Posted on 09/23/2017 in Fertility Treatment Options

Three Main Causes for the Creation of the DNA Damage of Spermatozoon

Three Main Causes for the Creation of the DNA Damage of Spermatozoon

Abstract: Three Main Causes for the Creation of the DNA Damage of Spermatozoon: Chromatin Remodelling by Topoisomerase, Oxidative Stress and Abortive Apoptosis. Consequences and Preventing Measures of the DNA Damage of Spermatozoon in Close Scientific Focus. Perspectives of Clinical Management of DNA Damage in the Male Germ Line. The article focuses on representing the basic overview of three main causes for the creation of the DNA damage of spermatozoon: chromatin remodelling by topoisomerase, oxidative stress and abortive apoptosis. There has been substantiated and explicated the consequences and preventing measures of the DNA damage of spermatozoon. As a result of studying the main causes for the creation of the DNA damage of spermatozoon there have been revealed perspectives of clinical management of DNA damage in the male germ line.

INTRODUCTION

A comprehensive high–quality semen analysis is an essential first line investigation for the infertile couple. Traditionally, the diagnosis of male infertility is based upon microscopic assessment and analysis of sperm concentration, motility and morphology as routine indicators of human semen quality. These indicators provide fundamental information about sperm production upon which clinicians base their initial diagnosis. Semen quality is conventionally determined according to the number, motility and morphology of spermatozoa in an ejaculate. An improved understanding of sperm morphology and physiology with special emphasis on the role of integrity of the male gamete in both fertilization and embryogenesis, has led experts in reproductive medicine to an increased demand on sperm examination techniques: a good sperm examination technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte. It was theoretically established and proved through the scientific investigations that male factor represents ∼40% of all infertility cases worldwide, with additional male infertility cases possibly being misdiagnosed as idiopathic infertility [Kim and Lipshutz, 1999; Schultz and Williams, 2002]. While conventional semen evaluation by light microscopy provides a good estimate of sperm quality and fertility in most cases, more accurate, biochemical tests are sought in reproductive medicine to better evaluate the sperm quality and to properly diagnose male infertility [Eliasson, 2003].

It is well recognized that spermatozoa from infertile men possess multiple structural and functional defects. Under in–vitro conditions, these abnormalities are manifested primarily as an inability to interact with the oocyte, particularly with the zona pellucida. Such sperm–zona pellucida binding dysfunction, alone or in association with other defects, leads to a failed or sub-optimal fertilizing capacity [Liu and Gordon Baker, 1992; Oehninger et al., 1992, 1997]. Other demonstrated sperm dysfunctions associated with male infertility include isolated sperm motility defects [Mortimer et al., 1986], inadequacies in the ability of the spermatozoa to acrosome react after binding to the zona pellucida [Liu and Baker, 1994] and failures of sperm–oocyte fusion [Aitken et al., 1991]. The advent and success of intracytoplasmic sperm injection (ICSI) has removed numerous barriers of sperm selection that were mandatory for choosing those cells demonstrating superior morphological and functional attributes in assisted reproduction. Because of the concern of transmission of genetic disease through intracytoplasmic sperm injection (ICSI), recent attention has re–focused on the genomic integrity of the male gamete.

However, even with appropriate quality assurance, traditional semen parameters provide a limited degree of prognostic and diagnostic information and their predictive power is highest primarily at the lower ranges of the spectrum [Lefièvre et al., 2007; Lewis, 2007]. Sperm production is only part of the story. Sperm chromatin and DNA integrity is essential to ensure that the fertilizing sperm can support normal embryonic development of the zygote. To better inform treatment pathways and, more importantly, to ensure the “top–quality” embryo or embryos from assisted reproductive technologies (ART), the experts in reproductive medicine urgently require tests of sperm function, including the normalcy of sperm DNA, that provide high quality and robust diagnostic and prognostic information.

The clinical significance of DNA damage in the male germ line has been the subject of much discussion and the source of some confusion. There is an extensive literature addressing the relationship between DNA damage in spermatozoa and fertility, defined in a variety of ways and under a variety of circumstances including natural conception [Giwercman et al., 2010], IVF [Simon et al., 2010, 2013], ICSI [Zini, 2011; Simon et al., 2013] and IUI [Bungum et al., 2007]. The general conclusion from these data is that there appears to be a general relationship between DNA damage and fertility but the correlations are weak and of variable significance. Together with this, at present, the issues of researching the essence of male’s infertility: its causes, testing and treatment have become an interdisciplinary research mainstream focus for both paradigms of modern andrology and male reproductive endocrinology. Furthermore, in the connection with the above, the key objective of modern andrology and male reproductive endocrinology is to determine characteristic malformations of the sperm DNA ultrastructure in patients with severe subfertility undergoing intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) treatment therapy cycles.

(1)           Insight into composition and ultrastructure of the spermatozoon 

In general, composition and ultrastructure of the spermatozoon (sperm cell) can be represented as head, neck, mid–piece and tail. The sperm plasma membrane covers the head and runs to the tip of the tail. The major part of the head is occupied by a dense compact nucleus containing highly condensed paternal DNA which is capped by an acrosome and the plasma membrane. The acrosome is divided into a cap region and an equatorial region and consists of inner and outer acrosome membranes within which is the acrosomal matrix. The acrosome is a Golgi–derived organelle covering more than two–thirds of the sperm nucleus. Its membranes contain hydrolytic enzymes which play an essential role during the acrosome reaction and oocyte penetration. Below the acrosome is the post–acrosomal segment of the head where sperm egg membrane fusion usually occurs. The acrosome reaction usually takes place by vesiculation of surface membranes when multiple fusions occur between the plasma membrane and the outer acrosome membrane [Nikolettos et al., 1999].

The major components of the neck are the connecting piece just behind the nucleus and the proximal centriole which consists of nine triplet microtubules (i.e. the sperm centriole has 9+0 organization of microtubules), forming a circle with dense material within the circle and outside the centriole, called the sperm centrosome or peri–centriolar material. Segmented columns surround the proximal centriole on either side. The centriole is, after the nucleus, the most important sperm organelle for initiation of the intra–ooplasmic fertilization process, being responsible for the formation of the sperm aster [Van Blerkom and Davis, 1995].

The sperm mid–piece is the driving force of the spermatozoon and consists of a central axoneme consisting of microtubules extending from the proximal centriole to the distal tip of the tail. The microtubules of the axonemal complex are arranged in a characteristic pattern 9 + 2, i.e. nine sets of double microtubules at the periphery surrounding two single microtubules in the centre [Nikolettos et al., 1999]. Outside the peripheral doublets are nine dense fibres. Cranially, these fibres fuse with the connecting piece. The fibres facilitate sperm movement, mediated by protein phosphorylation [Tash and Means, 1983], and serve as a protector against damage during sperm transit through the male and female reproductive tracts. The movement of the tail is mediated through action of the dynein arms, resulting in sliding of the axonemal microtubules alongside one another. In the mid–piece, axoneme and outer dense fibres are ensheathed by mitochondria which are helically organized in 11–13 gyres [Zamboni et al., 1971]. Flagellar motility requires ATP, which originates from the mid–piece mitochondria and is hydrolysed by ATPase of the dynein arms in the presence of magnesium. A cytoplasmic droplet apparently indicates immature spermatozoa. The mid–piece ends with a thickening of the plasmalemma, the annulus [Nikolettos et al., 1999].

The tail consists of a principle piece and the terminal piece or end–piece. The proximal region of the principle piece has a dense fibrous sheath and seven dense fibres surrounding the axoneme. The fibres become reduced in thickness and in number in a distal direction throughout the principle piece. The terminal piece begins at the end of the fibrous sheath of the principal piece. In the proximal part of the terminal piece the double tubules of axoneme remain in typical

arrangement. In the distal part of the terminal piece the 9 + 2 order of the axoneme disappears and the tubules form a single bundle without distinct organization [Pedersen 1970].

The spermatozoon is a highly regionalized cell with localized membrane domains that have specific functions [Martinez and Morros, 1996]. The domains have heterogeneous fluidity, shape, diffusion coefficients and composition of phospholipids, glycolipids and steroids [Ladha, 1998]. There are five specialized regions: the acrosomal, equatorial, postacrosomal, midpiece and tail region [Bedford and Hoskins, 1990]. The spermatozoon membrane is a dynamic system undergoing many changes, often domain specific, as the spermatozoon passes through the reproductive tract. These maturational changes are thought to be essential for eventual fertilization of the oocyte [Joanna V. Abraham–Peskir et al., 2002].

Sperm undergo membrane changes as they mature in the epididymis. During this period, the spermatozoon’s surface is modified by integration of proteins, glycoproteins and lipids such as phosphatidylcholine [Haidl and Opper, 1997]. These lipids are significant for induction of progressive motility as well as for subsequent functions and processes such as capacitation and the acrosome reaction. Water loss from sperm also occurs as they pass through the epididymis, consequently, sperm are good osmometers. Furthermore, the packed cell volume of sperm is sensitive to osmotic pressure. The osmotic water permeability coefficient of sperm membranes is very high whilst the associated activation energy is low [Noiles et al., 1993], suggesting the presence of a porous membrane.

Cytoplasm necessary for spermatogenesis is normally eliminated from spermatids while they are still in contact with the Sertoli cell as a structure commonly called the residual body, which forms on severance of the cytoplasmic stalk. Any residual cytoplasm is eliminated from the spermatozoon flagellum at a later time, during the period within the epididymis and before the release of sperm. Cytoplasm retained abnormally as a sac or droplet at the spermatozoon midpiece is termed the cytoplasmic droplet. Its presence on sperm in semen indicates aberrant spermatogenesis and is associated with sub–fertility [Jouannet et al., 1988; Keating et al., 1997]. Thus, in freshly ejaculated semen, the incidence of sperm with a cytoplasmic droplet is low [Keating et al., 1997; Laudat et al., 1998].

The assessment of sperm morphology, an integral component of the basic semen evaluation, has been demonstrated to aid the clinician in the management of male infertility [Oehninger and Kruger, 1995]. It is clear that the evaluation of a single sperm feature or function may not provide enough power for the prediction of fertilization or implantation outcome. This reflects the complexity and multiplicity of events leading to sperm–oocyte interaction and conception. It is clear that the evaluation of a single sperm feature or function may not provide enough power for the prediction of fertilization or implantation outcome. This reflects the complexity and multiplicity of events leading to sperm–oocyte interaction and conception. Notwithstanding these considerations, sperm morphology assessed by strict criteria [Kruger et al., 1986] has been shown by multiple authors to have a high predictive value not only for the outcome of advanced assisted reproductive technologies [in–vitro fertilization (IVF) and gamete intra–Fallopian transfer (GIFT)] but also for those of intrauterine insemination and in–vivo reproduction [Kruger et al., 1988; Oehninger et al., 1988; Hinting et al., 1990; Enginsu et al., 1991; Kobayashi et al., 1991; Check et al., 1992; Eggert–Kruse et al., 1993; Grow et al., 1994; Ombelet et al., 1994; Toner et al., 1994; Grow and Oehninger, 1995; Coetzee et al., 1998; Duran et al., 1998]. A few studies, however, have not supported those findings [Coates et al., 1992; Matorras et al., 1995; Karabinus and Gelety, 1997].

The fusion of the spermatozoon with the oocyte, requires both gametes to be structurally normal, viable and functionally competent. The spermatozoon in particular must possess effective motility, as well as penetrating, fusiogenic and fertilizing capacities. The evaluation of the three most important semen parameters, i.e. sperm concentration, progressive motility and morphology, defines the capability of spermatozoa to effect oocyte fertilization [Kupker et al., 1998]. In particular, a link was established between sperm morphological characteristics and infertility. That sperm morphology plays a key role in the fertilization process was deduced by studies involving in–vitro fertilization (IVF). Sperm morphology as assessed by strict criteria is an excellent biomarker of sperm dysfunction(s) that assists the clinician in determining the source of male infertility and in predicting the outcome of assisted reproductive technologies [Acosta et al., 1989; Franken et al., 1990; Oehninger et al., 1990, 1992].

(2)           Underlying factors for the unrecognized derangements of the fertilizing capacity of spermatozoa, regardless of sperm morphology: sperm decondensation defects and DNA anomalies as the main causes of infertility, miscarriage and birth defects

Despite the ever–increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. Consequently, it was hypothesized and proved that sperm DNA integrity is one of the important determinants of normal fertilization and embryo development. However, sperm with DNA damage are capable of fertilizing an egg [Aitken et al., 1998a; Lopes et al., 1998; Gandini et al., 2004], which may explain why studies evaluating the relationship between high DNA damage and pregnancy rates have only found a modest effect on conception rates with conventional IVF and little, if any effect with intracytoplasmic sperm injection (ICSI) [Henkel et al., 2003; Larson–Cook et al., 2003; Gandini et al., 2004; Virro et al., 2004; Check et al., 2005; Zini et al., 2005a,b; Borini et al., 2006; Benchaib et al., 2007; Bungum et al., 2007; Collins et al., 2008; Frydman et al., 2008; Lin et al., 2008]. For the purposes of this paper we define sperm DNA damage as fragmentation of the sperm DNA, in the form of double or single–strand breaks which have been induced in the DNA prior to, or post, ejaculation.

Basically, sperm DNA damage is defined as fragmentation of the sperm DNA, in the form of double or single–strand breaks which have been induced in the DNA prior to, or post, ejaculation.

The scientists Tesarik J., Greco E. and Mendoza C. in their study “Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation” suggest that paternal effects on early development, prior to the activation of the embryonic genome, are mediated by centrosome dysfunction or deficiency of oocyte–activating factors and are not associated with high frequency of sperm with DNA damage. However, increased sperm DNA damage has been associated with a ‘late paternal effect’ during the activation of male gene expression and hence could give rise to an increased risk of miscarriage [Tesarik et al., 2004a].

In contrast, other studies report marked reductions in all the major early check points: fertilization, embryo quality and pregnancy rates following IVF in couples with high levels of sperm DNA damage as measured by the Comet assay [Simon et al., 2010, 2011].

What is essential to emphasize is that the major contributor to infertility, miscarriage and birth defects is DNA damage in the male germ line [Aitken et al., 2009]. This concept represents that it should be incontrovertible that DNA damage in the male germ line has the potential to disrupt the viability and developmental normality of the embryo. In support of this theory, a great deal of correlative data has been represented in recent years suggesting that DNA damage in spermatozoa is associated with the impairment of oocyte fertilization, the preimplantation development of the embryo and subsequent progress of pregnancy to term [Aitken et al., 2009; Zini and Sigman 2009]. Additionally, it should be noted that these adverse results are not consistently observed across all studies [Zini and Sigman 2009], consequently there is some scepticism about the clinical significance of such damage. However, these adverse outcomes are not consistently observed across all studies [Zini and Sigman, 2009], generating some scepticism about the clinical significance of such damage.

Sakkas D. and Alvarez J.G. in their study “Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis” postulated that DNA damage in sperm can be induced by six main mechanisms: (I) apoptosis during spermatogenesis; (II) strand breaks during chromatin remodelling during spermiogenesis; (III) post–testicular DNA fragmentation induced by oxygen free radicals during transit through the male reproductive tract; (IV) DNA fragmentation induced by endogenous endonucleases; (V) DNA damage induced by radiotherapy and chemotherapy and (VI) DNA damage induced by environmental factors such as smoking and air pollution [Sakkas and Alvarez, 2010].

There are a number of assays which are used to analyze DNA damage. Some tests measure DNA damage directly, such as TdT–mediated–dUTP nick–end labelling (TUNEL) and COMET at a neutral pH. Other tests are indirect, such as the sperm chromatin structure assay (SCSA), acridine orange test, sperm chromatin dispersion (SCD test) at acid and COMET at alkaline pH. It is worth noting that when used clinically the reported percentages of sperm with DNA damage have varied meanings between different techniques: most methods score a percentage of sperm above a certain detectable damage threshold, not a percentage of DNA damaged within a given cell; the exception to this is the COMET assay which scores the percentage of DNA damage per sperm and returns an average level of damage per sperm for the population. In COMET almost all sperm in even a fertile sperm donor population are observed to have some level of detectable damage [Simon et al., 2011]. When interpreting this miscarriage data, it is therefore important to understand that in any population of sperm the DNA damage levels per cell are heterogenous.

In general, sperm DNA fragmentation is a possible predictive parameter for male fertility status as it presupposes DNA damage in the male germ line. Sperm DNA fragmentation consists of single and double–stranded DNA breaks, frequently occurring in semen of subfertile male patients [Lopes et al., 1998; Irvine et al., 2000; Muratori et al., 2000]. Despite the origin and the mechanisms responsible for such genomic anomaly are not yet clarified, it has been proposed that sperm DNA fragmentation could be a good parameter to predict the male fertility status as an alternative or in addition to poorly predictive standard parameters presently determined in routine semen analysis [Lewis, 2007; Erenpreiss et al., 2006]. Indeed, sperm DNA breakage reflects, but not exactly overlaps, standard semen parameters [Lopes et al., 1998; Irvine et al., 2000; Muratori et al., 2000] suggesting that it is partially independent from semen quality. Results of studies aimed to establish whether the amount of sperm DNA fragmentation could predict the outcome of Assisted Reproduction Techniques (ARTs) are conflicting. The fact whether or not the amount of sperm DNA fragmentation negatively impacts on fertilization, embryo development and pregnancy rate is still matter of controversy [O’Brien and Zini, 2005; Li et al., 2006; Tarozzi et al., 2007]. Such conflicting results have been ascribed to different causes [Makhlouf and Niederberger, 2006], including poor criteria for couples recruitment, different sperm populations used for DNA fragmentation detection (unprocessed semen or selected sperm), and different techniques used to determine DNA damage [Evenson and Wixon, 2006; Li et al., 2006]. Concerning the latter point, one of the most popular technique employed to detect DNA fragmentation is terminal deoxynucleotidyl transferase (TdT)–mediated fluorescein–dUTP nick end labeling (TUNEL) coupled to flow cytometry [O’Brien and Zini, 2005], which allows detection of the phenomenon in a great number of cells.

In works by modern scientists investigating the impact of sperm DNA fragmentation on reproduction, the prevailing idea is that sperm with damaged DNA, even if retaining the ability to fertilize the oocyte [Ahmadi and Ng, 1999], affect the subsequent steps resulting in increased failure of embryo development and miscarriage [Agarwal and Allamaneni, 2004; Lewis and Aitken, 2005; Li et al., 2006]. However, data on the relationship between DNA damage and assisted reproductive technology (ART) outcome are very conflicting [O’Brien and Zini, 2005; Li et al., 2006]. In this controversial scenario, Alvarez (2005) suggested that only a deeper knowledge of the phenomenon of sperm DNA fragmentation can help in solving the issue. This author suggests that it is the time to consider, beside the amount of sperm DNA damage as a whole, if, and which, other variables affect the outcome of ART, including the DNA regions that are damaged (i.e. introns versus exons), the efficacy of the oocyte DNA repairing systems and the types of DNA damage (and thus the mechanism responsible for it) [Alvarez, 2005].

This inconsistency should not generate complacency amongst infertility specialists for a number of reasons. Firstly, DNA damage in spermatozoa is but one factor among many that will ultimately determine the outcome of a given pregnancy. In the case of DNA damage to the male germ line, much will depend on the type of damage induced, when it was induced, the region of the genome affected and the ability of the embryo to repair the damage before initiation of the S–phase that precedes the first mitotic division [Aitken et al., 2004, 2009]. Even if the embryo is not completely effective in repairing the genetic damage brought into the zygote by the fertilizing spermatozoon, the chances that a phenotype will be generated in the F1 generation are highly remote. For example, dominant genetic conditions such as achondroplasia reflect the genetic deterioration of male germ cells as a consequence of ageing [Crow, 2000].

Additionally, what is essential to mention, that of the many putative causes thus far suggested for aberrant sperm function, nuclear DNA (nDNA) damage is the most studied and is increasingly being acknowledged as a crucial factor affecting embryo quality, development and implantation [Morris et al., 2002; Henkel et al., 2003; Speyer et al., 2010]. The identification, therefore, of sperm with intact nDNA is of great importance to the success of any artificial reproduction treatment (ART) [Tomlinson et al., 2001; Duran et al., 2002; Benchaib et al., 2003]. In light of such considerations, any kind of DNA damage in the germ line should be regarded as a potential risk factor for the development of normal embryos that must be addressed in the name of ‘best practice’, if for no other reason. In this context, two questions are critical: how does the DNA damage arise and how should such damage be clinically managed? On the other hand, the clear correlation we found between sperm DNA fragmentation, and poor semen quality is in agreement with studies employing different techniques [Irvine et al., 2000; Saleh et al., 2003] or selected sperm preparations [Sun et al., 1997; Muratori et al., 2000] to reveal DNA breakage.

Even if the origin of sperm DNA fragmentation is not yet definitively clear, several mechanisms, alternative or concurrent, have been hypothesized. These mechanisms include the failure of germ cell apoptosis to complete [i.e. abortive apoptosis, Sakkas et al., 1999], an impairment in sperm chromatin packaging during spermiogenesis [Sakkas et al., 1995; Marcon and Boissonneault, 2004] and the imbalance between reactive oxygen species production and antioxidant defense in semen [Agarwal et al., 2003].

(3)           Origins of DNA damage in spermatozoa: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N. 

To date, three major mechanisms for the creation of DNA damage in the male germ line have been proposed involving chromatin remodelling by topoisomerase, oxidative stress and abortive apoptosis. It should be recognized however, that these proposed mechanisms are not mutually exclusive and, in reality, DNA damage may arise from combinations of all three mechanisms, and the possibility of different variations of such DNA damage predetermines the appearance of complex abnormalities in further embryo development. In the connection with the above, the key objective of this paper is to reveal the origins and the peculiarities of the DNA damage in spermatozoa [Aitken and De Iuliis, 2010].

(3.1)       Chromatin remodelling and DNA strand breaks in the male germ line: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N. 

One of the first hypotheses to be advanced concerning the origins of DNA damage in the male germ line, focused on the physiological strand breaks created by topoisomerase during spermiogenesis as a means of relieving the torsional stresses created as DNA is condensed and packaged into the differentiating sperm head [Sakkas et al., 1999; Marcon and Boissonneault, 2004]. Normally these strand breaks are marked by a histone phosphorylation event (gamma–H2AX; H2A histone family, member X) and fully resolved by topoisomerase before spermatozoa are released from the germinal epithelium during spermiogenesis [Aitken and De Iuliis, 2010]. Should the testes be subjected to a genotoxic stress of some kind, such as mild testicular heating or gamma irradiation, then the DNA damage induced is reflected by an increased incidence of gamma H2AX–positive foci in precursor germ cells as the DNA strand breaks are marked for repair [Hamer et al., 2003; Forand et al., 2004]. If this repair process is impaired for some reason, then, the expected outcome might be the existence of spermatozoa possessing high levels of DNA damage associated with the persistent expression of gamma–H2AX. To the author’s knowledge such persistent gamma–H2AX expression has not been reported to date for spermatozoa exhibiting high levels of DNA damage. However, a recent publication suggested that mature ejaculated human spermatozoa retain the H2AX phosphorylation machinery, presumably as a means of marking DNA strand breaks for subsequent repair in the oocyte [Li et al., 2008]. In this scientific study, treatment of spermatozoa with adriamycin resulted in the creation of double strand breaks and the concomitant expression of gamma–H2AX along with DNA maintenance/repair proteins RAD50 and 53BP1 [Li et al., 2008]. Similar results have also been reported by the same group in spermatozoa exposed to oxidative stress, such that when spermatozoa were exposed to hydrogen peroxide then H2AX phosphorylation was induced in a time– and dose–dependent manner [Li et al., 2006]. That a transcriptionally and translationally silent spermatozoon with such tightly compacted, histone–depleted chromatin, possesses the capacity to detect and mark DNA strand breaks for repair by phosphorylating H2AX is fascinating and deserves further attention. At face value such a concept runs contrary to the widely held belief that the chromatin with these cells is inert and once damaged has to wait until fertilization for repair to be effected by the embryo during a post fertilization round of DNA repair that unequivocally does involve activation of the gamma–H2AX signalling pathway [Aitken et al., 2004].

(3.2)       Oxidative stress as a major factor in male infertility: the concepts and conclusions represented in the study “The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies” written by Aitken R.J., Bronson R., Smith T.B. and De Iuliis G.N.

There is considerable evidence which points towards oxidative stress as a major factor in male infertility [Lewis and Agbaje, 2008; Tremellen, 2008; Agarwal et al., 2009; Kefer et al., 2009]. Reactive oxygen species (ROS) are principally produced by leucocytes and sperm cytoplasm [Aitken et al., 1998b]. Morphologically normal sperm will produce less reactive oxygen species (ROS) than immature sperm as the latter contain more cytoplasm. Normally, the amount of reactive oxygen species (ROS) produced is counterbalanced by endogenous antioxidant activity, but if this balance is impaired then extensive DNA damage can occur. Subfertile men appear to have lower levels of antioxidative activity than fertile men [Fraga et al., 1996; Lewis et al., 1997; Tremellen et al., 2007]. Antioxidants (such as vitamins C and E, folate, zinc, selenium, carnitine and carotenoids) are scavengers of reactive oxygen species (ROS) and therefore they have been proposed as a treatment to reverse the adverse impact of high reactive oxygen species (ROS) concentrations on semen parameters. A recent meta-analysis (Ross et al., 2010) showed an improvement in sperm motility and pregnancy rates, both spontaneous and assisted, with antioxidant use. Recently, a Cochrane Review [Showell et al., 2011] showed a statistically significant increase in live birth rate and pregnancy rate with the use of antioxidants. However, only three trials reported on live birth rate and no recommendation could be made on individual antioxidants. Some studies have also suggested an improvement in sperm motility and decreased ROS production when antioxidants are added to sperm in vitro [Pang et al., 1993; Oeda et al., 1997; Okada et al., 1997]. However, there should be some caution employed when using antioxidants as one study reported >20% increase in sperm decondensation [Menezo et al., 2007]. Perturbation of sperm chromatin structure may cause changes in paternal gene expression during preimplantation development as a result of asynchronous chromosome condensation, as well as cytoplasmic fragments in the embryo. Also excessive levels of antioxidants can be harmful; ascorbate can increase the chance of miscarriage and ascorbate and α tocopherol can, either singly or in combination, decrease sperm motility [Donnelly et al., 1999]. Therefore, although antioxidant therapy is promising, further research is required and its use should be employed with a degree of caution.

There are only two ways in which DNA strand breakage can occur – free radical attack and enzymatic cleavage. The finding that male infertility is associated with an increase in reactive oxygen species (ROS) generation originating from the spermatozoa and, occasionally, leukocytes [Aitken and Clarkson, 1987; Alvarez et al., 1987; Aitken et al., 1989; Baker and Aitken, 2004, 2005] suggested that an oxidative attack on the DNA backbone might be a possibility in defective human spermatozoa. Since such attacks are preferentially focused on guanine residues, the oxidative base adduct, 8–hydroxyl–2’–deoxyguanosine (8OHdG), was targeted and found to be present in high amounts in the spermatozoa of infertile patients [Kodama et al., 1997]. The importance of sperm preparation techniques in the genesis of this damage suggested that oxidative DNA damage could be induced in mature spermatozoa following ejaculation; it did not have to originate in the testes or epididymis [Twigg et al., 1998]. Accordingly, oxidative DNA damage can be readily induced in vitro, in otherwise normal spermatozoa, by exposure to an oxidative stress [Twigg et al., 1998; Sierens et al., 2002; Sawyer et al., 2003]. Furthermore, in careful dose–dependent studies it was demonstrated that the DNA in the sperm nucleus was more vulnerable to oxidative attack than the mechanisms regulating motility or sperm–oocyte fusion [Aitken et al., 1998]. Hence, while all aspects of sperm function will ultimately succumb to oxidative stress, DNA is particularly vulnerable. As a result, it is quite feasible to imagine situations, such as paternal smoking, where the DNA is oxidatively damaged but the spermatozoa are still competent to fertilize the oocyte and deliver their damaged payload into the oocyte [Aitken et al., 2013].

Interestingly, studies involving the chemical induction of oxidative DNA damage have emphasized that the highly compacted nuclear genome present in spermatozoa is actually quite resistant to oxidative stress when compared with somatic cells, compensating in some way for the intrinsic lack of antioxidant defence enzymes in these cells and their ineptitude at DNA repair [Sawyer et al., 2003]. When the DNA is poorly compacted, this protection, which is dependent on the close association of DNA with cysteine rich protamines [Bennetts and Aitken, 2005; Enciso et al., 2011], is lost and the cells become very susceptible to oxidative DNA damage. In keeping with this model, excellent correlations have been observed between DNA fragmentation, 8OHdG formation and impaired protamination of the sperm nucleus [De Iuliis et al., 2009].

The nature of chromatin compaction in the sperm nucleus has been excellently reviewed by Ward (2010). According to this model, the DNA is compacted into doughnut–shaped toroids that contain ∼50 kb of DNA in a semicrystalline state. Interspersed between these toroids are interlinker regions of DNA that form a close association with the nuclear matrix. Within the toroids, extensive creation of intra– and inter–molecular disulphide bridges within and between protamines during epididymal transit generates a chromatin structure that is relatively resistant to damage once the spermatozoa have achieved a state of maturity [Sawyer et al., 2003]. By contrast, the inter–toroid linker regions are histone rich and are, therefore, particularly vulnerable to attack by nucleases [Villani et al., 2010; Ward, 2010] and, presumably, reactive oxygen species (ROS). In normally compacted sperm chromatin, the DNA cleavage would, therefore, preferentially occur at the interlinker regions. However, in defective spermatozoa the chromatin is relatively histone rich [Foresta et al., 1992] creating additional areas of vulnerability that may extend into the toroid regions of the DNA in order to generate the close correlations that have been observed between poor protamination and DNA damage [De Iuliis et al., 2009b; Simon et al., 2011a, b]. Such observations have led to a ‘two–step’ hypothesis of DNA damage in spermatozoa, whereby poor protamination of sperm chromatin during spermiogenesis is held to create a state of vulnerability that is subsequently exploited in attacks on DNA integrity that are largely mediated by reactive oxygen species (ROS) [Aitken and De Iuliis, 2010]. Such a model is clearly consistent with the correlations highlighted above between DNA fragmentation, 8OHdG formation and chromatin protamination. Indeed, these associations are so strong that oxidative damage to sperm DNA can be seen as an indirect deflection of the quality of sperm chromatin remodelling during spermiogenesis [Aitken et al., 2013].

The idea that oxidative stress is responsible for the DNA damage observed in spermatozoa gathered pace after 1998, with several authors confirming the presence of significantly elevated levels of 8OHdG in the spermatozoa of infertile patients and finding weak, often inconsistent, correlations (r = ∼0.3–0.4) with conventional measures of semen quality [Shen et al., 1999; Barroso et al., 2000; Loft et al., 2003]. It was also about this time that oxidative stress in defective spermatozoa was found to be associated with markers of apoptosis including phosphatidylserine exteriorization [Barroso et al., 2000] and caspase activation [Wang et al., 2003]. Although apoptosis is conventionally associated with the release of endonucleases and the widespread induction of DNA fragmentation, this is not the case with spermatozoa [Aitken et al., 2013].

The primary reasons why spermatozoa suffer from oxidative stress are as numerous as they are complex and interdependent. They include a loss of antioxidant protection [Smith et al., 1996], the presence of free–radical–generating phagocytes in the immediate vicinity of the spermatozoa [Aitken et al., 1992; Saleh et al., 2002] and reactive oxygen species (ROS) generation by the spermatozoa themselves either from their mitochondria [Koppers et al., 2011] or, possibly, from NADPH oxidases [Aitken et al., 1997; Donà et al., 2011]. In order to integrate these observations together into a single coherent scheme, it may be instructive to reflect that reactive oxygen species (ROS) generation by spermatozoa can, itself, be induced by reactive oxygen species (ROS) in a self–perpetuating cycle [de Lamirande and Lamothe, 2009; Aitken et al., 2012]. Thus, if spermatozoa are exposed to hydrogen peroxide or are co–incubated with free–radical–generating leukocytes, then reactive oxygen species (ROS) generation by spermatozoa is stimulated [Saleh et al., 2002]. It is also important to emphasize that oxidative DNA damage (8OHdG) is correlated with other criteria for assessing DNA damage including SCSA and TUNEL [Oger et al., 2003; De Iuliis et al., 2009a, b] and is, therefore, central to the aetiology of sperm DNA damage, not merely a facet of it. Somehow, the experts have to integrate these findings on reactive oxygen species (ROS), oxidative stress, apoptosis, chromatin compaction, oxidative DNA damage and DNA fragmentation into a single coherent scheme that accounts for most, if not all, of the observations made on this phenomenon [Aitken et al., 2013].

A general scheme for the generation of DNA strand breaks in spermatozoa was presented by Aitken et al. (2013). According to this proposal a variety of genetic, metabolic, lifestyle and environmental factors can perturb the later stages of spermiogenesis resulting in the generation of defective spermatozoa characterized by poorly compacted chromatin. These defective cells will ultimately experience a state of oxidative stress driven by the mitochondrial reactive oxygen species (ROS) generated when they default to the intrinsic apoptotic cascade [Aitken et al., 2013]. The reactive oxygen species (ROS) then attack the poorly compacted chromatin generating oxidized DNA base adducts such as 8–hydroxy, 2’deoxyguanosine (8OHdG). The spermatozoon responds with oxoguanine glycosylase (OGG1) which clips the base adduct out of the chromatin generating an abasic site and a single–strand break. These changes ultimately destabilize the DNA leading to high levels of fragmentation, possibly aided by nucleases of intracellular [Sotolongo et al., 2005] or extracellular [Boaz et al., 2008] origin. PUFA = polyunsaturated fatty acid, RFEMR = radio frequency electromagnetic radiation [Aitken et al., 2013].

The basic tenet of this hypothesis is that spermatozoa experiencing oxidative stress default to an apoptotic pathway that begins by triggering enhanced reactive oxygen species (ROS) generation by the mitochondria and culminates in DNA fragmentation and cell death [Aitken et al., 2013]. As indicated above, the pathways leading to oxidative stress are numerous. They may involve local or systemic antioxidant depletion [Aitken, 1995; Gharagozloo and Aitken, 2011], exposure to radiofrequency electromagnetic radiation or heat, [De Iuliis et al., 2009a], poor differentiation during spermiogenesis resulting in excess retention of residual cytoplasm [Gomez et al., 1996] and poor chromatin compaction [De Iuliis et al., 2009b], exposure to heavy metals such as cadmium [Xu et al., 2003], prolonged culture in vitro [Muratori et al., 2003] or reproductive toxicants of various kinds [Barratt et al., 2010; Aitken and Curry, 2011]. The net effect of any of these sperm stressors is to initiate the intrinsic apoptotic cascade in these cells [Aitken et al., 2013].

The trigger here is a failure to fully maintain the phosphorylation status of PI3–kinase/AKT1 [Koppers et al., 2011]. This pathway appears to be critical to the maintenance of sperm survival because it prevents these cells from defaulting to an apoptotic state [Aitken et al., 2013]. Prosurvival factors such as insulin or prolactin serve to enhance the phosphorylation status of PI3 kinase/AKT and in this way can prolong the survival of these cells [Pujianto et al., 2010]. However, if PI3 kinase is inhibited with compounds such as wortmannin, the cells rapidly default to an apoptotic cascade characterized by rapid motility loss, mitochondrial ROS generation, caspase activation in the cytosol, phosphatidylserine exposure on the cell surface, cytoplasmic vacuolization and oxidative DNA damage [Koppers et al., 2011].

According to Aitken et al. (2013) scientific investigation, the fact that this apoptotic pathway starts with the activation of mitochondrial reactive oxygen species (ROS) generation is significant, since it explains how a wide variety of different suboptimal conditions can culminate in a state of oxidative stress. Thus, any condition that can diminish the phosphorylation status of PI3 kinase/AKT can trigger an apoptotic response by spermatozoa and one of the first signs that apoptosis has been induced is the release of mitochondrial reactive oxygen species (ROS). From this point onwards, oxidative stress becomes a self–perpetuated cascade of reactive oxygen species (ROS)–induced reactive oxygen species (ROS) production from which there is no escape. As soon as oxidative stress is initiated, the high polyunsaturated fatty acid (PUFA) content of spermatozoa ensures the rapid activation of a lipid peroxidation cascade that generates small–molecular–mass lipid aldehydes such as 4HNE, acrolein and malondialdehyde. These aldehydes are electrophilic and will rapidly form covalent bonds with the nucleophilic centres of susceptible proteins [Aitken et al., 2013]. One of the major targets for these electrophilic lipid aldehydes turns out to be the proteins of the mitochondrial electron transport chain (ETC), including succinic acid dehydrogenase [Aitken et al., 2012]. Adduction of these proteins interferes with the regulated transport of electrons along the electron transport chain (ETC), leading to the adventitious formation of superoxide anion. The latter then dismutates to hydrogen peroxide, inducing the production of yet more electrophilic lipid aldehydes that again target the ETC—and so the cycle continues. Hence, whether the initial insult to the spermatozoon is developmental, environmental or a consequence of some pathological process such as infection or diabetes, the net result is the activation of apoptosis and the creation of oxidative stress—all roads lead to an oxidative Damascus [Aitken et al., 2013].

Consequently, the intrinsic apoptotic cascade in spermatozoa also was represented. As long as protein kinase B (AKT1) is phosphorylated, the spermatozoa are viable and potential mediators of apoptosis such as Bcl–2 associated death promoter (BAD) are held in an inactivated, phosphorylated state with their keeper protein 14–3–3. As soon as AKTI is inactivated, as a result of the absence of prosurvival factors such as insulin or the presence of disruptive elements (electromagnetic radiation, toxic metabolites, environmental pollutants), pro–apoptotic factors like BAD become activated by dephosphorylation and the intrinsic apoptotic cascade is initiated. The latter involves the generation of reactive oxygen species (ROS) by the mitochondria via a self–perpetuating cycle in which lipid peroxidation generates electrophilic aldehydes that bind to the mitochondrial ETC, triggering more ROS generation and further lipid peroxidation. The net result of this oxidative stress is to induce oxidative base damage in the sperm DNA. SDH = succinic acid dehydrogenase [Aitken et al., 2013].

Several hours after the activation of mitochondrial reactive oxygen species (ROS), other classical markers of the apoptotic cascade become expressed including caspase activation and phosphatidylserine externalization [Koppers et al., 2011]. The appearance of these markers should then be associated with the release of endonucleases from the mitochondria (e.g. endonuclease G) or the activation of these enzymes in the cytosol (e.g. caspase activated DNase) followed by their migration into the sperm nucleus and enzymatic cleavage of the DNA. Although this would be a rational corollary of apoptosis in a somatic cell featuring a typical interphase nucleus surrounded by a sea of cytoplasm and mitochondria, this logic does not extend to spermatozoa [Aitken et al., 2013].

One of the latter’s unique characteristics is a highly compartmentalized architecture in which the nucleus, located in the sperm head, is physically separated from the mitochondria and a majority of the cytoplasm concentrated in the sperm midpiece [Aitken et al., 2013]. As a consequence of this arrangement, the endonucleases released and activated during apoptosis remain resolutely locked in the midpiece of the cell and never gain access to the nucleus [Koppers et al., 2011]. Even if an endonuclease did manage to gain access to the sperm nucleus, it would take some time to permeate such a dense structure and induce widespread DNA damage. The only components of the apoptotic cascade that are generated in the midpiece and can impact upon chromatin integrity in the sperm head are reactive oxygen species (ROS), such as hydrogen peroxide—a powerful, membrane-permeant oxidant capable of inducing significant damage to DNA in the sperm nucleus [Aitken et al., 2013]. The small molecular mass of such oxidants also enables them to penetrate into areas of the chromatin from which nucleases such as DNAse 1 would be excluded because of their bulk [Villani et al., 2010]. These considerations explain why most of the DNA damage in spermatozoa is oxidative [De Iuliis et al., 2009b]. Furthermore, if the primary lesion is a failure of normal chromatin compaction during spermiogenesis and the oxidative attack associated with apoptosis is subsequent to spermiation, and possibly ejaculation, there may be ample opportunities to prevent or at least limit the level of oxidative DNA damage sustained by the spermatozoa through the careful use of antioxidants and appropriate sperm preparation techniques [Greco et al., 2005; Aitken et al., vol. 26, 2011; Gharagozloo and Aitken, 2011; Aitken et al., 2013].

Spermatozoa are highly compartmentalized cells in which the nucleus is physically separated from the mitochondria and a majority of the cytoplasm. As a result, when these cells undergo apoptosis, endonucleases activated in the cytoplasm or released from the mitochondria remain resolutely locked in the midpiece of the cell (stained dark blue). The only elements of the apoptotic cascade that can impact on DNA integrity are the ROS produced by the sperm mitochondria [Aitken et al., 2013].

The only other mechanism for inducing DNA damage in spermatozoa would be the persistence of unresolved DNA nicks from the chromatin remodelling that occurs during spermiogenesis [Aitken et al., 2013]. Under normal circumstances, the strand breaks induced by topoisomerase to relieve the torsional stresses associated with DNA compaction would become labelled with gamma H2AX and repaired [Leduc et al., 2008]. It is theoretically possible that if this repair process were to be impaired in any way then the strand breaks would persist in the mature gamete. Furthermore, defects in the topoisomerase system might be associated with a failure to remodel sperm chromatin adequately, leaving spermatozoa with unresolved topoisomerase-mediated strand breaks and persistent gamma H2AX foci that would also be vulnerable to oxidative stress and apoptosis [Aitken et al., 2013]. The persistence of these gamma H2AX foci would not necessarily be associated with any signs of oxidative stress, although the latter has been proposed to directly stimulate H2AX phosphorylation in spermatozoa [Li et al., 2006]. If this is the case, then it will always be very difficult to determine whether the DNA damage seen in ejaculated spermatozoa is induced by oxidative stress arising during the terminal stages of spermiogenesis or following the release of the spermatozoa from the germinal epithelium at spermiation. Similarly, it will be difficult to determine conclusively whether any involvement of apoptosis in the aetiology of oxidative stress and DNA damage precedes or succeeds spermiation. Further studies of the persistence of gamma H2AX foci in spermatozoa and their association with oxidative stress/apoptosis markers and criteria for normal spermiogenesis such as the efficiency of chromatin compaction or the retention of excess levels of sperm cytoplasm will be needed to address this point [Aitken et al., 2013]. From a diagnostic perspective, it may not really matter when sperm DNA becomes attacked. The important question that needs to be addressed is the sensitivity and interrelatedness of the large number of assays that are currently used to detect DNA in patients’ semen samples [Aitken et al., 2013].

It was also established that spermatozoon is vulnerable to free radical attack and the induction of a lipid peroxidation process that disrupts the integrity of the plasma membrane and impairs sperm motility [Jones et al., 1978, 1979]. This susceptibility stems from the presence of targets for free radical attack in these cells including a superabundance of polyunsaturated fatty acids. The presence of unsaturated fatty acids in the plasma membrane is necessary to create the membrane fluidity required by the membrane fusion events associated with fertilization, particularly acrosomal exocytosis and fusion with the oolemma. Thus, as much as 50% of the fatty acid in a spermatozoon is docosahexaenoic acid with six double bonds per molecule [Jones et al., 1979]. Unfortunately, such highly unsaturated fatty acids are particularly prone to oxidative attack because the conjugated nature of the double bonds facilitates such processes as hydrogen abstraction, which initiates the lipid peroxidation cascade. The latter can be promoted by the presence of transition metals such as iron and copper that can vary their valency state by gaining or losing electrons. Significantly, there is sufficient free iron and copper in seminal plasma to promote lipid peroxidation once this process has been initiated [Kwenang et al., 1987]. Such transition metals can also promote the ability of reactive oxygen species (ROS) to attack another important substrate in spermatozoa – the DNA present in the sperm nucleus and mitochondria. Mitochondrial DNA is particularly vulnerable to free radical attack because it is essentially unprotected [Sawyer et al., 2001]. This vulnerability makes mitochondrial DNA a particularly sensitive marker for monitoring oxidative stress in the male germ line. However, since this DNA makes no contribution to the functionality of the spermatozoon or the subsequent development of the embryo, such damage has little biological meaning. Sperm nuclear DNA, on the other hand, is much harder to damage because it is tightly compacted with protamines that are further stabilized by the creation of inter– and intra–molecular disulphide bonds [Sawyer et al., 2003]. Nevertheless, free radicals can still attack this material, engaging in H–abstraction reactions with the ribose unit and inducing the formation of DNA base adducts. Both of these processes greatly destabilize the DNA structure and may ultimately result in the formation of DNA strand breaks. It has been known for some time that the spermatozoa of subfertile patients contain particularly high levels of 8–hydroxy–2’–deoxyguanosine (8OHdG), the major oxidized base adduct formed when DNA is subjected to attack by ROS [Kodama et al., 1997]. The experts Aitken R.J. and De Iuliis G.N. in their study “On the possible origins of DNA damage in spermatozoa” have recently not only confirmed this observation but also found the presence of 8OHdG adducts in human spermatozoa to be highly correlated with DNA strand breaks, as assessed with a TUNEL assay. Indeed, the correlation between DNA strand breaks and 8OHdG formation is so strong that it would reasonable to conclude that oxidative stress is one of the major contributors to DNA damage in the male germ line [Aitken and De Iuliis, 2010].

The scientists Aitken R.J. and De Iuliis G.N. hypothesized that the source of the oxidative stress responsible for creating DNA damage in the germ line could theoretically involve a number of factors including: (I) a loss of antioxidant protection in the male reproductive tract, (II) infection (III) xenobiotic exposure (IV) intrinsic radical production by spermatozoa [Aitken and De Iuliis, 2010].

Loss of antioxidant protection: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N. 

Antioxidant protection is particularly critical for spermatozoa because these cells are relatively deficient in ROS–scavenging enzymes as a consequence of the limited volume, and restricted distribution, of cytosolic space. As a result, these cells are particularly dependent on the antioxidant protection offered by the male reproductive tract. This is of major importance in the epididymis where spermatozoa will spend several days completing the first stages of their post–testicular maturation [Aitken and De Iuliis, 2009]. In order to protect the spermatozoa during their sojourn in the epididymis this organ secretes a complex array of antioxidant factors into the lumen of the epididymal tubules including small molecular mass free radical scavengers (vitamin C, uric acid, taurine, thioredoxin) and highly specialized extracellular antioxidant enzymes, including unique isoforms of superoxide dismutase and glutathione peroxidase, particularly glutathione peroxidase 5 (GPx5) [Vernet et al., 1996]. GPx5 is an unusual glutathione peroxidase in that it is solely expressed in the caput epididymis under androgenic control. It is also unusual in that it lacks a selenocysteine residue while still retaining its antioxidant properties [Vernet et al., 1996, 2004]. This protein associates with the sperm surface during epididymal transit and protects the spermatozoa from peroxide mediated attack as they are undergoing maturation [Vernet et al., 1996; Drevet, 2006].

Whether defects in the antioxidant protection afforded by seminal plasma is as important as the contribution made by epididymal plasma is uncertain. Unlike the epididymis, sperm spend very little time in seminal plasma. In the case of seminal plasma, the antioxidant capacity of this fluid can provide an important insight into the level of oxidative stress a given subject might be under, as in the low antioxidant capacity recorded for male smokers [Fraga et al., 1996]. In non–smoking males there is also some data to suggest that DNA damage in spermatozoa is associated with a reduction in the antioxidant capacity of semen as reflected in the levels of, for example, vitamin C [Song et al., 2006] carnitine [De Rosa et al., 2005] and co–enzyme Q10 [Mancini et al., 2005]. Similarly, the total antioxidant capacity of semen has been measured and been shown to be negatively associated with oxidative stress and fertility status [Pasqualotto et al., 2008a, b; Mahfouz et al., 2009]. Although seminal plasma is richly endowed with antioxidants that can, in vitro, protect spermatozoa from oxidative stress and DNA damage [Twigg et al., 1998; Potts et al., 2000] whether it plays a major role in vivo is open to debate. Many authors have observed that as reactive oxygen species (ROS) generation in semen goes up, seminal antioxidant capacity goes down [Song et al., 2006; Pasqualotto et al., 2008a, b; Tremellen, 2008]. It could be argued that the oxidative stress in the ejaculate was generated by the decline in antioxidant protection. However, it is just as likely that any reduction in the antioxidant status of seminal plasma is a consequence of oxidative stress, not its cause. In other words reactive oxygen species (ROS) production in the ejaculate consumes antioxidant equivalents from seminal plasma lowering the level of protection that can be afforded to the viable cells in the ejaculate [Aitken and De Iuliis, 2010]. In this context, the major culprits responsible for lowering the antioxidant capacity of semen are not the spermatozoa but infiltrating leucocytes [Aitken and Baker, 1995; Sharma et al., 2001].

Infection and leukocytic infiltration: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N. 

Every semen sample contains leukocytes, particularly neutrophils and macrophages. Because these cells are professional generators of reactive oxygen species (ROS) they can make a very significant contribution to the overall levels of reactive oxygen species (ROS) in semen [Aitken and West, 1990]. Indeed, if semen is simply diluted to remove its antioxidant activity and then cellular reactive oxygen species (ROS) generation is measured, a highly significant correlation with seminal leukocyte concentrations is observed, reflecting the fact that on a cell–by–cell basis, leukocytes are log orders of magnitude more active in the generation of reactive oxygen species (ROS) than spermatozoa [Aitken et al., 1995a, b]. Since the leukocytes are sometimes entering the seminal fluids in an activated, free radical–generating state, they are potentially capable of inducing oxidative damage in the spermatozoa. Whether this is the case depends on a number of factors such as: (I) the number and sub–type of leukocytes involved, (II) when, where and how they were activated and (III) how efficient the male reproductive tract fluids were in protecting the spermatozoa from oxidative stress. In as much as infection is the major cause of leukocytic infiltration into the male tract, in a vast majority of cases the first time the spermatozoa will come into contact with the leukocytes should be at ejaculation. At this moment, the spermatozoa should be well protected by the antioxidants present in seminal plasma [Aitken and De Iuliis, 2010]. As a result, leukocytic infiltration into the ejaculate, even to the point of leukocytospermia, should have little impact on the functionality of the spermatozoa or the levels of DNA damage in their nuclei [Aitken et al., 1995a, b; Henkel et al., 2003; Moskovtsev et al., 2007]. However, it should be noted that where the infection is chronic [Kullisaar et al., 2008] or where it is epididymal in origin [Haidl et al., 2008], then a state of oxidative stress can arise which is associated with the induction of significant DNA damage [Alvarez et al., 2002].

Leucocytes may also be instrumental in creating iatrogenic sperm DNA damage in assisted conception cycles, when the protective action of seminal plasma is removed and the spermatozoa are inadvertently co–cultured with contaminating leukocytes in media that may contain catalytic amounts of transition metals [Gomez and Aitken, 1996]. Under these circumstances, there is every possibility that leukocyte derived reactive oxygen species (ROS) will impede oocyte fertilization and development. Indeed a good prediction of in vitro fertilization success has been secured using sperm morphology and leukocyte contamination (measured with an N–formyl–methionyl–leucyl–phenylalanine provocation test) as the only independent variables in a multiple regression equation [Sukcharoen et al., 1995].

Exposure to redox–cycling compounds: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N. 

Oxidative stress and DNA damage could also be induced in the male germ line by xenobiotics that either redox cycle or activate free radical production by the spermatozoa. At present, there is very little information available on the impact of xenobiotics on free radical generation and oxidative DNA damage in spermatozoa [Aitken and De Iuliis, 2010]. Recent analyses of the impact of quinones and catechol estrogens on free radical production by spermatozoa indicated that these cells have the one electron reduction/oxidation machinery needed to activate such compounds and initiate reactive oxygen species (ROS) generation [Bennetts et al., 2008; Hughes et al., 2009]. Moreover, these studies suggest that such redox cycling activity is perfectly capable of inducing significant DNA damage in vitro. Whether patients come into contact with such compounds in sufficient quantities to induce oxidative DNA damage in the germ line, is not known at the present time. Preliminary studies are certainly suggestive [Bonde et al., 2008] particularly in the context of chemotherapy [Barton et al., 2007] but much more extensive analyses are required before general conclusions can be drawn.

(3.3)       Oxidative damage and chromatin remodelling: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N.

The induction of DNA damage may involve more than just the generation of reactive oxygen species (ROS). An important element on the oxidative stress equation is also the susceptibility of the sperm DNA to damage. Chromomycin (CMA3) has been widely used as a reagent for assessing the efficiency of chromatin remodelling during spermiogenesis. This compound competes with protamines for binding sites in the minor groove of GC–rich DNA, so that the more deficient the protamination, the greater the degree CMA3 fluorescence. Using this probe the scientists by Aitken R.J. and De Iuliis G.N. have found a very tight relationship between the efficiency of sperm chromatin protamination and the degree of oxidative DNA damage [De Iuliis et al., 2009]. This finding is in keeping with a number of studies implicating poor chromatin remodelling in the origins of DNA damage in spermatozoa [Bianchi et al., 1993; Aoki et al., 2006; Zini et al., 2007]. Moreover, these findings corroborate independent clinical data revealing that both fertilization rate and preimplantation embryonic development are negatively correlated with CMA3 fluorescence in populations of spermatozoa [Sakkas et al., 1998].

In light of these data the experts Aitken R.J. and De Iuliis G.N. have represented the most essential conceptions in their two–step hypothesis for the origins of DNA damage in the germ line [Aitken et al., 2009]. According to this hypothesis the first step in the DNA damage cascade has its origins in spermiogenesis when the DNA is being remodelled prior to condensation. Defects in the chromatin remodelling process result in the production of spermatozoa that are characterized by an overall reduction in the efficiency of protamination, an abnormal protamine 1 to protamine 2 ratio and relatively high nucleohistone content [Sakkas et al., 1998; Carrell et al., 2008; De Iuliis et al., 2009]. These defects in the chromatin remodelling process create a state of vulnerability, whereby the spermatozoa become susceptible to oxidative damage. In the second step of this DNA damage cascade, the chromatin is attacked by reactive oxygen species (ROS) [Aitken and De Iuliis 2010].

A two-step hypothesis for the origins of DNA damage in spermatozoa: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N.

In Step 1 a disruption of spermiogenesis generates defective sperm cells characterized by vulnerable chromatin. In Step 2, this vulnerable DNA is attacked by reactive oxygen species (ROS) leading to the formation of oxidized DNA base adducts and strand breaks. Although the reactive oxygen species (ROS) that mediate this attack could come from external sources such as activated leukocytes, the scientists proposed that the most common source of oxidative attack is in the form of H2O2 released from the spermatozoa’s own mitochondria. This could be part of a process of controlled senescence similar to apoptosis. HSP2A, heat shock protein 2A diaperone [Aitken and De Iuliis 2010].

The reactive oxygen species (ROS) mediating such an attack could, as indicated above, result from impaired antioxidant defenses, redox cycling xenobiotics, or free radicals generated by infiltrating leukocytes. However, a central tenet of our hypothesis is that, in a majority of cases, the reactive oxygen species (ROS) that attack the DNA come from the spermatozoa themselves and, specifically, their mitochondria [Aitken and De Iuliis 2010]. These poorly remodelled, vulnerable cells bear many of the hallmarks of cellular immaturity, particularly the retention of excess residual cytoplasm resulting in elevated cellular levels of several biochemical markers for the cytoplasmic space including creatine kinase [Aitken and De Iuliis 2010], glucose-6-phosphate dehydrogenase, superoxide dismutase and lactic acid dehydrogenase [Huszar et al., 1988, 1990; Casano et al., 1991; Aitken et al., 1994, 1996a; Gomez et al., 1996]. In addition, the experts Aitken R.J. and De Iuliis G.N. anticipated that such immature cells will possess abnormal levels of the chaperone HSP2A, impaired zona binding abilities, poor protamination and a high cellular content of unsaturated fatty acids [Ollero et al., 2001; Zini et al., 2007; Sati et al., 2008]. Given the importance of sperm mitochondria as a source of reactive oxygen species (ROS) [Koppers et al., 2008], the scientists Aitken R.J. and De Iuliis G.N. also proposed that the oxidative stress that damages sperm DNA is created by their mitochondria [Aitken and De Iuliis 2010]. They indicated with a special emphasis that mechanisms responsible for the activation of mitochondrial reactive oxygen species (ROS) generation were and are unknown. However, they hypothesized that one of the contributors to this activity might be the instigation of a limited version of the intrinsic apoptotic cascade involving the activation of mitochondrial reactive oxygen species (ROS) generation [Aitken and De Iuliis 2010]. These functionally defective, vulnerable, free radical–generating, DNA–damaged, apoptotic cells exhibiting cytoplasmic retention and a high polyunsaturated fatty acid content probably correspond to the ‘immature’ cells described by Huszar’s group [Huszar et al., 1990; Sati et al., 2008]. Unfortunately the term ‘immature’ is traditionally used to describe normal spermatozoa that have left the testes but are yet to complete their maturation in the epididymis. A better term for these cells might be ‘dysmature’ indicating an unspecified disruption in the maturation of these cells during spermiogenesis [Aitken and De Iuliis 2010].

A final thought, presented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J. and De Iuliis G.N. with respect to the mechanisms generating DNA damage is that although oxidative damage alone will create DNA strand breaks, it is also possible that the strong relationship between oxidative stress and DNA damage is indirect. Thus oxidative stress may serve to activate an endonuclease, which then induces the strands breaks associated with this process. In somatic cells it is perfectly possible for endonucleases released from the mitochondria (such as endonuclease G) or activated in the cytosol (caspase-activated deoxyribonuclease) to move into the nucleus during apoptosis and cleave the intra-nucleosomal DNA. However, in spermatozoa the physical separation of the mitochondria and cytoplasmic space from the sperm nucleus means that such mechanisms cannot be operative. Nonetheless, an alternative possibility is that the sperm chromatin contains endogenous nucleases that can be activated by stress [Aitken and De Iuliis 2010]. Recent evidence for such a chromatin-associated nuclease has been secured [Boaz et al., 2008]. It may also be significant that sperm chromatin contains at east two different forms of topoisomerase, which appear to exhibit features that distinguish them from the somatic isoforms, in terms of their molecular masses and DNA decatenation activities [Har–Vardi et al., 2007].

Sperm apoptosis: the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” written by Aitken R.J., De Iuliis G.N.

Central to this model of DNA damage in spermatozoa is the tenuous proposal that spermatozoa have to undergo a regulated form of cell death with similarities (but also differences) to the intrinsic apoptotic cascade. Spermatozoa must be capable of controlled senescence because following insemination there is a massive infiltration of leukocytes (largely neutrophils and macrophages) into the lower female tract in order to phagocytose the millions of moribund, senescent spermatozoa that did not progress to the site of fertilization [Aitken and De Iuliis 2010]. Clearly, this phagocytosis has to be silent. Thus it is vital that the phagocytes removing these cells do not generate an oxidative burst or produce pro–inflammatory cytokines that would otherwise generate a full–blown inflammatory response in response to insemination [Aitken and De Iuliis 2010]. There are many examples of silent phagocytosis in the literature and a common feature is the expression of apoptotic markers, such as phosphatidylserine, on the surface of the phagocytosed cell [Kurosaka et al., 2003].

In accordance with the scientific position of Aitken R.J. and and De Iuliis G.N. (2010), a thorough analysis of this apoptotic process in spermatozoa is beyond the scope of their brief review. In essence, the scientists emphasized that this process must be different from somatic cell apoptosis for a number of reasons including (I) these cells are transcritionally and translationally silent and so cannot undergo programmed cell death in the conventional sense, ‘regulated cell death’ might be a more appropriate term (II) the chromatin has a reduced nucleosome content due to extensive protamination and so cannot exhibit the characteristic DNA laddering seen in somatic cells (III) as discussed above, the physical architecture of these cells prevents endonucleases activated in the cytoplasm or released from the mitochondria from physically accessing the DNA [Aitken and De Iuliis 2010]. That said spermatozoa are capable of exhibiting some of the hallmarks of apoptosis including caspase activation and phosphatidylserine exposure on the surface of the cell [Weng et al., 2002]. Another element of this process which appears to be functional is the generation of ROS by sperm mitochondria [Koppers et al., 2008].

(4)           Clinical management of DNA Damage in the Male Germ Line, proposed by the scientists Aitken R.J. and De Iuliis G.N. in accordance with the results of their experimental investigations: : the concepts and conclusions represented in the study “On the possible origins of DNA damage in human spermatozoa” 

Combating oxidative stress in vitro: in an in vitro fertilization setting it is probable that contaminating leukocytes have a much greater impact on the functionality of viable spermatozoa than dead or moribund spermatozoa [Plante et al., 1994]. In this context, the oxidative stress created by the presence of activated leukocytes could be neutralized using a number of different strategies.

Leucocytes removal: firstly, contaminating leukocytes could be selectively and efficiently removed from human sperm suspensions using magnetic beads or ferrofluids coated with an antibody against the common leukocyte antigen–CD45 [Aitken et al., 1996b]. Depleting human sperm populations of leukocytes in this manner has been found to significantly enhance their capacity for fertilization [Aitken et al., 1996b].

Limit exposure to transition metals: secondly, the culture medium in which the in vitro fertilization is conducted could be carefully selected to avoid the presence of transition metals that would otherwise only serve to stimulate the ROS–mediated damage [Gomez and Aitken, 1996].

Antioxidant supplementation: thirdly, the culture medium could also be supplemented with antioxidants to scavenge any reactive oxygen species (ROS) that are generated by the leukocytes before they have an opportunity to interact with the surface of the spermatozoa. Experimental studies involving the co-culture of human spermatozoa with activated neutrophils have demonstrated the effectiveness of reduced glutathione, N–acetylcysteine, hypotaurine and catalase in this regard [Baker et al., 1996]. Glutathione and hypotaurine have also been shown to protect spermatozoa from hydrogen peroxide mediated stress by Donnelly et al. (2000). However, if this strategy is pursued, great care must be taken in selecting the most appropriate antioxidants for clinical use. Furthermore, since reactive oxygen species (ROS) play an important role in regulating the signal transduction cascades that drive sperm capacitation, we should ensure that any antioxidants employed in vitro do not compromise the fertilizing potential of these cells [De Lamirande and Gagnon, 1993; Aitken et al., 1995a, b].

Combating oxidative stress in vivo: if oxidative stress is such a prominent feature of DNA damage in spermatozoa then surely antioxidant administration should be part of the cure. Recent analyses of DNA damage in spermatozoa following exposure to various antioxidant preparations in vivo have provided some support for this concept [Greco et al., 2005; Tremellen, 2008]. Additional studies are now required involving the careful selection of patients exhibiting high levels of oxidative DNA damage in their germ line, using robust recruitment criteria such as the cellular expression of 8OHdG.

CONCLUSION

The major purpose of surveying DNA damage in spermatozoa does not entirely rest on the ability of this criterion to predict fertility. Such assays also provide important information about the underlying quality of spermatogenesis and the risk that damaged genetic material will be transmitted to the baby. There can be no doubt that DNA damage in the paternal germ line has the potential to generate mutations in the embryo that will affect the progress of pregnancy and the health and well-being of the baby. In this context, it is no surprise that DNA damage in spermatozoa is significantly correlated with impaired preimplantation embryo development as well an increase in the incidence of miscarriage in the ensuing pregnancy [Razavi et al., 2003; Zini and Sigman, 2009; Robinson et al., 2012].

Spermatozoa from infertile men have been shown to contain various nuclear alterations, including an abnormal chromatin structure, chromosomes with microdeletions, aneuploidy and DNA strand breaks. Hofmann and Hilscher (1991) reported on abnormalities of chromatin condensation secondary to retention of histones using the aniline blue staining technique (Hofmann and Hilscher, 1991). Using the sperm chromatin structure assay first described by Evenson et al. (Evenson et al., 1980), Tejada et al. related denatured DNA frequency with decreased fertility (Tejad et al., 1984). This test utilizes the metachromatic properties of acridine orange to distinguish between single (low pH- or heat-denatured) and double-stranded (native) DNA (Evenson et al., 1999). Aravindan et al. (1997) showed that human sperm cells with DNA denaturation also contain DNA strand breaks as evaluated by the alkaline comet (single-cell microgel electrophoresis) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling of DNA) assays (Aravindan et al., 1997). Manicardi et al. also reported a strong correlation between DNA damage (assessed by nick translation and terminal transferase assays) and underprotamination as examined by chromomycin (Manicardi, et al., 1995, 1998) .

Various scientific theories as the results of the investigations’ conclusions have been proposed to explain the origin of DNA damage in spermatozoa. Damage could occur at the time of, or be the result of, DNA packing during the transition of histone to protamine complex at spermiogenesis (late spermatid stage) [McPherson and Longo, 1992; Sailer et al., 1995]. Alternatively, DNA fragmentation could be the result of free radical–induced damage. DNA fragmentation in ejaculated spermatozoa (assessed by TUNEL and comet assays) has been correlated with oxidative stress (endogenous generation or exogenous stimulus) and with impaired sperm functional competence, including poor fertilization rates in in vitro fertilization (IVF) therapy cycles [Sun et al., 1997; Aitken et al., 1998; Lopes et al., 1998a,b]. Reactive oxygen species (ROS) cause peroxidative damage to the sperm plasma membrane [Alvarez et al., 1987; Aitken et al., 1989a,b; Iwasaki et al., 1992]. In addition, reactive oxygen species (ROS) are also known to attack DNA, inducing strand breaks and other oxidative–based damage in spermatozoa [Hughes et al., 1996, 1999; Kodoma et al., 1997; Twigg et al., 1998a,b]. Free radical–induced DNA damage of spermatozoa has also been associated with antioxidant depletion, presence of transition metals in the sperm culture medium, leukocyte contamination, redox cycling xenobiotics and testicular heating [Fraga et al., 1996; Lloyd et al., 1997; Shen et al., 1997; Hughes et al., 1998; Jarow, 1998; Twigg et al., 1998a,b].

Thirdly, nuclear damage could be the consequence of apoptosis. Gorczyca et al. demonstrated presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal sperm cells [Gorczyca et al., 1993]. These authors speculated that activation of the endogenous endonucleases that cause extensive DNA damage, characteristic of apoptosis in somatic cells, might be responsible for functional elimination of (possibly defective) germ cells from the reproductive pool.

Various cell types in which apoptosis is occurring, or has been induced, show a TUNEL signal that has been associated with endonuclease digestion of DNA into oligonucleosomal fragments which exhibit a characteristic ladder–like pattern when separated by electrophoresis [Gavrieli et al., 1991; Kerr, 1993]. Furthermore, most cells induced to undergo apoptosis exhibit binding of the protein annexin V to the outer leaflet of the plasma membrane due to translocation of phosphatidylserine [Martin et al., 1995]. The presence of phosphatidylserine as measured by annexin V binding is another marker of apoptosis and its detection has been reported to precede other alterations such as DNA digestion, changes in nuclear and cytoplasmic organization, and cellular fragmentation into apoptotic bodies [Van Blerkom and Davis, 1998].

It must be remembered that it is not currently possible to examine the degree of DNA damage of a spermatozoon and then to use it clinically. The degree of DNA damage of a spermatozoon cannot be assessed by any currently available method without rendering that spermatozoon unsuitable for clinical use. The single cell gel electrophoresis assay [COMET assay; Ostling and Johanson, 1984], permits assessment of DNA damage in individual somatic cells. The Comet assay has been soon modified by the scientists to enable assessment of sperm DNA, with results which are highly reproducible [Hughes et al., 1997]. Briefly, sperm membranes are lysed, protein bonds are broken and the DNA is unwound. During electrophoresis, broken strands of DNA are drawn out and a spermatozoon with damaged DNA gives the appearance of a “comet”. The head of the comet contains undamaged DNA, which does not migrate with electrophoresis, and the comet tail contains damaged DNA. The DNA is stained with ethidium bromide to facilitate analysis. Sperm comets are viewed with a fluorescence microscope and 50 images are captured onto a television monitor and then analyzed (using an image analysis package), to calculate the length and fluorescent intensity of the tail. This is proportional to the amount of damaged DNA (the comet tail) that the spermatozoon contains, and is expressed as a percentage of the total DNA of that spermatozoon. Thus, the percentages of damaged and undamaged DNA of individual spermatozoa, as well as the total damage in an ejaculate, can be determined [Steele et al., 1999].

DNA damage in spermatozoa is associated with a range of adverse clinical consequences including infertility, miscarriage and morbidity. The origins of this damage are not clearly understood but in light of recent findings we have advanced a two–step hypothesis for its possible cause. The first step involves a defect in spermiogenesis as a consequence of which cells are prematurely released from the germinal epithelium in a dysfunctional state. These ‘dysmature’ cells are distinguished by excess residual cytoplasm, significant nucleohistone presence in the chromatin, aberrant protamination, high levels of unsaturated fatty acids, and poor zona binding potential. Defective chromatin remodelling renders these cells particularly susceptible to oxidative attack and the induction of DNA damage. Moreover, these cells are proposed to have a tendency to default to a programmed pro–senescence pathway resembling apoptosis, which involves the elevated generation of reactive oxygen species (ROS) by the sperm mitochondria. Of course this model may not apply in all cases of DNA damage and other mechanisms may also contribute to the overall pathological picture. Nevertheless, it provides a theoretical framework with which to further investigate this important pathological process [Aitken and De Iuliis, 2010].

Clearly, there is no direct relationship between the status of DNA in a sperm nucleus and the fertilizing potential of the cell. The sperm nucleus is densely compacted, inert to the point of transcriptional silence and plays no active role in the processes of capacitation and fertilization [Aitken, 2013]. The scientists Aitken R.J., Gordon E., Harkiss D., Twigg J.P., Milne P., Jennings Z., Irvine D.S. have demonstrated this directly by testing the fertilizing potential of spermatozoa in vitro, while subjecting these cells to increasing levels of oxidative attack [Aitken et al., vol.59, 1998]. This study revealed that the DNA in the sperm nucleus is more sensitive to oxidative damage than the mechanisms regulating sperm fertilization. Indeed, at moderate levels of oxidative stress the DNA was extensively damaged and yet the spermatozoa exhibited an enhanced capacity for fertilization as a consequence of the redox regulation of sperm capacitation [Aitken et al., vol.59, 1998]. Furthermore, the entire field of paternally mediated reproductive risk is dependent on the fact that spermatozoa with damaged DNA, as a consequence of paternal age, lifestyle or inadvertent toxicant exposure, can still fertilize oocytes and initiate development. If there is a relationship between sperm DNA damage and the fertilizing potential of these cells, it must be indirect [Aitken et al., vol.59, 1998; Aitken et al., 2013].

One possible mechanism for such an indirect effect would be that spermatozoa experiencing very high levels of oxidative stress not only suffer from DNA fragmentation but also exhibit collateral damage to the sperm plasma membrane as a result of extensive lipid peroxidation [Aitken et al., 2013]. The latter would then be expected to precipitate a loss of motility and a reduced competence for sperm–oocyte fusion [Aitken et al., 1989]. In this context, there is an extensive literature linking measurements of oxidative stress and fertility both in vitro [du Plessis et al., 2010; Succu et al. 2011] and in vivo [Aitken et al., 1991; Sikka, 2001]. Such an oxidative stress model would explain why DNA damage in spermatozoa tends to be correlated with fertility in situations where the functional competence of the spermatozoa is severely tested (natural conception, IUI and IVF), whereas this association is weakened when ICSI is used to achieve fertilization and the spermatozoon is but a passenger in the insemination process [Thomson et al, 2011; Simon et al., 2013].

Thus, DNA damage in spermatozoa is just one attribute of sperm quality and not one that will be inevitably or inextricably linked with fertility [Aitken et al., 2013]. Perfectly normal spermatozoa, in terms of both their appearance and function, may still carry DNA damage, creating a problem when it comes to selecting spermatozoa for ICSI [Avendaño and Oehninger, 2011]. The significance of DNA damage in spermatozoa is not about predicting fertility but rather about its potential to modify the genetic constitution of the embryo. It is absolutely incontrovertible that DNA damage in the father’s germ line can influence embryonic development [Aitken et al., 2013]. Indeed, there is entire toxicology literature describing tests such as ‘the dominant lethal assay’ which are completely dependent on the way in which toxicants can influence embryogenesis by working through the father’s germ line [Singer et al., 2006]. Epidemiologically, the link between childhood cancer and paternal exposure to environmental or lifestyle factors must also involve a similar chain of cause–and–effect between DNA damage in spermatozoa and an increased genetic/epigenetic mutational load [Lee et al., 2009; Milne et al., 2012; Peters et al., 2013].

The mechanism by which DNA damage in spermatozoa influences the mutational load carried by the embryo/baby probably involves a significant degree of collusion with the oocyte. As soon as spermatozoa fertilize the oocyte, the oocyte surveys the amount of DNA damage present in the sperm chromatin and immediately launches into a round of DNA repair [Aitken et al., 2013] that precedes S–phase of the first mitotic division [Shimura et al., 2002]. If the oocyte makes a mistake, or is inefficient in effecting this repair, then the potential exists to create a mutation that will be present in every cell of the body [Aitken and Krausz, 2001; Aitken et al., 2004]. Such mechanisms could plausibly contribute to the increased burden of disease born by assisted reproductive technology (ART) children [Hansen et al., 2002; Gosden et al., 2003]. In addition to an inflated incidence of birth defects, infants produced by assisted reproductive technology (ART) are also significantly more likely to be admitted to a neonatal intensive care unit, to be hospitalized and to stay in hospital longer than their naturally conceived counterparts [reviewed by Aitken and Curry, 2011]. Thanks to the DNA–repair capacity of the oocyte, the risk of overt birth defects in assisted reproductive technology (ART) children is extremely low [Aitken et al., 2013]. Nevertheless, we cannot use this information to become complacent about the safety of ART treatments involving the use of severely damaged DNA of paternal origin [Gandini et al., 2004]. In this circumstance, absence of evidence is not evidence of absence. Only a very small percentage of DNA encodes functional genes and only a very few of those genes will generate an overt phenotype when damaged. This is why spontaneous major birth defects are so rare [Aitken et al., 2013]. However, all because a baby looks phenotypically normal, the experts cannot conclude that he/she is not carrying harmful genetic or epigenetic mutations that will cause disease later in life or in subsequent generations [Halliday, 2012].

The major purpose of surveying DNA damage in spermatozoa does not entirely rest on the ability of this criterion to predict fertility. Such assays also provide important information about the underlying quality of spermatogenesis and the risk that damaged genetic material will be transmitted to the embryo. There can be no doubt that DNA damage in the paternal germ line has the potential to generate mutations in the embryo that will affect the progress of pregnancy and the health of the baby in the future. In this context, it is no surprise that DNA damage in spermatozoa is significantly correlated with impaired preimplantation embryo development as well an increase in the incidence of miscarriage in the ensuing pregnancy [Razavi et al., 2003; Zini and Sigman, 2009; Robinson et al., 2012].

Future studies could address the fundamental molecular basis of sperm chromatin remodelling so that insights might be gained into the mechanisms responsible for the aberrant spermiogenesis seen in infertile males. Furthermore, since poor protamination (a spermiogenesis defect) and the retention of excess residual cytoplasm (a spermiation defect) are commonly encountered in defective spermatozoa, the relationship between these two processes needs to be clarified, including the triggers responsible for timing the release of spermatozoa from the germinal epithelium. The triggers for mitochondrial reactive oxygen species (ROS) generation also need to be determined and the relationship between this process and the induction of ‘apoptosis’, carefully investigated [Aitken and De Iuliis, 2010]. The role of other sources of reactive oxygen species (ROS) generation such as NOX 5 in the creation of oxidative stress in the germ line also need to be critically investigated [Bánfi et al., 2001; Baker and Aitken, 2004; De Iuliis et al., 2006]. Finally, important questions are raised by the heterogeneous nature of spermatozoa, which need to be explored in more depth [Muratori et al., 2008]. Thus there is still uncertainty as to whether indices of sperm quality such as morphology or DNA damage are of diagnostic value because they tell us about the quality of an individual gamete or because they reflect the underlying quality of the spermatogenic process. This issue has particular relevance to the practice of ICSI, where emphasis is placed on selecting spermatozoa that appear to be normal [Aitken and De Iuliis, 2010]. It may be significant that Sperm Chromatin Structure Assay values are of diagnostic significance when measured in unfractionated semen, but of no diagnostic value when performed on the washed selected cells used for insemination [Bungum et al., 2008]. Such data clearly suggest that measures of sperm DNA damage are telling us as much about the quality of the underlying spermatogenic process as the fertilizing potential of individual spermatozoa [Bungum et al., 2008; Zini and Sigman, 2009].

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