Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment
Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment
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DOI：
10.1093/humrep/deu040
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发表时间：
2014-05-01
影响因子：
6.1
通讯作者：
Carrell, D. T.
中科院分区：
文献类型：
--
作者：
Simon, L.;Liu, L.;Carrell, D. T.
在辅助生殖治疗（ART）中，通过三种不同的检测方法测量的精子DNA损伤、精子核蛋白含量与临床结果之间是否存在关联？在我们的单机构研究中，通过末端脱氧核苷酸转移酶介导的dUTP镍端标记（TUNEL）和Comet测定的精子DNA损伤与ART结果显著相关。鱼精蛋白异常表达与精子DNA损伤和男性不育有关。许多研究表明，精子DNA损伤与抗逆转录病毒治疗结果之间存在显著关系。到目前为止，还没有大型研究在同一研究人群中提供DNA损伤测试的直接比较。因此，每种方法的预后价值仍然未知。对2011年4月至2013年3月期间在美国犹他州大学生殖医学中心接受抗逆转录病毒治疗的238名不育夫妇进行横断面研究。接受抗逆转录病毒治疗的男性精子使用碱性彗星测定法、TUNEL和流式细胞仪染色质评价（FCCE）测定法检测DNA损伤。用苯胺蓝染色法分析组蛋白保留，用酸性尿素凝胶电泳法分析鱼精蛋白含量（蛋白P1和P2）和比例。计算各精子DNA检测对临床妊娠的预测价值。组蛋白滞留与精子DNA损伤（P 0.001）、胚胎质量下降（P 0.005）和临床妊娠（P 0.001）相关。通过TUNEL测定（15.04 1.16比8.79 0.56,P 0.001）和碱性彗星测定（72.79 2.49比55.86 2.29,P 0.001），未怀孕夫妇的精子中DNA损伤的平均百分比明显高于怀孕夫妇。临床妊娠与FCCE测定的DNA断裂指数无相关性（12.97 1.46比14.93 1.65;P 0.379）。在分析的鱼精蛋白参数中，只有P1/P2比值与精子数量（P 0.013）、男性年龄（P 0.037）、成熟度（P 0.049）和囊胚质量（P 0.012）相关。Comet法和TUNEL法测定组蛋白保留率和精子DNA损伤与受精率（p0.05）、胚胎质量（p0.05）和着床率（p0.05）相关。这项研究的一个潜在缺点是它是横断面的。一般来说，在这类研究中，可能导致这种结果的变量不止一个。分析精子是这个等式的一部分；还有一些女性因素也有可能影响抗逆转录病毒治疗的结果。因此，鉴于女性因素在不孕症中的重要作用，正常的精子DNA完整性和蛋白化并不一定确保抗逆转录病毒治疗的临床妊娠。因此，女性因素会降低精子DNA检测的预后价值。此外，我们使用天然精液而不是制备的精子可能会增加DNA损伤的医源性。精子核蛋白的改变影响精子DNA的完整性。此外，根据目前的数据集，TUNEL和Comet分析似乎比FCCE更能预测ART的成功。这项工作没有收到任何个人或直接的财政支持。作者声明没有利益竞争。
Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)?Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study.Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown.Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013.Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated.Histone retention was associated with sperm DNA damage (P 0.001), reduced embryo quality (P 0.005) and clinical pregnancies (P 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 1.16 versus 8.79 0.56; P 0.001) and alkaline Comet assay (72.79 2.49 versus 55.86 2.29; P 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 1.46 versus 14.93 1.65; P 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P 0.013), mens age (P 0.037), maturity (P 0.049) and blastocyst quality (P 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P 0.05), embryo quality (P 0.05) and implantation rate (P 0.05).A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage.Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE.No personal or direct financial support has been received for any of this work. The authors declare no competing interests.N/A.