Single-cell RNA sequencing reveals transcriptomic landscape and potential targets for human testicular ageing.

STUDY QUESTION: What is the molecular landscape underlying the functional decline of human testicular ageing? SUMMARY ANSWER: The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing. WHAT IS KNOWN ALREADY: Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing. STUDY DESIGN, SIZE, DURATION: Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors. PARTICIPANTS/MATERIALS, SETTING, METHODS: scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell-cell communication analysis was performed using CellChat. MAIN RESULTS AND THE ROLE OF CHANCE: The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing. LARGE SCALE DATA: The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349. LIMITATIONS, REASONS FOR CAUTION: Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest.

A data-driven analysis to discover research hotspots and trends of technologies for PFAS removal.

The frequent detection of persistent per- and polyfluoroalkyl substances (PFAS) in organisms and environment coupled with surging evidence for potential detrimental impacts, have attracted widespread attention throughout the world. In order to reveal research hotspots and trends of technologies for PFAS removal, herein, we performed a data-driven analysis of 3975 papers and 436 patents from Web of Science Core Collection and Derwent Innovation Index databases up to 2023. The results showed that China and the USA led the way in the research of PFAS removal with outstanding contributions to publications. The progression generally transitioned from accidental discovery of decomposition, to experimentation with removal effects and mechanisms of existing methods, and finally to enhanced defluorination and mechanism-driven design approaches. The keywords co-occurrence network and technology classification together revealed the main knowledge framework, which was constructed and correlated through contaminants, substrates, materials, processes and properties. Moreover, adsorption was demonstrated to be the dominant removal process among the current studies. Subsequently, we concluded the principles, advances and drawbacks of enrichment and separation, biological methods, advanced oxidation and reduction processes. Further exploration indicated the hotspots such as alternatives and precursors for PFAS ("genx": 1.258, "f-53b": 0.337), degradable mineralization technologies ("photocatalytic degrad": 0.529, "hydrated electron": 0.374), environment-friendly remediation technologies ("phytoremedi": 0.939, "constructed wetland": 0.462) and combination with novel materials ("metal-organic framework": 1.115, "layered double hydroxid": 0.559) as well as computer science ("molecular dynamics simul": 0.559, "machine learn"). Furthermore, the future direction of technological innovation might lie in high-performance processes that minimize secondary pollution, the development of recyclable and renewable treatment agents, and collaborative control strategies for multiple pollutants. Overall, this study offers comprehensive and objective review for researchers and industry professionals in this field, enabling rapid access to knowledge guidance and insights into research frontiers.

Single-cell RNA sequencing reveals the local cell landscape in mouse epididymal initial segment during aging.

BACKGROUND: Morphological and functional alterations in aging reproductive organs result in decreased male fertility. The epididymis functions as the transition region for post-testicular sperm maturation. And we have previously demonstrated that the epididymal initial segment (IS), a region of the reproductive tract essential for sperm maturation and capacitation, undergoes considerable histological changes and chronic immune activation in mice during aging. However, the local aging-associated cellular and molecular changes in the aged epididymal IS are poorly understood. RESULTS: We conducted single-cell RNA sequencing analysis on the epididymal IS of young (3-month-old) and old (21-month-old) mice. In total, 10,027 cells from the epididymal IS tissues of young and old mice were obtained and annotated. The cell composition, including the expansion of a principal cell subtype and Ms4a4bHiMs4a6bHi T cells, changed with age. Aged principal cells displayed multiple functional gene expression changes associated with acrosome reaction and sperm maturation, suggesting an asynchronous process of sperm activation and maturation during epididymal transit. Meanwhile, aging-related altered pathways in immune cells, especially the "cell chemotaxis" in Cx3cr1Hi epididymal dendritic cells (eDCs), were identified. The monocyte-specific expression of chemokine Ccl8 increased with age in eDCs. And the aged epididymal IS showed increased inflammatory cell infiltration and cytokine secretion. Furthermore, cell-cell communication analysis indicated that age increased inflammatory signaling in the epididymal IS. CONCLUSION: Contrary to the general pattern of lower immune responses in the male proximal genital tract, we revealed an inflammaging status in mouse epididymal initial segment. These findings will allow future studies to enable the delay of male reproductive aging via immune regulation.

Hotspots and trends of biological water treatment based on bibliometric review and patents analysis.

In order to reveal the hotspots and trends of biological water treatment from the perspectives of scientific and technological innovation, both of the bibliometric review and patents analysis were performed in this study. The Web of Science Core Collection database and Derwent Innovation Index database recorded 30023 SCI papers and 50326 patents, respectively were analyzed via information visualization technology. The results showed that China ranked the first in both papers and patents, while the United States and Japan had advantages in papers and patents, respectively. It was concluded through literature metrology analysis that microbial population characteristics, biodegradation mechanism, toxicity analysis, nitrogen and phosphorus removal and biological treatment of micro-polluted wastewater were the research hotspots of SCI papers. Activated sludge process and anaerobic-aerobic combined process were the two mainstream technologies on the basis of patent technology classification analysis. Technology evolution path of biological water treatment was also elucidated in three stages based on the citation network analysis. Furthermore, the future directions including research on the law of interaction and regulation of biological phases and pollutants and the technology innovations towards the targeted biotransformation or selective biodegradation of pollutants and resource reuse of wastewater were prospected.

[Human urine-derived stem cell-derived exosomes improve diabetic erectile dysfunction in rats].

Objective: To investigate the improving effect of human urine-derived stem cell-derived exosomes (USC-Exo) on the endothelial function and erectile function of male rats with diabetic ED (DED) and explore their action mechanism. METHODS: USC-Exo were extracted from the culture medium of USC by ultracentrifugation and identified. Cavernous sinus endothelial cells (CCEC) were collected from SD male rats and cultured in endothelial cell growth medium-2 (EGM-2) (the normal control group), EGM-2 + L-glucose at 25 mM (the high glucose group), EGM-2 + L-glucose at 25 mmol/L) + USC-Exo at 10 µg/ml (the Exo group), and EGM-2 + L-glucose at 25 mmol/L + USC-Exo at 10 µg/ml) + 3-methyladenine at 2 mmol/L (the 3-MA group), respectively. Changes of the autophagic flux in the CCECs transfected with mRFP-GFP-LC3 adenovirus were detected under the fluorescence microscope. The proliferation and tube-forming ability of the cells were assessed by CCK8 and Matrigel assays, respectively. DED was induced by intraperitoneal injection of streptozotocin in 10 of the rats, which were equally and randomly divided into a DED and an Exo group, and another 5 normal male rats were taken as controls. The rats in the normal and DED groups were injected intracavernously with 100 µl of PBS, and those in the Exo group with 100 µl of USC-Exo at the concentration of 1 µg/µl. Four weeks after treatment, the maximum intracavernous pressure (ICPmax) and mean arterial pressure (MAP) were measured, the endothelial marker CD31 detected by immunofluorescence assay, the expressions of the CD31, Beclin1 and LC3 I/II proteins examined by Western blot, and the number of autophagosomes in the cavernous endothelial cells determined under the transmission electron microscope. RESULTS: USC-Exo significantly increased the number of autophagosomes in the CCEC in the high glucose group compared with that in the normal controls (39.5 ± 6.2 vs 12.5 ± 5.4, P < 0.05). The expression of Beclin1 and proliferation of the CCEC were significantly higher in the Exo than in the high glucose group (P < 0.05). The autophagy inhibitor 3-MA evidently reversed the increasing effect of USC-Exo on the proliferation of the CCEC. The tube-forming ability of the CCEC was significantly increased in the Exo group compared with that in the high glucose group (15.3 ± 3.2 vs 6.3 ± 2.1, P < 0.05), which was also reversed in the 3-MA group. Both ICPmax and the ICPmax/MAP ratio were significantly higher in the Exo than in the DED group (ï¼»86.6 ± 12.6ï¼½ vs ï¼»37.9 ± 10.9ï¼½ mmHg, P < 0.05; 89.3 ± 14.1 vs 41.7 ± 11.5, P < 0.05), and so were the expressions of CD31, Beclin1 and LC3 I/II (P< 0.05) and the number of autophagosomes in the cavernosal endothelial cells (3.7 ± 0.6 vs 1.0 ± 1.0, P < 0.05). CONCLUSIONS: USC-Exo can significantly improve the endothelial and erectile functions of DED rats by increasing the autophagy of cavernosal endothelial cells.

[Compound Chamomile and Lidocaine Hydrochloride Gel applied at different time-windows for premature ejaculation: A prospective single-center randomized controlled study].

OBJECTIVE: To investigate the efficacy and safety of Compound Chamomile and Lidocaine Hydrochloride Gel (CCLH) (Kamistad) applied at different time-windows on premature ejaculation (PE). METHODS: This prospective study included 72 PE patients treated by application of CCLH to the glans and penile body in our hospital from February to October 2021. According to the time of drug administration before insertion into the vagina, we randomly divided the patients into a 5-minute group (n = 39) and a 15-minute group (n = 33). Before and after 1 and 2 weeks of treatment, we compared the intravaginal ejaculation latency time (IELT), PE diagnostic tool (PEDT) score, quality of life, and adverse reactions between the two groups of patients. RESULTS: Totally 62 of the patients completed the follow-up, 35 in the 5-minute group and 27 in the 15-minute group, and all showed significant improvement in IELT (P < 0.01) and PEDT score (P < 0.05) after treatment compared with the baseline. No allergic reactions, such as redness and swelling, developed at the application site in any of the patients, and no adverse significant effect was observed on the erectile hardness in 61 of the cases. Six cases showed increased erectile hardness instead. Fifty-seven of the patients experienced no obvious penile numbness or reduced sexual satisfaction, and all could complete their sexual activities. CONCLUSION: Compound Chamomile and Lidocaine Hydrochloride Gel applied at different time-windows is effective on PE, with a 5-minute rapid onset of action before intercourse, and no obvious adverse effects.

Outcome prediction of microdissection testicular sperm extraction based on extracellular vesicles piRNAs.

PURPOSE: Microdissection testicular sperm extraction (micro-TESE) could retrieve sperm from the testicles to help the non-obstructive azoospermia (NOA) patients to get their biological children, but also would cause damage to the testicles. Therefore, it is necessary to preoperatively predict the micro-TESE outcome in NOA patients. For this purpose, we aim to develop a model based on extracellular vesicles' (EVs) piRNAs (EV-piRNAs) in seminal plasma. METHODS: To identify EV-piRNAs that were associated with spermatogenic ability, small RNA-seq was performed between the NOA group (n = 8) and normal group (n = 8). Validation of EV-piRNA expression in seminal plasma EVs and testicles tissues was used to select EV-piRNAs for the model. Candidate EV-piRNAs were further selected by LASSO regression analysis. Binary logistic regression analysis was used for the models' calculation formula. ROC analysis and Hosmer-Lemeshow test was used to assess the models' performance in the training (n = 20) and validation (n = 25) cohorts. RESULTS: We identified 8 EV-piRNAs which were associated with spermatogenic ability. Two EV-piRNAs (pir-60351 and pir-61927) were selected by LASSO regression analysis. Finally, we developed a favorable model based on the expression of pir-61927 with good discrimination wherein the AUC was 0.82 (95% CI: 0.63~1.00, p = 0.016) in the training cohort and 0.83 (95% CI: 0.66~1.00, p = 0.005) in the validation cohort, as well as good calibration. CONCLUSIONS: A favorable model based on the expression of pir-61927 in seminal plasma EVs was established to predict the micro-TESE outcome in NOA patients.

Exosome circ_0044516 promotes prostate cancer cell proliferation and metastasis as a potential biomarker.

Circular RNAs (circRNAs) are important regulators in cancer growth and progression. Exosomes carry various molecules including RNA, protein, and lipid from one cell to another cell. But the role of circRNAs from the exosomes from prostate cancer patients are not elucidated. In this study, circ_0044516 was found upregulated in prostate cancer and the roles and molecular mechanism of Hsa_circ_0044516 (circ_0044516) was investigated. Firstly, the exosomes of prostate cancer patients were collected for human circRNAs microarray to screen the circRNA expression profile. There were 35 significantly expressed circRNAs with more than fivefolds from microarray analysis. Circ_0044516 was verified to be significantly upregulated in the exosomes from prostate cancer patients and the cell lines. Further investigation demonstrated that circ_0044516 downregulation inhibited the proliferation and metastasis of prostate cancer cells. By bioinformatics and luciferase reporter assays, circ_0044516 was verified to downregulate miR-29a-3p expression and negatively related to miR-29a-3p expression levels in prostate cancer. In a summary, the study indicated that circ_0044516 played an important role in prostate cancer cell survival and metastasis, which suggested that an oncogenic role of circ_0044516 in prostate cancer.

A panel of extracellular vesicle long noncoding RNAs in seminal plasma for predicting testicular spermatozoa in nonobstructive azoospermia patients.

STUDY QUESTION: Whether the testis-specific extracellular vesicle (EV) long noncoding RNAs (lncRNAs) in seminal plasma could be utilized to predict the presence of testicular spermatozoa in nonobstructive azoospermia (NOA) patients? SUMMARY ANSWER: Our findings indicate that the panel based on seminal plasma EV lncRNAs was a sensitive and specific method in predicting the presence of testicular spermatozoa and may improve clinical decision-making of NOA. WHAT IS KNOWN ALREADY: The adoption of sperm retrieval techniques, especially microdissection testicular sperm extraction (mTESE), in combination with ICSI has revolutionized treatment for NOA. However, there are no precise and noninvasive methods for predicting whether there are testicular spermatozoa in NOA patients before mTESE. STUDY DESIGN, SIZE, DURATION: RNA sequencing was performed on seminal plasma EVs from 6 normozoospermic men who underwent IVF due to female factor and 5 idiopathic NOA patients who failed to obtain testicular spermatozoa by mTESE and were diagnosed as having Sertoli cell-only syndrome by postoperative pathology. A biomarker panel of lncRNAs was constructed and verified in 96 NOA patients who underwent mTESE. Decision-making process was established based on the panel in seminal plasma EVs from 45 normozoospermia samples, 43 oligozoospermia samples, 62 cryptozoospermia samples, 96 NOA samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA sequencing was done to examine altered profiles of EV lncRNAs in seminal plasma. Furthermore, a panel consisting of EV lncRNAs was established and evaluated in training set and validation sets. MAIN RESULTS AND THE ROLE OF CHANCE: A panel consisting of nine differentially expressed testis-specific lncRNAs, including LOC100505685, SPATA42, CCDC37-DT, GABRG3-AS1, LOC440934, LOC101929088 (XR_927561.2), LOC101929088 (XR_001745218.1), LINC00343 and LINC00301, was established in the training set and the AUC was 0.986. Furthermore, the AUC in the validation set was 0.960. Importantly, the panel had a unique advantage when compared with models based on serum hormones from the same group of NOA cases (AUC, 0.970 vs 0.723; 0.959 vs 0.687, respectively). According to the panel of lncRNAs, a decision-making process was established, that is when the score of an NOA case exceeds 0.532, sperm retrieval surgery may be recommended. LIMITATIONS, REASONS FOR CAUTION: In the future, the sample size needs to be further expanded. Meanwhile, the regulatory functions and mechanism of lncRNAs in spermatogenesis also need to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: When the score of our panel is below 0.532, subjecting the NOA patients to ineffective surgical interventions may not be recommended due to poor sperm retrieval rate. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81871110, 81971314 and 81971759); the Guangdong Special Support Plan-Science and Technology Innovation Youth Top Talents Project (2016TQ03R444); the Science and Technology Planning Project of Guangdong Province (2016B030230001 and 201707010394); the Key Scientific and Technological Program of Guangzhou City (201604020189); the Pearl River S&T Nova Program of Guangzhou (201806010089); the Transformation of Scientific and Technological Achievements Project of Sun Yat-sen University (80000-18843235) and the Youth Teacher Training Project of Sun Yat-sen University (17ykpy68 and 18ykpy09). There are no competing interests related to this study. TRIAL REGISTRATION NUMBER: N/A.

Intratunical injection of human urine-derived stem cells derived exosomes prevents fibrosis and improves erectile function in a rat model of Peyronie's disease.

We aimed to evaluate the effects of intratunical injection of exosomes derived from human urine-derived stem cells (USC-exo) on plaque formation and erectile function in a transforming growth factor-ß1 (TGF-ß1) induced Peyronie's disease (PD) rat model. Twenty-four SD rats were randomly assigned equally to three groups: (I) Sham group (50 µl phosphate-buffered saline [PBS] injected into the tunica albuginea [TA]), (II) PD group (0.5 µg TGF-ß1 in 50 µl PBS injected into the TA) and (III) USC-exo group (0.5 ug TGF-ß1 plus 100 µg USC-exo injected into the TA at the same day). The maximum intracavernous pressure (ICPmax ) and mean arterial pressure (MAP) of each group were evaluated 4 weeks after injection. The plaque formation, fibrosis, matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) in the TA were evaluated. Four weeks after injection, USC-exo group showed more significantly enhanced ICPmax and ICPmax /MAP than PD group (p < .05). USC-exo could significantly ameliorate the TA fibrosis that could be associated with the inhibition of transdifferentiation of fibroblasts into myofibroblasts, decreased expression of TIMPs (TIMP-1, 2, 3) and increased activity of MMPs (MMP-1, 3, 9) in the TA. According to these findings, USC-exo can be a new candidate for the prevention of PD.

Impact on using cryopreservation of testicular or epididymal sperm upon intracytoplasmic sperm injection outcome in men with obstructive azoospermia: a systematic review and meta-analysis.

PURPOSE: To determine whether there was a significant impact on using cryopreservation of testicular or epididymal sperm upon the outcomes of intracytoplasmic sperm injection (ICSI) in patients with obstructive azoospermia (OA). METHOD: Systematic review and meta-analysis of 20 retrospective studies in databases from January 1, 1995, to June 1, 2020. RESULT: Twenty articles were included in this study. A total of 3602 (64.1%) of 5616 oocytes injected with fresh epididymal sperm were fertilized, compared with 2366 (61.2%) of 3862 oocytes injected with cryopreserved sperm (relative risk ratio (RR) 0.96, 95% confidence interval (CI) (0.90, 1.02), P > 0.05). A total of 303 (44.1%) of 687 ICSI cycles using fresh epididymal sperm resulted in a clinical pregnancy, compared with 150 (36.6%) of 410 ICSI cycles using cryopreserved epididymal sperm (RR 0.84, 95% CI (0.72, 0.97), P < 0.05). In the testis, a total of 2147 (68.7%) of 3125 oocytes injected with fresh sperm were fertilized, compared with 1623 (63.5%) of 2557 oocytes injected with cryopreserved sperm (RR 0.97, 95% CI (0.90, 1.06), P > 0.05). A total of 151 (47.8%) of 316 ICSI cycles using fresh testicular sperm resulted in a clinical pregnancy, compared with 113 (38.2%) of 296 ICSI cycles using cryopreserved sperm (RR 0.87, 95% CI (0.72, 1.05), P > 0.05). CONCLUSIONS: In men with OA, there was a statistical lower clinical pregnancy rate (CPR) by using frozen epididymal sperm compared with fresh epididymal sperm, but showing no difference on fertilization rate (FR). Additionally, FR and CPR were not affected by whether the retrieved testicular sperm was frozen or fresh.

[Proposals for the diagnosis and treatment of andrologic diseases during the novel coronavirus pneumonia epidemic].

In December, 2019, several cases of novel coronavirus pneumonia (NCP) were reported in Wuhan, Hubei. Since then, more and more NCP cases, confirmed or suspected, have been found in China and other parts of the world, and the virus is now showing a tendency towards a wider spread. During the NCP epidemic, all medical workers are confronted with special challenges in the diagnosis and treatment of various diseases and required of even more accurate therapeutic protocols as well as stricter observation of the principles for the prevention and control of NCP. Therefore, the Andrology Branch of Chinese Medical Association convened relevant experts to summarize the special points for andrologic clinicians to attend to in the diagnosis and treatment of male diseases during the NCP epidemic.

Free testosterone value before radical prostatectomy is related to oncologic outcomes and post-operative erectile function.

PURPOSE: To investigate whether free testosterone (FT) prior to radical prostatectomy was related to post-operative oncologic outcomes, erectile function and continence. METHODS: The data of 586 patients with available information underwent treatment in our center was retrospectively reviewed. Total testosterone (TT) was tested by chemiluminescence immunoassay, and FT value was calculated using Vermeulen's formula. Post-operative continence and erectile function were evaluated by the requirement of pad and the IIEF-5 score at 12 months. RESULTS: The median TT and FT value was 344 ng/dL (interquartile, IQR 314-374) and 6.9 ng/dL (IQR 6.4-7.3), and 106 patients (18.1%) and 152 patients (25.9%) were evaluated as having low TT and low FT based on current guidelines. Low TT and FT value were both related to older age (both p < 0.001), concomitant diabetes (p = 0.018 & 0.049), higher possibility of pre-operative erectile dysfunction (ED, both p < 0.001), higher pre-operative PSA value (both p < 0.001), higher clinical stage (both p < 0.001) and higher Gleason score in biopsy (both p < 0.001). Low FT was related to higher risk for pT3 (p = 0.020) and high Gleason score (p = 0.011) in logistic regression. The median follow-up duration was 52 moths (IQR 29-67) and FT was found to be an independent risk factor for biochemical recurrence (p = 0.005). In logistic regression TT was related to pre-operative ED (p = 0.010) and FT was related to post-operative ED (p = 0.001). CONCLUSION: Low FT value before radical prostatectomy was related to adverse pathological outcomes, biochemical recurrence and post-operative ED.

Phosphorylated mixed lineage kinase domain-like protein in human seminal plasma: A potential novel biomarker of spermatogenic function.

Keeping the self-renewal and differentiation of spermatogonial stem cell (SSC) in balance is essential for maintaining spermatogenesis. However, whether the cell death of SSC also plays a vital role in the human being remains unknown. To explore the necroptosis of SSC, the activation marker of necroptosis, phosphorylated mixed lineage kinase domain-like protein (pMLKL) in testes was evaluated by immunofluorescence staining and Western blot. Meanwhile, a total of 81 semen samples were divided based on the sperm concentration (>15, 10-15, 5-10 and 0-5 million/ml) to study the relationships between pMLKL levels and sperm counts. We found that the pMLKL was increased in the ageing human testes (p < 0.05). Moreover, the seminal pMLKL expression was decreased in groups with sperm concentration 0-5 and 5-10 million/ml when compared with normal sperm concentration in young men (p < 0.05). Further analysis revealed that pMLKL showed an age-related increased expression in men aged 22-60 years with normal sperm concentration. These data demonstrated that the necroptosis of SSC was important for the spermatogenic function and would raise in advance on the point of testicular hypofunction. In conclusion, the pMLKL may serve as a potential biologically seminal indicator for the spermatogenic function in men.

[Oxidative stress induces damage to epididymal epithelial tight junction protein ZO-1 and impairs epididymal function in varicocele rats].

OBJECTIVE: To investigate oxidative stress-mediated damage to the epididymal epithelial tight junction protein ZO-1 and its impact on epididymal function in varicocele rats. METHODS: We randomly divided 45 male adolescent SD rats into three groups of equal number: sham operation (left renal vein exposed and isolated), experimental (left renal vein constricted and collaterals of the left spermatic vein fully ligated), and treatment (60-day intragastric administration of vitamin E at 150 mg/kg/d after modeling). At 60 days after modeling, we observed the histological changes in the left epididymis, detected the expressions of ZO-1 and other tight junction-related proteins by real-time quantitative PCR, immunohistochemistry, immunofluorescence staining and Western blotting, determined sperm motility, and measured the levels of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), methylene dioxyamphetamine (MDA) and α-glucosidase (α-Glu) in the epididymal tissue of the rats. RESULTS: Compared with the rats of the sham operation group, those of the experimental group showed disorganized epithelial structure and decreased number of epithelial cells in the left epididymis, with some epithelial cells desquamated into the lumen. The expression of ZO-1 was significantly lower in the experimental than in the sham operation group (P < 0.05) but markedly upregulated after VE treatment (P < 0.05). In comparison with the sham operation group, the animals in the experimental group exhibited remarkably increased content of MDA in the epididymal tissue (ï¼»0.41 ± 0.05ï¼½ vs ï¼»1.21 ± 0.18ï¼½ nmol/mg prot, P < 0.05) but decreased levels of SOD (ï¼»814.65 ± 73.64ï¼½ vs ï¼»298.62 ± 67.84ï¼½ U/mg prot, P < 0.05), T-AOC (ï¼»0.84 ± 0.07ï¼½ vs ï¼»0.24 ± 0.04ï¼½ nmol/mg prot, P < 0.05) and α-Glu (ï¼»11.72 ± 2.72ï¼½ vs ï¼»5.82 ± 1.24ï¼½ U/mg prot, P < 0.05). VE treatment, however, remarkably reduced the content of MDA (ï¼»0.69 ± 0.12ï¼½ nmol/mg prot) and elevated the levels of SOD (ï¼»497.73 ± 48.03ï¼½ U/mg prot), T-AOC (ï¼»0.42 ± 0.06ï¼½ nmol/mg prot) and α-Glu (ï¼»9.11 ± 1.91ï¼½ U/mg prot) as compared with those in the experimental group (all P < 0.05). The percentage of progressively motile sperm was significantly lower in the experimental than in the sham operation group (ï¼»31.33 ± 6.32ï¼½% vs ï¼»71.21 ± 5.21ï¼½%, P < 0.05), but markedly increased after VE treatment (ï¼»60.68 ± 5.31ï¼½%, P < 0.05). CONCLUSIONS: Varicocele reduces the expression of the EETJ protein ZO-1 and impairs epididymal function via oxidative stress, while vitamin E can effectively upregulate the ZO-1 expression and improve epididymal function by decreasing oxidative stress in the epididymis of varicocele rats.

[Protective effect of urine-derived stem cells on erectile dysfunction in rats with cavernous nerve injury].

OBJECTIVE: To investigate the protective effect of human urine-derived stem cells (USCs) on erectile function and cavernous structure in rats with cavernous nerve injury (CNI). METHODS: Sixty adult male SD rats with normal sexual function were randomly divided into four groups of equal number: sham operation, bilateral CNI (BCNI) model control, phosphate buffered saline (PBS), and USC. The BCNI model was established in the latter three groups of rats by clamping the bilateral cavernous nerves. After modeling, the rats in the PBS and USC groups were treated by intracavernous injection of PBS at 200 µl and USCs at 1×106/200 µl PBS respectively for 28 days. Then, the maximum intracavernous pressure (mICP) and the ratio of mICP to mean arterial pressure (mICP/MAP) of the rats were calculated by electrical stimulation of the major pelvic ganglions, the proportion of nNOS- or NF200-positive nerve fibers in the total area of penile dorsal nerves determined by immunohistochemical staining, the levels of endothelial cell marker eNOS, smooth muscle marker α-SMA and collagen I detected by Western blot, and the smooth muscle to collagen ratio and the cell apoptosis rate in the corpus cavernosum measured by Masson staining and TUNEL, respectively. RESULTS: After 28 days of treatment, the rats in the USC group, as compared with those in the PBS and BCNI model control groups, showed significant increases in the mICP (ï¼»81 ± 9.9ï¼½ vs ï¼»31 ± 8.3ï¼½ and ï¼»33 ± 4.2ï¼½ mmHg, P <0.05), mICP/MAP ratio (0.72 ± 0.05 vs 0.36 ± 0.03 and 0.35 ± 0.04, P <0.05), the proportions of nNOS-positive nerve fibers (ï¼»11.31 ± 4.22ï¼½% vs ï¼»6.86 ± 3.08ï¼½% and ï¼»7.29 ± 4.84ï¼½% , P <0.05) and NF200-positive nerve fibers in the total area of penile dorsal nerves (ï¼»27.31 ± 3.12ï¼½% vs ï¼»17.38 ± 2.87ï¼½% and ï¼»19.49 ± 4.92ï¼½%, P <0.05), the eNOS/GAPDH ratio (0.52 ± 0.08 vs 0.31 ± 0.06 and 0.33 ± 0.07, P <0.05), and the α-SMA/GAPDH ratio (1.01 ± 0.09 vs 0.36 ± 0.05 and 0.38 ± 0.04, P <0.05), but a remarkable decrease in the collagen I/GAPDH ratio (0.28 ± 0.06 vs 0.68 ± 0.04 and 0.70 ± 0.10, P <0.05). The ratio of smooth muscle to collagen in the corpus cavernosum was significantly higher in the USC than in the PBS and BCNI model control groups (17.91 ± 2.86 vs 7.70 ± 3.12 and 8.21 ± 3.83, P <0.05) while the rate of cell apoptosis markedly lower in the former than in the latter two (3.31 ± 0.83 vs 9.82 ± 0.76, P <0.01; 3.31 ± 0.83 vs 9.75 ± 0.91, P <0.05). CONCLUSIONS: Intracavernous injection of USCs can protect the erectile function of the rat with cavernous nerve injury by protecting the nerves, improving the endothelial function, alleviating fibrosis and inhibiting cell apoptosis in the cavernous tissue.

UCA1 involved in the metformin-regulated bladder cancer cell proliferation and glycolysis.

Despite great scientific advances have been achieved in cancer treatment in recent years, the death rate of bladder cancer has been staying at a high level. Metformin, a widely-used and low-cost diabetes medicine, might have the potential of anticancer. The aim of this study was to evaluate the effects of metformin on bladder cancer cells and to identify potential molecular targets and signaling pathways. Bladder cancer 5637 cells transfected with either pcDNA/UCA1 vector or pcDNA3.1 empty vector were treated with various doses of metformin for different periods of time, and then cell proliferation and glycolysis were assessed. Reverse transcription polymerase chain reaction and Western blotting were applied to examine the expression of long non-coding RNA UCA1 and mammalian target of rapamycin-signal transducer and activator of transcription pathway molecules. We found metformin inhibited bladder cancer cell proliferation in a dose- and time-dependent manner. UCA1-overexpressed 5637 cells showed increased proliferation and glycolysis compared with control cells. Metformin downregulated both endogenous and exogenous UCA1 expression, leading to the inhibition of mammalian target of rapamycin-signal transducer and activator of transcription 3-hexokinase 2 signaling pathway. Our study provided the first evidence that metformin inhibited proliferation and glycolysis in cancer cells through regulation of long non-coding RNA UCA1. The discovery also suggested the important roles of long non-coding RNA in chemoprevention, which is a property of metformin.

Subinguinal microsurgical varicocelectomy with intraoperative microvascular Doppler ultrasound leads to the pain-free outcome after surgery.

OBJECTIVE: To determine efficacy of intraoperative microvascular Doppler ultrasound in assisting subinguinal microsurgical varicocelectomy for pain relief in the treatment of painful varicoceles, compared to the microsurgery without Doppler ultrasound. METHODS: Total 153 patients underwent microsurgical varicocelectomy were randomly allocated to two groups: Groups 1 and 2 included 82 and 71 patients monitored with and without using intraoperative microvascular Doppler ultrasound, respectively. The assessments were compared between two groups, including intraoperative parameters (vessel numbers and operative time) and postoperative outcomes (pain resolution, complications and recurrence). RESULTS: The average numbers of internal spermatic veins ligated (13.87±6.43 vs 11.72±5.66) and arteries preserved (1.96±0.87 vs 1.73±0.86) were significantly greater in Group 1 with Doppler ultrasound. Precisely, the smaller size of the internal spermatic veins was ligated and the more encircled arteries were also preserved in Group 1. In two groups with and without using Doppler ultrasound, 56 (68.3%) and 36 (50.7%) patients experienced a complete resolution of pain, 21 (25.6%) and 29 (40.9%) patients experienced partial resolution, whereas 5 (6.1%) and 6 (8.5%) patients experienced no change in the chronic pain, respectively. Thus, patients in Group 1 had a better outcome in chronic pain resolution (P = 0.033). The operative time, complications and recurrence rate were not different between the two groups. CONCLUSIONS: Subinguinal microsurgical varicocelectomy is an effective method to treat painful varicoceles. With the assistance of Doppler Ultrasound monitoring, greater numbers of vessels were identified and a better outcome of pain resolution was achieved.

[Anatomical variability of the left spermatic vein and establishment of the experimental left varicocele model in adolescent rats].

OBJECTIVE: To identify the anatomical variability of the left spermatic vein (LSV) and determine its effect on the induction of experimental left varicocele (ELV) in adolescent rats. METHODS: We equally randomized 30 adolescent male SD rats to groups A (LSV collaterals fully ligated and the left renal vein constricted), B (only the left renal vein constricted), and C (sham operation), observed the courses of the LSVs and measured their diameters. At 30 days after operation, we analyzed the changes in the left kidneys and the diameters of the LSVs. RESULTS: Irregular collaterals were observed in 90% of the LSVs and no abnormal changes were found in the left kidneys after surgery. The postoperative LSV diameter was remarkably increased in group A as compared with the baseline ([1.47 +/- 0.15 ] vs [0.16 +/- 0.08] mm, P < 0.01), but showed no significant difference in group B ([0.31 +/- 0.49] vs [0.15 +/- 0.07] mm, P > 0.05) and C ([0.17 +/- 0.07] vs [0.16 +/- 0.06] mm, P > 0.05), and it was significantly longer in A than in B (P < 0.01). The success rate of ELV induction was 100% in group A and 10% in group B, but no varicocele was observed in group C. CONCLUSION: Correct identification of the anatomical course of the LSV and ligation of its irregular collaterals are essential for the establishment of a stable and consistent ELV model.

[Suprapubis-assisted umbilical laparoendoscopic mini-dual-site surgery for varicocele: a report of 80 cases].

OBJECTIVE: To study the safety, effectiveness and feasibility of suprapubis-assisted umbilical laparoendoscopic mini-dual-site surgery (SAU-LEMDS) in the treatment of varicocele. METHODS: This study included 80 varicocele patients aged 24 - 44 (mean 28.5 +/- 2.6) years, 25 cases of grade I, 45 cases of grade II and 10 cases of grade III, 58 cases in the left side, 6 in the right and 16 in both sides, and all with asthenospermia. The patients were treated by SAU-LEMDS under subarachnoid anesthesia combined with general anesthesia in a supine position with a head-down-feet-up slope of 15 degrees. Two 5 mm trocars were inserted bilaterally at the umbilical edge, one with a 5 mm 30 degrees laparoscope placed in it, and another into the abdominal cavity below the pubic hairline with a 5 mm laparoendoscopic clipper placed in it. The operation procedure was similar to that of standard laparoscopic ligation of spermatic veins, with reservation of the spermatic artery and double-ligation of spermatic veins. And the procedure was repeated for the contralateral lesion in the bilateral cases. Postoperative follow-up was conducted for the incidences of orchiatrophy and testicular hydrocele and changes of seminal parameters. RESULTS: All the operations were successful, with the mean operation time of (10 +/- 5.0) min (range 8 to 25 min) for the unilateral cases and (18 +/- 6.5) min (range 15 to 30 min) for the bilateral cases, the mean blood loss of (1.5 +/- 0.5) ml (range 1 to 2 ml), and the mean postoperative hospital stay of (2 +/- 0.5) d (range 1.5 to 3 d). The patients were followed up for 6 -24 (12 +/- 2.5) months, which showed significant improvement in sperm motility as compared with the baseline ([28.53 +/- 5.21] vs [19.62 +/- 3.56]%, P < 0.05), with 28 cases (35.0%) restored to normal. Recurrence was found in 4 cases (5.0%). Testicular hydrocele occurred in 7 cases (8.75%), but orchiatrophy in none. The scars in the umbilicus and suprapubis were invisible because of the wrinkles and pubic hair. CONCLUSION: SAU-LEMDS is safe, effective and feasible for the treatment of varicocele. It is superior to umbilical laparoendoscopic single-site surgery (U-LESS) for its less invasiveness, simpler operation, and better cosmetic appearance.