Fertility specialists' views, behavior, and attitudes towards the use of endometrial scratching in Italy.

BACKGROUND: Endometrial scratching (ES) or injury is intentional damage to the endometrium performed to improve reproductive outcomes for infertile women desiring pregnancy. Moreover, recent systematic reviews with meta-analyses and randomized controlled trials demonstrated that ES is not effective, data on the safety are limited, and it should not be recommended in clinical practice. The aim of the current study was to assess the view and behavior towards ES among fertility specialists throughout infertility centers in Italy, and the relationship between these views and the attitudes towards the use of ES as an add-on in their commercial setting. METHODS: Online survey among infertility centers, affiliated to Italian Society of Human Reproduction (SIRU), was performed using a detailed questionnaire including 45 questions with the possibility to give "closed" multi-choice answers for 41 items and "open" answers for 4 items. Online data from the websites of the infertility centers resulting in affiliation with the specialists were also recorded and analyzed. The quality of information about ES given on infertility centers websites was assessed using a scoring matrix including 10 specific questions (scored from 0 to 2 points), and the possible scores ranged from 0 to 13 points ('excellent' if the score was 9 points or more, 'moderate' if the score was between 5 and 8, and 'poor' if it was 4 points or less). RESULTS: The response rate was of 60.6% (43 questionnaires / 71 infertility SIRU-affiliated centers). All included questionnaires were completed in their entirety. Most physicians (~ 70%) reported to offer ES to less than 10% of their patients. The procedure is mainly performed in the secretory phase (69.2%) using pipelle (61.5%), and usually in medical ambulatory (56.4%) before IVF cycles to improve implantation (71.8%) without drugs administration (e.g., pain drugs, antibiotics, anti-hemorrhagics, or others) before (76.8%) or after (64.1%) the procedure. Only a little proportion of infertility centers included in the analysis proposes formally the ES as an add-on procedure (9.3%), even if, when proposed, the full description of the indications, efficacy, safety, and costs is never addressed. However, the overall information quality of the websites was generally "poor" ranging from 3 to 8 and having a low total score (4.7 ± 1.6; mean ± standard deviation). CONCLUSIONS: In Italy, ES is a procedure still performed among fertility specialists for improving the implantation rate in IVF patients. Moreover, they have a poor attitude in proposing ES as an add-on in the commercial setting.

Indole and 2,4-Thiazolidinedione conjugates as potential anticancer modulators.

BACKGROUND: Thiazolidinediones (TZDs), also called glitazones, are five-membered carbon ring molecules commonly used for the management of insulin resistance and type 2 diabetes. Recently, many prospective studies have also documented the impact of these compounds as anti-proliferative agents, though several negative side effects such as hepatotoxicity, water retention and cardiac issues have been reported. In this work, we synthesized twenty-six new TZD analogues where the thiazolidinone moiety is directly connected to an N-heterocyclic ring in order to lower their toxic effects. METHODS: By adopting a widely applicable synthetic method, twenty-six TZD derivatives were synthesized and tested for their antiproliferative activity in MTT and Wound healing assays with PC3 (prostate cancer) and MCF-7 (breast cancer) cells. RESULTS: Three compounds, out of twenty-six, significantly decreased cellular viability and migration, and these effects were even more pronounced when compared with rosiglitazone, a well-known member of the TZD class of antidiabetic agents. As revealed by Western blot analysis, part of this antiproliferative effect was supported by apoptosis studies evaluating BCL-xL and C-PARP protein expression. CONCLUSION: Our data highlight the promising potential of these TZD derivatives as anti-proliferative agents for the treatment of prostate and breast cancer.

Secretome Analysis of Hypoxia-Induced 3T3-L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity.

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte-secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3-L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin-1 and -2, and matrix metalloproteinase-11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3-L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.

Correction to: Insulin and osteocalcin: further evidence for a mutual cross-talk.

Correction to: Endocrine https://doi.org/10.1007/s12020-017-1396-0 The article Insulin and osteocalcin: further evidence for a mutual cross-talk, written by Francesco L. Bilotta, Biagio Arcidiacono, Sebastiano Messineo, Marta Greco, Eusebio Chiefari, Domenico Britti, Tomoko Nakanishi, Daniela P. Foti, Antonio Brunetti, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 2 September 2017 without open access.

HMGA1 is a novel transcriptional regulator of the FoxO1 gene.

PURPOSE: The forkhead transcription factor (FoxO1) is a master transcriptional regulator of fundamental cellular processes ranging from cell proliferation and differentiation to inflammation and metabolism. However, despite its relevance, the mechanism(s) underlying FoxO1 gene regulation are largely unknown. We have previously shown that the chromatin factor high-mobility group A1 (HMGA1) plays a key role in the transcriptional regulation of glucose-responsive genes, including some that are involved in FoxO1-mediated glucose metabolism. Here we investigated the impact of HMGA1 on FoxO1 gene expression. METHODS: FoxO1 protein and gene expression studies were performed by Western blot analysis combined with qRT-PCR of material from human cultured cells and EBV-transformed lymphoblasts, and from primary cultured hepatocytes from wild-type and Hmga1 -/- mice. Reporter gene assays and chromatin immunoprecipitation for binding of HMGA1 to the endogenous FoxoO1 locus were performed in cells overexpressing HMGA1 and in cells pretreated with siRNA targeting HMGA1. RESULTS: HMGA1 increased FoxO1 mRNA and protein expression in vitro, in cultured HepG2 and HEK-293 cells by binding FoxO1 gene promoter, thereby activating FoxO1 gene transcription. Forced expression of HMGA1 in primary cultured hepatocytes from Hmga1 -/- mice and in EBV-transformed lymphoblasts from subjects with reduced expression of endogenous HMGA1 increased FoxO1 mRNA and protein levels. CONCLUSION: These findings may contribute to the understanding of FoxO1 gene regulation and its role in metabolism.

Insulin and osteocalcin: further evidence for a mutual cross-talk.

PURPOSE: In the last few years, bone has been recognized as an endocrine organ that modulates glucose metabolism by secretion of osteocalcin, an osteoblast-specific hormone, that influences fat deposition and blood sugar levels. To date, however, very few in vitro models have been developed to investigate, at the molecular levels, the relationship between glucose, insulin and osteocalcin. This study aims at covering this gap. METHODS: We studied osteogenic differentiation, osteocalcin gene expression, and osteblast-mediated insulin secretion, using cultured MG-63 human osteoblast-like cells that underwent glucotoxicity and insulin resistance. In addition, we investigated whether a correlation existed between hyperglycemia and/or insulin resistance and total osteocalcin serum concentrations in patients. RESULTS: While insulin and low glucose increased osteocalcin gene expression, disruption of insulin signaling in MG-63 osteoblasts and high glucose concentration in cell culture medium decreased osteocalcin gene transcription and reduced osteogenic differentiation. Concomitantly, insulin secretion was significantly impaired in rat INS-1 ß-cells treated with conditioned medium from insulin resistant MG-63 cells or cells exposed to high glucose concentrations. Also, chronic hyperglycemia, but not insulin resistance, inversely correlated with circulating osteocalcin levels in patients. CONCLUSION: Our results further support the existence of an endocrine axis between bone, where osteocalcin is produced, and pancreatic ß-cells, and add new insights into the molecular details of this relationship. These findings may contribute to the understanding of osteocalcin regulation and its role in metabolism.

Expression of matrix metalloproteinase-11 is increased under conditions of insulin resistance.

AIM: To investigate matrix metalloproteinase-11 (MMP-11) expression in adipose tissue dysfunction, using in vitro and in vivo models of insulin resistance. METHODS: Culture of mouse 3T3-L1 preadipocytes were induced to differentiation into mature 3T3-L1 adipocytes. Cellular insulin resistance was induced by treating differentiated cultured adipocytes with hypoxia and/or tumor necrosis factor (TNF)-α, and transcriptional changes were analyzed in each condition thereafter. For the in vivo studies, MMP-11 expression levels were measured in white adipose tissue (WAT) from C57BL/6J mice that underwent low fat diet or high-fat feeding in order to induce obesity and obesity-related insulin resistance. Statistical analysis was carried out with GraphPad Prism Software. RESULTS: MMP-11 mRNA expression levels were significantly higher in insulin resistant 3T3-L1 adipocytes compared to control cells (1.46 ± 0.49 vs 0.83 ± 0.21, respectively; P < 0.00036). The increase in MMP-11 expression was observed even in the presence of TNF-α alone (3.79 ± 1.11 vs 1 ± 0.17, P < 0.01) or hypoxia alone (1.79 ± 0.7 vs 0.88 ± 0.1, P < 0.00023). The results obtained in in vitro experiments were confirmed in the in vivo model of insulin resistance. In particular, MMP-11 mRNA was upregulated in WAT from obese mice compared to lean mice (5.5 ± 2.8 vs 1.1 ± 0.7, respectively; P < 3.72E-08). The increase in MMP-11 levels in obese mice was accompanied by the increase in typical markers of fibrosis, such as collagen type VI alpha 3 (Col6α3), and fibroblast-specific protein 1. CONCLUSION: Our results indicate that dysregulation of MMP-11 expression is an early process in the adipose tissue dysfunction, which leads to obesity and obesity-related insulin resistance.

Cooperation between HMGA1 and HIF-1 Contributes to Hypoxia-Induced VEGF and Visfatin Gene Expression in 3T3-L1 Adipocytes.

The architectural transcription factor high-mobility group AT-hook 1 (HMGA1) is a chromatin regulator with implications in several biological processes, including tumorigenesis, inflammation, and metabolism. Previous studies have indicated a role for this factor in promoting the early stages of adipogenesis, while inhibiting adipocyte terminal differentiation, and decreasing fat mass. It has been demonstrated that hypoxia - through the hypoxia-inducible factor 1 (HIF-1) - plays a major role in triggering changes in the adipose tissue of the obese, leading to inhibition of adipocyte differentiation, adipose cell dysfunction, inflammation, insulin resistance, and type 2 diabetes. To examine the possible cooperation between HMGA1 and HIF-1, herein, we investigated the role of HMGA1 in the regulation of Visfatin and VEGF, two genes normally expressed in adipose cells, which are both responsive to hypoxia. We demonstrated that HMGA1 enhanced Visfatin and VEGF gene expression in human embryonic kidney (HEK) 293 cells in hypoxic conditions, whereas HMGA1 knockdown in differentiated 3T3-L1 adipocytes reduced these effects. Reporter gene analysis showed that Visfatin and VEGF transcriptional activity was increased by the addition of either HMGA1 or HIF-1 and even further by the combination of both factors. As demonstrated by chromatin immunoprecipitation in intact cells, HMGA1 directly interacted with the VEGF gene, and this interaction was enhanced in hypoxic conditions. Furthermore, as indicated by co-immunoprecipitation studies, HMGA1 and HIF-1 physically interacted with each other, supporting the notion that this association may corroborate a functional link between these factors. Therefore, our findings provide evidence for molecular cross-talk between HMGA1 and HIF-1, and this may be important for elucidating protein and gene networks relevant to obesity.

Gene profiling reveals specific molecular pathways in the pathogenesis of atherosclerosis and cardiovascular disease in antiphospholipid syndrome, systemic lupus erythematosus and antiphospholipid syndrome with lupus.

OBJECTIVE: To identify shared and differential molecular pathways involved in the pathogenesis of atherosclerosis (AT) and cardiovascular disease (CVD) in systemic lupus erythematosus (SLE), primary antiphospholipid syndrome (APS) and APS associated with SLE (APS plus SLE). METHODS: 129 patients (42 APS, 31 APS plus SLE and 56 SLE) and 61 healthy donors were included. Microarray expression profiling was performed in monocytes. RT-PCR of selected genes and western blot were used to validate microarray data. Clinical and inflammatory parameters were also analysed. RESULTS: Compared with controls, 555, 1224 and 518 genes were differentially expressed in monocytes from SLE, APS plus SLE and APS patients, respectively. Approximately 25-30% of differentially expressed genes were related to AT and CVD. Each disease displayed a specific AT/CVD/Inflammation-related gene signature. Compared with SLE, APS showed alterations in mitochondria biogenesis and function and oxidative stress. Besides the interferon signature, found in APS plus SLE and SLE patients, various genes mediating atherosclerotic/inflammatory signalling were also differentially expressed in APS plus SLE. IgG-anticardiolipin (aCL) titres independently predicted both atherosclerotic and thrombosis in APS plus SLE. Moreover, a significant correlation of IgG-aCL titres with mRNA levels of certain inflammatory molecules in monocytes was further noticed. In vitro treatment of monocytes with IgG-aCL promoted an increase in the expression of the genes most significantly changed in APS plus SLE versus healthy donors. CONCLUSIONS: Gene expression profiling allows the segregation of APS, APS plus SLE and SLE, with specific signatures explaining the pro-atherosclerotic and pro-thrombotic alterations in these highly related autoimmune diseases.