父源印记基因H19印记控制区域DNA甲基化与少、弱精子症相关性分析  

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

作　　者：李建波[1] 李博[1] 梁新新[1] 王珺[1] 马夜肥[1] 张永琦[1] 刘正[1] 闵保华[1] 马旭辉[1] 王晓红[1] 

机构地区：[1]第四军医大学唐都医院妇产科生殖医学中心,陕西西安710038

出　　处：《中华男科学杂志》2013年第6期511-517,共7页National Journal of Andrology

基　　金："十二五"国家科技支撑计划(2012BAI32B01);"十二五"全军医学科研项目(BWS11J072)~~

摘　　要：目的:研究印记基因H19印记控制区域的DNA甲基化程度与男性少、弱精子症的相关性。方法:通过染色体核型分析和Y染色体微缺失检测排除染色体因素干扰,进一步借助精液常规参数、伊红染色以及精子形态等指标,筛选出18例单一因素少精子症患者(浓度<15×106/ml,其余指标均正常)和20例单一因素弱精子症患者(前向运动精子百分率<32%,其余指标均正常)用于DNA甲基化检测;提取精子样本全基因组DNA,进行亚硫酸氢盐处理、PCR扩增目的基因片段并测序;通过BIQ Analyzer软件对测序结果进行质控和DNA甲基化程度分析。20例正常生育男性精液标本作为对照组。结果:与对照组甲基化丢失率[(0.30±0.06)%]相比,少精子症患者的H19印记控制区域的DNA甲基化丢失程度显著增高[(9.19±2.45)%],尤其当精子浓度<3×106/ml时,差异达到极显著水平(P<0.01)。在弱精子症患者中H19印记控制区域的DNA甲基化丢失程度[(0.30±0.07)%]和模式均与对照组无显著差异(P=0.62)。进一步重点分析了CTCF6位点的DNA甲基化状态,少精子症患者的DNA甲基化丢失程度[(2.67±0.75)%]显著高于对照组[(0.05±0.03)%]和弱精子症组[(0.03±0.02)%],而后两者之间无显著差异(P=0.35)。结论:H19印记控制区域的DNA甲基化程度的降低与少精子症密切相关,且降低程度与精子浓度呈显著负相关,而与精子活力无关。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 factorinduced oligozoospermia （sperm concentration 〈 15 ×10^6/m]） 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 bisulrite, 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 in- creased significantly in the oligozoospermia patients （ [ 9.19 ± 2.45 ] %, P 〈 0.05 ）, especially in the severe oligozoospermia males with sperm concentration 〈 3 ×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 asthenozo- ospermia 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.

关 键 词：印记基因H19 DNA甲基化 男性不育 少精子症 弱精子症 

分 类 号：R698.2[医药卫生—泌尿科学]