Impact of waterpipe and tobacco cigarette smoking on global DNA methylation and nuclear proteins genes transcription in spermatozoa: a comparative investigation.

BACKGROUND: Waterpipe smoking is harmful and dangerous, and it is a growing threat to public health. OBJECTIVES: This study was performed to evaluate the influence of waterpipe smoking on global DNA methylation, DNA fragmentation, and protamine deficiency in spermatozoa compared to cigarette heavy smokers and nonsmokers, and to determine whether the transcription levels of spermatozoa nuclear proteins genes 'PRM1, PRM2, and H2BFWT' in waterpipe smokers are different compared to cigarette heavy smokers and nonsmokers. METHODS: A total of 900 semen samples were collected from males with a mean age of 32.5 ± 6.3 years (300 waterpipe smokers, 300 cigarette heavy smokers, and 300 nonsmokers). The nucleic acids were isolated from purified spermatozoa, and then the global DNA methylation and transcription levels of the PRM1, PRM2, and H2BFWT genes were assessed using ELISA and qPCR, respectively. RESULTS: A significant increase was found in the level of global DNA methylation (8.6 ± 0.6 ng/µl vs. 7.1 ± 0.6 ng/µl and 4.7 ± 0.6 ng/µl, p < 0.001), protamine deficiency (72.8 ± 15.3 vs. 51.7 ± 19.2 and 15.3 ± 5.9%, p < 0.001), and DNA fragmentation (73.4 ± 13.4 vs. 50.5 ± 18.9 and 9.3 ± 4.3%, p < 0.001) in waterpipe smokers compared to cigarette heavy smokers and nonsmokers. A significant increase was shown in the transcription levels of PRM1, PRM2, and H2BFWT genes in waterpipe smokers compared to cigarette heavy smokers and nonsmokers (p < 0.001). A down-regulation was found in the transcription level of these genes in different smoker groups compared to nonsmokers (<0.001). CONCLUSION: This study suggests that waterpipe smoking is more harmful than cigarette smoking on semen parameters, global DNA methylation, and transcription of nuclear protein genes.

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

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 < 0.0001). A significant decline was shown in transcription levels of protamine and histone genes in hubble-bubble compared to nonsmokers (P < 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.

Investigation of the Predictive Factors of Diminished Ovarian Reserve in Women Aged Less Than 40 Years and Undergoing ICSI Cycle.

Diminished ovarian reserve (DOR) is one of the primary causes of poor ICSI outcomes. Therefore, this study was performed to speculate which of the following parameters: AMH, AFC, and women's age can be used as a predictor factor of the DOR in women aged < 40 years. This prospective study enrolled 500 women suffering from idiopathic infertility problems and who underwent GnRH antagonist multiple-dose stimulation protocol. The women were divided into two groups: normal fertility (FSH ≤ 10 mIU/mL, n = 300) and DOR (FSH > 10 mIU/mL, n = 200). At the time of the study, the average of women age was 29.3 ± 5.7 years. A significant reduction was found in AMH level, AFC, number of mature, immature oocytes, fertilized oocytes, embryos transferred, and ß-hCG level in the DOR group compared to the normal fertility group (P < 0.001). Conversely, a significant increase was shown in the age of the DOR group compared to the normal fertility group (30.8 ± 5.8 vs. 28.2 ± 5.4, respectively; P < 0.001). A significant negative association was found between the AFC, the number of mature oocytes, fertilized oocytes, embryos transferred, and the basal level of FSH in the DOR group (P < 0.01). The receiver operating characteristics (ROC) demonstrated that AMH level and AFC had the highest accuracy, followed by age in the prediction of DOR (P < 0.001) with a cut-off value of ≤ 1.2 ng/mL, ≤ 4.5, and > 29.5 years, respectively. This study exhibited that the levels of AMH and AFC are the best biomarkers, followed by age for the prediction of DOR in women < 40 years old. Furthermore, AMH is the only independent factor that is significantly related to DOR in women.

Cigarette heavy smoking alters DNA methylation patterns and gene transcription levels in humans spermatozoa.

Tobacco smoking is considered the most common reason of death and infertility around the world. This study was designed to assess the impact of tobacco heavy smoking on sperm DNA methylation patterns and to determine whether the transcription level of ALDH3B2, PTGIR, PRICKLE2, and ALS2CR12 genes is different in heavy smokers compared to non-smokers. As a screening study, the 450 K array was used to assess the alteration in DNA methylation patterns between heavy smokers (n = 15) and non-smokers (n = 15). Then, four CpGs that have the highest difference in methylation level (cg16338278, cg08408433, cg05799088, and cg07227024) were selected for validation using deep bisulfite sequencing in an independent cohort of heavy smokers (n = 200) and non-smokers (n = 100). A significant variation was found between heavy smokers and non-smokers in the methylation level at all CpGs within the PRICKLE2 and ALS2CR12 gene amplicon (P < 0.001). Similarly, a significant variation was found in the methylation level at nine out of thirteen CpGs within the ALDH3B2 gene amplicon (P < 0.01). Additionally, eighteen CpGs out of the twenty-six within the PTGIR gene amplicon have a significant difference in the methylation level between heavy smokers and non-smokers (P < 0.01). The study showed a significant difference in sperm global DNA methylation, chromatin non-condensation, and DNA fragmentation (P < 0.001) between heavy smokers and non-smokers. A significant decline was shown in the transcription level of ALDH3B2, PTGIR, PRICKLE2, and ALS2CR12 genes (P < 0.001) in heavy smokers. In conclusion, heavy smoking influences DNA methylation at several CpGs, sperm global DNA methylation, and transcription level of the PRICKLE2, ALS2CR12, ALDH3B2, and PTGIR genes, which affects negatively the semen parameters of heavy smokers.