Varicocele is the abnormal dilatation of the veins
in the pampiniform plexus. While its incidence is
reported to be 4.4-22.6% in the general population,
an incidence of 21-41% in infertile patients and
75-81% in secondary infertile patients has been reported
Several theories have been suggested to explain
the relationship between varicocele and infertility.
The theories accepted include: the increase of arterial
blood flow and testicular temperature caused
by varicocele (
In the last phase of spermatogenesis, spermatid
nuclear remodeling and condensation is associated with the displacement of nuclear histones by transition
proteins and then by protamines. Protamines
provide sperm head condensation and DNA stabilization
and allow for denser packaging of DNA
in spermatozoon than histones (
While the incidence of sperm with DNA damage is
approximately 10% in fertile males, the corresponding
rate is about 20-30% in infertile males (
In this experimental study, after obtaining approval
from the Ethics Committee, a total of 30
adult male Wistar albino rats were randomized into
three groups. In group 1, 10 rats underwent a sham
operation and were classified as the control group.
Experimental varicoceles were created in both the
10 rats in group 2 and the 10 rats in group 3 (a total
of 20 rats), using the method described by Saypol
et al. (
After four weeks the rats in group 2 were sacrificed, while the rats in group 3 underwent a varicocelectomy and were sacrificed four weeks later to observe the effects of the varicocelectomy. The total number of rats was maintained by replacing the rats that did not develop a varicocele or those that died before the sacrifice with new ones.
All rats were fed using standard 8 mm pellet feed and maintained in a constant environment with a 12: 12 hour light: dark cycle. All animals were given ad libitum water and feed. Room temperature was 22 ± 2˚C and humidity rate was 50 ± 10%.
The surgical procedure was performed according to rules of antisepsis. The body temperature of the animals was monitored with a rectal thermometer; we attempted to maintain the temperature be kept at 37˚C.
The rats were weighed. For general anesthesia, ketamine (10 mg/100 g) and chlorpromazine (1 mg) was administered intraperitoneally.
In the rats that underwent the experimental varicocele, an 0.8 mm-thick metal wire probe was placed along the renal vein. A 4-0 silk suture was ligated around the renal vein and metal wire probe. When the metal wire probe was removed, a nearly 50% narrowing of the renal vein was observed. After tying the ligature the probe was removed, which produced an approximately 50% decrease in renal vein diameter. In the sham group, the rats underwent a similar procedure, but we did not ligate the renal vein.
After four weeks we checked for the occurrence of varicocele. At the end of four weeks, dilated left spermatic veins were seen in 18 of 20 rats that underwent laparotomy. In 10 rats, dilated spermatic vein was ligated using 4/0 silk and cut (varicocelectomy). Tunica albuginea was opened at the fourth week in groups 1 and 2. In the eighth week in group 3, tunica albuginea was opened, testicular tissue was obtained, and the rats were sacrificed by cervical dislocation. The testicular tissue samples obtained were put in hydroxyethyl piperazineethanesulfonic acid (HEPES) buffered medium (G-IVF, Vitrolife, Sweden).
Sperm DNA fragmentation was assessed with a Halomax® kit (Halotech DNA, Spain). An aliquot of testicular tissue was diluted to 15-20×106/ml in phosphate-buffered saline (PBS). Agarose (in eppendorf tubes provided with the kit) was placed in a water bath at 95˚C -100˚C for 5 minutes to fuse the agarose, and then in a water bath at 37˚C until the temperature equilibrated. After 5 minutes of incubation at 37˚C, 25 μL of the diluted tissue sample was added to a 50 μl agarose-filled eppendorf tube at 37˚C and mixed with the fused agarose. 1.5-2 μL of tissue-agarose mixture was pipetted onto slides precoated with agarose (provided in the kit) and then covered with a 24 × 24 mm coverslip. The slides were placed on a cold plate in the refrigerator at 4˚C for 5 minutes to allow the agarose to produce a microgel that had the sperm cells embedded. The coverslips were removed and slides were immersed horizontally in a lysis solution (LS) prepared by mixing 7 μL of reducing agent (RA) with 1 mL of base lysis solution (BLS) provided in the kit and incubated for 5 minutes. After washing for 5 minutes with distilled water, the slides were dehydrated in increasing concentrations of ethanol (70-100%) for 2 minutes and then left to dry. Slides were stained with a mixture of Wright’s staining solution (Merck, Germany) and PBS (Merck, Germany) (1:1) for 5-10 minutes, washed with water, and then dried. A total of 100 spermatozoa per sample were analyzed by brightfield microscopy (×100 objective). Sperm with large, spotty halos of chromatin dispersion were classified as ’spermatozoa having DNA fragmentation' and those with small, compact halos of chromatin dispersion were classified as ’spermatozoa having no fragmentation'. DNA Fragmentation Index (DFI) was calculated and the groups were compared for DFI.
All data were analyzed by SPSS software (Version 13.0, SPSS Inc., Chicago, IL, USA). Statistical analysis was performed using the Mann-Whitney U test. P<0.05 was considered statistically significant.
After staining the slides with Wright’s solution
some of the sperm cells presented a halo structure,
which indicated the presence of fragmented DNA;
some had no halo (DNA intact; Figs
Sperm cells with DNA fragmentation seen with the halo structure surrounding the head.
Sperm cells with no DNA fragmentation seen without the halo structure surrounding the head.
Median sperm DFI was 17.6 (range: 7.6) in the right testicle, 18.3 (range: 6.8) in the left testicle, and 18.3 (range: 7.9) in both testicles of the control group. No statistically significant difference was found between the right and left testicles in the control group (p>0.05).
Median sperm DFI was 30.7 (range: 8.8) in the right testicle, 31.8 (range: 9.6) in the left testicle, and 31.8 (range: 10.1) in both testicles of the varicocele group. No statistically significant difference was found between the right and left testicles in the varicocele group (p>0.05).
Median sperm DFI was 27.1 (range: 8.1) in the right testicle, 28.6 (range: 8.9) in the left testicle, and 27.1 (range: 9.0) in both testicles of the varicocelectomy group. No statistically significant difference was found between the right and left testicles in the varicocelectomy group (p>0.05).
A statistically significant difference was found
between the three groups in terms of sperm DFI in
both the right and left testicles (
Because there was no significant difference between right and left testicles in the three groups, the differences between the control and varicocele groups (p=0.028), control and varicocelectomy groups (p=0.036), and varicocele and varicocelectomy groups (p=0.048) were statistically significant when the right and left testicles were evaluated together.
Sperm DNA fragmentation rates
|Control group (n=10)||Varicocele group (n=10)||Varicocelectomy group (n=10)||P value|
* P values of right (R), left (L), and both (B) testes. Control group-varicocele group: p=0.026 (R), p=0.028 (L), p=0.028 (B); Con-trol group-varicocelectomy group: p=0.032 (R), p=0.032 (L), p=0.036 (B); Varicocele group-varicocelectomy group: p=0.046 (R), p=0.046 (L), p=0.048 (B).
The relationship between varicocele and infertility
has not been fully elucidated. All patients with varicocele
are not infertile and all patients who undergo
varicocelectomy do not become fertile. Although it
has been known for many years that varicocele may
impair spermatogenesis (
This study aimed to examine the effects of varicocele and varicocelectomy on sperm DNA damage. While no statistically significant difference was found between the right and left testicles in all three groups, a statistically significant difference was found in the three groups in terms of sperm DFI.
Marmar have suggested that a varicocele could be
a secondary lesion that accompanies an underlying
genetic disorder, which impairs spermatogenesis and
contributes to infertility (
In the experimental varicocele model created by
Barqawi et al. they evaluated apoptosis at the 7th,
14th, and 28th day of the study in the testicles of rats
and found maximum apoptosis on the 28th day (
One of the interesting features of the varicocele is
that, even in a unilateral varicocele, both testicles develop
The integrity of sperm DNA is important for male
fertility potential. Evenson et al. have reported that
the probability of spontaneous pregnancy was lower
in couples in whom the sperm DNA damage rate was
above 30% (
The sperm chromatin dispersion (SCD) test is a
reliable method used to evaluate mammalian sperm
DNA fragmentation (
Diagnosis and follow up of infertile patients is generally
performed using semen analysis. Although semen
analysis provides valuable information, it has
some limitations. Semen parameters show both biological,
intra-, and inter-observer variations (
Although sperm DNA damage has been known for
many years, the acceptable rate of sperm with DNA
damage in humans is controversial. Evenson et al.
have determined that the likelihood of spontaneous
pregnancy was lower in couples with a DFI>30%,
reasonable in couples with a DFI between 15-30%,
and higher in those with a DFI<15% (
It has been suggested that sperm DNA damage
could be a late outcome of increased ROS levels, and
therefore its resolution could take time (
The limitations of this study include the small number of rats used due to ethical reasons, observations of sperm DNA damage examined after different time periods following varicocelectomy, and the lack of an evaluation on the effects of sperm DNA damage on pregnancy rates.
The results of this study demonstrate that varicocele causes increased sperm DNA damage and varicocelectomy decreases this damage. Thus, it should be considered that sperm DNA damage which increased by varicocele may be decreased by varicocelectomy.