[Impact of mobile phone radiation on the quality and DNA methylation of human sperm in vitro].

OBJECTIVE: To investigate the influences of mobile phone radiation on the quality and DNA methylation of human sperm in vitro. METHODS: According to the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, we randomly selected 97 male volunteers with normal semen parameters and divided each semen sample from the subjects into two equal parts, one exposed to mobile phone radiation at 1950 M Hz, SAR3. 0 W/kg for 3 hours while the other left untreated as the control. We obtained routine semen parameters as well as the acrosomal reaction ability, apoptosis and DNA methylation of sperm, and compared them between the two groups. RESULTS: Compared with the control, the radiation group showed significantly decreased progressive sperm motility ([36.64 ± 16.93] vs [27.56 ± 16.92]%, P < 0.01) and sperm viability ([63.72 ± 16.35] vs [54.31 ± 17.35]%, P < 0.01) and increased sperm head defects ([69.92 ± 4.46] vs [71.17 ± 4.89]%, P < 0.05), but no significant differences in sperm acrosomal reaction ([66.20 ± 6.75] vs [64.50 ± 3.47]%, P > 0.05). The early apoptosis rate of sperm cells was remarkably higher in the radiation group ([6.89 ± 9.84]%) than in the control ([4.44 ± 5.89]%) (P < 0.05). However, no statistically significant differences were found between the control and radiation groups in the DNA methylation patterns of the paternal imprinting gene H19 ICR ([0.60 ± 0.02] vs [1.40 ± 0.03]%, P > 0.05) or the maternal imprinting gene KvDMR1 ([0.00 ± 0.00] vs [1.80 ± 0.031%, P > 0.05). CONCLUSION: Mobile phone radiation reduces the progressive motility and viability of human sperm and increases sperm head defects and early apoptosis of sperm cells.

Altered DNA methylation patterns of the H19 differentially methylated region and the DAZL gene promoter are associated with defective human sperm.

DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ) and asthenozoospermia (AZ) usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control), with only complete methylation and mild hypomethylation(<50% unmethylated CpGs) identified, and there was no significant difference in the occurrence of these two methylation patterns between AZ and NZ (P>0.05). However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs ) and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05%) and AZ (0.58±1.77%) (P<0.001).Additional, our data indicated the occurrence of >20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05). In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.

[Correlation of DNA methylation status of imprinted gene H19 ICR with oligozoospermia and asthenozoospermia].

OBJECTIVE: To study the correlation of the DNA methylation status of the imprinted gene H19 imprinting control region (ICR) with oligozoospermia and asthenozoospermia. METHODS: We eliminated chromosomal abnormality as the cause of male infertility in the subjects by karyotype analysis and detection of Y-chromosome microdeletions, and identified 18 cases of single factor-induced oligozoospermia (sperm concentration < 15 x 10(6)/ml) and 20 cases of single factor-induced asthenozoospermia (progressively motile sperm <32%) by computer-aided sperm analysis (CASA). Then we extracted genome-wide sperm DNA, treated it with bisul- fite, subjected the target gene fragments to PCR amplification and sequencing. Lastly, we analyzed the DNA methylation status of the target genes with BIQ Analyzer and processed the data using SPSS17.0. RESULTS: The DNA methylation level of the H19 ICR was increased significantly in the oligozoospermia patients ([9.19 +/- 2.45]%, P < 0.05), especially in the severe oligozoospermia males with sperm concentration < 3 x 10(6)/ml (P < 0.01), as compared with that of the 20 fertile control men ([0.30 +/- 0.06]%). However, no significant differences were found in the level ([0.30 +/- 0.07]%) and pattern of the DNA methylation of the H19 ICR (P = 0.62). Further analysis of the DNA methylation status of the CTCF-6 binding sites indicated that the DNA methylation degree was significant higher in the oligozoospermia men ([2.67 +/- 0.75]%) than in the fertile control ([0.05 +/- 0.03]%) or the asthenozoospermia group ([0.03 +/- 0.02]%), with no significant differences between the latter two (P = 0.35). CONCLUSION: The reduced DNA methylation of the H19 ICR is negatively correlated with sperm concentration but not associated with sperm motility.

Connexin37 mRNA expression in in vivo and in vitro mouse oocyte.

To evaluate gene expression of Connexin37 (Cx37) in oocytes from in vitro follicles at different stages, mouse preantral follicles were isolated and cultured for 12 days in vitro. Compared with in vitro follicles, follicles grown in vivo were collected at day 14 (d14), d16, d18, d20, d22 and d24 with the same stages for gene expression of Cx37 in oocytes. Our results showed that Cx37 mRNA increased along with follicular development, reached the highest level at the onset of antrum cavity formation and decreased after antrum formation in both in vivo and in vitro mouse oocytes. However, Cx37 mRNA was significant higher (p < 0.01) in in vitro cultured oocytes than in vivo oocytes. Moreover, significantly higher levels of Cx37 mRNA were found in oocytes from in vitro disrupted follicles (p < 0.01) and non-grown follicles (p < 0.05) than those from normal follicles with a similar size. These data determine temporal gene expression of Cx37 in oocytes from follicules at different stages and indicate that the gene expression level of Cx37 in oocytes could be evaluated as a criterion to the regulatory mechanism of Cx37 in an in vitro model.