Mauri A L, Oliveira J B A, Baruffi R L R, Petersen C G, Vagnini L D, Massaro F C, Silva L F I, Nicoletti A P M, Franco J G
Centre for Human Reproduction Prof Franco Jr., Ribeirao Preto, Brazil.
Int J Androl. 2011 Dec;34(6 Pt 1):594-9. doi: 10.1111/j.1365-2605.2010.01119.x. Epub 2010 Oct 24.
The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-strand or normal double-strand DNA in spermatozoa with extruded nuclear chromatin (ENC) selected by high magnification. Fresh semen samples from 55 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and ENC were selected at 8400× magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation was not significantly different (p = 0.86) between spermatozoa with ENC (19.6%) and those with NN (20%). However, the percentage of spermatozoa with detectable denatured-stranded DNA in the ENC spermatozoon group (59.1%) was significantly higher (p < 0.0001) than in the NN group (44.9%). The high level of denatured DNA in spermatozoa with ENC suggests premature decondensation and disaggregation of sperm chromatin fibres. The results show an association between ENC and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high-magnification intracytoplasmic sperm injection.
本研究的目的是确定通过高倍镜选择的具有核染色质挤出（ENC）的精子中DNA片段化的程度以及变性单链或正常双链DNA的存在情况。55例患者的新鲜精液样本通过不连续分离浓度梯度制备。在8400倍放大倍数下选择具有正常细胞核（NN）和ENC的精子，并置于不同载玻片上。通过TUNEL检测确定DNA片段化。通过吖啶橙荧光法鉴定变性和双链DNA。具有ENC的精子（19.6%）和具有NN的精子（20%）之间的DNA片段化无显著差异（p = 0.86）。然而，ENC精子组中可检测到变性双链DNA的精子百分比（59.1%）显著高于NN组（44.9%）（p < 0.0001）。具有ENC的精子中高水平的变性DNA表明精子染色质纤维过早解聚和分散。结果显示ENC与精子DNA损伤之间存在关联，并支持在高倍镜下进行常规形态学选择和注射活动精子的胞浆内单精子注射。
