无精子症患者精液Y染色体微缺失检测与分析

目的 建立用无精子症患者精液进行Y染色体微缺失检查的简便方法,并用于检测我国无精子症患者Y染色体微缺失的发生情况和位点.方法 无精子症患者241例,取精液标本(其中51例有血液标本),45例正常精液标本作为对照,快速裂解提取DNA,用4组多重PCR检测分布于AZFa、AZFb、AZFc区共15个序列标签位点(STS)的缺失.PCR产物测序证实无精子症患者精液为模板扩增的准确性,琼脂糖凝胶电泳观察精液标本和相应血液标本PCR产物的一致性.结果 所有多重PCR反应均成功进行,产物测序证实为目的 片段.正常精液未检测到Y染色体微缺失,而无精子症患者共检测到26例(10.8%)Y染色体微缺失,均有欧洲男科学会(EAA)和欧洲分子遗传学质量控制体系(EMQN)推荐STS位点的缺失:AZFa区、AZFb区各2例(各占7.7%),3例(11.5%)位于AZFb+AZFc区,19例(73.1%)位于AZFc区.血液标本检测结果与精液标本检测结果完全一致.结论 用无精子症患者精液进行Y染色体微缺失检查是可行、可靠的.初步表明EAA/EMQN推荐STS位点适合我国无精子症患者Y染色体微缺失检查.

Detection of Y chromosome microdeletions in semen of patients with azoospermia: study of 241 cases

Objective To set up a simple and reliable method to screen Y chromosome microdeletions in semen of azoospermic patients.And to explore the incidence and loci of Y chromosome microdeletions in Chinese azoospermia.Methods Two hundred and forty-one semen samples.51 containing blood.Were collected from 241 Chinese azoospermic patients.45 normal semen samples and 1 anticoagulated blood sample from female were used as controls.DNA was quickly abstracted by incubating the cells with a lysis buffer containing polymerase chain reaction(PCR)buffer and protease K,and was used to detect the deletion of 15 kinds of sequence tagged site(STs)distributed in AZFa,AZFb,and AZFc by 4 sets of multiplex PCR.Agarose electrophoresis was used to observe and compare the PCR products from the semen samples and their corresponding blood samples.And the PCR products from l semen sampie were confirmed by sequencing.Results All muhiplex PCR reactions were amplified successfullv.The sequencing of the PCR products from the semen samples confirmed the PCR reactions.No microdeletion was detected in the 45 normal semen samples.Microdeletion was found in 26 out of the 241 semen samples of azoospermic patients(10.8％):2 patients(7.7％)had the deletions located in AZFa,2 patients(7.7％)in AzFb,3 patients (11.5％)in both AZFb+AZFc,and other 19 patients(73.1％)in AZFc.The detection results of the blood samples were completely consistent with those of the semen samples.The STS deletion recommended by European Academy of Andrology(EAA)and European Molecular Genetics Quality Network(EMQN)were all found in these 26 cases.Conclusions The semen samples of azoospermie patients present a convenient,reliable,and noninvasive substitute for blood in screening of Y chromosome microdeletions.And can be employed in study and clinical examination.The EAA/EMQN recommendations Mlow the detection of complete AZF deletions in Chinese azoospermia.