Inter-center variation in the efficiency of sperm DNA damage reduction following density gradient centrifugation

Carmen López-Fernández, Stephen D. Johnston, Altea Gosálbez, Jose Luís Fernández, Juan G. Álvarez, Jaime Gosálvez

Centro ANDROGEN, A Coru?a, Spain.
Department of Biology, Genetics Unit, University Autónoma of Madrid, Madrid, Spain.
Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, A Coru?a, Spain.
School of Agriculture and Food Science, The University of Queensland, Gatton, Australia.

DOI: 10.4236/ns.2013.57A003                

Abstract

This was a prospective multicenter study aiming at comparing the efficiency of sperm selection by density gradient centrifugation (DGC) in reducing sperm DNA fragmentation (SDF) in different ART centers. The study was designed using 290 semen samples collected from 10 different ART centers performing artificial insemination, in vitro fertilization and blind assessment of SDF at the University facilities. The results showed that while there was a significant reduction in the SDF levels in sperm isolated from the gradient centrifuged pellet (DGC) compared to neat semen samples (NSS), there was also significant inter-center variability in the efficiency to reduce SDF values by DGC (78.5% to 29.2%). Surprisingly, for some patients, the level of SDF actually increased following sperm selection. The main conclusions derived from this study were that 1) isolation of sperm from the gradient pellet by DGC must be performed using validated, optimized protocols; 2) routine comparison of SDF values in NSS semen and in processed sperm after DGC or swim-up must be recommended as part of the internal quality control (QC) of ART laboratories to test the efficacy of sperm processing; and 3) SDF values in processed spermatozoa should be obtained to compare with the pregnancy rate when insemination or fertilization is about to be performed, otherwise, attempts to predict pregnancy outcome from SDF could be biased or are essentially meaningless.

Keywords

Assisted Reproductive Technology; Fertility; Sperm DNA Fragmentation; Male Factor; Semen Processing

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Lopes, S., Sun, J.G., Jurisicova, A., Meriano, J. and Cas per, R.F. (1998) Sperm deoxyribonucleic acid fragmenta tion is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertility and Sterility, 69, 528-532. doi:10.1016/S0015-0282(97)00536-0
[2]	Evenson, D.P., Jost, L.K., Marshal, D., Zinaman, M.J., Clegg, E., Purvis, K., De Angelis, P. et al. (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Human Reproduction, 14, 1039-1049. doi:10.1093/humrep/14.4.1039
[3]	Spano, M., Bonde, J.P., Hjollund, H.I., Kolstad, H.A., Cordelli, E. and Leter, G. (2000) Sperm chromatin damage impairs human fertility. The Danish first pregnancy planner study team. Fertility and Sterility, 73, 43-50.
[4]	Bungum, M., Humaidan, P., Spano, M., Jepson, K., Bun gum, 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. doi:10.1093/humrep/deh280
[5]	Collins, J.A., Barnhart, K.T. and Schlegel, P.N. (2008) Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertility and Sterility, 89, 823-831. doi:10.1016/j.fertnstert.2007.04.055
[6]	Bakos, H.W., Thompson, J.G., Feil, D. and Lane, M. (2008) Sperm DNA damage is associated with assisted reproductive technology pregnancy. International Journal of Andrology, 31, 518-526. doi:10.1111/j.1365-2605.2007.00803.x
[7]	Castilla, J.A, Zamora, S., Gonzalvo, M.C., Luna Del Castillo, J.D., Roldan-Nofunentes, J.A., Clavero, A. et al. (2010) Sperm chromatin structure assay and classical se men parameters: Systematic review. Reproductive Bio medicine Online, 20, 114-124. doi:10.1016/j.rbmo.2009.10.024
[8]	Gandini, L., Lombardo, F., Paoli, D., Caruso, F. Eleuteri, P., Criminna, R. et al. (2004) Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Human Reproduction, 19, 1409-1417. doi:10.1093/humrep/deh233
[9]	Li, Z., Wang, L., Cai, J. and Huang, H. (2006). Correlation of sperm DNA damage with IVF and ICSI outcomes: A systematic review and meta-analysis. Journal of Assisted Reproduction and Genetics, 23, 367–376. doi:10.1007/s10815-006-9066-9
[10]	Virro M.R., Larson-Cook, K.L. and Evenson, D.P. (2005) Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in vitro fertilization and intracytoplamic sperm injection cycles. Fertility and Sterility, 81, 1289-1295. doi:10.1016/j.fertnstert.2003.09.063
[11]	Simon, L., Castillo, J., Oliva, R. and Lewis, S.E. (2011) Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes. Reproductive Biomedicine Online, 23, 724-734. doi:10.1016/j.rbmo.2011.08.010
[12]	Santiso, R., Tamayo, M., Gosálvez, J., Meseguer, M. Garrido, N and Fernandez, J.L. (2010) Swim-up procedure selects spermatozoa with longer telomere length. Mutation Research, 688, 88-90. doi:10.1016/j.mrfmmm.2010.03.003
[13]	Tampaichitr, N., Agulnik, A., Seibel, M. and Taymor, M. (1988) Comparison of the in vitro fertilization rate by human sperm capacitated by multiple-tube swim-up and Percoll gradient centrifugation. Journal of in Vitro Fertilization and Embryo Transfer, 5, 119-122. doi:10.1007/BF01131172
[14]	Zini, A., Finellia, A., Phanga, D. and Jarvi, K. (2000) Influence of semen processing technique on human sperm DNA integrity. Urology, 56, 1081-1084. doi:10.1016/S0090-4295(00)00770-6
[15]	Bungum, M., Spano, M., Humaidan, P., Eleteri, P., Rescia, M. and Giwercman, A. (2008) Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART. Hu man Reproduction, 23, 4-10. doi:10.1093/humrep/dem353
[16]	Enciso, M., Iglesias, M., Galán, I., Sarassa, J. Gosalvez, A. and Gosalvez, J. (2011) The ability of sperm selection techniques to remove single or double-strand DNA damage. Asian Journal of Andrology, 13, 764-768. doi:10.1038/aja.2011.46
[17]	Mortimer, D. (1991) Sperm preparation techniques and iatrogenic failures of in vitro fertilization. Human Reproduction, 6, 173-176.
[18]	Gosálvez, J., López-Fernández, C., Fernández, J.L., Gou rand, A. and Holt, W.V. (2011) Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Molecular Reproduction and Development, 78, 951-961. doi:10.1002/mrd.21394
[19]	Gosálvez J., López-Fernández, C. and Fernández, J.L. (2011) Sperm Chromatin Dispersion (SCD) test: Technical aspects and clinical applications. In: Zini, A., Agarwal, A., Eds., Sperm DNA damage: Biological and clinical applications in male infertility and assisted reproduction. Springer Verlag, New York, 151-170.
[20]	Menezo, Jr. Y., Russo, G.L., Tosti, E., Moutassim, S. and Benkhalifa, M. (2007) Expression profile of genes coding for DNA repair in human oocytes using pangenomic microarrays, with a special focus on ROS linked decays. Journal of Assisted Reproduction and Genetics, 24, 513-520. doi:10.1007/s10815-007-9167-0
[21]	Meseguer, M., Santiso, R., Garrido, N., Garcia-Herrero, S., Remohi, J. and Fernandez, J.L. (2011) Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality. Fertility and Sterility, 95, 124-128. doi:10.1016/j.fertnstert.2010.05.055
[22]	Nunez-Calonge, R., Caballero, P., López-Fernández, C., Guijarro, J.A., Fernandez, J.L., Johnston, S. and Gosalvez, J. (2012) An improved experimental model for understanding the impact of sperm DNA fragmentation on human pregnancy following ICSI. Reproductive Science, 19, 1163-1168. doi:10.1177/1933719112459238
[23]	Gosálvez, J., De La Torre, J., López-Fernández, C., Perez-Gutierrez, L., Ortega, L., Caballero, P. and Nunez, R. (2010) DNA fragmentation dynamics in fresh versus frozen thawed 1 plus gradient-isolated human spermatozoa. Systems Biology in Reproductive Medicine, 56, 27-36. doi:10.3109/19396360903515430
[24]	Parmegiani, L., Cognigni, G.E., Bernardi, S., Troilo, E., Ciampaglia, W. and Filicori, M. (2010) “Physiologic ICSI”: Hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality. Fertility and Sterility, 93, 598-604. doi:10.1016/j.fertnstert.2009.03.033
[25]	Tavalaee, M., Deemeh, M.R., Arbabian, M. and Nasr Esfahani, M.H. (2012) Density gradient centrifugation before or after magnetic-activated cell sorting: Which technique is more useful for clinical sperm selection? Journal of Assisted Reproduction and Genetics, 29, 31-38. doi:10.1007/s10815-011-9686-6
[26]	Ebner, T., Shebl, O., Moser, M., Arzt, W. and Tews, G. (2011) Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strand break-free spermatozoa. Reproductive Biomedicine On line, 22, 37-43. doi:10.1016/j.rbmo.2010.09.004
[27]	Ollero, M., Gil-Guzman, E., Lopez, M.C., Sharma, R.K., Agarwal, A., Larson, K. et al. (2001) Characterization of subset of human spermatozoa at different stages of maturation: Implications in the diagnosis and treatment of male infertility. Human Reproduction, 16, 1912-1921. doi:10.1093/humrep/16.9.1912
[28]	Agarwal, A., Makker, K. and Sharma, R. (2008) Clinical relevance of oxidative stress in male factor infertility: An update. American Journal Reproductive Immunology, 59, 2-11. doi:10.1111/j.1600-0897.2007.00559.x
[29]	Aitken, R.J. and De Iuliis, G.N. (2010) On the possible origins of DNA damage in human spermatozoa. Molecular Human Reproduction, 16, 3-13. doi:10.1093/molehr/gap059
[30]	López-Fernández, C., Johnston, S.D., Fernández, J.L., Wilson, R.J. and Gosalvez, J. (2010) Fragmentation dy namics of frozen-thawed ram sperm DNA is modulated by sperm concentration. Theriogenology, 74, 1362-1370. doi:10.1016/j.theriogenology.2010.06.006
[31]	Gosálvez, J., Cortés-Gutierrez, E., López-Fernádez C., Fernandez, J.L., Caballero, P. and Nunez, R. (2009) Sperm DNA fragmentation dynamics in fertile donors. Fertility and Sterility, 92, 170-173. doi:10.1016/j.fertnstert.2008.05.068
[32]	Calamera, J.C., Buffone, M.G., Doncel, G.F., Brugo-Ol medo, S., De Vincentiis, S., Calamera, M.M. et al. (2010) Effect of thawing temperature on the motility recovery of cryopreserved human spermatozoa. Fertility and Sterility, 93, 789-794. doi:10.1016/j.fertnstert.2008.10.021