Department of Obstetrics and Gynaecology, BC Women's Hospital and Health Centre, University of British Columbia, Room D414B, D6-4500 Oak Street, Vancouver, British Columbia, Canada V6H 3N1.
Reproduction. 2011 Jun;141(6):749-57. doi: 10.1530/REP-11-0008. Epub 2011 Mar 9.
Male factor infertility has been associated with abnormal DNA methylation at imprinted genes. Little information is available on the status of imprinting in the sperm of men with azoospermia, including the association between aberrant imprinting and obstructive azoospermia (OA) or non-OA (NOA). Analysis of DNA methylation at imprinted genes in the sperm of men undergoing vasectomy reversal would aid determination of whether aberrant imprinting is associated with obstruction. Testicular sperm was retrieved from testicular biopsies obtained from men with azoospermia (N=18), including OA (N=10), NOA (N=5), and unknown pathology (N=3), and from men undergoing vasectomy reversal (N=17). Sperm was also obtained from proven fertile men (N=9). DNA methylation was investigated at multiple CpG sites within the differentially methylated regions (DMRs) of three imprinted genes, H19, IG-GTL2 and MEST, using bisulphite sequencing. Unique clones representative of single cells were analyzed. We found a significant decrease in DNA methylation at the H19 DMR in testicular sperm of azoospermic men compared with proven fertile men. The decrease was also significant between OA and proven fertile men, and between men undergoing vasectomy reversal and proven fertile men, suggesting that aberrant DNA methylation may be associated with obstruction. Changes in DNA methylation at IG-GTL2 and MEST DMRs among groups were not significant. Our data suggest that imprinting abnormalities may be associated with obstruction and may occur in response to changes in testicular environment and not only spermatogenesis failure, as previously reported. Methylation at the H19 DMR was particularly prone to modification in testicular sperm.
男性因素不育与印迹基因的异常 DNA 甲基化有关。关于无精子症男性精子印迹的状态，包括异常印迹与梗阻性无精子症（OA）或非梗阻性无精子症（NOA）之间的关系，信息有限。分析行输精管复通术男性精子中的印迹基因 DNA 甲基化有助于确定异常印迹是否与梗阻有关。从无精子症男性（N=18），包括 OA（N=10）、NOA（N=5）和未知病理学（N=3）的睾丸活检中获取睾丸精子，以及从行输精管复通术的男性（N=17）中获取精子。还从已证实的生育男性（N=9）中获取精子。使用亚硫酸氢盐测序法，在三个印迹基因（H19、IG-GTL2 和 MEST）的差异甲基化区域（DMR）内的多个 CpG 位点研究 DNA 甲基化。分析了代表单细胞的独特克隆。我们发现与已证实的生育男性相比，无精子症男性睾丸精子中的 H19 DMR 的 DNA 甲基化显著降低。OA 与已证实的生育男性之间，以及行输精管复通术的男性与已证实的生育男性之间的差异也具有统计学意义，这表明异常 DNA 甲基化可能与梗阻有关。各组间 IG-GTL2 和 MEST DMR 中的 DNA 甲基化变化无统计学意义。我们的数据表明，印迹异常可能与梗阻有关，并且可能是对睾丸环境变化的反应，而不仅仅是以前报道的精子发生失败。H19 DMR 的甲基化特别容易在睾丸精子中发生改变。
