Archivio Italiano di Urologia e Andrologia 2013; 85, 18

INTRODUCTION
Varicocele is one of the major causes of infertility in men,
present in between 15-20% of the general male popula-
tion (1, 2). It’s predominant in adolescents but found in
41% infertile men and 80% of those with secondary infer-
tility (2). This anatomical abnormality due to the dilation
of the venous plexus which covers the testicles (pampini-
form plexus) is probably one of the most common causes
of oligoasthenozoospermia (1, 3). The pathogenetic mech-
anism through which varicocele causes testicular dysfunc-
tion and subsequent alterations in spermatogenesis, is,
however, not completely understood. Although various
factors may be involved (venous stasis which leads to tes-
ticular hypoxia) the increasing of internal testicular tem-

ORIGINAL PAPER

The study of spermatic DNA fragmentation
and sperm motility in infertile subjects

Giuseppina Peluso 1, Alessandro Palmieri 2, Pietro Paolo Cozza 1, 
Giancarlo Morrone 1, Paolo Verze 2, Nicola Longo 2, Vincenzo Mirone 2

1 U.O.S. of Andrology and Physiophatology of Reproduction-A.O. of Cosenza, Italy; 
2 Urological Clinic, University Federico II of Naples, Italy.

Introduction: Although the pathophysiology of the testicular damage associated with
varicocele remains unclear, sperm DNA damage has been identified as a potential
explanation for this cause of male infertility. The current study was designed to
determine the extent of sperm nuclear DNA damage in patients with varicocele, and
to examine its relationship with parameters of seminal motility.

Materials and method: Semen samples from 60 patients with clinical varicocele and 90 infertile
men without varicocele were examined. Varicocele sperm samples were classified as normal or
pathological according to the 1999 World Health Organizzation guidelines. Sperm DNA damage
was evalutated using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test.
Results: The DNA fragmentation index (DFI: percentage of sperm with denatured nuclei)
 values was significantly higher in patients with varicocele, either with normal or abnormal
(DFI 25.8 ± 3.2 vs 17.4 ± 2.8 - P < 0,01) semen profiles. In addition, an inverse correlation
was found between spermatic motility and the degree of spermatic DNA fragmentation in
patients with clinical varicocele.
Conclusions: Varicocele is associated with high levels of DNA-damage in spermatozoa. In
addition, in subjects with varicocele, abnormal spermatozoa motility is associated with high-
er levels of sperm DNA fragmentation. DNA fragmentation may therefore be an essential
additional diagnostic test that should be recommended for patients with clinical varicocele.

KEY WORDS: Spermatic DNA fragmentation; Oxidative stress; Varicocele; Male infertility.

Submitted 18 March 2013; Accepted 30 March 2013 No conflict of interest declared

Summary

perature is probably the most likely link between varico-
cele and infertility. In fact, elevated scrotal temperature
caused by vascular defects can cause altered production of
spermatogenetic cells by germinal epithelium (1, 3). In
fact, induced varicocele in laboratory animals leads to ele-
vated intratesticular temperature and sperm dysfunction
with decreased sperm motility (1, 3).
Another link between varicocele and infertility may arise
from impairment of the hypothalamic-gonadal axis or
oxidative stress (OS) (4, 5). In fact, studies evaluating the
role of oxidative stress in male infertility have recently
shown that OS could be considered as an important cause
of sperm dysfunction in varicocele infertile men (6-8),

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The study of spermatic DNA fragmentation and sperm motility in infertile subjects

and attending to Andrology Service for infertility diag-
nosis and other andrological problems. Once consensus
had been granted to be involved in this study, patients
were given a thorough andrological analysis according
to the guidelines provided by WHO 2001 which includ-
ed complete anamnesis, scrotal scan, scrotal Doppler
ultrasound, hormonal profile based on serum/blood lev-
els basal gonadotrophines (FSH, LH and total blood
Testo sterone), urethral sample for common microbes,
two spermiograms at a week’s interval with a minimum
of three days since sexual activity. Considering the
results of these analysis, it was possible to identify 80
patients with varying degrees of clinical varicocele but
predominantly unilateral. All patients with urinary tract
infections,  leucocytospermia, hypogonadism (testicular
volume < 15 ml), a history of excess of cigarette, alco-
hol or drug use were excluded from the study. The con-
trol group consisted of 100 healthy males with normal
genitalia and normal seminal parameters, as defined by
the WHO 2001 guidelines.
For each patient included in the study, the spermatozoon
population was determined. In addition, sperm DNA
fragmentation was quantified using the sperm chromatin
dispersion (SCD) test (Halosperm kit-Indas Labora tories,
Madrid Spain) which allows to express the spermatic
DNA fragmentation as a percentage index (DFI: DNA
Fragmentation Index). On the basis of the usual semino-
logic criteria, 34 patients with varicocele showed isolat-
ed asthenospermia, whereas spermatic concentration
and morphology have normal values.
Patients with severe dispermia in conjunction with
abnormal standard semen parameters such as oligoas-
thenotheratozoospermia (OAT) where 3%: in such case
high levels of damaged sperm DNA are usually observed,
consequently they were excluded from this study.
Patients considered in this phase of the study were there-
fore characterized by asthenozoospermia with various
degrees of motility, ranging from 5-45% of the progres-
sive linear a+b motility, according to the cut-off of nor-
mal sperm motility established by WHO, where a+b is
greater than or equal to 50% values.
The degree of DNA damage in spermatozoa of these
patients with only altered motility parameter was then
correlated with the a+b motility seminal parameter.

ANALYSIS OF SPERM DNA FRAGMENTATION
Spermatozoon DNA fragmentation was quantified using
Halosperm (Diasint-CGA, Florence-Italy) which has been
used in the well-developed sperm chromatin dispersion
(SCD) test (16, 17). The basis of the technology lies in
the differential response offered by the nuclei of sperma-
tozoa with fragmented DNA compared to those with
their DNA intact.
The controlled denaturation of the DNA followed by the
extraction of the nuclear proteins, gives rise to partially
deproteinized nucleoids in which the DNA loops
expand, forming halos of chromatin dispersion.
The nucleoid, which corresponds to the massively depro-
teinized nucleus of the spermatozoon, is composed of
two parts: spermatozoon nucleus silhouette, called the
“core”, positioned centrally, and a peripheral halo of chro-

with negative impact on sperm plasma membranes which
contain higher amounts of polyunsaturated fatty acids
(PUFA) which easily experience lipid peroxidation by
ROS (9, 10). It is a result of cascade of events including
lipid peroxidation (LPO) of sperm plasma membrane that
ultimately affect an axonemal protein phosphorylation
and sperm immobilization (9-11). These spermatozoa
would therefore not only be particularly susceptible to
reactive oxygen species (ROS) (12), with compromised
membrane fluidity and integrity and greatly reduced
motility (11-13). Metanalysis studies support this
hypothesis, demonstrating that infertile patients with
varicocele have higher levels of ROS when compared to
other typologies of infertility and controls (14, 15). In
addition, the seminal plasma of varicocele patients exhib-
ited reduced total antioxidant capacity (TAC) (7, 15-18).
Recently, various studies have also demonstrated that an
elevated presence of DNA fragmentation is present in the
spermatozoon nuclei of infertile patients with clinical
varicocele (19, 18). Interestingly, DNA damage are higher
when compared to spermatozoon of patients suffering
from other types of infertility; moreover the spermatozoon
of infertile varicocele patients exhibit higher levels of ROS,
suggesting correlation between oxidative damage and DNA
fragmentation. Studies using rat models have confirmed
this, demonstrating that nitric oxide (NO) released by
endothelial cells of dilated spermatic veins and peroxyni-
trites generated from reaction with superoxide radicals
cause intracellular oxidative damage (20-22), particularly
regarding membrane lipid peroxidation, thus altering the
integrity of chromatin in spermatozoon nuclei which may
be a direct expression of such oxidative damage (17). In
addition, sperms of varicocele patients exhibit elevated lev-
els of 8-hydroxy-2 deoxyguanosine which are associated
with a deficiency in the pro-oxidant defense system which
would cause oxidative damage to the DNA by modifying
the base, DNA strand breaks and chromatin cross linking,
since spermatozoa have limited defense mechanisms
against oxidative attack on their DNA mainly due to its
exclusive structural composition for the complex packag-
ing arrangement of DNA (18, 23, 24).
Other indications of increased ROS in patients with varic-
ocele are elevated quantities of cytoplasmic droplets in the
young spermatozoa. The droplets are indicative of imma-
ture and functionally defective spermatozoon (25-27)
and, containing high concentrations of cytoplasmic
enzymes such as G6PDH and SOD, are additional sources
of ROS (21, 26). Together, these studies indicate a positive
correlation between spermatozoon immaturity, elevated
levels of ROS and increase of concentration of mature
spermatozoa with damaged DNA in ejaculates of these
patients (21, 25-27).
Therefore, in this study, DNA fragmentation in sperma-
tozoa of infertile individuals diagnosed with clinical
varicocele was quantified with respect to infertile men
and controls. In addition, the amount of DNA fragmen-
tation was correlated to sperm motility.

MATERIALS AND METHODS
One hundred and fifty subjects analyzed in this study
ranged in age between 20-50 with the median age of 35

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10

Sperm cells with very small halos, without halos, and
without halo-degraded contain fragmented DNA.
Nucleoids that do not correspond to sperm cells were sep-
arately scored.

STATISTICAL ANALYSIS
All data were calculated as average + standard deviation
on experiments which were repeated and analyzed sta-
tistically using the statistical program SPSS. The statisti-
cal tests used were Student’s t for continuous values and
c2 for parametric values. In addition, statistical correla-
tion was used to test non-linear regression with evalua-
tion of the correlation coefficient.

RESULTS
The concentration of nemasperms in varicocele patients
included in the study, was significantly lower than that of
controls (19.8 + 6.5 e 39.7 + 7.1 respectively: P < 0.01.
Motility (type a+b) considered was also significantly
lower than that of controls (30.4 + 9.7 and 49.9 + 8.5
respectively: P < 0.01). In addition, the frequency of
DNA damage in nemasperms of infertile patients with
varicocele (% DFI) was statistically higher comparised
with infertile patients without varicocele (25.8 + 3.2 and
17.4 + 2.8 respectively: P < 0.01: figure 1). Besides the
DFI of patients with varicocele was higher compared to
values in patients with varicocele and infertile patients
for other causes. As a whole the values measured is
reported in Figure 1.
Subsequently the values were grouped together, for
everyone of the two categories of patients, referred to
threshold values, established equal to 15, 20, 25, 30, 35,
and 45. These values referred to conditions in a range
from normality to extreme pathological condition.
Therefore the average values of groups under each
threshold value were calculated, and the comparison is
showed in the Figure 2.
The average of these groups were evaluated for differences

matin/DNA dispersion. Likewise, when DNA fragmenta-
tion is present, the nucleoid do not exhibit a dispersion
hallow or, if present, is negligible.
The tail of the spermatozoon is visible and serves as an
important morphological parameter to distinguish the
nuclei of nemaspermic cells from others.
An aliquot of each sperm sample was diluted in phos-
phate buffer solution (PBS) to a concentration of 5 mil-
lion/ml; 25 μl of each sample was mixed and resuspend-
ed in fused agarose microgel, as provided by the kit. 20 μl
of semen-agarose mix was pipetted onto an agarose pre-
coated slide, provided in the kit, and covered with a 22-x
22-mm coverslip. The slide was placed on a cold plate in
the refrigerator (4°C) for 5 minutes to allow the agarose to
produce a microgel with the sperm cells trapped. The
coverslip was gently removed and the slide immediately
immersed horizontally in a denatured solution, previous-
ly prepared by mixing 80 μl of HCL from an Eppendorf
tube in the kit, with 10 ml of distilled water, and incu-
bated for 7 minutes at room temperature before transfer
to 10 ml of lysing solution and left to incubate for 25 min-
utes. After washing 5 minutes in a tray with
abundant distilled water, the slides were dehydrated in
increasing ethanol bath (70%-90%-100%) for 2 minutes
each and air dried at room temperature. For the latter, the
slides were horizontally covered with a mix of Wright’s
solution (Merck, Darmstadt, Germany) and phosphate
buffer solution (Merck) (1:1) for 5 to 10 minutes, with
continuous airflow. 
Then the slides were briefly washed in tap water and
allowed to dry. Strong staining is preferred to easily visu-
alize the periphery of the dispersed DNA loop halos. A
minimum of 500 spermatozoa for sample were scored
under the 100x objective of the microscope.

SCORING CRITERIA
The categorization of the different halo sizes is per-
formed using the minor diameter of the core from the
own nucleoid as a reference to which the halo width is
compared.
Five SCD patterns were estab-
lished (28):
a) Sperm cells with large

halos: those whose halo
width is similar or higher
than the smallest diameter
of the core.

b) Sperm cells with medium-
size halos: the halo size is
between those with high
and with very small halo.

c) Sperm cells with very
small-size halo: the halo
width is similar or smaller
than one third of the minor
diameter of the core.

d) Sperm cells without a halo.
e) Sperm cells without a

halo-degraded: similar to
point d), but weakly or
irregularly stained.

Figure 1.

Comparison of DNA Fragmentation Index (DFI) experimentally evaluated 
for infertile men affected from varicocele (dots) or other causes (linees).

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The study of spermatic DNA fragmentation and sperm motility in infertile subjects

such as varicocele-DFI infer-
tile-DFI. Such differences were
correlated to the DFI values of
the two categories and revealed
that such differences were
always positive and higher for
patients with varicocele (Fi -
gure 3).
Therefore, the most significant
data of our study came from
the analysis of correlation
between the DFI values calcu-
lated and progressive a+b
motility values expressed in %
and calculated in patients with
varicocele associated with the
condition of isolated astheno-
zoospermia. 
Statistical analysis of the data
found a semi-empirical corre-
lation of 0.9982 between the
index of percentage fragmen-
tation (DFI) and percentage
sperm motility according to
the:
Eq.1: DFI = 49,48*EXP (-
0,022*motility), as seen in
Figure 4.

DISCUSSION
While varicocele is one of the
most common adrological
pathologies in the general
population, it is particularly
common in infertile men (2).
That varicocele negatively
influences spermatic function
is well documented (29, 30),
although the exact underlying
mechanisms are still not
understood. In fact, infertility
may be associated to a variety
of spermatogenetic conditions
ranging from normozoosper-
mia to moderate oligoas-
thenoteratozoospermia (OAT),
to azoospermia (4, 5).
Recently, several authors have
suggested that human patients
with varicocele have a signifi-
cantly higher DNA fragmentation index (27). Studies
show that varicocele samples contain a higher proportion
of spermatozoa with abnormal DNA and immature chro-
matin than those from fertile men as well as infertile men
without varicocele (31). A cause of this phenomena may
be the increased production of ROS in varicocele patients
which is significantly higher in patients diagnosed with 2°
and 3° degree varicocele in whom altered sperm motility
is common in these patients (8, 32).
An enhancement in OS, both due to an increase in ROS
production and a decrease in the antioxidant capacity, has

been reported in men with varicocele (16, 17, 33, 34).
NO and peroxynitrite, a potent oxidant ROS, have been
demonstrated to be produced in high concentrations in
the dilated spermatic veins, so they could be main con-
tributors to the high OS level in varicocele (20, 22, 35). In
addition NO can improve sperm DNA fragmentation that
is associated with infertility in men with varicocele (36).
Besides the dilated veins, ROS may be released in the
seminiferous tubules by the cytoplasmic droplets
retained in immature spermatozoa, which seem to be fre-
quent in the sperm samples from infertile men with

Figure 2.

Comparison of DNA Fragmentation Index (DFI) experimental evaluated 
from different groups of infertile men. 

For each group, the threshold value has been selected on the basis 
of different relevance of the pathology.

Figure 3.

Detailed comparison of the relevance of the varicocele 
on the DNA Fragmentation Index (DFI) for infertile men. 

It’s evident that DFI% is higher for varicocele infertile men.

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12

varicocele (37). Immaturity is a consequence of defec-
tive spermiogenesis that could also lead to differences
in disulfide crosslinking and in susceptibility toward
DNA fragmentation (38, 39).
Because all the varicocele samples in this study showed
abnormal standard semen parameters, we compared
them with those from idiopathic infertility, either nor-
mozoospermic patients and from patients with abnormal
semen parameters, all attending the infertility clinic.
Significant differences were found between the 3 groups
in the frequency of sperm cells with fragmented DNA
using the Halosperm kit, particulary between the varico-
cele group and the infertile group, except those samples
with more intense and combined abnormalities that
could have more frequency of sperm cells with frag-
mented DNA.
Results of our study, on the higher frequency of DNA
fragmentation presented in the sperm cells of infertile
patients with varicocele compared to patients suffering
from other typologies of infertility and the fertile con-
trols, supporting the hypothesis which has been pro-
posed by other authors.
Besides, a higher proportion was evidenced in our varic-
ocele samples in relation to the fertile controls.
In addition, we also found a high inverse correlation
between low sperm motility and index of DNA fragmen-
tation in sperms of varicocele patients and patients man-
ifesting isolated asthenozoospermia.
These findings suggest that a common pathological
mechanisms underlies this pathological condition. This
mechanism may be the presence of ROS or other types of
agents which compromise the energetic metabolism of
gametes (32, 40). 
These can react negatively upon sperm membranes, prob-
ably by disrupting the equilibrium between antioxidants
and prooxidant. In addition, these can also act upon
genomic integrity and chromatin structure of the sperma-

tozoon, damaging sperm cell
DNA (36, 41).
The fact that sperms of subjects
with varicocele exhibit higher
levels of DNA damage with
respect to sperms from other
typologies of infertility, even
when normal seminal parame-
ters are attained, clearly
demonstrate the importance of
studying sperm DNA fragmen-
tation (29). This evaluation can
be included as a routine test in
the clinical management of
patients with varicocele, fol-
lowed by suggestions regarding
the potential negative effects
that the elevated presence of
damaged DNA may have on
eventual future fertility (30).
Future research is needed to
better understand the exact
mechanism by which DNA is
damaged in the spermatozoon
of varicocele patients so that

treatment to repair varicocele may be successful in the
treatment of infertility and the reduction of such damage.

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Correspondence

Giuseppina Peluso, MD (Corresponding Author)
Via G. Verdi 82/d - 87036 Rende (CS), Italy
pina.peluso@libero.it

Pietro Paolo Cozza, MD
Giancarlo Morrone, MD
U.O.S. of Andrology and Physiophatology of Reproduction
A.O. of Cosenza, Italy

Alessandro Palmieri, MD
Paolo Verze, MD
Nicola Longo, MD
Vincenzo Mirone, MD
Urological Clinic, University Federico II of Naples, Italy

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