Clinical relevance of sperm DNA damage in assisted reproduction
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RBMOnline - Vol 14. No 6. 2007 746-757 Reproductive BioMedicine Online; www.rbmonline.com/Article/2765 on web 17 April 2007 746 © 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK Nicoletta Tarozzi obtained her PhD in 2004 at the University of Modena (Italy), working at the Genetics Laboratory of the Animal Biology Department. In 2004 she joined the team of Tecnobios Procreazione, Centre for Reproductive Health in Bologna. Dr Tarozzi has published several papers in the field of sperm DNA damage. Current research interests focus on male infertility, especially as regards molecular biology of the male germ cell and structure of sperm chromatin. Dr Nicoletta Tarozzi Nicoletta Tarozzi 1 , Davide Bizzaro 2 , Carlo Flamigni 3 , Andrea Borini 1,4 1 Tecnobios Procreazione, Centre for Reproductive Health, Via Dante 15, I-40125 Bologna; 2 Institute of Biology and Genetic, University Polytechnic of Marche, Via Brecce Bianche, I-60131 Ancona; 3 University of Bologna, I-40125 Bologna, Italy 4 Correspondence: e-mail: [email protected] Abstract Many studies have shown how a ‘paternal effect’ can cause repeated assisted reproduction failures. In particular, with increasing experience of intracytoplasmic sperm injection (ICSI), it became evident that spermatozoa from some patients repeatedly fail to form viable embryos, although they can fertilize the oocyte and trigger early preimplantation development. Many authors have shown how this paternal effect can be traced back to anomalies in sperm chromatin organization: the spermatozoa of subfertile men are characterized by numerical abnormalities in spermatozoal chromosome content, Y chromosome microdeletions, alterations in the epigenetic regulation of paternal genome and non-specific DNA strand breaks. In particular, pathologically increased sperm DNA fragmentation is one of the main paternal-derived causes of repeated assisted reproduction failures in the ICSI era. The intention of this review is to describe nuclear sperm DNA damage, with emphasis on its clinical significance and its relationship with male infertility. Assessment of sperm DNA damage appears to be a potential tool for evaluating semen samples prior to their use in assisted reproduction, helping to select spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception. Keywords: assisted reproduction outcome, diagnosis, DNA fragmentation, human spermatozoa, male infertility, treatment protocols The integrity of the paternal genome plays a key role in maintaining human reproductive potential: the impact of an altered paternal genome on conception is certainly at least as important as that of the maternal genome. However, while the role of oocytes has been well established, the influence of male germ cells on conception is still largely to be clarified. It has been stressed that a so-called ‘paternal effect’ on the failure to conceive is basically ascribable to anomalies in sperm chromatin organization (Seli and Sakkas, 2005). The DNA of human spermatozoa is compacted in linear arrays organized as loop domains, each of which coils to form a ‘doughnut’ or toroid, a structure made extremely stable and compact by the replacement of about 85% of histones by protamines, which are smaller and more basic (Ward and Coffey, 1991). The spermatozoa of subfertile males reveal structural changes in this organization, such as epigenetic alterations, single or double DNA strand breaks, wrong number of chromosomes and/or chromosome Y microdeletions (reviewed in Seli and Sakkas, 2005). Among these, DNA fragmentation would seem to be one of the main causes of decreased reproductive ability of men, in natural as well as in assisted reproduction (Sun et al., 1997; Duran et al., 2002; Benchaib et al., 2003; Henkel et al., 2003, 2004; Larson-Cook et al., 2003; Lewis et al., 2004; Seli et al., 2004; Borini et al., 2006). Therefore, it is important to investigate a number of issues: (i) the mechanism underlying sperm DNA fragmentation, (ii) the DNA damage assessment methods, (iii) the relationship between DNA fragmentation in male germ cells Review Clinical relevance of sperm DNA damage in assisted reproduction Introduction
Transcript of Clinical relevance of sperm DNA damage in assisted reproduction
RBMOnline - Vol 14. No 6. 2007 746-757 Reproductive BioMedicine Online; www.rbmonline.com/Article/2765 on web 17 April 2007- Vol 14. No 6. 2007 746-757 Reproductive BioMedicine Online; www.rbmonline.com/Article/2765 on web 17 April 2007- Vol 14. No 6. 2007 746-757 Reproductive BioMedicine Online; www.rbmonline.com/Article/
746
© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Nicoletta Tarozzi obtained her PhD in 2004 at the University of Modena (Italy), working at the Genetics Laboratory of the Animal Biology Department. In 2004 she joined the team of Tecnobios Procreazione, Centre for Reproductive Health in Bologna. Dr Tarozzi has published several papers in the fi eld of sperm DNA damage. Current research interests focus on male infertility, especially as regards molecular biology of the male germ cell and structure of sperm chromatin.
Dr Nicoletta Tarozzi
Nicoletta Tarozzi1, Davide Bizzaro2, Carlo Flamigni3, Andrea Borini1,4
1Tecnobios Procreazione, Centre for Reproductive Health, Via Dante 15, I-40125 Bologna; 2Institute of Biology and Genetic, University Polytechnic of Marche, Via Brecce Bianche, I-60131 Ancona; 3University of Bologna, I-40125 Bologna, Italy4Correspondence: e-mail: [email protected]
Abstract
Many studies have shown how a ‘paternal effect’ can cause repeated assisted reproduction failures. In particular, with increasing experience of intracytoplasmic sperm injection (ICSI), it became evident that spermatozoa from some patients repeatedly fail to form viable embryos, although they can fertilize the oocyte and trigger early preimplantation development. Many authors have shown how this paternal effect can be traced back to anomalies in sperm chromatin organization: the spermatozoa of subfertile men are characterized by numerical abnormalities in spermatozoal chromosome content, Y chromosome microdeletions, alterations in the epigenetic regulation of paternal genome and non-specifi c DNA strand breaks.In particular, pathologically increased sperm DNA fragmentation is one of the main paternal-derived causes of repeated assisted reproduction failures in the ICSI era. The intention of this review is to describe nuclear sperm DNA damage, with emphasis on its clinical signifi cance and its relationship with male infertility. Assessment of sperm DNA damage appears to be a potential tool for evaluating semen samples prior to their use in assisted reproduction, helping to select spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception.
Keywords: assisted reproduction outcome, diagnosis, DNA fragmentation, human spermatozoa, male infertility, treatment protocols
The integrity of the paternal genome plays a key role in maintaining human reproductive potential: the impact of an altered paternal genome on conception is certainly at least as important as that of the maternal genome. However, while the role of oocytes has been well established, the infl uence of male germ cells on conception is still largely to be clarifi ed.
It has been stressed that a so-called ‘paternal effect’ on the failure to conceive is basically ascribable to anomalies in sperm chromatin organization (Seli and Sakkas, 2005). The DNA of human spermatozoa is compacted in linear arrays organized as loop domains, each of which coils to form a ‘doughnut’ or toroid, a structure made extremely stable and compact by the replacement of about 85% of histones by protamines, which are smaller and more basic (Ward and Coffey, 1991).
The spermatozoa of subfertile males reveal structural changes in this organization, such as epigenetic alterations, single or double DNA strand breaks, wrong number of chromosomes and/or chromosome Y microdeletions (reviewed in Seli and Sakkas, 2005).
Among these, DNA fragmentation would seem to be one of the main causes of decreased reproductive ability of men, in natural as well as in assisted reproduction (Sun et al., 1997; Duran et al., 2002; Benchaib et al., 2003; Henkel et al., 2003, 2004; Larson-Cook et al., 2003; Lewis et al., 2004; Seli et al., 2004; Borini et al., 2006). Therefore, it is important to investigate a number of issues: (i) the mechanism underlying sperm DNA fragmentation, (ii) the DNA damage assessment methods, (iii) the relationship between DNA fragmentation in male germ cells
Review
Clinical relevance of sperm DNA damage in assisted reproduction
Introduction
and assisted reproduction outcome, and (iv) the development of appropriate treatment protocols.
The objective of this review is to deepen these issues, with special reference to the biological meaning and clinical importance of sperm DNA damage in assisted reproduction.
Sperm nuclear DNA damage and its origin
A number of theories have been advanced to explain the occurrence of sperm DNA damage in the ejaculate, and three of these seem more convincing than others: oxidative stress, defi ciencies in chromatin packaging and abortive apoptosis.
Oxidative stress
Oxidative damage is described as damage caused by molecular oxygen. While oxygen is essential to aerobic life, it becomes toxic when administered in high concentrations, as it may produce reactive species (oxygen paradox). Free radicals, in particular, cause benefi cial or detrimental effects to sperm structure and function, depending on their nature and concentration (Aitken and Baker, 2004).
Male germ cells are especially vulnerable: oxidative stress induces lipid peroxidation in the membrane, which is rich in unsaturated fatty acids, thus decreasing its fl uidity (Jones et al., 1979), and also infl icts damage to mitochondrial and genomic DNA (Aitken and Krausz, 2001). Such special susceptibility to oxidative stress is due to the lack of DNA repair systems and antioxidants in spermatozoa (Aitken et al., 2003; Olsen et al., 2005).
Oxidative stress is caused by the imbalance between the antioxidant ability of seminal plasma and the production of reactive oxygen species (ROS). The antioxidant ability of seminal plasma is due to the secretion of antioxidant molecules, such as glutathione peroxidase, superoxide dismutase, vitamin C, alpha tocopherol, hypotaurine, albumin and pyruvate, by the male reproductive tract (Perry et al., 1993; van Overveld et al., 2000; Twigg et al., 1998). The antioxidant ability of seminal plasma and the strong sperm chromatin compactness are the only defence mechanisms of male germ cells against free radicals. Both spermatozoa and leukocytes, on the other hand, can produce free radicals. Spermatozoa that have not completed cytoplasm extrusion contain a high amount of cytoplasmic enzymes, such as glucose-6-phosphate dehydrogenase, lactic acid dehydrogenase and creatine kinase (Casano et al., 1991; Aitken et al., 1994, 1996), which can generate ROS (Gomezet al., 1996). In particular, it has been postulated that in terms of pathology the key enzyme is glucose-6-phosphate dehydrogenase, which controls the rate of glucose oxidation generating NADPH needed for ROS production (Gomez et al., 1996). Studies on IVF patients have revealed a strong negative correlation between the presence of cytoplasmic residues in male germ cells and fertilization rate (Keating et al., 1997). Leukocytes are another source of ROS, hydrogen peroxide included (Aitken et al., 1994), which can easily penetrate plasma and nuclear membranes and induce DNA damage.
Though many authors underline the importance of oxidative stress in the aetiology of sperm DNA damage and male subfertility (Smith et al., 1996; Barbieri et al., 1999; Hendin et al., 1999; Agarwal and Saleh, 2002; Agarwal and Said, 2003), no consensus exists regarding a clear direct correlation between oxidative stress and reproductive outcome: Hammadeh and colleagues (Hammadeh et al., 2006) have shown an inverse correlation between ROS concentration and fertilization rate in 48 patients, but the authors have also found that ROS concentrations were not statistically different between men whose partners achieved a pregnancy (n = 15) and those who were unsuccessful (n = 33). Moreover, in a recent publication (Veritet al., 2006), the authors did not fi nd any difference between idiopathic infertile men (n = 30) and controls (n = 20) in regard to various parameters of seminal plasma antioxidant ability. These results may be explained considering that oxidative stress is not a direct effect of ROS concentration or of the antioxidant ability of seminal plasma, but is caused by the imbalance between this two factors: therefore some patients might have high ROS concentration and low seminal fl uid antioxidant ability, while others may have high ROS concentration and high antioxidant ability, which may counteract each other.
‘When’ ROS generate sperm DNA damage is still an open question: probably during epididymal maturation, as the exposure time of spermatozoa to reactive oxygen species is longer (Henkel et al., 2003). A recent study (Greco et al., 2005b) has shown that DNA fragmentation in testicular spermatozoa is signifi cantly less than in ejaculated spermatozoa (4.5 versus 23%), and this corresponds to higher pregnancy rates in ICSI when testicular spermatozoa are used rather than those in the ejaculate (44.4 versus 5.6%). Similar results were obtained comparing testicular and epididymal spermatozoa (Steele et al., 1999). The statement that spermatozoa are less fragmented in the testis is highly controversial, especially when considering abortive apoptosis or dysfunction of topoisomerase II as possible causes of DNA damage. These discrepancies are discussed in the section ‘Strategies to reduce sperm DNA damage’.
Alterations in sperm chromatin packagingDuring male germ cell maturation, transient DNA double strand breaks occur naturally (Sakkas et al., 1999a): these DNA strand breaks have been shown to agree with the chromatin remodelling steps both in rodents (McPherson and Longo, 1993; Smith and Haaf, 1998; Wayne et al., 2002) and humans (Marcon and Boissonneault, 2004).
The enzyme topoisomerase II, in particular, introduces DNA double strand breaks transiently, allowing the passage of one double strand through the other, and subsequently reseals them (Wang et al., 1990): this process enables the replacement of histones by protamines and determines the correct chromatin packaging. In particular, the chromatin of mammalian spermatozoa is stabilized by disulphide bridges between protamines, which contain large amounts of cysteine residues (Calvin and Bedford, 1971); the formation of disulphide bridges causes sperm chromatin organization in a more compact structure, and is believed to protect DNA from physical and chemical damage until the spermatozoa have reached the oocyte. Topoisomerase II appears to be the only enzyme 747
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responsible for the transient DNA double strand breaks, and its activity has been associated with histone hyperacetylation (Laberge and Boissonneault, 2005). These physiological DNA breaks are usually resealed by topoisomerase II at the spermatid stage of spermatogenesis.
It has been proposed that endogenous nicks in the DNA of ejaculated spermatozoa may be produced by the incomplete maturation of germ cells during spermatogenesis: altered topoisomerase II activity, especially as to nick repair, might result in altered chromatin structure and residual breaks in the DNA of a number of spermatozoa (Bianchi et al., 1993; McPherson and Longo, 1993; Manicardi et al., 1995; Sailer et , 1995; Sailer et , 1995; Saileral., 1995).
Abortive apoptosis
In mammalian testis, germ cells expand clonally before meiosis and differentiation. Such clonal expansion is balanced by cell death in their progeny: it has been estimated that about 75% of the potential spermatozoa are eliminated from the population of maturing male germ cells (Huckins, 1978).
Cell death takes place mainly by apoptosis (Blanco-Rodriguez and Martinez-Garcia, 1996, 1998; Rodriguez et al., 1997; Wang et al., 1998), a physiological and continuous process characterized by a complex cascade of events: a number of studies carried out mainly in mice (Furuchi et al., 1996; Rodriguez et al., 1997; Huynh et al., 2002; Russell et al., 2002) have shown that pro- and anti-apoptotic factors play a critical role in spermatogenesis, maintaining male germ cell homeostasis. In particular, it would seem that Sertoli cells can start and regulate germ cell apoptosis via the apoptosis stimulating fragment (Fas) system, that is characterized by the interaction between Fas protein and Fas ligand (Richburg, 2000). The complex cascade of events involved in apoptosis also includes the activation of endonucleases, which induce DNA strand breaks (Sakkas et al., 1999a).
Apoptosis appears to play two primary roles in spermatogenesis. Firstly, it is essential in limiting the population of germ cells to a number that can be supported by Sertoli cells, thus ensuring normal spermatogenesis. It has also been proposed that programmed cell death may be responsible for the selective depletion of abnormal germ cells, i.e. cells with abnormal morphology, altered biochemical function or DNA damage (Sakkas et al., 1999b).
Evidence has been presented suggesting that ‘abortive apoptosis’ events may occur in males with abnormal seminal parameters, with a high concentration of molecular markers of apoptosis, such as Fas or p53, in ejaculated spermatozoa (Sakkas et al., 1999b, 2002). Abortive apoptosis may therefore be a cause of sperm DNA damage: malfunctioning or incomplete apoptosis might fail to destroy cells earmarked for elimination from the overall pool; the population of spermatozoa in the ejaculate would therefore present an array of anomalies typical of apoptotic cells.
However, the actual role of apoptosis in the selective elimination of abnormal germ cells is still to be proven: some studies would seem to invalidate the abortive apoptosis hypothesis by stressing
the lack of correlation between apoptotic markers and sperm DNA damage (Barroso et al., 2000; Muratori et al., 2000).
Detection of sperm DNA damage
Regardless of the cause of sperm DNA damage, the discovery of male subfertility cases due to alterations of the paternal genetic material raises new issues. In particular, poor information is available about the possible consequences on the reproductive outcome and, especially, on the progeny of fertilization obtained using spermatozoa with an abnormal chromatin organization. Hence some authors have suggested that tests to assess the integrity of the paternal genome should be introduced into the routine andrological laboratory work-up (De Jonge, 2002; Perreault et al.Perreault et al.Perreault , 2003).
A variety of tests have been described to analyse the integrity of the paternal genome (reviewed in Oehninger and Kruger, 2007), including tests for the assessment of sperm nuclear maturity and chromatin condensation and tests for the direct analysis of DNA damage. Table 1 shows a list of the available tests, with a brief description of the underlying principles, detection methods, and their advantages and disadvantages.
The sperm chromatin structure assay (SCSA), the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay and the Comet assay are currently the most widely used tests to measure sperm DNA damage and assess subfertility in natural as well as medically assisted reproduction.
SCSA
The SCSA is a fl uorescence-activated cell sorter test that measures the susceptibility of sperm DNA to heat- or acid-induced DNA denaturation in situ, followed by staining with acridine orange (Evenson and Jost, 2000). As with the acridine orange test, it is based on the use of acridine orange, but the detection is done by fl ow cytometry, which makes it possible to measure large amounts of spermatozoa per sample; the technique is therefore simple and highly reproducible (Evensonet al., 2002). Acridine orange is a metachromatic dye that fl uoresces red when associated with denatured DNA and green when associated with normal, double-stranded DNA. The SCSA measures several parameters (Evenson et al., 2002): DNA fragmentation index (DFI) represents the sperm fraction with detectable denaturable single-stranded DNA mainly due to DNA breaks and the highly DNA stainable cells (HDS) describes the sperm fraction with increased double-stranded DNA accessibility to the metachromatic dye, mainly due to altered replacement of histones with protamines.
Sperm DNA damage assessed by SCSA is a more constant parameter over a longer period of time compared with the traditional sperm evaluation parameters (Zini et al., 2001), and this is the reason why this test is held useful in epidemiological studies (Spano et al., 1998; Bungum et al., 2004). Furthermore, several clinical studies have shown its usefulness in evaluating male fertility (Evenson et al., 2002; Larson et al., 2000; Spanoet al., 2000; Bungum et al., 2004; Virro et al., 2004).
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Tabl
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Tes
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to a
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n ad
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Si
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stra
nd D
NA
Fl
uore
scen
ce m
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scop
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Rel
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to T
UN
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ssay
; spe
cifi c
for s
ingl
e st
rand
DN
A b
reak
s Sp
ecia
l equ
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tra
nsla
tion
brea
ks
TUN
EL a
ssay
Si
ngle
and
dou
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Fluo
resc
ence
mic
rosc
opy/
C
linic
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sign
ifi ca
nt; h
igh
sens
itivi
ty a
nd sp
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ge n
umbe
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men
t; m
ore
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sp
erm
atoz
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ount
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ex
pens
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ssay
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and
dou
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scen
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scop
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UN
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ssay
; che
ap; h
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sens
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st
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chr
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NA
Fl
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linic
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igh
sens
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ge n
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truct
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Mea
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labe
lling
.
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TUNEL assay
The TUNEL assay is widely used for the direct assessment of sperm DNA fragmentation. This technique is based on the addition of a tail of marked nucleotides on 3’-OH of DNA breaks by means of an enzymatically catalysed reaction, using the terminal deoxynucleotide transferase (Gorczyca et al.the terminal deoxynucleotide transferase (Gorczyca et al.the terminal deoxynucleotide transferase (Gorczyca , 1993). The proportion of DNA-damaged spermatozoa can be measured by microscopy or fl ow cytometry. The TUNEL assay resembles the nick translation in situ in a number of technical aspects, but can reveal both single and double strand damage. However, it cannot quantify the magnitude of damage in individual cells.
The TUNEL assay is sophisticated, expensive and time consuming; however, its popularity is justifi ed by good quality control parameters, such as a low intra- and inter-observer variability (Barroso et al., 2000). Furthermore, sperm DNA fragmentation, measured by TUNEL assay, is a parameter with good stability over time and it can be taken as a baseline both in fertile and subfertile men (Sergerie et al., 2005a). The use of the TUNEL assay in fl ow cytometry also makes it possible to evaluate a very high number of cells, thus enhancing reproducibility and simplifying the technique. Thanks to its specifi city and reproducibility, it is broadly used to assess sperm DNA fragmentation and its value as an indicator of male fertility (Sergerie et al., 2005b) and in predicting the outcome of assisted reproduction has been amply demonstrated (Sun et al., 1997; Lopes et al., 1998; Duran et al., 2002; Benchaib et al., 2003; Henkel et al., 2003, 2004; Seli et al., 2004; Borini et al., 2006).
Comet assay
The single cell gel electrophoresis (Comet assay) is used to assess DNA damage and repair due to several factors in a wide variety of mammalian cells; studies on the effects of UV radiation, carcinogens, toxic substances and radiotherapy as well as studies on tumour regression utilize the Comet assay (Singh et al., 1988; Fairbairn et al., 1995; Olive et al., 1998), a technique also used for the analysis of sperm DNA fragmentation (Lewis et al., 2004). In the Comet assay, the cells are put in agarose gel and treated to destroy plasma and nuclear membranes and digest proteins; an electrophoretic fi eld is then applied to cause the migration of damaged DNA through the agarose. DNA-fragmented cells are visualized by DNA-specifi c dyes and appear as comets, where tail length and signal intensity are related to the degree of DNA fragmentation, while non-fragmented DNA does not migrate from the nucleus (Collins et al., 1997). The Comet parameters (head density, Comet tail length, Comet moment) are analysed by means of appropriate software. The Comet assay can be performed in a neutral or alkaline environment: in the fi rst case only DNA double strand damage is revealed, while in the second single and double strand damages and alkali-labile sites are made visible. This technique also measures the magnitude of DNA damage in individual cells.
The Comet assay is cheap and one of the most sensitive techniques available to measure DNA damage; it is also related to the results of the TUNEL assay (Aravindan et al., 1997). One disadvantage is the lack of standardized protocols, which makes it diffi cult to fully understand and relate the results of different authors. Furthermore, the alkaline Comet assay produced large comets in all cells, presumably due to the presence of alkaline labile
sites in spermatozoa (Singh et al., 1989); this makes it diffi cult to discriminate between endogenous and induced DNA breaks. The clinical importance of the Comet assay in assessing male infertility and sperm function, however, has been demonstrated (Irvine et al., 2000; Chan et al., 2001; Donnelly et al., 2001), as its predictive value in assisted reproduction outcome (Lewis et al., 2004).
Sperm DNA damage and assisted reproduction outcomeThe experience gained in assisted reproduction has revealed that the germ cells of some male patients repeatedly fail to form viable embryos in spite of successful fertilization (Hammadeh et al.embryos in spite of successful fertilization (Hammadeh et al.embryos in spite of successful fertilization (Hammadeh , 1996; Sanchez et al., 1996). In particular, the expression ‘paternal effect’ describes cases where apparently normal preimplantation embryos are produced, but they fail to implant or are lost soon after the detection of pregnancy; sperm DNA fragmentation seems to play an important role in inducing this paternal effect (Tesariket al., 2004; Tesarik, 2005). Such evidence has promoted studies aiming to analyse the real link between sperm DNA fragmentation and reproductive outcome.
Table 2 shows the works published from 1997 to date in which the authors discuss the relationship between sperm DNA fragmentation, assessed by SCSA, TUNEL assay or Comet assay, and assisted reproduction outcome, assessed as fertilization, embryo/blastocyst development, pregnancy and pregnancy loss. The analysis of these studies reveals that sperm DNA fragmentation and fertilization do not seem to be closely related, whereas there is a strong link between sperm DNA damage, embryo/blastocyst development and pregnancy.
Specifi cally, most of the works show no clear correlations between sperm DNA fragmentation and fertilization rate, using SCSA (Larson et al., 2000; Gandini et al., 2004; Virro et al., 2004), TUNEL assay (Benchaib et al., 2003; Henkel et al., 2003, 2004; Greco et al., 2005a; Borini et al., 2006), or Comet assay (Morris et al., 2002; Lewis et al., 2004; Nasr-Esfahani et al., 2005). In a few works, this relationship appears more evident (Sun et al., 1997; Lopes et al., 1998; Saleh et al., 1998; Saleh et al., 1998; Saleh , 2003).
In contrast, the negative correlation between embryo/blastocyst development and sperm DNA damage becomes manifest using all three damage assessment techniques: SCSA (Virro et al., 2004), TUNEL assay (Benchaib et al., 2003; Seli et al., 2004) and Comet assay (Morris et al., 2002; Tomsu et al., 2002; Nasr-Esfahani et al., 2005).
A clear negative correlation is also found between sperm DNA fragmentation and pregnancy using SCSA (Larson et al., 2000; Larson-Cook et al.Larson-Cook et al.Larson-Cook , 2003; Evenson and Wixon, 2006), TUNEL assay (Benchaib et al., 2003; Henkel et al., 2003, 2004; Grecoet al., 2005a; Borini et al., 2006) and Comet assay (Lewis et al., 2004). Few works show no correlation between sperm DNA damage and pregnancy (Gandini et al., 2004; Huang et al., 2005).
A recent study has shown (Borini et al., 2006) that sperm DNA fragmentation and pregnancy loss, defi ned as miscarriage or biochemical pregnancy, are closely related. This work stresses: (i) the strong correlation between sperm DNA fragmentation 750
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Table 2. Relationship between sperm DNA fragmentation, assessed by sperm chromatin structure assay (SCSA), TUNEL assay or Comet assay, and assisted reproduction technology (ART) outcome.
Authors Assisted Assay Reproductive reproduction outcome procedure
Sun et al., 1997 IVF TUNEL DNA fragmentation negatively related to fertilization and embryo cleavage rateLopes et al., 1998 ICSI TUNEL Fertilization rate negatively related to DNA fragmentationLarson et al., 2000 ICSI SCSA DNA denaturation lower in men that initiated pregnancy; no pregnancies when DNA fragmentation >27%Tomsu et al., 2002 IVF Comet Head and tail Comet parameters useful in prediction of embryo qualityMorris et al., 2002 IVF, ICSI Comet DNA damage in ICSI cycles is associated with impairment of post-fertilization embryo cleavageDuran et al., 2002 IUI TUNEL No pregnancies if fragmentation >12%Saleh et al., 2003 IUI, IVF, ICSI SCSA Both fertilization and embryo development negatively related to DNA denaturationBenchaib et al., IVF, ICSI TUNEL No correlation between DNA fragmentation and embryo quality; 2003 in ICSI no pregnancy when DNA fragmentation >20%Henkel et al., 2003 IVF, ICSI TUNEL No association between DNA damage and fertilization rate; DNA fragmentation is related to pregnancyLarson-Cook IVF, ICSI SCSA Sperm DNA fragmentation higher in couples with negative et al., 2003 pregnancy outcomeLewis et al., 2004 ICSI Comet Nuclear DNA fragmentation closely related to pregnancy in ICSI. No relationship with fertilization rateNasr-Esfahani ICSI Comet, Correlation between lower potential to reach blastocyst stage et al., 2005 CMA3 and sperm DNA fragmentation; sperm DNA fragmentation does not preclude fertilizationGandini et al., 2004 IVF, ICSI SCSA No correlation between SCSA parameters and fertilization and pregnancy ratesVirro et al., 2004 IVF, ICSI SCSA Fertilization rate is not statistically different between high and low DFI groups; high DFI related to low blastocyst rateSeli et al., 2004 IVF TUNEL Correlation between sperm DNA damage and blastocyst developmentBungum et al., 2004 IUI, IVF, ICSI SCSA IUI group: chance of pregnancy/delivery signifi cantly higher in group with low DNA damage; no statistically signifi cant difference in the outcomes after IVF/ICSIHenkel et al., 2004 IVF TUNEL No association between DNA damage and fertilization rate; DNA fragmentation is related to pregnancyGreco et al., 2005a ICSI TUNEL Decrease in the percentage of sperm DNA fragmentation improve ICSI outcome. No relationship with fertilization rateHuang et al., 2005 IUI, IVF, ICSI TUNEL Association between sperm DNA fragmentation and fertilization rate; no association with pregnancy rateEvenson and IUI, IVF, ICSI SCSA High sperm DNA fragmentation is signifi cantly predictive for Wixon, 2006 reduced pregnancy success using IUI, IVF and, to a lesser extent, in ICSIBorini et al., 2006 IVF, ICSI TUNEL Sperm DNA fragmentation affects embryo post-implantation development: sperm DNA damage is related to pregnancy loss; in ICSI no relationship with fertilization rate
DFI = DNA fragmentation index; ICSI = intracytoplasmic sperm injection; IUI = intrauterine insemination; IVF = in-vitro fertilization.
Review - Relevance of sperm DNA damage in IVF - N Tarozzi et al.
and sperm concentration, motility and morphology (P < 0.01); (ii) the higher predictive value of sperm DNA fragmentation on the reproductive outcome compared with conventional semen assessment techniques; and (iii) the close link between sperm DNA fragmentation and pregnancy and pregnancy loss in ICSI. In particular, in the ICSI group a highly signifi cant difference in clinical pregnancy rates was found between patients with high and low sperm DNA damage (10 versus 45%; P = 0.007); patients with high sperm DNA fragmentation also had higher pregnancy loss rates compared with patients with low sperm DNA fragmentation (62.5 versus 0%; P = 0.009), with no biochemical pregnancies or miscarriages in the low sperm DNA fragmentation group; the same relationship was not found in IVF. These fi ndings would seem to be supported by studies concerned with the infl uence of the paternal effect on the aetiology of early miscarriage (Slama et al., 2005), with special reference to the higher risk of miscarriage due to paternal age and hence sperm chromatin anomalies. Some authors have also analysed DNA damage, using the TUNEL assay, in patients with a history of unexplained recurrent pregnancy loss (RPL), showing a signifi cantly higher sperm DNA damage in RPL patients (Carrell et al., 2003).
The lack of correlation between fertilization rate and high sperm DNA fragmentation underlined by the majority of authors suggests that sperm DNA damage, while not precluding fertilization, may affect blastocyst formation and/or successful embryo development (Ahmadi and Ng, 1999). It is now accepted that in the fi rst few steps of development there is a maternal regulation, while the expression of the paternal genes begins at the stage of four to eight cells: during the development of the embryo to the blastocyst stage the genome is activated, transcription activity has already started, and the paternal genome begins to contribute signifi cantly to the embryo function (Seli et al., 2004); it is therefore at this stage that alterations in paternal DNA may compromise successful embryo development or blastocyst formation. In particular, the results of a recent study (Tesarik et al., 2004) have shown that a ‘late’ adverse paternal effect on embryo development may become manifest even though there are no morphological anomalies at the zygote stage: high levels of sperm DNA fragmentation are in fact frequently associated with repeated assisted reproduction failures without any apparent impairment of zygote and cleaving embryo morphology.
The correlation, inverse and direct respectively, between sperm DNA damage and pregnancy and pregnancy loss rates is also explained by paternal genome anomalies blocking the correct embryo development (Borini et al., 2006). The oocyte, in fact, although capable of repairing DNA single strand breaks, may make ‘mistakes’ if high levels of DNA double strand breaks are present, thus causing genetic mutations that may subsequently block or modify embryo development and lead to pregnancy loss (Braude et al., 1988).
The analysis of the different works also shows an intriguing matter: the predictive value for reduced pregnancy success of sperm DNA fragmentation tests may be different considering IVF or ICSI treatments and considering the type of the assay used. In particular using a direct sperm DNA damage test, such as TUNEL or Comet assay, the predictive value appears stronger in ICSI patients compared with IVF (Morris et al., 2002; Benchaib et al., 2003; Borini et al., 2006). This may be
explained considering the aetiology of infertility: it has been previously reported that the highest level of TUNEL or Comet positivity is usually found in patients with poorest semen quality, i.e. ICSI candidates (Sun et al., 1997; Irvine et al., 2000; Benchaib et al., 2003; Borini et al., 2006), in which male factor is the determinant in subfertility; reasonably, in this group of patients the beginning and the progression of pregnancy are mainly related to variables of male origin, such as DNA fragmentation. On the contrary, in IVF patients, a decrease in pregnancy success may arise from others variables, such as female factors, and the relationship between sperm DNA damage and reproductive outcome may be softer. It was also suggested that in IVF, by a kind of ‘natural’ sperm selection, spermatozoa with abnormal morphology, low motility and DNA damage have a low fi tness in oocyte fertilization (Boriniet al., 2006); this hypothesis is supported by studies showing that genetically altered spermatozoa can be identifi ed by the zona pellucida (Menkveld et al., 1991; van Dyk et al., 2000). On the contrary, the predictive value of SCSA for reduced pregnancy success decreases in ICSI patients compared with IVF (Bungum et al., 2004; Evenson and Wixon, 2006). It was postulated that this fi nding may be related to SCSA technical characteristics: the sperm chromatin structure assay identifi es the spermatozoa with abnormal chromatin packaging, defi ned as susceptibility to acid-induced DNA denaturation in situ; therefore, SCSA parameters refer to the chromatin status of the whole cell population, not excluding, for example, the presence of a subpopulation with chromatin alterations but without signifi cant DNA fragmentation (TUNEL and Comet negative) (Bungum et al., 2004). Host and colleagues (Host et al., 2004). Host and colleagues (Host et al., 2004). Host and colleagues (Host , 2000) suggested that the technician who performs ICSI attempts to choose spermatozoa with normal morphology, reducing the risk of introducing spermatozoa with abnormal chromatin packaging, that is related to some sperm head anomalies (Fawcett et al.that is related to some sperm head anomalies (Fawcett et al.that is related to some sperm head anomalies (Fawcett , 1971); this may be the reason for the reduced predictive value of SCSA in ICSI treatments. However, this statement may be questioned, since SCSA values have been proven to be poorly related to the traditional sperm evaluation parameters, such as sperm morphology, motility and count (Evenson et al., 1999; Spano et al., 2000). Therefore, probably the different technical characteristics of sperm DNA damage tests, and thus the different information arising, may explain these discrepancies between SCSA and direct sperm DNA damage tests, such as TUNEL or Comet assay.
Finally, though many works stress the importance of sperm DNA damage assessment as a prognostic parameter of reproductive outcome, there are few published studies that have found no link between sperm DNA damage and assisted reproduction outcome (Gandini et al., 2004; Huang et al., 2005). For understanding these apparent discrepancies, it is essential to analyse the clinical context in which the observations were made: the same sperm DNA fragmentation value may be compatible or not with pregnancy, depending on other variables of both female and male origin (such as maternal age and the presence of other pathological conditions) that can be detailed by a severe study of the couple’s history of infertility (Ménézo, 2006; Tesarik et al., 2006). For example, a very important variable is the ability of the oocyte to repair sperm DNA damage, which is strongly related to maternal age (Ménézo, 2006). Furthermore the recommended values of sperm DNA fragmentation to discriminate between good and poor prognosis patients show a high degree of variability (reviewed 752
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in Tesarik et al., 2006). This is probably due to the different tests used (SCSA, TUNEL assay or Comet assay), the various testing modalities applied (microscopy or fl ow cytometry), the standardization diffi culties of the techniques and the sample analysed (raw or treated sample). For example, the predictive ability of the sperm DNA fragmentation test, performed on raw samples, decreases when spermatozoa are prepared using techniques such as density gradient centrifugation (Sakkas et al., 2000; Tomlinson et al., 2001; O’Connell et al., 2003; Seli and Sakkas, 2005). This stresses the need to assess sperm DNA integrity in the appropriate context: sperm DNA fragmentation in raw semen with reference to natural conception and sperm DNA fragmentation in post-preparation samples in relation to assisted reproduction (Tomlinson et al., 2001). In general, it can be stated that sperm DNA fragmentation tests do not yield completely black or white results and an absolute cut-off value not compatible with pregnancy is far from being established (Spano et al., 2005).
Strategies to reduce sperm DNA damageIn view of the impact of sperm DNA fragmentation on reproductive outcome, the development and correct application of treatment methods and strategies to minimize DNA damage in the population of spermatozoa used in assisted reproduction is essential.
One strategy to reduce the proportion of DNA-fragmented spermatozoa is the appropriate preparation of the semen. Density gradient centrifugation, swim-up and glass wool fi ltration are examples of techniques used to prepare the seminal fl uid that have been shown to yield signifi cantly higher sperm DNA integrity compared with that of native semen (Larson et al., 1999; Donnelly et al., 2000; Sakkas et al., 2000; Zini et al., 2000).
A new technique based on the electrophoretic separation of sperm has recently been proposed for the selection of male germ cells to be used in assisted reproduction (Ainsworth et al., 2005). The postulate of this system is that higher quality spermatozoa are more electronegative (Kirchhoff and Schroter, 2001; Giuliani et al., 2004), and they can be separated from the other contaminating electronegative cells based on their small cross-sectional size (Ainsworth et al., 2005). These authors have shown that the cell suspension obtained by electrophoretic separation contains motile, viable and morphologically normal spermatozoa with a low degree of DNA fragmentation measured by the TUNEL assay. Therefore, a re-examination of the techniques available for semen preparation is highly recommended to see which is the most suitable to select sperm cell populations with the lowest degree of DNA damage.
One of the main causes of sperm DNA damage is oxidative stress (Aitken and Krausz, 2001); another strategy proposed to enhance sperm DNA integrity is proper antioxidant treatment. Numerous in-vivo and in-vitro works have studied the ability of antioxidant treatments to manage male subfertility, stressing their positive effects on sperm DNA damage (reviewed in Agarwal et al., 2004).
In particular, one of the systems to reduce oxidative stress is increasing the scavenging capacity of seminal plasma by oral
antioxidant treatment before assisted reproduction, as described in a recent work by Greco and colleagues (Greco et al., 2005a). Patients with high sperm DNA fragmentation, assessed by the TUNEL assay, were treated with antioxidants (1 g vitamin C and 1 g vitamin E daily) for 2 months after one failed ICSI attempt and submitted once more to ICSI. The authors compared DNA fragmentation values before and after antioxidant treatment: a substantial improvement in sperm genome integrity was found in most patients and a signifi cant increase in clinical pregnancy (7 versus 48%) and implantation rates (2 versus 20%) was recorded, although in this study some patients did not react to the antioxidant therapy and a few reports show the ineffectiveness of therapy with vitamin C and vitamin E (Rolfet al., 1999)
Therefore, in patients in whom oxidative stress is the cause of sperm DNA damage, adequate oral antioxidant treatment seems a simple strategy to enhance sperm genome integrity and, as a result, reproductive outcome. Further research is necessary to design standard and reliable oral antioxidant treatment protocols and to develop alternative strategies for non-responder patients.
It has been stated that ROS can most probably damage male germ cells in the epididymis, where the time of sperm cell exposure to ROS is longer (Henkel et al., 2003). Hence, an idea of a further strategy to obtain undamaged male germ cells is that based on the collection of spermatozoa directly from the testis for use in ICSI.
A recent study (Greco et al., 2005b) has compared the reproductive outcome of two subsequent ICSI attempts using ejaculated and testicular spermatozoa in 18 patients. The authors have shown that TUNEL-assessed DNA damage is defi nitely lower in testicular rather than ejaculated spermatozoa and that clinical ICSI outcomes are improved using testicular spermatozoa even though there are no differences in fertilization, cleavage rate and embryo morphology: the pregnancy rate using testicular spermatozoa was 44.4 versus 5.6% using ejaculated spermatozoa, and the implantation rate was 20.7 versus 1.8%. However, these results are not in agreement with those of other studies (Nicopoullos et al., 2004): a meta-analysis by Nicopoullos and colleagues comparing ICSI outcomes using different sources of spermatozoa (epididymal, testicular, fresh and frozen−thawed) concludes that, in men with the same aetiology, ICSI outcomes do not differ. If the cause of sperm DNA damage is abortive apoptosis or alterations in topoisomerase II activity, there is no reason for a positive effect of the testicular spermatozoa. Considering the contrasting results reported in the literature, further research is recommended.
Finally, another strategy for the treatment of patients with sperm DNA fragmentation is high-magnifi cation ICSI (Bartoov et al., 2003; Hazout et al.2003; Hazout et al.2003; Hazout , 2006), which is based on the observation that spermatozoa with apparently normal morphology at standard ICSI magnifi cation may show a variety of structural anomalies, including intranuclear vacuoles that appear to be associated with alterations in chromatin packaging (Berkovitzet al., 2005). High magnifi cation is made possible by a ×100 oil-immersion objective lens and an inverted microscope equipped with Nomarski differential interference contrast optics combined with a digitally enhanced secondary magnifi cation system (Bartoov et al., 2002). 753
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ICSI performed by this system has been shown signifi cantly to increase pregnancy rates compared with conventional ICSI (Bartoov et al., 2003). This is confi rmed by a recent study (Hazoutet al., 2006) utilizing the same technique in 125 patients, 72 of whom also had their sperm DNA fragmentation analysed by the TUNEL assay: a signifi cant improvement in ICSI outcome was obtained in patients with both high DNA fragmentation and low sperm DNA damage. So high-magnifi cation ICSI, even though it is time-consuming and requires considerable investment, appears to be a good strategy in patients with high sperm DNA fragmentation.
Conclusions
The awareness that alterations at different levels in the organization of the paternal genome can impair reproductive potential and assisted reproduction outcome has led to a more accurate assessment of the function and integrity of male germ cells. In particular, the analysis of sperm DNA integrity is being used with increasing frequency as an independent measure of semen quality as it offers more accurate diagnostic and prognostic information than the traditional parameters of semen evaluation.
Further research is necessary to standardize the methods of DNA damage evaluation and apply them as useful pregnancy predictors in assisted reproduction. Greater knowledge is also needed on the causes of sperm DNA damage to design appropriate treatment protocols and new strategies to enhance the genomic integrity of male germ cells, thus contributing to optimize assisted reproduction outcome.
References
Agarwal A, Said T 2003 Role of sperm chromatin abnormalities and DNA damage in male infertility. Human Reproduction Update 9, 331–345.
Agarwal A, Saleh RA 2002 Role of oxidants in male infertility: rationale, signifi cance, and treatment. Urologic Clinics of North America 29, 817–827.
Agarwal A, Nallella KP, Allamaneni SS et al. 2004 Role of antioxidants in treatment of male infertility: an overview of the literature. Reproductive BioMedicine Online 8, 616–627.
Ahmadi A, Ng SC 1999 Fertilizing ability of DNA-damaged spermatozoa. Journal of Experimental Zoology 284, 696–704.
Ainsworth C, Nixon B, Aitken RJ 2005 Development of a novel electrophoretic system for the isolation of human spermatozoa. Human Reproduction 20, 2261–2270.
Aitken RJ, Baker MA 2004 Oxidative stress and male reproductive biology. Reproduction, Fertility and Development 16, 581–588.
Aitken RJ, Krausz C 2001 Oxidative stress, DNA damage and the Y chromosome. Reproduction 122, 497–506.
Aitken RJ, Baker MA, Sawyer D 2003 Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease. Reproductive BioMedicine Online 7, 65–70.
Aitken RJ, Buckingham DW, Carreras A et al. 1996 Superoxide dismutase in human sperm suspensions: relationship with cellular composition, oxidative stress, and sperm function. Free Radical Biology and Medicine 21, 495–504.
Aitken RJ, Krausz C, Buckingham DW 1994 Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation and the presence of leucocytes and precursor germ cells in human sperm suspensions. Molecular Reproduction and Development 39, 268–279.
Aravindan GR, Bjordahl J, Jost LK et al. 1997 Susceptibility of
human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identifi ed by single-cell electrophoresis. Experimental Cell Research 236, 231–237.
Barbieri ER, Hidalgo ME, Venegas A et al. 1999 Varicocele-associated decrease in antioxidant defenses. Journal of Andrology 20, 713–717.
Barroso G, Morshedi M, Oehninger S 2000 Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Human Reproduction 15, 1338–1344.
Bartoov B, Berkovitz A, Eltes F et al. 2003 Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertility and Sterility 80, 1413–1419.
Bartoov B, Berkovitz A, Eltes F et al. 2002 Real-time fi ne morphology of motile human sperm cells is associated with IVF-ICSI outcome. Journal of Andrology 23, 1–8.
Benchaib M, Braun V, Lornage J et al. 2003 Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Human Reproduction 18, 1023–1028.
Berkovitz A, Eltes F, Yaari S et al. 2005 The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Human Reproduction 20, 185–190.
Bianchi PG, Manicardi GC, Bizzaro D et al. 1993 Effect of deoxyribonucleic acid protamination on fl uorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biology of Reproduction 49, 1083–1088.
Blanco-Rodriguez J, Martinez-Garcia C 1998 Apoptosis precedes detachment of germ cells from the seminiferous epithelium after hormone suppression by short-term oestradiol treatment of rats. International Journal of Andrology 21, 109–115.
Blanco-Rodriguez J, Martinez-Garcia C 1996 Spontaneous germ cell death in the testis of the adult rat takes the form of apoptosis: re-evaluation of cell types that exhibit the ability to die during spermatogenesis. Cell Proliferation 29, 13–31.
Borini A, Tarozzi N, Bizzaro D et al. 2006 Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Human Reproduction 21, 2876–2881.
Braude P, Bolton V, Moore S 1988 Human gene expression fi rst occurs between the four- and eight-cell stages of preimplantation development. Nature 332, 459–461.
Bungum M, Humaidan P, Spano M et al. 2004 The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Human Reproduction 19, 1401–1408.
Calvin HI, Bedford JM 1971 Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. Journal of Reproduction and Fertility. Suppl 13, 65–75.
Carrell DT, Liu L, Peterson CM et al. 2003 Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Archives of Andrology 49, 49–55.
Casano R, Orlando C, Serio M et al. 1991 LDH and LDH-X activity in sperm from normospermic and oligozoospermic men. International Journal of Andrology 14, 257–263.
Chan PJ, Corselli JU, Patton WC et al. 2001 A simple Comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes. Fertility and Sterility 75, 186–192.
Collins AR, Dobson VL, Dusinska M et al. 1997 The Comet assay: what can it really tell us? Mutation Research 375, 183–193.
De Jonge C 2002 The clinical value of sperm nuclear DNA assessment. Human Fertility (Cambridge, England) 5, 51–53.
Donnelly ET, Steele EK, McClure N et al. 2001 Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Human Reproduction 16, 1191–1199.
Donnelly ET, O’Connell M, McClure N et al. 2000 Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Human Reproduction 15, 1552–1561.754
RBMOnline®
Duran EH, Morshedi M, Taylor S et al. 2002 Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Human Reproduction 17, 3122–3128.
Evenson D, Jost L 2000 Sperm chromatin structure assay is useful for fertility assessment. Methods in Cell Science 22, 169–189.
Evenson D, Wixon R 2006 Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reproductive BioMedicine Online 12, 466–472.
Evenson DP, Larson KL, Jost LK 2002 Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. Journal of Andrology 23, 25–43.
Evenson DP, Jost LK, Marshall D et al. 1999 Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Human Reproduction 14, 1039–1049.
Fawcett DW, Anderson WA, Phillips DM 1971 Morphogenetic factors infl uencing the shape of the sperm head. Developmental Biology 26, 220–251.
Fairbairn DW, Olive PL, O’Neill KL 1995 The Comet assay: a comprehensive review. Mutation Research 339, 37–59.
Furuchi T, Masuko K, Nishimune Y et al. 1996 Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia. Development 122, 1703–1709.
Gandini L, Lombardo F, Paoli D et al. 2004 Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Human Reproduction 19, 1409–1417.
Giuliani V, Pandolfi C, Santucci R et al. 2004 Expression of gp20, a human sperm antigen of epididymal origin, is reduced in spermatozoa from subfertile men. Molecular Reproduction and Development 69, 235–240.
Gomez E, Buckingham DW, Brindle J et al. 1996 Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. Journal of Andrology 17, 276–287.
Gorczyca W, Traganos F, Jesionowska H et al. 1993 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. Experimental Cell Research 207, 202–205.
Greco E, Romano S, Iacobelli M et al. 2005a ICSI in cases of sperm DNA damage: benefi cial effect of oral antioxidant treatment. Human Reproduction 20, 2590–2594.
Greco E, Scarselli F, Iacobelli M et al. 2005b Effi cient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Human Reproduction 20, 226–230.
Hammadeh ME, Radwan M, Al-Hasani S et al. 2006 Comparison of reactive oxygen species concentration in seminal plasma and semen parameters in partners of pregnant and non-pregnant patients after IVF/ICSI. Reproductive BioMedicine Online 13, 696–706.
Hammadeh ME, Al-Hasani S, Stieber M et al. 1996 The effect of chromatin condensation (aniline blue staining) and morphology (strict criteria) of human spermatozoa on fertilization, cleavage and pregnancy rates in an intracytoplasmic sperm injection programme. Human Reproduction 11, 2468–2471.
Hazout A, Dumont-Hassan M, Junca AM et al. 2006 High-magnifi cation ICSI overcomes paternal effect resistant to conventional ICSI. Reproductive BioMedicine Online 12, 19–25.
Hendin BN, Kolettis PN, Sharma RK et al. 1999 Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. Journal of Urology 161, 1831–1834.
Henkel R, Hajimohammad M, Stalf T et al. 2004 Infl uence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertility and Sterility 81, 965–972.
Henkel R, Kierspel E, Hajimohammad M et al. 2003 DNA fragmentation of spermatozoa and assisted reproduction technology. Reproductive BioMedicine Online 7, 477–484.
Host E, Lindenberg S, Smidt-Jensen S 2000 The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstetricia et Gynecologica Scandinavica 79, 559–563.
Huang CC, Lin DP, Tsao HM et al. 2005 Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates. Fertility and Sterility 84, 130–140.
Huckins C 1978 The morphology and kinetics of spermatogonial degeneration in normal adult rats: an analysis using a simplifi ed classifi cation of the germinal epithelium. Anatomical Record 190, 905–926.
Huynh T, Mollard R, Trounson A 2002 Selected genetic factors associated with male infertility. Human Reproduction Update 8, 183–198.
Irvine DS, Twigg JP, Gordon EL et al. 2000 DNA integrity in human spermatozoa: relationships with semen quality. Journal of Andrology 21, 33–44.
Jones R, Mann T, Sherins R 1979 Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertility and Sterility 31, 531–537.
Keating J, Grundy CE, Fivey PS et al. 1997 Investigation of the association between the presence of cytoplasmic residues on the human sperm midpiece and defective sperm function. Journal of Reproduction and Fertility 110, 71–77.
Kirchhoff C, Schroter S 2001 New insights into the origin, structure and role of CD52: a major component of the mammalian sperm glycocalyx. Cells, Tissues, Organs 168, 93–104.
Laberge RM, Boissonneault G 2005 On the nature and origin of DNA strand breaks in elongating spermatids. Biology of Reproduction73, 289–296.
Larson KL, DeJonge CJ, Barnes AM et al. 2000 Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Human Reproduction15, 1717–1722.
Larson KL, Brannian JD, Timm BK et al. 1999 Density gradient centrifugation and glass wool fi ltration of semen remove spermatozoa with damaged chromatin structure. Human Reproduction 14, 2015–2019.
Larson-Cook KL, Brannian JD, Hansen KA et al. 2003 Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertility and Sterility 80, 895–902.
Lewis SE, O’Connell M, Stevenson M et al. 2004 An algorithm to predict pregnancy in assisted reproduction. Human Reproduction19, 1385–1394.
Lopes S, Sun JG, Jurisicova A et al. 1998 Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertility and Sterility 69, 528–532.
Manicardi GC, Bianchi PG, Pantano S, et al. 1995 Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biology of Reproduction 52, 864–867.
Marcon L, Boissonneault G 2004 Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specifi city and link to chromatin remodeling. Biology of Reproduction 70, 910–918.
McPherson S, Longo FJ 1993 Chromatin structure-function alterations during mammalian spermatogenesis: DNA nicking and repair in elongating spermatids. European Journal of Histochemistry 37, 109–128.
Ménézo YJ 2006 Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment. Reproductive BioMedicine Online 12, 616–621.
Menkveld R, Franken DR, Kruger TF et al. 1991 Sperm selection capacity of the human zona pellucida. Molecular Reproduction and Development 30, 346–352.
Morris ID, Ilott S, Dixon L et al. 2002 The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Human Reproduction 17, 990–998.
Muratori M, Piomboni P, Baldi E et al. 2000 Functional and ultrastructural features of DNA-fragmented human sperm. Journal of Andrology 21, 903–912. 755
Review - Relevance of sperm DNA damage in IVF - N Tarozzi et al.
RBMOnline®
Review - Relevance of sperm DNA damage in IVF - N Tarozzi et al.
Nasr-Esfahani MH, Salehi M, Razavi S et al. 2005 Effect of sperm DNA damage and sperm protamine defi ciency on fertilization and embryo development post-ICSI. Reproductive BioMedicine Online11, 198–205.
Nicopoullos JD, Ramsay JW, Almeida PA et al. 2004 Assisted reproduction in the azoospermic couple. International Journal of Obstetrics and Gynaecology 111, 1190–1203.
O’Connell M, McClure N, Powell LA et al. 2003 Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation. Fertility and Sterility 79 (Suppl. 1), 754–762.
Oehninger SC, Kruger TF 2007 Male Infertility Diagnosis and Treatment. Informa Healthcare, London, p. 468.
Olive PL, Johnston PJ, Banath JP et al. 1998 The Comet assay: a new method to examine heterogeneity associated with solid tumors. Nature Medicine 4, 103–105.
Olsen AK, Lindeman B, Wiger R et al. 2005 How do male germ cells handle DNA damage? Toxicology and Applied Pharmacology 207, 521–531.
Perreault SD, Aitken RJ, Baker HW et al. 2003 Integrating new tests of sperm genetic integrity into semen analysis: breakout group discussion. Advances in Experimental Medicine and Biology 518, 253–268.
Perry AC, Jones R, Hall L 1993 Isolation and characterization of a rat cDNA clone encoding a secreted superoxide dismutase reveals the epididymis to be a major site of its expression. Biochemical Journal 293 (Pt 1), 21–25.
Richburg JH 2000 The relevance of spontaneous- and chemically-induced alterations in testicular germ cell apoptosis to toxicology. Toxicology Letters 112–113, 79–86.
Rodriguez I, Ody C, Araki K et al. 1997 An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO Journal 16, 2262–2270.
Rolf C, Cooper TG, Yeung CH et al. 1999 Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebo-controlled, double-blind study. Human Reproduction 14, 1028–1033.
Russell LD, Chiarini-Garcia H, Korsmeyer SJ et al. 2002 Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biology of Reproduction 66, 950–958.
Sailer BL, Jost LK, Evenson DP 1995 Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. Journal of Andrology 16, 80–87.
Sakkas D, Moffatt O, Manicardi GC et al. 2002 Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biology of Reproduction 66, 1061–1067.
Sakkas D, Manicardi GC, Tomlinson M et al. 2000 The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Human Reproduction 15, 1112–1116.
Sakkas D, Mariethoz E, Manicardi G et al. 1999a Origin of DNA damage in ejaculated human spermatozoa. Reviews of Reproduction 4, 31–37.
Sakkas D, Mariethoz E, St John JC 1999b Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Experimental Cell Research251, 350–355.
Saleh RA, Agarwal A, Nada EA et al. 2003 Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertility and Sterility 79 (Suppl. 3), 1597–1605.
Sanchez R, Stalf T, Khanaga O et al. 1996 Sperm selection methods for intracytoplasmic sperm injection (ICSI) in andrological patients. Journal of Assisted Reproduction and Genetics 13, 228–233.
Seli E, Sakkas D 2005 Spermatozoal nuclear determinants of reproductive outcome: implications for ART. Human Reproduction
Update 11, 337–349.Seli E, Gardner DK, Schoolcraft WB et al. 2004 Extent of nuclear
DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Fertility and Sterility 82, 378–383.
Sergerie M, Laforest G, Boulanger K et al. 2005a Longitudinal study of sperm DNA fragmentation as measured by terminal uridine nick end-labelling assay. Human Reproduction 20, 1921–1927.
Sergerie M, Laforest G, Bujan L et al. 2005b Sperm DNA fragmentation: threshold value in male fertility. Human Reproduction 20, 3446–3451.
Singh NP, Danner DB, Tice RR et al. 1989 Abundant alkali-sensitive sites in DNA of human and mouse sperm. Experimental Cell Research 184, 461–470.
Singh NP, McCoy MT, Tice RR et al. 1988 A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–191.
Slama R, Bouyer J, Windham G et al. 2005 Infl uence of paternal age on the risk of spontaneous abortion. American Journal of Epidemiology 161, 816–823.
Smith A, Haaf T 1998 DNA nicks and increased sensitivity of DNA to fl uorescence in situ end labelling during functional spermiogenesis. Biotechniques. 25, 496–502.
Smith R, Vantman D, Ponce J et al. 1996 Total antioxidant capacity of human seminal plasma. Human Reproduction 11, 1655–1660.
Spano M, Seli E, Bizzaro D et al. 2005 The signifi cance of sperm nuclear DNA strand breaks on reproductive outcome. Current Opinion in Obstetrics and Gynecology 17, 255–260.
Spano M, Bonde JP, Hjollund HI et al. 2000 Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertility and Sterility 73, 43–50.
Spano M, Kolstad AH, Larsen SB et al. 1998 The applicability of the fl ow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios. Human Reproduction 13, 2495–2505.
Steele EK, McClure N, Maxwell RJ et al. 1999 A comparison of DNA damage in testicular and proximal epididymal spermatozoa in obstructive azoospermia. Molecular Human Reproduction 5, 831–835.
Sun JG, Jurisicova A, Casper RF 1997 Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biology of Reproduction 56, 602–607.
Tesarik J 2005 Paternal effects on cell division in the human preimplantation embryo. Reproductive BioMedicine Online 10, 370–375.
Tesarik J, Mendoza-Tesarik R, Mendoza C 2006 Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment. Reproductive BioMedicine Online 12, 715–721.
Tesarik J, Greco E, Mendoza C 2004 Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Human Reproduction 19, 611–615.
Tomlinson MJ, Moffatt O, Manicardi GC et al. 2001 Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Human Reproduction 16, 2160–2165.
Tomsu M, Sharma V, Miller D 2002 Embryo quality and IVF treatment outcomes may correlate with different sperm Comet assay parameters. Human Reproduction 17, 1856–1862.
Twigg J, Irvine DS, Houston P et al. 1998 Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective signifi cance of seminal plasma. Molecular Human Reproduction 4, 439–445.
van Dyk Q, Lanzendorf S, Kolm P et al. 2000 Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound. Human Reproduction 15, 1529–1536.
van Overveld FW, Haenen GR, Rhemrev J et al. 2000 Tyrosine as important contributor to the antioxidant capacity of seminal plasma. Chemico-Biological Interactions 127, 151–161.
Verit FF, Verit A, Kocyigit A et al. 2006 No increase in sperm DNA damage and seminal oxidative stress in patients with idiopathic infertility. Archives of Gynecology and Obstetrics 274, 339–344.756
RBMOnline®
Virro MR, Larson-Cook KL, Evenson DP 2004 Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertility and Sterility 81, 1289–1295.
Wang JC, Caron PR, Kim RA 1990 The role of DNA topoisomerases in recombination and genome stability: a double-edged sword? Cell 62, 403–406.
Wang RA, Nakane PK, Koji T 1998 Autonomous cell death of mouse male germ cells during fetal and postnatal period. Biology of Reproduction 58, 1250–1256.
Ward WS, Coffey DS 1991 DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biology of Reproduction 44, 569–574.
Wayne CM, Sutton K, Wilkinson MF 2002 Expression of the pem homeobox gene in Sertoli cells increases the frequency of adjacent germ cells with deoxyribonucleic acid strand breaks. Endocrinology 143, 4875–4885.
Zini A, Bielecki R, Phang D et al. 2001 Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertility and Sterility75, 674–677.
Zini A, Finelli A, Phang D et al. 2000 Infl uence of semen processing technique on human sperm DNA integrity. Urology 56, 1081–1084.
Paper based on contribution presented at the Tecnobios Procreazione Symposium 2006 and 2nd International Conference on the Cryopreservation of the Human Oocyte in Bologna, Italy, 5–7 October 2006.
Received 26 January 2007; refereed 13 February 2007; accepted 30 March 2007.
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