Oxidative origin of sperm DNA fragmentation in the adult varicocele

ABSTRACT Purpose: Sperm DNA fragmentation is a major cellular mechanism underlying varicocele-related male infertility. However, the type of DNA fragmentation - whether oxidative or of another nature - remains unknown. Thus, the aim of this study was to evaluate single- and double-stranded sperm DNA fragmentation, and oxidative-induced sperm DNA damage in men with varicocele. Materials and Methods: A cross-sectional study was performed, including 94 normozoospermic adults, of which 39 men without varicocele (controls) and 55 men with varicocele grades II or III, uni- or bilaterally. All men collected semen by masturbation. After semen analysis, the remaining volume was used for evaluation of three types of sperm DNA damage: (i) total DNA fragmentation, using an alkaline comet assay, (ii) double-stranded DNA fragmentation, using a neutral comet assay, and (iii) oxidative DNA damage, using an alkaline comet assay associated with the DNA glycosylase formamidopyrimidine enzyme. In each assay, percentage of sperm with any degree of DNA fragmentation, and with high DNA fragmentation were compared between the groups using an unpaired Student's t test or a Mann-Whitney test. Results: The varicocele group presented a higher rate of sperm with fragmented DNA (both any and high DNA fragmentation), considering single-stranded DNA fragmentation, double-stranded DNA fragmentation, or a combination of both, as well as oxidative- induced DNA fragmentation. Conclusions: Patients with varicocele have an increase in sperm DNA fragmentation levels, particularly in oxidative stress-induced sperm DNA damage.摘要 目的：精子DNA碎片化是精索静脉曲张相关男性不育的核心细胞学机制之一，但目前尚未明确此类DNA碎片化的具体类型——究竟为氧化应激介导，抑或其他类型。因此本研究旨在评估精索静脉曲张患者的精子单链与双链DNA碎片化水平，以及氧化诱导的精子DNA损伤情况。 材料与方法：本研究为横断面研究，共纳入94名精液参数正常的成年男性，其中39名为无精索静脉曲张者（对照组），55名为单侧或双侧II~III级精索静脉曲张患者。所有受试者通过手淫方式采集精液。完成精液常规分析后，剩余样本用于检测三类精子DNA损伤：(i) 总DNA碎片化：采用碱性彗星试验（comet assay）；(ii) 双链DNA碎片化：采用中性彗星试验；(iii) 氧化DNA损伤：采用联合DNA糖苷酶甲酰胺基嘧啶（formamidopyrimidine enzyme）的碱性彗星试验。在各检测试验中，分别统计存在任意程度DNA碎片化的精子占比与高DNA碎片化的精子占比，组间比较采用非配对学生t检验或曼-惠特尼检验（Mann-Whitney test）。 结果：无论以单链DNA碎片化、双链DNA碎片化、二者联合，抑或氧化诱导DNA损伤作为评估指标，精索静脉曲张组的精子DNA碎片化（任意程度与高程度）发生率均显著高于对照组。 结论：精索静脉曲张患者的精子DNA碎片化水平升高，其中以氧化应激介导的精子DNA损伤尤为显著。