Avaliação histeroscópica em pacientes com infertilidade

PURPOSE: to describe hysteroscopy findings in infertile patients. METHODS: this was a retrospective series of 953 patients with diagnosis of infertility evaluated by hysteroscopy. A total of 957 patients investigated for infertility were subjected to hysteroscopy, preferentially during the first phase of the menstrual cycle. When necessary, directed biopsies (under direct visualization during the exam) or guided biopsies were obtained using a Novak curette after defining the site to be biopsied during the hysteroscopic examination. Outcome frequencies were determined as percentages, and the χ2 test was used for the correlations. The statistical software EpiInfo 2000 (CDC) was used for data analysis. RESULTS: a normal uterine cavity was detected in 436 cases (45.8%). This was the most frequent diagnosis for women with primary infertility and for women with one or no abortion (p<0.05). Abnormal findings were obtained in 517 of 953 cases (54.2%), including intrauterine synechiae in 185 patients (19.4%), endometrial polyps in 115 (12.1%), endocervical polyps in 66 (6.0%), submucosal myomas in 47 (4.9%), endometrial hyperplasia in 39 (4.1%), adenomyosis in five (0.5%), endometritis (with histopathological confirmation) in four (0.4%), endometrial bone metaplasia in two (0.4%), and cancer of the endometrium in one case (0.1%). Morphological and functional changes of the uterus were detected in 5.6% of the cases, including uterine malformations in 32 (3.4%) and isthmus-cervical incompetence in 21 (2.2%). CONCLUSIONS: intrauterine synechiae were the most frequent abnormal findings in patients evaluated for infertility. Patients with a history of abortion and infertility should be submitted to hysteroscopy in order to rule out intrauterine synechiae as a possible cause of infertility.

Stromal upregulation of lateral epithelial adhesions : gene expression analysis of signalling pathways in prostate epithelium

BACKGROUND: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. METHODS: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. RESULTS: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). CONCLUSIONS: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

Engineering a multi-biofunctional composite using poly(ethylenimine) decorated graphene oxide for bone tissue regeneration

Toward preparing strong multi-biofunctional materials, poly(ethylenimine) (PEI) conjugated graphene oxide (GO_PEI) was synthesized using poly(acrylic acid) (PAA) as a spacer and incorporated in poly( e-caprolactone) (PCL) at different fractions. GO_PEI significantly promoted the proliferation and formation of focal adhesions in human mesenchymal stem cells (hMSCs) on PCL. GO_PEI was highly potent in inducing stem cell osteogenesis leading to near doubling of alkaline phosphatase expression and mineralization over neat PCL with 5% filler content and was approximate to 50% better than GO. Remarkably, 5% GO_ PEI was as potent as soluble osteoinductive factors. Increased adsorption of osteogenic factors due to the amine and oxygen containing functional groups on GO_ PEI augment stem cell differentiation. GO_ PEI was also highly efficient in imparting bactericidal activity with 85% reduction in counts of E. coli colonies compared to neat PCL at 5% filler content and was more than twice as efficient as GO. This may be attributed to the synergistic effect of the sharp edges of the particles along with the presence of the different chemical moieties. Thus, GO_ PEI based polymer composites can be utilized to prepare bioactive resorbable biomaterials as an alternative to using labile biomolecules for fabricating orthopedic devices for fracture fixation and tissue engineering.

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

[Adhesions and abdominal pain]

Adhesions occur with a high incidence after intra-abdominal surgery but can also develop due to infections, radiation or for idiopathic reasons. The formation of adhesions is initiated by tissue damage and is the result of peritoneal tissue repair involving the activation of the inflammatory system and the coagulation cascade. Acute small bowel obstruction is one of the most common complications and should be diagnosed rapidly using clinical examination and radiological imaging. A complete obstruction is life threatening and in a high percentage of patients requires rapid surgical intervention by laparotomy or laparoscopy depending on the clinical situation and the patients history. Despite numerous investigations, there is no reliable, commonly used method to prevent intra-abdominal adhesions. Minimizing tissue damage and foreign body exposure, avoiding spillage of intestinal and biliary contents as well as a laparoscopic approach seem to have a beneficial effect on the formation of intra-abdominal adhesions.

Three-dimensional magnetic resonance observation of cartilage repair tissue (MOCART) score assessed with an isotropic three-dimensional true fast imaging with steady-state precession sequence at 3.0 Tesla

INTRODUCTION: Cartilage defects are common pathologies and surgical cartilage repair shows promising results. In its postoperative evaluation, the magnetic resonance observation of cartilage repair tissue (MOCART) score, using different variables to describe the constitution of the cartilage repair tissue and the surrounding structures, is widely used. High-field magnetic resonance imaging (MRI) and 3-dimensional (3D) isotropic sequences may combine ideal preconditions to enhance the diagnostic performance of cartilage imaging.Aim of this study was to introduce an improved 3D MOCART score using the possibilities of an isotropic 3D true fast imaging with steady-state precession (True-FISP) sequence in the postoperative evaluation of patients after matrix-associated autologous chondrocyte transplantation (MACT) as well as to compare the results to the conventional 2D MOCART score using standard MR sequences. MATERIAL AND METHODS: The study had approval by the local ethics commission. One hundred consecutive MR scans in 60 patients at standard follow-up intervals of 1, 3, 6, 12, 24, and 60 months after MACT of the knee joint were prospectively included. The mean follow-up interval of this cross-sectional evaluation was 21.4 +/- 20.6 months; the mean age of the patients was 35.8 +/- 9.4 years. MRI was performed at a 3.0 Tesla unit. All variables of the standard 2D MOCART score where part of the new 3D MOCART score. Furthermore, additional variables and options were included with the aims to use the capabilities of isotropic MRI, to include the results of recent studies, and to adapt to the needs of patients and physician in a clinical routine examination. A proton-density turbo spin-echo sequence, a T2-weighted dual fast spin-echo (dual-FSE) sequence, and a T1-weighted turbo inversion recovery magnitude (TIRM) sequence were used to assess the standard 2D MOCART score; an isotropic 3D-TrueFISP sequence was prepared to evaluate the new 3D MOCART score. All 9 variables of the 2D MOCART score were compared with the corresponding variables obtained by the 3D MOCART score using the Pearson correlation coefficient; additionally the subjective quality and possible artifacts of the MR sequences were analyzed. RESULTS: The correlation between the standard 2D MOCART score and the new 3D MOCART showed for the 8 variables "defect fill," "cartilage interface," "surface," "adhesions," "structure," "signal intensity," "subchondral lamina," and "effusion"-a highly significant (P < 0.001) correlation with a Pearson coefficient between 0.566 and 0.932. The variable "bone marrow edema" correlated significantly (P < 0.05; Pearson coefficient: 0.257). The subjective quality of the 3 standard MR sequences was comparable to the isotropic 3D-TrueFISP sequence. Artifacts were more frequently visible within the 3D-TrueFISP sequence. CONCLUSION: In the clinical routine follow-up after cartilage repair, the 3D MOCART score, assessed by only 1 high-resolution isotropic MR sequence, provides comparable information than the standard 2D MOCART score. Hence, the new 3D MOCART score has the potential to combine the information of the standard 2D MOCART score with the possible advantages of isotropic 3D MRI at high-field. A clear limitation of the 3D-TrueFISP sequence was the high number of artifacts. Future studies have to prove the clinical benefits of a 3D MOCART score.

Groin pain after open FAI surgery: the role of intraarticular adhesions

Femoroacetabular impingement (FAI) is an established cause of osteoarthrosis of the hip. Surgery is intended to remove the cause of impingement with hip dislocation and resection of osseous prominences of the acetabular rim and of the femoral head-neck junction. Using the Merle d'Aubigné score and qualitative categories, recent studies suggest good to excellent outcomes in 75% to 80% of patients after open surgery with dislocation of the femoral head. Unsatisfactory outcome is mainly related to pain, located either in the area of the greater trochanter or in the groin. There are several reasons for persisting groin pain. Joint degeneration with joint space narrowing and/or osteophyte formation, insufficient correction of the acetabula, and femoral pathology are known factors for unsatisfactory outcome. Recently, intraarticular adhesions between the femoral neck and joint capsule have been identified as an additional cause of postoperative groin pain. The adhesions form between the joint capsule and the resected area on the femoral neck and may lead to soft tissue impingement. MR-arthrography is used for diagnosis and the adhesions can be treated successfully by arthroscopy. While arthroscopic resection improves outcome it is technically demanding. Avoiding the formation of adhesions is important and is perhaps best accomplished by passive motion exercises after the initial surgery.

Tissue spreading on implantable substrates is a competitive outcome of cell–cell vs. cell–substratum adhesivity

While the interactions of cells with polymeric substrata are widely studied, the influence of cell–cell cohesivity on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or “tug-of-war” between cell–cell and cell–substratum adhesions. We have generated both a “library” of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesivity. Cell–substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell–cell cohesivity was varied through the expression of the homophilic cohesion molecules N- and R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell–cell cohesivity on a series of copolymeric substrata having different levels of cell–substratum adhesivity. In these experiments, cell–cell cohesivity was measured by tissue surface tensiometry, and cell–substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescence-labeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell–cell cohesivity dramatically slowed the spreading rate of cell aggregates.

The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1.

The transmembrane protein-tyrosine-phosphatases (PTPases) LAR, PTP delta, and PTP sigma each contain two intracellular PTPase domains and an extracellular region consisting of Ig-like and fibronectin type III-like domains. We describe the cloning and characterization of human PTP sigma (HPTP sigma) and compare the structure, alternative splicing, tissue distribution, and PTPase activity of LAR, HPTP delta, and HPTP sigma, as well their ability to associate with the intracellular coiled-coil LAR-interacting protein LIP.1. Overall, these three PTPases are structurally very similar, sharing 64% amino acid identity. Multiple isoforms of LAR, HPTP delta, and HPTP sigma appear to be generated by tissue-specific alternative splicing of up to four mini-exon segments that encode peptides of 4-16 aa located in both the extracellular and intracellular regions. Alternative usage of these peptides varies depending on the tissue mRNA analyzed. Short isoforms of both HPTP sigma and HPTP delta were also detected that contain only four of the eight fibronectin type III-like domains. Northern blot analysis indicates that LAR and HPTP sigma are broadly distributed whereas HPTP delta expression is largely restricted to brain, as is the short HPTP sigma isoform containing only four fibronectin type III-like domains. LAR, HPTP delta, and HPTP sigma exhibit similar in vitro PTPase activities and all three interact with LIP.1, which has been postulated to recruit LAR to focal adhesions. Thus, these closely related PTPases may perform similar functions in various tissues.

Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

A novel RGD-independent cel adhesion pathway mediated by fibronectin-bound tissue transglutaminase rescues cells from anoikis

Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies alpha5beta1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Calpha, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.