Molecular karyotyping of human single sperm by array- comparative genomic hybridization.
Molecular karyotyping of human single sperm by array- comparative genomic hybridization.
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DOI：
10.1371/journal.pone.0060922
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Ferlin A
中科院分区：
文献类型：
--
作者：
Patassini C;Garolla A;Bottacin A;Menegazzo M;Speltra E;Foresta C;Ferlin A
目前还没有有效的方法来分析精子的整个基因组，包括非整倍体和结构染色体改变。在这里，我们描述了芯片比较基因组杂交（aCGH）在单个人类精子上的优化和应用。 aCGH程序涉及通过DNA微阵列筛选整个染色体补体，从而获得分子核型，目前用于研究和诊断临床实践（产前诊断、植入前遗传学诊断），但从未应用于精子。通过显微操作器从单个人类精子中提取DNA，通过全基因组扩增（WGA）进行解压缩和扩增，然后进行标记，与BAC阵列杂交，并通过微阵列扫描仪进行扫描。将该方案应用于来自正常精子捐献者的 129 个单精子，发现 7.8% 的精子具有不同的遗传异常，包括非整倍体和不同染色体的增益和丢失（不平衡精子）。相反，在三个月化疗周期结束时，来自霍奇金淋巴瘤男性的 130 个单精子中有 23.8% 不平衡。该方法的验证还包括对平衡易位男性的 43 个精子进行分析 [46,XY,t(2;12)(p11.2;q24.31)]，结果显示 18.6% 的精子中涉及易位的区域存在增益和缺失，16.3% 的精子中存在其他染色体的改变。该方法的未来应用可能会为正常受试者和产生不平衡精子风险较高的患者（例如不育男性、核型异常携带者、高龄男性、接受化疗的受试者以及在辅助生殖技术期间反复流产和反复失败的夫妇的伴侣）的精子发生和精子染色体畸变的生物学和病理生理学提供重要信息。
No valid method is currently available to analyze the entire genome of sperm, including aneuploidies and structural chromosomal alterations. Here we describe the optimization and application of array-Comparative Genomic Hybridization (aCGH) on single human sperm. The aCGH procedure involves screening of the entire chromosome complement by DNA microarray allowing having a molecular karyotype, and it is currently used in research and in diagnostic clinical practice (prenatal diagnosis, pre-implantation genetic diagnosis), but it has never been applied on sperm. DNA from single human sperm isolated by micromanipulator was extracted, decondensed and amplified by whole-genome amplification (WGA) and then labeled, hybridized to BAC array, and scanned by microarray scanner. Application of this protocol to 129 single sperm from normozoospermic donors identified 7.8% of sperm with different genetic anomalies, including aneuploidies and gains and losses in different chromosomes (unbalanced sperm). On the contrary, of 130 single sperm from men affected by Hodgkin lymphoma at the end of three months of chemotherapy cycles 23.8% were unbalanced. Validation of the method also included analysis of 43 sperm from a man with a balanced translocation [46,XY,t(2;12)(p11.2;q24.31)], which showed gains and losses corresponding to the regions involved in the translocation in 18.6% of sperm and alterations in other chromosomes in 16.3% of sperm. Future application of this method might give important information on the biology and pathophysiology of spermatogenesis and sperm chromosome aberrations in normal subjects and in patients at higher risk of producing unbalanced sperm, such as infertile men, carriers of karyotype anomalies, men with advanced age, subjects treated with chemotherapy, and partners of couples with repeated miscarriage and repeated failure during assisted reproduction techniques.
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