Subclinical varicocele is a condition in which varicose veins from the pampiniform plexus cannot be diagnosed by physical examination but need adjunctive diagnostic methods such as Doppler examination, color Doppler ultrasound, scrotal thermography, or venography [
Many hypotheses were postulated and investigated how varicocele could exert a harmful effect on spermatogenesis. These included semen oxidative stress state [
Sperm DNA integrity is considered an indicator of normal spermatogenesis and fertility potential in males [
The WHO threshold for leucospermia was previously determined by 1.0 × 106 WBC/mL or more [
Fractalkine (CX3CL1) is the solitary member of the CX3C chemokine subfamily [
Fractalkine is produced in seminal plasma in small amounts and correlates with sperm motility [
However, until now, to the best of our knowledge, no reported studies about spermatozoa fractalkine gene expression are published in spite of presence of data about its receptors.
So, in this work we aim at investigating the possible effect of low-level leucospermia on spermatozoa oxidative stress as well as sDNA fragmentation in patients with subclinical varicocele and apparently normal seminogram. Also, we aim at detecting the role of fractalkine and its receptors at the level of spermatozoal mRNA gene expression as a marker of spermatozoa inflammatory response in such patients.
The study is carried out on 125 participants: 80 individuals who were already diagnosed as patients with subclinical varicocele (45 fertile and 35 infertile) and 45 age-matched fertile volunteers with no clinical or sonographic signs of varicocele as a control group. All subjects gave written informed consent. All work was conducted in accordance with the Declaration of Helsinki (1964), and an approval was obtained from the Institutional Review Board (IRB) of Mansoura Faculty of Medicine.
The included infertile subjects have normal seminogram according to WHO [
This research excluded any patient with semen abnormality, leucospermia (>1 × 106/ml), or had infertility risk factor (gonadal toxins, cigarette smoking, use recreational drugs, alcohol intake, urogenital infection, clinically detected varicocele, undescended or small-sized testes, and cryptorchidism). Also, patients with any chronic disease (heart, kidney, or liver disease), endocrine disorder, acute or chronic inflammatory disease, or long-term medications (e.g., corticosteroids) were excluded.
Subclinical varicocele was diagnosed and graded by scrotal color Doppler ultrasonography according to classification of Sarteschi et al. [ Grade 1: venous reflux at the emergence of the scrotal vein only during the Valsalva maneuver; hypertrophy of the venous wall without stasis. Grade 2: supratesticular reflux only during the Valsalva maneuver; venous stasis without varicosities. Grade 3: peritesticular reflux during the Valsalva maneuver; overt varicocele with early-stage varices of the cremasteric vein. Grade 4: spontaneous basal reflux that increases during the Valsalva maneuver, possible testicular hypotrophy, overt varicocele, varicosities in the pampiniform plexus. Grade 5: spontaneous basal reflux that does not increase during the Valsalva maneuver, testicular hypotrophy, overt varicocele, varicosities in the pampiniform plexus.
The scrotal color Doppler ultrasonography maneuver was done according to the American Institute of Ultrasound in Medicine (AIUM) [
Semen samples were collected from the subjects attending the Infertility Clinic of Andrology Unit, Mansoura University Hospital. After sexual abstinence (3–5 days), semen samples were collected by masturbation.
Seminal fluid was left for 1 hour at 37°C for liquefaction. Then, it was transferred to a test tube, and ejaculation volume was recorded. Sperms count and motility (total and progressive) were assessed with the motility/concentration module of the computer-assisted semen analysis (CASA) system using MiraLab–Egypt (Mira 9000 sperm Analyzer CASA software).
Morphology was investigated by smear preparation and sperm Mac stain method (Fertipro, Belgium) recommended by WHO [
One milliliter semen sample, after liquefaction, was added into tube with 2 ml RNAlater reagent (Sigma). Then, cells were pelleted by centrifugation. Following the manufacturer’s instructions, total RNA was extracted from the sperm pellet using TriFast TM reagent (PeqLab. Biotechnologie GmbH, Carl-Thiersch Str. 2B 91052 Erlangen, Germany, Cat. No. 30-2010). Remaining DNAs were eliminated by digestion with DNase I (Sigma). Extracted RNA concentration and purity were determined by NanoDrop™ 2000 Spectrophotometer (Thermo Scientific, USA). Confirmation of the extracted RNA purity was done by formaldehyde agarose gel electrophoresis (2%) and ethidium bromide staining, to present 2 sharp bands (28S and 18S rRNA).
According to manufacturer’s instructions, reverse transcription (RT) of the isolated RNA was carried out using Maxima First Strand cDNA Synthesis Kit for qRT-PCR (ThermoScientific, USA, cat No #K1641). The synthesized cDNA was stored at −20°C until use for qRT-PCR.
Primers for gene-specific qRT-PCR (purchased from Oligo™ Macrogen) were designed using the Primer3 software (v. 0.4.0) (
The qRT-PCR reactions (25
Relative quantification for fractalkine and CX3CR1 gene expression in semen samples was determined by the comparative ΔΔCT method.
DNA fragmentation analysis was done by agarose gel electrophoresis [
Seminal plasma MDA [
Data were tabulated, coded, and analyzed with the computer program SPSS (Statistical Package for Social Science) version 17.0. Descriptive statistics were presented as mean and standard deviation (mean ± SD). For statistical comparison, ANOVA (analysis of variance) test (for >2 groups of numerical parametric data) followed by post hoc was used. Pearson correlation coefficient test was used for different parameter correlation. The sensitivity and specificity were examined at different cutoff points using ROC curve analysis to determine the best cutoff point as well as the diagnostic power of each test.
The present study included 125 subjects with a mean age of 30.7 ± 11.3 years. The subjects were divided into 3 groups: 45 healthy subjects as controls and 80 patients with subclinical varicocele (45 fertile and 35 infertile). The subclinical varicocele patients were grade I or II by color Doppler ultrasound.
There are significant decrease in the quality of semen (concentration, normal morphology, motility, and vitality) and significant increase in the leucocytes count in the two subclinical varicocele groups in comparison to the control group. Also, there are significant decrease in the quality of semen and significant increase in the leucocytes count in the infertile group in comparison to the fertile group, but all parameters are still within normal values according to the normal reference ranges of WHO (2010) (Table
Semen parameters of all studied groups.
Control | Fertile |
Infertile | |
---|---|---|---|
Number of cases | 45 | 45 | 35 |
Volume (ml) | 3.61 ± 0.73 | 4.26 ± 1.14a | 5.02 ± 1.02b,c |
Concentration (106/ml) | 117.11 ± 30.85 | 98.23 ± 17.09a | 71.22 ± 19.04b,c |
Normal morphology (%) | 54.28 ± 18.29 | 37.09 ± 12.60a | 16.49 ± 4.20b,c |
% Motility (PR) | 69.62 ± 13.91 | 50.50 ± 9.41a | 33.58 ± 8.01b,c |
Vitality (%) | 56.51 ± 11.41 | 44.49 ± 10.11a | 30.44 ± 8.07b,c |
WBCs (106/ml) | 0.26 ± 0.08 | 0.57 ± 0.14a | 0.75 ± 0.13b,c |
Data are represented in the form of mean ± SD; asignificance between control group and fertile–subclinical varicocele group; bsignificance between control group and infertile–subclinical varicocele group; csignificance between fertile–subclinical varicocele group and infertile–subclinical varicocele group; PR: progressive motility.
Our results show that there are significant increases in MDA and 8-OHdG levels in subclinical varicocele groups in comparison to control and in the infertile group in comparison to the fertile group (Figures
Oxidative stress state, DNA fragmentation, and fractalkine expression in subclinical varicocele patients. (a) Levels of MDA. (b) TAC. (c) 8-OHdG level. (d) DNA Fragmentation. (e) Fractalkine gene expression. (f) CX3CR1 gene expression. Data are represented in the form of mean ± SD.
There is a significant increase in the DNA fragmentation in subclinical varicocele groups in comparison to control and in the infertile group in comparison to the fertile group (Figure
Moreover, results of the present study (Figure
Correlation between fractalkine expression and other parameters.
The effectiveness of fractalkine expression and 8-OHdG in discriminating fertile from infertile men with different clinical diagnoses was studied by generating receiver operating characteristic (ROC) curves (Figure
ROC curve analysis of 8-hydroxy-2-deoxyguanosine (8-OHdG) and seminal leucocytes count (WBCs).
ROC curve analysis of 8-OHdG and seminal leucocytes count.
AUC (CI 95%) | Cutoff value | Sensitivity % | Specificity % | PPV % | NPV % | Accuracy % | |
---|---|---|---|---|---|---|---|
8-OHdG | 0.938 (0.89–0.98) | 16.62 | 87.5 | 91.1 | 94.6 | 80.4 | 88.8 |
Seminal leucocytes count | 0.988 (0.975–1.00) | 0.519 | 80.0 | 100.0 | 100.0 | 73.8 | 87.2 |
AUC: area under the curve, CI: confidence interval, PPV: positive predictive value, NPV: negative predictive value.
Testicular dysfunctions that are associated with varicocele include elevated intratesticular temperature, developing testicular hypoxia, testicular gonadotoxins, and seminal of oxidants accumulation as well as evident production of anti-sperm antibodies. The documented pathophysiologic effects of varicocele could be suppressed activity of testicular DNA polymerase enzyme, induction of testicular apoptosis, and oxidative stress. Moreover, Sertoli and Leydig cell dysfunction and hormonal disorders were also reported [
The first objective of our study is to investigate the possible causative or associated relationship of low-level leucospermia and spermatozoa oxidative stress as well as sDNA fragmentation in patients with subclinical varicocele and apparently normal seminogram.
It is evident in the current study that subclinical varicocele is associated with spermatozoa oxidative stress which is presented by increased seminal plasma MDA and 8-OHdG with a significant increase in the percentage of sDNA fragmentation. On the other hand, there is a marked decrease in seminal TAC. The seminogram parameters also show a significant decrease in contrast to the controls in spite of the fact that it is still within normal level according to WHO [
The nonspecific seminal stress pattern in men with varicocele (either clinical or subclinical like our target group) is previously reported by Zümrütbaş et al. [
Leucocytes and abnormal sperms are considered major sources of ROS in semen. Both are prominent features of varicocele [
Spermatozoa membrane and nuclear DNA damage caused by increased ROS with defective antioxidant defect could play a role in development of poor sperm quality including motility and fertilizing ability [
Spermatozoa DNA fragmentation is associated with poor sperm function and quality regardless of the semen parameters. In most of the cases, seminogram shows a normal pattern on examination with CASA [
Also, we tested the specificity and sensitivity of low-level leucospermia as causative pathophysiologic mechanism in our target studied group (subjects with subclinical varicocele either fertile or infertile). ROC curve analysis revealed 100% specificity and 80.0% sensitivity at a cutoff level of 0.519 × 106/ml. The result of the current study supports Agarwal et al. [
Like our study, Agarwal et al. [
The concomitant presence of subclinical varicocele, low-level leucospermia, and sperm nuclear DNA fragmentation could play an important pathophysiologic mechanism of subfertility predisposition or even affect the male fertility potentials as presented in our study and documented previously by Agarwal et al. [
The five proposed and studied mechanisms of varicocele-induced delayed male fertility (hypoperfusion leading to hypoxia, heat stress, oxidative stress, hormonal imbalance, and exogenous toxins) still do not provide a full understanding. So, genetic and molecular factors might have a role in clarifying pathogenesis of varicocele-associated infertility [
The debate about the role of inflammation in varicocele pathogenesis of male subfertility took a long time of discussion. But it is documented that remarkable increase of ROS levels which can cause an inflammatory response detrimental to testicular tissue. It has been shown that varicocele increased ROS stress in a time-dependent manner. Varicocele-induced inflammation negatively impacted Sertoli cell physiologic function and may induce maturation arrest of spermiogenesis [
Many previous studies dealt with many seminal cytokines and inflammatory mediators in case of varicocele as nuclear factor-kappa B (NF-
The novelty of our study is investigating spermatozoa fractalkine signaling pathway gene expression at the level of mRNA. There are no published data about this issue is documented.
Our results revealed increased spermatozoa mRNA expression of fractalkine and its coupled receptors (CX3CR1) in individuals with subclinical varicocele which is significantly higher in the infertile subgroup when compared to those of the fertile group. Their expression levels are positively correlated with MDA, 8-OHdG, WBCs count, and sperm nuclear DNA fragmentation % while it is negatively correlated with seminal TAC.
These results could prove their involvement in the pathophysiology of varicocele-induced spermatozoa subclinical inflammation and pathogenesis in male subfertility in such individuals.
The present study could conclude that subclinical varicocele induces seminal and spermatozoal subclinical inflammatory response in the form of low-level leucospermia and increased mRNA expression of the fractalkine signaling pathway. This inflammatory response leads to increased spermatozoal ROS production, oxidative stress, and nuclear DNA fragmentation. All of these interplay mechanisms could cooperate in the pathogenesis of delayed fertility in males with subclinical varicocele.
The authors declare that no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. The authors also declare that they have no conflicts of interest in connection with this paper.