(086) Determination Of Diagnostic Molecular Markers In Non-Obstructive Azoospermia- Male Infertility Via Next Generation Sequencing Methods

Abstract

Abstract Introduction 1 in 6 couples are infertile and male factor contributes 49%. As an Urologist, azoospermia especially non obstructive azoospermia(NOA) poses a great clinical challenge for prediction of fertility. The basic understanding of any disease starts from its physiology which is determined by genes. With the advent of entire human genomic sequencing it has become easier than decades ago to identify genes that are causally linked to specific disease. Objective Hence Studying the molecular basis esp. functional genomics of NOA, a type of male infertility with failed spermatogenesis at various stages, can help in understanding the molecular mechanism of spermatogenesis, discover potential molecules for further clinical research and utility in diagnostics, prognostics and therapeutics and also identification of target molecules for research towards the development of new male contraceptives Methods Sequencing and analysis was carried out using TruSeq RNA Library Prep Kit v2andusing Illumina HiSeq 2000.Alignments, identification of transcripts and the chimeric/transplice molecules, and their quantification were performed by Kallisto software. Differential expression analysis was done using the Bioconductor package limma. Apart from the standard data analysis procedures for identifying transcripts expressed differently in NOA, a novel scoring system has been applied to rank the transcripts based on the 'Strength of Association' (StA) with NOA. Up-and down-regulated transcripts were hierarchically arranged based on their StA scores Testicular biopsies were collected from 18,7 2 and 2 donors, respectively, with NOA, Obstructive Azoospermia (OA), Congenital Bilateral Absence of the Vas Deferens (CBAVD), and Varicocele conditions, following ethical procedures approved by the Institutional Biosafety Committee (IBSC) at Institute of Bioinformatics & Applied Biotechnology (IBAB) and Ankur-Manipal hospital, Bengaluru. The samples were stored in RNA-later solution (Ambion, cat # AM7024), according to the manufacturer's guidelines. RNA was extracted using Ribo Pure kit (AM1924). Results The current study derived the first list of genes and transcripts that are arranged hierarchically based on their Strength of Association (StA) with NOA. Cluster analysis showed that the overall gene expression profiles in NOA were different compared to control samples with normal spermatogenesis - irrespective of sub-types within each set. More importantly, the observations supported the decision to group normal testis-samples with those from other conditions where spermatogenesis occurs normally, viz., VA, OA and CBAVD. The grouping not only enhanced the number of control samples, but also helped to eventually identify candidate markers that are more unique to NOA. Based on the 100% consistency of differential expression observed across 18 NOA and 22 control samples, via RNA-seq and/or the RT-qPCR results, 16mRNAs are being suggested as promising candidates for reliable diagnosis of NOA. Similarly,3chimeric transcripts have also been short-listed as potential diagnostic markers Conclusions The current transcriptomic analysis of the testes from donors with NOA has been used to identify the genes and the corresponding alternatively spliced mRNA-isoforms associated with the disorder. These molecules have been hierarchically listed based on their strength of association with the NOA condition, which would also suggest the association with spermatogenesis in most cases, particularly those down-regulated in NOA. Such an association, of several genes and most of the alternatively spliced forms of mRNAs, is being reported for the first time. If these 19 markers could be used in RT-qPCR assays, individually or in combination with each other, they could avoid the need for open surgeries for the detection of NOA. Disclosure No