“Profili di espressione dei microRNA nelle differenti forme di azoospermia”

Azoospermia, defined as the absence of spermatozoa in ejaculate after centrifugation, results from testicular dysfunction (non-obstructive) or obstruction of extratesticular ducts (obstructive). Different conditions, both congenital and acquired, can cause testicular failure. MicroRNAs (miRNAs) recently emerged as a different class of short endogenous single-stranded RNAs; free miRNAs were discovered in different body fluids. Although miRNAs play an essential role in spermatogenesis, little is known about seminal plasma miRNAs in azoospermic men. The aim of this work is to analyze the expression of microRNAs in normospermic and azoospermic men and to evaluate the molecular differences between various conditions of azoospermia. In this study, we evaluated the expression of testicular miR 509-5p, 122, 34b and 34c in: • Klinefelter (KS): 40 sera and 40 seminal plasma • Obstructive Azoospermia (OA): 12 sera and 60 seminal plasma • Non-Obstructive Azoospermia (NOA): 45 sera and 60 seminal plasma • XX male: 13 sera • AZF delected: 22 sera • Normospermic patients as control (CTRL): 40 sera and 40 seminal plasma Semen analyses have been performed according to the WHO criteria (2010) and seminal and serum total RNAs have been extracted with TRIzol-LS Reagent. We performed qRT-PCR and we used the relative quantitative method of 2-ΔΔCt to measure the dynamic change of specific selected miRNAs. Seminal plasma - MiRNAs in seminal plasma are downregulated in all azoospermic conditions. The comparison of KS and NOA vs OA shows a significant downregulation of studied miRNAs in OA patients. All the azoospermic patients show a significant downregulation of miRNAs. In particular, because of mir 509-5p and mir 122 are downregulated in OA versus KS or NOA and have been previously identified in testicular tissue, we hypothesize that miR 509-5p and 122 are mainly produced in testicular fraction. According to literature, miRNAs contribute to the regulation of genes involved in the spermatogenic processes and their downregulation can be caused by a progressive modification of testicular parenchyma, leading to an impairement of cell differentiation control. Serum- In NOA, AZF delected and XX males patients serum miR 34b and 34c are upregulated, while any significant differences are shown in the other cases. Interestingly, NOA shows an opposite expression profile than seminal plasma. This trend can be explained by assuming the transition of these small molecules from the testis to the bloodstream because of an alteration of testicular parenchyma. In conclusion, our study represents the first miRNAs analysis in different types of azoospermia