During spermiogenesis, histones are replaced by PROTAMINEs (P1 and P2), resulting in sperm chromatin condensation followed by a halt in gene expression of haploid spermatids and spermatozoa. As a consequence, PROTAMINE deficiency and aberrant P1/P2 ratio have a profound effect on both fertilization and embryo development. However, reports on the effect of the P1/P2 ratio on fertilization and embryo development after intracytoplasmic sperm injection (ICSI) are contradictory among human and animal studies. The question yet to be answered is which type of PROTAMINE deficiency is most common among PROTAMINE deficient samples. The main aim of this study was to investigate the possible correlations of the P1/P2 ratio with PROTAMINE deficiency, fertilization, embryo quality, and embryo development in ICSI patients. It was carried out in 71 patients. Chromomycin A3 (CMA3) staining was used to determine PROTAMINE deficiency. Since this procedure does not indicate the type of PROTAMINE deficiency, the P1/P2 ratio was evaluated by nuclear protein extraction, acetic acid urea polyacrylamide gel electrophoresis, and analysis of protein bands with software. Polyclonal anti-P1 and anti-P2 antibodies were used to confirm P1 and P2 presence. Our results showed a statistically significant negative correlation of fertilization rate with PROTAMINE deficiency and P1/P2 ratio. However, no significant correlation was observed between PROTAMINE deficiency and P1/P2 ratio. Therefore, it can be concluded that altered P1/P2 ratio effects fertilization rate and embryo quality which subsequently may affect implantation and pregnancy outcome

Background: Although aberrant PROTAMINE (PRM) ratios have been observed in infertile men, the mechanisms that implicit the uncoupling of PRM1 and PRM2 expression remain unclear. To uncover these mechanisms, in this observational study we have compared the PRM1/PRM2 mRNA ratio and mRNA contents of two regulatory factors of these genes. Materials and Methods: In this experimental study, sampling was performed by a multi-step method from 50 non-obstructive azoospermic and 12 normal men. After RNA extraction and cDNA synthesis, real-time quantitative polymerase chain reaction (RTQPCR) was used to analyze the PRM1, PRM2, Y box binding protein 2 (YBX2) and JmjC-containing histone demethylase 2a (JHDM2A) genes in testicular biopsies of the studied samples.Results: The PRM1/PRM2 mRNA ratio differed significantly among studied groups, namely 0.21±0.13 in azoospermic samples and -0.8±0.22 in fertile samples. The amount of PRM2 mRNA, significantly reduced in azoospermic patients. Azoospermic men exhibited significant under expression of YBX2 gene compared to controls (P<0.001). mRNA content of this gene showed a positive correlation with PRM mRNA ratio (R=0.6, P=0.007). JHDM2A gene expression ratio did not show any significant difference between the studied groups (P=0.3). We also observed no correlation between JHDM2A mRNA content and the PRM mRNA ratio (R=0.2, P=0.3).Conclusion: We found significant correlation between the aberrant PRM ratio (PRM2 under expression) and lower YBX2 mRNA content in testicular biopsies of azoospermic men compared to controls, which suggested that downregulation of the YBX2 gene might be involved in PRM2 under expression. These molecules could be useful biomarkers for predicting male infertility.

Background: Globozoospermia is a severe form of teratozoospermia (incidence < 0.1%) in infertile men that is characterized by round headed sperm and acrosomeless in semen.Objective: To compare the semen parameters, PROTAMINE deficiency, and apoptosis in ejaculated spermatozoa between globozoospermic and normozoospermic men.Materials and Methods: Thirty six semen samples were divided into two groups including 15 infertile men with total globozoospermic (> 90% round-headed sperm) and 21 healthy donors with normal spermograms as controls. Semen analysis was performed according to World Health Organization criteria (2010). Sperm PROTAMINE deficiency was assessed using Chromomycin A3 (CMA3) staining and the rate of apoptotic spermatozoa was evaluated with TUNEL assay.Results: Sperm concentration, motility, and normal morphology in globozoospermic men were significantly decreased compared with controls (p<0.05). The rate of CMA3-reacted spermatozoa (CMA3+) in globozoospermic men was higher than controls (65.93±11.77 vs. 21.24±7.37, respectively, p<0.0001). The rate of apoptotic spermatozoa (TUNEL positive) were significantly increased in globozoospermic cases with respect to the controls (17.60±10.72 and 5.95±3.02, respectively, p<0.0001). There was no significant correlation between sperm PROTAMINE deficiency and apoptosis in globozoospermic men.Conclusion: Globozoospermic samples contain a higher proportion of spermatozoa with abnormal chromatin packaging and DNA fragmentation than normozoospermic samples. Therefore, in addition to absence of acrosome in the spermatozoa of globozoospermic patients, the high percentage of spermatozoa with immature chromatin and apoptotic marker may be considered as the other etiologies of infertility in these patients.