CAIS molecular pathways unveil Testosterone/Estradiol ratio to germ cell tumor risk in non-obstructive azoospermia

Context. Non-obstructive azoospermia (NOA) is the most severe form of male infertility affecting 1% of all men, with a clinical picture characterized by no sperm production, hyalinization of the basal membrane of the seminiferous tubules, primary hypogonadism and earlier onset of age-related comorbidities compared with fertile men. NOA is also characterized by etiologic heterogeneity and the non-genetic form has higher incidence of testicular germ cell cancer (TGCC) compared to the forms with genetic abnormalities. Objective. We aimed to establish molecular pathways in the testicular somatic cells that are either shared or specific for non-genetic and genetic forms of NOA, as Complete Androgen Insensitivity Syndrome (CAIS) and Klinefelter Syndrome (KS). Methods. Single cell RNAseq of the testis somatic cells of an individual with CAIS, and data integration with published scRNA-seq datasets of testis with normal spermatogenesis, NOA, KS and germinal testicular cancer. Detailed clinical data of the CAIS patient, Testosterone and Estradiol levels in age-matched men (120 fertile, 155 infertile, 116 NOA, 18 KS, and 343 with TGCC). Results. In all conditions, Leydig cells are immature and senescent, but those of NOA associated with primary hypogonadism depict the highest expression of transcripts associated with the seminoma microenvironment, including estrogen-responsive genes. An oncological transcriptional signature in the Leydig cells has been confirmed at the systemic levels by showing a prognostic role of the decreasing Testosterone/Estradiol ratio for TGCC in men with non-genetic NOA. Conclusion. This study offers molecular insights into the prediction of TGCC in persons with NOA and eligibility for the use of aromatase inhibitors. Overall design: Single cell RNAseq of the testicular somatic cells of an individual with CAIS was obtained. This sample was integrated with published scRNA-seq datasets of testis with normal spermatogenesis, NOA, KS and germinal testicular cancer. We re-analyzed count matrices for 3 men with NOA (Alfano et al. 2021 - GSE154535 = GSM4673006, GSM4673007, GSM4673008 ), 2 men with KS (Mahyari et al. 2021 - GSE169062 = GSM5175967, GSM5175968, GSM5175972, GSM5175973), and 9 healthy men (3 from Guo et al. 2018 - GSE112013 = GSM3052917-GSM3052918, GSM3052919-GSM3052920, GSM3052921-GSM3052922) , 2 from Sohni et al. 2019 - GSE124263 = GSM3526588, GSM3526590 , and 4 young adults from Guo et al. 2022 - GSE182786 = GSM5536958, GSM5536959, GSM5536960, GSM5536961) using R scripts. All gene annotations were updated to GENCODE v31 using Ensembl IDs for consistency across versions. All these samples were integrated with our CAIS samples using the standard Seurat v4 workflow. To identify cell populations, the Seurat v4 graph-based clustering approach (using the FindNeighbours and FindClusters functions) was applied with a resolution of 0.2, and cell identities were assigned as previously described in our previous work (Alfano M, Tascini AS, Pederzoli F, Locatelli I et al. Aging, inflammation and DNA damage in the somatic testicular niche with idiopathic germ cell aplasia. Nat Commun 2021 Sep 1;12(1):5205. PMID: 34471128) *************************************************************** The table below lists GEO accessions reused/reanalyzed for this study. ***************************************************************