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.
Background: Although age is an important factor in female fertility, not much date were focused on the relationship between age and ovarian response and in vitro fertilization (IVF) outcome. However, the female reproductive capacity varies with age.Objective: To assess the impact of age on ovarian response and IVF outcome during controlled ovarian hyperstimulation in women from Gaza Strip. Methods: This prospective cohort study consisted of 75 women attending IVF at Al-Basma Fertility Center in Gaza City. The number of oocytes and embryos were recorded for each female and the occurrence of pregnancy was followed for three months. The obtained data were computer analyzed using SPSS statistical package version 18.Results: The mean age of the study population was 29.2±5.9 years. The total number of oocytes was significantly decreased with increasing age (F=3.932 and P=0.024).In this context Pearson correlation test exhibited negative significant correlation between women age and the number of mature oocyte (r=-0.276, P=0.017). There was an inverse relationship between age and ovarian response (F=6.773 and P=0.001), showing good response (9-16 oocytes) at mean age of 26.7±5.0 years. When related to women age, IVF outcome showed that the chance of getting pregnant increased with decreased age (F=4.278 and p= 0.018). Conclusion: The ovarian response and the chance of getting pregnancy were diminishing with ageing, implying that maternal age should by consider during IVF program.DOI: http://dx.doi.org/10.3329/jom.v15i1.19858 J Medicine 2014; 15: 36-40
Aim: To assess serum testosterone and gonadotropins in Sertoli cell only syndrome patients from Gaza Strip.Methods: Based on testicular biopsy, a cross section of 74 Sertoli cell only syndrome patients were enrolled in the study. Age matched 44 fertile men were served as controls. Patients and controls were questioned for their medical history. Blood samples were drawn and serum testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were determined by enzyme-linked immunosorbent assay. Data were computer analyzed using SPSS/PC, version 18.0.Results: Varicocele and hormonal problems were significantly more frequent among patients than controls (P<0.05). Serum testosterone was significantly lower in patients compared to controls (1.7±1.3 versus 5.0±2.2 ng/ml, P=0.000). In contrast, LH and FSH were significantly higher in patients than controls (12.8±9.7 and 20.8±14.8 mlU/ml versus 6.3±3.1 and 7.7±3.9 mlU/ml, P=0.000, respectively). Hypergonadotrophic hypogonadism and hypogonadotrophic hypogonadism patients showed lower levels of testosterone compared to the normal reference value (0.9±0.5 and 0.5±0.4 ng/ml versus 2.0-7.0 ng/ml). Higher levels of LH and FSH were recorded in hypergonadotrophic hypogonadism (24.5±2.6 and 37.4±6.7 mlU/ml) compared to the reference values of 2.0-13.0 and 2.5-10.0 mlU/ml, respectively whereas LH and FSH levels were lower in hypogonadotrophic hypogonadism (0.6±0.4 and 0.6±0.5 mlU/ml, respectively). In this context, all hypergonadotrophic hypogonadism and hypogonadotrophic hypogonadism patients showed abnormal levels of testosterone, LH and FSH.Conclusions: Abnormal levels of serum testosterone, LH and FSH, particularly in hypergonadotrophic hypogonadism and hypogonadotrophic hypogonadism were identified in infertile men with Sertoli cell only syndrome from Gaza Strip.J MEDICINE January 2017; 18 (1) : 21-26
The purpose of this study was to evaluate the aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity. A total of 108 males (65 males suffering from reduced fecundity as cases and 43 proven fertile males as a control) were included in the study. Thirty samples were subjected to 450K arrays as a screening phase, and then, three CpG sites located in the following genes: TYRO3, CGβ and FAM189A1 were selected to validate on 78 samples using deep bisulphite sequencing. A significant difference in the methylation level was found between cases and controls at all CpGs in TYRO3 gene-related amplicon (CpG1, p ≤ .003, CpG2, p ≤ .0001, CpG3, p ≤ .003 and CpG4, p ≤ .030) and CpG1 in CGβ gene-related amplicon (p ≤ .0001). Besides, a significant difference was found at two CpGs (CpG1, p ≤ .004 and CpG2, p ≤ .002) tested in the FAM189A1 gene-related amplicon. A significant correlation was found between the methylation level at CpG1 in the FAM189A1 gene and the different types of sperm motility. In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was showed. In addition, a relationship between variations in the methylation level of these CpGs and sperm motility has been observed.
This study was designed to evaluate the DNA methylation patterns and gene expression in spermatozoa from subfertile males. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then three CpG sites (cg07227024, cg05813498 and cg23081194) have the highest difference in methylation levels and located within ALS2CR12, GAA and UBE2G2 genes respectively; these were selected for further analysis using deep bisulphite sequencing and qPCR in 136 samples (64 proven fertile males "controls" and 72 subfertile males "cases"). A significant difference in the methylation level was found between cases and controls at two CpGs, six CpGs and three CpGs in ALS2CR12, GAA and UBE2G2 gene-related amplicon respectively. Besides, the qPCR results showed a significant change in the expression levels of GAA, UBE2G2 and ALS2CR12 gene in cases compared to the controls (p ≤ .00001). In conclusion, the methylation levels at CpGs in GAA, UBE2G2 and ALS2CR12 gene amplicons were significantly different in subfertile compared to proven fertile males. In addition, a significantly different was showed in the expression levels of GAA, UBE2G2 and ALS2CR12 genes in subfertile males compared to proven fertile males.
The aim of this study was to assess the stability of nuclear and mitochondrial DNA (n-DNA and mt-DNA) of spermatozoa under freeze-thawing and to find out the correlation between them and their association with standard sperm parameters. Forty-three semen samples were collected from fertile (G.1; n = 29) and sub-fertile (G.2; n = 14). N-DNA fragmentation was determined by TUNEL assay and mt-DNA using caspase 3 staining. Each semen sample was frozen at -196°C by the programmed freezer. Freeze-thawing decrease vitality, total motility and membrane integrity from (43.02 ± 22.74%; 31.63 ± 18.15%; 51.5 ± 24.82%) to (22.71 ± 17.3%; 9.21 ± 6.61%; 34.64 ± 19.92% respectively [p < .001]). G.1 native spermatozoa stained positive with TUNEL and caspase 3 were (14.85 ± 17.6% and 5.8 ± 11.59%) and increased after freeze-thawing to 27.54 ± 19.74% (p = .004) and 7.3 ± 6.13% (p = .01) respectively. In G.2, TUNEL and caspase 3 were (19.84 ± 17.52% and 7.53 ± 8.56%) and increased to (29.48 ± 16.97% [p = .03] and 10.21 ± 11.73%). In conclusion, freeze-thawing process affects not only semen parameters but also n-DNA and mt-DNA. Therefore, n-DNA and mt-DNA could be used as sensitive parameters for assessment of the cryodamage of 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.
Background: Intracytoplasmic sperm injection needs sufficient oocytes of high quality in order to increase the rate of fertilization and pregnancy. This study was designed to investigate the influence of maternal age on the ICSI outcomes in women undergoing to first ICSI cycle and to evaluate the influence of maternal age on global DNA methylation.Methods: A total of 242 females were included in this study with a mean age of 30.5±7.3 years. The participants were divided into three groups depending on women's age≤25, N=70; 26-35, N=102 and>35, N=70). The genomic DNA was isolated from the blood samples, then the global DNA methylation was evaluated using ELISA.Results: A significant reduction has been found in the level of anti-Müllerian hormone (AMH), total number of the collected oocyte, mature oocytes, fertilized oocytes and number of embryos transferred in the older females compared to the younger group (p<0.001). While a significant increase has been found in global DNA methylation level in the older females compared to the younger group (p<0.001). A positive significant correlation has been found between global DNA methylation level and maternal age (p<0.001). In contrast, a negative significant correlation has been shown between AMH level, mature oocytes and maternal age (p<0.001).Conclusions: Maternal age has a significant influence on the number of mature oocytes, number of embryos transferred and global DNA methylation. The pregnancy chance is more in the age group less than 35 years.
an increase in global spermatozoa DNA methylation and a strong bias toward regional loss of methylation at sites known to be impacted by aging (7).It has been reported that male age was associated with alterations in sperm DNA methylation levels at 1,698 CpGs and 1,146 regions, which were associated AbstractObjective: This study was performed to (I) evaluate the potential effect of advanced paternal age on global DNA methylation in spermatozoa, and (II) to investigate the association between the outcome of intracytoplasmic sperm injection (ICSI), semen parameters, and advanced paternal age. Material and Methods:This study comprised 230 semen samples collected from males with a mean age of 38.2±8.5 years.Medical records were used to gather clinical information related to the female partner.The participants were divided into three groups depending on age: age <30 years; age 30-40 years; and age >40 years.The DNA was extracted from purified spermatozoa.Then the sperm global DNA methylation, sperm DNA fragmentation, and chromatin decondensation were evaluated by an ELISA, TUNEL, and Chromomycin A3 staining, respectively. Results:The sample counts were n=50 (21.8%), n=90 (39.1%) and n=90 (39.1%) for the <30, 30-40 and >40 year age-groups, respectively.A significant variation was found in the age of males included in this study (p<0.001).There was a significant reduction in sperm count, total motility, and non-progressive motility in the older group compared to the younger group (p<0.001).There was also a significant elevation in chromatin decondensation, DNA fragmentation, and global DNA methylation of spermatozoa in the older age group (p<0.001).Finally, there was a significant positive correlation between the percentage of non-motile sperm, sperm chromatin decondensation, DNA fragmentation, global DNA methylation status, and paternal age (p<0.001). Conclusion:These results suggest that advanced paternal age increased the DNA fragmentation, chromatin decondensation, and global DNA methylation level in human spermatozoa, which negatively affects the ICSI outcomes in couples undergoing ICSI cycles.(
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