ARSci  Vol.3 No.4 , November 2015
Increased DNA Fragmentation in Patients with Infertility in Dakar (Senegal)
Abstract: The presence of DNA fragmentation in semen is associated with poor results in ART (Assisted Re-productive Technology). This has led to the introduction of sperm DNA integrity testing in the clinical assessment of male infertility. The TUNEL assay is one of the most commonly used tests to measure DNA fragmentation in research studies, but it is now being optimized for routine clinical use. So, the objectives of our study were to install the TUNEL assay in our laboratory and to use it to assess the degree of DNA fragmentation in our patients dealing with couple infertility. Forty seven (47) and 25 samples from patients followed up for infertility and known fertile men were analyzed between August 2013 and July 2015 respectively. The sperm samples were evaluated according to the World Health Organization Guidelines. Sperm DNA damage was assessed by the TUNEL assay, with a standardized protocol that we adapted for reading using fluorescence microscopy instead of flow cytometry. The Sperm DNA Fragmentation Index was significantly higher in patients with infertility compared to the controls (22.2% ± 5.6% vs. 16.7% ± 0.7%) with a p < 0.05. This Index was also high in patients with varicocele compared to the control group (22.8% ± 9.2 vs. 16.7% ± 0.7%) (p < 0.01). After establishing the ROC curve (Receiver Operating Characteristics curve), we found a 15% threshold of DNA fragmentation, separating the patients with infertility from the control group. In our study, a 15% threshold positivity of DNA fragmentation using the TUNEL assay was highly associated with infertility. The results indicate that high level DNA fragmentation is associated with infertility.
Cite this paper: Diallo, M. , Faye, O. , Diallo, A. , Diallo, Y. and Diao, B. (2015) Increased DNA Fragmentation in Patients with Infertility in Dakar (Senegal). Advances in Reproductive Sciences, 3, 97-105. doi: 10.4236/arsci.2015.34012.

[1]   Aitken, R.J., Irvine, D.S. and Wu, F.C. (1991) Prospective Analysis of Sperm-Oocyte Fusion and Reactive Oxygen Species Generation as Criteria for the Diagnosis of Infertility. American Journal of Obstetrics and Gynecoly, 164, 542- 551.

[2]   Benchaib, M., Braun, V., Lornage, J., Hadj, S., Salle, B., Lejeune, H. and Guérin, J.F. (2003) Sperm DNA Fragmentation Decreases the Pregnancy Rate in an Assisted Reproductive Technique. Human Reproduction, 18, 1023-1028.

[3]   Bungum, M., Humaidan, P., Spano, M., Jepson, K., Bungum, L. and Giwercman, A. (2004) The Predictive Value of Sperm Chromatin Structure Assay (SCSA) Parameters for the Outcome of Intrauterine Insemination, IVF and ICSI. Human Reproduction, 19, 1401-1408.

[4]   Carrell, D.T., Liu, L., Peterson, C.M., Jones, K.P., Hatasaka, H.H., Erickson, L. and Campbell, B. (2003) Sperm DNA Fragmentation Is Increased in Couples with Unexplained Recurrent Pregnancy Loss. Archives of Andrology, 49, 49-55.

[5]   Zini, A., Blumenfeld, A., Libman, J. and Willis, J. (2005) Beneficial Effect of Microsurgical Varicocelectomy on Human Sperm DNA Integrity. Human Reproduction, 20, 1018-1015.

[6]   Ahmadi, A. and Ng, S.C. (1999) Fertilizing Ability of DNA-Damaged Spermatozoa. Journal of Experimental Zoology, 284, 696-704.<696::aid-jez11>;2-e

[7]   Sakkas, D. and Alvarez, J.G. (2010) Sperm DNA Fragmentation: Mechanisms of Origin, Impact on Reproductive Outcome, and Analysis. Fertility and Sterility, 93, 1027-1036.

[8]   Sharma, R.K., Sabanegh, E., Mahfouz, R., Gupta, S., Thiyagarajan, A. and Agarwal, A. (2010) TUNEL as a Test for Sperm DNA Damage in the Evaluation of Male Infertility. Journal of Urology, 76, 1380-1386.

[9]   Sergerie, M., Laforest, G., Bujan, L., Bissonnette, F. and Bleau, G. (2005) Sperm DNA Fragmentation: Threshold Value in Male Fertility. Human Reproduction, 20, 3446-3451.

[10]   Henkel, R., Kierspel, E., Hajimohammad, M., Stalf, T., Hoogendijk, C., Mehnert, C., Menkveld, R, Schill, W.B. and Kruger, T.F. (2004) DNA Fragmentation of Spermatozoa and Assisted Reproduction Technology. Reproductive Biomedicine Online, 7, 477-484.

[11]   Benchaib, M., Lornage, J., Mazoyer, C., Lejeune, H., Salle, B. and Guerin, J.F. (2007) Sperm Deoxyribonucleic Acid Fragmentation as a Prognostic Indicator of Assisted Reproductive Technology Outcome. Fertility and Sterility, 87, 93- 100.

[12]   Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari, G., Verlengia, C., Dondero, F. and Lenzi, A. (2000) Study of Apoptotic DNA Fragmentation in Human Spermatozoa. Human Reproduction, 15, 830-839.

[13]   WHO (2010) Laboratory Manual for the Examination and Processing of Human Semen. WHO Press, 5, 113.

[14]   Avendaño, C. and Oehninger, S. (2011) DNA Fragmentation in Morphologically Normal Spermatozoa: How Much Should We Be Concerned in the ICSI Era? Journal of Andrology, 32, 356-363.

[15]   Komiya, A., Kato, T., Kawauchi, Y., Watanabe, A. and Fuse, H. (2014) Clinical Factors Associated with Sperm DNA Fragmentation in Male Patients with Infertility. Scientific World Journal, 2014.

[16]   Practice Committee of the American Society for Reproductive Medicine (2013) The Clinical Utility of Sperm DNA Integrity Testing: A Guideline. Fertility and Sterility, 99, 673-677.

[17]   Choi, W.S. and Kim, S.W. (2013) Current Issues in Varicocele Management: A Review. The World Journal of Men’s Health, 31, 12-20.

[18]   Saleh, R.A., Agarwal, A., Nada, E.A., El-Tonsy, M.H., Sharma, R.K., Meyer, A., Nelson, D.R. and Thomas, A.J. (2003) Negative Effects of Increased Sperm DNA Damage in Relation to Seminal Oxidative Stress in Men with Idiopathic and Male Factor Infertility. Fertility and Sterility, 79, 1597-1605.

[19]   Blumer, C.G., Fariello, RM., Restelli, A.E., Spaine, D.M., Bertolla, R.P. and Cedenho, A.P. (2008) Sperm Nuclear DNA Fragmentation and Mitochondrial Activity in Men with Varicocele. Fertility and Sterility, 90, 1716-1722.

[20]   Wang, X., Sharma, R.K., Sikka, S.C., Thomas Jr., A.J., Falcone, T. and Agarwal, A. (2003) Oxidative Stress Is Associated with Increased Apoptosis Leading to Spermatozoa DNA Damage in Patients with Male Factor Infertility. Fertility and Sterility, 80, 531-535.

[21]   Smith, R., Kaune, H., Parodi, D., Madariaga, M., Rios, R., Morales, I. and Castro, A. (2006) Increased Sperm DNA Damage in Patients with Varicocele: Relationship with Seminal Oxidative Stress. Human Reproduction, 21, 986-993.

[22]   Smit, M., Romijn, J.C., Wildhagen, M.F., Veldhoven, J.L., Weber, R.F. and Dohle, G.R. (2010) Decreased Sperm DNA Fragmentation after Surgical Varicocelectomy Is Associated With Increased Pregnancy Rate. Journal of Urology, 183, 270-274.

[23]   Li, F., Yamaguchi, K., Okada, K., Matsushita, K., Ando, M., Chiba, K., Yue, H. and Fujisawa, M. (2012) Significant Improvement of Sperm DNA Quality after Microsurgical Repair of Varicocele. Systems Biology in Reproductive Medecine, 58, 274-277.

[24]   Kadioglu, T.C., Aliyev, E. and Celtik, M. (2014) Microscopic Varicocelectomy Significantly Decreases the Sperm DNA Fragmentation Index in Patients with Infertility. BioMed Research International, 2014.

[25]   Werthman, P., Wixon, R., Kasperson, K.B.S. and Evenson, D.P. (2008) Significant Decrease in Sperm Deoxyribonucleic Acid Fragmentation after Varicocelectomy. Fertility and Sterility, 90, 1800-1804.

[26]   Pasqualotto, F.F., Sharma, R.K., Nelson, D.R., Thomas, A.J. and Agarwal, A. (2000) A Relationship between Oxidative Stress, Semen Characteristics, and Clinical Diagnosis in Men Undergoing Infertility Investigation. Fertility and Sterility, 73, 459-464.

[27]   Zini, A., Finelli, A., Phang, D. and Jarvi, K. (2000) Influence of Semen Processing Technique on Human Sperm DNA Integrity. Urology, 56, 1081-1084.

[28]   Agarwal, A., Saleh, R.A. and Bedaiwy, M.A. (2003) Role of Reactive Oxygen Species in the Pathophysiology of Human Reproduction. Fertility and Sterility, 79, 829-843.

[29]   Moustafa, M.H., Sharma, R.K., Thornton, J., Mascha, E., Abdel-Hafez, M.A., Thomas Jr., A.J. and Agarwal, A. (2004) A Relationship between ROS Production, Apoptosis and DNA Denaturation in Spermatozoa from Patients Examined for Infertility. Human Reproduction, 19, 129-138.

[30]   Higuchi, Y. (2003) Chromosomal DNA Fragmentation in Apoptosis and Necrosis Induced by Oxidative Stress. Biochemical Pharmacology, 66, 1527-1535.

[31]   Kwon, Y.W., Masutani, H., Nakamura, H., Ishii, Y. and Yodoi, J. (2003) Redox Regulation of Cell Growth and Cell Death. Biological Chemistry, 384, 991-996.

[32]   Sills, E.S., Fryman, J.T., Perloe, M., Michels, K.B. and Tucker, M.J. (2004) Chromatin Fluorescence Characteristics and Standard Semen Analysis Parameters: Correlations Observed in Andrology Testing among 136 Males Referred for Infertility Evaluation. Journal of Obstetrics and Gynaecoly, 24, 74-77.

[33]   Cohen-Bacrie, P., Belloc, S., Ménézo, Y.J., Clement, P., Hamidi, J. and Benkhalifa, M. (2009) Correlation between DNA Damage and Sperm Parameters: A Prospective Study of 1633 Patients. Fertility and Sterility, 91, 1801-1805.

[34]   Saleh, R.A., Agarwal, A., Nelson, D.R., Nada, E.A., El-Tonsy, M.H., Alvarez, J.G., Thomas Jr., A.J. and Sharma, R.K. (2002) Increased Sperm Nuclear DNA Damage in Normozoospermic Infertile Men: A Prospective Study. Fertility and Sterility, 78, 313-318.

[35]   Muratori, M., Piomboni, P., Baldi, E., Filimberti, E., Pecchioli, P., Moretti, E., Gambera, L., Baccetti, B., Biagiotti, R., Forti, G. and Maggi, M. ( 2000) Functional and Ultrastructural Features of DNA-Fragmented Human Sperm. Journal of Andrology, 21, 903-912.

[36]   Erenpreiss, J., Jepson, K., Giwercman, A., Tsarev, I., Erenpreisa, J. and Spano, M. (2004) Toluidine Blue Cytometry Test for Sperm DNA Conformation: Comparison with the Flow Cytometric Sperm Chromatin Structure and TUNEL Assays. Human Reproduction, 19, 2277-2282.

[37]   Domínguez-Fandos, D., Camejo, M.I., Ballescà, J.L. and Oliva, R. (2007) Human Sperm DNA Fragmentation: Correlation of TUNEL Results as Assessed by Flow Cytometry and Optical Microscopy. Cytometry A, 71, 1011-1018.

[38]   Muratori, M., Forti, G. and Baldi, E. (2008) Comparing Flow Cytometry and Fluorescence Microscopy for Analyzing Human Sperm DNA Fragmentation by TUNEL Labeling. Cytometry A, 73, 785-787.

[39]   Kalyuzhny, A.E. (2011) Combination of TUNEL Assay with Immunohistochemistry for Simultaneous Detection of DNA Fragmentation and Oxidative Cell Damage. Methods in Molecular Biology, 682, 15-27.

[40]   Mitchell, L.A., De Iuliis, G.N. and Aitken, R.J. (2011) The TUNEL Assay Consistently Underestimates DNA Damage in Human Spermatozoa and Is Influenced by DNA Compaction and Cell Vitality: Development of an Improved Methodology. International Journal of Andrology, 34, 2-13.

[41]   Sergerie, M., Mieusset, R., Daudin, M., Thonneau, P. and Bujan, L. (2006) Ten-Year Variation in Semen Parameters and Sperm Deoxyribonucleic Acid Integrity in a Healthy Fertile Man. Fertility and Sterility, 86, 1513.e11-1513.e18.