Varicocele is defined by abnormal tortuosity and
pampiniform plexus venous dilation within the
spermatic cord and is the most common surgically
correctible cause of male infertility (
A relationship between infertility and the generation
of reactive oxygen species (ROS) has recently
been established and widely studied (
In addition, oxidative stress has been shown to
affect the integrity of the sperm’s genome by causing
high frequencies of single- and double-strand
DNA breaks, often detected in the ejaculates of infertile
In this experimental study,twenty-four mature male Wistar rats, 10 weeks old that weighed 200 ± 14 g were used. The rats were purchased from the Animal Resources Center, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran and were acclimatized in an environmentally controlled room (temperature: 20-23°C and a 12 hours light/12hours dark schedule). Food and water were available ad libitum. In this study, all experiments conducted on the animals were in accordance with Urmia University’s guidance from the Ethical Committee for Research on Laboratory Animals. Following one week acclimation, the animals were assigned to four groups (n=6) of control-sham and test groups. The test group was further divided into 3 subgroups according to the study termination (4, 6 and 8 months) after varicocele induction.
In the test groups, the left varicocele was induced
as previously reported (
Animals were euthanized on 4, 6 or 8 months following varicocele induction, by a special CO2 device. The testes were dissected out, fixed in 10% formalin fixative for histological investigations and subsequently embedded in paraffin. Sections (5-6 μm) were stained with Iron-Weigert (Pajohesh Asia, Iran) for detection of germinal cell nuclei in the testis. The sections were analyzed under light microscope at two magnifications (×400 and ×1000).
To estimate tubular differentiation index (TDI), the percentage of seminiferous tubules (STs) that contained more than three layers of differentiated germinal cells from spermatigonia type A were considered negative. Those which showed more than three and/or four layers were considered TDI positive. From each sample, 20 sections (6 μm) were prepared and analyzed.
We used the repopulation index (RI) to calculate
the ratio of active spermatogonia (spermatogonia
type B) with light nuclei to inactive spermatogonia
(spermatogonia type A) with dark nuclei as seen
by Iron-Weigert staining in STs from 20 prepared
sections per sample, as described earlier (
Epididymides were carefully separated from the testicles under x20 magnification with a stereo zoom microscope (Model TL2, Olympus Co., Tokyo, Japan). The epididymis was divided into 3 segments: head, body and tail. The epididymal tail was trimmed and minced in 5 mL Hams F10 medium. After 20 minutes, the ground epididymal tissue was separated from the released spermatozoa and counted. Smears were prepared by eosinnegrosin to evaluate dead, abnormal and morphologically immature sperm (MIS). Sperm which stained red were considered non-viable; those with cytoplasmic residuals were considered morphologically immature.
Aniline blue staining was performed in order to
evaluate sperm nuclear maturity (or protamine deficiency).
The proportion of motile spermatozoa
was determined by counting 100 cells in randomly
selected fields for each sample at 37°C (
The comet assay (Cat No. 1435723/34, Sigma Co., St. Louis, MO, USA) was used to investigate DNA fragmentation A ×40 objective device (Olympus, Germany) was linked to an epi-fluorescent microscope (Model GS7, Nikon Co., Japan) and used for visualization of DNA damage. Light green spots with typical tails were considered to be damaged DNA and spots with no tails were marked as normal DNA.
To evaluate DNA double strand breaks, an acridine
orange staining kit (Sigma Co., St. Louis, MO,
USA) was used. Samples were analyzed by epifluorescent
microscope (Model GS7, Nikon Co.,
Japan). Sperm samples for acridine orange staining
were provided as previously described (
Samples that contained spermatozoa were prepared
from the sperm suspensions as mentioned
earlier. The samples were incubated at 37°C under
5% CO2 (CO2 Incubator, LEEC, England) for 3
hours. Then, as previously described, 0.1 ml from
superficial sperm and/or 0.1 ml from sediment
sperm of suspensions in one tube were added to
150 μl of tissue culture medium (TCM) that contained
the oocytes (
Mature female rats were injected subcutaneously with 7.5 IU pregnant mare’s serum hMG (hMG, Netherlands) 48 hours prior to an i.p. injection of 100 IU human chorionic gonadotropin hCG (Teikoku Zohki Co., korea). Rats were euthanized with a special CO2 device 24 hours after hCG injection. The oviducts were removed and the ampullar portion was placed into a plastic dish that contained PBS (pH=7.2). The oocytes that were in cumulus masses were dissected out of the oviducts and introduced into tissue culture medium 199 (TCM 199, Sigma Co., USA). A drop of medium with 2 oocytes was allocated with a 10 μl sperm suspension (total: 80,000 sperm). For each animal, a total of 20 oocytes were divided into 10 drops.
To determine the effect of varicocele on oxidative
stress, total antioxidant capacity (TAOC) of
the control-sham and test groups were measured.
The assessment was performed based on the ferric
reduction antioxidant power (FRAP) assay
The total sulfhydryl level in serum was measured
according to Hu and Dillared method (
Statistical analyses were performed on all numerical data by two-way ANOVA and SPSS software version 13.00. All values were expressed as mean ± SD. P<0.05 was considered statistically significant.
Light microscopic analyses of the seminiferous
tubules (STs showed most tubules with germinal
epithelium dissociation and disruption of the cellular
junction between sertoli and spermatogenesis
cells. More than 33.50 ± 5.80% of STs in the left
testes and 21.25 ± 2.21% in the right testes .showed
dissociated epithelium after 4 months of varicocele
induction. This phenomenon increased over time; after 6 months, 41.50 ± 3.10% of STs, and at
8 months, 52.25 ± 1.70%, of STs demonstrated cellular
dissociation in the left testes. The right testes
of the test groups showed 34.25 ± 3.09% and 38.12
± 1.93% dissociation in the germinal epithelium of
tubules at 6 and 8 months after varicocele initiation,
respectively. No histological alterations were
noted in the control-sham group and the STs manifested
with normal cellular junction (
Photomicrograph of rat testis. A. Control-sham group. The seminiferous tubules with normal cellular junction (S) and interstitial connective tissue without any edema are seen (I). B. Left varicocelized testis. The dissociated germinal epithelium in seminiferous tubules (S) with edema in the interstitial connective tissue is observed (I). Iron-Weigert staining, (A × 100 and B × 400).
Histological observations demonstrated that most
STs had negative TDI, which further deteriorated with
time in the test groups. The majority of STs showed
depletion and reduction of germinal cell layers after 8
months. Right testes of the test animals showed relatively
moderate destruction in STs when compared to
the left testes. No histological abnormality was observed
in the control-sham group (
Special nucleus staining (Iron-Weigert) was performed
in order to differentiate inactive cells (spermatogonia
type A) from active cells (spermatogonia type
B). The results showed that after varicocele induction
the RI was negative in most test group STs and the
number of STs with negative RI increased over time
in both the right and left testes. Although pathologic
RI was present in both testes, the right testes of the
test animals showed lower negative RI in comparison
to the left (
Photomicrograph of rat testis. A. Control-sham group. Note the seminiferous tubules with normal cellular junction (S) and the interstitial connective tissue with no edema (I). B. Right testes of varicocelized rats. Note the seminiferous tubules with negative tubular differentiation index (S). The spermatogenesis regions (arrowheads) are indicative of early maturation from the previous cycle. C. Left testis of varicocelized group. The seminiferous tubules (S) are completely depleted with no detectable spermatic maturation. Vascular thrombosis (T) is also observed in interstitial connective tissue. Iron-Weigert staining, (A × 100; B × 400 and C × 400).
Effect of varicocele on mean average of tubular differentiation index and maturation arrest percentage in the right and left testes of test and control-sham groups
|Tubular differentiation index (TDI, %)|
|Groups||Left testicle||Right testicle|
|2.25 ± 1.00||2.75 ± 0.95|
|23.25 ± 3.59*b'||14.5 ± 3.31*a'|
|33.5 ± 3.41*b||23.25 ± 3.59*b'|
|37.87 ± 2.65*c||32.50 ± 3.10*c'|
|0.7±0.44||0.75 ± 0.28|
|33.6±3.57*a||25.50 ± 1.91*a'|
|39.2±1.92*||38.5 ± 1.29*|
|44.6±3.20*b||40.25 ± 1.70*b'|
Stars indicate significant differences (p≤0.05) between the control-sham and test groups in the same column. Letters in the same rows represent significant differences (p≤0.05) between the left and right testes in same months. All data are presented as mean ± SD.
Hemocytometric evaluations for sperm count
showed that after varicocele induction the sperm
number reduced significantly (p<0.05); this reduction
increased with time. Eight month varicoceleinduced
cases manifested severely decreased sperm
count; one of the six rats in this group was azoospermic.
The right testes of varicocele-induced animals
showed relatively higher sperm counts in all test
groups when compared to the left testes. Aniline
blue staining for sperm nucleus maturity revealed
that the ratio of immature sperm with light stained
nuclei increased remarkably in varicocele-induced
Light microscopic analyses for sperm motility showed that the percentage motility decreased over time in varicocele-induced rats. Sperm delivered from the right testes of varicocelized animals exhibited relatively higher sperm motility in comparison to the left testes.
According to the results from eosin-negrosin staining, there was lower sperm viability in varicocele- induced rats, with the highest sperm mortality seen in the 8 month varicocelized animals. The percentage of viable sperm was also higher in the right varicocele group when compared with the left varicocele group. Data for sperm count and morphology are presented in table 2.
Effect of varicocele on: mean average for sperm count (S/C), sperm normality (S/M), sperm viability (S/V), morphological immature sperm (MIS) and nuclear immature sperm (NIS) in the different test groups and control-sham group
|Parameters||Control-sham||4 months||6 months||8 months|
|72.50 ± 3.78||42.50 ± 2.08*a||38.00 ± 2.16*b||31.75 ± 2.36*c|
|92.25 ± 1.70||91.00 ± 1.41*a||39.75 ± 1.70*b||33.75 ± 2.62*c|
|92.33 ± 2.08||57.34 ± 5.03*a||44.66 ± 4.16*b||32.34 ± 2.51*c|
|16.20 ± 4.43||50.80 ± 1.64*a||59.20 ± 1.66*b||70.20 ± 1.78*c|
|9.25 ± 0.95||48.75 ± 2.21*a||57.00 ± 1.82*b||62.25 ± 1.70*c|
|73.75 ± 3.30||54.50 ± 3.41*a'||43.75 ± 2.06*b'||37.25 ± 1.70*c'|
|91.25 ± 1.50||56.75 ± 1.70*a'||44.00 ± 3.65*b'||36.00 ± 4.08*c'|
|92.60 ± 3.05||70.66 ± 3.05*a'||61.34 ± 3.05*b'||44.67 ± 4.16*c'|
|15.80 ± 5.63||48.60 ± 1.67*a'||55.60 ± 2.60*b'||62.80 ± 2.77*c'|
|9.26 ± 1.70||42.50 ± 2.51*a'||46.25 ± 2.36*b'||53.72 ± 3.30*c'|
Stars indicate significant differences (p<0.05) between the control-sham and test groups in the same row.
Different letters in the same columns are significant differences (p<0. 05) between the right and left testes in the same month. All data are presented as mean ± SD.
Effect of varicocele on repopulation index in: (A) Left testis and (B) right testis. Repopulation index percentage of spermatogonia type A reduced over time in all varicoceleinduced rats and the spermatogonia type B increased by the time. Ø and stars indicate significant differences (p ≤ 0.05) between data for spermatogonia types A and B, respectively. All data are presented as mean ± SD.
Effect of varicocele on mean average for DNA fragmentation and DNA double-strand breaks in the test groups and control-sham group
|DNA fragmentation (comet assay,%)|
|Groups||Left testes||Right testes|
Stars indicate significant differences (p < 0.05) between the control-sham and test groups in the same column. Letters in the same rows indicate significant differences (p ≤ 0.05) between the left and right testes in the same months. All data are presented as mean ± SD.
Light microscopic architecture from sperm; A1; Abnormal sperm with dense blue stained mature nucleus, A2; Normal sperm with unstained cytoplasm in head section, B1; Normal sperm with light stained immature nucleus, and B2; Abnormal sperm; note the sperm in the left side of the figure with cytoplasmic droplet (arrows) and the dead sperm with eosin-stained cytoplasm (below, right hand side). Aniline-blue (A-1, B-1) and eosin-negrosin (A-2, B-2) stainings, (× 400).
Results from the comet assay showed an elevated
ratio of sperm DNA fragmentation in the varicoceleinduced
animals that increased over time. Sperm from
the left varicocelized testes had higher DNA fragmentation
when compared with the right testes in the same
animals and the control-sham group (
Epi-fluorescent architecture of rat sperms by Comet assay. A. Sperms from control group; the green spots without any tails are normal sperm. B. Sperms from right testes; The green spots without tails (arrowhead) and spots with tails (arrow) indicate the DNA fragmentation. In panel B both normal and fragmented DNA are seen in sperms collected from the right testes of varicocelized rats, C. Sperms collected from the left testes of varicocelized rats with intensive DNA fragmentation; (Comet assay technique, × 100).
Acridine-orange staining showed that the number
of sperm with double-strand DNA breaks was significantly
(p<0.05) higher in the varicocelized
groups with the highest rate of DNA breaks seen
in rats with 8 months varicocelized testes. The percentage
of DNA breaks was significantly (p<0.05)
lower in sperm from the right testes of varicocelized
animals when compared to samples from the left
Effect of varicocele on double strand DNA in rat sperm. A. Epi-fluorescent architecture of the control-sham group. The light green stained nuclei are indicative of the normal double-strand DNA in this group. B. Light microscopic architecture of sperm from right testes of varicoceleinduced rats. The normal sperm with light green stained nucleolus (arrows) and sperm with damaged DNA with light yellow stained nucleolus (arrowheads) are presented. C. Light microscopic architecture of sperm from left testis of varicocele-induced rat, the sperm with light yellow stained nucleolus represent remarkable DNA damage (Acridineorange staining, × 400).
Effect of varicocele on means average of 4- and 8-cell embryos, moru- la and blastocysts after in vitro insemination of oocytes with superficial and sediment sperm contents of the right and left testes from the test and control- sham groups
|Groups||4-cell embryo||Left testes8-cell embryo||Morula||Blastocyst|
|27.80 ± 2.94||24.20 ± 0.83||22.06 ± 1.67||20.50 ± 1.04|
|13.66 ± 2.25*a||12.20 ± 1.64*b||11.40 ± 0.84*c||11.20 ± 0.83*d|
|5.50 ± 1.16*||5.80 ± 1.30*||5.60 ± 1.07*c||5.40 ± 2.50*d|
|4.83 ± 0.75*a||3.40 ± 1.34*b||3.00 ± 0.70*c||2.80 ± 0.83*d|
|27.16 ± 1.47||24.40 ± 1.14||22.60 ± 1.14||19.83 ± 0.75|
|11.80 ± 1.48*a'||10.60 ± 1.14*b'||9.20 ± 0.83*c'||9.00 ± 1.41*d'|
|5.00 ± 1.41*||4.20 ± 1.09*||4.00 ± 0.70*c'||3.80 ± 0.44*d'|
|1.40 ± 0.89*a'||1.30 ± 0.83*b'||1.20 ± 1.09*c'||0.60 ± 0.54*d'|
|4-cell embryo||8-cell embryo||Morula||Blastocyst|
|27.60 ± 1.81||24.80 ± 1.48||20.80 ± 0.83||19.80 ± 1.14|
|14.00 ± 1.00#e||12.37 ± 1.52#f||12.00 ± 1.00#g||11.57 ± 0.95#h|
|8.33 ± 1.15#e||8.00 ± 1.00#f||7.66 ± 0.57#g||7.00 ± 0.81#h|
|6.34 ± 0.57#e||5.25 ± 0.50#f||4.60 ± 0.57#g||4.25 ± 0.95#h|
|27.33±1.03||24.83 ± 1.47||21.50 ± 1.04||19.84 ± 1.47|
|13.25±1.25#e'||12.10 ± 0.84#f'||10.62 ± 0.47#g'||9.98± 0.14#h'|
|7.50±1.29#e'||6.75 ± 0.95#f'||5.70 ± 0.94#g'||5.00 ± 0.08#h'|
|4.50±1.29#e'||9.25 ± 0.95#f'||2.20 ± 0.54#g'||2.25 ± 0.51#h'|
Stars and # indicate significant differences (p < 0.05) between data in the same column with control-sham rats; different letters indicate significant differences (p < 0.05) between data of superficial sperm and sediment sperm contents. All data are presented as mean ± SD. A total of 20 oocytes were used for each sperm sample and the percentages in the table were calculated from 20 oocytes.
The results for
Results indicated that both TAOC and TTM
significantly (p<0.05) decreased after varicocele
induction for 4, 6 and 8 months compared to the
control-sham group. Accordingly, animals in the 8
month varicocele-induced group showed remarkable
diminished antioxidant capacity of serum
Effect of varicocele on IVF and 2-cell embryo after invitro fertilization in the control and varicocele-induced rats: A. Left testicle data, B. right testicle data. Stars indicate significant differences (p ≤ 0.05) between all data for different months following varicocele induction with each test group and the control group. All data are presented as mean ± SD.
Effect of varicocele on the serum level of TAOC (A) and TTM (B); Stars indicate significant differences (p ≤ 0.05) between control and test groups. All data are presented as mean ± SD.
In the present study we showed that in varicoceleinduced rats there were early MI, negative TDI and RI due to disrupted spermatogenesis in both the left and right testes. The negative impact of varicocele increased over time. Our further analyses illustrated that following varicocele-induction, in addition to a reduction in sperm motility, sperm protamine-histone transition, DNA and plasma membrane integrity were impaired with a subsequent loss of motility that increased over time. Ultimately, our results showed that the quality of sperm content reduced with time, which in turn reduced the IVF rate.
Varicocele is a disorder with an incidence of 10%
to 20% in the general population (
According to our findings, varicocele does not
exert its pathologic effect immediately, but rather it
takes time to see the various pathological features
associated with varicocele. In many pathological
conditions, apoptosis of germinal cells in STs is the
main cause of germ cell loss and this impairment
can also influence sertoli cells (
In damaged spermatogenesis, the cytoplasmic
extrusion mechanisms do not function normally
In the current study, samples obtained from 4
month varicocelized rats contained MIS with cytoplasmic
droplets (residual cytoplasm). The rate
of immature sperm occurrence was found to correlate
with imbalance in oxidative defense of the
8 month varicocelized rats. Therefore, the rate of
immature sperm was higher in the samples with
lowest TTM and TAOP. Evidence has shown that
any disruption in the physiological function of
the cytosolic enzyme glucose-6-phosphate-dehydrogenase,
the enzyme that mediates some physiological
bioactivities involved in sperm maturity,
can influence sperm maturity processes in varicocele
A number of studies have suggested that the presence
of spermatozoa with damaged DNA may be
the result of impaired chromatin packing or indicative
of apoptosis (
Reports have indicated that disorders which have
resulted in failure of epididymal sperm maturation
also impair sperm fertilizing ability (
According to our results the unsaturated fatty acids in plasma membrane of sperms were severely damaged after varicocele induction. These unsaturated fatty acids are essential for fluidity for the plasma membrane in order to participate in membrane fusion events associated with fertilization. When the associated double bonds with unsaturated fatty acids deform, the membrane fluidity decreases and it leads to a consequent loss of sperm function. Of note, the lowest results for IVF were manifested after 8 months.
The ability of the embryo to survive appears to
be negatively correlated with the level of DNA
fragmentation in the germ line (
Our data suggest that following varicocele induction severe damage occurs in the process of spermatogenesis which influence produced spermatozoa quality, maturation (testicular and epididimal) and DNA integrity. On the other hand, increasing impaired and abnormal sperm leads to remarkable ROS stress that can result in sperm plasma membrane disintegrity. Ultimately these impairments affect sperm capacitation. It may be concluded that for IVF purposes either sperm samples from varicocele patients should not be used or sperm samples that are collected early could be used after testing for DNA integrity.