Validation of a set of proteins in sperm from fertile and infertile men

Abstract

Although semen analysis is essential during infertility investigation, and may provide information about male fertile potential, it is insufficient as a diagnostic method of male infertility. Thus, additional methods for its evaluation are required. In this context, evaluating the sperm proteome of fertile and infertile men, as well as the phosphorylated proteins (regulators of post-testicular sperm function) altered in association with infertility, would be valuable. Taking this into consideration, a comparative sperm proteomic analysis between fertile and infertile men was previously performed by Dr. Ashok Agarwal and colleagues, to identify potential sperm biomarkers of infertility, as well as to unravel the underlying molecular mechanisms related to male infertility. The differentially expressed proteins involved with the most important biological processes identified in the fertile group were proposed as biomarkers of male fertility (CCT6B, UQCRC2, GAA, NDUFS1, ATP1A4, RUVBL1, ODF2, and AK7). On the other hand, the Histone H2B type 1-A (HIST1H2BA) protein was overexpressed in the infertile group and suggested as a sperm biomarker of this condition. Therefore, the aim of the present study is to evaluate the proposed biomarkers and their phosphorylated forms in sperm samples from fertile and infertile men, in order to validate these proteins as sperm biomarkers of male fertility or infertility. The expression of these proteins will be evaluated by Western blotting, immunocytochemistry and/or Selected Reaction Monitoring (SRM) in 20 fertile men and 20 infertile men (previously collected and stored by Dr. Agarwal's group), and in smokers, men with varicocele or obesity, and men without infertility conditions (n=120, prospectively collected by our group for my PhD study funded by FAPESP, process number 2014/11493-0). Protein quantification data will then be analyzed by univariate (unpaired Student's t test) and multivariate (logistic regression and ROC curve analysis) statistical analyses. Based on the findings obtained with this study, we expect to validate these proteins as effective sperm biomarkers of male fertility/infertility, which would be extremely helpful during male infertility clinical management. (AU)