Role of protamine 1 and protamine 2 in spermatozoa of men with borderline infertility

Abstract Background Protamines are diminutive, positively charged, testis-specific nuclear proteins that replace somatic cell histones during the latter phases of spermiogenesis. Variations in sperm protamine mRNA expression, or the proportion of protamine 1 (PRM1) and 2 (PRM2), can cause inadequate chromatin condensation, leading to DNA fragmentation, which is known to be related to male subfertility and infertility and is also important for human embryonic development. Objective The objective of the current study was to assess the levels of protamine mRNA expression and their ratio in the germ cells of men with borderline Infertility. Methods A total of 279 infertile and 32 fertile men were subjected to semen analysis following the 6 th edition of the World Health Organization (WHO) guidelines. Sperm RNA isolation was performed, and complementary DNA (cDNA) was prepared. The levels of protamine transcripts and their ratios were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results The expression study showed significant difference in PRM1 and PRM2 ΔCt values across infertile subgroups compared to controls, with the exception of PRM1 in the normozoospermia group. Based on the corresponding 2 −ΔΔCt values, the difference indicated significant downregulation of PRM1 and PRM2 mRNA transcripts in the infertile subgroups, whereas the normozoospermic group demonstrated a distinct expression pattern, including a significant upregulation in PRM2 expression. Volcano plot analysis based on ΔCt values demonstrated that the highest difference in PRM1 expression in the asthenozoospermic and teratozoospermic group, while PRM1 expression in the normozoospermic group remained close to baseline. The PRM1/PRM2 mRNA transcript ratio was significantly ( p < 0.001) elevated in all infertile categories. Receiver Operating Characteristic (ROC) analysis of the PRM1/PRM2 mRNA transcript ratio revealed modest discriminatory performance(Area Under the Curve (AUC) = 0.74), with an ideal cutoff of 1.29 giving 67.38% sensitivity and 78.12% specificity. Conclusion Altered PRM1 and PRM2 mRNA expression levels, along with an increased PRM1/PRM2 transcript ratio in infertile individuals, emphasize impaired protamine homeostasis as a key molecular characteristic of male infertility. The PRM1/PRM2 mRNA transcript ratio exhibited moderate diagnostic performance (AUC = 0.74), indicating its potential value as a supplementary biomarker for the evaluation and risk stratification of male infertility with conventional semen analysis.