D-chiro-inositol in clinical practice: A perspective from The Experts Group on Inositol in Basic and Clinical Research (EGOI).

BACKGROUND: D-chiro-inositol is a natural molecule that, in association with its well-studied isomer myo-inositol, may play a role in treating various metabolic and gynecological disorders. OBJECTIVES: This perspective seeks to explore the mechanisms and functions of D-chiro-inositol, laying the foundations to discuss its use in clinical practice, across dysmetabolism, obesity, and hormonal dysregulation. METHODS: A narrative review of all the relevant papers known to the authors was conducted. OUTCOME: D-chiro-inositol acts through a variety of mechanisms, acting as an insulin sensitizer, inhibiting the transcription of aromatase, in addition to modulating white adipose tissue/brown adipose tissue trans differentiation. These different modes of action have potential applications in a variety of therapeutic fields including: PCOS, dysmetabolism, obesity, hypoestrogenic/hyperandrogenic disorders, and bone health. CONCLUSIONS: D-chiro-inositol mode of action has been studied in detail in recent years, resulting in a clear differentiation between D-chiro-inositol and its isomer myo-inositol. The insulin sensitizing activities of D-chiro-inositol are well understood; however, its potential applications in other fields, in particular obesity and hyperestrogenic/hypoandrogenic disorders in men and women, represent promising avenues of research that require further clinical study.

Glass wool column filtration for stallion sperm selection: A comparative analysis with the single-layer colloid centrifugation.

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Association between meteorological variables and semen quality: a retrospective study.

Spermatogenesis is a temperature-dependent process, and high summer temperatures have been linked to lower sperm concentration and count. However, reports describing the association between other meteorological variables and semen quality are scarce. This study evaluated the association between semen quality and temperature, humidity, pressure, apparent temperature (AT), temperature-humidity index (THI), simplified wet-bulb global temperature (sWBGT), and sunshine duration. Semen samples were obtained at the Laboratorio de Andrología y Reproducción (LAR, Argentina), from men undergoing routine andrology examination (n=11657) and computer-assisted sperm analysis (n=4705) following WHO 2010 criteria. Meteorological variables readings were obtained from the Sistema Meteorológico Nacional. Sperm quality parameters were negatively affected in summer when compared to winter. Additionally, there was a significant decrease in sperm kinematics between winter and spring. Branch and bound variable selection followed by multiple regression analysis revealed a significant association between semen quality and meteorological variables. Specifically, changes in sunshine duration and humidity reinforced the prognosis of semen quality. Highest/lowest sunshine duration and humidity quantiles resulted in decreased sperm concentration, count, motility, vitality and membrane competence, nuclear maturity, and sperm kinematics associated to highest sunshine duration and lowest humidity. Findings from this report highlight the relevance of environmental studies for predicting alterations in male reproductive health associated to variations in meteorological variables, especially considering the current climate changes around the planet due to global warming and its consequences for human health.

Deficiency of fibroblast growth factor 2 (FGF-2) leads to abnormal spermatogenesis and altered sperm physiology.

In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.

Identification of a Novel Human E-Cadherin Splice Variant and Assessment of Its Effects Upon EMT-Related Events.

Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them acquisition of a fibroblastic-like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 232: 1368-1386, 2017. © 2016 Wiley Periodicals, Inc.

Epithelial and neural cadherin expression in the mammalian reproductive tract and gametes and their participation in fertilization-related events.

Mammalian fertilization involves a series of well-orchestrated cell-cell interaction steps between gametes, as well as among spermatozoa and somatic cells of both the male and female reproductive tracts. Cadherins are Ca(2+)-dependent glycoproteins that have been involved in cellular adhesion and signaling in somatic cells. Taking into account that Ca(2+) ions are required during fertilization, the involvement of these proteins in adhesion events during this process can be anticipated. This report presents an overview on two members of classical cadherins, Epithelial (E-) and Neural (N-) cadherin in reproductive biology. Its provides evidence of studies done by several research groups about the expression of E- and N-cadherin during spermatogenesis, oogenesis and folliculogenesis, and their involvement in gamete transport in the reproductive tracts. Moreover, it describes current knowledge of E- and N-cadherin presence in cells of the cumulus-oocyte complex and spermatozoa from several mammalian species, and shows gathered evidence on their participation in different steps of the fertilization process. A brief summary on general information of both proteins is also presented.

Secretory Leukocyte Protease Inhibitor (SLPI) expression downregulates E-cadherin, induces ß-catenin re-localisation and triggers apoptosis-related events in breast cancer cells.

BACKGROUND INFORMATION: Epithelial cadherin (E-cadherin) is involved in cell-cell adhesion through its extracellular domain, whereas the intracellular domain interacts with adaptor proteins, i.e. ß-catenin, links E-cadherin to the actin cytoskeleton and participates in signal transduction events. E-cadherin protects mammary epithelial cells from apoptosis and its loss during tumour progression has been documented. Secretory Leukocyte Protease Inhibitor (SLPI) has anti- and pro-tumourigenic activities but its role in breast cancer has not been fully elucidated. Notwithstanding its relevance, how SLPI affects E-cadherin in breast cancer is still unknown. This study evaluated the effect of SLPI upon E-cadherin/ß-catenin expression and apoptosis-related markers in murine (F3II) and human (MCF-7) breast tumour cells either treated with exogenous recombinant human SLPI (rhSLPI) or stably transfected with a plasmid encoding its sequence. RESULTS: Addition of rhSLPI to F3II cells caused a decrease (P < 0.05) in E-cadherin transcript and protein levels. Similar results were observed in SLPI-stable F3II transfectants (2C1), and treatment of 2C1 cells with a siRNA toward SLPI restored E-cadherin to control levels. SLPI-expressing cells showed disruption of E-cadherin/ß-catenin complex and increased (P < 0.05) percentage of cells depicting nuclear ß-catenin localisation. Associated to these changes, 2C1 cells showed increased Bax/Bcl-2 ratio and p21 protein levels, decreased c-Myc protein levels and decreased Cyclin D1 and Claudin-1 transcript levels. No differences in N- and P-cadherin were observed between SLPI-transfected cells and controls. Addition of rhSLPI to MCF-7 cells or stable transfection with SLPI caused a decrease (P < 0.05) in E-cadherin expression (transcript/protein) and its redistribution to the cytoplasm, as well as ß-catenin re-localisation to the cell nucleus. CONCLUSIONS: Expression of SLPI was associated to a decrease in E-cadherin expression and re-localisation of E-cadherin to the cell cytoplasm and ß-catenin to the cell cytoplasm and nucleus, and had pro-apoptotic and cell cycle-arrest effects.

Impact of heat waves on semen quality: A retrospective study in Argentina between 2005 and 2023.

Heat waves, defined as periods with daily temperatures surpassing the historical average for a specific region, have become more frequent worldwide in recent years. Previous studies have reported a negative association between temperature and semen quality, but the focus has mainly been on Asian and European populations. The study included 54,926 men (18-60 years) undergoing routine semen analysis between 2005 and 2023 at CEUSA-LAEH andrology unit, in Buenos Aires, Argentina. Hourly temperature readings were provided by the Servicio Meteorológico Nacional. R programming (R Studio v2022.07.2) was used to define heat waves, calculate key characteristics, visualize results, and perform statistical tests at the IBYME laboratory. During the period studied, a total of 124 days had heat waves (defined after at least 3 consecutive days with 32.3 °C and 22 °C). Men exposed to heat waves during spermatogenesis exhibited lower sperm number (concentration and count; P < 0.0001) and decreased normal morphology (percentage of normal sperm and normal motile count; P < 0.05) compared to those not exposed. These differences were most pronounced between semen samples from years with several heat waves (2013, 2023) and none (2005, 2007, 2016), displaying 4-5 times higher fold changes (P < 0.05). Further analysis employing multiple regression revealed a significantly negative association between semen quality and heat wave length, suggesting that a prolonged exposure may be more detrimental than an acute exposure. Subsequent analysis focusing on prolonged exposure (≥6-days heat wave) during spermatogenesis revealed a negative (P < 0.05) association between early exposure (spermatocytogenesis: 64-90 days prior semen collection) and semen quality. This study underscores the negative association between early exposure to heat waves during sperm development and semen quality, raising concerns about its possible association with the worldwide declining male fertility. A comprehensive collaborative approach is crucial, involving global governmental policies, sustainable practices, and coordinated efforts across scientific, healthcare, and policy domains.

A 2-step remote TUNEL approach for sperm DNA fragmentation assessment. Analysis in donors and patients.

OBJECTIVES: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters. STUDY DESIGN: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318). RESULTS: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients. CONCLUSIONS: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.

Frequency, morbidity and equity - the case for increased research on male fertility.

Currently, most men with infertility cannot be given an aetiology, which reflects a lack of knowledge around gamete production and how it is affected by genetics and the environment. A failure to recognize the burden of male infertility and its potential as a biomarker for systemic illness exists. The absence of such knowledge results in patients generally being treated as a uniform group, for whom the strategy is to bypass the causality using medically assisted reproduction (MAR) techniques. In doing so, opportunities to prevent co-morbidity are missed and the burden of MAR is shifted to the woman. To advance understanding of men's reproductive health, longitudinal and multi-national centres for data and sample collection are essential. Such programmes must enable an integrated view of the consequences of genetics, epigenetics and environmental factors on fertility and offspring health. Definition and possible amelioration of the consequences of MAR for conceived children are needed. Inherent in this statement is the necessity to promote fertility restoration and/or use the least invasive MAR strategy available. To achieve this aim, protocols must be rigorously tested and the move towards personalized medicine encouraged. Equally, education of the public, governments and clinicians on the frequency and consequences of infertility is needed. Health options, including male contraceptives, must be expanded, and the opportunities encompassed in such investment understood. The pressing questions related to male reproductive health, spanning the spectrum of andrology are identified in the Expert Recommendation.

Corrigendum: FXYD5/Dysadherin, a biomarker of endometrial cancer myometrial invasion and aggressiveness: its relationship with TGF-ß1 and NF-κB pathways.

[This corrects the article DOI: 10.3389/fonc.2019.01306.].

Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model.

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Expression analysis of epithelial cadherin and related proteins in IBH-6 and IBH-4 human breast cancer cell lines.

Epithelial cadherin (E-cadherin) is a 120 kDa cell-cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as beta-catenin. Loss of E-cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down-regulation of E-cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH-6 and IBH-4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E-cadherin and related proteins in IBH-6 and IBH-4 cell lines was evaluated. In IBH-6 and IBH-4 cell extracts, only an 89 kDa E-cadherin form (Ecad89) was detected, which is truncated at the C-terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E-cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH-6 and IBH-4 cells showed an intracellular scattered E-cadherin localization. beta-catenin accompanied E-cadherin localization, and actin stress fibers were identified in both cell types. E-cadherin mRNA levels were remarkably low in IBH-6 and IBH-4 cells. The E-cadherin mRNA and genomic sequence encoding exons 14-16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up-regulation of Twist mRNA was found in both cell lines. In conclusion, IBH-6 and IBH-4 breast cancer cells show down-regulation of E-cadherin expression with aberrant protein localization, and up-regulation of Twist; these features can be related to their invasive/metastatic characteristics.

Identification of early stage recurrence endometrial cancer biomarkers using bioinformatics tools.

Endometrial cancer (EC) is the sixth most common cancer in women worldwide. Early diagnosis is critical in recurrent EC management. The present study aimed to identify biomarkers of EC early recurrence using a workflow that combined text and data mining databases (DisGeNET, Gene Expression Omnibus), a prioritization algorithm to select a set of putative candidates (ToppGene), protein­protein interaction network analyses (Search Tool for the Retrieval of Interacting Genes, cytoHubba), association analysis of selected genes with clinicopathological parameters, and survival analysis (Kaplan­Meier and Cox proportional hazard ratio analyses) using a The Cancer Genome Atlas cohort. A total of 10 genes were identified, among which the targeting protein for Xklp2 (TPX2) was the most promising independent prognostic biomarker in stage I EC. TPX2 expression (mRNA and protein) was higher (P<0.0001 and P<0.001, respectively) in ETS variant transcription factor 5­overexpressing Hec1a and Ishikawa cells, a previously reported cell model of aggressive stage I EC. In EC biopsies, TPX2 mRNA expression levels were higher (P<0.05) in high grade tumors (grade 3) compared with grade 1­2 tumors (P<0.05), in tumors with deep myometrial invasion (>50% compared with <50%; P<0.01), and in intermediate­high recurrence risk tumors compared with low­risk tumors (P<0.05). Further validation studies in larger and independent EC cohorts will contribute to confirm the prognostic value of TPX2.

Ephrin-B1 Is a Novel Biomarker of Bladder Cancer Aggressiveness. Studies in Murine Models and in Human Samples.

Bladder cancer (BC) is the ninth most common cancer worldwide, but molecular changes are still under study. During tumor progression, Epithelial cadherin (E-cadherin) expression is altered and ß-catenin may be translocated to the nucleus, where it acts as co-transcription factor of tumor invasion associated genes. This investigation further characterizes E-cadherin and ß-catenin associated changes in BC, by combining bioinformatics, an experimental murine cell model (MB49/MB49-I) and human BC samples. In in silico studies, a DisGeNET (gene-disease associations database) analysis identified CDH1 (E-cadherin gene) as one with highest score among 130 BC related-genes. COSMIC mutation analysis revealed CDH1 low mutations rates. Compared to MB49 control BC cells, MB49-I invasive cells showed decreased E-cadherin expression, E- to P-cadherin switch, higher ß-catenin nuclear signal and lower cytoplasmic p-Ser33-ß-catenin signal, higher Ephrin-B1 ligand and EphB2 receptor expression, higher Phospho-Stat3 and Urokinase-type Plasminogen Activator (UPA), and UPA receptor expression. MB49-I cells transfected with Ephrin-B1 siRNA showed lower migratory and invasive capacity than control cells (scramble siRNA). By immunohistochemistry, orthotopic MB49-I tumors had lower E-cadherin, increased nuclear ß-catenin, lower pSer33-ß-catenin cytoplasmic signal, and higher Ephrin-B1 expression than MB49 tumors. Similar changes were found in human BC tumors, and 83% of infiltrating tumors depicted a high Ephrin-B1 stain. An association between higher Ephrin-B1 expression and higher stage and tumor grade was found. No association was found between abnormal E-cadherin signal, Ephrin-B1 expression or clinical-pathological parameter. This study thoroughly analyzed E-cadherin and associated changes in BC, and reports Ephrin-B1 as a new marker of tumor aggressiveness.

Characterization of the molecular changes associated with the overexpression of a novel epithelial cadherin splice variant mRNA in a breast cancer model using proteomics and bioinformatics approaches: identification of changes in cell metabolism and an increased expression of lactate dehydrogenase B.

BACKGROUND: Breast cancer (BC) is the most common female cancer and the leading cause of cancer death in women worldwide. Alterations in epithelial cadherin (E-cadherin) expression and functions are associated to BC, but the underlying molecular mechanisms have not been fully elucidated. We have previously reported a novel human E-cadherin splice variant (E-cadherin variant) mRNA. Stable transfectants in MCF-7 human BC cells (MCF7Ecadvar) depicted fibroblast-like cell morphology, E-cadherin wild-type downregulation, and other molecular changes characteristic of the epithelial-to-mesenchymal transition process, reduced cell-cell adhesion, and increased cell migration and invasion. In this study, a two-dimensional differential gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS) protein identification and bioinformatics analyses were done to characterize biological processes and canonical pathways affected by E-cadherin variant expression. RESULTS: By 2D-DIGE and MS analysis, 50 proteins were found differentially expressed (≥ Δ1.5) in MCF7Ecadvar compared to control cells. Validation of transcript expression was done in the ten most overexpressed and underexpressed proteins. Bioinformatics analyses revealed that 39 of the 50 proteins identified had been previously associated to BC. Moreover, metabolic processes were the most affected, and glycolysis the canonical pathway most altered. The lactate dehydrogenase B (LDHB) was the highest overexpressed protein, and transcript levels were higher in MCF7Ecadvar than in control cells. In agreement with these findings, MCF7Ecadvar conditioned media had lower glucose and higher lactate levels than control cells. MCF7Ecadvar cell treatment with 5 mM of the glycolytic inhibitor 2-deoxy-glucose led to decreased cell viability, and modulation of LDHB expression in MCF7Ecadvar cells with a specific small interfering RNA resulted in decreased cell proliferation. Finally, a positive association between expression levels of the E-cadherin variant and LDHB transcripts was demonstrated in 21 human breast tumor tissues, and breast tumor samples with higher Ki67 expression showed higher LDHB mRNA levels. CONCLUSIONS: Results from this investigation contributed to further characterize molecular changes associated to the novel E-cadherin splice variant expression in BC cells. They also revealed an association between expression of the novel variant and changes related to BC progression and aggressiveness, in particular those associated to cell metabolism.

FXYD5/Dysadherin, a Biomarker of Endometrial Cancer Myometrial Invasion and Aggressiveness: Its Relationship With TGF-ß1 and NF-κB Pathways.

Objective: Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models. Methods: FXYD5/Dys messenger RNA (mRNA) levels were determined in EC tissue and UA biopsies. FXYD5/Dys expression was evaluated in EC RNAseq data from The Cancer Genome Atlas (TCGA) and GENEVESTIGATOR tools. FXYD5/Dys impact on E-cadherin expression and cell behavior was assessed in EC Hec1a cells treated with transforming growth factor (TGF)-ß1, stably transfected with ETV5, and transiently transfected with FXYD5/Dys small interfering RNA (siRNA) or pcDNA3-FXYD5/Dys plasmid. Results: FXYD5/Dys was associated with EC aggressiveness, finding high mRNA levels in tumors depicting MI > 50%, Grade 3, and intermediate/high risk of recurrence. FXYD5/Dys was highly expressed at the tumor invasive front compared to the superficial area. Most results were recapitulated in UA biopsies. FXYD5/Dys modulation in Hec1a cells altered cell migration/adhesion and E-cadherin expression. TGF-ß1 treatment of Hec1a cells induced FXYD5/Dys expression. TCGA-UCEC RNAseq analysis revealed a positive correlation between FXYD5/Dys, TGF-ß1, and plasminogen activator inhibitor (PAI)-1 mRNA levels. FXYD5/Dys induced nuclear factor (NF)-κB pathway activation in Hec1a cells. FXYD5/Dys mRNA levels positively correlated with transcriptional activation of NF-κB p65-regulated genes. Survival analysis revealed patient segregation into low- and high-risk groups, the latter depicting the highest FXYD5/Dys, PAI-1, tumor necrosis factor (TNF)-α, and TGF-ß1 mRNA levels and shorter survival rates. Conclusion: FXYD5/Dys is a novel biomarker of EC progression related to TGF-ß1 and NF-κB pathways that collectively promote tumor dissemination and result in poor patient prognosis.

Impact of age, clinical conditions, and lifestyle on routine semen parameters and sperm kinematics.

OBJECTIVE: To assess the impact of aging on routine semen and computer-assisted sperm analysis (CASA) motility parameters according to the current World Health Organization guidelines; and to evaluate the effect of obesity and lifestyle (alcohol consumption, cigarette smoking) in older men's semen. DESIGN: Blind cross-sectional study. SETTING: Research laboratory and andrology and reproduction laboratory. PATIENT(S): A population of 11,706 men. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen analysis: routine (semen volume, sperm concentration and count, motility, vitality, morphology, hypo-osmotic swelling test, round and peroxidase-positive cell concentration) and CASA (straight-line velocity, curvilinear velocity, average path velocity, linearity, straightness, beat cross frequency, wobble, amplitude of lateral head displacement, and mean angular displacement) parameters; and body mass index. RESULT(S): A negative correlation was found between age and routine semen parameters: volume, sperm count, motility, vitality, total motile spermatozoa and normal-motile spermatozoa, round cell concentration, and hypo-osmotic swelling test values. Several CASA variables (straight-line velocity, curvilinear velocity, average path velocity, beat cross frequency, amplitude of lateral head displacement, and mean angular displacement) were also negatively affected. Using 40 years as a cut-off value, significant differences in most parameters correlated to age. In a selected subpopulation of men unexposed to known fertility-compromising factors, the same evaluations were performed, finding some parameters still decreased. Although obesity exerted a significant deleterious effect on older patients' semen quality, alcohol consumption and cigarette smoking mildly affected it. CONCLUSION(S): Male aging, with the contribution of unhealthy conditions, are paramount effectors of sperm quality deterioration.