Sertoli cells are the most important somatic cells contributing to the microenvironment (named niche) for spermatogonial stem cells (SSCs). They produce amounts of crucial growth factors and structure proteins that play essential roles in the complex processes of male SSCs survival, proliferation, and differentiation. It has been suggested that Sertoli cell abnormalities could result in spermatogenesis failure, eventually causing azoospermia in humans. However, to the end, the gene expression characteristics and protein functions of human Sertoli cells remained unknown. In this study, we aimed to evaluate the effect of fibroblast growth factor-5 (FGF5), a novel growth factor downregulated in Sertoli cells from Sertoli cell-only syndrome (SCOS) patients compared to Sertoli cells from obstructive azoospermia (OA) patients, on SSCs. We compared the transcriptome between Sertoli cell from SCOS and OA patients. Then, we evaluated the expression of FGF5, a growth factor which is downregulated in SCOS Sertoli cells, in human primary cultured Sertoli cells and testicular tissue. Also, the proliferation effect of FGF5 in mice SSCs was detected using EDU assay and CCK-8 assay. To investigate the mechanism of FGF5, Phospho Explorer Array was performed. And the results were verified using Western blot assay. Using RNA-Seq, we found 308 differentially expressed genes (DEGs) between Sertoli cells from SCOS and OA patients. We noted and verified that the expression of fibroblast growth factor-5 (FGF5) was higher in Sertoli cells of OA patients than that of SCOS patients at both transcriptional and translational levels. Proliferation assays showed that rFGF5 enhanced the proliferation of mouse SSCs line C18-4 in a time- and dose-dependent manner. Moreover, we demonstrated that ERK and AKT were activated and the expression of Cyclin A2 and Cyclin E1 was enhanced by rFGF5. The distinct RNA profiles between Sertoli cells from SCOS and OA patients were identified using RNA-Seq. Also, FGF5, a growth factor that downregulated in SCOS Sertoli cells, could promote SSCs proliferation via ERK and AKT activation.

Vascular endothelial growth factor (VEGF) plays fundamental roles in testicular development; however, its function on testicular regeneration remains unknown. The objective of this study was to explore the roles VEGF/VEGFR2 signaling plays in mouse germ cells and in mouse testicular regeneration. VEGF and the VEGFR2 antagonist SU5416 were added to culture medium to evaluate their effects on spermatogonial stem cell line (C18-4 cells) proliferation. Testicular cells obtained from newborn male ICR mice were grafted into the dorsal region of male BALB/c nude mice. VEGF and SU5416 were injected into the graft sites to assess the effects of the VEGF and VEGFR2 signaling pathways on testicular reconstitution. The grafts were analyzed after 8 weeks. We found that VEGF promoted C18-4 proliferation in vitro, indicating its role in germ cell survival. HE staining revealed that seminiferous tubules were reconstituted and male germ cells from spermatogonia to spermatids could be observed in testis-like tissues 8 weeks after grafting. A few advantaged male germ cells, including spermatocytes and spermatids, were found in SU5416-treated grafts. Moreover, VEGF enhanced the expression of genes specific for male germ cells and vascularization in 8-week grafts, whereas SU5416 decreased the expression of these genes. SU5416-treated grafts had a lower expression of MVH and CD31, indicating that blockade of VEGF/VEGFR2 signaling reduces the efficiency of seminiferous tubule reconstitution. Collectively, these data suggest that VEGF/VEGFR2 signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization.

The aim of this study is to assess the value of contrast-enhanced ultrasound (CEUS) as a new non-invasive approach to locate the testicular area in which spermatogenesis is most likely to be found in non-obstructive azoospermic testes and to evaluate the accuracy of CEUS as a predictor of successful sperm retrieval. CEUS was performed in 120 nonobstructive azoospermia (NOA) patients. Microdissection testicular sperm extraction (M-TESE) was performed on the best and poorest perfusion areas selected by CEUS and on conventional areas. In the 187 testicles that underwent M-TESE, the sperm retrieval rates (SRRs) in the best perfusion area and poorest perfusion area over the maximal longitudinal section and conventional area were 63.1, 34.7 and 47.1%. According to receiver operating characteristic (ROC) analysis, the arrival times (AT) ≤27 s, time-to-peak intensity (TTP) ≤45 s, and peak intensity (PI) ≥11 dB were the best predictors of positive sperm retrieval. The location of the best perfusion area was able to guide M-TESE to improve the success rates. Testicle CEUS is suggested to be performed in all patients with NOA. If AT≤27 s, TTP ≤ 45 s or PI≥11 dB are found in the best perfusion area, M-TESE is strongly recommended.

KASH5 is an essential component of the LINC (linker of the nucleoskeleton and cytoskeleton) complex that regulates chromosome movements and nuclear envelope (NE) remodeling in mouse spermatocytes during meiosis prophase I, but its expression and function in human cells, as well as its association with male infertility are largely unknown. In this study, a novel heterozygous copy number variation (CNV) (seq [GRCh37] del(19) (19q13.33) chr19: g.49894043-49903011del) and a heterozygous loss of function variant (NM_144688: c.979_980del: p.R327Sfs*21) in human KASH5 were identified in a non-obstructive azoospermia (NOA)-affected patient and in his infertile sister by whole-exome sequencing and CNV array. Spermatogenesis in the proband was arrested at zygotene-like stage with a deficiency in homolog pairing and synapsis. KASH5 protein expression in human spermatocytes was evaluated and reported first in this study. Single-cell RNA sequencing demonstrated that the LINC complex and associated genes in human and mouse shared a similar expression pattern, indicating a conserved mechanism in the regulation of chromosome movements and NE remodeling. Kash5 knockout mouse displayed similar phenotypes, including a meiotic arrest at a zygotene-like stage and impaired pairing and synapsis. Collectively, we have identified novel rare variants within human KASH5 in patients with NOA and meiosis arrest. Our study expands the knowledge of KASH5 and associated proteins in regulating human meiosis prophase I progress and provides new insight into the genetic etiology of NOA.

Aging has a significant negative impact on human testicular function; steroidogenesis is gradually impaired, and testosterone replacement therapy still has many risks. Low-intensity pulsed ultrasound (LIPUS) has been used as a novel non-invasive treatment for male erectile dysfunction and other fields, and has been shown to increase testosterone levels in animal models. Testosterone is synthesized and secreted by Leydig cells (LCs), and the serum testosterone level decreases after aging due to the LCs senescence. However, the effect of LIPUS on human senescent LCs has not been reported. In this study, human senescent LCs were isolated and stimulated with different energy intensities in vitro, and cell morphology, cell apoptosis, cell proliferation, cell senescence levels, lipid droplet number, testosterone and INSL3 secretion levels were tested and analyzed. Quantitative Polymerase Chain Reaction (QPCR) and Western Blot were performed to compare cell senescence characteristics and the expression profile of key pathways of testosterone secretion, and transcriptome analysis was performed to explore the signaling pathways of LCs alteration after LIPUS stimulation. It was safe and effective to stimulate LCs with the 75 mW/cm² energy of LIPUS in vitro, which not only improved the senescence phenotype, but also effectively enhanced the secretory function of LCs in vitro, and increased the expression of key pathways of the testosterone synthesis pathway. These results suggest that LIPUS could be used as a novel treatment to human senescent LCs with decreased testosterone secretion levels in vitro.

Background. Previously published predictive models for microdissection testicular sperm extraction (micro‐TESE) were generally assumed patients with nonobstructive azoospermia (NOA) a homogenous population, i.e., the laboratory predictors were associated with sperm retrieval rate (SRR) in a similar way among different subpopulations. In addition, previous studies primarily regarded the success of sperm retrieval as the sole endpoint, although live birth is the ultimate goal for the couples. Objectives. The main objective is to develop and evaluate the clinical benefit of a model predicting the clinical outcome of micro‐TESE in heterogeneous population with NOA. The outcome of pregnancy was taken into account via assessing the association between the predicted outcome of micro‐TESE and pregnancy. Materials and Methods. A development cohort of 1,292 patients with NOA and an external validation cohort of 530 patients were included. Sperm retrieval was performed using micro‐TESE. Clinical outcomes, including sperm retrieval, clinical pregnancy, and live birth, were collected. We developed a model using the machine learning method random forest and provided a web‐based calculator. Results. The SRR was 38.1% (492/1,292) in the development cohort and 48.5% (257/530) in the validation cohort. The final model includes etiology, AMH, sperm retrieval surgical history, testicular volume, FSH, LH, and age as predictors (ordered by variable importance). The area under the curve of our model was 0.76 (0.74–0.79) in the development cohort and 0.75 (0.71–0.79) in the external validation cohort. The decision curve analysis showed that personalized model‐based surgical decision provides additional clinical benefit. The clinical pregnancy rate (CPR) and cumulative live birth rate (CLBR) were 45.3% (405/895) and 57.6% (338/587), respectively, in the overall population. For patients of different SRR, the CPR and CLBR of whom had successful sperm retrieval were similar. Discussion and Conclusion. Our model predicting the SRR of micro‐TESE was generalizable and easy to use. Predicted pregnancy outcomes like CPR and CLBR could also be derived from predicted SRR. A model‐based surgical decision after personalized consultation would be beneficial to patients with NOA.