmodifiable variable in the treatment of male infertility, it is outside the scope of this review to discuss related mechanisms.

      Obesity

      Obesity is associated with male factor infertility and abnormal semen parameters mainly due to hormonal aberrations and incidence of negative lifestyle factors (
      • Du Plessis S.S.
      • Cabler S.
      • McAlister D.A.
      • Sabanegh E.
      • Agarwal A.
      The effect of obesity on sperm disorders and male infertility.
      ,
      • Hammoud A.O.
      • Gibson M.
      • Peterson C.M.
      • Meikle A.W.
      • Carrell D.T.
      Impact of male obesity on infertility: a critical review of the current literature.
      ). DNA damage has also been investigated in numerous studies of obese men. One study of 305 subfertile men (n = 36 obese) demonstrated a significant increase in DNA fragmentation as measured by the Comet assay with obesity (
      • Fariello R.M.
      • Pariz J.R.
      • Spaine D.M.
      • Cedenho A.P.
      • Bertolla R.P.
      • Fraietta R.
      Association between obesity and alteration of sperm DNA integrity and mitochondrial activity.
      ). Analysis of men of subfertile couples (possibly not all men were subfertile) also showed a higher incidence of spermatozoa with DNA fragmentation in obese men (
      • Chavarro J.E.
      • Toth T.L.
      • Wright D.L.
      • Meeker J.D.
      • Hauser R.
      Body mass index in relation to semen quality, sperm DNA integrity, and serum reproductive hormone levels among men attending an infertility clinic.
      ,
      • Dupont C.
      • Faure C.
      • Sermondade N.
      • Boubaya M.
      • Eustache F.
      • Clement P.
      • Briot P.
      • Berthaut I.
      • Levy V.
      • Cedrin-Durnerin I.
      • Benzacken B.
      • Chavatte-Palmer P.
      • Levy R.
      Obesity leads to higher risk of sperm DNA damage in infertile patients.
      ). A study of 520 men presenting for semen analysis demonstrated a positive correlation between body mass index and DFI, with the mean DFI rising from 19.9% in normal body mass index men to 27.0% in obese men (
      • Kort H.I.
      • Massey J.B.
      • Elsner C.W.
      • Mitchell-Leef D.
      • Shapiro D.B.
      • Witt M.A.
      • Roudebush W.E.
      Impact of body mass index values on sperm quantity and quality.
      ). Finally, a study of normal volunteers from the general population (n = 50 obese) also confirmed the relationship between body mass index and DNA damage as measured by TUNEL (
      • La Vignera S.
      • Condorelli R.A.
      • Vicari E.
      • Calogero A.E.
      Negative effect of increased body weight on sperm conventional and nonconventional flow cytometric sperm parameters.
      ). Moderate exercise combined with diet may be recommended for weight loss (Table 2). Both moderate exercise and weight loss have correlated with improved semen parameters in a limited number of studies (
      • Reis L.O.
      • Dias F.G.
      Male fertility, obesity, and bariatric surgery.
      ,
      • Sharma R.
      • Biedenharn K.R.
      • Fedor J.M.
      • Agarwal A.
      Lifestyle factors and reproductive health: taking control of your fertility.
      ), although there have been no human studies to date that measured DNA fragmentation as an outcome.

      Antioxidants

      Antioxidants from the diet can act directly as antioxidants or are essential cofactors to the enzymic antioxidant systems SOD, CAT and GPX. As reviewed by
      • Lanzafame F.M.
      • La Vignera S.
      • Vicari E.
      • Calogero A.E.
      Oxidative stress and medical antioxidant treatment in male infertility.
      , seminal plasma contains both enzymic antioxidants and low-molecular-weight nonenzymic antioxidants such as ascorbic acid, α-tocopherol, uric acid, albumin, carnitine, carotenoids, flavonoids and coenzyme Q10 (CoQ10) (
      • Lanzafame F.M.
      • La Vignera S.
      • Vicari E.
      • Calogero A.E.
      Oxidative stress and medical antioxidant treatment in male infertility.
      ).

      Dietary antioxidant intake

      Food frequency questionnaires have been utilized to elucidate the association between dietary intakes and sperm parameters, including DNA damage. In one study, older men (>44 years) with greater intakes of vitamin C, vitamin E and zinc had the lowest levels of DNA fragmentation, similar to levels in younger men (
      • Schmid T.E.
      • Eskenazi B.
      • Marchetti F.
      • Young S.
      • Weldon R.H.
      • Baumgartner A.
      • Anderson D.
      • Wyrobek A.J.
      Micronutrients intake is associated with improved sperm DNA quality in older men.
      ). In a study of men attending an assisted reproduction clinic in the Netherlands (n = 161), a ‘health conscious’ diet was significantly associated with a lower risk of DNA fragmentation, specifically attributed to higher intakes of fruits and vegetables, which are excellent sources of antioxidants (
      • Vujkovic M.
      • de Vries J.H.
      • Dohle G.R.
      • Bonsel G.J.
      • Lindemans J.
      • Macklon N.S.
      • van der Spek P.J.
      • Steegers E.A.
      • Steegers-Theunissen R.P.
      Associations between dietary patterns and semen quality in men undergoing IVF/ICSI treatment.
      ); the study was adjusted for body mass index, smoking, age and vitamin supplementation. In a study of normal volunteers, the same association was not seen: there was no correlation between intakes of vitamin C, E and β-carotene and % DFI (
      • Silver E.W.
      • Eskenazi B.
      • Evenson D.P.
      • Block G.
      • Young S.
      • Wyrobek A.J.
      Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men.
      ). The authors note that this does not preclude the potential effects of antioxidant status on subfertile men.
      Diet is a major modifiable factor in the treatment of male infertility. Antioxidants undoubtedly play an important role in spermatozoa health, and dietary changes should be considered in any treatment regimen involving elevated DNA fragmentation (Table 3).
      Table 3Dietary sources of antioxidants.
      Vitamin CVitamin EZincSelenium
      PapayaSpinachSpinachHalibut
      Bell peppersSwiss chardShiitake mushroomTuna
      StrawberriesSunflower seedsCrimini mushroomCod
      BroccoliAlmondsOrganic lambShrimp
      PineappleAsparagusOrganic beefCrimin mushroom
      KiwiBell peppersScallopsMustard seeds
      OrangesCayenne pepperSesame seedsSardines
      CanataloupePapayaPumpkin seedsSalmon
      KaleKaleOatsTurkey
      CauliflowerBarley
      Adapted from

      World’s Healthiest Foods, 2013. World’s Healthiest Foods. Available from: <http://whfoods.com>.

      .

      Supplementary antioxidants

      Antioxidant therapy for DNA fragmentation has been tested in numerous trials. The overall view is that antioxidant treatment may play a relevant role, although some results have been inconsistent and large well-controlled trials are still required for conclusive evidence as to their efficacy (
      • Gharagozloo P.
      • Aitken R.J.
      The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy.
      ,
      • Lombardo F.
      • Sansone A.
      • Romanelli F.
      • Paoli D.
      • Gandini L.
      • Lenzi A.
      The role of antioxidant therapy in the treatment of male infertility: an overview.
      ,
      • Showell M.G.
      • Brown J.
      • Yazdani A.
      • Stankiewicz M.T.
      • Hart R.J.
      Antioxidants for male subfertility.
      ,
      • Zini A.
      • San Gabriel M.
      • Baazeem A.
      Antioxidants and sperm DNA damage: a clinical perspective.
      ). There is large heterogeneity to studies including design, antioxidants used singly or in combinations, cohort selection (aetiology of infertility) and outcome measurement. Many reviews have called out for relevant clinical outcomes to be included such as pregnancy or delivery rate. However, in the field of fertility, there are many variables that affect delivery rates in natural pregnancy and, particularly, after assisted reproduction treatment. For this review, we chose to only assess studies that directly measured the effect of antioxidants on DNA fragmentation in order to limit variable factors and to determine the effect, if any, of antioxidant intake on DNA damage in spermatozoa (Table 4).
      Table 4Comparison of studies of antioxidant treatment in DNA fragmentation.
      StudySupplementMeasurementStudy designOutcome
      • Fraga C.G.
      • Motchnik P.A.
      • Shigenaga M.K.
      • Helbock H.J.
      • Jacob R.A.
      • Ames B.N.
      Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.
      Vitamin C8-OHdG10 normal volunteers, uncontrolled, depletion to 5 mg/day, repletion to 250 mg/dayDNA damage increased upon depletion, restored by repletion
      • Greco E.
      • Iacobelli M.
      • Rienzi L.
      • Ubaldi F.
      • Ferrero S.
      • Tesarik J.
      Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment.
      1 g vitamin C, 1 g vitamin ETUNEL64 males with >15% damage, placebo controlled, double blindedDNA damage reduced from 22.1% to 9.1%
      • Greco E.
      • Romano S.
      • Iacobelli M.
      • Ferrero S.
      • Baroni E.
      • Minasi M.G.
      • Ubaldi F.
      • Rienzi L.
      • Tesarik J.
      ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment.
      1 g vitamin C, 1 g vitamin ETUNEL38 infertile males,>15% damage, uncontrolledDNA damage reduced from 24.8% to 8.2% in responsive group
      • Kodama H.
      • Yamaguchi R.
      • Fukuda J.
      • Kasai H.
      • Tanaka T.
      Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients.
      400 mg GSH, 200 mg vitamin C, 200 mg vitamin E8-OHdG14 males with DNA damage, uncontrolledModest decrease in DNA damage; possible marked decrease in responsive group
      • Menezo Y.J.
      • Hazout A.
      • Panteix G.
      • Robert F.
      • Rollet J.
      • Cohen-Bacrie P.
      • Chapuis F.
      • Clement P.
      • Benkhalifa M.
      Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect.
      400 mg vitamin C, 400 mg vitamin E, 33 mg zinc, 80 μg selenium, 18 mg β-caroteneSCSA58 males with DFI >15%, double centred, uncontrolledDFI decreased from 32.4% to 26.2%, high-density staining increased
      • Tremellen K.
      • Miari G.
      • Froiland D.
      • Thompson J.
      A randomised control trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-ICSI treatment.
      Menevit: 100 mg vitamin C, 400 IU vitamin E, 6 mg lycopene, 333 μg garlic oil, 25 mg zinc, 26 μg selenium, 500 μg folic acidTUNEL60 males with damage >25%, double-blind RCTDNA damage reduced from 37.9% to 33.3%; but from 40.03% to 32% in controls
      Results from patent submission for Menevit supplement (Tremellen, 2006).
      • Tunc O.
      • Thompson J.
      • Tremellen K.
      Improvement in sperm DNA quality using an oral antioxidant therapy.
      Menevit: as aboveTUNEL50 males with seminal oxidative stress, uncontrolledDNA damage reduced from 22.2% to 18.2%
      • Abad C.
      • Amengual M.J.
      • Gosalvez J.
      • Coward K.
      • Hannaoui N.
      • Benet J.
      • Garcia-Peiro A.
      • Prats J.
      Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.
      1500 mg l-carnitine, 20 mg CoQ10, 60 mg vitamin C, 10 mg vitamin E, 200 μg vitamin B9, 1 μg vitamin B12, 10 mg zinc, 50 μg seleniumSCD20 asthenteratozoospermic males, uncontrolledDNA damage reduced from 28.5% to 20.12%
      8-OHdG = 8-hydroxy-2-deoxyguanosine; DFI = DNA fragmentation index; GSH = glutathione; RCT = randomized controlled trial; SCD = sperm chromatin dispersion test; SCSA = sperm chromatin structure assay; TUNEL = TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling.
      a Results from patent submission for Menevit supplement (

      Tremellen, K., 2006. Methods and compositions for improving pregnancy outcome. US Patent 20090081177.

      ).

      Vitamin C

      Vitamin C is a powerful hydrophilic antioxidant in seminal plasma. The incidence of DNA fragmentation is higher in men with low concentrations of seminal ascorbic acid (
      • Song G.J.
      • Norkus E.P.
      • Lewis V.
      Relationship between seminal ascorbic acid and sperm DNA integrity in infertile men.
      ) while the incidence is lower in men with higher dietary intake (
      • Schmid T.E.
      • Eskenazi B.
      • Marchetti F.
      • Young S.
      • Weldon R.H.
      • Baumgartner A.
      • Anderson D.
      • Wyrobek A.J.
      Micronutrients intake is associated with improved sperm DNA quality in older men.
      ). One notable study examined the effects of ascorbic acid concentrations on DNA damage using measurement of 8-OHdG concentration. Concentrations of ascorbic acid in 10 normal individuals were maintained via a controlled diet (
      • Fraga C.G.
      • Motchnik P.A.
      • Shigenaga M.K.
      • Helbock H.J.
      • Jacob R.A.
      • Ames B.N.
      Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.
      ). While this was a very small study with no control group, it is of interest due to the model of depletion and repletion utilized. During an initial 32-day period of depletion, 8-OHdG concentrations almost doubled. This increase was followed by a 28-day period of marginal repletion where 8-OHdG further increased to 2.5-fold higher than the baseline value. Full repletion over 28 days then resulted in a reduction of DNA damage by 49%, although a period of 1 month may not have been sufficient to see the full effect. Of additional note in this study was that oral intake rapidly affected concentrations of ascorbic acid in seminal plasma. Upon depletion, seminal concentrations reduced by 71%, which was fully restored after the period of repletion.
      In another study, DNA fragmentation (Comet assay) was prevented in lead-exposed workers in India by administration of 1000 mg vitamin C 5 days/week for 3 months when administered prophylactically (
      • Vani K.
      • Kurakula M.
      • Syed R.
      • Alharbi K.
      Clinical relevance of vitamin C among lead-exposed infertile men.
      ). However, high doses of vitamin C may be linked with negative effects. Ascorbate can reduce disulphide bridges of cysteine residues (
      • Giustarini D.
      • Dalle-Donne I.
      • Colombo R.
      • Milzani A.
      • Rossi R.
      Is ascorbate able to reduce disulfide bridges? A cautionary note.
      ), potentially damaging the chromatin structure. A study of mixed antioxidants, including just 400 mg of ascorbic acid daily for 3 months, attributed this effect to the induction of chromatin decondensation in spermatozoa (
      • Menezo Y.J.
      • Hazout A.
      • Panteix G.
      • Robert F.
      • Rollet J.
      • Cohen-Bacrie P.
      • Chapuis F.
      • Clement P.
      • Benkhalifa M.
      Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect.
      ). In order to avoid oversupplementation, blood tests can be used to determine plasma concentrations of vitamin C, although seminal plasma concentrations are far higher (∼600 μmol/l compared with ∼40 μmol/l (
      • Frei B.
      • England L.
      • Ames B.N.
      Ascorbate is an outstanding antioxidant in human blood plasma.
      ,
      • Thiele J.J.
      • Friesleben H.J.
      • Fuchs J.
      • Ochsendorf F.R.
      Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen.
      )). Bioavailability from food sources including fruit and vegetables is good, and intake can restore suboptimal plasma concentrations rapidly (
      • Vissers M.C.
      • Carr A.C.
      • Pullar J.M.
      • Bozonet S.M.
      The bioavailability of vitamin C from kiwifruit.
      ).

      Vitamin C and E

      Oral vitamin E supplementation affords an increase in both plasma and seminal concentrations, with a nonsignificant difference found between intakes of 600, 800 and 1200 mg, favouring 800 mg (
      • Moilanen J.
      • Hovatta O.
      Excretion of alpha-tocopherol into human seminal plasma after oral administration.
      ). With regard to effects on DNA fragmentation, the only studies investigating vitamin E have used mixed antioxidants. Vitamin C and E can work synergistically, although ascorbate is present in much greater amounts in seminal fluid (
      • Lewis S.E.
      • Sterling E.S.
      • Young I.S.
      • Thompson W.
      Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men.
      ). The first publication using mixed antioxidants was a convincing placebo-controlled double-blind study (
      • Greco E.
      • Iacobelli M.
      • Rienzi L.
      • Ubaldi F.
      • Ferrero S.
      • Tesarik J.
      Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment.
      ). Men with varicocele, infection or inflammation and smokers were excluded. A 2-month period was used to assess post-testicular DNA damage. The mean DNA damage was markedly reduced from 22.1% to 9.1% in the treatment group, with no change in the placebo group. The second publication had a similar treatment design but no placebo group (
      • Greco E.
      • Romano S.
      • Iacobelli M.
      • Ferrero S.
      • Baroni E.
      • Minasi M.G.
      • Ubaldi F.
      • Rienzi L.
      • Tesarik J.
      ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment.
      ). Men were treated after one failed ICSI attempt and a second attempt was carried out immediately after antioxidant treatment. This study divided the group into ‘responsive’ and ‘nonresponsive’. Those responsive to antioxidant treatment (29/38, 76%) had a decrease in DNA damage from a mean of 24% to 8.2%, similar to the first study. It was suggested by the authors that alternative pathologies may be present in the ‘nonresponsive’ group and that this may also represent a source of variation in other studies, contributing to the heterogeneity of results seen in this field. It could also be argued that antioxidant treatment should be based on the individual rather than a blanket treatment regimen. With this approach, these trials suggest a very positive role for vitamin C and E in the treatment of DNA fragmentation. In addition, the clinical pregnancy rate increased remarkably and significantly after treatment, from 2/29 (both which ended in spontaneous abortion) to 14/29 (48%).
      A previous small study combined vitamin C and E with glutathione (
      • Kodama H.
      • Yamaguchi R.
      • Fukuda J.
      • Kasai H.
      • Tanaka T.
      Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients.
      ), although oral glutathione has poor bioavailability (
      • Allen J.
      • Bradley R.D.
      Effects of oral glutathione supplementation on systemic oxidative stress biomarkers in human volunteers.
      ). This study demonstrated a modest decrease in 8-OHdG values in infertile men after 2 months. The authors suggest that lack of controls and high interpatient variability do not conclusively show efficacy. However, the data shows a 50% response rate, whereby there was a more notable decrease in oxidative damage in seven participants, including the male with the highest 8-OHdG value. If the group was divided into ‘responsive’ and ‘nonresponsive’ in a manner similar to
      • Greco E.
      • Romano S.
      • Iacobelli M.
      • Ferrero S.
      • Baroni E.
      • Minasi M.G.
      • Ubaldi F.
      • Rienzi L.
      • Tesarik J.
      ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment.
      , the results may have shown a greater mean decrease in responders alone. In addition, the quantities of vitamins were much lower in this study and so the modest response may still be relevant.

      Zinc and selenium

      Zinc is an integral element in the development of spermatozoa and DNA synthesis and an important antioxidant in seminal fluid (
      • Ebisch I.M.
      • Thomas C.M.
      • Peters W.H.
      • Braat D.D.
      • Steegers-Theunissen R.P.
      The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility.
      ). It can prevent iron- and copper-mediated lipid peroxdiation and works synergistically with α-tocopherol in this regard (
      • Zago M.P.
      • Oteiza P.I.
      The antioxidant properties of zinc: interactions with iron and antioxidants.
      ). It is also required for Cu/Zn superoxide dismutase, which is the type of SOD found in high amounts in the male reproductive tract (
      • Mruk D.D.
      • Silvestrini B.
      • Mo M.Y.
      • Cheng C.Y.
      Antioxidant superoxide dismutase–a review: its function, regulation in the testis, and role in male fertility.
      ). Substantial zinc deficiency is common worldwide (
      • Wessells K.R.
      • Brown K.H.
      Estimating the global prevalence of zinc deficiency: results based on zinc availability in national food supplies and the prevalence of stunting.
      ). Testing for blood concentrations of zinc may identify deficiency as seen in oligozoospermic and azoospermic men (
      • Ali H.
      • Baig M.
      • Rana M.F.
      • Ali M.
      • Qasim R.
      • Khem A.K.
      Relationship of serum and seminal plasma zinc levels and serum testosterone in oligospermic and azoospermic infertile men.
      ), although in some cases concentrations may be normal in blood but decreased in seminal fluid under local conditions of oxidative stress (
      • Chia S.E.
      • Ong C.N.
      • Chua L.H.
      • Ho L.M.
      • Tay S.K.
      Comparison of zinc concentrations in blood and seminal plasma and the various sperm parameters between fertile and infertile men.
      ). Testing for seminal oxidative stress is not routine and protocols need to be established and tested before this becomes a reliable parameter on which to base antioxidant therapy (
      • Deepinder F.
      • Cocuzza M.
      • Agarwal A.
      Should seminal oxidative stress measurement be offered routinely to men presenting for infertility evaluation?.
      ). Hair mineral analysis may be useful for both zinc and selenium status assessment, although results require careful interpretation and more robust trials are required to prove its reliability (
      • Ashton K.
      • Hooper L.
      • Harvey L.J.
      • Hurst R.
      • Casgrain A.
      • Fairweather-Tait S.J.
      Methods of assessment of selenium status in humans: a systematic review.
      ,
      • Lowe N.M.
      • Fekete K.
      • Decsi T.
      Methods of assessment of zinc status in humans: a systematic review.
      ,
      • Namkoong S.
      • Hong S.P.
      • Kim M.H.
      • Park B.C.
      Reliability on intra-laboratory and inter-laboratory data of hair mineral analysis comparing with blood analysis.
      ).
      Selenium is an essential component of the GPX selenoproteins (
      • Tinggi U.
      Selenium: its role as antioxidant in human health.
      ). Outright selenium deficiency is rare in the USA (

      U.S. Department of Agriculture (USDA), Agriculture Research Service, 2009/2010. What we eat in America–the National Health and Nutrition Examination Survey (NHANES). Available from <http://www.ars.usda.gov/Services/docs.htm?docid = 18349>.

      ). Conversely, the National Diet and Nutrition Survey in the UK showed that intakes were approximately 70% of the recommended nutrient intake, with half of women and a fifth of men below the lower recommended limit (

      Food Standards Agency and Department of Health, 2008/2009. The National Diet and Nutrition Survey (NDNS). Available from: <http://www.food.gov.uk/multimedia/pdfs/publication/ndnsreport0809.pdf>.

      ). Intake from food can also be underestimated. Widespread deficiency of selenium in soil can result in lower selenium concentrations in foodstuffs such as meat, with daily intakes varying greatly across countries (

      European Commission Scientific Committee on Food, 2000. Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Selenium. Available from: <http://ec.europa.eu/food/fs/sc/scf/out80g_en.pdf>.

      ,
      • Niskar A.S.
      • Paschal D.C.
      • Kieszak S.M.
      • Flegal K.M.
      • Bowman B.
      • Gunter E.W.
      • Pirkle J.L.
      • Rubin C.
      • Sampson E.J.
      • McGeehin M.
      Serum selenium levels in the US population: Third National Health and Nutrition Examination Survey, 1988–1994.
      ).
      A study of mixed antioxidants including zinc and selenium by
      • Menezo Y.J.
      • Hazout A.
      • Panteix G.
      • Robert F.
      • Rollet J.
      • Cohen-Bacrie P.
      • Chapuis F.
      • Clement P.
      • Benkhalifa M.
      Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect.
      demonstrated a significant reduction of DNA fragmentation, but a worrying increase in high-density staining, indicating chromatin decondensation. After 3 months of supplementation the mean DFI decreased from 32.4% to 26.2%, below the important threshold of 30%. However, high-density staining increased from 17.5% to 21.5%, which was attributed to the ability of vitamin C to reduce disulphide bridges, as already discussed. The authors themselves have observed a threshold of 28% for high-density staining beyond which no pregnancy can be achieved. Thus they recommend that antioxidant treatment is not given to men with high-density staining >20% while below this, the risk–benefit ratio is in favour of antioxidants. It could also be argued that, while zinc and selenium may be beneficial, limiting the amount of vitamin C could prevent the increase in chromatin decondensation.
      Two additional studies have been published by an Australian group using the Menevit mixed supplement formulation, which includes vitamins C and E, zinc and selenium (
      • Tremellen K.
      • Miari G.
      • Froiland D.
      • Thompson J.
      A randomised control trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-ICSI treatment.
      ,
      • Tunc O.
      • Thompson J.
      • Tremellen K.
      Improvement in sperm DNA quality using an oral antioxidant therapy.
      ). The first study by
      • Tremellen K.
      • Miari G.
      • Froiland D.
      • Thompson J.
      A randomised control trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-ICSI treatment.
      was a double-blind randomized control trial. The outcomes reported in this publication included fertilization, embryo quality and pregnancy success. There was a significant increase in pregnancy success in the treatment group, with clinical pregnancy detected in 38.5% of embryos transferred versus 16% in the control group. The measurements of DNA fragmentation were not reported in this publication but a patent submitted for the Menevit supplement gave further information (

      Tremellen, K., 2006. Methods and compositions for improving pregnancy outcome. US Patent 20090081177.

      ). There was a nonsignificant reduction of DNA fragmentation in the treated group from 37.9% to 33.3% but a greater reduction was seen in the placebo group, from 40.3% to 32%. This was attributed to the statistical ‘regression-towards-the-mean’ phenomenon. In any case, the decrease in DNA fragmentation cannot explain the increase in pregnancy rates in this study.
      The subsequent study by this group claim to avoid the ‘regression-towards-the-mean’ phenomenon by selecting men with proven levels of seminal oxidative stress rather than those chosen for extremes of the primary outcome measured (
      • Tunc O.
      • Thompson J.
      • Tremellen K.
      Improvement in sperm DNA quality using an oral antioxidant therapy.
      ). This trial was uncontrolled and, given the results of the previous study by the same group, the evidence presented was weak. In any case, the mean decrease in DNA fragmentation was from 22.2% to 18.2%. Interestingly, however, a subgroup of participants whose partners subsequently became pregnant after IVF/ICSI (13/36, 36%) did have a more marked response, from 22% to 13.3%, indicating a possible link between improvement of DNA fragmentation and pregnancy outcome.

      l-Carnitine and coenzyme Q10

      Carnitines are found at high concentrations in the epididymis and are key to spermatozoal maturation and metabolism (
      • Agarwal A.
      • Said T.M.
      Carnitines and male infertility.
      ). l-Carnitine is also a powerful antioxidant, which can prevent lipid peroxidation, scavenge O2 and H2O2 and inhibit iron-mediated ROS production (
      • Gulcin I.
      Antioxidant and antiradical activities of l-carnitine.
      ). A systematic review by
      • Zhou X.
      • Liu F.
      • Zhai S.
      Effect of l-carnitine and/or l-acetyl-carnitine in nutrition treatment for male infertility: a systematic review.
      indicated that supplementation can improve pregnancy rates and more specifically motility. CoQ10 is a strong antioxidant in its reduced form (ubiquinol) and can prevent oxidative damage to lipids and DNA (
      • Littarru G.P.
      • Tiano L.
      Bioenergetic and antioxidant properties of coenzyme Q10: recent developments.
      ). Specifically in seminal fluid, a significant negative correlation between ubiquinol concentrations and lipid peroxidation products has been demonstrated (
      • Alleva R.
      • Scararmucci A.
      • Mantero F.
      • Bompadre S.
      • Leoni L.
      • Littarru G.P.
      The protective role of ubiquinol-10 against formation of lipid hydroperoxides in human seminal fluid.
      ).
      Only one study was found which included l-carnitine and CoQ10 in the investigation of DNA fragmentation (
      • Abad C.
      • Amengual M.J.
      • Gosalvez J.
      • Coward K.
      • Hannaoui N.
      • Benet J.
      • Garcia-Peiro A.
      • Prats J.
      Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.
      ). DNA fragmentation was significantly reduced from 28.5% to 20.12%. While this study was uncontrolled, concomitant improvements in other semen parameters suggest that this therapy was successful in this cohort rather than being an artefact. However, as with mixed antioxidants, there is no way to determine which played a significant role in altering the parameters.

      Recommendations

      As summarized in Table 5, medically modifiable factors such as leukocytospermia and varicocele should be assessed and addressed by the appropriate physician. Lifestyle factors are easily modifiable without risk in men presenting with DNA fragmentation. Lifestyle changes can therefore be readily recommended even in the absence of conclusive evidence. Dietary changes may also be carried out under the supervision of a dietician or nutritional therapist, assessing the current intakes of antioxidants particularly and incorporating foods high in vitamin C, vitamin E, zinc and selenium. Supplements should also be advised where deficiency has been detected by relevant testing. Antioxidant therapies should always be individually tailored according to deficiency or risk. DNA fragmentation should be retested after a 3-month period of treatment to allow for a full cycle of spermatogenesis.
      Table 5Modifiable factors in the treatment of oxidative stress causing DNA fragmentation.
      Medical factorLifestyle changeDietary changeSupplement
      Treatment of leukocytospermiaSmoking cessationHealthy dietVitamin C
      Surgical repair of varicoceleAvoid xenobiotic sources of reactive oxygen speciesIncrease in fruit/vegetables and sources of antioxidantsVitamin E
      Avoid heavy/toxic metalsWeight lossMixed antioxidants?
      Avoid testicular heat
      Avoid testicular mobile phone radiation

      Conclusion

      DNA fragmentation is underevaluated in male infertility, but represents an extremely important parameter indicative of infertility and potential outcome of assisted reproduction treatment. It is also important to consider the health of offspring produced by assisted reproduction procedures that bypass natural hurdles such as ICSI, allowing men with high levels of DNA damage to reproduce. Oxidative stress is the major cause of DNA fragmentation in male infertility but may be modifiable in many cases. Sources of oxidative stress as discussed herein should be analysed in men exhibiting DNA fragmentation and avoided or limited as part of a treatment protocol. Using antioxidant supplementation has shown an overall benefit, most especially vitamins C and E, but caution is advised whether because of the risk of oversupplementing or of supplementing any nutrients without determining deficiency or necessity due to the delicate balance of reduction and oxidation required for fertility and ultimately successful fertilization and ensuing pregnancy.

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