An Alternative Application of Magnetic-Activated Cell Sorting: CD45 and CD235a Based Purification of Semen and Testicular Tissue Samples.

Magnetic activated cell sorting (MACS) is a well-known sperm selection technique, which is able to remove apoptotic spermatozoa from semen samples using the classic annexinV based method. Leukocytes and erythrocytes in semen samples or in testicular tissue processed for in vitro fertilization (IVF) could exert detrimental effects on sperm. In the current study, we rethought the aforementioned technique and used magnetic microbeads conjugated with anti-CD45/CD235a antibodies to eliminate contaminating leukocytes and erythrocytes from leukocytospermic semen samples and testicular tissue samples gained via testicular sperm extraction (TESE). With this technique, a 15.7- and a 30.8-fold reduction could be achieved in the ratio of leukocytes in semen and in the number of erythrocytes in TESE samples, respectively. Our results show that MACS is a method worth to reconsider, with more potential alternative applications. Investigations to find molecules labeling high-quality sperm population and the development of positive selection procedures based on these might be a direction of future research.

Oxido-Reduction Potential as a Method to Determine Oxidative Stress in Semen Samples.

There are different estimates for the incidence of infertility. Its occurrence may vary from area to area, but on average, it affects 15% of couples and 10-12% of men worldwide. Many aspects of infertility can be linked to reactive oxygen species (ROS) and the process of oxidative stress (OS). The association between poor semen quality and OS is well known. Unfortunately, there is no accepted protocol for the diagnosis and treatment of OS in andrology. Oxido-reduction potential (ORP) measurement is a new method for determining the ratio between oxidant and antioxidant molecules. Currently, ORP measurement is one of the fastest and most user-friendly methods of andrological OS determination and our goals were to confirm published correlations between ORP values and sperm parameters, examine how sperm concentration influences these results, and investigate whether intracellular ROS formations are also manifested in the ORP values or not after artificial ROS induction. Intracellular ROS formations were induced by menadione (superoxide anion inducer), hydrogen peroxide, and tert-butyl hydroperoxide (lipid peroxidation inducer) treatments; sperm parameters like motility and viability were determined with an SCA Scope system, and ORP changes were recorded by the Mioxsys system. Significant correlations were noticed among the ORP, spermatozoa concentration, motility, progressive motility, and viability. Nevertheless, only the ORP value after normalization with the sperm count correlated with these parameters. Due to normalization, very low and very high sperm concentrations can give misleading results. The means of the non-normalized ORP values were almost the same. All of the applied treatments resulted in decreases in the viability, motility, and progressive motility, and interestingly, altered ORP levels were detected. In addition, it was determined that seminal plasma had a significant protective effect on spermatozoa. The elimination of seminal plasma caused higher sensitivity of spermatozoa against used OS inducers, and higher ORP levels and decreased viabilities and motilities were measured. The ORP level could be a good indicator of male OS; however, in cases of low and high sperm counts, its result can be misleading. Overall, the conclusion can be drawn that ORP determination is a suitable method for detecting intracellular ROS accumulation, but it has limitations that still need to be clarified.

Estrogenic and Non-Estrogenic Disruptor Effect of Zearalenone on Male Reproduction: A Review.

According to some estimates, at least 70% of feedstuffs and finished feeds are contaminated with one or more mycotoxins and, due to its significant prevalence, both animals and humans are highly likely to be exposed to these toxins. In addition to health risks, they also cause economic issues. From a healthcare point of view, zearalenone (ZEA) and its derivatives have been shown to exert many negative effects. Specifically, ZEA has hepatotoxicity, immunotoxicity, genotoxicity, carcinogenicity, intestinal toxicity, reproductive toxicity and endocrine disruption effects. Of these effects, male reproductive deterioration and processes that lead to this have been reviewed in this study. Papers are reviewed that demonstrate estrogenic effects of ZEA due to its analogy to estradiol and how these effects may influence male reproductive cells such as spermatozoa, Sertoli cells and Leydig cells. Data that employ epigenetic effects of ZEA are also discussed. We discuss literature data demonstrating that reactive oxygen species formation in ZEA-exposed cells plays a crucial role in diminished spermatogenesis; reduced sperm motility, viability and mitochondrial membrane potential; altered intracellular antioxidant enzyme activities; and increased rates of apoptosis and DNA fragmentation; thereby resulting in reduced pregnancy.

Magnetic-Activated Cell Sorting as a Method to Improve Necrozoospermia-Related Asthenozoospermic Samples.

According to some statistics, absolute asthenozoospermia affects every 1 in 5000 men. Although this incidence rate does not appear to be too high, it is extremely important to address the phenomenon because it can drastically reduce the chances of pregnancy, even with assisted reproduction. The biggest problem with absolute asthenozoospermia is that it is difficult to distinguish between live and dead sperm cells, and fertilization with non-viable spermatozoa may contribute to the failure of an assisted reproduction cycle. Nowadays, DNA fragmentation (DF) is a crucial parameter of semen analysis, and in this paper, we provide evidence of the correlation between DF and vitality. For this purpose, the main semen parameters were investigated by a CASA system (concentration, motility, progressive motility, vitality and DF). In the necrozoospermic group (vitality < 58%), all the measured parameters showed significant differences compared to normal vitality. Concentration (30.1 M mL−1 vs. 13.6 M mL−1), motility (31.9% vs. 18.3%), and progressive motility (24.3% vs. 12.7%) were significantly decreased, while DF was significantly increased (17.4% vs. 23.7%). Based on the connection between vitality decrement and DF increment, DF lowering methods, such as magnetic-activated cell sorting, have been hypothesized as novel methods for the elimination of dead spermatozoa.

Application of Mass Spectrometry Imaging in Uro-Oncology: Discovering Potential Biomarkers.

A growing need is emerging worldwide for new molecular markers which could enhance the accuracy of diagnostic and therapeutic methods for detecting urogenital cancers. Mass spectrometry imaging (MSI) is a very promising tool in this regard. In this review, we attempt to provide a subjective summary of the latest publications on potential biomarkers of renal, bladder, prostate, and testicular malignancies detected with MSI through the eyes of a clinical urologist.

Involvement of Mitochondrial Mechanisms and Cyclooxygenase-2 Activation in the Effect of Desethylamiodarone on 4T1 Triple-Negative Breast Cancer Line.

Novel compounds significantly interfering with the mitochondrial energy production may have therapeutic value in triple-negative breast cancer (TNBC). This criterion is clearly fulfilled by desethylamiodarone (DEA), which is a major metabolite of amiodarone, a widely used antiarrhythmic drug, since the DEA previously demonstrated anti-neoplastic, anti-metastasizing, and direct mitochondrial effects in B16F10 melanoma cells. Additionally, the more than fifty years of clinical experience with amiodarone should answer most of the safety concerns about DEA. Accordingly, in the present study, we investigated DEA's potential in TNBC by using a TN and a hormone receptor positive (HR+) BC cell line. DEA reduced the viability, colony formation, and invasive growth of the 4T1 cell line and led to a higher extent of the MCF-7 cell line. It lowered mitochondrial transmembrane potential and induced mitochondrial fragmentation. On the other hand, DEA failed to significantly affect various parameters of the cellular energy metabolism as determined by a Seahorse live cell respirometer. Cyclooxygenase 2 (COX-2), which was upregulated by DEA in the TNBC cell line only, accounted for most of 4T1's DEA resistance, which was counteracted by the selective COX-2 inhibitor celecoxib. All these data indicate that DEA may have potentiality in the therapy of TNBC.

Metoidioplasty: Surgical Options and Outcomes in 813 Cases.

Introduction: Metoidioplasty is a variant of phalloplasty for transmen that includes the creation of the neophallus from a hormonally enlarged clitoris, urethral lengthening and scrotoplasty. The procedure results in male appearance of genitalia, voiding in standing position and preserved sexual arousal, but without possibility for penetrative intercourse. We evaluated outcomes of metoidioplasty at our center, based on latest surgical refinements. Methods: During the period of 14 years (from February 2006 to April 2020), 813 transmen with mean age of 24.4 years and mean body mass index of 24.6, underwent one stage metoidioplasty. Hysterectomy was simultaneously performed in 156, and mastectomy in 58 cases. Hysterectomy, mastectomy and metoidioplasty were done as a one-stage procedure in 46 transmen. Patients are divided in 5 groups, depending on the type of urethroplasty. Postoperative questionnaires were used to evaluate cosmetic and functional outcomes, as well as patients' satisfaction. Results: Follow-up ranged from 16 to 180 months (mean 94 months). Mean surgery time was 170 minutes and mean hospital stay was 3 days. Length of the neophallus ranged from 4.8 cm to 10.2 cm (mean 5.6 cm). Urethroplasty was complication-free in 89.5% of cases, and ranged between 81% to 90.3% in different groups. Urethral fistula and stricture occured in 8.85% and 1.70% of cases, respectively. Other complications included testicular implant rejection in 2%, testicular displacement in 3.20% and vaginal remnant in 9.60% of cases. From 655 patients who answered the questionnaire, 79% were totally satisfied and 20% mainly satisfied with the result of surgery. All patients reported voiding in standing position and good sexual arousal of the neophallus, without possibility for penetrative intercourse due to small size of the neophallus. Conclusion: Metoidioplasty has good cosmetic and functional outcomes, with low complication rate and high level of patients' satisfaction. In transmen who request total phalloplasty after metoidioplasty, all available phalloplasty techniques are feasable.

Impaired Vitamin D Signaling Is Associated With Frequent Development of Renal Cell Tumor in End-stage Kidney Disease.

BACKGROUND/AIM: End-stage kidney disease is characterized by chronic inflammation and frequent development of cancer. The level of circulating vitamin D is generally low in patients with end-stage renal disease (ESRD). Experimental studies have implicated the role of dysfunctional vitamin D metabolism in tumorigenesis. PATIENTS AND METHODS: We analyzed the expression of vitamin D receptor (VDR), cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1), the key genes involved in vitamin D signaling, in kidneys from patients with ESRD, tissue microarrays containing ESRD-associated renal cell tumors, as well as in their precursor lesions by immunohistochemistry. RESULTS: Kidneys from patients with ESRD showed strong structural rearrangement with only few tubules and epithelial cell groups embedded in fibrotic-inflammatory stroma. Only an estimated 1-3% of the epithelial cells showed positive staining with antibodies to VDR, CYP27B1 and CYP24A1, which contrasted with the 100%, 40-50% and 40-50% of positively stained cells, respectively, found in normal kidneys. Down-regulation of the vitamin D signaling proteins was found in patients with renal cancer, with the exception of tumors and their precursors occurring exclusively in ESRD. CONCLUSION: The significantly reduced activity of CYP27B1 in kidney from patients with ESRD explains the low level of circulating vitamin D. We suggest that the lack of anti-tumorigenic effect of vitamin D is a crucial factor in the frequent development of unique types of renal cell cancer in in patients with ESRD.

M2 Macrophage Marker Chitinase 3-Like 2 (CHI3L2) Associates With Progression of Conventional Renal Cell Carcinoma.

BACKGROUND/AIM: In spite of early detection, appoximately 15% of the small renal cell carcinomas (RCC) will develop metastasis within 5 years follow-up. The aim of this study was to identify new biomarkers to estimate the postoperative relapse of the most common conventional RCC. PATIENTS AND METHODS: Tissue multi arrays of conventional RCC without metastasis at the time of operation from a cohort of 634 patients were analysed by immunohistochemistry for expression of the chitinase 3-like protein 2 (CHI3L2). Cancer specific survival of patients was estimated with Kaplan-Meier analysis, univariate and multivariate Cox regression models. RESULTS: Kaplan-Meier analysis estimated a shorter cancer-free survival for patients with CHI3L2 positive tumors. In multivariate analysis, the CHI3L2 positivity associated with a 3.5 times higher risk for tumor relapse (p<0.001). CONCLUSION: Expression of CHI3L2 in tumor cells of conventional RCC define a group of patients at high risk for postoperative progression.

Dual role of KRT17: development of papillary renal cell tumor and progression of conventional renal cell carcinoma.

Expression of KRT17 has been described in multi-layered epithelia as well as in tumors derived from these cells. In cancers arising from KRT17 negative single layered epithelia neo-expression of KRT17 has been associated with tumor progression. To obtain more insight into the biology of kidney cancers we have investigated KRT17 expression by immunohistochemistry in normal kidney, in papillary preneoplastic lesions and in 151 papillary and 692 conventional renal cell carcinomas placed on tissue microarray. We found a positive staining in ureteric bud and collecting duct cells in foetal kidney, in all papillary preneoplastic lesions and also in 77% of the 151 papillary renal cell tumors indicating a continuos KRT17 expression during tumor development. The neo-expression of KRT17 in conventional renal cell carcinomas, which derives from KRT17 negative proximal tubules showed a significant correlation with postoperative tumor relapse (RR=2.50; 95% CI=1.59-3.94; p<0.001). In conclusion, the continuous expression of KRT17 from emerging fetal kidney tubules and microscopic pre-neoplastic lesions towards papillary renal cell tumors and its neo-expression in aggressive growing conventional renal cell carcinomas reflects the multiple function of KRT17 in kidney cancers with distinct natural history. This should be taken into account in clinical managements and therapy.

FOXI1 Immunohistochemistry Differentiates Benign Renal Oncocytoma from Malignant Chromophobe Renal Cell Carcinoma.

BACKGROUND/AIM: Renal oncocytoma (RO) and chromophobe renal cell carcinoma (chRCC) are suggested to develop from α- and ß-intercalated (IC) cells of the collecting duct expressing solute carrier family 4 member 1 (SLC4A1) and SLC26A4 under control of forkhead box 1 (FOXI1) transcription factor. The aim of this study was to clarify the possible cellular origin and of RO and chRCC. MATERIALS AND METHODS: Immunohistochemistry for aquaporin 2 (AQP2), FOXI1, SLC4A1 and SLC16A4 was applied to distinct types of renal cell tumors. RESULTS: Nuclear FOXI1 staining occurred in 96% of 83 ROs, in 3% of 90 chRCCs and none of the other tumor types. The α-IC cell marker SLC4A1 was seen in 60% of RO and 11% of chRCC, whereas staining for the ß-IC cell marker SLC26A4 was negative in all but one tumor. CONCLUSION: Although the origin of RO remains unclear, our findings suggest that FOXI1 immunohistochemistry is useful in differential diagnosis of RO from chRCC with overlapping histology.

Embryonal Origin of Metanephric Adenoma and its Differential Diagnosis.

BACKGROUND/AIM: The association of Wilms' tumor (WT), papillary renal cell tumor (PRCT) and mucinous tubular and spindle cell carcinoma (MTSCC) with embryonal rests has already been documented, but the cellular origin of metanephric adenoma (MA) is not yet known. The aim of this study was to understand their developmental evolution and find diagnostic markers. MATERIALS AND METHODS: CD57, KRT7, AMACR, SCEL, WT1 and CDH17 expression was analysed by immunohistochemistry in the four types of tumors and the associated pre-neoplastic lesions. RESULTS: Immunohistochemistry was able to differentiate WT, MA, MTSCC and PRCT. A phenotypic correlation between MA and perilobar nephrogenic rest associated with WT was identified. CONCLUSION: Perilobar nephrogenic rest and MA arise from differentiation arrested cells of the proximal domain of the S-shape body. We propose that WT1, MA, MTSCC and PRCT derive from different forms of maturation arrested embryonal rests.

Shift of Keratin Expression Profile in End-stage Kidney Increases the Risk of Tumor Development.

BACKGROUND/AIM: Pre-neoplastic lesions and renal cell tumors of distinct pheno- and genotypes occur frequently in end-stage kidneys. The aim of this study was to investigate the role of KRT7 and KRT19 in this process, the expression of which was previously detected by Affymetrix GeneChip analysis. PATIENTS AND METHODS: Twelve end-stage kidneys were analyzed to find pre-neoplastic lesions and tumors and expression of KRT7 and KRT19 was examined by immunohistochemistry. RESULTS: A total of 17 tumors, 149 pre-neoplastic lesions, 179 simple or proliferative cysts >2 mm were identified. Diffuse expression of KRT7 and KRT19 was seen in all end-stage kidneys as well as in the vast majority of cysts, pre-neoplastic lesions and tumors. CONCLUSION: Our data indicates that de novo expression of KRT7 and KRT19 resulting in altered plasticity and stem cell characteristics of epithelial cells might be a crucial factor in increasing the risk of tumor development in end-stage kidneys.

Desethylamiodarone-A metabolite of amiodarone-Induces apoptosis on T24 human bladder cancer cells via multiple pathways.

Bladder cancer (BC) is a common malignancy of the urinary tract that has a higher frequency in men than in women. Cytostatic resistance and metastasis formation are significant risk factors in BC therapy; therefore, there is great interest in overcoming drug resistance and in initiating research for novel chemotherapeutic approaches. Here, we suggest that desethylamiodarone (DEA)-a metabolite of amiodarone-may have cytostatic potential. DEA activates the collapse of mitochondrial membrane potential (detected by JC-1 fluorescence), and induces cell death in T24 human transitional-cell bladder carcinoma cell line at physiologically achievable concentrations. DEA induces cell cycle arrest in the G0/G1 phase, which may contribute to the inhibition of cell proliferation, and shifts the Bax/Bcl-2 ratio to initiate apoptosis, induce AIF nuclear translocation, and activate PARP-1 cleavage and caspase-3 activation. The major cytoprotective kinases-ERK and Akt-are inhibited by DEA, which may contribute to its cell death-inducing effects. DEA also inhibits the expression of B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and reduces colony formation of T24 bladder carcinoma cells, indicating its possible inhibitory effect on metastatic potential. These data show that DEA is a novel anti-cancer candidate of multiple cell death-inducing effects and metastatic potential. Our findings recommend further evaluation of its effects in clinical studies.

Expression of inflammatory lipopolysaccharide binding protein (LBP) predicts the progression of conventional renal cell carcinoma - a short report.

BACKGROUND: The mortality of conventional renal cell carcinoma (RCC) correlates directly with the presence or postoperative development of metastases. The aim of this study was to identify new markers associated with the postoperative progression of conventional RCC. METHODS: Tissue microarrays (TMA) of conventional RCC from a cohort of 414 patients were analysed by immunohistochemistry for expression of the lipopolysaccharide binding protein (LBP), which was identified as a candidate biomarker through Affymetrix U133 Plus 2.0 array analysis. Univariate and multivariate Cox regression models were addressed to cancer-specific survival in association with age, sex, clinicopathological parameters and LBP expression. The survival time of the patients was estimated by Kaplan-Meier analyses, and comparisons of survival curves were made using the Log rank test. RESULTS: Univariate analysis revealed an association of patient survival with all clinicopathological parameters tested and LBP expression. In multivariate analysis only T classification, grade and LBP staining showed a significant association with postoperative cancer-specific survival (p < 0.001). LBP expression was found to be associated with a poor patient survival in Kaplan-Meier analyses. The estimated median survival time for patients with tumours showing LBP expression was 74 months, whereas the overall survival time was 142 months. CONCLUSION: LBP expression in conventional RCC defines a group of patients at a high risk of postoperative progression and may help to direct optimized active surveillance and timely adjuvant therapy.

Linalool-induced oxidative stress processes in the human pathogen Candida albicans.

The present study investigated the linalool (Lol)-induced effects in acute toxicity tests in the human pathogen Candida albicans (C. albicans). Lol treatments induced reduced germ tube formation of the pathogen, which plays a crucial role in the virulence. In comparison with the untreated control, the exposure of 107 cells ml-1 to 0.7 mM or 1.4 mM Lol for one hour induced 20% and 30% decrements, respectively, in the colony-forming ability. At the same time, these treatments caused dose-dependent decrease in the levels of superoxide anion radical and total reactive oxygen species, while there was 1.5 and 1.8-fold increases in the concentrations of peroxides and lipid peroxides, respectively, indicating oxidative stress induction in the presence of Lol. Lol treatments resulted in different adaptive modifications of the antioxidant system. In 0.7 mM-treated cells, decreased specific activities of superoxide dismutase and catalase were detected, while exposure to 1.4 mM Lol resulted in the up-regulation of catalase, glutathione reductase and glutathione peroxidases.

Cytoplasmic expression of ß-catenin is an independent predictor of progression of conventional renal cell carcinoma: a simple immunostaining score.

AIMS: The aims of this study were to investigate the potential of ß-catenin as a biomarker for predicting cancer-specific survival, and to find a reproducible mode of evaluation of immunohistochemistry. METHODS AND RESULTS: ß-Catenin expression was analysed by immunohistochemistry in a cohort of 488 patients with conventional renal cell carcinoma (RCC) operated on between 2000 and 2010. The association between ß-catenin expression and cancer-specific survival was assessed with univariate and multivariate Cox regression models in relation to conventional clinical pathological prognostic factors, and by Kaplan-Meier survival analysis with the log rank test. The univariate Cox regression model revealed an association of cytoplasmic ß-catenin positivity and pathological variables with cancer-specific death. The multivariate Cox regression model analysis of tumours without metastatic disease at the first presentation identified the T-classification (P < 0.001) and cytoplasmic ß-catenin positivity as risk factors for postoperative tumour progression. Specifically, cytoplasmic ß-catenin expression was an independent factor indicating an unfavourable prognosis, with a four-fold higher risk of cancer-specific death (relative risk 4.017; 95% confidence interval 2.489-6.482; P < 0.001). The median survival time for patients with tumours showing cytoplasmic accumulation of ß-catenin was 48 months, whereas the overall survival time was 166 months. CONCLUSIONS: Cytoplasmic ß-catenin expression is an independent prognostic factor for conventional RCC, and may help to identify patients with a high risk of cancer-specific death and to direct optimized active surveillance or adjuvant therapy.

Lack of TMEM27 expression is associated with postoperative progression of clinically localized conventional renal cell carcinoma.

PURPOSE: In spite of early detection of conventional renal cell carcinoma (RCC) by widespread use of abdominal imaging, approximately 10-15 % of patients will die due to disease. The aim of this study was to identify new biomarkers predicting the postoperative progression of conventional RCC. METHODS: Tissue multiarrays (TMA) of conventional RCC from a cohort of 486 patients were analysed by immunohistochemistry for expression of the transmembrane protein TMEM27, which was identified as a candidate biomarker by Affymetrix U133 Plus 2.0 array. Univariate and multivariate Cox regression models were addressed to assess cancer-specific survival in association with clinicopathological variables and TMEM27 expression. Cancer-specific survival time was estimated with Kaplan-Meier analysis, and the comparison of survival curves was made with the log-rank test. RESULTS: The Kaplan-Meier survival analysis indicated a poor disease-specific survival rates for tumours without TMEM27 staining. Univariate analysis revealed an association of patient survival with T stadium, grade, stage and size of tumour and TMEM27 expression in all cases as well as in the cohort of patients with postoperative tumour progression. In multivariate analysis, only T stadium and TMEM27 staining showed a significant association with postoperative cancer-specific death (p < 0.001). CONCLUSIONS: Lack of expression of the TMEM27 in conventional RCC defines a group of patients at high risk for cancer-related death.

Impact of parenchymal loss on renal function after laparoscopic partial nephrectomy under warm ischemia.

PURPOSE: To elucidate the impact of renal parenchymal loss and the ischemic reperfusion injury (RI) on the renal function after laparoscopic partial nephrectomy (LPN) under warm ischemia (WI). METHODS: Thirty-five patients with a single polar renal mass ≤4 cm and normal contralateral kidney underwent LPN. Transperitoneal LPN with WI using en bloc hilar occlusion was performed. The total differential renal function (T-DRF) using 99mTc-dimercaptosuccinic acid was evaluated preoperatively and postoperatively over a period of 1 year. A special region of interest (ROI) was selected on the non-tumorous pole of the involved kidney, and was compared with the same ROI in the contralateral kidney. The latter comparison was defined as partial differential renal function (P-DRF). Any postoperative decline in the P-DRF of the operated kidney was attributed to the RI. Subtraction of the P-DRF decline from the T-DRF decline was attributed to the parenchymal loss caused by the resection of the tumor and suturing of the normal parenchyma. RESULTS: The mean WI time was 22 min, and the mean weight of resected specimen was 18 g. The mean postoperative eGFR declined to 87 ml/min/1.73 m2 from its baseline mean value of 97 ml/min/1.73 m2 (p value = 0.075). Mean postoperative T-DRF and P-DRF of the operated kidney declined by 7 and 3 %, respectively. CONCLUSIONS: After LPN of small renal mass, decline in renal function is primarily attributed to parenchymal loss caused by tumor resection and suturing of the normal parenchyma rather than the RI.