Construction and external validation of a 5-gene random forest model to diagnose non-obstructive azoospermia based on the single-cell RNA sequencing of testicular tissue.

Non-obstructive azoospermia (NOA) is among the most severe factors for male infertility, but our understandings of the latent biological mechanisms remain insufficient. The single-cell RNA sequencing (scRNA-seq) data of 432 testicular cells isolated from the patient with NOA was analyzed, and the cell samples were grouped into 5 cell clusters. A sum of 455 cell markers was identified and then included in the protein-protein interaction network. The Top 5 most critical genes in the network, including CCT8, CDC6, PSMD1, RPS4X, RPL36A, were selected for the diagnosis model construction through the random forest (RF). The RF model was a strong classifier for NOA and obstructive azoospermia (OA), which was validated in the training cohort (n = 58, AUC = 1) and external validation cohort (n = 20, AUC = 0.9). We collected the seminal plasma samples and testicular biopsy samples from 20 OA and 20 NOA cases from the local hospital, and the gene expression was detected via Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) and Immunohistochemistry. The RF model also exhibited high accuracy (AUC = 0.725) in the local cohort. In summary, a novel gene signature was developed and externally validated based on scRNA-seq analysis, providing some new biomarkers to uncover the underlying mechanisms and a promising clinical tool for diagnosis in NOA.

Down regulating PHGDH affects the lactate production of sertoli cells in varicocele.

BACKGROUND: Although varicocele is considered to be one of the leading causes of male infertility, the precise mechanism underlying how varicocele leads to male infertility is not completely understood. We found the lactate concentration on the varicocele side of the patients was decreased compare with peripheral venous blood. In the testicles, the lactate produced by the sertoli cells through the glycolysis pathway provides most of the energy needed for spermatogenesis, the reduction of lactate will affect spermatogenesis. The objective of this study was to investigate the mechanism of this abnormal energy metabolism phenomenon in varicocele. METHODS: In this study, we collected the testicular tissue from patients with varicocele, the glycolysis related proteins PHGDH was identified by iTRAQ proteomics technology. Experimental rat varicocele model was constructed according to our new clip technique, the mRNA and protein expression levels of PHGDH were examined with qRT-PCR and Western blotting. We constructed a sertoli cell of PHGDH down-regulation model, and then detected the glucose consumption, LDH activities and lactate production in the sertoli cells. Western blot was conducted to investigate the effects of PHGDH on the expression of phosphoserine phosphatase (PSPH) and Pyruvate kinase M2 (PKM2). Flow cytometry was used to detect the cell apoptosis and cell cycle in sertoli cells. RESULTS: The results showed that testicular protein PHGDH was down-regulated in patients with varicocele and in experimental rat varicocele model. Down-regulation of PHGDH in sertoli cells significantly decreased the glucose consumption, LDH activities and lactate production in the sertoli cells, indicating that the low expression of PHGDH ultimately led to a decrease in lactate production by affecting the glycolysis. The Western blot results showed that the down-regulation of PHGDH significantly reduced the expression of pathway protein PSPH and PKM2, leading to the reduction of lactate production. Moreover, PHGDH knockdown can promote apoptosis and inhibit cell cycle to affect cell growth. CONCLUSIONS: Overall, we conformed that varicocele lead to the decreasing of testis lactate production. Down-regulation of PHGDH in sertoli cells may mediate the process of abnormal glucose metabolism. Our study provide new insight into the mechanisms underlying metabolism-associated male infertility and suggests a novel therapeutic target for male infertility.

[rotective effect of bone marrow mesenchymal stem cells-derived exosomes against testicular ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the protective effect of bone marrow mesenchymal stem cells (BMSCs)-derived exosomesagainst testicular ischemia-reperfusion injury (IRI) in rats. METHODS: Rat BMSCs were isolated, cultured and identified in theprimary culture. The exosomes were extracted from the BMSCs and characterized using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Twenty-four healthy male SD rats were randomly divided into shamoperation group, testicular IRI with saline treatment group and IRI with exosome treatment group. The contralateral testes ofthe rats were collected for pathological observation, aseessment of superoxide dismutase (SOD) and malondialdehyde (MDA), and detection of HMGB1, caspases-3 and cleaved caspase-3 expressions using Western blotting. RESULTS: We successfullyobtained exosomes from rat BMSCs. Testicular IRI significantly impaired testicular spermatogenesis, which was markedlyimproved by treatment with the exosomes (P < 0.05). Testicular IRI also caused significant increase in the protein expression ofHMGB1, caspase-3 and cleaved caspase-3 in the testicular tissue, and treatment with the exosomes obviously amelioratedthese changes (P < 0.05). CONCLUSIONS: BMSCs-derived exosomes protects against testicular IRI due to the anti-oxidant, antiinflammatory and anti-apoptosis activities of the exosomes.