P-008 Sperm DNA methylation landscape in infertile men with clinical varicocele before and after treatment
Authors: Arya D , Singh D , Gajbhiye R , Pawar P , Tandon D , Kothari P 
Published: July 2024

Abstract

Study question
As varicocele leads to poor semen parameters, does it also affect sperm DNA methylation levels? Does varicocele treatment help in epigenetics restoration in the sperm?

Summary answer
Varicocele leads to aberrant sperm DNA methylation and varicocele treatment helps in the partial restoration of sperm DNA methylation levels.

What is known already
Varicocele affects 15% to 20% of the adult male population and 20% of men with varicocele are reported to be infertile. Treatment modalities for varicocele improve semen parameters, yet more than 50% of cases remain infertile. High oxidative stress (OS) has been implicated as a key factor in varicocele-associated infertility. OS during spermatogenesis may lead to aberrant epigenetic modifications in spermatozoa. In view of the crucial role of sperm epigenome in its functionality, this study aims to evaluate sperm genome-wide DNA methylation by whole-genome bisulfite sequencing (WGBS) before and after varicocele treatment in men with varicocele.

Study design, size, duration
This is a case-control study, which includes (n = 28) healthy fertile men and (n = 35) infertile men with clinical varicocele. Study participants were recruited from ICMR-NIRRCH and, LTMG Hospital, Mumbai. According to the inclusion and exclusion criteria, study participants were recruited from December 2020 to November 2023. Varicocele patients were followed up, and semen samples (n = 18) were recollected after 3-6 months of varicocele treatment.

Participants/materials, setting, methods
WGBS was used to identify differentially methylated CpG (DMC) sites in sperm of varicocele group compared to controls. 5 samples from each group were pooled for WGBS. DMCs identified with ≥25% methylation change and ≤0.05 q-value. DMCs within genes relevant to sperm function were selected for validation in the study population by pyrosequencing. Differentially methylated Genes (DMGs) were identified based on the location of DMC and used for pathway analysis using KEGG and DAVID tools.

Main results and the role of chance
Varicocele group had significantly lower semen parameters compared to controls, which were found to be improved after varicocele treatment. WGBS of sperm DNA was done and DMC sites were obtained in varicocele group compared to controls. A total of 6414 DMCs were obtained, of which, 3730 were hypermethylated and 2684 were hypomethylated. Based on genomic context, differential methylation was observed in introns, exons, promoters and intergenic regions. WGBS analysis gave a total of 1484 DMGs. KEGG and DAVID pathway analysis revealed that the PI3K-Akt signaling, MAPK signaling, cAMP signaling, calcium signaling, signal transduction pathway, cell adhesion molecules, and tight junction, were affected in the varicocele group. The DMGs SPATA6L, H2AX, SOD2, BCL2, NF1, PRKN, CDKN1B, BCR, P2RX7, RNF212B obtained are involved in spermatogenesis, regulation of cell cycle, apoptotic signaling pathway, cell differentiation, regulation of ROS metabolic process, response to heat, mitochondria regulation, regulation of gene expression, etc. Pyrosequencing validation of a few selected DMCs reflects the same methylation difference (hypo or hyper) as obtained by the WGBS pooled sample. Significant hypermethylation and hypomethylation were observed in the H2AX DMC and CDKN1B DMC, respectively, in the varicocele population; however, after varicocele treatments, these methylation levels seem to be restored.

Limitations, reasons for caution
The limitations of this study are the small number of patients and pooled sperm DNA sample WGBS analysis in the study population. However, a pooling sample strategy is considered more efficient than a random sample selection strategy, as pooling minimizes the amount of information lost below the detection threshold.

Wider implications of the findings
This study identified differentially methylated genes relevant to spermatogenesis and sperm functions in the case of varicocele compared to fertile controls and gave deeper insights into the possibility of epigenetic restoration after varicocele treatment. This study provides insight into the molecular aetiology of varicocele associated infertility.

Trial registration number
Not Applicable