精子DNA碎片指数与精浆外泌体代谢物的关联分析

Seminal plasma exosomes and sperm DNA fragmentation index:A metabolomics analysis

目的:探讨精子DNA碎片指数(DFI)高和低的两组男性精浆中外泌体包含的代谢物水平差异。方法:精浆外泌体代谢组学分析选择健康已婚已育,且DFI≤15%的5例男性作为对照组,匹配婚育情况、年龄及体质量指数(BMI)后选择DFI≥30%的5例男性为病例组。高效液相色谱串联质朴检测代谢物,多元统计分析差异代谢物,KEGG分析代谢产物富集通路。根据纳入排除标准另选11例男性作为对照组和20例男性作为病例组,液-质联用检测两组精浆中氨基酸和肉碱差异。结果:经过一级、二级分析及限定条件筛选后得到23种与精子DNA完整性相关的、存在精浆外泌体中的代谢物,包括9种有机酸,2种氨基酸中间代谢物,酰基肉碱、嘌呤、烟酸及其他中间产物等11种。KEGG富集分析发现23种代谢物主要参与鞘脂类信号通路、烟酸和烟酰胺代谢通路、精氨酸和脯氨酸代谢通路。进一步验证发现两组研究对象精浆中氨基酸水平无差异,病例组精浆中酰基肉碱水平低于对照组,其中游离肉碱、丙酰肉碱、3-羟基丁酰肉碱与丙二酰肉碱二者之和、3-羟基异戊酰肉碱与丁二酰肉碱之和、异戊(烯)酰肉碱的水平显著低于对照组(P<0.05)。结论:两组精浆外泌体中有机酸、氨基酸和酰基肉碱等代谢物水平存在明显差异。精浆中酰基肉碱水平与DFI高低显著相关,具有作为定量、快速检测的可评估精子DNA损伤程度的指标。

Objective:To investigate the difference in the levels of metabolites in the seminal plasma exosomes(SPE)of men with a high sperm DNA fragmentation index(DFI)from those with a low DFI.Methods:We performed a sperm exosomal metabolomics analysis of 5 healthy married men with DFI≤15%(the control group)and another 5 with DFI≥30%and matched in marital status,age and body mass index with the controls(the case group).Using high-performance liquid chromatography and mass spectrum,we examined the metabolites,observed their difference,and analyzed the metabolite enrichment pathway by Kyoto encyclopedia of genes and genomes(KEGG).According to the inclusion and exclusion criteria,we also selected 11 men in the control group and 20 men in the case group,and detected the differences in the seminal plasma amino acid and carnitine between the two groups using liquid measurement systems.Results:After primary and secondary analyses and qualified screening,23 metabolites related to sperm DNA integrity were obtained,including 9 organic acids,2 amino acid intermediate metabolites,and 11 acylcarnitine,purine,niacin and other intermediate products.KEGG enrichment analysis showed that 23 metabolites were mainly involved in the sphingoid signaling pathway,niacin and niacinamide metabolic pathway,and arginine and proline metabolic pathway.Further verification revealed no difference in the level of seminal plasma amino acid between the two groups,and significantly lower levels of seminal plasma acylcarnitine,free carnitine,propionylcarnitine,3-hydroxybutyrylcarnitine and malonylcarnitine,3-hydroxyisovalerylcarnitine and succinylcarnitine,and isoamyl(enylcarnitine)in the case group than in the controls(P<0.05).Conclusion:There are significant differences in the levels of the metabolites organic acids,amino acids and acylcarnitine in the SPE of males with a high DFI from those with a low DFI.The level of seminal plasma acylcarnitine is significantly correlated with sperm DFI,which can be used as an indicator in quantitative and rapid assessment of the degree of sperm DNA damage.