NRF2启动子甲基化与男性不育

目的探讨抗氧化基因NRF2启动子上游CpG岛内408⁹94 bp位置处的甲基化水平与男性不育之间的关系。方法酚氯仿法手提精子DNA,建立重亚硫酸盐修饰精子DNA后克隆测序法(即BSP-克隆测序法),选择正常男性及不育中少、弱、畸精症男性患者每份精液样本DNA的抗氧化基因NRF2的三个片段,每个片段选择15994 bp位置处的甲基化水平与男性不育之间的关系。方法酚氯仿法手提精子DNA,建立重亚硫酸盐修饰精子DNA后克隆测序法(即BSP-克隆测序法),选择正常男性及不育中少、弱、畸精症男性患者每份精液样本DNA的抗氧化基因NRF2的三个片段,每个片段选择15²0个正确的克隆结果并比较其甲基化水平。结果不育男性少、弱精组和正常精液组其抗氧化基因NRF2启动子上游40820个正确的克隆结果并比较其甲基化水平。结果不育男性少、弱精组和正常精液组其抗氧化基因NRF2启动子上游408⁹94 bp处99%甲基化位点均为非甲基化状态,正常男性精子DNA第7个甲基化位点缺失率(18.14%)明显比少弱精组(24.9%)缺失率低(P<0.05)。结论虽然正常生育男性及不育男性少、弱精患者抗氧化基因NRF2启动子上游408994 bp处99%甲基化位点均为非甲基化状态,正常男性精子DNA第7个甲基化位点缺失率(18.14%)明显比少弱精组(24.9%)缺失率低(P<0.05)。结论虽然正常生育男性及不育男性少、弱精患者抗氧化基因NRF2启动子上游408⁹94 bp区均呈现非甲基化状态但其第7个甲基化位点缺失可能与精子成熟障碍有关。

NRF2 promoter methylation and male infertility

Objective To compare the methylation levels of NRF2 gene promoter in sperm cells between the normozoospermia males and oligoasthenoteratozoospermia males. Methods The sperm DNA was extracted by proteinase-K-chloroform method. The bisulfite mix kit was used to process sperm DNA and 15-20 positive clones were selected and analyzed by PCR for sequencing analysis. Results Although the final statistical analysis found that more than 99%of the CPG loci were unmethylated, while loss rate of the seventh methylation loci in normal sperm（18.14%） was much lower than oligoasthenoteratozoospermia group（24.9%）, P〈0.05. Conclusions Both normozoospermia men and oligoasthenoteratozoospermia patients had largely unmethylated loci in the upstream 408 bp to 994 bp of NRF2 antioxidant gene promoter regions. However, the seventh methylation sites of sperm may be associated with sperm maturation.