During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 ( ): immature sperm <20% and G2 ( ): immature sperm ≥20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities ( ) and microcephalic sperm ( ). We founded significant correlation between sperm immaturity and acrosome abnormalities ( ; ). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility. 1. Introduction Routine semen analysis is the basic analysis in the exploration of male infertility. It provides useful data concerning sperm count, sperm motility and viability, sperm morphology, performance of genital glands, and ejaculation. Although, the contribution of the semen analysis is limited on the assessment of some important criteria implicated in the sperm functional potential. In fact, it is well established that the maturity of sperm chromatin is essential for fertilizing capacity of spermatozoa and embryonic development [1–3]. The proportion of sperm with abnormal chromatin condensation in the ejaculate could be a prognostic factor in assessing the chances of fertilization and pregnancy [4]. Indeed, during spermiogenesis, sperm nuclear is completely reorganized and undergoes characteristic rearrangements with an important compaction [4–6]. This chromatin condensation involves a replacement of somatic and testis—specific nucleoproteins: histones by transition proteins then by more basic proteins named protamines P1 and P2 [5, 7] with a proper ratio of protamine 1 to protamine 2 (P1/P2) [8]. These protamines pack
References
[1]
D. Sakkas, F. Urner, D. Bizzaro et al., “Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development,” Human Reproduction, vol. 13, no. 4, supplement, pp. 11–19, 1998.
[2]
M. E. Hammadeh, M. Stieber, G. Haidl, and W. Schmidt, “Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program,” Andrologia, vol. 30, no. 1, pp. 29–35, 1998.
[3]
A. D. Esterhuizen, D. R. Franken, J. G. H. Lourens, E. Prinsloo, and L. H. Van Rooyen, “Sperm chromatin packaging as an indicator of in-vitro fertilization rates,” Human Reproduction, vol. 15, no. 3, pp. 657–661, 2000.
[4]
C. Roux, C. Tripogney, C. Joanne, et al., “Sperm chromatin packaging as an indicator of in-vitro fertilization rates,” Gynécologie Obstétrique et Fertilité, vol. 32, no. 9, pp. 792–798, 2004.
[5]
J.-P. Dadoune, “Expression of mammalian spermatozoal nucleoproteins,” Microscopy Research and Technique, vol. 61, no. 1, pp. 56–75, 2003.
[6]
J. P. Dadoune, “The nuclear status of human sperm cells,” Micron, vol. 26, no. 4, pp. 323–345, 1995.
[7]
A. Hekmatdoost, N. Lakpour, and M. R. Sadeghi, “Sperm chromatin integrity: etiologies and mechanisms of abnormality, assays, clinical importance, preventing and repairing damage,” Avicenna Journal of Medical Biotechnology, vol. 1, no. 3, pp. 147–160, 2009.
[8]
D. T. Carrell, B. R. Emery, and S. Hammoud, “The aetiology of sperm protamine abnormalities and their potential impact on the sperm epigenome,” International Journal of Andrology, vol. 31, no. 6, pp. 537–545, 2008.
[9]
J. W. Hou, D. Chen, and R. S. Jeyendran, “Sperm nuclear maturity in spinal cord-injured men: evaluation by acidic aniline blue stain,” Archives of Physical Medicine and Rehabilitation, vol. 76, no. 5, pp. 444–445, 1995.
[10]
A. Agarwal and T. M. Said, “Role of sperm chromatin abnormalities and DNA damage in male infertility,” Human Reproduction Update, vol. 9, no. 4, pp. 331–345, 2003.
[11]
J. Auger, M. Mesbah, C. Huber, and J. P. Dadoune, “Aniline blue staining as a marker of sperm chromatin defects associated with different semen characteristics discriminates between proven fertile and suspected infertile men,” International Journal of Andrology, vol. 13, no. 6, pp. 452–462, 1990.
[12]
J. P. Dadoune, M. J. Mayaux, and M. L. Guihard-Moscato, “Correlation between defects in chromatin condensation of human spermatozoa stained by aniline blue and semen characteristics,” Andrologia, vol. 20, no. 3, pp. 211–217, 1988.
[13]
O. Hingst, S. Blottner, and C. Franz, “Chromatin condensation in cat spermatozoa during epididymal transit as studied by aniline blue and acridine orange staining,” Andrologia, vol. 27, no. 5, pp. 275–279, 1995.
[14]
M. E. Hammadeh, T. Zeginiadov, P. Rosenbaum, T. Georg, W. Schmidt, and E. Strehler, “Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility,” Archives of Andrology, vol. 46, no. 2, pp. 99–104, 2001.
[15]
A. R. Talebi, M. R. Moein, N. Tabibnejad, and J. Ghasemzadeh, “Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests,” Andrologia, vol. 40, no. 4, pp. 245–251, 2008.
[16]
M. R. Sadeghi, M. Hodjat, N. Lakpour, et al., “Effects of sperm chromatin integrity on fertilization rate and embryo quality following intracytoplasmic sperm injection,” Avicenna Journal of Medical Biotechnology, vol. 1, no. 3, pp. 173–180, 2009.
[17]
T. Kazerooni, N. Asadi, L. Jadid et al., “Evaluation of sperm's chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion,” Journal of Assisted Reproduction and Genetics, vol. 26, no. 11-12, pp. 591–596, 2009.
[18]
A. M. Andreetta, J. C. Stockert, and C. Barrera, “A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures,” International Journal of Andrology, vol. 18, no. 1, supplement, pp. 23–28, 1995.
[19]
N. Hofmann and B. Hilscher, “Use of aniline blue to assess chromatin condensation in morphologically normal spermatozoa in normal and infertile men,” Human Reproduction, vol. 6, no. 7, pp. 979–982, 1991.
[20]
C. Foresta, M. Zorzi, M. Rossato, and A. Varotto, “Sperm nuclear instability and staining with aniline blue: abnormal persistance of histones in spermatozoa in infertile men,” International Journal of Andrology, vol. 15, no. 4, pp. 330–337, 1992.
[21]
WHO World Health Organization, Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, Cambridge University Press, 4th edition, 1999.
[22]
N. Salsabili, A. Mehrsai, B. Jalalizadeh, et al., “Correlation of sperm nuclear chromatin condensation staining method with semen parameters and sperm functional tests in patients with spinal cord injury, varicocele, and idiopathic infertility,” Urology Journal, vol. 3, no. 1, pp. 32–37, 2006.
[23]
A. Sadek, A. S. A. Almohamdy, A. Zaki, M. Aref, S. M. Ibrahim, and T. Mostafa, “Sperm chromatin condensation in infertile men with varicocele before and after surgical repair,” Fertility and Sterility, vol. 95, no. 5, pp. 1705–1708, 2011.
[24]
H. S. Kim, M. J. Kang, S. A. Kim, et al., “The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis,” Clinical and Experimental Reproductive Medicine, vol. 40, no. 1, pp. 23–28, 2013.
[25]
V. W. Aoki, L. Liu, and D. T. Carrell, “Identification and evaluation of a novel sperm protamine abnormality in a population of infertile males,” Human Reproduction, vol. 20, no. 5, pp. 1298–1306, 2005.
[26]
A. Wong, S. S. Chuan, W. C. Patton, J. D. Jacobson, J. Corselli, and P. J. Chan, “Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones,” Fertility and Sterility, vol. 90, no. 5, pp. 1999–2002, 2008.
[27]
Y. S. Park, M. K. Kim, S. H. Lee, et al., “Efficacy of testicular sperm chromatin condensation assay using aniline blue-eosin staining in the IVF-ET cycle,” Clinical and Experimental Reproductive Medicine, vol. 38, no. 3, pp. 142–147, 2011.
[28]
S. Leiva, M. Loyola, A. M. Agar, and E. Bustos-Obregón, “Evaluation of DNA/protein status and nuclear maturity of human sperm,” Cytobios, vol. 78, no. 312, pp. 7–18, 1994.
[29]
D. R. Franken, C. J. Franken, H. de la Guerre, and A. de Villiers, “Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining,” Andrologia, vol. 31, no. 6, pp. 361–366, 1999.
[30]
L. A. Lazaros, G. A. Vartholomatos, E. G. Hatzi et al., “Assessment of sperm chromatin condensation and ploidy status using flow cytometry correlates to fertilization, embryo quality and pregnancy following in vitro fertilization,” Journal of Assisted Reproduction and Genetics, vol. 28, no. 10, pp. 885–891, 2011.
[31]
A. Zini, S. Phillips, A. Courchesne et al., “Sperm head morphology is related to high deoxyribonucleic acid stainability assessed by sperm chromatin structure assay,” Fertility and Sterility, vol. 91, no. 6, pp. 2495–2500, 2009.
[32]
F. Boitrelle, F. Ferfouri, J. M. Petit et al., “Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation,” Human Reproduction, vol. 26, no. 7, pp. 1650–1658, 2011.
[33]
I. M. Adham, K. Nayernia, E. Burkhardt-G？ttges et al., “Teratozoospermia in mice lacking the transition protein 2 (Tnp2),” Molecular Human Reproduction, vol. 7, no. 6, pp. 513–520, 2001.
[34]
N. Torregrosa, D. Domínguez-Fandos, M. I. Camejo et al., “Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients,” Human Reproduction, vol. 21, no. 8, pp. 2084–2089, 2006.
[35]
K. Tseden, O. Topaloglu, A. Meinhardt et al., “Premature translation of transition protein 2 mRNA causes sperm abnormalities and male infertility,” Molecular Reproduction and Development, vol. 74, no. 3, pp. 273–279, 2007.
[36]
L. Nanassy, L. Liu, J. Griffin, and D. T. Carrell, “The clinical utility of the protamine 1/protamine 2 ratio in sperm,” Protein and Peptide Letters, vol. 18, no. 8, pp. 772–777, 2011.
[37]
G. N. de Iuliis, L. K. Thomson, L. A. Mitchell et al., “DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress,” Biology of Reproduction, vol. 81, no. 3, pp. 517–524, 2009.
[38]
R. T. Schulte, D. A. Ohl, M. Sigman, and G. D. Smith, “Sperm DNA damage in male infertility: etiologies, assays, and outcomes,” Journal of Assisted Reproduction and Genetics, vol. 27, no. 1, pp. 3–12, 2010.
[39]
R. J. Aitken and G. N. De Iuliis, “On the possible origins of DNA damage in human spermatozoa,” Molecular Human Reproduction, vol. 16, no. 1, Article ID gap059, pp. 3–13, 2009.
[40]
J. Erenpreiss, J. Bars, V. Lipatnikova, J. Erenpreisa, and J. Zalkalns, “Comparative study of cytochemical tests for sperm chromatin integrity,” Journal of Andrology, vol. 22, no. 1, pp. 45–53, 2001.
[41]
D. Sakkas, E. Mariethoz, G. Manicardi, D. Bizzaro, P. G. Bianchi, and U. Bianchi, “Origin of DNA damage in ejaculated human spermatozoa,” Reviews of Reproduction, vol. 4, no. 1, pp. 31–37, 1999.
[42]
R. Henkel, M. Hajimohammad, T. Stalf et al., “Influence of deoxyribonucleic acid damage on fertilization and pregnancy,” Fertility and Sterility, vol. 81, no. 4, pp. 965–972, 2004.
[43]
D. Sakkas, G. Manicardi, P. G. Bianchi, D. Bizzaro, and U. Bianchi, “Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa,” Biology of Reproduction, vol. 52, no. 5, pp. 1149–1155, 1995.
[44]
F. Morel, S. Mercier, C. Roux, T. Elmrini, M. C. Clavequin, and J. L. Bresson, “Interindividual variations in the disomy frequencies of human spermatozoa and their correlation with nuclear maturity as evaluated aniline blue staining,” Fertility and Sterility, vol. 69, no. 6, pp. 1122–1127, 1998.
[45]
L. Sati, L. Ovari, D. Bennett, S. D. Simon, R. Demir, and G. Huszar, “Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation,” Reproductive BioMedicine Online, vol. 16, no. 4, pp. 570–579, 2008.
[46]
L. óvári, L. Sati, J. Stronk, A. Borsos, D. C. Ward, and G. Huszar, “Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies,” Fertility and Sterility, vol. 93, no. 7, pp. 2255–2261, 2010.
[47]
E. Kovanci, T. Kovacs, E. Moretti et al., “FISH assessment of aneuploidy frequencies in mature and immature human spermatozoa classified by the absence or presence of cytoplasmic retention,” Human Reproduction, vol. 16, no. 6, pp. 1209–1217, 2001.
[48]
F. Morel, C. Roux, and J. L. Bresson, “Disomy frequency estimated by multicolour fluorescence in situ hybridization, degree of nuclear maturity and teratozoospermia in human spermatozoa,” Reproduction, vol. 121, no. 5, pp. 783–789, 2001.
[49]
S. Razavi, M. H. Nasr-Esfahani, M. Mardani, A. Mafi, and A. Moghdam, “Effect of human sperm chromatin anomalies on fertilization outcome post-ICSI,” Andrologia, vol. 35, no. 4, pp. 238–243, 2003.
[50]
R. Sanchez, E. Villagran, J. Risopatron, and R. Celis, “Evaluation of nuclear maturity in human spermatozoa obtained by sperm-preparation methods,” Andrologia, vol. 26, no. 3, pp. 173–176, 1994.
[51]
D. le Lannou and Y. Blanchard, “Nuclear maturity and morphology of human spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure,” Journal of Reproduction and Fertility, vol. 84, no. 2, pp. 551–556, 1988.
[52]
M. E. Hammadeh, A. Kühnen, A. S. Amer, P. Rosenbaum, and W. Schmidt, “Comparison of sperm preparation methods: effect on chromatin and morphology recovery rates and their consequences on the clinical outcome after in vitro fertilization embryo transfer,” International Journal of Andrology, vol. 24, no. 6, pp. 360–368, 2001.
[53]
D. Sakkas, G. C. Manicardi, M. Tomlinson et al., “The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies,” Human Reproduction, vol. 15, no. 5, pp. 1112–1116, 2000.
[54]
R. R. Henkel, D. R. Franken, C. J. Lombard, and W.-B. Schill, “Selective capacity of glass-wool filtration for the separation of human spermatozoa with condensed chromatin: a possible therapeutic modality for male-factor cases?” Journal of Assisted Reproduction and Genetics, vol. 11, no. 8, pp. 395–400, 1995.
[55]
A. R. Talebi, S. Vahidi, A. Aflatoonian, et al., “Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions,” Andrologia, vol. 44, supplement 1, pp. 462–470, 2012.
[56]
C. Jeulin, D. Feneux, and C. Serres, “Sperm factors related to failure of human in-vitro fertilization,” Journal of Reproduction and Fertility, vol. 76, no. 2, pp. 735–744, 1986.
[57]
A. D. Esterhuizen, D. R. Franken, J. G. H. Lourens, C. van Zyl, I. I. Müller, and L. H. Van Rooyen, “Chromatin packaging as an indicator of human sperm dysfunction,” Journal of Assisted Reproduction and Genetics, vol. 17, no. 9, pp. 508–514, 2000.
[58]
D. Y. Liu and H. W. G. Baker, “Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding, and fertilization rates in vitro,” Fertility and Sterility, vol. 58, no. 6, pp. 1178–1184, 1992.
[59]
M. H. Nasr-Esfahani, S. Razavi, and M. Mardani, “Relation between different human sperm nuclear maturity tests and in vitro fertilization,” Journal of Assisted Reproduction and Genetics, vol. 18, no. 4, pp. 219–225, 2001.
[60]
E. E. Mohamed and M. A. Mohamed, “Effect of sperm chromatin condensation on the outcome of intrauterine insemination in patients with male factor infertility,” Journal of Reproductive Medicine, vol. 57, no. 9-10, pp. 421–426, 2012.
[61]
M.-H. Lin, R. Kuo-Kuang Lee, S.-H. Li, C.-H. Lu, F.-J. Sun, and Y.-M. Hwu, “Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates,” Fertility and Sterility, vol. 90, no. 2, pp. 352–359, 2008.
[62]
R. Oliva, “Protamines and male infertility,” Human Reproduction Update, vol. 12, no. 4, pp. 417–435, 2006.
[63]
C. Cho, W. D. Willis, E. H. Goulding et al., “Haploinsufficiency of protamine-1 or -2 causes infertility in mice,” Nature Genetics, vol. 28, no. 1, pp. 82–86, 2001.
[64]
M. E. Hammadeh, S. Al-Hasani, S. Doerr et al., “Comparison between chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their relationship to ICSI outcome,” Human Reproduction, vol. 14, no. 2, pp. 363–367, 1999.
[65]
J. Auger, D. Schoevaert, I. Negulesco, and J. Dadoune, “The nuclear status of human sperm cells by TEM image cytometry: nuclear shape and chromatin texture in semen samples from fertile and infertile men,” Journal of Andrology, vol. 14, no. 6, pp. 456–463, 1993.
