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: 

1. Apoptosis during spermatogenesis; 
2. Strand breaks during chromatin remodelling during spermiogenesis; 
3. Post–testicular DNA fragmentation induced by oxygen free radicals during transit through the male reproductive tract; 
4. DNA fragmentation induced by endogenous endonucleases; 
5. DNA damage induced by radiotherapy and chemotherapy and 
6. 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 achondropblocked="font-size: 16px;">[250] Zini A., Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J. Androl., 2009; 30: 219–229.