1. health and fitness
2. disease
3. cancer

Review article
Journal of Andrological Sciences 2009;16:77-90
Report from the International Congress
of Andrology (ICA) 2009 Satellite Symposium
“Sperm DNA Damage: from Research to Clinic”
F. Lanzafame, S. La Vignera*, P. Asero*
Centro Territoriale di Andrologia, Azienda Sanitaria Provinciale 8, Siracusa; * U.O.C. Andrologia ed Endocrinologia
della Riproduzione, Ospedale Garibaldi (centro), Università di Catania
Summary
Key words
Sperm DNA • Sperm epigenetic • Male
infertility • Oxidative stress
Several factors are related to sperm DNA damage and not all mechanism are
known. Multiple sources have been proposed including abortive apoptosis,
abnormal chromatin packaging during the transition from round to elongated
spermatids and oxidative stress. Moreover, many enviromental conditions
are related to reproductive toxicity, including structural and functional alterations of human sperm and particularly sperm DNA damage.
Finally, several evidences suggest that infiammation/infection in the male reproductive tract may impair fertility leading to improve of DNA fragmentation.
In the last years, several evidences showed like sperm DNA damage may to
related to infertility leading to the insight that the assessment of sperm DNA
integrity could be considered a potential new semen quality biomarker.
Introduction
As reflected by the proceedings of the recent ICA 2009 Satellite Symposium held in Rome, Italy, modern andrology takes a wider responsibility for sperm DNA damage related to male infertility. The ‘poor’
quality of sperm DNA is an important factor affecting male reproductive ability.
The presence of DNA breaks in sperm chromatin may decrease the
capability of sperm to fertilize both in natural and in assisted procreation 1. Sperm DNA fragmentation may lead to disorders of pregnancy
and may have a harmful effect on embryo development 1. The presence
of DNA breaks in paternal genome is also one of the factors increasing
the risk of genetic defects in offspring 2-14. It seems that because of its
extremely strong condensation and consequently its unique structure
of chromatin, sperm DNA fragmentation is hardly possible. However,
from many clinical and experimental studies it appears that sperm
genome may be susceptible to toxic influence from many different
endogenous and exogenous factors 2 4 6. The susceptibility is a result
of sperm poor ability to repair damaged genetic material, as well as
Corresponding author:
Paola Asero, Sezione di Endocrinologia, Andrologia e Medicina Interna, Dipartimento di Scienze Biomediche, Ospedale Garibaldi, piazza S.M. Gesù, 95123 Catania, Italia
– Tel. +39 340 9691968 – E-mail: [email protected]
77
F. Lanzafame, et al.
its insufficient antioxidative defence preventing DNA
damage caused by radical oxygen species (ROS).
Undoubtly the susceptibility of sperm genome to
the damage is associated with biochemical and
morphological maturity of sperm, which depends on
normal spermatogenesis.
These conditions have been discussed during the
ICA 2009 Satellite Symposium of Andrology from a
panel of expert scientists who exposed the presentations, following reported:
1. environmental hormones and male reproduction
(J.P. Bonde, Denmark);
2. gene environmental interaction: the impact of
persistent organohalogen pollutants on sperm
characteristics and genital malformations (Y. Giwercman, Sweden);
3. cryopreservation of sperm DNA (S. Lewis, Northern Ireland);
4. epigenetic control in male germ cells: the chromatoid body as an RNA-processing center
(P. Sassone-Corsi, USA);
5. effect of chemotherapy and folate pathway deficiencies on the sperm epigenome (J. Trasler,
Canada);
6. our genome in the male germ line: is it safe?
(A. Grootegoed, The Netherlands);
7. clinical significance of sperm DNA fragmentation
assays (M. Spanò, Italy);
8. sperm aneuploidy and ART offspring (L. Gianaroli, Italy);
9. Y chromosome rearrangements: their cellular
origin and clinical consequences (S. Krausz,
Italy);
10.mtDNA and sperm function (J. St John, UK);
11.sperm chromatin packaging and DNA methylation: relevance to ART (D. Carrel, USA);
12.defining sites susceptible to DNA damage within
the sperm nucleus: the nuclear matrix connection (S. Krawetz, USA);
13.characteristic histone modifications and timing
of histone to protamine switch in Drosophila
sperm chromatin (A. Awe, Germany);
14.persistence of DNA damage and its consequence
for utagenesis in male germ cells of OGG1 -/- Big
Blue mice exposed to benzo(a)pyrene (A.K. Olsen,
Norway);
15.cellular mechanism underlying the effects of
partenal acrylamide-exposure on preimplantation development in mice (S. Shahzadi, Norway);
16.epydidimal glutathione peroxidase 5 contributes
to the maintenance of sperm DNA integrity and
to embryo viability (J. Drevet, France);
17.male-to-female sex-ratio is potentially correlated
to air pollution levels (J. Hallak, Brazil);
18.analytical investigation on TUNEL/P1 assay for
the determination of sperm DNA fragmentation:
pitfalls and possible solutions (M. Muratori, Italy);
19.impact of environmental exposure to perfluorinated compounds on sperm DNA quality
(L. Governini, Italy);
20.chromomycin A3 staining vs TUNEL assay: different prognostic value on ART outcome (M. Nadalini, Italy);
21.apoptosis and sperm DNA fragmentation in infertile patients with Chlamydia and Mycoplasms
infection (S. Alvarez, Mexico);
22.inflammatory mediators induce apoptosis in
ejaculated spermatozoa in in vitro conditions
(M. Fraczek, Poland);
23.ROS induced damage and its clinical significance (J. Aitken, Australia);
24.diagnostic tests for monitoring scrotal hyperthermia (A. Ledda, Italy);
25.testicular hyperthermia and the pathways leading to DNA breakage (C. Wang, USA);
26.pathways of ROS generation in male germ cells:
insights generated by proteomics (J. Aitken,
Australia);
27.genital tract inflammation and its consequences
on sperm DNA (A. Calogero, Italy);
28.antioxidants and sperm DNA damage (A. Zini,
Canada).
Origin of DNA damage in spermatozoa
There are several mechanisms which can damage
sperm DNA. Defective sperm chromatin packaging,
Table I. Biological events related to chromatin condensation impairment.
Chromatin damage causes
References
Chromatin structure alterations
Evenson et al., 15 1999; Spano et al., 2000 16; Zini et al., 2001 17
DNA fragmentation
Hughes et al., 1996 18; Irvine et al., 2000 19
DNA oxidation
Shen et al., 2000 20
Protamine deficiency
Gatewood et al., 1990 21; Carrell et al., 2001 22; Zhang et al., 2006 23
78
Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic”
Table II. Consequences of sperm DNA poor integrity.
1. Fertilization impairment
2. Embryo development disorder
3. Implantation impairment
4. Spontaneous abortion
5. Risk-danger of genetic defects in blastomeres
6. Epigenetic modification errors
7. Cancers
8. Infertility
apoptosis and oxidative stress are the most important aetiological factors which disrupt DNA integrity.
Sperm chromatin condensation impairment
Infertile men have higher levels of sperm DNA chromatin damage than fertile men. DNA chromatin damage are due to several biological events which are
reported in the Table I.
DNA fragmentation and sperm apoptosis
Different pathological condition can lead to DNA
fragmentation resulting in germ cell apoptosis. Several type of sperm DNA alteration can occur including
whole and segmental-chromosomal aneuploidies,
mutations 24, trinucleotide repeat-length variations 25,
defects in the imprinting profiles 26 27 and unspecific
DNA breaks 28.
The pathological condition associated to DNA fragmentation and sperm apoptosis will be discussed
following.
Oxidative stress
Among the principal process that lead to DNA fragmentation and sperm apoptosis there is oxidative
stress (OS).
In recent years, OS and the role of ROS in the physiopathology of human sperm function and male infertility have been emphasized. Indeed, spermatozoa,
from the time that they are produced in the testes to
ejaculation and in the female reproductive tract, are
constantly exposed to oxidizing environments. They
are extremely sensitive to ROS because of their high
content of polyunsaturated fatty acids (PUFA) and
their inability to repair DNA damages 20 29.
Sperm DNA damage is due (in part) to OS. Male infertility is associated with high ROS levels and these
are associated with sperm DNA damage.
OS can cause both loss of motility and DNA damage
in sperm 30-32.
There is evidence that infertile men present substantially more sperm DNA damage than fertile men
and human sperm DNA damage may adversely
affect reproductive outcomes. This is particularly
relevant for the couples undergoing to assisted
reproduction technologies (ARTs) (these technologies often bypass the barriers to natural selection)
since there is some uncertainty regarding the safety
of utilizing DNA-damaged spermatozoa for these
purposes.
Therefore, it is important to identify strategies that
may reduce sperm DNA damage. At present, there
is some evidence to suggest that antioxidant may be
useful for these reasons.
Recent studies suggest that oral antioxidants therapies, can protect sperm and improve their function
by increasing the antioxidant levels 33-35. Menezo et
al. 36 have shown that although vitamins (vitamins
C, E, zinc, selenium and β carotene) can reduce
sperm DNA fragmentation however, it also led to an
unexpected negative effect: an increase in sperm
decondensation. Although in vitro studies have demonstrated a beneficial effect of antioxidant supplements in protecting sperm from oxidative DNA injury,
the beneficial effect of dietary antioxidants on sperm
DNA integrity has not been clearly demonstrated.
Sperm DNA damage and pathological conditions
Sperm DNA damage can be attributed to various
pathological conditions including: some diseases,
many environmental conditions, sperm preparation
protocols and the last but not least is inflammation/
infection in the male reproductive tract.
Some diseases and/or their treatment are related
to sperm DNA damage and also cancer is among
them. Most common form of cancer affect men on
reproductive age.
Cancer treatments, which are necessarily based on
the use of DNA damaging compounds, are examples
of iatrogenic induced sperm DNA damage. Today,
the vast majority of childhood cancer patients are
treated by chemo or radiotherapy. Among the several long-term complications of oncological treatments
in cancer survivors, an important one is certainly
sperm DNA damage.
The knowledge of the kinetics of the induction
and removal of DNA lesions would be important.
Frias et al. 37 using multiprobe FISH (fluorescence
in situ hybridization) techniques observed that, in
Hodgkin’s disease patients, chemotherapy induced
a transient increase (up to 5 times) of sperm chromosomal aneuploidy which came back to normal in
the following 2-3 years. The same trend has been
79
F. Lanzafame, et al.
observed by Robbins et al. 38 in testicular cancer patients, the most frequent malignant disease in young
men. Testicular cancer can be treated in 90-95% of
cases. Spano et al. 16 evaluated the impact of Chemotherapy (TC) on sperm DNA integrity using both
SCSA (sperm chromatin structure assay) and TUNEL
(TdT-mediated-dUTP nick end labeling) assay. Semen was collected at specific intervals of time up to
5 years after treatment. Compared with pretreatment
values, radiotherapy induced a transient increase in
DNA Fragmentation Index (DFI) 1-2 years after treatment and a following normalization after 3-5 years.
After chemotherapy, the transient increase at 1 year
was less pronounced and DFI was even reduced
as compared to pre-treatment values, possibly
because of some selective effects. The question if
cancer condition per se is associated with a higher
rate of defective sperm is still debatable.
Among the pathological conditions related to sperm
DNA damage, one of the most common is varicocele.
Varicocele is one of the main cause of male infertility 39-41; afflicting about the 15% of the general
popolation, about the 35% of men with primary infertility and more of the 80% of men with secondary
infertility 42 43.
Different studies suggest that men with varicocele,
even with normal seminal parameters or with a
documented previous fertility, risk a progressive loss
of testicular function and fertility 44 45.
In the last years, studies about the role of OS in
male infertility showed that infertile men with varicocele have elevated concentration of sperm-derived
ROS 46 47 and different studies showed that this
population of men have an increased levels of seminal OS as indicated from elevated ROS levels and
a reduced total antioxidant capacity (TAC) suggesting that the spermatic dysfunction should be also
associated to the OS 46-51. Moreover, the OS affect
the spermatic DNA integrity leading, with elevated
frequency, to DNA single and double strand breaks,
which often is found in the ejaculate of infertile
men 19 52 53-54.
It has been hypothisized that spermatic dysfunction shoul be associated with an increase of scrotal
themperature concern to venous reflow.
Human scrotal/testicular thermoregulation is a complex process that maintains the testes temperature
at levels compatible with a normal spermatogenesis.
Sinha Hikim et al. demonstrated that mild testicular
hyperthermia induces accelerated germ cell apoptosis predominantly via the mitochondrial-depen80
dent death pathway 55 56 hyperthermia lead to DNA
breakage and DNA breakage leading to germ cell
apoptosis. In fact different studies, in rodents, monkey and men, showed that transient testicular hyperthermia induced DNA fragmentation resulting in
germ cell apoptosis. The heat stress is mediated by
the stress kinases such as p38 MAPK (Mitogen-activated protein kinase) 57 leading to phosphorylation
and inactivation of the pro-survival protein BCL-2,
cytochrome c and DIABLO release from the mitochondria, induction of the caspase cascade leading
to DNA breakage and germ cell death 58. Cessation
of heat treatment results in rapid recovery of spermatogenesis 59.
Environmental conditions
Recent studies suggested that exposure to particular
compounds is associated with reproductive toxicity,
including structural and functional alterations of human sperm and particularly sperm DNA damage.
Environmental factors include a lot of organic and
chemical compounds, chemical solvents, pesticides,
heavy metals; polychlorinated biphenyls (PCBs) and
other persistent organic pollutants (POPs). Among
these agents endocrine disruptors chemicals (EDCs)
may mimic, block or modulate the normal system of
hormones. EDCs are hormonally active compounds,
that can interfere with the endocrine system. EDCs
are estrogen-like and/or anti-androgenic chemicals
that have potentially effects on male reproductive
axis, resulting in infertility. EDCs may mimic, block
or modulate the: synthesis; release; transport; metabolism; binding; elimination of hormones 42. Much
attention has focused on changing trends in male
reproductive parameters in relation to EDC exposure
and an association has been postulated between the
global decline in semen quality and the increased exposure to these environmental chemicals 60. Among
these, the perfluorinated compounds (PFCs), are suspected to play an adverse effects on human fertility.
perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) are the most well know members of
the PFC chemical group. PFCs are characterised by
chains of carbon atoms, which are strongly bonded
with fluorine atoms. These are very persistent in the
environment and extremely resistent to degradation.
Indeed, these are heat stable and repel both water
and oil 61. PFCs have been discovered as global pollutants; they are widely used as industrial surfactants
and in various commercial applications.
The properties that makes PFCs so effective in industrial product are also the reason why they tend
to persist in the environment. The acute toxicity of
Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic”
PFCs is moderate, but they have various potential
health effect.
We are always expose to PFCs and the levels of
these compounds in our bodies may never be completely removed.
Moreover, PFCs can play a potential developmental
toxicity 62. These molecules infact are involved in alterations of hypotalamus-hypophisis-gonadal axis.
A number of recent studies suggest possible associations of exposure to PFCs with altered functions of reproductive system. PFOS and PFOA act
as endocrine disruptors with direct effects on sex
hormone levels, resulting in lower testosterone and
higher estradiol levels 63 64. Governini et al. 65 evaluated PFC contamination in three different organic
samples: whole blood, seminal plasma and sperm
cell fraction, from subfertile men. PFC contamination was present in 42.40% of subjects. Particularly,
PFC contamination was significantly more frequent
among men with abnormal semen parameters in
comparison to normospermic subjects.
The results obtained in this study suggest a negative interference of PFCs on sperm quality in accord
to the study of Muratori et al. where, the results
showed that PFC “positive” subjects had the highest degree of DNA fragmentation and diffuse sperm
structural anomalies, mainly related to apoptosis 66.
In conclusion, PFCs could induce spermatogenetic
imbalances by raising the levels of sperm DNA fragmentation and decreasing sperm quality.
POPs can have hormone like or antihormone like effects and can act via sex hormone receptors such as
AR. AR mutations are responsible to profound effects
on phenotype. AR gene polymorphisms concern CAGrepeat and GGN-repeat. Polymorphisms for this gene
are frequent (> 1% of the population) and can be related to small effects on phenotype. So far, only limited
evidence link GGN number to male subfertility; one
study indicated an increase of infertility in males with
GGN ≥ 24 67, while any correlation has been reported
between CAG length and sperm concentration 68. Indeed, no correlation between POP and sperm number
have been shown 69, whereas CAG length modifies the
impact of POP on sperm number 68.
Lichtenfels et al., demonstrate that male-to-female
sex ratio is potentially correlated to air pollution levels. Increased levels of air pollution and a decrease
in the human and mouse male-to-female ratio in São
Paulo, Brazil has been reported 70. In conlusion, AR
polymorphisms and dioxin receptor related (AHRR)
genes were shown to modify the effect of POPs regard to sperm concentration as well as Y:X ratio.
Another toxic environmental factor is Benzo(a)pyrene
(BaP) which is believed to induce both bulky DNA
adducts (NER) as well as oxidative DNA lesions.
BaP is known to cause DNA adducts, and is also
believed to increase ROS mediated oxidative DNA
damage. The latter is well documented in vitro, but
very scarce data exist from in vivo studies. The study
of Olsen et al. 71 obtained specific information on the
susceptibility of each spermatogenic cell stage.
By exposing the mice to BaP and waiting for 120
days before sacrifice, the isolated epididymal sperm
emanated from exposed stem cell spermatogonia,
reflecting the susceptibility of these cells. By this
procedures, have been obtained specific information
about stem cell spermatogonia (120 days), differentiating spermatogonia (45 days), primary spermatocytes (31 days), round spermatids (16 days), and
spermatozoa (5 days). The results achieved from this
study showed that environmental agents have negative effects on spermatozoa. Agents such as BaP
leads to spermatozoa containing DNA lesions and
to spermatozoa containing de novo germ line mutations. These effects are seen both in the wild type,
and the repair system deficient mouse line studied.
Sperm with excessive DNA damage retain the ability
to fertilize oocytes, but the normal development of
the early embryo is often compromised, resulting in
reduced fertility or developmental toxicity. Shahzadi
et al. 72 studied the effects of acrylamide, a toxicant
that humans are exposed to, via heat-treated starchrich food. Acrylamide is a germ cell mutagen inducing clastogenic effects in cells of the male germline.
Mating acrylamide-exposed males with unexposed
females has been shown to result in reduced fertility and to induce pre and post-implantation loss.
In this study has been valuated the measurements
of acrylamide-induced DNA damage in individual
sperm and the induction of stress responses in the
early embryo. This study is part of a larger project,
the primary objective of which is to clarify mechanisms underlying negative effects of environmentally
induced paternal DNA modifications on early embryo
development. Epididymal spermatozoa were isolated at different time, following acrylamide exposure
for analysis of DNA lesions by the Comet assay. The
results indicate that the highest amount of damage
in cauda sperm results from an exposure seven days
earlier, a time point which has been associated with
induction of preimplantation loss. The Comet assay
showed an increased damage levels in the majority
of exposed cauda sperm. In contrast to testicular
cells, no Formamidopyrimidine DNA-glycosylase
(fpg)-sensitive DNA lesions (representing oxidated
purines) were observed in sperm following in vivo
81
F. Lanzafame, et al.
acrylamide exposure. Paternal acrylamide exposure influenced the early embryo cleavage rate and
induced the expression of DNA damage response
proteins like γH2AX and p53.
Sperm preparation protocols
Sperm cryopreservation is routinely used in a variety
of circumstances including assisted reproduction,
preradiation or chemotherapy treatment, as “fertility
insurance” for men undergoing vasectomy and for
storage of donor semen until seronegativity for HIV
and hepatitis is confirmed. It is also used for storage of sperm retrieved from azoospermic patients
who have undergone testicular sperm extraction or
percutaneous epididymal sperm aspiration to prevent the repetition of invasive biopsies. and in many
other circumstances in seminology laboratories.
Studies investigating the effects of cryopreservation
have largely been limited to conventional parameters. Reports include changes in sperm morphology including damage to mitochondria, acrosome
and flagellum. Unfortunately, the proportion of fully
functional sperm that retain intact membranes, tail
and mitochondrial activity after freeze-thawing is
often low. Sperm motility has also been shown to
be particularly sensitive to such damage but, while it
is generally accepted that sperm motility is reduced
by cryopreservation, the mechanism by which this
occurs is, yet unclear. However, since up to 40% of
post thawed sperm are still motile, these spermatozoa could be utilized. Only few trials have been
performed to improve cryopreservation procedures
and strategies for its limitation to more meaningful
diagnostic parameters such as DNA quality.
Sperm DNA is particularly susceptible to oxidative damage due to high content of PUFA acting
as substrates for ROS and for its lack of repairing
mechanism. Only few studies have been evaluated
the effects of cryopreservation on nuclear and mitochondrial DNA. The results of these studies are often
conflicting as following reported.
Watson et al. found that sperm chromatin structure
remains stable 73; Spano et al. and Royere et al.
found chromatin alterations 74 75 and Hammadeh et
al. found abnormal DNA condensation 76 77.
A study lead by Donelly et al. 78 found that the DNA
fragmentation percentage significatilly increase in
human semen after cryopreservation (Freeze-thaw)
respect to fresh semen. The increase is more evident
in infertile respect to fertile men.
Zribi et al. 79 demonstrated that cryopreservation
increased DNA fragmentation, the obtained results
82
showed that in infertile men, following cryoinjury, the
susceptibility of morphologically abnormal sperm to
damage is three fold higher than normozoospermic
samples. Besides DNA fragmentation correlates with
abnormal morphology.
Freezing process lead to intracellular ice formation
and dissolution and also osmotic stresses caused
by dehydration 80-82; freezing caused a GSH (glutathione) depletion (-78%) and a reduced SOD activity
(-50%) 83 the addition of thiols (GSH, Cysteine, NAC)
in post-thaw samples prevented the H2O2 mediated
motility decrease 84 and also pyruvate, metal chelators or catalase avoided this phenomenon 85 86.
Antioxidant addition, during cryopreservation have
fertility benefits, infact α tocopherol and ascorbate
increase viability and SOD and Catalase improve
embryo numbers 87.
During thawing process, The rapid warming prevents recrystallization 74. Novel methods of freezing
and novel cryoprotectants can lead to reduction of
cryoinjury to DNA, examples of those are vitrification and ultra rapid freezing. The processes involve
solidifying liquids without crystallization, and embryo
cryopreservation by vitrification 88-91 in liquid nitrogen
slush 92.
Isachenko et al. 93 evaluated DNA integrity and motility of human spermatozoa after standard slow
freezing versus cryoprotectant-free vitrification and
the results showed that human spermatozoa vitrified
with cryoprotectant have a lower percentage of DNA
fragmentation respect to human spermatozoa vitrified without cryoprotectant. Moreover, the results
demonstrated that the percentage of DNA fragmentation is lower in vitrified spermatozoa respect to
slowly-frozen spermatozoa and the use of cryoprotectant lead to a reduction of DNA fragmentation in
human spermatozoa after swim up, slow-frozen and
vitrification procedures.
Inflammation/infection in the male reproductive tract
A number of evidences suggest that urogenital inflammation may impair fertility. The inflammation
of the male accessory glands has been also called
male accessory gland infection (MAGI) 94.
MAGI are very frequent pathological conditions and
this-term include:
• uncomplicated forms: prostatitis;
• complicated forms: prostato-vesiculitis (PV);
prostato-vesiculo-epididymitis (PVE) and epididymal-orchitis (EO).
Many studies investigated the effects of MAGI on
Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic”
sperm conventional sperm parameters (density,
motility, normal form), the results of these studies
are often conflicting probably because many studies published up to the 1990s have not relied on the
above-mentioned, generally accepted classification
of prostatic diseases 95 96 and WHO criteria for MAGI,
thus making it impossible to differentiate between
patients with a real inflammatory prostatic process
and the so called ‘pelvic pain patients’ without inflammatory reaction in prostatic secretions. Even
recent investigations are contradictory 97.
The inflammatory-infective process may affect reproduction through the functional alteration of the
male accessory glands because they do not produce adequate amounts of nutrients and antioxidant
compound or they produce and release ROS and
cytokines that alter the microenvironment where
spermatozoa develop and mature.
Particularly, cytokines are mediators of the host response to inflammation, that may modulate the activities of prooxidative and scavenger systems which
also brings about the burst of ROS.
La Vignera et al. 98 evaluated seminal plasma cytokine levels in prostatitis and prostato-vesiculitis
and have been found significatively elevated levels
of TNF (tumor necrosis factor)-α, IL-6 and IL-10 in
prostatitis and prostato-vesiculitis respectively compared to normal controls.
TNF-α and sperm function were also assessed in some
other studies and the results showed that seminal plasma TNF-α concentration is higher in patients with bacterial or mycoplasma infections compared to normal
controls. Seminal plasma TNF-α concentrations are
increased in patients with leukocytospermia 99 while
TNF-α and IL-6 significantly reduces total and progressive motility in a time and dose-dependent manner 100.
Moreover, they increase nitric oxide production in a
dose-dependent manner too 100. Only few studies evaluated TNF-α effects on sperm DNA integrity. Said et
al. 101, measured the exposure of human spermatozoa
to varying concentrations of TNF-α and infliximab. The
results showed that spermatozoa quality declined following incubation with TNF-α in a dose (100, 300, 400,
500 pg/ml, and 2.5 mg/ml) and time-dependent manner. Sperm motility and DNA integrity were higher in
the samples incubated with TNF-α plus infliximab than
in the samples treated with TNF-α only. These results
suggest that, exposing spermatozoa to pathological
concentrations of TNF-α can result in significant loss
of their functional and genomic integrity 101. Moreover,
the inflammatory-infective process may affect reproduction also through the germ-spermatozoa interaction. The association of germs commonly observed
in genital infections include Chlamydia trachomatis
(C. trachomatis) and Mycoplasms. Some studies about
C. trachomatis infection showed that it is associated
with sperm parameters alterations 102-105. other studies
did not confirmed the above mentioned results 106-109.
This discrepancy could be also ascribed to the different methods used to identify the presence of C. trachomatis (cultural test and immunological test).
Data obtained from Hossenzadeh 110 suggest a negative effect on sperm motility and viability.
The apoptotic process related with elevated levels
of ROS and the damage in the spermatic DNA have
been associated with the presence of infectious
agents and may have a crucial influence on fertilization process 111.
In a study of Alvarez et al. 112 thirty seven patients,
grouped according to the microbiological results
Mycoplasms (+)/C. trachomatis (-), Mycoplasms (-)/
C. trachomatis (+) and Mycoplasms (+)/C. trachomatis (+) and a control group of eleven normozoospermic healthy men have been evaluated. The authors
assessed sperm apoptosis and spermatic DNA fragmentation. High levels of DNA breaks were associated with the presence of C. trachomatis and Mycoplasms. These, in combination, shown a synergistic
effect as inducers of damage to the DNA structure.
There is an association between high levels of apoptosis/DNA sperm fragmentation and seminal infection by C. trachomatis and Mycoplasms.
Satta et al., 113 found that Infection with C. trachomatis EB at the concentration of 300, 3000 or 30000
CFU had no effect on the percentage of sperm with
PS translocation after 6 h of incubation
A significant effect on this parameter was instead
observed after 24 h of incubation. C. trachomatis also
caused a statistically significant increase in the percentage of sperm with DNA fragmentation both after 6
and 24 h of incubation. The effect reached a statistical
significance only at the highest concentration (30000
EB) of C. trachomatis EB after 6 h of incubation, whereas it was effective at 3000 EB after 24 h of incubation.
The molecular mechanism by which C. trachomatis
induce sperm death is still unknown, some studies showed that LGV and serovar E extracted LPS
cause a marked reduction of sperm motility and a
concomitant increase of sperm death; Escherichia
Coli extracted LPS is about 500 times less powerful,
C. trachomatis EB have a stronger effect, suggesting
that other molecules may play an additional role 110.
An experimental model 114 evaluated LPS (0.1 mg
lipopolysaccharide (LPS)/kg body weight/day for 7
days) administered Imprinting Control Region (ICR)
mice. In this study, were examined sperm concen83
F. Lanzafame, et al.
tration and motility in the cauda epididymis as well
as immunohistochemical localization of Fas and
FasL and germ cell apoptosis. Sperm concentration and motility markedly fluctuated in LPS-treated
mice. The increase of apoptotic cells was common
in all post-LPS treatment groups, with a peak at 24h
after LPS injection. In contrast to the lack of Fas immunoreactivity in control testes, LPS-treated groups
demonstrated Fas in many germ cells, especially
spermatocytes and spermatids. Moreover FasL immunoreactivity was positive for some Sertoli cells,
Leydig cells and germ cells in both control and LPStreated mice. In conclusion These results suggest
that the Fas/FasL system mediates apoptosis of
germ cells in LPS-treated mice testes 114.
Hakimi et al., 115 studied Lipid A (the toxic component of LPS) and 3-deoxy-D-manno-octulosonic
acid (Kdo), a C. trachomatis LPS component. The
results demonstrated that Lipid A and Kdo cause
sperm death. Particularly, Lipid A and Kdo co-incubation causes apoptotic-like sperm death mediated
by caspase activation 115. It is clear that the inflammatory reactions are inevitably associated with the
oxidative stress phenomenon.
The results obtained from Fraczek et al. 116, suggest
that the bacterial invasion or local tissue damage is
accompanied by infiltrating leukocytes, especially
phagocytic cells connected with the production
and release of large amounts of ROS and biologically active substances, such as proinflammatory
cytokines. The cytokines, mediators of the host
response to inflammation, may modulate the activities of the prooxidative and antioxidative systems
which brings about the rush of ROS. When the ROS
amount overwhelm the potential of the antioxidative
defence, peroxidative damage to spermatozoa occurs, which in turn lead to sperm dysfunction that
results in infertility 116. Taking into consideration
that DNA fragmentation and apoptosis may result
from ROS-dependent activity, and that the inflammatory process is inseparably connected with OS,
Fraczek et al. 116, analyzed the effect of selected
inflammatory mediators on DNA fragmentation of
different sperm subpopulations and, particularly,
three sperm subpopulations exposed to inflammatory mediators (leukocytes, proinflammatory cytokines, bacteria).
The results indicated that, during the male reproductive tract infections, bacteria are the most important
inducers of DNA fragmentation in ejaculated spermatozoa.
84
Epigenetics and genetics sperm DNA
alterations
As before exposed it is clear that different pathological condition lead to the increase of OS and definitely, the OS phenomenon is inevitably associated
with epigenetics and/or genetics alterations. Deficiencies on sperm genome and epigenome could
be related to inpairment on male reproduction. DNA
methylation; chromatin modifications (acetylation
or methylation of specific residues on chromatin);
post-translational modifications of histone aminoterminal (histone code), are examples of epigenetic
processes and all this modifications are heritable
through cell division, yet reversible.
The biological roles of DNA methylation are genomic
imprinting; X-chromosome inactivation and repression of transposons. Altered methylation profiles are
associated with human diseases, some examples
of these are cancer imprinting diseases, Angelman
Syndrome, Prader-Willi Syndrome; Beckwith-Wiedemann Syndrome and immunodeficiency Syndrome
(ICF syndrome).
The DNA methylation patterns start in germ cells and
have important implications for health and disease.
Some studies show that epigenetic defects can be
also associated with infertility.
Marques et al., found that oligozoospermia is associated with methylation of H19 and methylation of
PEG1 117Kobayashi et al., found that oligozoospermia is associated with methylation of H19 and also
with GTL2 SNRPN, PEG1, LIT1, ZAC 118. Epigenetic
program start in gametes (prenatal and postnatal
phases) can be perturbed and ‘epimutations’ can be
transmitted.
Altered chromatin modifications can also be associated with infertility.
Protamines are sperm small nuclear basic proteins
(50-57 amino acids), with a high content of: arginine
(50%) and cysteine (10%) 119. In spermatozoa somatic
cell histones are replaced by the protamines to yield
chromatin condensation and related to compact
sperm cell size and transcriptional quiescence 119.
The protamines are a varied family of small argininerich proteins that are synthesized in the late-stage
spermatids of many animals and plants and bind
to DNA, condensing the spermatid genome into a
genetically inactive state. Human sperm protamine
are protamine-1 (P1) and protamine-2 (P2) Vertebrates have from one to fifteen protamine genes per
haploid genome, which are clustered together on the
same chromosome. Comparison of protamine gene
and amino-acid sequences suggests that the family evolved from specialized histones through prot-
Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic”
amine-like proteins to the true protamines. Structural
elements present in all true protamines are a series
of arginine-rich DNA-anchoring domains (often containing a mixture of arginine and lysine residues in
non-mammalian protamines) and multiple phosphorylation sites. The two protamines found in mammals,
P1 and P2, are the most widely studied. P1 packages
sperm DNA in all mammals, whereas protamine P2 is
present only in the sperm of primates, many rodents
and a subset of other placental mammals. P2, but
not P1, is synthesized as a precursor that undergoes
to proteolytic processing after binding to DNA and it
also binds a zinc atom, the function of which is not
known. P1 and P2 are soon phosphorylated after
their synthesis, but after binding to DNA most of the
phosphate groups are removed and cysteine residues are oxidized, forming disulfide bridges that link
the protamines together. Both P1 and P2 have been
shown to be required for normal sperm function in
primates and many rodents. Aoki et al. 120 evaluated
P1/P2 ratio distribution for fertile men and infertility patients and they showed a novel population of
infertile males with a reduced P1/P2 ratio. Aberrant
P1/P2 ratios arise from an abnormal concentration of
P1 and/or P2, either of which is associated with male
infertility. Moreover, in human spermatozoa, there
are different evidences that abnormal protamine
expression is associated with a low spermatozoa
concentration and motility 121-124.
Histone retention mainly involve three gene classes:
developmental promoters; miRNAs and imprinted
clusters. Particularly, the results obtained from
Aoki et al. 120 about TH2B showed its high relative
amount of retention in sperm (2% of genome bound
to Th2B, previously hypothesized to “poise” sperm
genome 124. Rangasamy et al. 125 demonstrated that
H2Az too, with a low amount, is retained in spermatic DNA. In conclusion the results obtained from different studies indicate that abnormal protamination
is associated with diminished sperm quality including elevated DNA damage and altered methylation
of some imprinted genes. Moreover, sperm from
fertile men retain about 5% of the genome bound to
histones, and an incomplete protamination is associated with increased histone retention; furthermore,
in fertile men, retained histones are generally associated with demethylated DNA regions in promoters of
embryonic developmental genes, imprinted genes,
and miRNAs. Particularly, TH2B is retained in gene
groups linked to sperm biology and H2Az with pericentromeric heterochromatin. Additionally, developmental genes are generally bound with H3K4me3
and H3K4me2 activating modifications. During sper-
miogenesis, human sperm chromatin undergoes
replacement of nuclear histones by protamines,
resulting in a highly condensed DNA. The replacement of nuclear histones by protamines has both the
goals, to pack DNA tightly and to protect DNA from
chemical and physical damage (i.e. ROS). One of the
potential consequence of abnormal protamination
is the greater susceptibility to DNA damage 126-128.
Both protamine deficiency and sperm DNA damage
are related to decreased reproductive ability of men,
in natural as well as in assisted reproduction 128 129.
Tarozzi et al. 129 carried out a study to evaluate the
impact of abnormal protamination on sperm parameters, sperm DNA fragmentation, ART outcome and
the link between protamine deficiency and seminal
plasma antioxidant ability. A significant negative
correlation was found between abnormal protamination and sperm parameters, including sperm DNA
integrity (p < 0.001). The results showed a close relationship among sperm protamination, fertilization
and pregnancy only in IVF procedures (p = 0.004
and p < 0.04, respectively) while ICSI demonstrated
a correlation between DNA integrity and pregnancy
(p = 0.031). Finally, authors found a negative correlation between chromatin underprotamination and
seminal plasma antioxidant ability (p < 0.01). The
results of this study underline that, despite sperm
abnormal protamination and DNA fragmentation are
positively correlated, they affect the reproductive
outcome in different manners. Particularly authors
found good prognostic value of CMA3 analysis only
in IVF, whereas DNA fragmentation analysis is of
prognostic value only for ICSI outcome. The results
obtained, also provided data supporting the idea of
a relationship between a defective antioxidant system activity and the impairment of chromatin packaging. The DNA fragmentation induced in sperm
emerging from the testes as a result of aberration
chromatin repackaging and aberrant free radical
generation. Damage can be trasmitted because mutagenic change present in fertilizing spermatozoon
lead to the failure of the oocyte to repair the DNA
damage. Among them, Y chromosome deletion are
frequent because Y chromosome is very susceptible
to DNA deletion 130. The Y chromosome structure
predisposes it to deletions, segmental duplications
and to copy number variations. Three regions on the
Y chromosome called azoospermia factor (AZF) regions (AZFa, AZFb, AZFc) contain genes involved in
spermatogenesis 131. Their complete removal following deletions causes impairment of spermatogenesis. Therefore, they are considered clear causes
of spermatogenic failure 132. Two others structural
85
F. Lanzafame, et al.
variations are known on the Y chromosome with
potential effect on spermatogenesis, the partial
AZFc deletions/duplications and the copy number
variation of the TSPY cluster. Among partial AZFc
deletions the gr/gr deletions has been reported as a
genetic risk factor for impaired sperm production 133.
Given that the number of genes removed by the gr/
gr deletion is half that the classical AZFc deletion its
effect on spermatogenesis seems to be milder. Thus
we should consider it as a co-factor for spermatogenic impairment with variable penetrance. On the
other hand its pathogenic effect may also be related
to distinct Y-linked or non-Y genetic factors. Krausz
et al. have carried out a multicenter study to investigate the contribution of Y-chromosomal factors
to the extensive and puzzling phenotypic variation
exhibited by gr/gr deletion carriers, which ranges
from normal spermatogenesis to azoospermia 134.
The genetic factors examined included the known
AZFc structural variants associated with this deletion (removal of different DAZ and CDY1 gene copies), deletion followed by duplication and the more
general Y chromosome background. The obtained
results, showed significant geographic differences in
the deletion subtypes distribution, which may affect
the outcome of case control association studies in
different geographic areas. However, the phenotypic
variation of gr/gr carriers in men of European origin
seems to be largely independent from the Y chromosomal background 134.
Conclusion
The integrity of the genetic material is a prerequisite
for normal fertilization and transmission of paternal
genetic information. Finely tuned differentiation steps
of the male germ cell line, during its active lifetime,
ensure this goal and any derailment is thought to be
crucial, especially during spermiogenesis when repair
system fade leaving DNA more vulnerable. Causes of
sperm DNA damage are numerous, and not all mechanism are known. Multiple (testicular and extra-testicular) sources have been proposed including abortive
apoptosis, abnormal chromatin packaging during the
transition from round to elongated spermatids, and
OS. Genetic defects that may be transmitted through
sperm are different and include whole and segmentalchromosomal aneuploidies, mutations, trinucleotide
repeat-length variations, defects in the imprinting
profiles and unspecific DNA breaks.
Sperm DNA damage can be associated with reduced
rates of fertilization in vivo, by natural conception or
intrauterine insemination. Less unequivocal informa86
tion exists regarding the link between DNA strand
breaks and in vitro fertility. Unexplained recurrent
pregnancy loss has also been suggested to be associated with a variety of DNA anomalies including
sperm DNA fragmentation. moreover the abortion
rate was found to be higher in apparently fertile couples where the partner’s sperm had poor chromatin
quality. In the last 10 years, the assessment of sperm
DNA integrity has emerged as a strong candidate to
be included in the list of potential new semen quality
biomarkers. In spite of those insights, the incluson of
the sperm DNA integrity tests into the andrological
practice is still under examination.
References
1
2
3
4
5
6
7
8
9
10
11
12
13
Zini A, Meriano J, Kader K, Jarvi K, Laskin CA, Cadesky
K. Potential adverse effect of sperm DNA damage on embryo qualità after ICSI. Hum Reprod 2005;20:3476-80.
Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive
oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79:829-43,
Aitken RJ, Ryan AL, Baker MA, McLaughlin EA. Redox
activity associated with the maturation and capacitation of mammalian spermatozoa. Free Radic Biol Med
2004;36:994-1010.
Aitken RJ, Skakkebaek NE, Roman SD. Male reproductive health and the environment. Med J Aust
2006;185 414-5.
Aitken RJ, De Iuliis GN. Value of DNA integrity assays
for fertility evaluation. Reprod Fertil 2007;65:81-92.
Blumer CG, Fariello RM, Restelli AE, Spaine DM, Bertolla RP, Cedenho AP. Sperm nuclear DNA fragmentation and mitochondrial activity in men with varicocele.
Fertil Steril 2007;90:1716-22.
Chemes EH, Rawe YV. Sperm pathology: a step beyond descriptive morphology. Origin, characterization
and fertility potential of abnormal sperm phenotypes in
infertile men. Hum Reprod 2003;9:405-28.
Henkel R, Kierspel E, Hajimohammad M, Stalf T,
Hoogendijk C, Mehnert C, et al. DNA fragmentation of
spermatozoa and assisted reproduction technology.
Reprod Biomed Online 2003;7:477-84.
Host E, Lindenberg S, Smidt-Jensen S. The role of DNA
strand breaks in human spermatozoa used for IVF and
ICSI. Acta Obstet Gynecol Scand 2000;79:559-63.
Lewis SEM, Aitken RJ. DNA damage to spermatozoa
has impacts on fertilization and pregnancy. Cell Tissue
Res 2005;322:33-41.
Seli E, Sakkas D. Spermatozoal nuclear determinants
of reproductive outcome: implications for ART. Hum
Reprod 2005;11:337-49.
Shitara H, Kaneda H, Sato A, Inoue K, Ogura A, Yonekawa H, et al. Selective and continuous elimination
of mitochondria microinjected into mouse eggs from
spermatids, but not from liver cells, occurs throughout
embryogenesis. Genetics 2000;156:1277-84.
Sutovsky P, Manandhar G, Wu A, Oko R. Interactions of
sperm perinuclear theca with the oocyte: implications for
Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic”
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
oocyte activation, anti-polyspermy defense, and assisted
reproduction. Microsc Res Tech 2003;61:362-78.
Tesarik J. Paternal effects on cell division in the human preimplantation embryo. Reprod Biomed Ondine
2005;10:370-5.
Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg
E, Purvis K, et al. Utility of the sperm chromatin assay
as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999;14:1039-49.
Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli
E, Leter G, et al. Sperm chromatin damage impairs human fertility. Fertil Steril 2000;73, 43-50.
Zini A, Bielecki R, Phang D, Zenzes MT. Correlations
between two markers of sperm DNA integrity, DNA
denaturation and DNA fragmentation, in fertile and
infertile men. Fertil Steril 20001;75:674-7.
Hughes CM, Lewis SEM, McKelvey-Martin VJ, Thompson W. A comparison of baseline and induced DNA
damage in human spermatozoa from fertile and infertile
men, using a modified comet assay. Molecular Human
Reproduction 1996;8:613-9.
Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA,
Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000;21:33-44.
Shen H, Ong C. Detection of oxidativde DNA damage in human sperm and its association with sperm
function and male infertility. Free Radic Biol Med
2000;28:529-36.
Gatewood JM, Cook GR, Balhorn R, Schmid CW,
Bradbury EM. Isolation of four core histones from human sperm chromatin representing a minor subset of
somatic histones. J Biol Chem 1990;265:20662-6.
Carrell DT, Liu L. Altered protamine 2 expression is
uncommon in donors of known fertility, but common
among men with poor fertilizing capacity, and may
reflect other abnormalities of spermiogenesis. J Androl
2001;22:604-10.
Zhang X, San Gabriel M, Zini A. Sperm nuclear histone
to protamine ratio in fertile and infertile men: evidence
of heterogeneous subpopulations of spermatozoa in
the ejaculate. J Androl 2006;27:414-20.
Jansen G, Willems P, Coerwinkel M, Nillesen W,
Smeets H, Vits L, C. Höweler, et al. Gonosomal mosaicism in myotonic dystrophy patients: involvement of
mitotic events in (CTG)n repeat variation and selection
against extreme expansion in sperm. Am J Hum Genet
1994;54:575-85.
Patrizio P, Leonard DG, Chen KL, Hernandez-Ayup S,
Trounson AO. Larger trinucleotide repeat size in the
androgen receptor gene of infertile men with extremely
severe oligozoospermia. J Androl 2001;22:444-8.
Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C,
Utsunomiya T. Aberrant DNA methylation of imprinted
loci in sperm from oligospermic patients. Hum Mol Gen
2007;16:2542-51.
Kobayashi H, Hiura H, John RM, Sato A, Otsu E, Kobayashi N, et al. DNA methylation errors at imprinted
loci after assisted conception originate in the parental
sperm. Eur J Hum Gene 2009 May 27.
Laberge RM, Boissonneault G. On the nature and origin of DNA strand breaks in elongating spermatids. Biol
Reprod 2005;73:289-96.
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Griveau JF, Le Lannou D. Reactive oxygen species and
human spermatozoa: physiology and pathology. Int J
Androl 1997;20:61-9.
Whittington K, Harrison SC, Williams KM, Day JL,
McLaughlin EA, Hull MG, et al. Reactive oxygen species (ROS) production and the outcome of diagnostic
tests of sperm function. Int J Androl 1999;22:236-42.
Nakamura H, Kimura T, Nakajima A, Shimoya K, Takemura M, Hashimoto K, et al. Detection of oxidative
stress in seminal plasma and fractionated sperm from
subfertile male patients. Eur J Obstet Gynecol Reprod
Biol 2002;105:155-60.
Sikka SC. Relative impact of oxidative stress on male
reproductive function. Curr Med Chem 2001;8:851-62.
Fraga CG, Motchnik PA, Shigenaga MK, Helbock HJ,
Jacob RA, Ames BN. Ascorbic acid protects against
endogenous oxidative DNA damage in human sperm.
Proc Natl Acad Sci USA 1991;88:11003-6.
Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S,
Tesarik J. Reduction of the incidence of sperm DNA
fragmentation by oral antioxidant treatment. J Androl
2005;26:349-53.
Lanzafame F, La Vignera S, Vicari E, Calogero AE. Oxidative stress and antioxidant medical treatment in male
infertility. RBM on line accepted for pubblication.
Menezo YJ, Hazout A, Panteix G, Robert F, Rollet J,
Cohen-Bacrie P, et al. Antioxidants to reduce sperm
DNA fragmentation: an unexpected adverse effect.
Reprod Biomed Online 2007;14:418-21.
Frias S, Van Hummelen P, Meistrich M, Lowe X, Hagemeister FB, Shelby MD, et al. NOVP Chemotherapy
for Hodgkin’s disease transiently induces sperm aneuploidies associated with the major clinical aneuploidy
syndromes involving chromosomes X, Y,18 and 21.
Cancer Res 2003;63:44-51.
Robbins WA, Meistrich ML, Moore D, Hagemeister FB,
Weier HU, Cassel MJ, et al. Chemotherapy induces
transient sex chromosomal and autosomal aneuploidy
in human sperm. Nat Genet 1987;16:74-8.
Schlesinger MH, Wilets IF, Nagler HM. Treatment outcome after varicocelectomy. A critical analysis. Urol
Clin North Am 1994;21:517-29.
Benoff S, Gilbert BR. Varicocele and male infertility:
part I. Preface. Hum Reprod 2001;7:47-54.
Hauser R, Paz G, Botchan A, Yogev L, Yavetz H.
Varicocele: effect on sperm functions. Hum Reprod
2001;7:482-5.
World Health Organization. WHO Laboratory manual
for the examination of human semen and sperm-cervical mucus interaction. 3rd edn. Cambridge, UK: Cambridge University Press 1992.
Belloli G, D’Agostino S, Pesce C, Fantuz E. Varicocele
in childhood and adolescence and other testicular
anomalies: an epidemiological study. Pediatr Med Chir
1993;15:159-62.
Cozzolino DJ, Lipshultz LI. Varicocele as a progressive
lesion: positive effect of varicocele repair. Hum Reprod
2001;7:55-8.
Marmar JL. The pathophysiology of varicoceles in the
light of current molecular and genetic information. Hum
Reprod 2001;7:461-72.
Barbieri ER, Hidalgo ME, Venegas A, Smith R, Lissi
87
F. Lanzafame, et al.
47
48
49
50
51
52
53
54
55
56
57
58
59
60
88
EA. Varicocele-associated decrease in antioxidant defenses. J Androl 1999;20:713-7.
Hendin BN, Kolettis PN, Sharma RK, Thomas AJ Jr,
Agarwal A. Varicocele is associated with elevated
spermatozoal reactive oxygen species production and
diminished seminal plasma antioxidant capacity. J Urol
1999;161:1831-4.
Pasqualotto FF, Sharma RK, Nelson DR, Thomas AJ, Agarwal A. Relationship between oxidative
stress, semen characteristics, and clinical diagnosis
in men undergoing infertility investigation. Fertil Steril
2000;73:459-64.
Zini A, Garrels K, Phang D. Antioxidant activity in the semen of fertile and infertile men. Urology 2000;55:922-6.
Wang X, Sharma RK, Gupta A, George V, Thomas AJ,
Falcone T, et al. Alterations in mithocondria membrane
potential and oxidative stress in infertile men: a prospective observation study. Fertil Steril 2003;80:844-50.
Meucci E, Milardi D, Mordente A, Martorana GE, Giacchi E, De Marinis L, et al.Total antioxidant capacity
in patients with varicocele. Fertil Steril 2003;79 (Suppl.
3):1577-83.
Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM,
Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res
1996;13;351:199-203.
Saleh RA, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl
2002;23:737-52.
Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas A, et al. Relationship between
ROS production, apoptosis and DNA denaturation in
spermatozoa from patients examined for infertility. Hum
reprod 2004;19:129-38.
Sinha Hikim AP, Lue Y, Diaz-Romero M, Yen PH, Wang
C, Swerdloff RS. Deciphering the pathways of germ
cell apoptosis in the testis. J Steroid Biochem Mol Bio
2003;85:175-82.
Sinha Hikim AP, Lue Y, Yamamoto CM, Vera Y, Rodriguez S, Yen PH, et al. Key apoptotic pathways for heat
–induced programmed germ cell death in the testis.
Endocrinology 2003;144:3167-75.
Vera Y, Erkkila K, Wang C, Nunez C, Kyttanen S, Lue
Y, et al. Involvement of p38 mitogen activated protein
kinase and inducile nitric oxide synthase in apoptotic
signaling of murine and human male germ cells after hormone deprivation. Mol Endocrinol 2006;20:1597-609.
Jia Y, Hikim AP, Lue YH, Swerdloff RS, Vera Y,
Zhang XS, et al. Signaling pathways for germ cell
death in adult cynomolgus monkeys (Macaca fascicularis) induced by mild testicular hyperthermia
and exogenous testosterone treatment. Biol Reprod
2007;77:83-92.
Wang C, Cui YG, Wang XH, Jia Y, Sinha HA, Lue YH,
et al. Transient scrotal hyperthermia and lrvonorgestrel
anhance testosterone-induced spermatogenesis suppression in men through increased germ cell apoptosis.
J Clin Endocrinol Metab 2007;92:3292-304.
Sharpe RM, Irvine DS. How strong is the evidence
of a link between environmental chemicals and adverse effects on human reproductive health? BMJ
2004;328:447-51.
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
Parks L, Ostby J, Lambright C, Abbott B, Klinefelter
GD, Barlow N, et al. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the
male rat. Toxicol Sci 2000;58:339-49.
Midash O, Schettgen T, Angerer J. Pilot study on the
perfluorooctanesulfonate and perfluorooctanoate exposure of the German general population. Int J Hyg
Environ Health 2006;209:489-96.
Sharpe RM, Skakkebaek NE. Are estrogens involved in
falling sperm counts and disorders of the male reproductive tract? Lancet 1993;351:1392-5.
Sikka SC, Wang R. Endocrine disruptors and estrogenic effects on male reproductive axis. Asian J Androl
2008;10:134-45.
Governini L, Guerranti C, Focardi S, Cuppone AM,
Stendardi A , DeLeo V, et al. Impact of environmental
exposure to perfluorinated compounds on sperm DNA
quality. Systems Biology in Reproductive Medicine
2009;55.
Muratori M, Marchiani S, Maggi M, Forti G, Baldi E. Origin and biological significance of DNA fragmentation in
human spermatozoa. Front Biosci 2006;1;11:1491-9.
Lundin KB, Giwercman A, Richthoff J, Abrahamsson
PA, Giwercman YL. No association between mutations
in the human androgen receptor GGN repeat and intersex conditions. Mol Hum Reprod 2003;9:375-9.
Giwercman A, Rylander L, Rignell-Hydbom A, Josson BA, Pedersen HS, Ludwicki JK, et al. Androgen
receptor gene CAG repeat length as a modifier of the
association between persistent organohalogen pollutant exposure markers and semen characteristics.
Pharmacogenet Genomics 2007;17:391-401.
Toft G, Rignell-Hydbom A, Tyrkiel E, Shvets M, Giwercman A, Lindh CH, et al. Semen quality and exposure
to persistent organochlorine pollutants. Epidemiology
2006;17:450-8.
Lichtenfels AJ, Gomes JB, Pieri PC, El Khouri Miraglia SG, Hallak J, Saldiva PH. Increased levels of air
pollution and a decrease in the human and mouse
male-to-female ratio in São Paulo, Brazil. Fertil Steril
2007;87:230-2.
Olse AK, Andreassen A, Singh R, Duale N, Wiger
R, Brunborg G. Persistence of DNA damage and its
consequence for mutagenesis in male germ cells og
OGG1 (-/-) Big Blue mice exposed to benzo(a)pyrene.
Systems Biology in Reprodutive Medicine 2009;55.
Shahzadi S, Sipinen V, Brevik A, Brunborg G, Lindemann. Cellular mechanisms underlying the effects of
paternal acrylamide-exposure on preimplantation development in mice. Syst Biol Reprod Med 2009;55.
Curry MR, Watson PF. Osmotic effects on ram and
human sperm membranes in relation to thawing injury.
Cryobiology 1994;31:39-46.
Spanò M, Cordelli E, Leter G, Lombardo F, Lenzi A,
Gandini L. Nuclear chromatin variations in human
spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin
structure assay. Mol Hum Reprod 1999;5:29-37.
Royere D, Hamamah S, Nicolle JC, Lansac J. Chromatin alterations induced by freeze-thawing influence
the fertilizing ability of human sperm. Int J Androl
1991;14:328-32.
Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic”
76
77
78
79
80
81
82
83
84
85
86
87
88
89
Hammadeh ME, Askari AS, Georg T, Rosenbaum P,
Schmidt W. Effect of freeze-thawing procedure on
chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and
subfertile men. Int J Androl 1999;22:155-62.
Hammadeh ME, Kühnen A, Amer AS, Rosenbaum P,
Schmidt W. Comparison of sperm preparation methods: effect on chromatin and morphology recovery
rates and their consequences on the clinical outcome
after in vitro fertilization embryo transfer. Int J Androl
2001;24:360-8.
Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated
spermatozoa from fertile and infertile men before and
after cryopreservation. Hum Reprod 2001;16:1191-9.
Zribi N, Chakroun NF, El Euch H, Gargouri J, Bahloul A,
Keskes LA. Effects of cryopreservation on human sperm
deoxyribonucleic acid integrity. Fertil Steril 2008 Nov 20
[Epub ahead of print].
Thompson-Cree MEM, McClure N, Donnelly ET, Steele
KE, Lewis SME. Effects of cryopreservation on testicular sperm nuclear DNA fragmentation and its relationship with assisted conception outcome following ICSI
with testicular spermatozoa. Reprod Biomed Online
2003;7:449-55.
Devireddy RV, Swanlund DJ, Roberts KP, Pryor JL,
Bischof JC. The effect of extracellular ice and cryoprotective agents on the water permeability parameters of
human sperm plasma membrane during freezing. Hum
Reprod 2000;15:1125-35.
Clulow JR, Mansfield LJ, Morris LH, Evans G, Maxwell
WM. A comparison between freezing methods for the
cryopreservation of stallion spermatozoa. Anim Reprod
Sci 2007;108:298-308.
Bilodeau JF, Chatterjee S, Sirard MA, Gagnon C. Levels of antioxidant defenses are decreased in bovine
spermatozoa after a cycle of freezing and thawing. Mol
Reprod Dev 2000;55:282-8.
Bilodeau JF, Blanchette S, Gagnon C, Sirard MA.
Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology
2001;15;56:275-86.
Bilodeau JF, Chatterjee S, Sirard MA, Gagnon C. Cryopreservation of bovine semen decreases antioxidant
defenses in spermatozoa. Biol Reprod 1999;60 (Suppl.
1):102.
Bilodeau JF, Blanchette S, Cormier N, Sirard MA. Reactive oxygen species-mediated loss of bovine sperm
motility in egg yolk Tris extender: protection by pyruvate, metal chelators and bovine liver or oviductal fluid
catalase. Theriogenology 2002;57:1105-22.
Roca J, Rodriguez MJ, Gil MA, Carvajal G, Garcia
EM, Cuello C, et al. Survival and in vitro fertility of
boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase. J Androl Andrology
2005;26:15-24.
Hunter JE, Bernard A, Fuller B, Amso N, Shaw RW.
Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures
and cryopreservation: a comparison of two techniques.
Hum Reprod 1991;6:1460-5.
Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O. Vitrification demonstrates
significant improvement versus slow freezing of human
blastocysts. Reprod Biomed Online 2005;11:53-7.
90
Zheng WT, Zhuang GL, Zhou CQ, Fang C, Ou JP, Li
T, et al. Comparison of the survival of human biopsied
embryos after cryopreservation with four different
methods using non-transferable embryos. Hum Reprod
2005;20:1615-8.
91
Liebermann J, Tucker MJ. Vitrifying and warming of
human oocytes, embryos, and blastocysts: vitrification
procedures as an alternative to conventional cryopreservation. Methods Mol Biol 2004;254:345-64.
92
Yavin S, Aroyo A, Roth Z and Arav A. Embryo cryopreservation in the presence of low concentration of
vitrification solution with sealed pulled straws in liquid
nitrogen slush. Hum Reprod 2009;24:797-804.
93
Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van Der Ven H. Cryoprotectant-free
cryopreservation of human spermatozoa by vitrification
and freezing in vapor: effect on motility, DNA integrity,
and fertilization ability. Biol Reprod 2004;71:1167-73.
94
World Health Organization. WHO Laboratory Manual
for the Examination of Human Semen and sperm–cervical mucus interaction. 4th edn. Cambridge, UK: Cambridge University Press 1999.
95
Giamarellou H, Tympanidis K, Bitos NA, Leonidas E,
Daikos GK. Infertility and chronic prostatitis. Andrologia
1984;16:417-22.
96
Christiansen E, Tollefsrud A, Purvis K. Sperm quality in
men with chronic abacterial prostatovesiculitis verified
by rectal ultrasonography. Urology 1991;38:545-9.
97
Weidner W, Schiefer HG, Brähler E. Refractory chronic
bacterial prostatitis: a re-evaluation of ciprofloxacin
treatment after a median followup of 30 months. J Urol
1991;146:350-2.
98
La Vignera S, Calogero AE, Castiglione R, D’Agata R,
Giammusso B, Condorelli R, et al. IL-6, TNF-alfa, IL-10
in the seminal plasma of patients with bacterial male
accessory gland infections after sequential therapy.
Minerva Urol Nefrol 2008;60:141-5.
99
Omu AE, Al-Qattan F, Al-Abdul-Hadi FM, Fatinikun MT,
Fernandes S. Seminal immune response in infertile men
with leukocytospermia: effect on antioxidant activity. Eur
J Obstet Gynecol Reprod Biol 1999;86:195-202.
100
Lampiao F, du Plessis SS. TNF-alpha and IL-6 affect
human sperm function by elevating nitric oxide production. Reprod Biomed Online 2008;17:628-31.
101
Said TM, Agarwal A, Falcone T, Sharma RK, Bedaiwy
MA, Li L. Infliximab may reverse the toxic effects induced
by tumor necrosis factor alpha in human spermatozoa:
an in vitro model. Fertil Steril. 2005;83:1665-73.
102
Leib Z, Bartoov B, Eltes F, Servadio C. Reduced semen quality caused by chronic abacterial prostatitis: an
enigma or reality? Fertil Steril 1994;61:1109-16.
103
Krieger JN, Berger RE, Ross SO, Rothman I, Muller
CH. Seminal fluid findings in men with nonbacterial
prostatitis and prostatodynia. J Androl 1996;17:310-8.
104
Wolff H, Neubert U, Zebhauser M, Bezold G, Korting
HC, Meurer M. Chlamydia trachomatis induces an
inflammatory response in the male genital tract and
is associated with altered semen quality. Fertil Steril
1991;55:1017-9.
105
Motrich RD, Cuffini C, Oberti JP, Maccioni M, Rivero
89
F. Lanzafame, et al.
106
107
108
109
110
111
112
113
114
115
116
117
118
119
90
VE. Chlamydia trachomatis occurrence and its impact
on sperm quality in chronic prostatitis patients. J Infect
2006;53:175-83.
Sanocka-Maciejewska D, Ciupińska M, Kurpisz M.
Bacterial infection and semen quality. J Reprod Immunol 2005;67:51-6.
Drach GW, Fair WR, Meares EM, Stamey TA.
Classification of benign diseases associated with
prostatic pain: prostatitis or prostatodynia? J Urol
1978;120:266
Nickel JC, Nigro M, Valiquette L, Anderson P, Patrick
A, Mahoney J et al. Diagnosis and treatment of prostatitis in Canada. Urology 1998;52:797-802.
Weidner W, Krause W, Ludwig M. Relevance of
male accessory gland infection for subsequent fertility with special focus on prostatitis. Hum Reprod
1999;5:421-32.
Hosseinzadeh S, Brewis IA, Eley A, Pacey AA. Coincubation of human spermatozoa with Chlamydia
trachomatis serovar E causes premature sperm death.
Hum Reprod 2001;16,293-9.
Hosseinzadeh S, Eley A, Pacey AA. Semen quality of
men with asymptomatic chlamydial infection. J Androl
2004;25:104-9.
Alvarez S, Gallegos G, Ramos B, Ancer J, Salazar R.
Apoptosis and sperm DNA fragmentation in infertile patients with Chlamydia and Mycoplasms infection. Syst
Biol Reprod Med 2009;55.
Satta A, Stivala A, Garozzo A, Morello A, Perdichizzi A,
Vicari E, Salmeri M, Calogero AE. Experimental Chlamydia trachomatis infection causes apoptosis in human
sperm. Hum Reprod 2006;21:134-7.
Kajihara T, Okagaki R, Ishihara O. LPS-induced transient testicular dysfunction accompanied by apoptosis
of testicular germ cells in mice. Med Mol Morphol
2006;39:203-8.
Hakimi H, Geary I, Pacey A, Eley A. Spermicidal activity
of bacterial lipopolysaccharide is only partly due to lipid
A. J Androl 2006;6:774-9.
Fraczek M, Dworacki G, Szumala-Kakol A, Sanocka D,
Zeromski J, Kurpisz M. Inflammatory mediators induce
apoptosis in ejaculated spermatozoa. Fertil Steril 2009
(in press).
Marques CJ, Costa P, Vaz B, Carvalho F, Fernandes
S, Barros A, et al. Abnormal methylation of imprinted
genes in human sperm is associated with oligozoospermia. Molecular Hum Reprod 2008;14:67-74.
Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C,
Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Mol
Hum Genet 2007;16:2542-51.
Balhorn R. The protamine family of sperm nuclear proteins. Genome Biology 2007;8:227.
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
Aoki VW, Liu L, Carrell DT. Identification and
evaluation of a novel sperm protamine abnormality in a population of infertile males. Hum Reprod
2005;20:1298-306.
Barroso G, Morshedi M, Oehninger S. DNA fragmentation, plasma membrane translocation of phosphatidylserine and OS, low sperm counts and reduced fertilizing capacità. Hum Reprod 2000;15:1338-44.
Aoki VW, Liu L, Jones KP, Hatasaka HH, Gibson
M, Peterson CM, et al. Sperm protamine 1/protamine 2 ratios are related to IVF pregnancy rates
and predictive of sperm fertilizing ability. Fertil Steril
2006;86:1408-15.
Oliva R. Protamines and male infertility. It is demostrated that the 5% of sperm genome packaged with
histones. Hum Reprod 2006;12:417-35.
Hammoud S, Liu L, Carrell DT. Protamine ratio and the
level of histone retention in sperm selected from a density gradient preparation. Andrologia 2009;41:88-94.
Rangasamy D, Berven L, Ridgway P, Tremethick DJ.
Pericentric heterochromatin becomes enriched with
H2A.Z during early mammalian development. EMBO J
2003;22:1599-607.
Carrell DT, Emery BR, Hammoud S. Altered protamine
expression and diminished spermatogenesis: what is
the link? Hum Reprod Update 2007;13:313-27.
Oliva R. Protamines and male infertility. Hum Reprod
Update 2006;12:417-35.
Tarozzi N, Bizzaro D, Flamigni C, Borini A. Clinical
relevance of sperm DNA damage in assisted reproduction. Reprod Biomed Online 2007;14:746-57.
Tarozzi N, Nadalini M, Stronati A, Bizzaro D, Dal Prato
L, Coticchio G. Anomalies in sperm chromatin packaging: implications for assisted reproduction techniques.
Reprod Biomed Online 2009;18:486-95.
Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction. 2001
Oct;122(4):497-506.Click here to read
Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum
HS, Hillier L, Brown LG, et al. The male-specific region
of the human Y chromosome is a mosaic of discrete
sequence classes. Nature 2003;423:810-1.
Krausz C, Degl’Innocenti S. Y chromosome and male
infertility. Front Biosci 2006;1:3049-61.
Repping S, de Vries JWA, van Daalen SKM, Korver
CM, Leschot NJ, van der Veen F. The use of spermHALO-FISH to determine DAZ gene copy number. Mol
Hum Reprod 2003;9:183-8.
Krausz C, Giachini C, Xue Y, O’Bryan MK, Gromoll J,
Rajpert-de Meyts E, et al. Phenotypic variation within
European carriers of the Y-chromosomal gr/gr deletion
is independent of Y-chromosomal background. J Med
Genet 2009;46:21-31.