Reactive oxygen species-induced alterations in H19-Igf2 methylation patterns, seminal plasma metabolites, and semen quality
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This study was conducted in order to investigate the effects of reactive oxygen species (ROS) levels on the seminal plasma (SP) metabolite milieu and sperm dysfunction.
Semen specimens of 151 normozoospermic men were analyzed for ROS by chemiluminescence and classified according to seminal ROS levels [in relative light units (RLU)/s/106 sperm]: group 1 (n = 39): low (ROS < 20), group 2 (n = 38): mild (20 ≤ ROS < 40), group 3 (n = 31): moderate (40 ≤ ROS < 60), and group 4 (n = 43): high (ROS ≥ 60). A comprehensive analysis of SP and semen parameters, including conventional semen characteristics, measurement of total antioxidant capacity (TAC), sperm DNA fragmentation index (DFI), chromatin maturation index (CMI), H19-Igf2 methylation status, and untargeted seminal metabolic profiling using nuclear magnetic resonance spectroscopy (1H-NMR), was carried out.
The methylation status of H19 and Igf2 was significantly different in specimens with high ROS (P < 0.005). Metabolic fingerprinting of these SP samples showed upregulation of trimethylamine N-oxide (P < 0.001) and downregulations of tryptophan (P < 0.05) and tyrosine/tyrosol (P < 0.01). High ROS significantly reduced total sperm motility (P < 0.05), sperm concentration (P < 0.001), and seminal TAC (P < 0.001) but increased CMI and DFI (P < 0.005). ROS levels have a positive correlation with Igf2 methylation (r = 0.19, P < 0.05), DFI (r = 0.40, P < 0.001), CMI (r = 0.39, P < 0.001), and trimethylamine N-oxide (r = 0.45, P < 0.05) and a negative correlation with H19 methylation (r = − 0.20, P < 0.05), tryptophan (r = − 0.45, P < 0.05), sperm motility (r = − 0.20, P < 0.05), sperm viability (r = − 0.23, P < 0.01), and sperm concentration (r = − 0.30, P < 0.001).
Results showed significant correlation between ROS levels and H19-Igf2 gene methylation as well as semen parameters. These findings are critical to identify idiopathic male infertility and its management through assisted reproduction technology (ART).
KeywordsSperm DNA fragmentation Sperm DNA methylation Oxidative stress Reactive oxygen species Total antioxidant capacity
The authors would like to thank all patients and their family members who voluntarily participated in this study. In addition, we thank the director of the Phytochemistry and the Medicinal Chemistry Research Center at Shahid Beheshti University of Medicinal Sciences, SBMU (Iran, Tehran) and the Reproductive Center of Cleveland Clinic (Cleveland, USA) for their assistance.
Mahsa Darbandi (data interpretation, study design, execution, analysis, manuscript drafting and revision), Sara Darbandi (data interpretation, study design, execution, analysis, manuscript drafting and revision), Ashok Agarwal (data interpretation, study design, analysis, manuscript review, revision and critical discussion), Saradha Baskaran (data interpretation, manuscript preparation, review and revision), Sulagna Dutta (data interpretation, manuscript preparation, review and revision), Pallav Sengupta (data interpretation, manuscript preparation, review and revision), Hamid Reza Khorram Khorshid (data interpretation, study design, manuscript drafting), Sandro Esteves (data interpretation, manuscript review and revision), Kambiz Gilany (acquisition of data, analysis), Mehdi Hedayati (data interpretation, study design, execution), Fatemeh Nobakht (acquisition of data, execution, analysis), Mohammad Mehdi Akhondi (data interpretation, study design), Niknam Lakpour (acquisition of data, study design, execution), and Mohammad Reza Sadeghi (data interpretation, study design, manuscript drafting and critical discussion). All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
Research involving human participants
All applicable international, national, and/or institutional guidelines for the use of human tissues were followed. All procedures performed in studies involving human subjects were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
Informed consent was obtained from all individual participants included in the study.
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