Weighted gene coexpression network analysis reveals ESR1, FLNA and Furin as hub genes for DEHP-induced prepubertal testicular injury.

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor that accumulates in organisms in various ways and induces male reproductive system disorders. In this study, we established a testicular injury model by gavage with different concentrations of DEHP. The testes were then collected for RNA sequencing (RNA-seq), and the results were analyzed by bioinformatics and verified by experiments. Our research results show that different concentrations of DEHP interfere with testicular development differently. Weighted gene coexpression network analysis (WGCNA) generated sixteen modules and identified the turquoise module as key. Then, estrogen receptor 1 (ESR1), filamin A (Flna) and Furin were identified as hub genes. qPCR and immunohistochemistry results revealed that all three hub genes were upregulated. We detected the locations of these genes by immunohistochemistry. ESR1 was mainly located in Leydig cells; Flna immunostaining is observed in the Leydig and some germ cells and Furin staining was seen in almost all types of testicular cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment mainly in MAPK signaling pathways, p53 signaling pathways, HIF-1 signaling pathways, protein processing in the endoplasmic reticulum, apoptosis, the cell cycle, RNA degradation, etc. This is the first study using WGCNA to investigate the mechanism of DEHP-induced injury in the prepubertal testis, providing new research angles to further understand the mechanism of DEHP-induced injury in the prepubertal testis.

Multiple transcriptomic profiling: p53 signaling pathway is involved in DEHP-induced prepubertal testicular injury via promoting cell apoptosis and inhibiting cell proliferation of Leydig cells.

Di(2-ethylhexyl) phthalate (DEHP) is a widely-used plasticizer and has long been recognized as an endocrine-disrupting chemical with male reproductive toxicities. DEHP exposure at the prepubertal stage may lead to extensive testicular injury. However, the underlying mechanisms remain to be elucidated. In the present study, we gavaged male C57BL/6 mice with different concentrations of DEHP (0, 250, and 500 mg/kg-bw·d) from postnatal day 22-35, and exposed TM3 Leydig cells with 0, 100, 200, 300, and 400 µM of MEHP (bioactive metabolite of DEHP) for 12-48 h. RNA sequencing was performed both in testicular tissue and TM3 cells. The results showed that DEHP disrupts testicular development and reduces serum testosterone levels in male prepubertal mice. Bioinformatic analysis and experimental verification have revealed that DEHP/MEHP induces cell cycle arrest in TM3 cells and increases apoptosis both in vivo and in vitro. Furthermore, the p53 signaling pathway was found to be activated upon DEHP/MEHP treatment. The inhibition of p53 by pifithrin-α significantly reduced MEHP-induced injuries in TM3 cells. Cumulatively, these findings revealed the involvement of the p53 signaling pathway in DEHP-induced prepubertal testicular injury by promoting cell apoptosis and inhibiting cell proliferation of Leydig cells.

Complications Following Primary Repair of Non-proximal Hypospadias in Children: A Systematic Review and Meta-Analysis.

Purpose: The aim of this study was to systematically review the literature on the complications and postoperative outcomes of children with non-proximal hypospadias. Methods: Electronic databases including PubMed, Embase, and Cochrane Library CENTRAL were searched systematically from January 1990 to June 2020 for the literature that reported the postoperative outcomes of patients with non-proximal hypospadias. Non-proximal hypospadias encompassed distal and mid-penile hypospadias. Results: We included 44 studies involving 10,666 subjects. Urethrocutaneous fistula (UCF) was the most common complication with an incidence of 4.0% (95% CI, 3.1-5.0%). Incidence of overall complications was 8.0% (95% CI, 6.3-9.8%). Meta-regression analysis revealed that length of urethral stent indwelling (coefficient 0.006; 95% CI, 0.000-0.011; p = 0.036) and penile dressing (coefficient 0.010; 95% CI, 0.000-0.021; p = 0.048) were two risk factors for UCF. Multivariate meta-regression analysis did not identify any independent risk factors for UCF. No differences were found between stent and stentless groups in non-proximal hypospadias regarding incidences of UCF (OR, 0.589; 95% CI, 0.267-1.297), meatal stenosis (OR, 0.880; 95% CI, 0.318-2.437), and overall complications (OR, 0.695; 95% CI, 0.403-1.199). No differences were found between foreskin preservation and circumcision in terms of complications either. Conclusions: UCF is the most common complication following hypospadias repair with an incidence of 4.0%. Independent risk factors for UCF were not identified in the current research. Distal hypospadias repair without stent indwelling is not likely to compromise the postoperative outcome. Further studies should be designed to explore the differences between different surgical approaches and the potential risk factors for complications following hypospadias repair.