Андрология и генитальная хирургия. 2019; 20: 39-44
Фрагментация ДНК сперматозоидов у мужчин разного возраста
https://doi.org/10.17650/2070-9781-2019-20-4-39-44Аннотация
Цель исследования – определить содержание сперматозоидов с одно- и двунитевыми разрывами ДНК в образцах эякулята у пациентов разных возрастных групп.
Материалы и методы. Исследован уровень фрагментации ДНК в 300 образцах эякулята, полученных от 266 пациентов с нарушением фертильности. В 1-ю группу включены 150 образцов, полученных от 131 пациента моложе 45 лет (21–44 лет), во 2-ю группу – 150 образцов, полученных от 135 пациентов старше 45 лет (45–68 лет). Средний возраст мужчин составил 34,8 ± 3,9 и 48,6 ± 3,1 года соответственно. Количество сперматозоидов с фрагментированной ДНК оценивали методом флуоресцентного мечения одно- и двунитевых разрывов ДНК в мазках эякулята (методом TUNEL). Повышенным считали количество сперматозоидов с фрагментированной ДНК >15 %. Стандартное спермиологическое исследование выполняли у 117 и 97 мужчин 1-й и 2-й групп соответственно.
Результаты. Количество сперматозоидов с фрагментированной ДНК варьировало в образцах эякулята от 1,5 до 64,5 %. Среднее количество сперматозоидов с разрывами ДНК в 1-й группе (12,0 ± 6,0 %) статистически значимо ниже, чем во 2-й группе (16,1 ± 8,3 %, p <0,05). Среднее количество сперматозоидов в эякуляте в 1-й группе статистически значимо больше (267,0 ± 198,7 млн), чем во 2-й группе (201,0 ± 162,9 млн, p = 0,02).
Заключение. У мужчин в возрасте старше 45 лет содержание сперматозоидов с фрагментированной ДНК статистически значимо больше, чем у мужчин моложе 45 лет (p <0,05), что может косвенно свидетельствовать о повышенном уровне активных форм кислорода в семенной плазме.
Список литературы
1. Брагина Е.Е., Арифулин Е.А., Лазарева Е.М. и др. Нарушение конденсации хроматина сперматозоидов и фрагментация ДНК сперматозоидов: есть ли корреляция? Андрология и генитальная хирургия 2017;18(1):48–61. DOI: 10.17650/2070-9781-2017-18-1- 48-61.
2. WHO laboratory manual for the examination and processing of human semen. 5th edn. Geneva: World Health Organization, 2010. 271 р.
3. Zini A., Libman J. Sperm DNA damage: clinical significance in the era of assisted reproduction. CMAJ 2006;175(5): 495–500. DOI: 10.1503/cmaj.060218.
4. Vagnini L., Baruffi R.L., Mauri A.L. et al. The effects of male age on sperm DNA damage in an infertile population. Reprod Biomed Online 2007;15(5):514–9. DOI: 10.1016/s1472-6483(10)60382-3.
5. Plastira K., Msaouel P., Angelopoulou R. et al. The effects of age on DNA fragmentation, chromatin packaging and conventional semen parameters in spermatozoa of oligoasthenoteratozoospermic patients. J Assist Reprod Genet 2007;24(10):437–43. DOI: 10.1007/s10815-007-9162-5.
6. Sati L., Ovari L., Bennett D. et al. Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation. Reprod Biomed Online 2008;16(4):570–9. DOI: 10.1016/s1472-6483(10)60464-6.
7. Petersen C.G., Mauri A.L., Vagnini L.D. et al. The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples. JBRA Assist Reprod 2018;22(4):323–30. DOI: 10.5935/1518-0557.20180047.
8. Brahem S., Mehdi M., Elghezal H., Saad A. The effects of male aging on semen quality, sperm DNA fragmentation and chromosomal abnormalities in an infertile population. J Assist Reprod Genet 2011;28(5):425–32. DOI: 10.1007/s10815-011-9537-5.
9. Colin A., Barroso G., Gómez-López N. et al. The effect of age on the expression of apoptosis biomarkers in human spermatozoa. Fertil Steril 2010;94(7):2609–14. DOI: 10.1016/j.fertnstert.2010.04.043.
10. Moskovtsev S.I., Willis J., Mullen J.B. Age-related decline in sperm deoxyribonucleic acid integrity in patients evaluated for male infertility. Fertil Steril 2006;85(2): 496–9. DOI: 10.1016/j.fertnstert.2005.05.075.
11. Moskovtsev S.I., Willis J., White J., Mullen J.B. Sperm DNA damage: correlation to severity of semen abnormalities. Urology 2009;74(4):789–93. DOI: 10.1016/j.urology.2009.05.043.
12. Wyrobek A.J., Eskenazi B., Young S. et al. Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm. Proc Natl Acad Sci USA 2006;103(25):9601–6. DOI: 10.1073/pnas.0506468103.
13. Singh N.P., Muller C.H., Berger R.E. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril 2003;80(6):1420–30. DOI: 10.1016/j.fertnstert.2003.04.002.
14. Schmid T.E., Eskenazi B., Baumgartner A. et al. The effects of male age on sperm DNA damage in healthy non-smokers. Hum Reprod 2007;22(1):180–7. DOI: 10.1093/humrep/del338.
15. Nicoletti I., Migliorati G., Pagliacci M.C. et al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139(2):271–9. DOI: 10.1016/0022-1759(91)90198-o.
16. Winkle T., Rosenbusch B., Gagsteiger F. et al. The correlation between male age, sperm quality and sperm DNA fragmentation in 320 men attending a fertility center. J Assist Reprod Genet 2009;26(1):41–6. DOI: 10.1007/s10815-008-9277-3.
17. Agarwal A., Said T.M. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9(4):331–45. DOI: 10.1093/humupd/dmg027.
18. Aitken R.J., Clarkson J.S., Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 1989;41(1):183–97. DOI: 10.1095/biolreprod41.1.183.
19. Zandieh Z., Vatannejad A., Doosti M. et al. Comparing reactive oxygen species and DNA fragmentation in semen samples of unexplained infertile and healthy fertile men. Ir J Med Sci 2018; 187(3):657–62. DOI: 10.1007/s11845-017-1708-7.
20. Xie D., Lu C., Zhu Y. et al. Analysis on the association between sperm DNA fragmentation index and conventional semen parameters, blood microelements and seminal plasma ROS in male patients with infertility. Exp Ther Med 2018;15(6):5173–6. DOI: 10.3892/etm.2018.6115.
Andrology and Genital Surgery. 2019; 20: 39-44
Sperm DNA fragmentation in men of different age
https://doi.org/10.17650/2070-9781-2019-20-4-39-44Abstract
The study objective is to analyze the content of spermatozoa with single and double-stranded DNA breaks in different age groups.
Materials and methods. The level of DNA fragmentation was studied in 300 ejaculate samples obtained from 266 sub- or infertile men. The group 1 included 150 samples obtained from 131 patients under the age of 45 (21–44 years), the group 2 included 150 samples obtained from 135 patients above the age of 45 (45–68 years). Mean ages were 34.8 ± 3.9 and 48.6 ± 3.1 years, respectively. The number of sperm with fragmented DNA was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method on ejaculate smears. The number of spermatozoa with >15 % of fragmented DNA was considered elevated. Standard semen analysis was performed in 117 and 97 men from the groups 1 and 2, respectively.
Results. The number of sperm with fragmented DNA varied in ejaculated samples from 1.5 to 64.5 %. Mean number of sperm with DNA breaks in the group 1 (12.0 ± 6.0 %) was significantly lower than in the group 2 (16.1 ± 8.3 %, p <0.05). Mean sperm count in the ejaculate of the group 1 (267.0 ± 198.7 million) was significantly higher than in the group 2 (201.0 ± 162.9 million, p = 0.02).
Conclusion. We revealed that in men over the age of 45 years, the percentage of spermatozoa with DNA fragmentation is higher than in men under 45 years of age, it may indirectly indicate an increased level of reactive oxygen species in the seminal plasma in older patients.
References
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2. WHO laboratory manual for the examination and processing of human semen. 5th edn. Geneva: World Health Organization, 2010. 271 r.
3. Zini A., Libman J. Sperm DNA damage: clinical significance in the era of assisted reproduction. CMAJ 2006;175(5): 495–500. DOI: 10.1503/cmaj.060218.
4. Vagnini L., Baruffi R.L., Mauri A.L. et al. The effects of male age on sperm DNA damage in an infertile population. Reprod Biomed Online 2007;15(5):514–9. DOI: 10.1016/s1472-6483(10)60382-3.
5. Plastira K., Msaouel P., Angelopoulou R. et al. The effects of age on DNA fragmentation, chromatin packaging and conventional semen parameters in spermatozoa of oligoasthenoteratozoospermic patients. J Assist Reprod Genet 2007;24(10):437–43. DOI: 10.1007/s10815-007-9162-5.
6. Sati L., Ovari L., Bennett D. et al. Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation. Reprod Biomed Online 2008;16(4):570–9. DOI: 10.1016/s1472-6483(10)60464-6.
7. Petersen C.G., Mauri A.L., Vagnini L.D. et al. The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples. JBRA Assist Reprod 2018;22(4):323–30. DOI: 10.5935/1518-0557.20180047.
8. Brahem S., Mehdi M., Elghezal H., Saad A. The effects of male aging on semen quality, sperm DNA fragmentation and chromosomal abnormalities in an infertile population. J Assist Reprod Genet 2011;28(5):425–32. DOI: 10.1007/s10815-011-9537-5.
9. Colin A., Barroso G., Gómez-López N. et al. The effect of age on the expression of apoptosis biomarkers in human spermatozoa. Fertil Steril 2010;94(7):2609–14. DOI: 10.1016/j.fertnstert.2010.04.043.
10. Moskovtsev S.I., Willis J., Mullen J.B. Age-related decline in sperm deoxyribonucleic acid integrity in patients evaluated for male infertility. Fertil Steril 2006;85(2): 496–9. DOI: 10.1016/j.fertnstert.2005.05.075.
11. Moskovtsev S.I., Willis J., White J., Mullen J.B. Sperm DNA damage: correlation to severity of semen abnormalities. Urology 2009;74(4):789–93. DOI: 10.1016/j.urology.2009.05.043.
12. Wyrobek A.J., Eskenazi B., Young S. et al. Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm. Proc Natl Acad Sci USA 2006;103(25):9601–6. DOI: 10.1073/pnas.0506468103.
13. Singh N.P., Muller C.H., Berger R.E. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril 2003;80(6):1420–30. DOI: 10.1016/j.fertnstert.2003.04.002.
14. Schmid T.E., Eskenazi B., Baumgartner A. et al. The effects of male age on sperm DNA damage in healthy non-smokers. Hum Reprod 2007;22(1):180–7. DOI: 10.1093/humrep/del338.
15. Nicoletti I., Migliorati G., Pagliacci M.C. et al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139(2):271–9. DOI: 10.1016/0022-1759(91)90198-o.
16. Winkle T., Rosenbusch B., Gagsteiger F. et al. The correlation between male age, sperm quality and sperm DNA fragmentation in 320 men attending a fertility center. J Assist Reprod Genet 2009;26(1):41–6. DOI: 10.1007/s10815-008-9277-3.
17. Agarwal A., Said T.M. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9(4):331–45. DOI: 10.1093/humupd/dmg027.
18. Aitken R.J., Clarkson J.S., Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 1989;41(1):183–97. DOI: 10.1095/biolreprod41.1.183.
19. Zandieh Z., Vatannejad A., Doosti M. et al. Comparing reactive oxygen species and DNA fragmentation in semen samples of unexplained infertile and healthy fertile men. Ir J Med Sci 2018; 187(3):657–62. DOI: 10.1007/s11845-017-1708-7.
20. Xie D., Lu C., Zhu Y. et al. Analysis on the association between sperm DNA fragmentation index and conventional semen parameters, blood microelements and seminal plasma ROS in male patients with infertility. Exp Ther Med 2018;15(6):5173–6. DOI: 10.3892/etm.2018.6115.
