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- Review -

Sperm chromatin structure and male fertility: biological and clinical aspects

J. Erenpreiss^1,2, M. Spano³, J. Erenpreisa², M. Bungum^1,4, A. Giwercman¹

Abstract

Aim: Sperm chromatin/DNA integrity is essential for the accurate transmission of paternal genetic information, and normal sperm chromatin structure is important for sperm fertilizing ability. The routine examination of semen, which includes sperm concentration, motility and morphology, does not identify defects in sperm chromatin structure. The origin of sperm DNA damage and a variety of methods for its assessment are described. Evaluation of sperm DNA damage appears to be a useful tool for assessing male fertility potential both in vivo and in vitro. The possible impact of sperm DNA defects on the offspring is also discussed. (Asian J Androl 2006 Jan; 8: 11-29)

Keywords: infertility; sperm; DNA damage; human

Correspondence to: Dr Juris Erenpreiss, Fertility Centre, Malmö University Hospital, Malmö SE 205 02. Sweden
Tel: +46-4033-7830, Fax: +46-4033-7043
E-mail: Juris.Erenpreiss@med.lu.se
Received 2005-04-18 Accepted 2005-11-12
DOI: 10.1111/j.1745-7262.2006.00112.x

Infertility affects approximately 15 % of couples trying to conceive and a male cause is believed to be a sole or contributing factor in approximately half of these cases [1]. In clinical practice, the traditional, manual-visual light microscopic methods for evaluating semen quality maintain their central role in assessment of male fertility potential. However, often a definitive diagnosis of male fertility cannot be made as a result of basic semen analysis. This consists of measuring seminal volume, pH, sperm concentration, motility, morphology and vitality [2]. Abnormalities in the male genome characterized by damaged sperm DNA may be indicative for male subfertility regardless of routine semen parameters [3, 4], and these parameters do not reveal sperm DNA defects.

Focus on the genomic integrity of the male, gamete has been intensified by the growing concern about transmission of genetic diseases through intracytoplasmic sperm injection (ICSI). This technique bypasses processes of natural selection during sperm-oocyte interaction, which are still present in conventional in vitro fertilization (IVF). There are concerns relating to potential chromosomal abnormalities, congenital malformations and developmental abnormalities in ICSI-born progeny [5-8].

2 Human sperm chromatin structure

In many mammals, spermatogenesis leads to the production of highly homogenous spermatozoa. For example, mouse sperm nuclei contain more than 95 % protamines in their nucleoprotein component [9]. This allows the mature sperm nuclei to adopt a volume 40 times less than that of normal somatic nuclei [10]. The final, very compact packaging of the primary sperm DNA filament is produced by DNA-protamine complexes, which, contrary to nucleosomal organization in somatic cells provided by histones, approach the physical limits of molecular compaction [11]. Human sperm nuclei, however, contain considerably fewer protamines (approximately 85 %) than those of bull, stallion, hamster and mouse [12, 13]. Human sperm chromatin, therefore, is less regularly compacted and frequently contains DNA strand breaks [14, 15].

To achieve this uniquely condensed state, sperm DNA must be organized in a specific manner, which differs substantially from that of somatic cells [10]. The fundamental packaging unit of mammalian sperm chromatin is a toroid containing 50-60 kb of DNA. Individual toroids represent the DNA loop-domains highly condensed by protamins and fixed at the nuclear matrix; toroids are cross-linked by disulfide bonds, formed by oxidation of sulfhydryl groups of cysteine present in the protamins [11, 16]. Thus, each chromosome represents a garland of toroids, and all 23 chromosomes are clustered by centromeres into a compact chromocenter positioned well inside the nucleus with telomere ends united into dimers exposed to the nuclear periphery [17, 18]. This condensed, insoluble and highly organized nature of sperm chromatin acts to protect genetic integrity during transport of the paternal genome through the male and female reproductive tracts. It also ensures that the paternal DNA is delivered in the form that sterically allows the proper fusion of two gametic genomes and enables the developing embryo to correctly express the genetic information [18-20].

In comparison with other species [21], human sperm chromatin packaging is exceptionally variable, both within and between men. This variability has been mostly attributed to its basic protein component. The retention of 15 % histones, which are less basic than protamines, leads to the formation of a less compact chromatin structure [13]. Moreover, in contrast to the bull, cat, boar and ram, whose spermatozoa contain only one type of protamine (P1), human and mouse spermatozoa contain a second type of protamine (P2), which is deficient in cysteine residues [22]. Consequently, the disulfide cross-linking responsible for more stable packaging is diminished in human sperm as compared to species containing P1 alone [23]. It is noteworthy that altered P1/P2 ratio and the absence of P2 are associated with human male fertility problems [24-31].

3 Origin of sperm DNA damage

DNA fragmentation is characterized by both single and double DNA strand breaks, and is particularly frequent in the ejaculates of subfertile men [15]. Oocytes and early embryos have been shown to repair sperm DNA damage [32, 33]. Consequently, the biological effect of abnormal sperm chromatin structure depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it.

Abnormal sperm chromatin/DNA structure is thought to arise from four potential sources: 1) deficiencies in recombination during spermatogenesis, which usually lead to cell abortion; 2) abnormal spermatid maturation (protamination disturbances); 3) abortive apoptosis; and 4) oxidative stress [14, 34].

3.1 Deficiencies in recombination

Meiotic crossing-over is associated with the genetically programmed introduction of DNA double strand breaks (DSBs) by specific nucleases of the SPO11 family [35]. These DNA DSBs should be ligated until the end of meiosis I. Normally the recombination checkpoint in the meiotic prophase does not allow meiotic division I to proceed until the DNA is fully repaired or ablates defective spermatocytes [35, 36]. A defective checkpoint may lead to persistent sperm DNA fragmentation in ejaculated spermatozoa. However, direct data for this hypothesis in humans is lacking.

3.2 Abnormal spermatid maturation

Stage-specific introduction of transient DNA strand breaks during spermiogenesis has been also described [37-39]. DNA breaks have been found in round and elongating spermatids. DNA breaks are necessary for transient relief of torsional stress, favoring casting off of the nucleosome histone cores, and aiding their replacement with transitional proteins and protamines during maturation in elongating spermatids [37, 39-41]. DNase I-hypersensitive sites were found to be localized throughout the maturing spermatid nuclei or in a graduated manner, increasing from the anterior to posterior pole of the spermatid nucleus, mirroring the pattern of chromatin re-packaging and condensation [40]. Subsequently, their native DNA superhelicity was found to decrease from the anterior to posterior pole as detected by the acridine orange test (AOT) [42]. Thus, chromatin re-packaging includes a sensitive step necessitating endogenous nuclease activity, which is evidently fulfilled by coordinated loosening of the chromatin by histone hyper-acetylation and introduction of breaks by topoisomerase II (topo II), capable of both creating and ligating breaks [40, 41]. Chromatin packaging around the new protamine cores is completed and DNA integrity restored during epididymal transit [42]. Although there is little evidence that spermatid maturation-associated DNA breaks are fully ligated, biologically broken DNA ends should not be allowed [43]. Ligation of DNA breaks is necessary not only for preserving the integrity of the primary DNA structure but also for reassembly of the important unit of genome expression, the DNA loop-domain. However, if these temporary breaks are not repaired, DNA fragmentation in ejaculated spermatozoa may occur.

In practice, in sperm DNA, contrary to somatic cell DNA, it is nearly impossible to distinguish single strand breaks from DSBs [44]. A huge radiation dosage of 30 Gy or more is necessary to produce detectable levels of X-ray-induced damage in elongated spermatids [45]. This is probably due to the uniquely tight chromatin packaging produced by protamines [38, 44]. The link between disturbances in chromatin packaging and the consequent occurrence of DNA strand breaks is also confirmed in knock-out mouse models defective in the expression of transition proteins and protamines [46-52].

It should be noted that elongated spermatids are enriched in both alkali-labile [53] and DNase I-hypersensitive sites [40], which evidently represent the same sensitive chromatin conformation. DNase I-sensitive sites are formed in pachytene in the chromatin domain containing protamine 1 (P1) and protamine 1 (P2) and the transition protein Tnp2 genes, in the histone-enriched region. This configuration is necessary to induce transcription of these genes, however, it is also preserved in mature sperm [54].

The other methodical approach showed that human sperm DNA, compared to leukocytes, is enriched in segments of partially denatured DNA, which can also be considered alkali-sensitive sites [55]. These sites represent potential DNA breaks if induced by any factors. Although protected by proper chromatin packaging [53], the relative spermatid DNA/chromatin conformational fragility may be responsible for the presence of higher levels of spontaneous DNA damage in sperm than in somatic cells [45]. In addition, elongating chromatids have a lower repair capacity for strand breaks [56].

Enzymatic activity involved in the creation of DNA breaks in spermatids has only been proven (by decatenating activity and specific inhibition) for topo II generating and ligating DSBs [37, 41, 57]. Re-modelling of chromatin by histone H4 hyperacetylation weakens the ionic interactions between the DNA and histone cores and is needed for topo II activity to be introduced in spermatids [57]. The presence of DNase I in acrosome vesicles, from their initial formation in early spermatids to their presence in mature sperm, was shown in rats [58]. The ability of spermatozoa to use it and to digest their own DNA, if exposed to stressful conditions, has been suggested [59].

3.3 Abortive apoptosis

An alternative etiology for the DNA DSBs in the spermatozoa of infertile patients can arise through an abor tive apoptotic pathway. Apoptosis of testicular germ cells occurs normally throughout life, controlling their overproliferation [60, 61]. It has been suggested that an early apoptotic pathway, initiated in spermatogonia and spermatocytes, is mediated by Fas protein. Fas is a type I membrane protein that belongs to the tumour necrosis factor-nerve growth factor receptor family [62, 63]. It has been shown that Sertoli cells express Fas ligand, which by binding to Fas leads to cell death through apoptosis [62], limiting the size of the germ cell population to numbers Sertoli cells can support [61]. Ligation of Fas ligand to Fas in the cellular membrane triggers the activation of caspases, therefore this pathway is also characterized as a caspase-induced apoptosis [64]. Men exhibiting deficiencies in their semen profile often possess a large number of spermatozoa bearing Fas. This fact prompts the suggestion that these dysfunctional cells are the product of an incomplete apoptotic cascade [14]. However, the contribution of aborted apoptosis in the DNA damage seen in the ejaculated spermatozoa is doubtful in cases where this process is initiated at the early stages of spermatogenesis. This is because that at the stage of DNA fragmentation apoptosis is an irreversible process [65] and these cells should be digested by Sertoli cells and removed from the pool of ejaculated sperm. Some studies have not found correlations between DNA damage and Fas expression [66], or, in contrast, have not revealed ultrastructural evidence for the association of apoptosis with DNA damage in sperm [67].

Alternatively, if the apoptotic cascade is initiated at the round spermatid phase, when transcription (and mitochondria) are still active, abortive apoptosis might be an origin of the DNA breaks. Bcl2 anti-apoptotic family gene member Bclw has been shown to be suppressing apoptosis in elongating spermatids [68].

Although many apoptotic biomarkers have been found in the mature male gamete, particularly in infertile men, their definitive association with DNA fragmentation remains elusive [69-78].

3.4 Oxidative stress

Reactive oxygen species (ROS) play an important physiological role, modulating gene and protein activities vital for sperm proliferation, differentiation and function. In the semen of fertile men the amount of ROS generation is properly controlled by seminal antioxidants. The pathogenic effects of ROS occur when they are produced in excess of the antioxidant capabilities of the male reproductive tract or seminal plasma [79]. Morphologically abnormal spermatozoa (with residual cytoplasm, in particular) and leukocytes are the main source of excess ROS generation in semen [79]. It seems that sperm DNA is more prone to leukocyte-induced ROS damage in infertile men with abnormal semen parameters likely possessing "masked" DNA damage and/or more fragile chromatin structure which are under the sensitivity threshold of the assays used for the sperm DNA damage assessment [80]. Such samples from infertile men frequently show depressed fertilization rates in vitro associated with the DNA damage [81].

Processes leading to DNA damage in ejaculated sperm are inter-related. For example, defective spermatid protamination and disulphide bridge formation because of inadequate oxidation of thiols during epididymal transit, resulting in diminished sperm chromatin packaging, makes sperm cells more vulnerable to ROS-induced DNA fragmentation. The origin and interaction of different sources of sperm DNA damage is shown schematically in Figure 1.

4 Assessment of sperm chromatin structure

Several assays have been developed to evaluate sperm chromatin/DNA integrity, and their capability to assess male fertility potential has been under active scrutiny [34, 82-86]. In general, all assays can be divided into three groups: 1) sperm chromatin structural probes, 2) tests for direct assessment of sperm DNA fragmentation, and 3) sperm nuclear matrix assays (see Table 1).

4.1 Chromatin structural probes using nuclear dyes

Chromatin structural probes using nuclear dyes are both sensitive and simple to use and therefore attractive for clinical use. Their cytochemical bases, however, are rather complex. Several factors influence the staining of the chromatin by planar ionic dyes: 1) secondary structure of DNA, 2) regularity and density of chromatin packaging, and 3) binding of DNA to chromatin proteins.

4.1.1 DNA secondary structure and conformation

Fragmented DNA is easily denatured [87]. However, even a single DNA strand break causes conformational transition of the DNA loop-domain from a supercoiled state to a relaxed state. Supercoiled DNA avidly takes up intercalating dyes (like acridine orange [AO]) because this reduces the free energy of torsion stress. In contrast, the affinity for intercalation is low in relaxed DNA and is lost in fragmented DNA. In this case, an external mecha nism of dye binding to DNA phosphate residues and dye polymerization (metachromasy) is favored [88, 89]. Nevertheless, fragmentation of DNA is not the only factor affecting the determination between metachromatic versus orthochromatic staining. Chromatin packaging density also influences this balance.

4.1.2 Chromatin packaging density

If the chromatin is regularly arranged and sufficiently densely packed, dye co-planar polymerization providing metachromatic shift (change of color) is favored [90, 91]. However, if the chromatin is packaged even more densely (as in normal sperm), the polymerization of the dye is hindered [92] and may even prevent dye binding, especially by large, bulky dyes at an unfavorable pH. The latter case is seen with aniline blue (AB) at low pH where it stains basic proteins loosely associated with DNA and is unable to bind to the chromatin of normal sperm, which is very densely packaged. Explanations of how protamine molecules interact to facilitate DNA condensation and toroid formation have only been published recently [93-95]. Substitution of histones to more cationic protamines occurring during spermiogenesis neutralizes DNA charge and decreases the accessibility of DNA-specific dyes. Thus, the fluorescence staining intensity of a haploid sperm is much lower than the fluorescence intensity of a haploid round spermatid. However, after removal of nuclear proteins (e.g., by acid extraction), the net gain of stainability of sperm DNA can vary depending on the chemical structure of the fluorescent probe and from the type binding the dye forms with the DNA substrate [96].

4.1.3 Chromatin proteins

Chromatin proteins affect the binding of DNA dyes in the way that they themselves bind differently to relaxed/fragmented or supercoiled DNA. DNA supercoiling requires covalent binding of some nuclear matrix proteins and tighter ionic interactions between DNA and chromatin proteins to support negative supercoils [97]. Relaxed/fragmented DNA has looser ionic interactions with chromatin proteins, which can be more easily displaced from the DNA, thus favoring external metachromatic binding of the dye to DNA phosphate groups. Both mechanisms of dye binding, external and intercalating, compete within each constraint loop-domain (toroid) depending on its conformational state.

Since the 1960s it has been known that DNA is more prone to denaturation by heat or low pH in sperm nuclei with abnormal chromatin structure [98, 99], as shown by AO. This test has been applied using flow cytometry as the sperm chromatin structural assay (SCSA) [100], which has been shown to have a predictive value for both in vivo and in vitro fertilization [101-104]. Tejada et al. [105] introduced the microscopic AOT, a simplified fluorescent microscopic method using acid fixative that does not require flow cytometry equipment. Both SCSA and AOT measure the susceptibility of sperm nuclear DNA to acid-induced conformational transition in situ by quantifying the metachromatic shift of AO fluorescence from green (native DNA) to red (denatured or relaxed DNA).

Chromatin proteins in sperm nuclei with impaired DNA appear to be more accessible to binding with the acidic dye, as found by the AB test [42, 106]. An increase in the ability to stain sperm by acid AB indicates a looser chromatin packaging and increased accessibility of the basic groups of the nucleoprotein. This is due to the presence of residual histones [107] and correlates well with the AOT [42, 108]. Chromomycin-A₃(CMA₃) is another staining technique, which has been used as a measure of sperm chromatin condensation anomalies. CMA₃ is a fluorochrome specific for GC-rich sequences and is believed to compete with protamines for association with DNA. The extent of staining is therefore related to the degree of protamination of mature spermatozoa [109, 110].

In turn, it can be inferred that the phosphate residues of sperm DNA in nuclei with loosely packed chromatin and/or impaired DNA will be more liable to binding with basic dyes. Such conclusions were also deduced from the results of staining with basic dyes, such as toluidine blue (TB), methyl green and Giemsa stain[42, 110-112].

The most widely used techniques for sperm chromatin structure assessment are the SCSA [100-104], AO [105, 113-115] and TB tests [42, 116-122].

4.2 Tests for direct assessment of sperm DNA fragmentation

The most widely used of these tests are in situ nick translation assays, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) and single-cell gel electrophoresis assay (COMET). Their basic principles are well described elsewhere [15, 109, 121-133] and are summarized in Table 1. Nick translation is a relatively simple assay for fluorescence microscopy that quantifies the incorporation of biotinylated dUTP at single-stranded DNA breaks in a reaction catalyzed by the template-dependent enzyme, DNA polymerase I. The TUNEL assay quantifies the same incorporation at breaks in double-stranded DNA using a reaction catalyzed by terminal deoxynucleotidyl transferase. TUNEL can be applied in both bright field and fluorescence microscopy, and also using flow cytometry. The COMET assay quantifies single- and/or double-stranded DNA breaks (dependant on the pH conditions, see Table 1), using single-cell electrophoresis of spermatozoa stained with a fluorescent DNA-binding dye. It is therefore suggested as a very sensitive assay for DNA damage evaluation.

4.3 Sperm nuclear matrix assays

Two similar assays have been described that can be allocated to this group. The sperm nuclear matrix stability assay and the sperm chromatin dispersion test are based on the ability of intact DNA deprived of chromatin proteins to loop around the sperm nucleus carcass [134-136]. These two recently described assays are at the developmental stage and no studies verifying their usefulness in routine clinical practice have been reported to date. However, published data show that germ-line mutations in the nuclear matrix protein may lead to deficient DNA repair and chromatin organisation [137], so matrix pathologies can impair fertility and should be considered in future.

The assays’ principles, as well as the advantages and disadvantages of assays from all three groups, are described in Table 1.

5 Clinical significance of sperm DNA damage

5.1 Relationship of DNA damage to other semen parameters

Relationships between sperm chromatin/DNA damage and conventional semen analysis parameters are summarized in Table 2.

Although some studies have reported either only a weak or no correlation between conventional semen parameters and sperm DNA damage, most of them do indicate that spermatozoa from patients with abnormal sperm count, morphology and motility have increased levels of DNA damage. It can be hypothesized that both testicular and extratesticular factors (see also Figure 1) contribute to the final load of sperm DNA damage in ejaculated sperm, therefore it is not surprising that different studies have found various correlation levels with other parameters of sperm quality. If we assume that DNA damage in a particular patient arises solely from the failure to repair DNA breaks introduced during sperma-togenesis, one could logically expect that it would also correlate well with other indices of spermatogenic failure, like oligozoospermia and teratozoospermia. Alternatively, if sperm DNA damage is mostly as a result of the adverse effects of ROS, then a relationship to sperm motility could be expected. This factor is also affected by ROS, due to the lipid peroxidation of sperm membranes rich with unsaturated fatty acids. In fact some studies report a correlation solely between sperm DNA damage and motility [140, 145]. However, it should be remembered that these processes are inter-related. Unrepaired DNA DSBs can lead to defective sperm packaging which, in turn, as a consequence of persistent DNA fragmentation or due to the other reasons, can cause increased access to ROS attack. It is therefore understandable that clear unimodel patterns are not evident among the various published reports when different study populations with varying causes of sperm DNA damage were investigated.

5.2 Natural conception

Available studies clearly indicate a significant impact on in vivo fertilization from sperm DNA damage. Many studies, using a variety of techniques, have shown significant differences in sperm DNA damage levels between fertile and infertile men [102, 103, 139-141, 147]. The probability of fertilization in vivo seems to be close to zero if the proportion of sperm cells with DNA damage exceeds 30 % as detected by SCSA [101, 102].

5.3 Intrauterine insemination (IUI)

The probability of fertilization by IUI also seems to be close to zero if the proportion of sperm cells with DNA damage exceeds 30 % by means of SCSA [104, 144]. In addition, it has been shown that when semen samples containing > 12 % sperm with fragmented DNA (as detected by TUNEL assay) were used for insemination, no pregnancies were achieved [148].

Therefore, sperm DNA damage assessment has a high predictive value for the outcome of both natural conception and IUI.

5.4 In vitro fertilization (IVF)

The results from assisted reproductive techniques (ART) (IVF and intracytoplasmic sperm injection [ICSI]) in connection to sperm DNA damage are more controversial.

Sperm DNA damage was reported to show a significant negative correlation with embryo quality in IVF cycles [149]. Several authors have also reported significant correlations between sperm DNA damage and blastocyst development following IVF [150], and sperm DNA damage and fertilization rates following IVF [151] and ICSI [128], even though sperm DNA damage may not necessarily preclude fertilization and pronucleus formation during ICSI [124]. It has also been reported that a sperm DNA fragmentation index (DFI) predictive threshold of 27 %, detected by SCSA, is necessary to obtain a successful pregnancy both by IVF and ICSI [152, 153]. However, these results could not be repeated either by the same authors [138] or by other research groups [104, 154, 155], demonstrating that successful pregnancies in IVF/ICSI cycles can even be obtained using semen samples with a high proportion of DNA damage. Nevertheless, a study by Virro et al. [138] showed that men with DFI ³ 30 % were at risk for low blastocyst rates and no ongoing pregnancies when IVF/ICSI were performed. The study by Bungum et al. [104] did not find such a difference between groups of men with low and high DFI proportions, however, it demonstrated that significantly higher clinical pregnancy rates (52.9 % vs. 22.2 %) and delivery rates (47.1 % vs. 22.2 %) were seen after ICSI compared with IVF when semen samples with high levels of sperm DNA damage were used. In this study, when DFI exceeded 27 % the odds ratio for a positive reproductive outcome after ICSI compared with standard IVF was 8 for biochemical pregnancy, 4 for clinical pregnancy and 3 for delivery. This data is in agreement with other reports showing that sperm DNA damage is more predictive in IVF and much less so in ICSI [151, 153, 156]. Apparently, sperm chromatin integrity, evaluated on neat semen, becomes particularly relevant when contact between the two gametes occurs in a more natural way when selective pressures operate to avoid the development of an embryo derived from sperm with a high load of genetic damage [157]. On the other hand, it is not surprising that the ICSI procedure, which bypasses normal egg-sperm interactions, and was initially developed for men with very seriously impaired semen parameters [158], allows even very low quality sperm to initiate a successful pregnancy. Pregnancies by ICSI using testicular spermatids have been reported [159-164], which stresses the fact that ICSI can lead to preg nancy regardless of traditional sperm quality parameters and sperm chromatin structural integrity. As it is likely that sperm with high DNA damage levels contributes to successful fertilization and in vitro development, the potential adverse effects when sperm with high loads of DNA damage are used still remain to be clarified.

5.5 Embryonal loss

Adverse male-mediated developmental outcomes can occur if the fertilizing sperm has a defective genome with, for example, DNA strand breaks. Depending on the severity of the genetic damage and the ability of the oocyte to repair it, the embryo may fail at any stages of pregnancy or might develop to term with abnormalities. Studies of miscarriages may be a feasible and sensitive approach to increase knowledge on male-mediated developmental toxicity. However, data on miscarriages as a possible consequence of sperm DNA damage is rather scarce. Whether conventional measures of semen quality are related to embryonic loss or not, sporadic but suggestive clues have been offered [165, 166]. It has been shown that the proportion of sperm with DNA damage (as detected by TUNEL) is significantly higher in men from couples with recurrent pregnancy loss(38.0 ± 4.2 %), compared with the general population (22.0 ± 2.0 %) or fertile donors (11.9 ± 1.0 %) [167]. It has also been reported that 39 % of miscarriages could be predicted using a combination of selected cut-off values for percentage spermatozoa with denaturated (likely fragmented) DNA and/or abnormal chromatin packaging as assessed by SCSA [101]. In this study, 7 of 18 men from couples that had experienced miscarriages had an increased sperm DNA fragmentation index or percentage of immature sperm cells as detected by SCSA. The study by Virro et al. [138] also showed an increased trend of spontaneous abortions following IVF/ICSI when sperms from men with high loads of damaged DNA, as detected by SCSA, were used. Recently, the SCSA test was performed on 106 male partners from couples failing to have a successful pregnancy despite at least two previous IVF attempts. Authors found that DFI ³ 30 % was associated with a trend for lower ongoing pregnancy rates especially related to a high miscarriage rate [155]. The activation of embryonic genome expression occurs at the four- to eight-cell stage in human embryos [168], suggesting that the paternal genome may not be effective until that stage, therefore we can speculate that an elevated level of sperm DNA strand breaks seems to be of importance in the later stages of embryonic development [169]. In conclusion, it is possible that sperm DNA damage assessment could be a good predictor of possible miscarriages, which are dependent on the male factor. However, the findings mentioned above should be supported by more extended studies.

5.6 Effect of sperm DNA quality on offspring

Sperm DNA damage can affect the health of the embryo, fetus, and offspring [165, 166, 170, 171]. A possible consequence of sperm DNA damage is infertility in the offspring [172-174].

One concern raised from studies of smokers is the increased risk of childhood cancer in the offspring of men with a high proportion of sperm DNA fragmentation in their semen. It was shown that the offspring of these men, whose ejaculates are under oxidative stress [109] and whose semen is characterized by high chromatin fragmentation, are four to five times more likely to develop childhood cancer than the children of non-smoking fathers [175]. Another study has demonstrated that 15 % of all childhood cancers are directly attributable to paternal smoking [176]. However, the linkage between sperm DNA damage and abnormalities in offspring is not confined to smokers. For example, powerful associations exist between childhood disease and paternal occupation [177].

Of particular concern is recent data showing that ICSI is able to overcome the normal barrier of high loads of sperm DNA damage and initiate a successful pregnancy when this would hardly be possible through natural conception, IUI, or even to some extent IVF. The safety of the ICSI procedure has been questioned [178], and findings from the latest studies [104, 140] provide further reason for concern. Aitken and Krausz [174] proposed that sperm DNA damage is promutagenic and can give rise to mutations after fertilization, as the oocyte attempts to repair DNA damage prior to the initiation of the first cleavage. Mutations occurring at this point will be fixed in the germline and may be responsible for the induction of not only such pathologies as described above (infertility and childhood cancer in the offspring), but also for a higher risk of imprinting diseases [179, 180]. So far, however, follow-up studies of children born after ICSI compared with children born after conventional IVF have not been conclusive regarding the risks of congenital malformations, imprinting diseases and health prob lems in general [5, 181-189]. The recent meta-analysis of 25 studies addressing the prevalence of birth defects in infants conceived following IVF and/or ICSI compared with spontaneously conceived infants demonstrated that two-thirds of these studies show a 25 % or greater increased risk of birth defects in infants conceived through ART [190].

6 Suggestions for a clinical approach

Without doubt the existing data justify the necessity to introduce sperm DNA damage assessment into the routine infertility investigation. Some cases of unexplained or idiopathic infertility, when a traditional semen analysis falls into normal range and no evident female reproductive system pathologies can be revealed, will probably meet an explanation. In addition, the ART method of choice can be recommended based on sperm DNA damage assessment. It is clear that the chance of conception using IUI is negligible if the sperm DFI as detected by SCSA exceeds 30 %, and these couples should be transferred to either IVF or ICSI. DFI can therefore be used as an independent predictor of pregnancy and birth in couples undergoing IUI [104]. In addition, an extended study by Bungum et al. (personal commu-nication), including a large study population from ART cycles, presents preliminary data [104] that exceeding the 30 % DFI threshold as detected by SCSA is not compatible with in vivo fertilization by means of IUI. They also report that even though high DFI does not exclude successful treatment by means of IVF, ICSI is far more successful compared with IVF in these cases.

Therefore, a considerable number of patients can benefit from improved male infertility diagnosis and prognosis by means of sperm DNA damage assessment, enabling them to avoid unnecessary medical interventions with a very low chance of success (IUI when DFI > 30 %), and giving them the opportunity to choose a method with the highest chance of success (ICSI when DFI > 30%). However, it should be kept in mind that IVF, and especially ICSI, are able to overcome the natural barriers of sperm DNA damage levels not compatible with fertilization under natural circumstances, and the consequences of this for the progeny are still not clear. Further studies are needed in order to investigate whether treatment modalities as administration of antioxidants (Greco et al., 2005[191]) to men with high DFI, can play a role in infertility treatment.

A suggestive clinical approach flow chart for infertile couples is shown in Figure 2.

It has to be mentioned that at the moment SCSA is the only method which has demonstrated clear clinically useful cut-off levels between fertile and infertile men [101, 102], and its prognostic value for ART has also been shown [104, 138]. The undisputed advantages of this technique are its robustness and small intra- and inter-assay variations [122, 145, Spano and Giwercman, unpublished data].

SCSA is not yet very common in andrological laboratories worldwide. However, alternative and cheaper tests of the same clinical value for measuring sperm DNA damage are not yet available. Our studies show that the TB test [42, 121, 122] has potential to become a robust assay and the search for clinically valuable predictive thresholds both in vivo and in vitro is currently under investigation.

Whether sperm DNA damage can be decreased by some treatment modalities, allowing these couples to switch from ICSI to IVF/IUI or even achieve a pregnancy in a natural way, remains to be elucidated.

7 Conclusion

Normal structure of sperm chromatin is essential for the fertilizing ability of spermatozoa in vivo. It is a relatively independent measure of semen quality that yields diagnostic and prognostic information complementary to, but distinct from, that obtained from standard sperm parameters (concentration, motility and morphology). Accumulated data allows sperm DNA damage assessment to be recommended among routine tests for infertility investigations. Several methods are used to assess sperm chromatin/DNA status. SCSA is currently the only method that has provided clear clinical cut-off levels and that can be recommended for a robust sperm DNA damage evaluation. The normality ranges and predictive thresholds for male fertility potential of the other assays discussed still need to be established or clarified. It seems that ART, especially ICSI, are able to overcome the natural barriers of sperm DNA damage levels not compatible with fertilization under natural circumstances. The consequences of this for the progeny are still not clear.

Acknowledgment

The authors are very grateful to Dr Stephen A. Beers from the Tenovus Research Laboratory, University of Southampton (Southampton, UK) for his assistance in editing the manuscript.

References

1 Oehninger S. Strategies for the infertile man. Semin Reprod Med 2001; 19: 231-7.

2 Centola GM, Ginsburg KA. Evaluation and Treatment of Infertile Male. Cambridge: Cambrige University Press; 1996.

3 Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998; 13: 896-900.

4 Sakkas D, Tomlinson M. Assessment of sperm competence. Semin Reprod Med 2000; 18: 133-9.

5 Hansen M, Kurinczuk JJ, Bower C, Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 2002; 346: 725-30.

6 Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS. Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med 2002; 346: 731-7.

7 Moll AC, Imhof SM, Cruysberg JR, Schouten-van Meeteren AY, Boers M, van Leeuwen FE. Incidence of retinoblastoma in children born after in-vitro fertilisation. Lancet 2003; 361: 309-10.

8 Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O, et al. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet 2003; 72: 218-9.

9 Bellve AR, McKay DJ, Renaux BS, Dixon GH. Purification and characterization of mouse protamines P1 and P2. Amino acid sequence of P2. Biochemistry 1988; 27: 2890-7.

10 Ward WS, Coffey DS. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 1991; 44: 569-74.

11 Fuentes-Mascorro G, Serrano H, Rosado A. Sperm chromatin. Arch Androl 2000; 45: 215-25.

12 Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CW. Sequence-specific packaging of DNA in human sperm chromatin. Science 1987; 236: 962-4.

13 Bench GS, Friz AM, Corzett MH, Morse DH, Balhorn R. DNA and total protamine masses in individual sperm from fertile mammalian subjects. Cytometry 1996; 23: 263-71.

14 Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4: 31-7.

15 Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000; 21: 33-44.

16 Ward WS. Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol Reprod 1993; 48: 1193-201.

17 Zalensky AO, Allen MJ, Kobayashi A, Zalenskaya IA, Balhorn R, Bradbury EM. Well-defined genome architecture in the human sperm nucleus. Chromosoma 1995; 103: 577-90.

18 Solov'eva L, Svetlova M, Bodinski D, Zalensky AO. Nature of telomere dimers and chromosome looping in human spermatozoa. Chromosome Res 2004; 12: 817-23.

19 Ward WS, Zalensky AO. The unique, complex organization of the transcriptionally silent sperm chromatin. Crit Rev Eukaryot Gene Expr 1996; 6: 139-47.

20 De Jonge CJ. Paternal contributions to embryogenesis. Reprod Med Rev 2000; 8: 203-14.

21 Lewis JD, Song Y, de Jong ME, Bagha SM, Ausio J. A walk through vertebrate and invertebrate protamines. Chromosoma 1999; 111: 473-82.

22 Corzett M, Mazrimas J, Balhorn R. Protamine 1 : protamine 2 stoichiometry in the sperm of eutherian mammals. Mol Reprod Dev 2002; 61: 519-27.

23 Jager S. Sperm nuclear stability and male infertility. Arch Androl 1990; 25: 253-9.

24 Balhorn R, Reed S, Tanphaichitr N. Aberrant protamine 1/protamine 2 ratio in sperm of infertile human males. Experientia 1988; 44: 52-5.

25 de Yebra L, Ballesca JL, Vanrell JA, Bassas L, Oliva R. Complete selective absence of protamine P2 in humans. J Biol Chem 1993; 268: 10553-7.

26 Bench G, Corzett MH, De Yebra L, Oliva R, Balhorn R. Protein and DNA contents in sperm from an infertile human male possessing protamine defects that vary over time. Mol Reprod Dev 1998; 50: 345-53.

27 de Yebra L, Ballesca JL, Vanrell JA, Corzett M, Balhorn R, Oliva R. Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels. Fertil Steril 1998; 69: 755-9.

28 Carrell DT, Liu L. Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis. J Androl 2001; 22: 604-10.

29 Mengual L, Ballesca JL, Ascaso C, Oliva R. Marked differences in protamine content and P1/P2 ratios in sperm cells from percoll fractions between patients and controls. J Androl 2003; 24: 438-47.

30 Nasr-Esfahani MH, Salehi M, Razavi S, Mardani M, Bahramian H, Steger K, et al. Effect of protamine-2 deficiency on ICSI outcome. Reprod Biomed Online 2004; 9: 652-8.

31 Aoki VW, Liu L, Carrell DT. Identification and evaluation of a novel sperm protamine abnormality in a population of infertile males. Hum Reprod 2005; 20: 1298-306.

32 Matsuda Y, Tobari I. Chromosomal analysis in mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) and methyl and ethyl methanesulfonate (MMS and EMS). Mutat Res 1988; 198: 131-44.

33 Genesca A, Caballin MR, Miro R, Benet J, Germa JR, Egozcue J. Repair of human sperm chromosome aberrations in the hamster egg. Hum Genet 1992; 89: 181-6.

34 Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003; 9: 331-45.

35 Bannister LA, Schimenti JC. Homologous recombinational repair proteins in mouse meiosis. Cytogenet Genome Res 2004; 107: 191-200.

36 Page AW, Orr-Weaver TL. Stopping and starting the meiotic cell cycle. Curr Opin Genet Dev 1997; 7: 23-31.

37 McPherson SM, Longo FJ. Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II. Dev Biol 1993; 158: 122-30.

38 Sakkas D, Manicardi G, Bianchi PG, Bizzaro D, Bianchi U. Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol Reprod 1995; 52: 1149-55.

39 Marcon L, Boissonneault G. Transient DNA strand breaks during mouse and human spermiogenesis: new insights in stage specificity and link to chromatin remodeling. Biol Reprod 2004; 70: 910-8.

40 McPherson SM, Longo FJ. Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev 1992; 31: 268-79.

41 Laberge RM, Boissonneault G. On the nature and origin of DNA strand breaks in elongating spermatids. Biol Reprod 2005; 73: 289-96.

42 Erenpreiss J, Bars J, Lipatnikova V, Erenpreisa J, Zalkalns J. Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 2001; 22: 45-53.

43 Kierszenbaum AL. Transition nuclear proteins during spermiogenesis: unrepaired DNA breaks not allowed. Mol Reprod Dev 2001; 58: 357-8.

44 Manicardi GC, Tombacco A, Bizzaro D, Bianchi U, Bianchi PG, Sakkas D. DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays. Histochem J 1998; 30: 33-9.

45 Fernandez JL, Goyanes VJ, Ramiro-Diaz J, Gosalvez J. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998; 82: 251-6.

46 Yu YE, Zhang Y, Unni E, Shirley CR, Deng JM, Russell LD, et al. Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice. Proc Natl Acad Sci USA 2000; 97: 4683-8.

47 Zhao M, Shirley CR, Yu YE, Mohapatra B, Zhang Y, Unni E, et al. Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice. Mol Cell Biol 2001; 21: 2243-55.

48 Boissoneault G. Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair. FEBS Letters 2002; 514: 111-4.

49 Cho C, Jung-Ha H, Willis WD, Goulding EH, Stein P, Xu Z, et al. Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice. Biol Reprod 2003; 69: 211-7.

50 Meistrich ML, Mohapatra B, Shirley CR, Zhao M. Roles of transition nuclear proteins in spermiogenesis. Chromosoma 2003; 111: 483-8.

51 Shirley CR, Hayashi S, Mounsey S, Yanagimachi R, Meistrich ML. Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice. Biol Reprod 2004; 71: 1220-9.

52 Zhao M, Shirley CR, Hayashi S, Marcon L, Mohapatra B, Suganuma L, et al. Transition nuclear proteins are required for normal chromatin condensation and functional sperm development. Genesis 2004; 38: 200-13.

53 Singh NP, Danner DB, Tice RR, McCoy MT, Collins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989; 184: 461-70.

54 Wykes SM, Krawetz SA. The structural organization of sperm chromatin. J Biol Chem 2003; 278: 29471-7.

55 Muriel L, Segrelles E, Goyanes V, Gosalvez J, Fernandez JL. Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis. Mol Hum Reprod 2004; 10: 203-9.

56 van Loon AA, Sonneveld E, Hoogerbrugge J, van der Schans GP, Grootegoed JA, Lohman PH, et al. Induction and repair of DNA single-strand breaks and DNA base damage at different cellular stages of spermatogenesis of the hamster upon in vitro exposure to ionizing radiation. Mutat Res 1993; 294: 139-48.

57 Laberge RM, Boissonneault G. On the nature and origin of DNA strand breaks in elongating spermatids. Biol Reprod 2005; 73: 289-96.

58 Stephan H, Polzar B, Rauch F, Zanotti S, Ulke C, Mannherz HG. Distribution of deoxyribonuclease I (DNase I) and p53 in rat testis and their correlation with apoptosis. Histochem Cell Biol 1996; 106: 383-93.

59 Ward MA, Ward WS. A model for the function of sperm DNA degradation. Reprod Fertil Dev 2004; 16: 547-54.

60 Billig H, Furuta I, Rivier C, Tapanainen J, Parvinen M, Hsueh AJ. Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology 1995; 136: 5-12.

61 Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J 1997; 16: 2262-70.

62 Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993; 75: 1169-78.

63 Krammer PH, Behrmann I, Daniel P, Dhein J, Debatin KM. Regulation of apoptosis in the immune system. Curr Opin Immunol 1994; 6: 279-89.

64 Said TM, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Hum Reprod Update 2004; 10: 39-51.

65 Zhivotovsky B, Kroemer G. Apoptosis and genomic instability. Nat Rev Mol Cell Biol 2004; 5: 752-62.

66 Muratori M, Piomboni P, Baldi E, Filimberti E, Pecchioli P, Moretti E, et al. Functional and ultrastructural features of DNA-fragmented human sperm. J Androl 2000; 21: 903-12.

67 Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000; 15: 1338-44.

68 Ross AJ, Waymire KG, Moss JE, Parlow AF, Skinner MK,
Russell LD, et al. Testicular degeneration in Bclw-deficient mice. Nat Genet 1998; 18: 251-6.

69 Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod 2002; 66: 1061-7.

70 Sutovsky P, Neuber E, Schatten G. Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay. Mol Reprod Dev 2002; 61: 406-13.

71 Muratori M, Maggi M, Spinelli S, Filimberti E, Forti G, Baldi E. Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J Androl 2003; 24: 253-62.

72 Sakkas D, Seli E, Bizzaro D, Tarozzi N, Manicardi GC. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod Biomed Online 2003; 7: 428-32.

73 Cayli S, Sakkas D, Vigue L, Demir R, Huszar G. Cellular maturity and apoptosis in human sperm: creatin kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm. Mol Hum Reprod 2004; 10: 365-72.

74 Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, et al. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril 2004; 81: 965-72.

75 Lachaud C, Tesarik J, Canadas ML, Mendoza C. Apoptosis and necrosis in human ejaculated spermatozoa. Hum Reprod 2004; 19: 607-10.

76 Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ, et al. Relationship between ROS production, apoptosis, and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod 2004; 19: 129-38.

77 Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha EJ, Thomas AJ, et al. Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol Reprod 2004; 71: 1828-37.

78 Sutovsky P, Hauser R, Sutovsky M. Increased levels of sperm ubiquitin correlate with semen quality in men from an andrology clinic population. Hum Reprod 2004; 19: 628-35.

79 Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW. Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. J Reprod Fertil 1992; 94: 451-62.

80 Erenpreiss J, Hlevicka S, Zalkalns J, Erenpreisa J. Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples. J Androl 2002; 23: 717-23.

81 Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997; 56: 602-7.

82 Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002; 23: 25-43.

83 Perreault SD, Aitken RJ, Baker HW, Evenson DP, Huszar G, Irvine DS, et al. Integrating new tests of sperm genetic integrity into semen analysis: breakout group discussion. Adv Exp Med Biol 2003; 518: 253-68.

84 Sakkas D, Manicardi GC, Bizzaro D. Sperm nuclear DNA damage in the human. Adv Exp Med Biol 2003; 518: 73-84.

85 Agarwal A, Allamaneni SSR. The effect of sperm DNA damage on assisted reproduction outcomes. Minerva Ginecol 2004; 56: 235-245.

86 Sharma RK, Said T, Agarwal A. Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J Androl 2004; 6: 139-45.

87 Darzynkiewicz Z. Acid-induced denaturation of DNA in situ as a probe of chromatin structure. Methods Cell Biol 1994; 41: 527-41.

88 Erenpreisa EA, Zirne RA, Zaleskaia ND, S'iakste TG. Effect of single-stranded breaks on the ultrastructural organization and cytochemistry of the chromatin in tumor cells. Biull Eksp Biol Med 1988; 106: 591-3.

89 Erenpreisa EA, Sondore OIu, Zirne RA. Conformational changes in the chromatin of tumor cells and the phenomenon of nuclear achromasia. Eksp Onkol 1988; 10: 54-7.

90 Sculthorpe HH. Metachromasia. Med Lab Sci 1978; 35: 365-70.

91 Erenpreisa J, Zaleskaya N. Effect of triton X-100 on cytochemical and ultrastructural pattern of chromatin. Acta Morphol Hung 1983; 31: 387-93.

92 Erenpreisa J, Freivalds T, Selivanova G. Influence of chromatin condensation on the absorption spectra of nuclei stained with toluidine blue. Acta Morphol Hung 1992; 40: 3-10.

93 Brewer LR, Corzett M, Balhorn R. Protamine-induced condensation and decondensation of the same DNA molecule. Science 1999; 286: 120-3.

94 Brewer L, Corzett M, Balhorn R. Condensation of DNA by spermatid basic nuclear proteins. J Biol Chem 2002; 277: 38895-900.

95 Brewer L, Corzett M, Lau EY, Balhorn R. Dynamics of protamine 1 binding to single DNA molecules. J Biol Chem 2003; 278: 42403-8.

96 Evenson D, Darzynkiewicz Z, Jost L, Janca F, Ballachey B. Changes in accessibility of DNA to various fluorochromes during spermatogenesis. Cytometry 1986; 7: 45-53.

97 Benyajati C, Worcel A. Isolation, characterization, and structure of the folded interphase genome of Drosophila melanogaster. Cell 1976; 9: 393-407.

98 Rigler R, Killander D, Bolund L, Ringertz NR. Cytochemical characterization of deoxyribonucleoprotein in individual cell nuclei. Techniques for obtaining heat denaturation curves with the aid of acridine orange microfluorimetry and ultraviolet microspectrophotometry. Exp Cell Res 1969; 55: 215-24.

99 Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR. Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 1975; 90: 411-28.

100 Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of
mammalian sperm chromatin heterogeneity to fertility. Science 1980; 210: 1131-3.

101 Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999; 14: 1039-49.

102 Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril 2000; 73: 43-50.

103 Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril 2003; 80: 895-902.

104 Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A. The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod 2004; 19: 1401-8.

105 Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 1984; 42: 87-91.

106 Auger J, Mesbah M, Huber C, Dadoune JP. Aniline blue staining as a marker of sperm chromatin defects associated with different semen characteristics discriminates between proven fertile and suspected infertile men. Int J Androl 1990; 13: 452-62.

107 Terquem T, Dadoune JP. Aniline blue staining of human spermatozoa chromatin. Evaluation of nuclear maturation. In: Adre J, ed. The Sperm Cell. The Hague: Martinus Nijhoff Publishers; 1983.

108 Liu DY, Baker HW. Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding, and fertilization rates in vitro. Fertil Steril 1992; 58: 1178-84.

109 Manicardi GC, Bianchi PG, Pantano S, Azzoni P, Bizzaro D, Bianchi U, et al. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol Reprod 1995; 52: 864-7.

110 Bianchi PG, Manicardi GC, Bizzaro D, Bianchi U, Sakkas D. Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biol Reprod 1993; 49: 1083-8.

111 Mello ML. Induced metachromasia in bull spermatozoa. Histochemistry 1982; 74: 387-92.

112 Andreetta AM, Stockert JC, Barrera C. A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures. Int J Androl 1995; 18 Suppl 1: 23-8.

113 Foresta C, De Carlo E, Mioni R, Zorzi M. Sperm nuclear chromatin heterogeneity in infertile subjects. Andrologia 1989; 21: 384-90.

114 Hoshi K, Katayose H, Yanagida K, Kimura Y, Sato A. The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil Steril 1996; 66: 634-9.

115 Duran EH, Gurgan T, Gunalp S, Enginsu ME, Yarali H, Ayhan A. A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro. Hum Reprod 1998; 13: 1235-9.

116 Bianchi PG, Manicardi GC, Bizzaro D, Bianchi U, Sakkas D. Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biol Reprod 1993; 49: 1083-8.

117 Mello ML. Induced metachromasia in bull spermatozoa. Histochemistry 1982; 74: 387-92.

118 Andreetta AM, Stockert JC, Barrera C. A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures. Int J Androl 1995; 18 Suppl 1: 23-8.

119 Beletti ME, Mello ML. Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology. Theriogenology 2004; 62: 398-402.

120 Potts RJ, Notarianni LJ, Jefferies TM. Extra-epididymal spermatozoa express nuclear abnormalities. Int J Androl 1999; 22: 282-8.

121 Erenpreisa J, Erenpreiss J, Freivalds T, Slaidina M, Krampe R, Butikova J, et al. Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry A 2003; 52: 19-27.

122 Erenpreiss J, Jepson K, Giwercman A, Tsarev I, Erenpreisa J, Spano M. Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod 2004; 19: 2277-82.

123 Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ. Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod 1998; 4: 439-45.

124 Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod 1998; 13: 1864-71.

125 Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res 1993; 207: 202-5.

126 Aravindan GR, Bjordahl J, Jost LK, Evenson DP. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp Cell Res 1997; 236: 231-7.

127 Henkel R, Kierspel E, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, et al. DNA fragmentation of spermatozoa and assisted reproduction technology. Reprod Biomed Online 2003; 7: 477-84.

128 Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in
intracytoplasmic sperm injection. Fertil Steril 1998; 69: 528-32.

129 Haines G, Marples B, Daniel P, Morris I. DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay. Adv Exp Med Biol 1998; 444: 79-91.

130 Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Mol Hum Reprod 1996; 2: 613-9.

131 Singh NP, Stephens RE. X-ray induced DNA double-strand breaks in human sperm. Mutagenesis 1998; 13: 75-9.

132 Shen H, Ong C. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 2000; 28: 529-36.

133 Lewis SE, O'Connell M, Stevenson M, Thompson-Cree L, McClure N. An algorithm to predict pregnancy in assisted reproduction. Hum Reprod 2004; 9: 1385-94.

134 Ankem MK, Mayer E, Ward WS, Cummings KB, Barone JG. Novel assay for determining DNA organization in human spermatozoa: implications for male factor infertility. Urology 2002; 59: 575-8.

135 Ward WS, Kimura Y, Yanagimachi R. An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development. Biol Reprod 1999; 60: 702-6.

136 Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003; 24: 59-66.

137 Sjakste N, Sjakste T. Nuclear matrix proteins and hereditary diseases. Genetika 2005; 41: 293-8.

138 Virro MR, Larson-Cook KL, Evenson DP. Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril 2004; 81: 1289-95.

139 Host E, Lindenberg S, Kahn JA, Christensen F. DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples. Acta Obstet Gynecol Scand 1999; 78: 336-9.

140 Gandini L, Lombardo F, Paoli D, Caponecchia L, Familiari G, Verlengia C, et al. Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod 2000; 15: 830-9.

141 Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril 2001; 75: 674-7.

142 Oosterhuis GJ, Mulder AB, Kalsbeek-Batenburg E, Lambalk CB, Schoemaker J, Vermes I. Measuring apoptosis in human spermatozoa: a biological assay for semen quality? Fertil Steril 2000; 74: 245-50.

143 Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003; 1: 1023-8.

144 Saleh RA, Agarwal A, Nada ES, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damge in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003; 79 Suppl 3: 1597-605.

145 Giwercman A, Richthoff J, Hjollund H, Bonde JP, Jepson K, Frohm B, et al. Correlation between sperm motility and sperm chromatin structure assay parameters. Fertil Steril 2003; 80: 1404-12.

146 Apedaile AE, Garrett C, Liu de Y, Clarke GN, Johnston SA, Baker HW. Flow cytometry and microscopic acridine orange test: relationship with standard semen analysis. Reprod Biomed Online 2004; 8: 398-407.

147 Saleh RA, Agarwal A, Nelson DR, Nada EA, El-Tonsy MH, Alvarez JG, et al. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 2002; 78: 313-8. Hum Reprod 2001; 16: 2160-5.

148 Duran EH, Morshedi M, Taylor S, Oehninger S. Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod 2002; 17: 3122-8.

149 Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, et al. Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Hum Reprod 2001; 16: 2160-5.

150 Seli E, Gardner DK, Schoolcraft WB, Moffatt O, Sakkas D. Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Fertil Steril 2004; 82: 378-83.

151 Hammadeh ME, Stieber M, Haidl G, Schmidt W. Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program. Andrologia 1998; 30: 29-35.

152 Larson KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 2000; 15: 1717-22.

153 Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril 2003; 80: 895-902.

154 Gandini L, Lombardo F, Paoli D, Caruso F, Eleuteri P, Leter G, et al. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum Reprod 2004; 19: 1409-17.

155 Check JH, Graziano V, Cohen R, Krotec J, Check ML. Effect of an abnormal sperm chromatin structural assay (SCSA) on pregnancy outcome following (IVF) with ICSI in previous IVF failures. Arch Androl 2005; 51: 121-4.

156 Host E, Lindenberg S, Smidt-Jensen S. The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand 2000; 79: 559-63.

157 Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization
and embryo development. Hum Reprod 2002; 17: 990-8.

158 Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17-8.

159 Silber SJ, Nagy Z, Liu J, Tournaye H, Lissens W, Ferec C, et al. The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility. Hum Reprod 1995; 10: 2031-43.

160 Kahraman S, Polat G, Samli M, Sozen E, Ozgun OD, Dirican K, et al. Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum Reprod 1998; 13: 104-10.

161 Vicdan K, Isik AZ, Delilbasi L. Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure. J Assist Reprod Genet 2001; 18: 78-86.

162 Urman B, Alatas C, Aksoy S, Mercan R, Nuhoglu A, Mumcu A, et al. Transfer at the blastocyst stage of embryos derived from testicular round spermatid injection. Hum Reprod 2002; 17: 741-3.

163 Khalili MA, Aflatoonian A, Zavos PM. Intracytoplasmic injection using spermatids and subsequent pregnancies: round versus elongated spermatids. J Assist Reprod Genet 2002; 19: 84-6.

164 Marh J, Tres LL, Yamazaki Y, Yanagimachi R, Kierszenbaum AL. Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes. Biol Reprod 2003; 69: 169-76.

165 Bonde JP, Hjollund HI, Henriksen TB, Jensen TK, Spano M, Kolstad H, et al. Epidemiologic evidence on biological and environmental male factors in embryonic loss. Adv Exp Med Biol 2003; 518: 25-35.

166 Perreault SD. Distinguishing between fertilization failure and early pregnancy loss when identifying male-mediated adverse pregnancy outcomes. Adv Exp Med Biol 2003; 518: 189-98.

167 Carrell DT, Liu L, Peterson CM, Jones KP, Hatasaka HH, Erickson L, et al. Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl 2003; 49: 49-55.

168 Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988; 332: 459-61.

169 Tesarik J, Greco E, Mendoza C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum Reprod 2004; 19: 611-5.

170 Brinkworth MH. Paternal transmission of genetic damage: findings in animals and humans. Int J Androl 2000; 23: 123-35.

171 Savitz DA. Paternal exposure to known mutagens and health of the offspring: ionizing radiation and tobacco smoke. Adv Exp Med Biol 2003; 518: 49-57.

172 Silber SJ, Repping S. Transmission of male infertility to future generations: lessons from the Y chromosome. Hum Reprod Update 2002; 8: 217-29.

173 Aitken RJ. The Amoroso Lecture. The human spermatozoon - a cell in crisis? J Reprod Fertil 1999; 115: 1-7.

174 Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001; 122: 497-506.

175 Ji BT, Shu XO, Linet MS, Zheng W, Wacholder S, Gao YT, et al. Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers. J Natl Cancer Inst 1997; 89: 238-44.

176 Sorahan T, Prior P, Lancashire RJ, Faux SP, Hulten MA, Peck IM, et al. Childhood cancer and parental use of tobacco: deaths from 1971 to 1976. Br J Cancer 1997; 76: 1525-31.

177 Sawyer DE, Aitken RJ. Male mediated developmental defects and childhood disease. Mol Hum Reprod 2000; 8: 107-26.

178 Schultz RM, Williams CJ. The science of ART. Science 2002; 296: 2188-90.

179 Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL, et al. Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 2002; 71: 162-4.

180 DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003; 72: 156-60.

181 Bonduelle M, Wilikens A, Buysse A, Van Assche E, Wisanto A, Devroey P, et al. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1996; 11 Suppl 4: 131-55.

182 Bonduelle M, Joris H, Hofmans K, Liebaers I, Van Steirteghem A. Mental development of 201 ICSI children at 2 years of age. Lancet 1998; 351: 1553.

183 Bonduelle M, Wilikens A, Buysse A, Van Assche E, Devroey P, Van Steirteghem AC, et al. A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1998; 13 Suppl 1: 196-207.

184 Bonduelle M, Aytoz A, Van Assche E, Devroey P, Liebaers I, Van Steirteghem A. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum Reprod 1998; 13: 781-2.

185 Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A, et al. Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod 2002; 17: 2600-14.

186 Bonduelle M, Ponjaert I, Steirteghem AV, Derde MP, Devroey P, Liebaers I. Developmental outcome at 2 years of age for children born after ICSI compared with children born after IVF. Hum Reprod 2003; 18: 342-50.

187 Kurinczuk JJ, Bower C. Birth defects in infants conceived by intracytoplasmic sperm injection: an alternative interpretation. BMJ 1997; 315: 1260-6.

188 Sutcliffe AG, Taylor B, Li J, Thornton S, Grudzinskas JG, Lieberman BA. Children born after intracytoplasmic sperm injection: population control study. BMJ 1999; 318: 704-5.

189 Wennerholm UB, Bergh C. Obstetric outcome and follow-up
of children born after in vitro fertilization (IVF). Hum Fertil (Camb) 2000; 3: 52-64.

190 Hansen M, Bower C, Milne E, de Klerk N, Kurinczuk JJ. Assisted reproductive technologies and the risk of birth defects-a systematic review. Hum Reprod 2005; 20: 328-38

191 Greco E, Romano S, Ferrero S, Baroni E, Minasi MG, Ubaldi F, et al. ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment. Hum Reprod 2005; 20: 2590-4.