MD  >> Vol. 7 No. 3 (September 2017)

    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

  • 全文下载: PDF(506KB) HTML   XML   PP.43-49   DOI: 10.12677/MD.2017.73008  
  • 下载量: 245  浏览量: 666   科研立项经费支持

作者:  

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

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Sperm Chromatin Dispersion (SCD) Test Acridine Orange Staining Test Sperm DNA Integrity Idiopathic Oligozoospermia Male Infertility

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。

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.

文章引用:
房振亚, 张美华, 李安娜, 邱毅. 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[J]. 医学诊断, 2017, 7(3): 43-49. https://doi.org/10.12677/MD.2017.73008

参考文献

[1] Virro, M.R., Larson-Cook, K.L. and Evenson, D.P. (2004) Sperm Chromatin Structure Assay (SCSA) Parameters Are Related to Fertilization, Blastocyst Development, and Ongoing Pregnancy In Vitro Fertilization and Intracytoplasmic Sperm Injection Cycles. Fertility and Sterility, 81, 1289-1295. https://doi.org/10.1016/j.fertnstert.2003.09.063
[2] Calogero, A.E., Condorelli, R.A., Russo, G.I. and Vignera, S. (2017) Conservative No Hormonal Options for the Treatment of Male Infertility: Antibiotics, Anti-Inflammatory Drugs, and Antioxidants. Biomed Research International, 8, Article ID: 4650182.
[3] 世界卫生组织. 人类精液及精子宫颈粘液相互作用实验室检验手册[M]. 第4版. 北京: 科学出版社, 1999.
[4] 世界卫生组织. 男性不育标准化检查与诊疗手册[M]. 北京: 人民卫生出版社, 2007.
[5] Fernández, J.L., Muriel, L., Rivero, M.T., et al. (2003) The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation. International Journal of Andrology, 24, 59-66.
[6] Fernández, J.L., Muriel, L., Goyanes, V., et al. (2005) Simple Determination of Human Sperm DNA Fragmentation with an Improved Sperm Chromatin Dispersion Test. Fertility and Sterility, 84, 833-842. https://doi.org/10.1016/j.fertnstert.2004.11.089
[7] Gallegos, G., Ramos, B., Santiso, R., et al. (2007) Sperm DNA Fragmentation in Infertile Men with Genitourinary Infection by Chlamydia Trachomatis and Mycoplasma. Fertility and Sterility, 90, 328-334. https://doi.org/10.1016/j.fertnstert.2007.06.035
[8] Tejada, R.I., Mitchell, J.C., Norman, A., et al. (1984) A Test for the Practical Evaluation of Male Fertility by Acridine Orange (AO) Fluorescence. Fertility and Sterility, 42, 87-91. https://doi.org/10.1016/S0015-0282(16)47963-X
[9] 李福平, 岳焕勋, 徐克惠, 等. 精子核成熟度与精液参数之间的关系[J]. 中华男科学杂志, 2007(13): 179-180.
[10] 吴周亚, 王冲, 王信心. 生育与不育男性精子核DNA成熟度的比较研究[J]. 生殖医学杂志, 1999(3): 40-42.
[11] Eggert-Kruse, W., Rohr, G., Kerbel, H., et al. (1996) The Acridine Orange Test: A Clinically Relevant Screening Method for Sperm Quality during Infertility Investigation? Human Reproduction, 11, 784-789. https://doi.org/10.1093/oxfordjournals.humrep.a019255
[12] Chohan, K.R., Griffin, J.T., Lafromboise, M., et al. (2006) Comparison of Chromatin Assays for DNA Fragmentation Evaluation in Human Sperm. International Journal of Andrology, 27, 53-59. https://doi.org/10.2164/jandrol.05068