多重连接探针在无精子症和严重少精子症患者AZF微缺失检测中的应用

目的:探讨多重连接探针扩增技术(MLPA)在无精子症及严重少精子症不育男性无精子因子(AZF)微缺失筛查中的应用可能。方法:提取147例无精子症或严重少精子症患者及154例正常对照男性外周血DNA,经95℃变性后与设计合成的AZF区域探针特异杂交,杂交产物经连接酶连接后用带有FAM荧光标记的通用引物扩增,毛细管电泳将产物分离生成MLPA图谱。所有样本同时行AZF序列标签位点(STS)的多重PCR分析。结果:病例组STS缺失检出率为15.0%(22/147),对照组中未检出STS缺失者;MLPA法于病例组中检出40例AZF区探针缺失患者,检出率为27.2%,其中包括22例STS缺失型患者。对照组中亦有20例AZF探针缺失者。结论:相比较传统的多重PCR,MLPA技术在AZF微缺失筛查中具有更佳的检测灵敏度,同时MLPA图谱所展现的高分辨的AZF区遗传学信息将有助于男性生精障碍病因学机制的深入探索。

Multiplex ligation-dependent probe amplification for detecting AZF microdeletions on the Y chromosome in infertile men with azoospermia or severe oligozoospermia

Objective: To investigate the possibility of applying multiplex ligation-dependent probe amplification(MLPA) to the detection of azoospermia factor(AZF) microdeletion on the Y chromosome in infertile men with azoospermia or severe oligozoospermia.Methods: DNA samples were obtained from 147 azoospermia or severe oligozoospermia patients and 154 normal controls.After denatured at 95 ℃,the samples were hybridized to the specific probes designed for the AZF region.With the ligase,the hybrid products were amplified by a pair of universal primers labeled with FAM fluorescence,and then separated by capillary electrophoresis for data analysis.Meanwhile all the samples were subjected to multiplex-PCR(mPCR) analysis for sequence-tagged sites(STS) in the AZF region.Results: STS deletion was detected in 22(15.0%) of the 147 patients but not in the normal controls.By MLPA,40(27.2%) of the patients were found with specific probe omission in the AZF region,as compared with 20 cases in the control group.Conclusion: Compared with mPCR,MLPA has a better sensitivity in detecting AZF microdeletions,and it provides more precise genetic information on the AZF regions,which may contribute to in-depth exploration into the etiological mechanism of impaired spermatogenesis.