1Department of Urology, Ota-Jinemed Hospital, Turkey
2Department of Histology and Embriyology, Biruni University, Turkey
3Department of Obstetrics and Gynecology, Balikesir Univesity, Turkey
4Department of Medical Biology and Genetics, Yeni Yuzyil University, Turkey
5Department of Obstetrics and Gynecology, Yeni Yuzyil University, Turkey
Submission: December 31, 2018; Published: February 08, 2019
*Corresponding author: Erkan Erdem, Department of Urology, Ota-Jinemed Hospital, Turkey
How to cite this article: Erkan E, Nazli E G O, Akin U, Cenk K, Meric K. Metformin Reduces the Extent of
Varicocele-Induced Damage in Testicular Tissue. Glob J
Reprod Med. 2019; 6(4): 555691. DOI:10.19080/GJORM.2019.06.555691.
Varicocele is a medical condition where retrograde flow of blood leads to increased hydrostatic pressure in testicular veins and the prevalence of varicocele is predicted to vary between 30 to 40% in infertile men. Metformin, major therapeutic agent in the treatment of type 2 diabetes mellitus, was shown to reduce oxidative stress and apoptosis in renal tubular epithelial cells and testis of animal models. However potential protective effects of metformin against varicocele-induced testicular damage has not been studied. We investigated the impact of metformin on spermatogenesis assessed with Johnsen score, seminiferous tubule integrity and apoptotic activity assessed with the expression of cleaved caspase 3 activity. A total of 36 male Wistar rats (6-week-old) were divided into six groups (n=6 for each group); (C) control group, S (sham group), V (varicocele-only group), V+M (varicocele + metformin group), V/E (varicocele + varicocelectomy group), V/E+M (varicocele + varicocelectomy + metformin group).
Metformin administration improved spermatogenesis, seminiferous tubule integrity and reduced apoptotic activity as manifested by the decreased expression of cleaved caspase 3 in rats with varicocele. However, metformin did not exhibit any additional benefit on these parameters in varicocelectomies rats. As Results, metformin treatment reduced the extent of damage to spermatogenesis in rats with varicocele, although no additional benefit was detected when administered following varicocele surgery.
Varicocele is an abnormal vascular dilatation of pampiniform plexus, commonly developing at puberty. Although underlying mechanisms remain poorly understood genetic background, anatomical aberrations, incompetence of venous valves, difference between the drainage of left and right testicular veins were suggested in the etiology . As left spermatic vein being longer than the right vein, it is more commonly incurred to increased hydrostatic pressure and dilatation. Compression of the left renal vein between the aorta and the superior mesenteric artery may also contribute to the disturbed intravenous pressure .
The prevalence of varicocele varies between 15-20 % in general population and 30-40% in infertile men, and 11-19% of adolescents [3-6]. It was reported that varicocele is a progressive disease and early diagnosis and treatment in youth may enhance fertility potential . Several contributing factors in the pathophysiology of varicocele have been proposed such as higher temperature of testis, the disorder of neuroendocrine
system, autoimmunity, accumulation of renal and adrenal metabolites, genetic and epigenetic factors, hypoxia and oxidative stress [8-10].
Varicocele represents a chronic process within the testicle, which is linked to increased reactive oxygen species (ROS) beyond physiologic limits and, subsequently, disrupting sperm membrane fluidity, causing DNA damage and necrosis . Moreover, superoxide dismutase 1, glutathione S-transferase M1 and T1 which are counteracting free superoxide radicals in cells have been reported to be decreased in men with varicocele, that may be important on disturbed sperm parameters . Apoptosis of germ cells was also demonstrated in the pathogenesis of varicocele-related infertility . Clinical findings suggest that surgical repair of varicocele may decrease seminal oxidative stress levels and sperm DNA fragmentation and, thus, may improve sperm quality . Therefore, surgical intervention seems to be a reliable option in the treatment of varicocele-related male infertility, although some controversial reports exist.
Additionally, anti-oxidant medications such as kallikrein, L-carnitine with L-acetyl carnitine, pentoxifylline, coenzyme Q10 have been used to improve the milieu in the testis in men with varicocele . Metformin is a major therapeutic agent in the treatment of type 2 diabetes mellitus as an insulin sensitizer, which decreases hepatic glucose output and increases peripheral
glucose uptake. Although its action was not fully elucidated,
metformin attenuated intracellular reactive oxygen species and
apoptosis in aortic endothelial cells, myocardium, renal tubular
cells and testicular cells [16-20].
Potential effects of metformin on varicocele-induced
testicular damage have not been studied in neither humans nor in
animal models. Thus, we investigated the impact of metformin on
spermatogenesis, testicular integrity, and apoptotic activity in the
testis of adolescent rats with experimentally-induced varicocele.
Thirty-six male adolescent Wistar rats (6-week-old) were
randomly and equally divided into six experimental groups.
Surgical procedures were carried out under anesthesia
with intraperitoneal injection of ketamine (50 mg/kg). The
experimental groups were as follows:
• (C) Control group; no surgical procedure was performed,
and testis was examined after removal.
• (S) Sham group, a midline incision was performed, and
testis was examined 8 weeks later.
• (V) Varicocele - only group: Experimental varicocele was
induced by partial ligation of left renal vein with
Silk suture at the area medial to the insertion of the adrenal
and spermatic vein into renal vein as described previously
• (V+M) Varicocele + metformin group: All rats were
treated with metformin (300 mg/kg per day by oral gavages)
for 8 weeks following induced varicocele.
• (V/E) Varicocele + varicocelectomy group:
Varicocelectomy was performed 4 weeks and the examination
of the testis 8 weeks after the induction of varicocele. No
medication was used.
• (V/E+M) Varicocele + varicocelectomy + metformin
group: Varicocelectomy was performed 4 weeks after the
induced varicocele. Metformin treatment (300 mg/kg per day
by oral gavages) was initiated after the induction of varicocele
and continued for 8 weeks. Left testes were examined 8 weeks
after the induction of varicocele in all varicocele - induced
groups. As maximum apoptotic activity initiates approximately
28 days after the induction of varicocele the procedure of
varicocelectomy was performed 4 weeks after the formation
of varicocele .
The testicular tissue was fixed in Bouin’s solution (75% picric
acid, 5% glacial acetic acid, and 25% formaldehyde) and embedded
in paraffin blocks. Sections (5 μm) were formed, deparaffinized,
and stained with hematoxylin and eosin. Spermatogenesis was
examined in each group using Johnsen’s score (a score of 1-10
was assigned to each tubule regarding epithelial maturation) as
described previously . Sections were examined in a random
order under a standard light microscope with 10x and 40x
magnification by a blinded histologist; unaware of which group
each rat belonged to. Histological grading was done by examining
approximately 80 randomly selected seminiferous tubules per
rat. Thus, a total of approximately 480 seminiferous tubules were
scored for each group.
A total of 103 randomly selected seminiferous tubules stained
with hematoxylin-eosin were analyzed in each group. The presence
of round spermatid stage (RSS) and primary spermatocyte stages
(PSS) were assessed as described previously and compared among
the groups .
Cleaved caspase-3 was used for immunohistochemical
staining. Testicular tissue samples were immediately fixed in 10%
neutral-buffered formalin, embedded in paraffin, and sectioned (5
μm). Sections were deparaffinized and blocked for endogenous
peroxidase activity with methanol containing 3% H2O2 for
10 m. Ultra V Block (Lab vision, Freemont, CA) for 7 m at room
temperature. Cleaved Caspase-3 (#9664, Cell Signaling, U.S.) was
applied at a dilution of 1: 500 and incubated overnight at +4 °C in
a humidified chamber for nonspecific binding. The sections were
washed in phosphate-buffered saline (PBS) and incubated with
biotinylated horse anti-rabbit IgG (3 mg/mL; Vector, Burlingame,
CA) at a 1: 500 dilution for 1 h at room temperature.
Antibodies were detected using a VECTASTAIN avidinbiotin
complex (Vector PK 4000) for 30 m at room temperature.
Antibody complexes were visualized after incubation with
3,3’-diaminobenzidine tetrahydrochloride (DAB, Bio-Genex,
San Ramon, CA.) and were mounted under glass coverslips in
Entellan (Merck) and then evaluated under a light microscope.
Immunohistochemical staining for cleaved caspase-3 was
analyzed by counting 100 seminiferous tubule cross-sections
in each group and expressed as the apoptotic index. In each
photomicrograph, the following parameter was measured with
ImageJ software: expression levels of cleaved-caspase-3 in both
groups at round spermatid stage (RSS) of testes. Each of this
parameter was measured 3 times for each image and the average
of the 9 measurements of each sample was used for the statistical
analysis. Histopathological features examined in rats with normal
testis and with sham, varicocele, varicocele+ metformin in a
subjective scoring (0 - not present; 1 - low grade; 2 - moderate
grade; 3 - high grade; 4 - very high grade).
Histopathological findings (Johnsen’s score) were assessed by
nonparametric Kruskal-Wallis test, and the mean Johnsen’s score
was used in the comparison of the groups. Multiple comparisons were made using Tukey’s procedure. p<0.05 was considered
statistically significant. Analysis of variance was used for statistical
analysis of the apoptotic index among the groups.
Johnsen’s score was significantly lower in V group (4.14±1.25)
compared to C group (9.1±0.3) or S group (9.0 ± 0.2) groups
(p<0.05). V+M group had significantly higher score (6.9±0.6) than
V group (p<0.05). V/E group and V/E+M group had similar Johnsen
scores (8.9 ± 1.02 and 9.2 ± 0.6). These findings suggest that the
administration of metformin resulted in 40.6% of improvement in
spermatogenesis in rats with varicocele. However, this favorable
effect was not observed when metformin was used along with
Histological and morphological changes in the testes of
rats were compared via hematoxylin and eosin staining and
degenerated tubules (DT) were only detected in V and V+ M
groups, not in C, S, V/E and V/E+M groups (Figure 1). Visual
assessment of the disorganized seminiferous tubules further
supported these findings as seen in Figure 2. Seminiferous
tubule degeneration scores were used for quantification of data
(Figure 2b). V group had significantly higher scores of RSS and
PSS compared to C and S group (2.6±0.8 and 3.7±0.4; 0.2±0.4 and
0.2±0.4; 0.9±0.6 and 0.6±0.7, respectively) (p<0.05). V/E group
had significantly lower RSS (0.7±0.8) and PSS (0.8±0.7) scores
than V group (p<0.05). V+M group had significantly lower RSS
and PSS scores (1.8±0.7 and 2.6±0.7, p<0.05) in comparison to
V group, implicating beneficial effects of metformin in rats with
varicocele. When compared to V/E group, V/E+M group did not
exhibit any difference in RSS (0.6±0.6) and PSS (1.4±0.5) scores,
suggesting the absence of additive positive effect of metformin in
Apoptotic activity was assessed by using cleaved caspase
3 expressions levels, staining of cleved caspase 3 positive
seminiferous tubules were shown in Figure 3a. Cleaved caspase
3 expressions were significantly higher in V group (3.5 ± 0.5)
compared to C (0) and S (0.2 ± 0.4) groups. V+M group had
significantly lower cleaved caspase 3 level (3.0 ± 0.7) than V group.
V/E group had lower cleaved caspase-3 expression levels (1.0 ±
0.7) compared to V group. Treatment of varicoceleectomy rats
with metformin (V/E+M) did not further reduce apoptotic activity
in the seminiferous tubules (1.75 ± 0.43) when compared to the
varicocelectomy group (V/E) (Figure 3b).
The present study demonstrates that metformin can
reduce the extent of testicular damage in rats with varicocele,
although having no effect in rats following varicocelectomy Spermatogenesis, seminiferous tubule integrity and the degree
of apoptosis were improved using metformin in the presence of
varicocele although it was not as remarkable as what was obtained
through varicocelectomy. A review of the literature revealed that
the impact of metformin on varicocele was not investigated in
humans or animal models until now.
Although it is a commonly identified abnormality not all men
with varicocele present with infertility. Some intrinsic factors
may render some men to become susceptible to varicocele, thus,
the best candidates who benefit from varicocelectomy yet to be
clarified. Since oxidative stress was shown to be important in
the pathophysiology of varicocele some agents have been used
to improve the milieu in the testis . A number of anti-oxidant
medications have been studied to relieve detrimental effects of
varicocele in the testis . These agents have been used either
alone or as an adjuvant therapy with surgery. However, surgery
remains the treatment of choice and there exists insufficient data to
recommend medical therapy in men with varicocele. Barekat et al.
 reported that administration of an antioxidant agent N-acetyl
cysytein as an adjunct therapy improved semen quality following
varicocelectomy . Tek et al.  demonstrated that vascular
endothelial growth factor decreased apoptosis in varicoceleinduced
rats as evidenced by diminished caspase-3 positive
cells . Both studies showed the benefit of these as adjunct
therapy following varicocelectomy. However, in the present study
metformin did not enhance the effect of varicocelectomy.
Minutoli et al.  demonstrated that neuronal apoptosis
inhibin factor and surviving expressions were significantly reduced
following varicocele induction and polydeoxyribonucleotide,
an agonist of adenosine A2A receptor, administration restored
testicular function . Several other studies detected increased
germ cell apoptosis in rats with varicocele [21,22,27]. However,
in another study, apoptosis was found to be decreased in germ
cells in the testes of infertile men with varicocele as compared
with normal men . It was speculated that the fixation of testis
in formaldehyde might have played a role in the different result.
In the present study, cleaved caspase 3 expression was used to assess apoptotic activity and it was found that metformin reduced
apoptotic activity in rats with varicocele, whereas no additional
effect was observed when metformin was administered after
Metformin is commonly used in type 2 diabetes mellitus and
polycystic ovarian disease as an insulin sensitizer . Also, it is
present in various tissues including myocardium, liver, pancreas,
thyroid, adipose tissue, hypothalamus, pituitary, and male and
female gonads [19,30,31]. It has been reported that metformin
is mainly transported into cells by organic cation transporters
as passive diffusion is limited . Although the mechanism of
action is not yet fully elucidated recent studies suggested that
metformin acts through AMP-activated protein kinase (AMPK)
pathway, inhibits the activity of the respiratory electron transport
chain in mitochondria, induces epigenetic modifications which in
part may explain long term effects and decreases oxidative stress
and apoptotic activity [16,19, 33-35].
Male reproductive system utilizes all these metabolic
pathways and is prone to be affected by metformin administration
[20,36,37]. Metformin was found to stimulate lactate production
which is important in the development of germ cells and show an
anti-apoptotic effect in rat Sertoli cells . It was also reported
that metformin reduced the apoptotic cells and caspase-3 level
in rat testis . The findings of the present study are consistent
with previous studies that metformin reduced apoptosis in testis
with varicocele. Yan et al.  reported that metformin improved
the semen parameters related to its effects on weight loss,
increased testicular weight and reduced testicular cell apoptosis
. On the other hand, Tartarin et al.  reported metformin at
concentration 10 times higher than therapeutic levels decreased
testosterone secretion and the number of Sertoli cells in rats
when it was administered during pregnancy . Faure et al.
 reduction in testicular weight and testosterone level were
observed in 6-week-old chickens treated with metformin for 3
Several groups demonstrated that post-operative
administration of metformin can exert protective effects in
male reproductive function in rat models . Bosman et al.  demonstrated that infertile hyperinsulinaemic men could
benefit from metformin treatment in combination with an
enriched antioxidant diet . Besides, metformin was reported
to act as a protective compound when used in the media for
cryopreservation of spermatozoa . In conclusion, metformin
reduces detrimental effects of varicocele, although no additional
benefit is expected following varicocelectomy. Further studies are
required to apply metformin for this indication in humans.
Abdelbaki SA, Sabry JH2, Al-Adl AM1, Sabry HH3(2017) The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: A prospective controlled study Arab J Urol. 2017 Jun; 15(2):131-139.