精子染色质扩散实验及吖啶橙染色检测精子DNA完整性研究

Medical Diagnosis 医学诊断, 2017, 7(3), 43-49

Published Online September 2017 in Hans. /journal/md

https:///10.12677/md.2017.73008

Sperm Chromatin Diffusion

Test and Detection of Sperm

DNA Integrity by Acridine

Orange Staining

Zhenya Fang, Meihua Zhang, Anna Li, Yi Qiu*

Key Laboratory of Birth Regulation and Control Technology of National Health and Family Planning Commission of China, The Maternal and Child Health Hospital of Shandong Province, Jinan Shandong

Received: Aug. 6th, 2017; accepted: Aug. 20th, 2017; published: Aug. 28th, 2017

Abstract

Objective: To analyze and compare the accuracy and utility of determining sperm DNA integrity by sperm chromatin dispersion (SCD) test and acridine orange staining test (AOT). Methods: The lev-el of DNA fragmentation was determined by SCD test and AOT in 32 adult healthy fertile men (control group) and 27 idiopathic oligozoospermia (IO) patients. Sperm nuclei with large DNA dispersion halos or with medium-sized halos were normal and nuclei with small-sized halos or no halo were abnormal. Sperm DNA strand breaks were tested by AOT of green for native DNA and red for denatured DNA. Results: The mean percentage of sperm nuclei with large halos, me-dium-sized halos, small-sized halos and no halo in patients with IO was (51.8 ± 17.1)%, (9.4 ±

8.6)%, (11.9 ± 5.8)% and (26.7 ± 10.6)%, but in control group it was (73.2 ± 6.2)%, (14.8 ± 5.7)%,

(6.8 ± 2.9)% and (5.3 ± 2.2)%, respectively, there was a significantly difference in the IO and con-

trol group (t = 4.256, P < 0.01; t = 5.1849, P < 0.05; t = 3.6967, P < 0.01; t = 5.7971, P < 0.01). IO pa-tients showed significantly higher DNA fragmentation (38.1% ± 9.5%) in their spermatozoa com-pared to healthy males (12.1% ± 5.2%, P < 0.01) determined by SCD test. No significant differences in the AOT outcome were seen between healthy males and IO patients (P > 0.05). Conclusion: The SCD is an effective test in sperm DNA fragmentation as a screening procedure to determine semen quality during basic infertility investigation for clinical use. The presence of sperm DNA fragmen-tation may lead to male infertility.

Keywords

Sperm Chromatin Dispersion (SCD) Test, Acridine Orange Staining Test, Sperm DNA Integrity,

Idiopathic Oligozoospermia, Male Infertility

*通讯作者。

房振亚 等

精子染色质扩散实验及吖啶橙染色检测精子DNA 完整性研究

房振亚，张美华，李安娜，邱 毅*

国家卫生计生委生育调控技术重点实验室，山东省妇幼保健院，山东 济南

收稿日期：2017年8月6日；录用日期：2017年8月20日；发布日期：2017年8月28日

摘 要

目的：采用精子染色质扩散(sperm chromatin dispersion, SCD)实验检测精子DNA 碎片，分析比较SCD 实验及吖啶橙染色实验(AOT)检测精子DNA 完整性的应用价值。方法：对32例正常成年已生育的男性和27例特发性少精子症(idiopathic oligozoospermia, IO)患者同时进行SCD 实验和AOT ，对检测结果进行分析。在SCD 实验中，DNA 完整性正常无损伤精子的DNA 扩散产生大晕环或中晕环，而DNA 损伤产生DNA 碎片的精子不产生或产生很小的晕环。AOT 染色实验中，正常精子DNA 为双链，染成绿色，不成熟或损伤精子DNA 为单链，染成红色、橙色或黄色。结果：SCD 实验检测结果为：IO 患者精子大晕环、中晕环、小晕环和无晕环精子百分率分别平均为(51.8 ± 17.1)%、(9.4 ± 8.6)%、(11.9 ± 5.8)%和(26.7 ± 10.6)%，健康对照组平均为(73.2 ± 6.2)%、(14.8 ± 5.7)%、(6.8 ± 2.9)%及(5.3 ± 2.2)%，二组比较，差异有统计学意义(t = 4.256, P < 0.01; t = 5.1849, P < 0.05; t = 3.6967, P < 0.01; t = 5.7971, P < 0.01)，DNA 损伤精子百分率，IO 患者为38.1% ± 9.5%，正常生育男性为12.1% ± 5.2%，二者比较有显著性差异(P < 0.01)。AOT 的检测结果为：IO 患者的单链DNA 精子比率和正常生育男性的比较，无显著差异(P > 0.05)。结论：精子DNA 完整性异常可导致男性不育。SCD 实验是一种有效的精子DNA 完整性检测方法。

关键词

精子染色质扩散(SCD)实验，精子吖啶橙染色实验，精子DNA 完整性，特发性少精子症，男性不育

Copyright © 2017 by author(s) and Hans Publishers Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY). /licenses/by/4.0/

1. 引言

精子DNA 是精子遗传物质的载体，近年来的研究表明精子DNA 的完整性能够影响精子的受精能力、受精卵的分裂以及胚胎的发育。精子DNA 完整性对自然受孕、人工授精、胚胎、胎儿、婴儿甚至成人的发育至关重要。研究表明精子DNA 完整性是优于常规精液分析的独立参数[1]，与辅助生殖技术结局有相关性[2]。因此，对男性不育患者进行精子DNA 完整性分析，有助于探讨或发现其不育的原因，为患者的预防和治疗提供咨询及诊断依据。

本研究对少精子症(idiopathic oligozoospermia, IO)患者同时应用精子染色质扩散(SCD)实验和吖啶橙染色实验(AOT)两种方法检测精子DNA 完整性，探讨男性不育机理，并通过比较两种方法的检测结果，