Varicocele is one of the most common causes of male
infertility. Approximately, 15% of healthy men and 40%
of infertile men suffer from varicocele (
In varicocele disease, heat stress induces undesirable
adverse effects on testis tissue, such as spermatogenesis
impairment, increase in production of reactive oxygen
species (ROS) and apoptosis (
Inflammation is an immune response to pathological
events, such as bacterial/viral infection and tissue damage
to protect other cells from injury (
In testis of rodents and primates, Sertoli cell is
responsible for NLRP3 expression and it is believed that
alteration in NLRP3 expression might impair fertility (
This study was performed from December 2017 to September 2018. Sample size was calculated by the following formula:
where type one (α) and type two errors (β) were 0.05 and 0.20
(power ¼; 85%), respectively according to previous studies
Semen samples were collected from varicocele and
control subjects by masturbation after at least 48 hours
of sexual abstinence. After liquefaction for 30 minutes,
semen parameters including volume, pH, concentration,
morphology and motility were analyzed according to the
World Health Organization criteria (
Semen samples were centrifuged at 1000 xg for 15
minutes at room temperature. The supernatant was then
collected and stored at -70°C for further analysis (
Concentrations of the mature IL-1β, IL-18 and caspase-1 (all from Abcam, USA) were measured by an enzymelinked immunosorbent assay (ELISA) kit following the manufacturer’s protocol. Seminal plasma samples were thawed at room temperature and placed in plates precoated with a specific monoclonal antibody for each of IL-1b, IL-18 or caspase-1. Each sample was duplicately assayed.
Seminal plasma samples were thawed at room
temperature. One microliter of seminal plasma from each
subject was mixed with loading buffer (containing a final
concentration of 50 mmol/l Tris-HCl pH=7.0, 2% sodium
dodecyl sulfate, 10% glycerol, 5% b-mercaptoethanol
and 0.002% bromophenol blue), and heated at 95°C
for 10 minutes. Protein concentrations were determined
using the BCA™ Protein Assay Kit (Pierce, Germany)
according to the manufacturer’s protocol. The same
amount of protein samples was loaded, separated on
8-12% (v/v) discontinuous sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred onto a polyvinylidenefluoride (PVDF)
membrane (Roche, Germany). After blocking with 5%
skimmed milk in Tris-buffered saline containing 0.05%
Tween 20 (TBS-T) for 1 hour at room temperature, PVDF
membranes were incubated with anti-ASC antisera
(Santa Cruz, USA, diluted 1:1000) or anti-NLRP3
antisera (Bioss, USA, diluted 1:1000) overnight at 4oC.
After washing with TBS-T, membranes were incubated
with a peroxidase-conjugated goat anti-rabbit (BioRad,
USA, diluted 1:500) secondary antibody for 2 hours at
room temperature. Visualization was performed using
the enhanced chemiluminescence method (ECL plus,
Pierce Scientific, USA) according to the manufacturer’s
protocol. For densitometric quantification, intensity of the
specific bands was normalized to β-actin (Bioss, USA,
diluted 1:1000) in the same blot using Image J software
(free Java software provided by the National Institute of
Health; Bethesda, USA) (
The results are expressed as means ± standard errors (SE). The Shaprio-wilktest was used to determine normal distribution. Independent sample t test was applied to check the matched factor between the case and control groups. Statistical significance between the mean values was determined by paired t test and P≤0.05 was considered statistically significant.
In this study, we analyzed relationship of sperm parameters with NLRP3, ASC, IL-18 and caspase-1 in both control and varicocele groups. We could not find any correlation in our study.
The semen volume (5.1 ± 1.8 ml in control vs. 4.06 ± 1.5 in varicocele) and pH (7.8 ± 0.2 in control vs. 7.7 ± 0.1 in varicocele) were not significantly different between the control and varicocele subjects. The median sperm concentration in the varicocele group (44 million/ml) was significantly lower than the control group (97 million/ml, P=0.0001).The median sperm total motility was equal in the both groups (61% in control vs. 55% in varicocele) while the sperm progressive motility in varicocele patients showed a significant decrease (P=0.0003) compared to the control subjects. In addition, varicocele patients had more abnormal sperm (especially abnormal head) morphology compared to the control group (99% in varicocele vs. 80% in control).
The levels of IL-1β, IL-18 and caspase-1 were
investigated in seminal plasma by ELISA. Our data
showed that IL-1β was significantly increased (P=0.03)
in seminal plasma of varicocele patients in comparison
with control subjects [optic densitometry (OD)=1 ± 0.016
vs. 0.94 ± 0.021, respectively; Fig.1A]. In these patients,
concentration of caspase-1 showed no obvious change
Measurement of the inflammatory cytokines in seminal plasma of
varicocele patients using ELISA.
OD; Optic densitometry and *; P≤0.05 compared to the controls.
To investigate whether inflammasome components are expressed and changed in seminal plasma of varicocele patients, we quantified respectively ASC and NLRP3 protein levels by Western blot. NLRP3 (P=0.005) and ASC (P=0.0002) protein levels were significantly elevated in varicocele patients versus control subjects (relative intensity of ASC was 2.02 ± 0.09 vs. 0.32 ± 0.28 and relative intensity of NLRP3 was 1.5 ± 0.13 vs. 0.56 ± 0.1, respectively, Fig. 2 A-C).
Analysis of inflammasome components in seminal plasma of
varicocele patients by Western blotting and subsequent measurement of
optical densities of immune-labelled bands.
In this study, quality of semen in all varicocele patients
was lower than the control group. Varicocele patients had
abnormal semen quality (
In this study our results showed that ASC and NLRP3 levels in semen of varicocele subjects were significantly elevated compared to the control subjects. Additionally, concentration of IL-1β was higher in varicocele versus control subjects, whereas IL-18 was decreased in seminal plasma of varicocele patients and caspase-1 was not changed. In addition, we could not find any significant correlation between sperm parameters and NLRP3 inflammasome components.
Sahin et al. (
In this study, we expected that IL-18 level would
be increased in varicocele subjects (
Western blot analysis showed high level of NLRP3 and
ASC protein expressions in seminal plasma of varicocele
patients. In our previuos work, we showed high levels of
ASC, NLRP3 and caspase 1 expression in the testis tissue
of varicocele-induced rats, three months after surgery (
Novelty is the strength of this study, as this is the first evidence for the existence and presence of the NLRP3 inflammasome in seminal plasma of varicocele patients. Thus, it highlights the importance of anti-inflammasome therapies to improve the fertility rate in varicocele patients. However, a limitation is about the control subjects. It was better to choose fertile varicocele subjects as control group. The other weak point is that the immunohistochemistry was not done in this study to localize this complex.
Findings obtained from this study suggest that NLRP3 activation occurs in varicocele and it might be responsible for pathological procedure occurring in varicocele patients. Details of the NLRP3 inflammasome activation process has not been clarified yet. At present, it is not clear whether NLRP3 is causally related to the onset of varicocele or the result of pathological damages appearing in the course of this gonadal disease. Further study is underway to determine time course of activation of inflammasome in varicocele disease.