Li Jian-Bo, Li Bo, Liang Xin-Xin, Wang Jun, Ma Ye-Fei, Zhang Yong-Qi, Liu Zheng, Min Bao-Hua, Ma Xu-Hui, Wang Xiao-Hong
Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, Shaanxi 710038, China.
Zhonghua Nan Ke Xue. 2013 Jun;19(6):511-7.
To study the correlation of the DNA methylation status of the imprinted gene H19 imprinting control region (ICR) with oligozoospermia and asthenozoospermia.
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.
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).
The reduced DNA methylation of the H19 ICR is negatively correlated with sperm concentration but not associated with sperm motility.
研究印记基因H19印记控制区（ICR）的DNA甲基化状态与少精子症和弱精子症的相关性。
通过核型分析和Y染色体微缺失检测排除染色体异常作为男性不育的原因，采用计算机辅助精子分析（CASA）鉴定18例单因素所致少精子症（精子浓度<15×10⁶/ml）患者和20例单因素所致弱精子症（前向运动精子<32%）患者。然后提取全基因组精子DNA，用亚硫酸氢盐处理，对目标基因片段进行PCR扩增和测序。最后，用BIQ Analyzer分析目标基因的DNA甲基化状态，并使用SPSS17.0处理数据。
与20名生育力正常的对照男性（[0.30±0.06]%）相比，少精子症患者H19 ICR的DNA甲基化水平显著升高（[9.19±2.45]%，P<0.05），尤其是精子浓度<3×10⁶/ml的严重少精子症男性（P<0.01）。然而，H19 ICR的DNA甲基化水平（[0.30±0.07]%）和模式在两组间无显著差异（P=0.62）。对CTCF-6结合位点的DNA甲基化状态进一步分析表明，少精子症男性（[2.67±0.75]%）的DNA甲基化程度显著高于生育力正常的对照（[0.05±0.03]%）或弱精子症组（[0.03±0.02]%），后两组间无显著差异（P=0.35）。
H19 ICR的DNA甲基化降低与精子浓度呈负相关，但与精子活力无关。
