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Objective: This study aimed to evaluate the effect of testicular cell conditioned medium (TCCM) on in vitro male germ cell differentiation and provide a proteomic profile of TCCM. Materials and Methods: TCCM was collected from 5-day-old mouse testicular tissues cultured in serum-free DMEM. Proteomic analysis was performed using liquid chromatography–tandem mass spectrometry. Germ cells were isolated from 13.5 days post-coitum (dpc) mouse fetal genital ridges and divided into three groups: (a) control (DMEM + 15% FCS), (b) 40% TCCM + 60% DMEM + 15% FCS, and (c) 60% TCCM + 40% DMEM + 15% FCS. Cells were cultured for 24 days. Gene expression of Oct4, Acr, Dazl, Vasa, Stra8, Prm1, and Gdf9 was measured using real-time PCR. Results: Proteomic analysis identified 26 proteins in TCCM. Notably, COL4A1, COL4A2, and HSPG2 are associated with the basement membrane and are essential for supporting extracellular matrix integrity, while FN1, FBN1, COL1A1, COL1A2, COL5A2, and COL3A12 are linked to the PI3K-AKT pathway, which regulates cell proliferation. In TCCM-treated groups, germ cells differentiated into spermatid-like cells by day 18 and sperm-like structures by day 24. Oct4, Dazl, Vasa, Stra8, Prm1, and Acr were expressed across all groups, but without statistically significant differences (p > 0.05), while Gdf9 was not expressed. Conclusion: The addition of TCCM, which contains extracellular matrix (ECM) proteins, enhanced in vitro differentiation of male germ cells into sperm-like structures, together with somatic cells from the genital ridge. These ECM proteins contribute to creating a microenvironment that closely mimics in vivo conditions.

Abstract Testicular cancer represents 1% to 1.5% of neoplasias in males and 5% of urologic tumors in general. The incidence of bilateral testicular tumors is 1-5%. Approximately one third of cases are diagnosed as synchronous, while the other two thirds are diagnosed as metachronous. Additionally, 5% of all patients diagnosed with testicular cancer may have contralateral intratubular germ cell neoplasia and may develop a contralateral germ cell tumor. However, few data are available regarding bilateral testicular germ cell tumors (BTGCTs). In this review, we aim to provide an overview of the incidence of BTGCTs and their pathological features and clinical outcomes

Androgen deficiency is a physical disorder that not only affects adults but can also jeopardize children's health. Because there are many disadvantages to using traditional androgen replacement therapy, we have herein attempted to explore the use of human umbilical cord mesenchymal stem cells for the treatment of androgen deficiency. We transplanted CM-Dil-labeled human umbilical cord mesenchymal stem cells into the testes of an ethane dimethanesulfonate (EDS)-induced male rat hypogonadism model. Twenty-one days after transplantation, we found that blood testosterone levels in the therapy group were higher than that of the control group (P = 0.037), and using immunohistochemistry and flow cytometry, we observed that some of the CM-Dil-labeled cells expressed Leydig cell markers for cytochrome P450, family 11, subfamily A, polypeptide 1, and 3-β-hydroxysteroid dehydrogenase. We then recovered these cells and observed that they were still able to proliferate in vitro. The present study shows that mesenchymal stem cells from human umbilical cord may constitute a promising therapeutic modality for the treatment of male hypogonadism patients.

Although the androgen receptor (AR) has been implicated in the promotion of apoptosis in testicular cells (TSCs), the molecular pathway underlying AR-mediated apoptosis and its sensitivity to environmental hormones in TSCs and induced pluripotent stem cells (iPSCs) remain unclear. We generated the iPSCs from bovine TSCs via the electroporation of OCT4. The established iPSCs were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4 to maintain and stabilize the expression of stemness genes and their pluripotency. Apoptosis signaling was assessed after exposure to mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate. Here, we report that iPSCs were more resistant to MEHP-induced apoptosis than were original TSCs. MEHP also repressed the expression of AR and inactivated WNT signaling, and then led to the commitment of cells to apoptosis via the cyclin dependent kinase inhibitor p21CIP1. The loss of the frizzed receptor 7 and the gain of p21CIP were responsible for the stimulatory effect of MEHP on AR-mediated apoptosis. Our results suggest that testicular iPSCs can be used to study the signaling pathways involved in the response to environmental disruptors, and to assess the toxicity of environmental endocrine disruptors in terms of the maintenance of stemness and pluripotency.

Malignant testicular germ cell tumors （TGCT） are the most frequent cancers in Caucasian males （20-40 years） with an 70% increasing incidence the last 20 years, probably due to combined action of （epi）genetic and （micro）environmental factors. It is expected that TGCT have carcinoma in situ （CIS） as their common precursor, originating from an embryonic germ cell blocked in its maturation process. The overall cure rate of TGCT is more than 90%, however, men surviving TGCT can present long-term side effects of systemic cancer treatment. In contrast, men diagnosed and treated for CIS only continue to live without these long-term side effects. Therefore, early detection of CIS has great health benefits, which will require an informative screening method. This review described the etiology and early pathogenesis of TGCT, as well as the possibilities of early detection and future potential of screening men at risk for TGCT. For screening, a well-defined risk profile based on both genetic and environmental risk factors is needed. Since 2009, several genome wide association studies （GWAS） have been published, reporting on single-nucleotide polymorphisms （SNPs） with significant associations in or near the genes KITLG, SPRY4, BAK1, DMRT1, TERT, ATF71P, HPGDS, MAD1L1, RFWD3, TEX14, and PPMIE, likely to be related to TGCT development. Prenatal, perinatal, and postnatal environmental factors also influence the onset of CIS. A noninvasive early detection method for CIS would be highly beneficial in a clinical setting, for which specific miRNA detection in semen seems to be very promising. Further research is needed to develop a well-defined TGCT risk profile, based on gene-environment interactions, combined with noninvasive detection method for CIS.

Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.

The serious coronavirus disease‐2019 (COVID‐19) was first reported in December 2019 in Wuhan, China. COVID‐19 is an infectious disease caused by severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS‐CoV‐2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single‐cell RNA‐sequencing (scRNA‐seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS‐CoV‐2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS‐CoV‐2. The expression level of ACE2 was related to the age, and the mid‐aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS‐CoV‐2 and may enable rapid deciphering male‐related reproductive disorders induced by COVID‐19.

Testicular germ cell tumors (TGCTs) are malignant neoplasms with a poor prognosis, and the absence of reliable biomarkers for patient stratification and diagnosis presents a significant challenge. We employed an integrated analysis of Single-Cell RNA-Sequencing and TCGA data to evaluate TNFSF10 as a potential biomarker for prognosis and immunotherapy in TGCTs. Our findings revealed that aberrant TNFSF10 expression was significantly associated with patient survival outcomes. Silencing TNFSF10 inhibited the proliferation, migration, and invasion of TGCT cells, highlighting its role in tumor progression. Moreover, TNFSF10 expression was closely correlated with various components of the tumor microenvironment, suggesting its involvement in both the biological behavior of TGCTs and their response to treatment. TNFSF10 emerges as a promising diagnostic and prognostic biomarker for stratification and targeted therapy in TGCTs, offering new prospects for personalized treatment strategies.

Cisplatin is used to treat a variety of malignancies, including testicular germ cell tumours (TGCTs). Although cisplatin‐based chemotherapy yields high response rates, a subset of patients develop cisplatin resistance, limiting treatment options and worsening prognosis. Therefore, there is a high clinical need for new therapeutic strategies targeting cisplatin‐resistant TGCTs. MicroRNA‐371a‐3p ( miR‐371 ), the new serum biomarker for TGCTs, shows significantly increased expression in cisplatin‐resistant TGCT cell lines compared to sensitive parental cell lines. However, the functional impact of miR‐371 on cisplatin sensitivity has not been investigated yet. To evaluate the impact of miR‐37 1 on cisplatin sensitivity, antagomirs were used to inhibit miR‐371 expression, resulting in a > 98% decrease in miR‐371 expression. Cisplatin sensitivity was significantly increased after miR‐371 inhibition in cisplatin‐resistant and corresponding parental TGCT cell lines, indicating a strongly reduced viability and increased apoptosis after cisplatin treatment in miR‐371 ‐inhibited cells. Our results suggest that miR‐371 may contribute to the development of cisplatin resistance in TGCTs. Interfering with miR‐371 expression can increase the cisplatin sensitivity of tumour cells, which may represent a promising approach to improve future therapeutic outcomes in patients with TGCTs, especially those with cisplatin‐resistant disease.

Germ cell tumours predominantly of the testis ((T)GCTs) are remarkably chemotherapy sensitive. However, a small proportion of patients fail to be cured with cisplatin‐based combination chemotherapy. miR‐371a‐3p is a new liquid biopsy biomarker for (T)GCTs. The aim of this study was to evaluate clinical utility of plasma miR‐371a‐3p level in patients starting systemic chemotherapy. Patients were included before the first cycle (N = 180) and second cycle (N = 101) of systemic first line chemotherapy, treated between July 2010 and May 2017. Plasma miR‐371a‐3p levels were measured with the ampTSmiR test and compared to disease characteristics and outcome. Pretreatment plasma miR‐371a‐3p levels were increased in 51.7% of cases and associated with number of metastatic sites, presence of lung, retroperitoneal, and mediastinal lymph node metastases, S – stage, IGCCCG risk group, and response to therapy. Patients with a negative pretreatment plasma level had better progression‐free survival (PFS) and overall survival (OS) compared to patients being positive for miR‐371a‐3p (hazard ratio [HR] = 0.26, 95% confidence interval [CI] 0.09‐0.71, P = 0.02 for PFS and HR = 0.21, 95% CI 0.07‐0.67, P = 0.03 for OS, respectively). Patients negative for miR‐371a‐3p in both samples had a superior PFS (HR = 0.10, 95% CI 0.01‐21.49, P = 0.02) and OS (HR = 0.08, 95% CI 0.01‐27.81, P = 0.008) compared to patients with miR‐371a‐3p positive in both samples (multivariate analyses were non‐significant). In total 68% of the patients were S0. This study demonstrates clinical value of plasma miR‐371a‐3p level in chemotherapy naïve (T)GCT patients starting first line of chemotherapy to predict prognosis.