RBMOnline - Vol 14. No 3. 2007 384-395 Reproductive BioMedicine Online; www.rbmonline.

com/Article/
www.rbmonline.com/Article/2730 on web 29 January 2007

Review
DNA damage of human spermatozoa in
assisted reproduction: origins, diagnosis,
impacts and safety
Dr Ozmen started studying obstetrics and gynaecology in 1998 at the University of Ankara.
Since his graduation from residency programme in 2003, he has been working on reproductive
medicine at the same centre. He is now continuing his research doctorate on assisted
reproduction at the University of Lbeck in Germany under the supervision of Professor
Safaa Al-Hasani. His major interest areas are cryopreservation and micromanipulation of
embryos, sperm DNA damage and endoscopic reproductive surgery.

Dr Ozmen
B Ozmen1,4, N Koutlaki 2,4, M Youssry3,4, K Diedrich4, S Al-Hasani4,5
1
Department of Obstetrics and Gynecology, University of Ankara, Ankara, Turkey; 2Department of Obstetrics and
Gynecology, Demokritus University of Thrace, Athens, Greece; 3Department of Obstetrics and Gynecology, University
of Alexandria, Alexandria, Egypt; 4University of Schleswig-Holstein, Department of Gynaecology and Obstetrics,
Reproductive Medicine Unit, University of Lbeck, Ratzeburger Allee 160, 23560 Lbeck, Germany
5
Correspondence: Tel: +494515002155; Fax: +494515004764; e-mail: sf_alhasani@hotmail.com

Abstract
Sperm DNA contributes half the offsprings genomic material and abnormal DNA can lead to derangements in the
reproductive process. Normal sperm genetic material is required for successful fertilization, as well as for further embryo
and fetal development that will result in a healthy child. Thus, the damage to sperm DNA is critical in assisted reproductive
techniques which are increasingly used to treat infertile couples. There has been improving data about the effects of human
sperm DNA damage or fragmentation. As well, increasing knowledge concerning the effects of DNA damage on embryo and
fetal development has been attained. This review aims to summarize the present knowledge on the impact of human sperm
cell DNA damage on male infertility and outcome in the context of safety.

Keywords: apoptosis, assisted reproduction, DNA damage, safety, sperm, outcome

Introduction
Assisted reproduction techniques have currently undertaken
a major role in treating infertility. Success rates depend on
a variety of factors most important being the structural and
functional integrity of the gametes used. Therefore, the exact
nature of infertility and its cause or pathogenesis should
be considered before initiation of treatment. Unidentied
factors may adversely affect the end results and add to the
nancial, social and emotional problems of the patients.
The positive relationship between poor sperm parameters
and DNA damage in human spermatozoa points to inherent
problems in spermatogenesis in specic patients. Various
hypotheses have been proposed as to the molecular
mechanism of sperm DNA damage. The most important
ones are abnormal chromatin packaging, oxidative stress and
384 apoptosis (Sakkas et al., 1999).
Semen samples that contain high levels of DNA damage are
often associated with decreased fertilization rates or embryo
cleavage after IVF and intracytoplasmic sperm injection
(ICSI) and may be linked to early embryo death. Although
the most normal appearing and motile spermatozoa are
selected, there is always a chance that spermatozoa
containing varying degrees of DNA damage may be used.
Thus, one of the main disadvantages of using assisted
reproduction is the bypass of the natural selection barriers
that are present throughout the female reproductive tract until
spermatozoa enters the oocyte (Chandley and Hargreave,
1996): spermatozoa with abnormal genomic material can
reach the genetic material of the oocyte with minimal (IVF)
or no effort (ICSI) at all. The miscarriage rate is higher
after ICSI, which possibly reects the fact that genomically
compromised spermatozoa are sometimes used and lead to

2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

irreparable DNA damage in the embryo (Carrell et al., 2003;
Agarwal and Said, 2005).
This review aims to summarize the present knowledge on the
impact of human sperm cell DNA damage, in the context of
the different damage origins, on outcome and prognosis of
male infertility, as well as the safety of using DNA-damaged
human spermatozoa in assisted reproduction.
Origins of DNA damage of human
sperm
Spermatogenesis is a complex process of male germ cell
proliferation and maturation from diploid spermatogonia
through meiosis to mature haploid spermatozoa (de Kretser et
al., 1998). However, damage of sperm DNA or its chromatin
structure can occur at any step of whole spermatogenesis
(Erenpreiss et al., 2006). The positive relationship between
poor sperm parameters and DNA damage in mature
spermatozoa points to inherent problems in spermatogenesis
in specic patients (Irvine et al., 2000; Agarwal and Said,
2005). Three theories have been proposed to explain DNA
anomalies in the ejaculated human spermatozoa. The rst
theory supports that DNA damage in mature spermatozoa is
associated with poor chromatin packaging or abnormal packing
due to underprotamination which results in the presence of
endogenous nicks in DNA (Manicardi et al., 1995; Sakkas et
al., 1999). The second theory proposes that the presence of
endogenous nicks is characteristic of programmed cell death
aiming to the functional elimination of possibly defective
germ cells from the genetic pool (Sakkas et al., 1999). Recent
models of apoptosis include receptor-mediated pathways and
intrinsic triggered apoptosis, as well as cytotoxic or stress
induced forms (Manicardi et al., 1995; Sakkas et al., 1999).
The last one is the oxidative stress mechanism that has been
studied extensively, and is caused by the overproduction of
reactive oxygen species (ROS) (Sharma and Agarwal, 1996;
Aitken and Krausz, 2001; Agarwal and Said, 2005; Lewis
and Aitken, 2005). All mechanisms described above, either
individually or together, have some bearing on the presence
of abnormal spermatozoa in the ejaculate, and they may or
may not be interrelated (Sakkas et al., 2003).
Abnormal and dysregulated chromatin
packing
Sperm chromatin structure, on contrary to somatic cells, is
tightly compacted due to the unique associations between
the DNA and sperm nuclear proteins. These nuclear proteins
are predominantly comprised of protamines which are highly
basic proteins. Thereafter the displacement of histones by
transition proteins and then by protamines, the spermatid
nucleus is remodelled and condensed in the nal stages of
spermatogenesis. The sperm DNA strands are tightly wrapped
around the protamine molecules (about 50 kb of DNA per
protamine) for forming tight and highly organized loops.
The compaction and stabilization is organized by inter- and
intramolecular disulphide cross-links between the cysteine-
rich protamines.
More than two-thirds of the chromatin structure of human
RBMOnline
Review - DNA damage of human spermatozoa - B Ozmen et al.
spermatozoa is thus packaged by protamines, only up to
15% of the DNA are less tightly compacted and packaged by
histones. It has been shown that infertile men have an increased
sperm histone:protamine ratio than fertile counterparts. This
alteration of histone:protamine ratio, that is also called
abnormal packing, increases susceptibility of sperm DNA
to external stresses due to poorer chromatin compaction.
Furthermore, complete deciency of protamine has been
demonstrated in about 515% of infertile men (Carrell and
Liu, 2001). Recent studies also underlined the link between
protamine deciency and sperm DNA damage that resulted
in poor fertilizing capacity (Nasr-Esfahani et al., 2005).
However there is a non-random selection of these histone-
bound DNA sequences or genes in normal sperm DNA, and
these are mainly presumed to be involved in fertilization and
early embryo development (Tesarik et al., 2002).
The mitochondrial DNA of human spermatozoa is a small,
circular DNA which is not bound to special proteins. It has
been demonstrated that sperm motility is directly related
to the mitochondrial volume within the sperm mid-piece.
The mitochondrial DNA exhibits a high rate of mutation
or deletions that have been associated with reduced sperm
motility. The inheritance of mitochondrial DNA is primarily
maternal and only in 1% of cases paternal transmission of
mitochondrial DNA mutations have been reported (Schwartz
and Vissing, 2002).
The role of apoptosis of human
spermatozoa in DNA damage
Apoptosis can be postulated to have two putative roles during
normal spermatogenesis: limitation of the germ cell population
to numbers that can be supported by the Sertoli cells and,
possibly, selective depletion of abnormal spermatozoa (Sakkas
et al., 2003). Throughout life, apoptosis eliminates cells that
are useless or potentially dangerous to the host such as aged,
infected, injured or mutated cells. In normal conditions, more
than half of the potential number of the mature germ cells is
lost, mostly due through apoptosis of spermatogonia and
spermatocytes (Dunkel et al., 1997).
During apoptosis the cells shrink and exhibit several typical
features, including cell membrane disruption, cytoskeletal
rearrangement, nuclear condensation, and intranucleosomal
DNA fragmentation (Kaufmann and Hengartner, 2001).
Apoptosis in the human spermatozoa is a result of DNA strand
breaks induced by a cascade of regulatory mechanisms with
infertility (Hst et al., 2000). The degradation of DNA into
fragments approximately 185 bp and its multiples in size is one
of the best characterized biochemical features of apoptotic cell
death and is used as the basis for the commonly used labelling
techniques for detecting apoptotic cells (Nagata, 2000).
Apoptotic cells are usually rapidly taken up and degraded by
neighbouring cells before their intracellular contents leak into
the extracellular space. In contrast, acute accidental injury may
lead to an uncontrolled form of cell death, called necrosis, which
is characterized by swelling and bursting of the dying cells with
an accompanying inammatory response (Raff, 1998)
However, dysregulation of this physiological germ cell
apoptosis thus itself might cause male infertility. Inappropriate 385
 Review - DNA damage of human spermatozoa - B Ozmen et al.
germ cell apoptosis may also result from external disturbances
such as alterations in hormonal support or exposure to toxic
chemicals or radiation (Sakkas et al., 1999). In this respect,
exposure to environmental toxicants and hormone-like
compounds has been suggested to cause declining sperm
counts and male fertility problems. Moreover, survival of
cancer patients treated with radiation and chemotherapeutic
drugs has increased, but the treatments may cause sperm cell
loss and infertility.
Apoptosis is thus characterized by a variety of changes
resulting in the special recognition and phagocytosis of
apoptotic cells. Some specic proteinases, called caspases
(cysteinyl aspartate-specic proteinases), have been claimed
to play a major role in the regulation of apoptosis. More than
a dozen of these specic proteinases have been reported to
be related to apoptosis in the human seminiferous epithelium
that are expressed as inactive proenzymes and participate in a
cascade triggered in response to pro-apoptotic signals. Among
these, caspase-3 is considered to be a major executioner
protease (Paasch et al., 2004a; Said et al., 2004). Caspases
share the ability to cleave their substrates on the carboxyl
side of aspartate residues (Kaufmann and Hengartner, 2001).
Cell-surface death receptors such as Fas or tumour necrosis
factor-a receptor 1 are activated by ligand binding, resulting
in the proteolytic activation of caspases, in the destruction of
vital proteins and nally in the death of the cell.
In addition, caspases activate other proteins needed for the
achievement of apoptosis such as caspase-activated DNase,
which is responsible for DNA fragmentation (Nagata,
2000). The Bcl-2 family proteins (Bcl-x, Bcl-w, Bax, Bak,
Bid, Bad), the tumour suppressor p53, the nuclear factor
B(NF-B) and the heat shock proteins have been shown
to be regulators of apoptosis (Barkett and Gilmore, 1999;
Antonson and Martinou, 2000; Hengartner, 2000; Gupta,
2001). Having in mind the ultimate purpose of apoptosis,
spermatozoa exhibiting apoptotic features should be
eliminated in the ejaculate. However, it has been found that
men with abnormal sperm parameters display higher levels of
apoptotic markers in the ejaculated spermatozoa. Sakkas et
al. (1999) showed that in men with reduced sperm parameters
the percentage of Fas-positive spermatozoa can be as high
as 50%. Several other studies have also found that other
apoptotic markers such as Bcl-x, p53 and annexin V are also
present on ejaculated human spermatozoa and show distinct
relationships with abnormal semen parameters (Barroso et
al., 2000; Sakkas et al., 2002). It has been postulated that in
these subfertile men spermatozoa that have been earmarked
to undergo apoptosis escape this process, so that the correct
clearance of spermatozoa via apoptosis is not occurring.
Therefore, production of ejaculated spermatozoa that possess
apoptotic markers (such as Fas positivity and DNA damage)
indicate that in some men with abnormal semen parameters
an abortive apoptosis has taken place (Sakkas et al., 1999).
The nal outcome is the production of spermatozoa that
possess a range of anomalies including abnormal levels of
apoptotic proteins and/or cytoplasmic retention, abnormal
chromatin packaging (indicated by low levels of protamine)
and the presence of DNA strand breaks. These abnormal
spermatozoa produced after spermatogenesis might be more
susceptible to environmental factors and oxidative attack
386 (Aitken et al., 2003).
Oxidative stress by reactive oxygen
species
The role of ROS as a cause of chromatin and DNA damage is well
recognized to play a major physiological role in differentiation
and function of sperm, as well as regulation or modulation of
sperm genetic material. They cause lipid peroxidation of sperm
plasma membranes, that results in alteration of protein activities
vital for sperm function and fertilizing capacity (Duru et al.,
2000). It has been reported that ROS might also cause high
frequencies of single- and double-strand DNA breaks (Twigg
et al., 1998a). Both superoxide (O2) and the hydroxyl radical
(OH) are known to be mutagenic and cause chromosome
deletions, dicentrics and sister chromatid exchanges (Twigg et
al., 1998a; Aitken and Krausz, 2001).
It has been shown that the amount of ROS generation is well
controlled by seminal antioxidants in the semen of fertile men.
Thus initially, the pathogenic effects of ROS presumed to occur
in cases of excessive production that cannot be tolerated by
antioxidant capabilities of the male reproductive tract or seminal
plasma (Aitken et al., 1992). Subsequently, it has been claimed
that there is not a signicant reduction in the total antioxidant
capacity associated with increased levels of ROS. Furthermore,
the pathological levels of ROS detected in the semen of infertile
men was reported to be more likely caused by increased ROS
production than by reduced antioxidant capacity of seminal
plasma (Zini et al., 1993).
Morphologically abnormal spermatozoa (with residual
cytoplasm, in particular) and leukocytes are the main source
of excess ROS generation in semen (Aitken et al., 1992).
Activated leukocytes are capable of producing 100-fold higher
amounts of ROS than non-activated leukocytes (Plante et al.,
1994). Sperm DNA thus 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
(Erenpreiss et al., 2002).
Human sperm chromatin becomes cross-linked under conditions
of oxidative stress and exhibits increased DNA strand breakage
(Twigg et al., 1998b). Inadequate oxidation is caused by
formation of defective spermatid protamination and disulphide
bridge of thiols during epididymal transit. This dysformation
results in diminished sperm chromatin packaging that makes
sperms more vulnerable to ROS-induced DNA fragmentation.
Recently, the oxidative stress caused by ROS is claimed to
be more likely to lead to sperm DNA damage in ejaculated
spermatozoa rather than apoptosis (Moustafa et al., 2004).
Methods for detection of human
sperm DNA damage
As sperm genetic integrity has been implicated in fertilization
and embryo development failures (Ahmadi and Ng, 1999; Cho
et al., 2003) several studies have been conducted analysing
sperm nuclear integrity in terms of DNA damage, its relationship
to male infertility and outcome and its inuence on mature
spermatozoon. Sperm DNA damage can be measured directly
RBMOnline

by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP
nick-end labelling (TUNEL) and single-cell gel electrophoresis
(COMET) assay. These methods are commonly used in
research applications for detecting apoptotic DNA and both
demonstrated good correlations with fertility outcome (Sharma
et al., 2004) but the subjectivity and variability of their results
does not currently allow their use as distinctive identiers of
samples with impaired fertility (Agarwal and Said, 2005).
A number of studies have proposed that the presence of
spermatozoa with damaged DNA is indicative of apoptosis. The
TUNEL technique identies DNA breaks by labelling 3OH
termini using exogenous terminal deoxynucleotidyl transferase
and was introduced by Gorczyca et al. (1993) to identify a
population of spermatozoa in the ejaculate that were believed to
be apoptotic. Numerous other studies using the same technique
have followed (Manicardi et al., 1995; Sun et al., 1997;
Lopes et al., 1998; Irvine et al., 2000). Muratori et al. (2000)
demonstrated that DNA fragmentation assessed by the TUNEL
method was not associated with an apoptosis-like phenomenon
in ejaculated spermatozoa and that DNA fragmentation should
be considered a sign of defective sperm maturation probably
dating back to the time of DNA packaging. Although Sakkas
et al. (2002) support that TUNEL positivity and apoptotic
markers do no always exist in unison in spermatozoa, however,
semen samples that had a low sperm concentration and poor
morphology were more likely to show high levels of TUNEL
positivity and Fas and p53 expression.
Using the TUNEL assay negative associations were found
between the percentage of spermatozoa with DNA fragmentation
and embryo cleavage rates after IVF (Sun et al., 1997). Lopes
et al. (1998) report a negative association between spermatozoa
with DNA fragmentation and ICSI fertilization rate. Benchaib
et al. (2003) found that a high proportion of spermatozoa with
fragmented DNA (>10%) was a negative factor for achievement
of pregnancy when ICSI was performed, but there was no
relationship when conventional IVF was carried out. Hst et al.
(2000) found that the level of DNA strand breaks was predictive
of the success rate in IVF but not in ICSI.
The COMET assay which uses single-cell gel electrophoresis
to analyse DNA fragmentation in individual cells was rst
introduced in 1984 by Ostling and Johanson (see Olive and
Banath, 1995) who used neutral buffer conditions to study
double-stranded DNA breaks. It was later modied using
alkaline electrophoresis buffers to increase the sensitivity
to both single and double stranded DNA breaks (Raman et
al., 2001). It is a useful technique because it allows for the
distinction between the different kinds of DNA fragmentation,
eg necrotic or apoptotic. Apoptotic cells produce teardrop
shaped comets due to the migration and accumulation of the
short DNA fragments, and the intensity of the tail represents the
amount of DNA fragments present (Duty et al., 2002). Apoptotic
DNA fragmentation is characterized by double-stranded DNA
breaks. Using the COMET assay Tomsu et al. (2002) noted that
the COMET head and tail DNA parameters could be considered
potentially useful predictors of embryo quality and IVF
outcomes, especially in couples with unexplained infertility.
It has also been shown that high loads of DNA damage were
predictive of embryo development failure after ICSI (Morris
et al., 2002). On the other hand, Abu-Hassan et al. (2005) do
not report any correlation between apoptosis levels assessed by
RBMOnline
Review - DNA damage of human spermatozoa - B Ozmen et al.
COMET assay and the outcome of ICSI as far as fertilization
and embryo quality are concerned.
Sperm chromatin structure assay (SCSA) is a quantitative
assessment of sperm chromatin integrity as susceptibility
of DNA to acid-induced denaturation in situ. Most methods
currently used to assess apoptosis and sperm DNA damage
lack a threshold between normal levels in the average fertile
population and the minimal levels of sperm DNA integrity
required for achieving pregnancy, except for the SCSA. The
method uses ow cytometry and the DNA damage is expressed
as the DNA fragmentation index (DFI). In clinical applications
the DFI not only distinguished fertile men from those who were
infertile, but also identied samples that were compatible with
pregnancies conceived (<2830%) (Agarwal and Said, 2005).
Two large independent studies (Evenson et al., 1999; Spano et
al., 2000) on the relationship between SCSA results and sperm
fertilization capacity have been carried out in the USA and
Europe. Both demonstrated that when >30% of spermatozoa
have abnormal chromatin as evaluated by SCSA, human
male infertility is hampered independent of sperm number,
morphology and motility. The categories proposed by Evenson
et al., (2002) for individual fertility potential according to DFI
fraction are: excellent <15%, good 1524%, fair 2530% and
poor >30% DFI, and if DNA staining is >15% the fertility
potential is downgraded at least one category. Aravindan et al.
(1997) established a signicant relationship between the SCSA,
COMET and TUNEL assays for human spermatozoa.
It has been shown that the loss of phospholipid asymmetry
leading to exposure of phosphatidylserine on the outside
of the plasma membrane is an early event of apoptosis. The
anticoagulant annexin V preferentially binds to negatively
charged phospholipids such as phosphatidylserine (Ricci et
al., 2002). By conjugating uorescein to annexin V it has been
possible to use the marker to identify apoptotic cells by ow
cytometry. During apoptosis the cells bind annexin V prior to
the loss of the plasma membranes ability to exclude propidium
iodide. Therefore, by staining cells with with a combination of
annexin V and propidium iodide it is possible to simultaneously
distinguish live, apoptotic and necrotic cell populations. This
method does not involve enzyme activity and does not require
cells to be previously xed. Accordingly, unlike the TUNEL
assay, this assay enables living spermatozoa to be evaluated
(Ricci et al., 2002). The method has produced conicting
results concerning the correlation of apoptotic spermatozoa
in the ejaculate and sperm parameters (Glander and Schaller,
1999; Osterhuis et al., 2000).
Impacts of human sperm DNA
damage
Impacts on outcomes of assisted
reproduction
Infertile men are reported to have a higher fraction of spermatozoa
with chromatin defects and DNA breaks, which may have a
possible negative impact on the assisted reproduction outcome
(Lopes et al., 1998; Larson et al., 2000; Sakkas et al., 2002;
Muratori et al., 2003). The identied relationship between
conventional semen parameters and sperm DNA fragmentation 387
 Review - DNA damage of human spermatozoa - B Ozmen et al.
(Lopes et al., 1998; Sakkas et al., 1999 Irvine et al., 2000; Zini et
al., 2001), is not strong enough to eliminate DNA fragmentation
as a potential source of infertility in normozoospermic men and
requires a distinct assessment of sperm DNA fragmentation in
male infertility evaluations (Larson-Cook et al., 2003).
Huang et al. (2005) found that sperm DNA fragmentation rates
>10% were correlated with lower fertilization rates but not with
pregnancy outcome. Hst et al. (2000) determined the incidence
of spermatozoa with DNA strand breaks in different groups
of infertile couples (tubal obstruction, unexplained infertility,
oligozoospermia and IVF, oligozoospermia and ICSI) and
related the DNA damage to the fertilization rates and outcome.
Sperm preparation was performed by the swim-up technique.
They presented negative correlations between the proportion of
spermatozoa with DNA strand breaks and the fertilization rates
in all groups except for the ICSI group. A possible explanation
for this might be that the person who processes ICSI selects gross
morphologically normal spermatozoa diminishing the chance of
injecting spermatozoa having DNA strand breaks. However, in
the groups of unexplained infertility and oligozoospermia and
IVF higher fertilization rates were seen when 4% spermatozoa
with DNA strand breaks were present (P < 0.05). Furthermore,
in the group with the unexplained infertility, the mean proportion
of spermatozoa with DNA strand breaks in couples with a
positive serum human chorionic gonadotrophin (HCG) was
signicantly lower compared with those with a negative serum
HCG (P < 0.05). No correlation between DNA strand breaks in
spermatozoa and the fertilization rate in the oligozoospermia and
ICSI group was found, while no impact of DNA stand breaks
on pregnancy rates was reported. Interestingly, the number of
spermatozoa with DNA strand breaks was signicantly higher
in the group of men where the females had tubal obstruction
compared with proven fertile men (Hst et al., 1999), which
suggests that even in the group with signicant female factors
a male factor may also be included. They suggest that if sperm
samples from couples with unexplained infertility exhibit more
than 4% DNA strand breaks in the spermatozoa, these couples
should have ICSI as the impact of DNA strand breaks will be
reduced.
The level of DNA breaks is conveniently expressed by the
DNA fragmentation index (DFI) (Larson et al., 2000; Evenson
et al., 2002). In two independent studies, DFI levels > 30%
40% were incompatible with fertility in vivo, whatever sperm
concentration, morphology and motility (Evenson et al., 1999;
Spano et al., 2000). Furthermore, although based on limited
numbers of patients, no pregnancy has been reported after
in-vitro procedures, both standard IVF and ICSI, when the
DFI in raw semen was more than 27% (Larson et al., 2000;
Larson-Cook et al., 2003; Saleh et al., 2003). Bungum et al.
(2004) examined the relationship between sperm chromatin
defects evaluated by SCSA and the outcome of IVF/ICSI and
intrauterine insemination (IUI). Two groups were studied for
each procedure; one with DFI 27% and a second with DFI
> 27% since a DFI of 27% previously was reported to be the
cut-off level for achieving a pregnancy by in-vitro techniques
(Larson-Cook et al., 2003). As far as IUI is concerned, there
were 22.2% biochemical pregnancies per insemination, 20.2%
clinical pregnancies per insemination and 17.6% deliveries per
started cycle for DFI 27%, while the respective rates for DFI
> 27% were 4.3%, 4.5% and 4.5%. In the IVF/ICSI patients
388 there were 51.2% biochemical pregnancies, 38.2% clinical
RBMOnline
pregnancies, 32.7% implantation rate and 31.4% deliveries
for DFI 27%, while for DFI > 27% the corresponding values
were 47.1%, 38.2%, 28.6% and 34.3%. It is important that this
study, contrary to previous reports (Larson et al., 2000; Larson-
Cook et al., 2003; Saleh et al., 2003), reports that DFI level
> 27% is compatible with pregnancy and delivery after both
IVF and ICSI, a result also conrmed in the study of Payne
et al. (2005) who reported that nine of 19 couples with DFI
> 27% achieved clinical pregnancy with IVF/ICSI. Thus, it
seems that in-vitro techniques are is able to compensate for the
impairment of sperm chromatin integrity, in particular if ICSI
is chosen as a fertilization method. In couples treated with IUI,
clinical pregnancy was seen in only one out of 23 cases when
the DFI exceed 27%. Duran et al. (2002) using the TUNEL
assay indicative for DNA fragmentation found that men with
a DNA fragmentation level > 12% had a decreased chance of
fathering a pregnancy following IUI. Saleh et al. (2003) using
the SCSA found signicantly higher DFI levels in the couples
who failed to obtain a pregnancy after IUI. Bungum et al.
(2004) demonstrated that even men with high levels of DNA
damage can become biological fathers when DFI exceeded
27%. However, signicantly higher clinical pregnancy rates
(52.9 versus 22.2) and delivery rates (47.1 versus 22.2%)
were seen after ICSI compared with IVF. With a DFI > 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 respectively,
results similar to those reported by Host et al. (Hst et al.,
2000) using the TUNEL assay for evaluation of DNA breaks.
After inclusion of other parameters as co-variates (sperm
concentration, percentage of progressively motile sperm,
number of previous treatments, age of the female) they showed
that DFI is an independent predictor of male fertility potential
in vitro. Larson-Cook et al. (2003) examined the relationship of
sperm nuclear DNA fragmentation as assessed by the SCSA test
with fertilization, embryo development, implantation rates and
pregnancy rates after conventional IVF and ICSI. Fertilization
rate (72.5 0.2%) was not related to DFI. This indicates that
normal fertilization does not ensure high quality DNA in the
paternal genome and supports previous studies that showed no
relationship between DNA fragmentation and fertilization rate
(Sakkas et al., 1996; Morris et al., 2002). On the contrary, other
investigators have shown a signicant negative correlation
between sperm DNA fragmentation and IVF (Sun et al., 1997)
and ICSI (Lopes et al., 1998) fertilization rates. In the study of
Larson-Cook et al. (2003) cleavage rates were not related to
SCSA parameters. Blastocyst formation rate (36.5 5.2%) was
also not signicantly related to SCSA parameters. All patients
who achieved pregnancy had DFI < 27% which contradicts
the results of Payne et al. (2005) reporting that only 2 out of
22 couples achieved clinical pregnancy when DFI was 9%.
One patient achieved a biochemical pregnancy with DFI >
27%, but subsequently lost the pregnancy before ultrasound.
DFI had a 100% specicity and positive predictive value for
failure to initiate an ongoing pregnancy. These results are
similar to those of other investigators who showed that DNA
damaged spermatozoa negatively affect preimplantation and
post-implantation embryonic development (Morris et al.,
2002), while in the study of Larson et al. (2000) it is suggested
that SCSA parameters in neat or washed spermatozoa are
not signicantly correlated with the fertilization and embryo
development rates.

Gandini et al. (2004) tried to determine possible relationships
between SCSA parameters evaluated on both neat semen
and the processed aliquot to be used in assisted reproduction
procedures and fertilization rate, embryo quality and pregnancy
rate following ICSI and IVF. No differences were seen in SCSA
parameter values between patients initiating pregnancies and
not doing so in either ICSI or conventional IVF. Pregnancies
and normal delivery were obtained even with high levels of
DFI. The mean DFI value for the nine men who fathered a
child (pregnancy rate 40.9%) was 32.1%. This value was not
different from that of the group of men not fathering a child
(25.1%). There was a statistically signicant difference between
percentage DFI in raw semen and in semen after swim-up (12
4% versus 5.5 4.3%, P < 0.001). Among infertile couples who
achieved pregnancy after ICSI, DFI ranged from 13.5 to 66.3%.
The results of this study were similar with those reported by
Larson-Cook et al. (2003) stating that fertilization rate, cleavage
rate and blastocyst formation rate were not signicantly related
to SCSA parameters and contradict the results of Saleh et al.
(2003) who found that DFI levels were negatively correlated
with fertilization and embryo quality after IVF and ICSI.
Virro et al. (2004) determined the relationship between SCSA
parameters and IVF/ICSI outcome. IVF and ICSI fertilization
rates were not statistically different between high and low DFI
groups. Men with high levels of DNA fragmentation (DFI
30%) were at greater risk for low blastocyst formation rates
and failure to initiate an ongoing pregnancy. In the same study
another SCSA parameter, high DNA stainability was related to
IVF but not to ICSI fertilization rates. Due to this reason the
authors suggest that men with DAN stainability >15% should
be treated with ICSI.
More recently, two meta-analyses have been published on sperm
DNA damage and assisted reproduction outcomes (Evenson and
Wixon, 2006; Li et al., 2006). One of them only addressed the
studies that diagnosed sperm DNA damage by SCSA (Evenson
and Wixon, 2006). As a result this meta-analysis showed that
in all IUI, IVF, and ICSI techniques clinical pregnancy and
outcomes were closely related to sperm DNA damage by means
of DFI. The other one suggested that sperm DNA damage seem
to signicantly decrease the chance of IVF clinical pregnancy
when only the studies that assessed sperm DNA damage by
TUNEL assay were included (Li et al., 2006). However, they
also indicated that neither IVF fertilization nor ICSI fertilization
nor ICSI clinical pregnancy is affected by sperm DNA damage.
As well, they claimed that their results also revealed sperm
DNA damage, when assessed by the SCSA, has no signicant
effect on the chance of clinical pregnancy after IVF or ICSI
treatment. Thus, there are still some controversies on the effect
of sperm DNA damage and outcomes due to two inconsistent
recent meta-analyses. However, the majority of studies indicate
that sperm DNA damage has negative effects on pregnancy rate,
embryo quality, live birth and early pregnancy loss (Table 1).
Impacts on traditional semen parameters
Semen from infertile men contain a high proportion of
spermatozoa with typical signs of apoptosis, including
morphological damage, nuclear DNA fragmentation and loss
of asymmetric distribution of phosphatidylserine at the plasma
membrane (Barroso et al., 2000; Gandini et al., 2000). Gandini
et al. (2000) studying the relationship between apoptosis and
seminal parameters, noted an increase of DNA fragmentation
RBMOnline
Review - DNA damage of human spermatozoa - B Ozmen et al.
in line with a decrease in sperm concentration and motility.
This correlation was even more evident with atypical forms
conrming that the results for morphology are strictly correlated
with sperm function. Examining the sperm morphological
aspect of cells showing DNA fragmentation they found that the
most common atypical forms were small and amorphous heads.
Amorphous heads seemed to be the most characteristic defect
in the spermatozoa before and after swim-up selection.
Siddighi et al. (2004) attempted to relate sperm parameters
associated with normal sperm physiology and function to
apoptosis assessed by a non-invasive dual stain DNA integrity
assay. The results showed differences in the percentage of
intact-DNA spermatozoa in individuals with normal WHO
sperm features (62 1.1%) compared with oligoasthenoteratoz
oospermia patients (38 5.3%). Individuals whose spermatozoa
had fertilizing capacity (capacitation index >7) had higher
percentages of intact DNA (60 1.3% versus 47 2.4%). The
percentages of intact-DNA spermatozoa were signicantly
correlated with total motility in semen (rr = 0.7), viability (r =
0.7), post-wash motility (rr = 0.6), rapid progression (r = 0.6),
intact acrosome (rr = 0.5) and strict morphology (r = 0.5). No
signicant correlation was found with sperm concentration.
Huang et al. (2005) evaluated sperm DNA fragmentation in
correlation with sperm parameters by use of TUNEL assay.
Sperm DNA fragmentation rates were signicantly higher in
patients with abnormal sperm parameters than in those with
normal sperm parameters.
In the study of Larson-Cook et al. (2003) the level of sperm
DNA fragmentation (percentage DFI) was not strongly
correlated to conventional semen parameters. Specically, only
three of the 10 men with DFI >27% had asthenozoospermia
(<50% motility) and/or oligozoospermia (<20 106 per ml) and
only two had abnormalities in both sperm concentration and
motility (oligoastheno-zoospermia).
In the study of Gandini et al. (2004) evaluation of sperm
chromatin structure was performed on semen samples
submitted to both swim-up and density gradient method. The
swim-up technique very effectively separated the fraction of
highly motile cells which were characterized by better sperm
chromatin integrity. Mean DFI was 12% in neat semen;
this gure dropped to 5.5% after swim-up, conrming the
evidence of previous experiments (Spano et al., 1999). The
swim-up procedure discriminates a subpopulation of highly
motile cells with improved morphology, consistently selecting
spermatozoa to give a notable improvement in sperm chromatin
features as evaluated by SCSA. On the contrary, the gradient
method, although powerful in its selection of motile and good
morphology spermatozoa (Sakkas et al., 2000; Tomlinson et
al., 2001), was less effective in enriching the cell population
with a higher fraction of spermatozoa with normal chromatin
structure; in fact DFI was 28.1% in neat semen and dropped to
24.2% after gradient density preparation.
In the study of Larson et al. (2000) no signicant relationship
between SCSA parameters in neat or washed spermatozoa and
WHO parameters was identied. SCSA parameters improved
following density-gradient preparation as reported previously
(Golan et al., 1997; Larson et al., 1999) but they were not
predictive of pregnancy outcome, indicating that elevated SCSA 389
 Review - DNA damage of human spermatozoa - B Ozmen et al.

Table 1. The results of studies on sperm DNA damage and outcomes of assisted
reproduction techniques.

Studies according to Pregnancy Embryo quality Early Live

method of diagnosis rate and development pregnancy birth
loss

TUNEL
Lopes et al., 1998
Hst et al., 2000
Tomlinson et al., 2001 Decrease No relation
Benchaib et al., 2003 Decrease Decrease
Henkel et al., 2004 Decrease Decrease, stops
Seli et al., 2004 No relation Decrease
Huang et al., 2005 No relation No relation
Benchaib et al., 2006 Decrease Decrease Increase Decrease
Borini et al., 2006 Decrease Increase Decrease
COMETa
Tomsu et al., 2002 Decrease Decrease
Morris et al., 2002 Decrease Increase Increase
Nasr-Esfahani et al., 2005b Decrease
SCSA or SCDc
Larson-Cook et al., 2003 Decrease No relation
Gandini et al., 2004 No relation Decrease
Virro et al., 2004 Decrease Decrease Decrease
Bungum et al., 2004 No relation
Payne et al., 2005 No relation
Zini et al., 2005 No relation Decrease
Check et al., 2005 Decrease Decrease Increase Decrease
Bungum et al., 2007d Decrease Decrease Decrease
Muriel et al., 2006e Decrease Decrease in
implantation rate

= not determined.
SCD = sperm chromatin dispersion test.
SCSA = sperm chromatin structure assay.
TUNEL = TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling.
a
COMET assay performed by single cell gel electrophoresis.
b
COMET and Chromomycin A3.
c
SCSA used except where noted otherwise.
d
SCSA with intrauterine insemination.
e
SCD.

values in neat spermatozoa reected chromatin abnormalities (Schultz and Williams, 2002). Aitken and Krausz (2001)
within the entire sperm population that were not eliminated by proposed that sperm DNA damage is promutagenic and can
sperm preparation techniques. give rise to mutations after fertilization as the oocyte attempts to
repair DNA damage prior to the initiation of the rst cleavage.
Mutations occurring at this point will be xed in the germline
The safety of using of DNA and may be responsible for infertility (Aitken and Krausz, 2001),
childhood cancer in the offspring (Fraga et al., 1996; Ji et al.,
damaged human spermatozoa in 1997; Aitken and Krausz, 2001) and imprinting diseases (Cox
assisted reproduction et al., 2002; De Baun et al., 2003). So far however, follow-up
studies of children born after ICSI compared with children born
after conventional IVF have not been conclusive regarding
DNA damaged human spermatozoa in the risks of congenital malformations and health problems in
ICSI and traditional IVF general (Bonduelle et al., 1996; 1998; 2003; Wennerholm and
Bergh, 2000; Hansen et al., 2002). It has been reported (Aitken
ICSI enables fertilization to take place in spite of severely and Krausz, 2001) that ICSI candidates usually have the highest
compromised semen characteristics and DNA damage. The level of sperm DNA damage. In the study of Bungum et al.
390 safety of the ICSI procedure in such cases has been questioned (2004) the men in the ICSI group had signicantly higher DFI

RBMOnline

levels compared with the men in the IVF group (median 18
versus 15%). Consequently, concern arises as to the fact that
the most efcient assisted reproduction techniques are used
to treat males with the highest level of sperm DNA damage.
However, evaluating DNA damage in the whole cell population
does not preclude sperm subpopulations with no signicant
DNA damage. Host et al. (Hst et al., 2000) suggested that the
technician who performs ICSI attempts to select spermatozoa
with normal morphology reducing the risk of introducing
spermatozoa with strand breaks. On the other hand, it has been
reported that traditional sperm parameters (concentration,
motility, morphology) are poorly correlated with DNA damage
evaluation (Evenson et al., 1999; Spano et al., 2000).
Data gained from the study of Larson-Cook et al. (2003) support
those from studies showing that ICSI overrides safeguards that
typically prevent spermatozoa with damaged DNA to fertilize
via spontaneous pregnancy or conception after conventional IVF
(Morris et al., 2002). The signicant decrease in implantation
and pregnancy rates using spermatozoa with high DFI indicates
that the damaged paternal genome is selected against during
embryonic development which provides a possible explanation
for the lack of evidence for an increased incidence of major
congenital malformations among children born after ICSI
(Bonduelle et al., 1996; Van Golde et al., 1999). The increased
risk for major and minor birth defects after ICSI described in
some studies (Kurinczuk and Bower, 1997) might be attributed
to parental background factors that required the use of ICSI and
not to the technique itself (Ludwig and Katalinic, 2000; Ludwig
and Diedrich, 2002).
Gandini et al. (2004) states that the biological impact of an
abnormal sperm chromatin structure depends on the combined
effects of the extent of DNA damage in the spermatozoa and
the capacity of the oocyte to repair that damage. Therefore, if
spermatozoa selected from samples with extensively damaged
DNA are used for IVF, the oocyte repair capacity may be
inadequate leading to a low rate of embryonic development
and high early pregnancy loss. However, although we are now
reasonably able to assess the damage level of a sperm population
we cannot assess the repair capability of the oocyte, neither can
the possibility of selection of spermatozoa with limited DNA
damage compensated by the oocyte repair capabilities, in a
sample characterized by a high DFI, be excluded.
Since genetically damaged spermatozoa may be able to fertilize
an oocyte when they are directly injected into it, it seems that the
development of new methods for identication, selection and use
of spermatozoa with intact DNA during ICSI would eliminate
the risk of inheriting genetic diseases to the ICSI offspring. In
the study of Hazout et al. (2006) the use of a high magnication
optical system to select spermatozoa to be used for ICSI (high
magnication ICSI) in couples with repeated conventional ICSI
failures improved clinical outcomes (pregnancy, implantation,
delivery and birth rates) without affecting biological outcomes
(fertilization and cleavage rates, embryo morphology). The
improvement of clinical ICSI outcomes was evident both in
patients with an elevated degree of DNA fragmentation and in
those with normal sperm DNA status. In the study of Said et
al. (2005) magnetic cell sorting using Annexin V microbeads
proved to effectively separate apoptotic and non apoptotic
spermatozoa, providing motile, viable and non apoptotic
spermatozoa. In addition, a recent study of the same group also
RBMOnline
Review - DNA damage of human spermatozoa - B Ozmen et al.
suggested that outcomes can be improved by using magnetic
cell sorting using annexin V microbeads (Said et al., 2006).
DNA damaged human spermatozoa in
cryopreservation
Today, cryopreservation has been accepted to be a main and
crucial procedure of assisted reproduction that enhances
outcomes along with reduction of costs caused by recurrent
procedures. Since its rst application took place in assisted
reproduction, the question of possible damage on cryopreserved
cells has been assumed. It has been clearly shown that
overall human sperm quality was found to deteriorate after
cryopreservation (Gandini et al., 2006). Animal studies also
showed that cryopreservation induces many changes in sperm
cells that include membrane disorders and programmed cell
death (Martin et al., 2004; Peris et al., 2004).
In animals, cryopreservation has been reported to facilitate
DNA damage in sperm cells (Martin et al., 2004). For example
in cattle, these procedures have been reported to act as an
apoptotic mechanism inducer in immature sperm cells (rather
than in mature spermatozoa). Authors named this abortive
process an apoptosis-like phenomenon. Duru et al. (2001)
reported that cryopreservation-thawing of human spermatozoa
from patients and donors was associated with membrane change,
as revealed by membrane translocation of phosphatidylserine,
while having no major impact on DNA fragmentation. It has
also been claimed that the DNA of spermatozoa obtained from
infertile men are more susceptible to be damaged by freeze-
thawing rather than the sperm DNA of fertile men (Donnelly et
al., 2001). On the contrary, de Paula et al. (2006) suggested that
cryopreservation induces apoptotic sperm DNA fragmentation
regardless of sperm concentration and the increase in DNA
fragmentation was found to be similar in both normozoospermic
and oligozoospermic men. However, men with oligozoospermia
presented with higher pre- and post-cryopreservation apoptotic
sperm DNA fragmentation. Recently, cryopreservation has also
been reported to be signicantly associated with activation of
caspases-3, -8, and -9, as well as disruption of the mitochondrial
membrane potential (Paasch et al., 2004b). In that study, it has
been demonstrated that annexin V-negative mature spermatozoa
showed no activated apoptosis during the cryopreservation
process, whereas apoptosis was induced both in annexin V-
positive mature spermatozoa and in immature spermatozoa
regardless of annexin V status. Authors, thus, suggested that
selection of annexin V-negative mature spermatozoa might be
of clinical relevance for fertility preservation of infertile men.
As well, it has been recently claimed that a general improvement
in nuclear maturity was seen in post-rise spermatozoa when
thawed spermatozoa were subjected to an extra swim-up round
(Gandini et al., 2006). Therefore in the light of these data,
apoptotic changes and possible sperm DNA damage especially
in infertile men after cryopreservation is not a myth.
It can be assumed that different procedures and techniques
of cryopreservation might alter the induction of apoptosis
and/or possible DNA damage of human spermatozoa caused
by cryopreservation. DNA damage of human spermatozoa
has been reported to be less with ash-freezing in liquid
nitrogen performed without the use of cryopreservative. This
technique gives the closest results to those reproduced by 391
 Review - DNA damage of human spermatozoa - B Ozmen et al.
cryopreservation of fresh human semen samples (Duty et al.,
2002). A study which compares the results of slow-rate freezing
and vitrication also showed that the vitrication of human
spermatozoa in the absence of conventional cryoprotectants is
indeed feasible (Isachenko et al., 2004a). Thus, DNA integrity of
vitried spermatozoa is comparable with the results obtained in
spermatozoa cryopreserved by standard slow-freezing/thawing.
As well, the same group also suggests that optimal regimes
for the cryoprotectant-free cryopreservation of spermatozoa
should not restricted to only very fast cooling, but a wide range
of cooling rates can be acceptable before storage in liquid
nitrogen (Isachenko et al., 2004b). On the contrary, Petyim and
Choavaratana (2006) advocated computer-controlled freezing
should be the recommended and rst choice method for routine
cryostorage of human spermatozoa when cryoprotective media
are used.
Conclusion
Imbalance in human spermatogenic and apoptotic pathways
adversely affect semen parameters, which contribute to male
infertility by means of sperm DNA damage. With the advent of
assisted reproduction procedures, traditional semen parameters
have become less important in the evaluation of sperm quality.
The assisted reproduction technique bypass natural selection
mechanisms, which increases the chance of spermatozoa with
abnormal genomic material fertilizing an oocyte. Although low
levels of sperm DNA damage can be repaired by the oocyte, the
risk of congenital malformations or health problems inherited
by the offspring is under consideration. Whereas the impact
of apoptotic DNA on fertilization rates remains controversial,
there is a wider agreement about its negative effects on embryo
development and pregnancy rates. In view of the changed
circumstances alternative methods which can evaluate sperm
quality such as assessment of DNA damage (e.g. magnetic
cell separation, high magnication optical system to select
spermatozoa) may positively affect the success rates and safety of
assisted reproduction techniques, providing vital, non apoptotic
spermatozoa. As well, standardization of cryopreservation
of human spermatozoa should be urgently performed due to
increased apoptosis indicated after cryopreservation. However,
vitrication without use of cryoprotective media can still be
used and be an alternative with similar damage like slow-rate
freezing.
References
Abu-Hassan D, Koester F, Schoepper B et al. 2005 COMET assay of
cumulus cells and spermatozoa DNA status, and the relationship
to oocyte fertilization and embryo quality following ICSI.
Reproductive BioMedicine Online 12, 447452.
Agarwal A, Said T 2005 Oxidative stress, DNA damage and apoptosis
in male infertility: a clinical approach. British Journal of Urology
International 95, 503507.
Ahmadi A, Ng SC 1999 Developmental capacity of damaged
spermatozoa. Human Reproduction 14, 22792285.
Aitken RJ, Krausz C 2001 Oxidative stress, DNA damage and the Y
chromosome. Reproduction 122, 497506.
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, 6570.
Aitken RJ, Buckingham D, West K et al. 1992 Differential
Contribution of leucocytes and spermatozoa to the generation
392 of reactive oxygen species in the ejaculates of oligozoospermic
patients and fertile donors. Journal of Reproductive Fertility 94,
451462.
Antonson B, Martinou JC 2000 The Bcl-2 protein family.
Experimental Cell Research 256, 5057.
Aravindan GR, Bjordhal J, Jost LK, Evenson DP 1997 Susceptibility
of human sperm to in situ DNA denaturation is strongly correlated
with DNA strand breaks identied by single-cell electrophoresis.
Experimental Cell Research 10, 231237.
Barkett M, Gilmore TD 1999 Control of apoptosis by Rel/NF-B
transcription factors. Oncogene 18, 69106924.
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, 13381344.
Benchaib M, Lornage J, Mazoyer C et al. 2006 Sperm
deoxyribonucleic acid fragmentation as a prognostic indicator of
assisted reproductive technology outcome. Fertility and Sterility
87, 93100. [Epub ahead of print].
Benchaib M, Braun V, Lornage J et al. 2003 Sperm DNA
fragmentation decreases the pregnancy rate in an assisted
reproductive technique. Human Reproduction 18, 10231028.
Bonduelle M, Ponjaert I, Steirteghem AV et al. 2003 Developmental
outcome at 2 years of age for children born after ICSI compared
with children born after IVF. Human Reproduction 18, 342350.
Bonduelle M, Joris H, Hofmans K et al. 1998 Mental development of
201 ICSI children at 2 years of age. Lancet 351, 1553.
Bonduelle M, Legein J, Buysse A et al. 1996 Prospective follow up
study of 423 children born after intracytoplasmic sperm injection.
Human Reproduction 11, 15581564.
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, 28762881.
Bungum M, Humaidan P, Axmon A et al. 2007 Sperm DNA integrity
assessment in prediction of assisted reproduction technology
outcome. Human Reproduction 22, 174179.
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, 14011408.
Carrell DT, Liu L 2001 Altered protamine 2 expression is uncommon
in donors of known fertility, but common among men with
poor fertilizing capacity, and may reect other abnormalities of
spermiogenesis. Journal of Andrology 22, 604610.
Carrell D, Wilcox A, Lowy L et al. 2003 Elevated sperm chromosome
aneuploidy and apoptosis in patients with unexplained recurrent
pregnancy loss. Obstetrics and Gynecology 101, 12291235.
Chandley AC, Hargreave TB 1996 Genetic anomaly and ICSI. Human
Reproduction 11, 930932.
Check JH, Graziano V, Cohen R et al. 2005 Effect of an abnormal
sperm chromatin structural assay (SCSA) on pregnancy outcome
following (IVF) with ICSI in previous IVF failures. Archives of
Andrology 51,121124.
Cho C, Jung Ha H, Willis WD et al. 2003 Protamine 2 deciency
leads to sperm DNA damage and embryo death in mice. Biology of
Reproduction 69, 211217.
Cox GF, Burger J, Lip V et al. 2002 Intracytoplasmic sperm injection
may increase the risk of imprinting defects. American Journal of
Human Genetics 71, 162164.
De Baun MR, Niemitz EL, Feinber AP 2003 Association of in vitro
fertilization with Beckwith Wiedemann syndrome and epigenetic
alterations of LIT1 and H19. American Journal of Human Genetics
72, 156160.
de Kretser DM, Loveland KL, Meinhardt A et al. 1998
Spermatogenesis. Human Reproduction 13 (Suppl. 1), 18.
de Paula TS, Bertolla RP, Spaine DM et al. 2006 Effect of
cryopreservation on sperm apoptotic deoxyribonucleic acid
fragmentation in patients with oligozoospermia. Fertility and
Sterility 86, 597600.
Dunkel L, Hirvonen V, Erkila K 1997 Clinical aspects of male germ
cell apoptosis during testis development and spermatogenesis. Cell
Death Differentiation 4, 171179.
RBMOnline

Duran EH, Morshedi M, Taylor S, Oehninger S 2002 Sperm DNA
quality predicts intrauterine insemination outcome a prospective
cohort study. Human Reproduction 17, 31223128.
Duru NK, Morshedi MS, Schuffner A, Oehninger S 2001
Cryopreservation-thawing of fractionated human spermatozoa is
associated with membrane phosphatidylserine externalization and
not DNA fragmentation. Journal of Andrology 22, 646651.
Duru NK, Morshedi M, Schuffner A, Oehninger S 2000 Semen
treatment with progesterone and/or acetyl-L-carnitine does
not improve sperm motility or membrane damage after
cryopreservation-thawing. Fertility and Sterility 74, 715720.
Duty SM, Singh NP, Ryan L et al. 2002 Reliability of the comet
assay in cryopreserved human sperm. Human Reproduction 17,
12741280.
Donnelly ET, Steele EK, McClure N, Lewis SE 2001 Assessment of
DNA integrity and morphology of ejaculated spermatozoa from
fertile and infertile men before and after cryopreservation. Human
Reproduction 16, 11911199.
Erenpreiss J, Spano M, Erenpreisa J 2006 Sperm chromatin structure
and male fertility: biological and clinical aspects. Asian Journal of
Andrology 8, 1129.
Erenpreiss J, Hlevicka S, Zalkalns J, Erenpreisa J 2002 Effect of
leukocytospermia on sperm DNA integrity: a negative effect in
abnormal semen samples. Journal of Andrology 23, 717723.
Evenson D, Wixon R 2006 Meta-analysis of sperm DNA
fragmentation using the sperm chromatin structure assay.
Reproductive BioMedicine Online 12, 466472.
Evenson DP, Larson KL, Jost LK 2002 Sperm chromatin structure
assay: its clinical use for detecting sperm DNA fragmentation in
male infertility and comparison with other techniques. Journal of
Andrology 23, 2543.
Evenson DP, Jost LK, Marshall D et al. 1999 Utility of the sperm
chromatin structure assay (SCSA) as a diagnostic and prognostic
tool in the human fertility clinic. Human Reproduction 14, 1039
1049.
Fraga CG, Motchnik PA, Wyrobek AJ et al. 1996 Smoking and low
antioxidant levels increase oxidative damage to sperm DNA.
Mutation Research 351, 199203.
Gandini L, Lombardo F, Lenzi A et al. 2006 Cryopreservation and
sperm DNA integrity. Cell Tissue Bank 7, 9198.
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, 14091417.
Gandini L, Lombardo F, Paoli D et al. 2000 Study of apoptotic DNA
fragmentation in human spermatozoa. Human Reproduction 15,
830839.
Glander HJ, Schaller J 1999 Binding of Annexin V to plasma
membranes of human spermatozoa: a rapid assay for detection
of membrane changes after cryostorage. Molecular Human
Reproduction 5, 109115.
Golan R, Shochat L, Weissenberg R et al. 1997 Evaluation of
chromatin condensation in human spermatozoa: a ow cytometric
assay using acridine orange staining. Molecular Human
Reproduction 3, 4754.
Gorczyca W, Traganos F, Jesionowska H, Darzynkievicz Z 1993
Presence of DNA strand breaks and increased sensitivity of DNA
in situ to degeneration in abnormal human sperm cells: analogy
to apoptosis of somatic cells. Experimental Cell Research 207,
202205.
Gupta S 2001 Molecular steps of death receptor and mitochondrial
pathways of apoptosis. Life Sciences 69, 29572964.
Hansen M, Kurinczuk JJ, Bower C, Webb S 2002 The risk of major
birth defects after intracytoplasmic sperm injection and in-vitro
fertilization. New England Journal of Medicine 346, 725730.
Hazout A, DumontHassan M, Junka AM et al. 2006 High
magnication ICSI overcomes paternal effect resistant to
conventional ICSI. Reproductive BioMedicine Online 12, 1925.
Hengartner MO 2000 The biochemistry of apoptosis. Nature 407,
770776.
Henkel R, Hajimohammad M, Stalf T et al. 2004 Inuence of
deoxyribonucleic acid damage on fertilization and pregnancy.
RBMOnline
Review - DNA damage of human spermatozoa - B Ozmen et al.
Fertility and Sterility 81, 965972.
Hst 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, 559563.
Hst E, Lindenberg S, Kahn JA, Christensen F 1999 DNA strand
breaks in human sperm cells: A comparison between men with
normal and oligozoospermic sperm samples. Acta Obstetricia et
Gynecologica Scandinavica 78, 336339.
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, 130140.
Irvine DS, Twigg JP, Gordon EL et al. 2000 DNA integrity in
human spermatozoa: relationships with semen quality. Journal of
Andrology 21, 3344.
Isachenko E, Isachenko V, Katkov II et al. 2004a DNA integrity
and motility of human spermatozoa after standard slow freezing
versus cryoprotectant-free vitrication. Human Reproduction 19,
932939.
Isachenko V, Isachenko E, Katkov II et al. 2004b Cryoprotectant-
free cryopreservation of human spermatozoa by vitrication
and freezing in vapor: effect on motility, DNA integrity, and
fertilization ability. Biology of Reproduction 71, 11671173.
Ji BT, Shu XO, Linet MS et al. 1997 Paternal cigarette smoking and
the risk of childhood cancer among offsprings of non smoking
mothers. Journal of National Cancer Institute 89, 238244.
Kaufmann SH, Hengartner MO 2001 Programmed cell death: alive
and well in the new millennium. Trends in Cellular Biology 11,
526534.
Kurinczuk JJ, Bower C 1997 Birth defects in infants conceived by
intracytoplasmic sperm injection: an alternative interpretation.
British Medical Journal 315, 12601265.
Larson KL, DeJonge CJ, Barnes AM et al. 2000 Sperm chromatin
structure assay parameters as predictors of failed pregnancy
following assisted reproductive techniques. Human Reproduction
15, 17171722.
Larson KL, Brannian JD, Timm BK et al. 1999 Density gradient
centrifugation and glass wool ltration of semen remove sperm
with damaged chromatin structure. Human Reproduction 14,
20152019.
Larson-Cook, K, Brannian J, Hansen K 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, 895902.
Lewis SE, Aitken RJ 2005 DNA damage to spermatozoa has impacts
on fertilization and pregnancy. Cell and Tissue Research 322,
3341.
Li Z, Wang L, Cai J, Huang H 2006 Correlation of sperm DNA
damage with IVF and ICSI outcomes: A systematic review and
meta-analysis. Journal of Assisted Reproduction and Genetics 23,
367376.
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, 528532.
Ludwig M, Diedrich K 2002 Follow up of children born after assisted
reproductive technologies. Reproductive BioMedicine Online 5,
317322.
Ludwig M, Katalinic A 2000 Malformation in fetuses and children
conceived after ICSI: results of a prospective cohort study.
Reproductive BioMedicine Online 5, 171178.
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, 864867.
Martin G, Sabido O, Durand P, Levy R 2004 Cryopreservation
induces an apoptosis-like mechanism in bull sperm. Biology of
Reproduction 71, 2837.
Morris ID, Ilott S, Dixon L, Brison DR 2002 The spectrum of DNA
damage in human sperm assessed by single cell gel electrophresis
(Comet assay) and its relationshipto fertilization and embryo
development. Human Reproduction 17, 990998. 393
 Review - DNA damage of human spermatozoa - B Ozmen et al.
Moustafa MH, Sharma RK, Thornton J et al. 2004 Relationship
between ROS production, apoptosis and DNA denaturation
in spermatozoa from patients examined for infertility. Human
Reproduction 19, 129138.
Muratori M, Maggi M, Spinelli S et al. 2003 Spontaneous DNA
fragmentation in Swim Up selected human spermatozoa during
long term incubation. Journal of Andrology 24, 253262.
Muratori M, Piomboni P, Baldi E et al. 2000 Functional and
ultrastractural features of DNA-fragmented sperm. Journal of
Andrology 21, 903912.
Muriel L, Garrido N, Fernandez JL et al. 2006 Value of the sperm
deoxyribonucleic acid fragmentation level, as measured by
the sperm chromatin dispersion test, in the outcome of in-vitro
fertilization and intracytoplasmic sperm injection. Fertility and
Sterility 85, 371383.
Nagata S 2000 Apoptotic DNA fragmentation. Experimental Cell
Research 256, 1218.
Nasr-Esfahani MH, Salehi M, Razavi S et al. 2005 Effect of sperm
DNA damage and sperm protamine deciency on fertilization and
embryo development post-ICSI. Reproductive BioMedicine Online
11, 198205.
Olive PL, Banath JP 1995 Sizing highly fragmented DNA in
individual apoptotic cells using the Comet assay and a DNA
crosslinking agent. Experimental Cell Research 221, 1926.
Osterhuis GJ, Mulder AB, Kalsbeek-Batenburg E et al. 2000
Measuring apoptosis in human spermatozoa: a biological assay for
semen quality. Fertility and Sterility 74, 245250.
Paasch U, Grunewald S, Agarwal A, Glandera HJ 2004a Activation
pattern of caspases in human spermatozoa. Fertility and Sterility
81 (suppl. 1), 802809.
Paasch U, Sharma RK, Gupta AK et al. 2004b Cryopreservation and
thawing is associated with varying extent of activation of apoptotic
machinery in subsets of ejaculated human spermatozoa. Biology of
Reproduction 71, 18281837.
Payne JF, Raburn DJ, Couchman GM et al. 2005 Redening the
relationship between sperm deoxyribonucleic acid fragmentation
as measured by the sperm chromatin structure assay and outcomes
of assisted reproductive techniques. Fertility and Sterility 84,
356364.
Peris SI, Morrier A, Dufour M, Bailey JL 2004 Cryopreservation of
ram semen facilitates sperm DNA damage: relationship between
sperm andrological parameters and the sperm chromatin structure
assay. Journal of Andrology 25, 224233.
Petyim S, Choavaratana R 2006 Cryodamage on sperm chromatin
according to different freezing methods, assessed by AO test.
Journal of the Medical Association of Thailand 89, 306313.
Plante M, de Lamirande E, Gagnon C 1994 Reactive oxygen species
released by activated neutrophils, but not by decient spermatozoa,
are sufcient to affect normal sperm motility. Fertility and Sterility
62, 387393.
Raff M 1998 Cell suicide for beginners. Nature 396, 119122.
Raman RS, Chan PJ, Corselli JU et al. 2001 Comet assay of cumulus
cell DNA status and the relationship to oocyte fertilization via
intracytoplasmic sperm injection. Human Reproduction 16,831
835.
Ricci G, Perticarari S, Fragonas E et al. 2002 Apoptosis in human
sperm: its correlation with semen quality and the presence of
leucocytes. Human Reproduction 17, 26652672.
Said T, Agarwal A, Grunewald S et al. 2006 Selection of nonapoptotic
spermatozoa as a new tool for enhancing assisted reproduction
outcomes: an in-vitro model. Biology o f Reproduction 74, 530
537.
Said T, Grunewald S, Paasch U et al. 2005 Advantage of combining
magnetic cell separation with sperm preparation techniques.
Reproductive BioMedicine Online 10, 740746.
Said TM, Paasch U, Glander HJ, Agarwal A 2004 Role of caspases in
male infertility. Human Reproduction Update 10, 3951.
Sakkas D, Seli E, Bizzaro D et al. 2003 Abnormal spermatozoa in the
ejaculate: abortive apoptosis and faulty nuclear remodeling during
spermatogenesis. Reproductive BioMedicine Online 7, 428432.
394 Sakkas D, Moffatt O, Manicardi G et al. 2002 Nature of DNA damage
in ejaculated human spermatozoa and the possible involvement of
apoptosis. Biology of Reproduction 66, 10611067.
Sakkas D, Manicardi G, 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, 11121116.
Sakkas D, Mariethoz E, Manicardi G et al. 1999 Origin of
DNA damage in ejaculated human spermatozoa. Reviews of
Reproduction 4, 3137.
Sakkas D, Urner F, Bianchi PG et al. 1996 Sperm chromatin
abnormalities can inuence decondensation after intracytoplasmic
sperm injection. Human Reproduction 11, 837843.
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, 15971605.
Schultz RM, Williams CJ 2002 The science of ART. Science 296,
21882190.
Schwartz M, Vissing J 2002 Paternal inheritance of mitochondrial
DNA. New England Journal of Medicine 347, 576580.
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,
378383.
Sharma RK, Agarwal A 1996 Role of reactive oxygen species in male
infertility. Urology 48, 835850.
Sharma RK, Said T, Agarwal A 2004 Sperm DNA damage and its
clinical relevance in assessing reproductive outcome. Asian
Journal of Andrology 6, 139148.
Siddighi S, Patton W, Jacobson J et al. 2004 Correlation of sperm
parameters with apoptosis assessed by dual uorescence DNA
integrity assay. Archives of Andrology 50, 311314.
Spano M, Bonde JP, Hijollund HI et al. 2000 Sperm chromatin
damage impairs human fertility. Fertility and Sterility 73, 4350.
Spano M, Cordelli E, Leter G et al. 1999 Nuclear chromatin variations
in human spermatozoa undergoing swim up and cryopreservation
evaluated by the ow cytometric sperm chromatin structure assay.
Molecular and Human Reproduction 5, 2937.
Sun JG, Jurisicova A, Casper RF 1997 Detection of deoxyribonucleic
acid in human sperm: correlation with fertilization in vitro. Biology
of Reproduction 56, 602607.
Tesarik J, Mendoza C, Greco E 2002 Paternal effects acting during the
rst cell cycle of human preimplantation development after ICSI.
Human Reproduction 17, 184189.
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, 21602165.
Tomsu M, Sharma V, Miller D 2002 Embryo quality and IVF
treatment outcomes may correlate with different sperm comet
assay parameters. Human Reproduction 17, 18561862.
Twigg JP, Irvine DS, Aitken RJ 1998a Oxidative damage to DNA
in human spermatozoa does not preclude pronucleus formation
at intracytoplasmic sperm injection. Human Reproduction 13,
18641871.
Twigg J, Fulton N, Gomez E et al. 1998b Analysis of the impact of
intracellular reactive oxygen species generation on the structural
and functional integrity of human spermatozoa: lipid peroxidation,
DNA fragmentation and effectiveness of antioxidants. Human
Reproduction 13, 14291436.
Van Golde R, Boada M, Veiga A et al. 1999 A retrospective follow
up study on intracytoplasmic sperm injection. Journal of Assisted
Reproduction and Genetics 16, 227232.
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, 12891295.
Wennerholm UB, Bergh C 2000 Obstetric outcome and follow up of
children born after in vitro fertilization (IVF). Human Fertility 3,
5264.
RBMOnline

Zini A, Meriano J, Kader K et al. 2005 Potential adverse effect
of sperm DNA damage on embryo quality after ICSI. Human
Reproduction 20, 34763480.
Zini A, Bielecki R, Phang D, Zenzes M 2001 Correlation between
two markers of DNA integrity and, DNA denaturation and DNA
fragmentation, in fertile and infertile men. Fertility and Sterility
75, 674677.
RBMOnline
Review - DNA damage of human spermatozoa - B Ozmen et al.
Zini A, de Lamirande E, Gagnon C 1993 Reactive oxygen species
in semen of infertile patients: levels of superoxide dismutase-
and catalase-like activities in seminal plasma and spermatozoa.
International Journal of Andrology 16,183188.
Received 30 January 2006; refereed 1 March 2006; revised and
resubmitted 8 January 2007; accepted 18 January 2007.
395