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.

Understanding endometriosis from an immunomicroenvironmental perspective / 中华医学杂志(英文版)

Endometriosis, a heterogeneous, inflammatory, and estrogen-dependent gynecological disease defined by the presence and growth of endometrial tissues outside the lining of the uterus, affects approximately 5-10% of reproductive-age women, causing chronic pelvic pain and reduced fertility. Although the etiology of endometriosis is still elusive, emerging evidence supports the idea that immune dysregulation can promote the survival and growth of retrograde endometrial debris. Peritoneal macrophages and natural killer (NK) cells exhibit deficient cytotoxicity in the endometriotic microenvironment, leading to inefficient eradication of refluxed endometrial fragments. In addition, the imbalance of T-cell subtypes results in aberrant cytokine production and chronic inflammation, which contribute to endometriosis development. Although it remains uncertain whether immune dysregulation represents an initial cause or merely a secondary enhancer of endometriosis, therapies targeting altered immune pathways exhibit satisfactory effects in preventing disease onset and progression. Here, we summarize the phenotypic and functional alterations of immune cells in the endometriotic microenvironment, focusing on their interactions with microbiota and endocrine and nervous systems, and how these interactions contribute to the etiology and symptomology of endometriosis.

Endometrioid adenocarcinoma with proliferated stromal cells, hyalinization and cord-like formations: A case report / 北京大学学报(医学版)

Corded and hyalinized endometrioid carcinoma (CHEC) is a morphologic variant of endo-metrioid adenocarcinoma. The tumor exhibits a biphasic appearance with areas of traditional low-grade adenocarcinoma merging directly with areas of diffuse growth composed of epithelioid or spindled tumor cells forming cords, small clusters, or dispersed single cells. It is crucial to distinguish CHEC from its morphological mimics, such as malignant mixed mullerian tumor (MMMT), because CHECs are usually low stage, and are associated with a good post-hysterectomy prognosis in most cases while the latter portends a poor prognosis. The patient reported in this article was a 54-year-old woman who presented with postmenopausal vaginal bleeding for 2 months. The ultrasound image showed a thickened uneven echo endometrium of approximately 12.2 mm and a detectable blood flow signal. Magnetic resonance imaging revealed an abnormal endometrial signal, considered endometrial carcinoma (Stage Ⅰ B). On hysterectomy specimen, there was an exophytic mass in the uterine cavity with myometrium infiltrating. Microscopically, most component of the tumor was well to moderately differentiated endometrioid carcinoma. Some oval and spindle stromal cells proliferated on the superficial surface of the tumor with a bundle or sheet like growth pattern. In the endometrial curettage specimen, the proliferation of these stromal cells was more obvious, and some of the surrounding stroma was hyalinized and chondromyxoid, which made the stromal cells form a cord-like arrangement. Immunostains were done and both the endometrioid carcinoma and the proliferating stroma cells showed loss of expression of DNA mismatch repair protein MLH1/PMS2 and wild-type p53 protein. Molecular testing demonstrated that this patient had a microsatellite unstable (MSI) endometrial carcinoma. The patient was followed up for 6 months, and there was no recurrence. We diagnosed this case as CHEC, a variant of endometrioid carcinoma, although this case did not show specific β-catenin nuclear expression that was reported in previous researches. The striking low-grade biphasic appearance without TP53 mutation confirmed by immunohistochemistry and molecular testing supported the diagnosis of CHEC. This special morphology, which is usually distributed in the superficial part of the tumor, may result in differences between curettage and surgical specimens. Recent studies have documented an aggressive clinical course in a significant proportion of cases. More cases are needed to establish the clinical behaviors, pathologic features, and molecular profiles of CHECs. Recognition of the relevant characteristics is the prerequisite for pathologists to make correct diagnoses and acquire comprehensive interpretation.

Lowered expression of CCN5 in endometriotic tissues promotes proliferation, migration and invasion of endometrial stromal cells / 南方医科大学学报

OBJECTIVE@#To explore the expression of CCN5 in endometriotic tissues and its impact on proliferation, migration and invasion of human endometrial stromal cells (HESCs).@*METHODS@#We collected ovarian endometriosis samples from 20 women receiving laparoscopic surgery and eutopic endometrium samples from 15 women undergoing IVF-ET for comparison of CCN5 expression. Cultured HESCs were transfected with a recombinant adenovirus Ad-CCN5 for CCN5 overexpression or with a CCN5-specific siRNA for knocking down CCN5 expression, and the changes of cell proliferation, migration and invasion were evaluated using CCK-8 assay, wound healing assay and Transwell chamber assay. RT-qPCR and Western blotting were used to examine the expression levels of epithelial-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin, Snail-1 and vimentin in HESCs with CCN5 overexpression or knockdown.@*RESULTS@#CCN5 expression was significantly decreased in ovarian endometriosis tissues as compared with eutopic endometrium samples (P < 0.01). CCN5 overexpression obviously inhibited the proliferation, migration and invasion of HESCs, significantly increased the expression of E-cadherin and decreased the expressions of N-cadherin, Snail-1 and vimentin (P < 0.01). CCN5 knockdown significantly enhanced the proliferation, migration and invasion of HESCs and produced opposite effects on the expressions of E-cadherin, N-cadherin, Snail-1 and vimentin (P < 0.01).@*CONCLUSION@#CCN5 can regulate the proliferation, migration and invasion of HESCs and thus plays an important role in EMT of HESCs, suggesting the potential of CCN5 as a therapeutic target for endometriosis.

Integrated optical density analysis of the immunohistochemical expression of the progesterone receptor in the uterine endometrium of prepubertal araucana sheep / Análisis por densidad óptica integrada de la expresión inmunohistoquímica del receptor de progesterona en el endometrio uterino de ovejas prepúberes de raza araucana

SUMMARY: Progesterone receptors are expressed in the reproductive organs of adult sheep, where they regulate morphofunctional and reproductive development. However, various studies have shown the presence of these receptors in the uterus of prepubertal females. It is not clear what role these receptors have at this level of development in uterine tissue. Therefore, it is relevant as a first step in the investigation, to determine the expression and histological distribution of the progesterone receptor in prepubertal sheep in order to determine possible functions at this level of reproductive development. Immunohistochemical analysis allows visualizing the specific presence of a protein in the cellular and histological context, however, the results displayed through digital images are qualitative data and subject to the observer's criteria. In this work, a quantitative analysis method of immunohistochemical expression of the progesterone receptor in ovine endometrium is presented, using digital analysis of images, by means of integrated optical density of digital photographs of histological sections processed with immunohistochemical methods. The results show the possibility of quantitatively evaluating the expression of progesterone receptors in the endometrial stroma and prepubertal endometrial glands by applying the integrated optical density analysis of digital images.

RESUMEN: Los receptores de progesterona se expresan en los órganos reproductores de ovejas adultas, donde regulan el desarrollo morfofuncional y reproductivo. Sin embargo, diversos estudios han demostrado la presencia de estos receptores en útero de hembras prepúberes. No está claro, el papel que estos receptores tienen en este nivel de desarrollo en tejido uterino. Por lo que, es relevante como primer paso en la investigación, determinar la expresión y distribución histológica del receptor de progesterona en ovejas prepúberes con el fin determinar posibles funciones en este nivel de desarrollo reproductivo. El análisis inmuno- histoquímico permite visualizar la presencia específica de una proteína en el contexto celular e histológico, sin embargo, los resultados visualizados a través de imágenes digitales, son datos cualitativos y sujeto al criterio del observador. En este trabajo se presenta un método de análisis cuantitativo de expresión inmunohistoquímica del receptor de progesterona en endometrio ovino, utilizando análisis digital de imágenes, mediante densidad óptica integrada de fotografías digitales de cortes histológicos procesados con métodos inmunohistoquímicos. Los resultados muestran la posibilidad de evaluar cuantitativamente la expresión de los receptores de progesterona en el estroma endometrial y las glándulas endometriales prepúberes aplicando el análisis de densidad óptica integrado de imágenes digitales.

Screening of Variants in the Transcript Profile of Eutopic Endometrium from Infertile Women with Endometriosis during the Implantation Window / Rastreio de variantes no perfil de tanscritos do endométrio eutópico de mulheres inférteis com endometriose durante a janela de implantação

Abstract Objective Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window. Methods Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease's context. Results None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment. Conclusion Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial

Resumo Objetivo Anormalidades no endométrio eutópico de mulheres com endometriose podem estar relacionadas à infertilidade associada à doença. Embora a análise prévia de sequenciamento de RNA não tenha evidenciado expressão diferencial em transcritos endometriais de pacientes com endometriose, outras alterações moleculares poderiam afetar a síntese de proteínas e a receptividade endometrial. Nosso objetivo foi rastrear mutações funcionais em transcritos de endométrios eutópicos de mulheres inférteis com endometriose e de controles durante a janela de implantação. Métodos Os dados do sequenciamento de RNA de biópsias endometriais coletados durante a janela de implantação de 17 pacientes (6 mulheres inférteis com endometriose, 6 controles inférteis, 5 controles férteis) foram analisados para a descoberta de variantes e a identificação de mutações funcionais. Um estudo direcionado das alterações encontradas foi realizado para compreender os dados no contexto da doença. Resultados Nenhuma das variantes identificadas foi comuma outras amostras dentro do mesmo grupo, assim como nenhuma mutação se repetiu entre pacientes com endometriose, controles inférteis e férteis. No grupo de endometriose, foram identificadas nove mutações deletérias preditas, mas apenas uma foi previamente associada a uma condição clínica sem impacto endometrial. Ao cruzar os genes mutados com os descritores endometriose e/ou endométrio, o gene CMKLR1 foi associado a resposta inflamatória na endometriose e a processos endometriais para estabelecimento da gravidez. Conclusão Apesar de nenhum padrão de mutação ter sido encontrado, ponderamos o pequeno tamanho da amostra e a análise dos dados de sequenciamento de RNA. Considerando o objetivo do estudo de triagem e a importância do gene CMKLR1 na modulação endometrial, este poderia ser um gene candidato para estudos adicionais que avaliem mutações no endométrio eutópico de pacientes com endometriose.

Increased expression levels of metalloprotease, tissue inhibitor of metalloprotease, metallothionein, and p63 in ectopic endometrium: an animal experimental study / Aumento nos níveis de expressão de metaloprotease, inibidor tecidual das metaloproteases, metalotioneina e p63 em endométrio ectópico: um estudo experimental animal

Abstract Objective To characterize the patterns of cell differentiation, proliferation, and tissue invasion in eutopic and ectopic endometrium of rabbits with induced endometriotic lesions via a well- known experimental model, 4 and 8 weeks after the endometrial implantation procedure. Methods Twenty-nine female New Zealand rabbits underwent laparotomy for endometriosis induction through the resection of one uterine horn, isolation of the endometrium, and fixation of tissue segment to the pelvic peritoneum. Two groups of animals (one with 14 animals, and the other with15) were sacrificed 4 and 8 weeks after endometriosis induction. The lesion was excised along with the opposite uterine horn for endometrial gland and stroma determination. Immunohistochemical reactions were performed in eutopic and ectopic endometrial tissues for analysis of the following markers: metalloprotease (MMP-9) and tissue inhibitor of metalloprotease (TIMP-2), which are involved in the invasive capacity of the endometrial tissue; and metallothionein (MT) and p63, which are involved in cell differentiation and proliferation. Results The intensity of the immunostaining for MMP9, TIMP-2, MT, and p63 was higher in ectopic endometria than in eutopic endometria. However, when the ectopic lesions were compared at 4 and 8 weeks, no significant difference was observed, with the exception of the marker p63, which was more evident after 8 weeks of evolution of the ectopic endometrial tissue. Conclusion Ectopic endometrial lesions seem to express greater power for cell differentiation and tissue invasion, compared with eutopic endometria, demonstrating a potentially invasive, progressive, and heterogeneous presentation of endometriosis.

Resumo Objetivo Caracterizar o padrão de diferenciação celular, proliferação e invasão tecidual em endométrio eutópico e ectópico de coelhas com lesões de endometriose induzidas por um modelo experimental 4 e 8 semanas após o procedimento de implantação endometrial. Métodos Vinte e nove coelhas fêmeas Nova Zelândia foram submetidas a laparotomia para indução de endometriose através da ressecção de um dos cornos uterinos, isolamento do endométrio e fixação do tecido no peritônio pélvico. Dois grupos de animais (14 animais em um grupo e 15 animais no outro) foram sacrificados 4 e 8 semanas após a indução da endometriose. A lesão foi excisada junto com o corno uterino contralateral para determinação da presença de glândulas e de estroma endometrial. Reações de imunohistoquímica foram realizadas no tecido endometrial eutópico e ectópico para análise dos seguintes marcadores: metaloprotease (MMP9) e inibidor tecidual da metaloprotease 2 (TIMP-2), os quais estão envolvidos na capacidade de invasão do tecido endometrial; e metalotioneina (MT) e p63, os quais estão envolvidos na diferenciação e proliferação celular. Resultados A intensidade da imunomarcação para MMP9, TIMP-2, MT e p63 foi mais alta nos endométrios ectópicos do que nos endométrios eutópicos. Contudo, quando as lesões foram comparadas entre 4 e 8 semanas, nenhuma diferença foi observada, com exceção do marcador p63, o qual foi mais evidente depois de 8 semanas de evolução do tecido endometrial ectópico. Conclusão Lesões endometriais ectópicas parecem expressar maior poder de diferenciação celular e de invasão tecidual comparadas com endométrios eutópicos, demonstrando o potencial de invasão, de progressão e de apresentação heterogênea da endometriose.

The impact of HSF on endometrium

Summary Objective: We conducted the research in order to explore the impact of hydrosalpinx fluid (HSF) on endometrium. Method: HSF group: 261 patients with HSF scheduled to undergo laparoscopic surgery 3 to 7 days after menstruation in our center. Hysteroscopy would also be performed in order to observe the endometrial morphology during the surgery. Sixty (60) patients would be randomly selected for endometrial biopsy in order to detect the inflammatory cytokines TNF-a and IL-2 mRNA. Non-HSF group: 210 patients with no evidence of HSF due to chronic salpingitis or pelvic adhesion. IVF-ET treatment was performed after eliminating the factor of male infertility and hysteroscopy was conducted before the treatment. Fifty (50) patients underwent endometrial biopsy in order to detect TNF-a and IL-2 mRNA. Results: Hysteroscopy was performed in 261 patients with HSF and 210 patients without HSF. The incidence rate of endometritis manifestation among these two groups of patients was 37.2% (97/261) and 20.5% (43/210), respectively. The incidence rate of endometritis in the patients with HSF is significantly higher than in the patients without HSF (p<0.05). Sixty (60) patients from the HSF group and 50 patients from the non-HSF group were regrouped according to inflammatory and normal manifestation after the endometrial biopsy. There were 49 patients in the inflammatory manifestation group and 61 patients in the normal manifestation group. RT-PCR technology was adopted to detect the expression of inflammatory cytokines TNF-a and IL-2 mRNA in endometrial tissue. The level of TNF-a mRNA expression in endometrial tissues with inflammatory manifestation was higher than in normal endometrium (76.75±11.95 vs. 23.45±9.75, p<0.01). There are significant differences between them. The level of IL-2 mRNA expression in endometrial tissues with inflammatory manifestation was higher than that found in normal endometrium (80.56±13.35 vs. 35.12±8.35, p<0.01). There are significant differences between them. Conclusion: Chronic endometritis is related to HSF and may therefore affect endometrial receptivity.

Estrogen signaling in the proliferative endometrium: implications in endometriosis / Sinalização pelo estrogênio no endométrio proliferativo: implicações na endometriose

SUMMARY Even though the physiological role of estrogen in the female reproductive cycle and endometrial proliferative phase is well established, the signaling pathways by which estrogen exerts its action in the endometrial tissue are still little known. In this regard, advancements in cell culture techniques and maintenance of endometrial cells in cultures enabled the discovery of new signaling mechanisms activated by estrogen in the normal endometrium and in endometriosis. This review aims to present the recent findings in the genomic and non-genomic estrogen signaling pathways in the proliferative human endometrium specifically associated with the pathogenesis and development of endometriosis.

RESUMO Embora esteja bem estabelecido o papel fisiológico do estrogênio no ciclo reprodutivo feminino e na fase proliferativa do endométrio, as vias de sinalização por meio das quais a ação do estrogênio é exercida no tecido endometrial são ainda pouco conhecidas. Nesse sentido, o avanço nas técnicas de cultura celular e a manutenção de células endometriais em cultivo possibilitaram a descoberta de novos mecanismos sinalizadores ativados pelo estrogênio no endométrio normal e na endometriose. Esta revisão tem o objetivo de apresentar as descobertas recentes envolvendo as vias de sinalização genômica e não genômica do estrogênio no endométrio proliferativo humano, especificamente associadas à patogênese e ao desenvolvimento da endometriose.

Gene expression of estrogen and oxytocin receptors in the uterus of pregnant and parturient bitches

In the canine species, the precise mechanisms of pregnancy maintenance and the initiation of parturition are not completely understood. The expression of genes encoding the receptors for estrogen (ERα mRNA) and oxytocin (OTR mRNA) was studied in the endometrium and myometrium during pregnancy and parturition in dogs. Real-time PCR was performed to quantify the levels of ERα mRNA and OTR mRNA in the uterus of bitches during early (up to 20 days of gestation), mid (20 to 40 days) and late pregnancy (41 to 60 days), and parturition (first stage of labor). All tissues expressed ERα and OTR mRNA, and are thus possibly able to respond to eventual estrogen and oxytocin hormonal stimuli. No statistically significant differences in the expression of ERα mRNA were verified in the endometrium and myometrium throughout pregnancy and parturition, but expression of OTR mRNA increased at both parturition and late pregnancy. We concluded that the increase of endometrial and myometrial OTR mRNA expression in dogs is not an event dependent on estrogenic stimulation. Moreover, the contractility response of the canine uterus to oxytocin begins during pregnancy and maintains myometrial activity. The expression of OTR mRNA in canine uterine tissues varied over time, which supports an interpretation that the sensitivity and response to hormone therapy varies during the course of pregnancy and labor. Further studies are needed to elucidate the factors underlying the synthesis of uterine oxytocin receptors and the possible role of ERβ rather than ERα in the uterine tissues during pregnancy and parturition in dogs.

Adiponectin effect on nitric oxide secretion by normal and endometriotic human endometrial stromal cells: in vitro study / Efecto de la adiponectica sobre la secreción de óxido nítrico por células estromales de endometrio humano, normales y con endometriosis: estudio in vitro

Endometriosis is an estrogen-dependent disease in reproductive age women. Adiponectin and Nitric oxide (NO) have an important role in physiologic functions especially in human reproductive system. Levels of NO increased in the endometriosis patients but serum adiponectin levels decreased in woman with endometriosis. The aim of this study was to determine adiponectin effect on nitric oxide secretion by cultured normal and endometriotic human endometrial stromal cells. In this experimental study, normal (n= 10) and endometriotic endometrial biopsies (n= 10) were taken in sterile condition. Stromal cells isolated and cultured in in DMEM/ F12 medium and treated with adiponectin concentrations (0, 10, 100, and 200 ng/ml) for 24 and 48 hours. NO assay was done on their supernatants by Greiss method. Data was analyzed by one way ANOVA and p<0.05 was considered significant. There was significant difference between endometriosis groups in NO secretion in all dose of adiponectin and time (p<0.05). In normal groups there was significant difference in 48 hours (p<0.05) but no significant change in 24 hours (p>0.05). Adiponectin effects nitric oxide secretion of cultured human endometriotic stromal cells.

La endometriosis es una enfermedad dependiente de estrógenos que se presenta en mujeres en edad reproductiva. La adiponectina y el óxido nítrico (ON) tienen un papel importante en las funciones fisiológicas, especialmente en el sistema reproductivo humano. Los niveles de ON aumentan en los pacientes con endometriosis, pero los niveles de adiponectina en suero disminuyen. El objetivo fue determinar el efecto de la adiponectina sobre la secreción de ON por las células estromales de endometrio humano, tanto normales como con endometriosis, en medio de cultivo. En este estudio experimental, las células estromales de endometrio normales (n= 10) y las biopsias de endometrio con endometriosis (n= 10) se tomaron en condiciones de esterilidad. Las células estromales fueron aisladas y cultivadas en un medio DMEM/F12, y se sometieron a distintas concentraciones de adiponectina (0, 10, 100, y 200 ng/ml) durante 24 y 48 horas. El ensayo con ON se realizó a los sobrenadantes obtenidos por el método de Greiss. Los datos recolectados fueron analizados por ANOVA de una vía y un valor p<0,05 se consideró significativo. Entre los grupos con endometriosis, en referencia a la secreción de ON, no hubo diferencia significativa en todas las dosis de adiponectina y los tiempos estipulados (p<0,05). En los grupos normales, hubo diferencia significativa a las 48 horas (p<0,05), pero ningún cambio significativo a las 24 horas (p>0,05). La adiponectina tiene efectos sobre la secreción de óxido nítrico por las células estromales endometriales humanas en cultivo.

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation

BACKGROUND: Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. RESULTS: Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERa) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. CONCLUSION: These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERa and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

Estudio inmunohistoquímico comparativo del receptor de estrógeno en tejido endometrial de ovejas razas Texel y Araucana / Comparative inmunohistochemical study of estrogen receptor in endometrial tissue from Texel and Araucana sheeps

La asociación de factores genéticos, nutricionales y ambientales afecta directamente la fertilidad de las ovejas sin embargo en condiciones de similitud ambiental, la raza es un factor determinante existiendo razas de alta fertilidad como por ejemplo Texel y Suffolk y otras de fertilidad estándar tales como Romney y Criolla Araucana. La fertilidad es modulada por las hormonas sexuales que actúan mediante la unión a receptores específicos. En ovinos el receptor de estrógeno alfa (ER-a) esta ampliamente distribuido en el sistema reproductivo y es responsable de la modulación de varios mecanismos asociados con la función del sistema reproductivo. Un factor posiblemente relacionado con la diferencia en la fertilidad entre razas de ovinos, es el nivel de expresión diferencial de estos receptores en el sistema reproductivo. En el presente estudio se realizo una comparación cuantitativa de la expresión inmunohistoquímica de ER-a se llevó en el endometrio de ovejas prepúberes de ovejas de alta fertilidad (Texel) y de fertilidad estándar (Araucana), mediante la medición de la densidad óptica integrada de la señal inmunohistoquímica en áreas específicas del endometrio. Los resultados indican una diferencia significativa entre la expresión del ER-a a favor de ovejas de raza Texel en todas las áreas de la raza del endometrio de ovejas prepúberes evaluadas. Esta expresión diferencial sugiere una posible relación entre la intensidad de la expresión de ER-a y la fertilidad en las razas ovinas estudiadas en este trabajo.

In breeding sheep high fertility rate is an important consideration factor. The association of genetic, nutritional and environmental conditions directly affects the fertility of ewes. There are high fertility sheep breeds (Texel, Suffolk) and other standard fertility breeds as (Romney, Criolla Araucana). Animal reproduction is modulated by sex hormones that act by binding to specific receptors. In sheep, alpha (ER-a) estrogen receptor is widely distributed in the reproductive system, modulating several mechanisms associated with reproductive system function. One factor possibly related to the difference in fertility between sheep breeds, is the differential expression level of these receptors in the reproductive system. In the present study a quantitative comparison of the immunohistochemical expression of ER-a was carried out in prepubertal sheep endometrium in high fertility (Texel) breed versus standard fertility (Araucana) breed, by measuring the integrated optical density in specific areas of the endometrium. Results indicate a significant difference between ER-a expression in endometrium off Texel breed ewes and Araucana breed ewes, and registered higher levels in all areas of evaluated Texel breed prepubertal ewes. This differential expression suggests a possible link between ER-a expression intensity and fertility in the breeds studied in this work.

Detección inmunohistoquímica del receptor de estrógeno en el endometrio de ovejas de alta prolificidad y de prolificidad estándar / Immunohistochemical detection of estrogen receptor in the endometrium of sheep high prolificacy and standard prolificacy

Uno de los factores que incide en la capacidad reproductiva de los ovinos es la presencia de receptores de estrógeno en el tracto genital, donde regulan la expresión de numerosos genes comprometidos en su desarrollo morfológico y funcional. Posiblemente el desempeño reproductivo de diferentes razas de ovejas podría estar determinado por la expresión diferencial de estos receptores. En el presente estudio, se evaluó comparativamente el nivel de expresión del receptor de estrógeno en tejido endometrial de ovejas prepúberes de alta prolificidad (raza Texel) y de prolificidad estándar (raza Araucana) mediante análisis inmunohistoquímico. Se concluye que el nivel de expresión del receptor de estrógeno en tejido estromal, epitelial, de revestimiento y glandular del endometrio de ovejas de raza Texel es significativamente mayor que en ovejas de raza Araucana. Esta diferencia en el nivel de expresión de estos receptores podría estar relacionado con la diferencia de prolificidad entre estas razas...

A factor that affects the reproductive capacity of sheep is the presence of estrogen receptors in the genital tract, where they regulate the expression of numerous genes involved in morphological and functional development. Possibly the reproductive performance of different breeds of sheep could be determined by the differential expression of these receptors. In the present study, we evaluated comparatively level of estrogen receptor expression in endometrial tissue ewe lambs high prolificacy (Texel) and standard prolificacy (Araucana) by immunohistochemical analysis. It is concluded that the level of estrogen receptor expression in stromal tissue, epithelial lining and endometrial glandular Texel Sheep is of greater intensity than in ewes Araucana, this difference in the expression level of these receptors could be related to the increased prolificacy of Texel breed...

Expresión de receptores de estrógenos y progesterona en el tracto genital de ovejas raza Texel / Expression of estrogen and progesterone receptors in the genital tract of Texel ewes

El comportamiento reproductivo de los ovinos varía entre las diferentes razas. Dentro de los factores que inciden en la capacidad reproductiva, el nivel de expresión de receptores de estrógenos y progesterona en su tracto genital parece tener un rol relevante. En el endometrio, oviducto y ovario los estrógenos y progesterona, regulan la expresión de numerosas proteínas comprometidas en su desarrollo morfofuncional. Factores genéticos como la raza estarian relacionados con los niveles de expresión de estos receptores. Las ovejas de raza Texel tienen elevados índices de fertilidad, son muy prolíficas y presentan un alto porcentaje de gestación múltiple lo cual podría tener relación con la expresión de estos receptores en el tracto reproductivo. El objetivo del presente estudio fue evaluar la expresión de receptores de estrógenos y progesterona en tracto genital de ovejas de raza Texel de alta prolificidad. La expresión de la proteína receptora de ambos receptores se detectó mediante análisis inmunohistoquímico y el nivel de expresión de los transcritos por RT-PCR en Tiempo Real Cuantitativo. Los resultados muestran expresión inmunohistoquímica del receptor de estrógeno principalmente en zonas glandulares y carunculares del endometrio. Se destaca además una menor expresión de ambos receptores en ovario, epitelio del oviducto y cervix. La expresión del receptor de progesterona a nivel inmunohistoquímico es bastante menor en cuanto a la señal destacándose marcas débiles en endometrio y ovarios. El nivel de expresión de los transcritos mantiene la misma distribución que las señales inmunohistoquimicas para ambos receptores. Concluimos que ambos receptores son expresados en el sistema reproductivo de ovejas de raza Texel en una distribución similar a lo encontrado en otras razas de ovejas quedando por definir si los niveles de expresión son similares en las distintas razas.

Reproductive performance of sheep varies between different races. Among the factors that affect the reproductive capacity, the level of expression of estrogen and progesterone receptors in the genital tract appears to have a relevant role. In the endometrium, oviduct and ovarian estrogen and progesterone regulate the expression of numerous proteins involved in morphofunctional development. Genetic factors such as race would be related to expression levels of these receptors. The Texel ewes have high fertility rates, are very prolific and have a high percentage of multiple births which could be related to the expression of these receptors in the reproductive tract. The aim of this study was to evaluate the expression of estrogen and progesterone receptors in the genital tract of ewes and Texel high prolificacy. The expression of the receptor protein of both receptors was detected by immunohistochemistry and the level of expression of the transcripts by Quantitative RT-PCR Real-Time. The results showed immunohistochemical expression of estrogen receptor mainly in areas of glandular and caruncular endometrium. It also highlights a lower expression of both receptors in ovarian tissue, epithelium of the oviduct and cervix. Expression of progesterone receptor immunohistochemical level is much lower with weaker signal in endometrium and ovaries. The expression level of transcripts remains the same distribution as immunohistochemical signals for both receptors. We conclude that both receptors are expressed in the reproductive system Texel ewes in a distribution similar to that found in other breeds of sheep, must be defined if the levels of expression are similar in different races.

Endometriose: papel da superfamília do fator transformador de crescimento beta / Endometriosis: the role of transforming growth factor β superfamily

A endometriose é uma condição ginecológica, que atinge mulheres em idade reprodutiva e pode ser causa de dor e infertilidade. A patogênese da doença é multifatorial e envolve a perda da capacidade de diferenciação das células endometrióticas, moléculas de adesão celular para adesão do endométrio ao peritônio, neoangiogênese, características do fluido peritoneal e alterações do sistema imune. A superfamília do fator transformador de crescimento β (TGF-β) parece exercer papéis importantes na implantação e manutenção do tecido ectópico na endometriose. Ativinas, inibinas, folistatina, hormônio anti-mülleriano e as proteínas morfogenéticas ósseas são membros da superfamília do TGF-β. Estas moléculas são expressas no endométrio humano e apresentam ações importantes na proliferação celular, diferenciação celular, função imune, regulação da apoptose e remodelamento dos tecidos, apresentando, por conseguinte, um importante papel no ciclo menstrual, decidualização do endométrio e no início da gestação. Este artigo objetiva rever os achados sobre tais proteínas no endométrio e seus possíveis papéis na gênese e fisiopatologia da endometriose

Endometriosis is a gynecological pathological entity typical of women in reproductive age, associated with pelvic pain and infertility. The pathogenesis of the disease is multifactorial and it involves loss of the endometriotic cell differentiation, cell adhesion, neo-angiogenesis, peritoneal fluid characteristics, and changes in the immune system. The transforming growth factor β (TGF-β) superfamily seems to play important roles in the implementation and maintenance of ectopic tissue in endometriosis. Activin, inhibin, follistatin, anti-Mullerian hormone, and bone morphogenetic proteins are members of the superfamily of TGF-β. The TGF-β and family members are expressed by human endometrium and act on cell proliferation, differentiation, immune function, apoptosis and tissue remodeling, playing a role in menstrual cycle, decidualization, and early pregnancy. The aim of this study is to review the findings about these molecules in the endometrium and their possible roles in the genesis and pathophysiology of endometriosis

Canonical and noncanonical Wnt pathways: a comparison between endometrial cancer type I and atrophic endometrium in Brazil / Vias Wnt canônica e não canônica: uma comparação entre o câncer endometrial tipo I e endométrio atrófico no Brasil

CONTEXT AND OBJECTIVE: The Wnt pathway is involved in tumorigenesis of several tissues. For this reason, we proposed to evaluate Wnt gene expression in endometrial cancer type I. DESIGN AND SETTING: Cross-sectional study on materials gathered from the tissue bank of the Department of Pathology, Universidade Federal de São Paulo. METHODS: Endometrial specimens were obtained from surgeries performed between 1995 and 2005 at São Paulo Hospital, Universidade Federal de São Paulo. The material was divided into two groups according to tissue type: Group A, atrophic endometrium (n = 15); and Group B, endometrial adenocarcinoma (n = 45). We compared the immunohistochemical expression of Wnt1, Frizzled-1 (FZD1), Wnt5a, Frizzled-5 (FZD5) and beta-catenin between endometrial cancer type I and atrophic endometrium. RESULTS: Regarding Wnt1, FZD1 and Wnt5a expression, no significant association was observed between the groups. A significant association was observed between the groups in relation to FZD5 expression (P = 0.001). The proportion of FZD5-positive samples was significantly higher in group A (80.0 percent) than in group B (31.1 percent). Regarding the survival curve for FZD5 in group B, we did not find any significant association between atrophic endometrium and endometrial adenocarcinoma. We also did not find any significant association regarding beta-catenin expression (P = 1.000). CONCLUSION: FZD5 is downregulated in endometrial adenocarcinoma, in comparison with atrophic endometrium.

CONTEXTO E OBJETIVO: A via Wnt está envolvida na tumorigênese de diversos tipos de tecidos. Por essa razão, propusemo-nos a avaliar a expressão de genes da família Wnt no câncer endometrial tipo I. TIPO DE ESTUDO E LOCAL: Estudo transversal com coleta de materiais do banco de tecidos do Departamento de Patologia da Universidade Federal de São Paulo. MÉTODOS: Amostras endometriais foram obtidas de cirurgias que ocorreram entre 1995 e 2005 no Hospital São Paulo, Universidade Federal de São Paulo. Foram separados dois grupos segundo o tipo de tecido obtido: grupo A, com endométrio atrófico (n = 15); e grupo B, com adenocarcinoma endometrial (n = 45). Comparamos a expressão imunoistoquímica de Wnt 1, Frizzled-1 (FZD1), Wnt 5a, Frizzled-5 (FZD 5) e beta-catenina entre câncer endometrial tipo I e endométrio atrófico. RESULTADOS: Na expressão do Wnt1, FZD1 e Wnt5a, não observamos associação significante entre os grupos. Na expressão do FZD5, encontramos associação significante entre os grupos (P = 0,001). A proporção de positividade do FZD5 foi significantemente maior no grupo A comparado ao grupo B (31,1 por cento). Em relação à curva de sobrevida para o FZD5 no grupo B, não tivemos associação significante entre endométrio atrófico e adenocarcinoma do endométrio. Também não observamos associação significante na expressão da beta-catenina (P = 1,000). CONCLUSÃO: FZD5 é downregulated no adenocarcinoma endometrial quando comparado ao endométrio atrófico.

Análisis de la expresión del receptor del estrógeno en endometrio de ovejas alimentadas con dieta normal y dieta suplementada / Analysis of the expression of estrogen receptors in the endometrium of ewes fed with normal and supplementary diet

Los efectos de la nutrición en reproducción ovina han sido estudiados por numerosos autores. Principalmente, el interés se ha centrado en los efectos de la calidad de la dieta ya que en ovinos sometidos a planos nutricionales bajos se ha observado anormalidades del embrión, fase lutea inadecuada, deficiencia en el aporte de progesterona al útero y falla en los mecanismos que comprenden el reconocimiento materno a la preñez. Existen trabajos sobre la relación alimentación-hormonas sin embargo la información existente sobre los receptores de hormonas esteroidales endometriales, indispensables para que ellas actúen, es muy escasa. El objetivo del presente estudio fue analizar comparativamente la expresión del receptor de estrógenos (RE) en el endometrio de ovejas en ciclo alimentadas con dieta suplementada y dieta normal. La expresión de la proteína receptora y del transcrito se detectó mediante análisis inmunohistoquímico y RT-PCR en tiempo real respectivamente. Los resultados muestran expresión inmunohistoquímica en zonas glandulares y carunculares, destacándose una intensa inmunorreacción en núcleos de células estromales y del epitelio glandular. Se detecto mayor expresión del transcrito del RE en endometrio de ovejas alimentadas con suplemento respecto a las que no recibieron suplemento alimenticio. Se discute el posible uso de esta información para aplicarla en programas de mejoramiento reproductivo en ovinos.

The effects of nutrition on sheep reproduction have been studied by many authors. The interest has focused on the effects of diet quality in sheepin in which low plans n have low plane nutrition can cause abnormalities of the ovum or the embryo, luteal inadequancy and failure of the supply of progesterone to the uterus or failurein the mechanisms involved maternal of pregnancy. There are papers about the relation food-hormones, however, the existing information on endometrial steroid hormone receptors, which are essential for them to act, is very scarce. The aim of this study was to comparatively analyze the expression of estrogen receptor (ER) in endometrial cycle of sheep fed diet supplemented and normal diet. The expression of the receptor protein and the transcript were detected by immunohistochemical analysis and RT real-time PCR, respectively. The results showed immunohistochemical expression of RE in glandular and carunculares areas, especially in an intense inmunorreacción stromal cell nuclei and glandular epithelium. Detected increased expression of transcripts of RE in sheep fed a supplement with respect to not receiving a dietary supplement. We discuss the possible use of this information for application in breeding sheeps programs.

Local immune regulatory effects of Bangdeyun on the endometrium of mice with embryo implantation dysfunction during the implantation time / 华中科技大学学报（医学）（英德文版）

This study examined the effects of Bangdeyun on the expressions of nuclear factor-kappaB (NF-kappaB), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the endometrium of mice with embryo implantation dysfunction (EID) during the implantation time (namely on pregnancy day 5, 6, 7 and 8) and explored the local immune regulatory effects of Bangdeyun. The gestational mice were randomly divided into normal group, model group and Bangdeyun-treated group. EID models of mice were established by using indomethacin. The endometrial expression of NF-kappaB was detected by immunohistochemistry and Western blotting. IFN-gamma and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that in the normal group, NF-kappaB and IFN-gamma were weakly expressed and IL-10 was strongly expressed in the endometrium during the whole implantation period. In the model group, the expressions of NF-kappaB and IFN-gamma were increased on pregnancy day 5, 6 and 7, and IL-10 expression decreased during the whole implantation time when compared with those in the normal group (P0.05). The expressions of NF-kappaB and IFN-gamma were much lower in the Bangdeyun-treated group than those in the model group on pregnancy day 5, 6 and 7 (P<0.01 for all), while the expression of IL-10 was much higher than in the model group during the whole implantation time (P<0.01). It was suggested Bangderun may favor a shift from Th1- to Th2-type immune response, therefore inhibiting the maternal immune rejection, inducing the immune tolerance and improving the fetal implantation.

Characterization of Calreticulin Expression in Mouse Endometrium during Embryo Implantation

Calreticulin (CRT), a Ca2+-binding storage protein and chaperone in the endoplasmic reticulum, modulates cell adhesiveness and integrin-dependent Ca2+ signaling. However, the role of CRT during implantation remains poorly understood. In the present study, we characterized the expression of CRT mRNA and the protein in mouse endometria from pregnancy DI to D7. Real-Time PCR and in situ hybridization results showed that the levels of CRT mRNA in the endometria of pregnant mice were significantly higher than those of non-pregnant mice (P<0.05), and increased gradually from pregnancy DI to D4, reaching the máximum level on D4, followed by a plateau from D4 to D7. Using immunofluorescence histochemistry and western blot, changes of CRT expression in the endometria of pregnant mice were consistent with the expression of CRT mRNA. Furthermore, antisense CRT oligodeoxynucleotide was injected into the uterus horns of pregnant mice (D3) to investígate its effect on embryo implantation. The result showed that the number of implanted embryos markedly decreased in the side of uterine horns receiving antisense CRT oligodeoxynucleotide(í><0.05). These findings suggest that CRT may play an important role in embryo implantation in mice.