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

Sperm Chromatin Diffusion Test and Detection of Sperm DNA Integrity by Acridine Orange Staining

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。

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 level 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, medium-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 control 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 patients showed significantly higher DNA fragmentation (38.1% ± 9.5%) in their spermatozoa compared 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 fragmentation may lead to male infertility.