Ryan Flannigan

BACKGROUND: Non-obstructive azoospermia (NOA) is defined by the complete absence of spermatozoa in repeated ejaculate samples. The vast majority of cases remain unexplained, hindering development of effective treatments. Existing studies mostly employed whole genome/exome and bulk RNA-sequencing, while evaluation of testicular cell-type specific abnormalities in NOA would lay a promising foundation to advance therapeutics research.OBJECTIVES: 1. To systematically review all available reports specifying cell-type-specific deviations in the testes of NOA males. 2. To investigate intratesticular heterogeneity in an idiopathic NOA case and elaborate on its cell-type-specific abnormalities using single-cell RNA sequencing (scRNA-seq). METHODS: For the systematic review, eligibility criteria comprised human studies published in English language specifying cell types of the reported abnormalities in NOA testes. All MEDLINE sources were searched. For the scRNA-seq project, testicular tissue specimens were obtained from an idiopathic NOA patient with intratesticular heterogeneity and a fertile male undergoing vasectomy reversal. Cell types were identified based on the scRNA-seq data and marker gene expression. Transcriptome dissimilarity, developmental progression, pathway activity, and cell-cell communication among the corresponding cell types of NOA and normal control were statistically assessed. Immunofluorescence and immunohistochemistry were used to evaluate cellular composition and developmental markers.RESULTS: The systematic review summarized a diverse range of cellular dysfunctions in most testis cell types in NOA. Reported abnormalities included DNA damage response in Sertoli cells, dysregulation of steroidogenic pathways in Leydig cells; myoid cell-related tubule wall thickening; increase in immune cells; increased apoptosis and potential changes in spindle formation in germ cells, and more. ScRNA-seq project identified hypospematogenesis and Sertoli-Cell Only phenotypes within the NOA testis; immaturity of Leydig and Sertoli cells expressing early developmental markers, e.g., MAFB; higher dissimilarity between somatic cells of the two NOA phenotypes than between those of individual states and normal control; HS germline developmental deviations, especially at the later spermatogenesis stages; significant increase in macrophages and activated T cells and enlarged interstitium in both phenotypes.CONCLUSIONS: Abnormalities in both somatic and germ cells are present in NOA testes, and they vary from patient to patient. A precision medicine approach may be necessary for treatment of NOA.

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