P-007 Unraveling the genetic basis of Idiopathic Azoospermia: Α transcriptome profiling analyses in a Greek Population

Abstract

Abstract Study question Are there differentially expressed genes (DEGs), in the testicular tissue of azoospermic males which could uncover tissue specific gene expression signatures, associated with idiopathic azoospermia? Summary answer The current findings link for the first time the pathophysiology of idiopathic azoospermia to the immune system and open a new era for further investigation What is known already Azoospermia is the most severe form of male infertility and affects 10% of the infertile men and 1% of the general male population which equals to several million men all over the world. A causal genetic factor is established in only 25% of the cases while in the remaining 75% there is no diagnosis and is termed idiopathic. Published data demonstrate that in idiopathic azoospermia heterogeneous genetic factors are the underlying cause. Clarifying the genetic basis of azoospermia will immensely improve our current clinical and treatment approaches for patient care at the levels of diagnosis, therapy, treatment, and safety Study design, size, duration During June 2020 and June 2021 testicular samples from 26 consented IVF patients were included in this study. The samples were divided in 7 pools based on the presence of testicular spermatozoa found. In particular, Pools 1-3 included testicular samples with high, average and very low presence of spermatozoa, Pools 4 & 5 samples with no spermatozoa found, and Pools 6-7 included only CF carriers. Pools 6 &7 were used as controls. Participants/materials, setting, methods Total RNA was isolated using the RΝeasy plus universal mini kit (Qiagen) and quantified using a photometer. Next, RNA sequencing was performed by Novogene, using the Illumina NovaSeq platform and the DEGs that were identified were processed using Gene Ontology (GO) functional enrichment analysis, pathway enrichment analysis and protein-protein interaction (PPI) network reconstruction using the Gene Ontology Resource, the KEGG database and the STRING database respectively. Main results and the role of chance In total RNA from up to 39.664 genes was detected and significant differences in gene expression (DEGs, log2fold change≥2, p-value < 0.05) were identified in the 7 pools analyzed. The top 10% of the protein coding DEGs from each comparison was further analyzed. Up to 1.900 protein coding genes were found to be common in at least two comparisons and exhibit consistent expression profiles. In particular, 920 genes were overexpressed in the “good quality” testicular samples (high & average presence of spermatozoa) while 940 genes were overexpressed in the “low quality” testicular samples (rare or no spermatozoa found). GO analysis revealed that the sexual reproduction, male gamete generation spermatogenesis, cilium movement and fertilization biological processes were enriched in the “good quality” testicular pools while the biological processes response to stimulus, response to stress, response to cytokine and defense response were overrepresented in the “low quality” testicular samples. The PPI network of the DEGs was reconstructed and hub proteins with a putative key role in the pathogenesis of azoospermia were identified. Limitations, reasons for caution While transcriptomics analysis is becoming a powerful analysis tool there are still technical limitations which may impact the accurate representation of the DEGs profiles found. The data obtained in this study represent the whole testicular tissue samples analyzed and they are not cell-type specific Wider implications of the findings The findings of this study demonstrate that in the “low quality” testicular samples, there is a significant under-expression of testis-specific genes directly involved in spermatogenesis and fertilization processes, an over-expression of the genes involved in the body’s immune defense system and advance our understanding in the pathophysiology of idiopathic azoospermia. Trial registration number Τ1EDK-02787