Report from the international congress of andrology (ica) 2009 Satellite Symposium

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
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.
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]
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,
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,
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,
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,
27.genital tract inflammation and its consequences
on sperm DNA (A. Calogero, Italy);
28.antioxidants and sperm DNA damage (A. Zini,
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
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
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
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
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
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
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
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.
Epigenetics and genetics sperm DNA
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
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
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.
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
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