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This study was conducted to assess the impact of hubble-bubble smoking on global DNA methylation, DNA fragmentation; protamine deficiency of spermatozoa, and to determine whether the transcription levels of the  protamine  and  histone  genes are different in hubble-bubble smokers compared to nonsmokers. Five hundred semen samples were collected from males with an average age of 32.2 ± 6.1 years (300 hubble-bubble smokers “60%” and 200 nonsmokers “40%”). The nucleic acid was isolated from purified sperm, then ELISA and qPCR were used to evaluate the global DNA methylation and transcription level of  protamine  and  histone , respectively. A significant elevation in global DNA methylation, protamine deficiency, and DNA fragmentation was found in hubble-bubble smokers compared to nonsmokers (P &lt; 0.0001). A significant decline was shown in transcription levels of  protamine  and  histone  genes in hubble-bubble compared to nonsmokers (P &lt; 0.0001). Additionally, a down-regulation in the transcription levels of  protamine  and  histone  was revealed in hubble-bubble compared to nonsmokers with fold change (0.0001 and 0.007, respectively). In conclusion, this study provided proof that hubble-bubble smoking has a negative impact on global DNA methylation, DNA fragmentation, protamine deficiency, and the transcription of  protamine  and  histone  genes in spermatozoa, and these findings influence negatively males’ fecundity.

Effect of hubble-bubble smoking on global DNA methylation and transcription levels of protamine and histone genes in human spermatozoa