Abstract: PD08-06
Sources of Funding: P50 HD076210, U1 1U01HD074542-01, Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation, Irena and Howard Laks Foundation_x000D_ _x000D_ This work was supported in part by the Urology Care Foundation Research Scholar Award Program and AUA New York Section Research Scholar Fund

Introduction

Non-obstructive azoospermia due to testicular failure can be histologically classified into 3 phenotypes: sertoli cell only (SCO), early maturation arrest (eMA), and late maturation arrest (lMA). Genetics of NOA is largely unknown and identification of genetic mutations leading to NOA has been inconclusive. In our study, we focused on large scale exome sequencing in testicular tissue from men with NOA to determine candidate genes for mechanistic, diagnostic and therapeutic evaluation._x000D_

Methods

Total RNA was extracted from tissue harvested from testicular biopsy. 37 patients with non-obstructive azoospermia (NOA) and 3 histologic phenotypes: SCO, eMA, lMA as well as normal controls (NL). RNA libraries were sequenced on an Illumina HiSeq 2000 platform. Results were mapped to the genome and transcriptome using TopHat (v2.0.8). Cufflinks was then used to quantify the number of reads. RNA Seq data was expressed as FPKMs and normalized using TMM. JMP genomics was used to identify differentially expressed (DE) transcripts at FDR = 0.001. Due expected loss of specific germ cells populations in lMA, eMA, and SCO syndrome, we focused our analysis on overexpressed transcripts in each histological category as compared to NL control. _x000D_ _x000D_

Results

Among 21,879 mapped transcripts 10,777 were DE between SCO and NLs. Using a contingency analysis, 6,169 genes were DE between eMA and normal when compared to SCO and normal DE (p<0.0001), and are likely involved in entry into meiosis and function of primary spermatocytes. Most overexpressed and DE genes include: JUN, BTG2, and RASAL1, which are expressed in the testis and previously described to regulate cell cycle and proliferation. 30 genes were DE between lMA and NL but not DE between NL and eMA, suggesting a mechanistic role in lMA as they are not due to loss of spermatids or spermatozoa. Here, most DE genes include SMIM8, SNF280C and OTUD6A and are exclusively expressed in testis based on GEO human tissues expression profile. To validate our analytical approach, we demonstrated a loss of SYCP3 (marker of primary spermatocytes) in SCO and down regulation of PRM1 (spermatids) in SCO, eMA, and lMA compared to NL. Expression of SOX9 (Sertoli marker) and INSL3 (Leydig marker) were similar across all groups._x000D_

Conclusions

Using whole exome sequencing to directly compare DE of genes among the differing phenotypes of SCO, eMA, and lMA have allowed us to identify active gene transcripts localized to specific stages of arrested spermatogenesis. Additionally, we identified a small subset of genes which are over expressed, potentially contributing to the pathology of NOA._x000D_ _x000D_

Funding

P50 HD076210, U1 1U01HD074542-01, Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation, Irena and Howard Laks Foundation_x000D_ _x000D_ This work was supported in part by the Urology Care Foundation Research Scholar Award Program and AUA New York Section Research Scholar Fund