Molecular mechanisms of spermatogenic failure at transcriptional and epigenetic level

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About half of the patients with male infertility presenting at the Centre of Reproductive Medicine and Andrology display severely abnormal sperm parameters (termed oligoasthenoteratozoospermia, OAT). To date, the underlying molecular mechanisms for this phenotype remain largely unknown. Unexpectedly, we found epimutations present in the undifferentiated germ cells (spermatogonia) of these OAT testes, which were not detected in mature, motile sperm. This suggests the existence of spermatogonial subpopulations and quality control checkpoints, preventing sperm with an abnormal epigenome from being produced. Furthermore, single cell RNA sequencing (scRNA-Seq) of testicular tissues from these men revealed changed dynamics of the spermatogonial compartment and an altered composition of the spermatogonial stem cell niche. Integration of methylome and transcriptome data indicated an association between differentially methylated regions in spermatogonia and differentially expressed genes specific for the stages of germ cell failure in OAT samples. Apart from these common alterations, we also found patient-specific changes that necessitate expansion of the study cohort.The overall objective of this application is to explore the molecular features (at transcriptional and DNA methylation level) that distinguish those spermatogonial subpopulations that contribute to the formation of mature sperm from those which fail to progress, and to assess the impact of the stem cell niche on these processes. We will capitalize on the unique access to rigorously characterized human testicular biopsies for the following research endeavour. scRNA-Seq (10x Genomics) will be performed on testicular tissues of infertile men with severe OAT compared to normal controls to generate the largest available dataset reflecting the transcriptional mechanisms of germ cell failure. To rigorously assess the differentially methylated regions in the spermatogonia of these men, ultra-high resolution targeted DNA methylation analysis will be performed. The relevance of these extensive datasets on cellular level will be corroborated by multi-spectral protein imaging in testicular tissues. By applying analysis at the epigenetic, transcriptional, and protein level we will gain an unparalleled and integrated view of the mechanisms guiding germ cell differentiation and quality control in human spermatogenesis which could result in better subclassification of the OAT syndrome.