Increased de novo DNA Methylation Enzymes in Sperm of Individuals with Varicocele.

This article was published in Cell J (Yakhteh), Vol 23, No 4, 2021, on pages 389-396, corresponding author asked us to add another address (5. Biomedical and Pharmaceutical Research Unit, QU Health, Qatar University, Doha, Qatar) to the fourth author's affiliation. The authors would like to apologies for any inconvenience caused.

Increased de novo DNA Methylation Enzymes in Sperm of Individuals with Varicocele.

Objective: Chronic genital heat-stress associated with varicocele leads to DNA hypo-methylation of spermatozoa. The objective of this study was comparing level of DNA methyl-transferases (DNMTs) in sperm of men suffering varicocele with fertile individuals. Materials and Methods: In this case-control study, semen samples were obtained from 35 infertile men with varicocele (grade II or III) and 26 fertile men. Sperm parameters were assessed according to World Health Organization (WHO) protocol. DNMTs enzymes level were assessed by flow cytometer and fluorescence microscope. mRNAs expression of these DNMTs were also assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). Results: DNMT1 and DNMT3A proteins were mainly localized in equatorial and mid-piece regions of sperm head, respectively, while DNMT3B protein appeared to be localized mainly in equatorial and anterior regions of sperm head. In contrast to DNMT1, expression and percentage of DNMT3A and DNMT3B at RNA and protein levels were significantly higher in the varicocele group compared to the fertile group (P<0.05). In addition, significant correlations were found between sperm concentration and motility as well as DNMT1 and DNMT3B proteins levels in the infertile individuals with varicocele (P<0.05). Additionally, significant correlations were observed between abnormal sperm morphology with DNMTs proteins in the infertile individuals with varicocele. Conclusion: Unlike DNMT1, which is involved in maintenance of DNA methylation at both RNA and protein levels, expression of de novo methylation enzymes (DNMT3A and DNMT3B) at both levels were increased in the varicocele group compared to the fertile group. Based on literature, this increase might be due to the dual roles played by DNMT3A and DNMT3B, as methyl-transferases in normal condition as well as dehydroxymethylases in stress condition, like varicocele. Although, this hypothesis needs further validation.

Comparison of main molecular markers involved in autophagy and apoptosis pathways between spermatozoa of infertile men with varicocele and fertile individuals.

Abnormal dilatation and tortuosity of the pampiniform plexus within the spermatic cord are termed varicocele which leads to impaired spermatogenesis due to heat-related oxidative stress and cell death. Previously, it was shown that both apoptosis and autophagy pathways were activated by heat in germ cells of mouse in vivo and in vitro. But, status of these pathways is not clear in chronic state of heat stress such as varicocele. Therefore, we aimed to access sperm apoptotic markers (active caspases 3/7 and DNA fragmentation), and autophagic markers (Atg7 and LC3 proteins) as primary outcomes, and also sperm parameters and protamine deficiency as secondary outcomes between 23 infertile men with varicocele and 16 fertile individuals. Sperm parameters were assessed according to World Health Organization 2010 protocol. Apoptotic markers (active caspases 3/7 and DNA fragmentation), autophagic markers (Atg7 and LC3 proteins), and protamine deficiency were evaluated by flow cytometry, fluorescence microscope, and western blotting techniques. Mean of autophagy and apoptosis markers, and also protamine deficiency have significantly increased in infertile men with varicocele compared to fertile individuals, but autophagy and apoptosis markers did not significantly correlate with each other. In conclusion, it seems that both apoptosis and autophagy pathways are independently active in spermatozoa of infertile men with varicocele.

An association between sperm PLCζ levels and varicocele?

PURPOSE: We aimed to compare the expression of phospholipase C ζ (PLCζ), as one of the main sperm factors involved in oocyte activation, at both RNA and protein levels in fertile men and those with varicocele. METHODS: This study included 35 individuals with male factor infertility presenting primary infertility with grade II and III unilateral varicocele and 20 fertile men without varicocele. Semen parameters were assessed according to WHO 2010. Sperm DNA fragmentation, relative expression of PLCζ at messenger RNA, and protein levels were evaluated by sperm chromatin structure assay (SCSA), real-time PCR, and Western blot analysis, respectively. RESULTS: The results of this study reveal that the mean relative expression of PLCζ was significantly lower in individuals with varicocele compared to fertile men at both transcription and translation levels. In addition, the percentage of DNA fragmentation was significantly higher in infertile men with varicocele compared to fertile men. CONCLUSIONS: The findings of the present study illustrate that one of the etiologies of reduced fertility associated with varicocele is the low expression of PLCζ. This effect could subsequently reduce the sperm ability to induce oocyte activation. Therefore, these results hold promise to modify our understanding of reproductive physiology of varicocele state.

DNA hypomethylation predisposes sperm to DNA damage in individuals with varicocele.

This study aimed to assess and compare global DNA methylation (GDM) between fertile men and infertile men with varicocele. In addition, we evaluated the correlations between DNA methylation with reactive oxygen species (ROS), protamine deficiency, and DNA integrity. Semen samples were collected from 44 men with grades II and III varicocele, and 15 fertile men for assessment of semen parameters, DNA methylation, DNA fragmentation, oxidative stress, and protamine deficiency. Samples were evaluated by the World Health Organization (WHO) criteria, immunostaining, the TUNEL assay, 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and chromomycin A3 (CMA3) staining. Semen parameters were significantly lower in individuals with varicocele compared to fertile men. The percentage of GDM and intensity of DCFH were reduced and the percentages of DCFH, TUNEL, and CMA3 positive sperm significantly increased in individuals with varicocele compared to fertile men. Correlation analysis revealed a negative significant relation between DNA methylation and DNA fragmentation, but not with the degree of protamine deficiency and ROS production. The results have shown that individuals with varicocele show increased DNA susceptibility to damage when DNA is hypomethylated. This phenomenon appears to be independent of ROS production.

Semen parameters and chromatin packaging in microsurgical varicocelectomy patients.

BACKGROUND: Varicocelectomy is considered as standard treatment for male infertility for clinical varicocele. The aim of this study is to address the effects of varicocelectomy on semen parameters, chromatin packaging, and pregnancy outcome. MATERIALS AND METHODS: This retrospective study was carried out between June 2006 and February 2011 on 145 infertile men with grade II or III varicocele. Microsurgical varicocelectomy was performed as part of patient management. Sperm count, motility, morphology, and chromatin packaging were assessed with a Makler counting chamber, light microscopy, Papanicoulaou and chromomycin A3 (CMA3) staining, respectively. In addition, we assessed spontaneous clinical pregnancy and miscarriage rates. RESULTS: The percentages of spontaneous cumulative pregnancies post-surgery were 33.1% (3 months), 42.06% (6 months), 46.2% (9 months), 48.9% (12 months), and 55.8% (after 12 months). Percentages of spontaneous cumulative miscarriage post-surgery were 2.46% (3 months), 4.93% (6 months), 4.93% (9 months), 6.17% (12 months), and 6.17 % (after 12 months). Both sperm parameters improved and the percentage of sperm protamine deficiency decreased significantly after varicocelectomy. CONCLUSION: These results confirm that varicocelectomy improves sperm parameters and chromatin packaging, thereby improving the chance of pregnancy. Positive aspects of this study include the large number of patients studied, duration of follow up, one surgeon who performed all of the surgeries, and type of surgery (microsurgery). The spontaneous pregnancy results also suggest that if pregnancy is not achieved within twelve months post-surgery, an alternative approach such as assisted reproductive technology (ART) treatment should be considered.