Franco J G, Baruffi R L R, Mauri A L, Petersen C G, Oliveira J B A, Vagnini L
Centre for Human Reproduction Professor Franco Jr, Ribeirão Preto, São Paulo, Brazil.
Reprod Biomed Online. 2008 Jul;17(1):42-5. doi: 10.1016/s1472-6483(10)60291-x.
The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at x8400 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 in spermatozoa with LNV (29.1%) was significantly higher (P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher (P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.
本研究的目的是确定通过高倍放大筛选出的具有大核空泡（LNV）的精子中DNA片段化程度以及变性单链或正常双链DNA的存在情况。通过不连续分离浓度梯度制备来自30名患者的新鲜精液样本。在8400倍放大倍数下选择具有正常细胞核（NN）和LNV的精子，并置于不同载玻片上。通过TUNEL法测定DNA片段化。通过吖啶橙荧光法鉴定变性和双链DNA。具有LNV的精子中的DNA片段化（29.1%）显著高于具有NN的精子（15.9%）（P<0.001）。因此，低分子量DNA片段（单链和寡核小体）中的基因组DNA切割以及高分子量DNA中的单链断裂（切口）在具有LNV的精子中更频繁发生。同样，具有LNV的精子中变性单链DNA的百分比（67.9%）显著高于具有NN的精子（33.1%）（P<0.0001）。具有LNV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LNV与精子中的DNA损伤之间存在关联，并支持在高倍放大下对活动精子进行常规形态学筛选和注射用于卵胞浆内单精子注射（ICSI）。
