Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

The role of KDR in intrauterine adhesions may involve the TGF-ß1/Smads signaling pathway

The aim of this study was to investigate the role of kinase-insert domain-containing receptor (KDR) in intrauterine adhesions (IUA) and its mechanism. The Case group consisted of 92 patients diagnosed with IUA, and the Control group included 86 patients with uterine septum who had normal endometrium verified with an uteroscope. In addition, 50 rats were randomly assigned into Control, Sham, Model, NC-siRNA, and KDR-siRNA groups. Rats in the Model, NC-siRNA, and KDR-siRNA groups were induced by uterine curettage and lipopolysaccharide (LPS) treatment to establish the IUA model. Then, immunohistochemistry was applied for detection of VEGF and KDR expression, HE staining was used for observation of the endometrial morphology and gland counting, Masson staining for measurement of the degree of endometrial fibrosis, and qRT-PCR and western blot for the expression of KDR, VEGF, MMP-9, as well as TGF-β1/Smads pathway-related proteins. Compared with the Control group, the mRNA and protein expressions of KDR were significantly higher in IUA endometrial tissues, and the expression of KDR was positively correlated to the severity of IUA. In addition, the injection of si-KDR increased the number of endometrial glands, reduced the area of fibrosis, inhibited mRNA and protein expression of KDR and VEGF, up-regulated the expression of MMP-9 and Smad7, and decreased the expression level of TGF-β1, p-Smad2, p-Smad3, and Smad4 in rats with IUA. Highly-expressed KDR was related to patients' severity of IUA, and silencing KDR may prevent the occurrence and development of IUA via TGF-β1/Smads signaling pathway and up-regulating the expression of MMP-9.

The comparison of methylene blue and vitamin E in prevention of abdominal postoperative adhesion formation in rat uterine horn models: Biochemical and histopathologic evaluation / Comparação do azul de metileno e vitamina E na prevenção de aderência abdominal pós-operatória em corno uterino de ratos: avalição bioquímica e histopatológica

Purpose: To compare the effects of vitamin E and 1 percent methylen blue solutions on prevention of experimentally induced adhesions in rats. Methods: Thirty seven female Spraque Dawley rats were randomized into four groups. First group was kept as sham operated group. An adhesion model was constituted on the left uterine horn of the other groups. The lesion areas of rats from the second, the third and the fourth groups were coated with 2 ml 0.9 percent saline solution (C group), 10 mg vitamin E (VE group) and 1 percent methylen blue solutions (MB group), respectively. Results: Histopathologically, adhesion scores, mononuclear cell infiltration, oedema and fibrosis were more prominent in the MB group compared with C and VE groups. There were no significant differences between the groups in tissue glutathione peroxidase (GPx), catalase (CAT) activities and glutation (GSH) level, these parameters were slightly increased in group with VE supplementation though. The administration of VE and MB significantly decreased NO (P<0.01) levels when compared to the C group. The level of malondialdehyde (MDA) in the VE group was significantly lower (P<0.05) than those of the Sh and C groups. Conclusion: Intraperitoneal methylen blue solutions treatments were more effective according to vitamin E in preventing the formation of intra-abdominal adhesion in a rat uterine horn model.

Objetivo: Comparar os efeitos da vitamina E e 1 por cento da solução de azul de metileno na prevenção de aderências induzidas em ratos. Métodos: Trinta e sete ratos fêmeas Spraque Dawley foram distribuídos em quatro grupos. O primeiro grupo foi mantido como grupo sham. O modelo de aderência foi realizado no corno uterino esquerdo nos outros grupos. As áreas da lesão dos ratos do segundo, terceiro e quarto grupos foram revestidas com 2 ml de solução salina 0,9 por cento (Grupo C), 10 mg de vitamina E (Grupo VE) e solução de azul de metileno 1 por cento (Grupo MB), respectivamente. Resultados: Histopatologicamente, o escore das aderências, infiltração celular mononuclear, edema e fibrose foram mais proeminentes no grupo MB em comparação aos grupos C e VE. Não houve diferença significante entre os grupos na peroxidase da glutatione do tecido (GPx), atividade da catalase (CAT) e o nível de glutation (GSH). Estes parâmetros foram ligeiramente aumentados no grupo com suplemento da VE. A administração da VE e do MB diminuiu significantemente os níveis quando quando comparada ao Grupo C. O nível de malondialdeído no grupo VE foi significantemente mais baixo do que nos grupos sham e C. Conclusão: A administração intraperitoneal da solução de azul de metileno foi mais eficaz de acordo com a vitamina E na prevenção de aderências intra-abdominais no corno uterino de ratos.

Estudio inmunocitoquímico de Receptores de Estrógeno Fracción Alfa y de Progesterona en Pólipos Endometriales / Immunocytochemistry Study of Estrogen Receiver Alfa Fraction and Progesterone in Endometrial Polyps

Los pólipos endometriales son causa frecuente de metrorragia y, en algunos casos, pueden ser responsables de esterilidad. Su patogenia es poco conocida, dando origen a masas sésiles o pedunculadas que protruyen en la cavidad uterina y que histológicamente corresponden a endometrio proliferativo de estroma fibroso dentro del cual se encuentran glándulas de tipo estrógenico en proporción variable que pueden mostrar irregularidad y, ocasionalmente, hiperplasia. La acción hormonal es fundamental para el desarrollo del endometrio tanto normal como patológico. Alteraciones cuantitativas a nivel de los receptores de estrógeno y progesterona podrían contribuir a la patogenia de los pólipos; siendo ellos el resultado de esta alteración. Su expresión podría corresponder a un aumento localizado de receptores de estrógeno (RE), de una disminución de receptores de progesterona (RP) o de ambos factores. Nuestro objetivo fue evaluar mediante método inmunocitoquímico, la presencia de receptores de estrógeno fraccion alfa y receptores de progesterona, en 9 pacientes portadoras de pólipos endometriales. La intensidad de la tinción fue evaluada como intensa, moderada, débil o negativa y comparada con muestras control de endometrio normal. Se observó un aumento de RE a en el estroma, en el epitelio glandular y en el endotelio de pequeños vasos que rodean a las glándulas endometriales. Los RP fueron negativos o no cuantificables, en el estroma y en el tejido glandular. Concluimos que los pólipos endometriales presentan una alteración de la distribución de los receptores con un aumento localizado de los REa en el estroma y en el epitelio glandular y una disminución de los RP, destacándose la presencia de REa en las células perivasculares y en el endotelio de pequeños vasos.

The endometrial polyps are a frequent cause of anormal bleeding and in same cases responsible of sterility. Their pathogenesis is poorly understand. They are masses sessiles or pedunculated that protrudes into the endometrial cavity.Thehistologic pattern resembles proliferative endometrium with fibrous stroma and estrogenic glands that can show irregularity and occasionally hyperplasia. The hormonal action is fundamentalfor the development of normal and pathologic endometrium. Quantitativealterations of the estrogen and progesterone receptors contributes to the pathogenesis of endometrial polyps. They could arise from localized overexpression of estrogen receptors (ER), reduced expression of progesterone receptors (PR) or both. This study was undertaken to evaluate with inmunocytochemistry the presence of estrogen receptors alfa and progesterone receptors in endometrial polyps of 9 patients. The intensity of staining was record on a scale high, moderate and weak or negative and compared with the staining in normal endometrium. High levels of ER a was seen in glandular epithelium, stroma and endothelial cells of capillaries that are surrounding the glands. The PR are negatives or very weak in stoma and glands. We conclude that endometrial polyps have a alterate distribution of the receptors with and increase number of stromal and glandular epitheliumlER alfa and a decrease of PRdetaching the large number of ER a in perivascular cells and in endotelial cells.

Determinaçäo dos receptores de estrogênio pelo método imunocitoquímico em fragmentos do epitélio de neovaginas de mulheres portadoras de agenesia vaginal / Determination of estrogen receptors by immunocitochemical method in fragments of epithelium of neovaginas in women with vaginal agenesis

OBJETIVOS: Observar as transformaçoes que ocorrem em fragmentos de membrana amniótica que foram utilizados para a confecçao de neovaginas em pacientes portadoras de agenesia útero-vaginal. CASUISTICA E METODOLOGIA: Foram 26 pacientes com agenesia utero-vaginal onde utilizou-se a técnica proposta por McIndoe. Para análise do epitélio da neovagina empregou-se método visando a determinaçao dos receptores de estrgênio citoplasmáticos (REc). Todos estes fragmentos de epitélio foram processados por método imunocitoquimico PAP (peroxidase x antiperoxidase). RESULTADOS: Estes receptores estrogênicos foram determinados em diversos graus de intensidade na camada profunda (CP), camada intermediária (CI) e na camada superficial (CS) dos epitélios de neovaginas. CONCLUSOES: O resultado obtido quanto ao grau de intensidade dos REc nas diversas camadas do epitélio vaginal de mulheres no menacme sao estatisticamente semelhantes e comparáveis no grau de intensidade aos fragmentos obtidos de epitélio de neovaginas confeccionadas com membrana amniótica.

Possible involvement of nitric oxide in estrogen-induced uterine edema in the immature rat

We examined whether nitric oxide mediates estrogen-induced uterine edema in the immature rat. Immature Wistar rats (19-21 days) received estradiol-17 beta (E2) in a single sc dose of 10 micrograms/animal and 6 h later the animals were sacrificed and the changes in uterine wet and dry weights were determined. E2 treatment caused a 93 per cent increase in uterine wet weight (control, N = 6, 39.88 +/- 3.2 mg; E2 treated, N = 6, 76.8 +/- 4.9 mg), but not in dry weight, suggesting that it induces uterine edema. Pretreatment with L-nitroarginine methyl ester (L-NAME), a competitive antagonist of nitric oxide synthetase, at doses of 10 and 20 mg/kg, ip, caused a dose-related reduction (59 and 86 per cent ) in the uterine wet weight increase induced by E2. Furthermore, L-arginine (300-600 mg/kg, sc), the nitric oxide precursor, was able to reverse L-NAME (20 mg/kg)-induced decreases in uterine weight by 47 and 62 per cent , respectively. The results suggest that nitric oxide is the principal mediator involved in estrogen-induced uterine edema in the immature rat