Negative effect of varicocele on sperm mitochondrial dysfunction: A cross-sectional study

Abstract Background Varicocele is an abnormal dilation and enlargement of the scrotal venous pampiniform plexus that impairs normal blood drainage and finally leads to infertility if not treated. Objective This study aimed to figure out the impact of mitochondria status through the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) assessment and its correlation with semen parameters to illuminate the impact of sperm mitochondria healthiness on normal sperm functionality. Materials and Methods This analytical cross-sectional study was conducted with 100 men including 50 cases in the normozoospermic group (normal) and 50 in an infertile group with the non-varicocelectomy operation (varicocele) referring to Infertility Research and Treatment Center, ACECR Khuzestan, Iran. Routine semen analysis was performed according to World Health Organization guidelines, DNA fragmentation index, the MMP assay, ATP content, and apoptosis were carried out for all samples. Results The results showed that the concentration, progressive motility, normal morphology, MMP, and ATP contents of sperm in varicocele were significantly lower than the normal group. In addition, the sperm DNA fragmentation index was significantly higher in the varicocele group in comparison with the normal group. Conclusion Reduction in MMP and ATP contents, besides the loss of sperm parameters quality and increase in sperm DNA fragmentation, were seriously implicating sperm mitochondria dysfunctionality in varicocele men.


Introduction
Varicocele is the most common male factor of infertility with abnormal dilation and enlargement of the pampiniform plexus, which drains blood from each testicle to the single testicular vein.
The decreased backflow of the vein is associated with hypoxia, which may be impaired testicular spermatogenesis, testicular volume, sperm parameters, and embryo implantation and pregnancy rate. Varicocele is the second most common cause of treatable infertility and occurs in around 15-20% of the general population. The incidence of varicocele increases with age (1).
It accounts for 35% of primary infertility, and up to 80% of secondary infertile cases (2,3). There is a controversy on the impact of varicocele before/after treatment of varicocele operation on sperm parameters and fertility. Numerous studies have shown that varicocelectomy not only improves semen parameters but also improves intra cytoplasmic sperm injection outcomes (4,5).
The exact mechanism of varicocele remains unclear. The hypoplasia and insufficient growth of the testis in adolescence due to congenital/acquired valve defects, venous obstruction, and any anatomical variations contributed to its pathogenesis. This affected the ultrastructure of the testis, and testicular hormone dysfunction through high testicular temperature, scrotal hyperthermia, and blood reflux of the testis vein, and it may cause pain and swelling of the testis that needs a proper medical examination to cure infertility in varicoceles cases (6,7). Recent studies have shown that there is a genetic downregulation in heat-shock proteins in varicoceles required for the neutralization of oxidative stress (OS).
Testicular hyperthermia can induce the OS and increase reactive oxygen species (ROS) products (i.e., hydroxyl, peroxyl, hydroperoxyl radicals, superoxide, and nitric oxide) and induce sperm dysfunction (8,9). An imbalance between ROS production and antioxidant protectant leads to OS and damage to lipids and proteins of the nucleic acids, induces sperm DNA and mitochondria nucleic acids fragmentation, damaged chromatin strands, and its crosslinks during the spermatogenesis and nuclear protamination in the varicocele's patients. (10,11    Written consent was obtained from the participants and a questionnaire containing personal information was completed for each person.

Statistical analysis
Statistical analysis was performed using SPSS 22 (IBM Corp., Armonk, NY, USA). Descriptive statistics mean, and standard deviation was used in the study. The Kolmogorov-Smirnov test was used to examine the normality of the quantitative variables.
The results of the test showed that the variables were abnormal. Therefore, a nonparametric test was adopted to analyze the data. The Mann-Whitney U test was used to compare the statistical differences between 2 groups. The Spearman correlation was used to analyze the correlations between DFI, MMP, ATP, apoptosis, and semen parameters. The significance level was set at p < 0.05.

Results
Among 500 men included in the current study,

Discussion
The pathogenesis of varicocele is not clearly Our purpose in the present study was to illuminate that MMP and ATP contents assessments are essential tests for evaluating sperm mitochondrial function. They significantly correlate with DFI and apoptosis and also with semen parameters.
In this study, sperm parameters such as sperm concentration, motility, and sperm morphology were reduced in varicocele compared to normal subjects.

Conclusion
The present study shows that the varicocele has a negative effect on sperm concentration, motility,