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.

Can endometrial arylsulfatase A activity predict the onset of endometrial polyps over the years? / A atividade da arilsulfatase endometrial A pode prever a aparição de pólipos endometriais ao longo dos anos?

PURPOSE: To assess if arylsulfatase A activity (ASA) and sulfatide (SL) concentration in the human endometrium can be predictive of the development of endometrial polyps over the years, since ASA activity reflects the endometrial sensitivity to hormones. METHODS: ASA activity and SL concentration were determined by biochemical procedures on endometrial samples collected between 1990 and 1994 in non-menopausal women. These women underwent a new endometrial sampling following the clinical indication some years after the first endometrial sampling. The histological assessment of the second endometrial specimens found four patients with normal endometrial pattern and 10 patients with one or more endometrial polyps. ASA activity/years elapsed and SL concentration/years elapsed were compared using two tailed Mann-Whitney test for unpaired data between patients with normal pattern and patients with endometrial polyps. RESULTS: Median ASA activities were 2.62 (normal pattern) versus 1.85 (endometrial polyps) nmol hydrolized substrate/min. Median activity/years elapsed is higher in patients with second endometrial sample presenting normal pattern (p=0.006) and median SL concentration/years elapsed does not differ significantly among groups, even if median SL concentration seems to be higher in patients who subsequently developed polyps (1031 µg/g of fresh tissue versus 341,5 µg/g of fresh tissue). CONCLUSIONS: ASA activity can predict the onset of endometrial polyps over the years.

OBJETIVO: Avaliar se a atividade da arilsulfatase A (ASA) e a concentração de sulfatida (SL) no endométrio humano pode ser preditivo em relação ao desenvolvimento de pólipos endometriais ao longo dos anos, posto que atividade da ASA reflete a sensibilidade do endométrio aos hormônios. MÉTODOS: A atividade da ASA, assim como a concentração de SL, foi determinada por meio de procedimentos bioquímicos em amostras de endométrio coletadas entre 1990 e 1994, em mulheres que não se encontravam na menopausa. Essas mulheres foram submetidas a uma nova amostragem endometrial após indicação clínica alguns anos depois da primeira amostragem endometrial. A avaliação histológica dos segundos espécimes endometriais permitiu identificar quatro pacientes com padrão endometrial normal e 10 com um ou mais pólipos endometriais. A atividade da ASA/anos depois e a concentração de SL/anos depois foram comparadas, utilizando o teste bilateral U de Mann-Whitney para dados não pareados entre as pacientes com padrão normal e as pacientes com pólipos endometriais. RESULTADOS: A ativitade da ASA foi 2,62 (padrão normal) em comparação com 1,85 (endometrial pólipos) de substrato hidrolisado/min. A atividade da ASA/anos depois é maior em pacientes com segunda amostra endometrial a apresentarem um padrão normal (p=0,006), e a concentração mediana de SL/anos depois não difere de forma significativa entre os grupos, apesar de a concentração mediana de SL parecer maior em pacientes que posteriormente desenvolveram pólipos (1031 µg/g de tecido fresco em comparação com 341,5 µg/g de tecido fresco). CONCLUSÕES: A atividade da ASA pode prever a aparição de pólipos endometriais ao longo dos anos.

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...

Histochemical study of early embryo implantation in rats / Estudio histoquímico de la implantación temprana en ratas

Embryo implantation is the process that results in attachment of the conceptus to the uterine wall. In this histochemical study, we investigated the early stage of embryo implantation in rats by morphological analysis and by the detection of total proteins and glycosaminoglycans using hematoxylin-eosin, toluidine blue at pH 4.0 (TB), and Xylidine Ponceau at pH 2.5 (XP). In non-pregnant females, the uterine layers could be clearly distinguished and showed the normal histology of the organ. In pregnant females, an increase in the number of cells and a reduction in the interstitial space were observed in the endometrium close to the implantation sites. The blastocyst was partially inserted in the endometrium, with the observation of the inner cells mass around the blastocyst cavity surrounded by trophoblastic cells. TB staining revealed mild metachromatic basophilia, which was more evident in the endometrial stroma around the implantation site. Histochemical staining with XP was also more intense in the stroma close to the site of implantation. On the other hand, histochemical staining with either TB or XP was more discrete at sites distant from the conceptus. This study demonstrated changes in the endometrial stroma in areas adjacent to the site of embryo implantation, with variations in glycosaminoglycans and proteins as demonstrated by the detection of anionic and cationic radicals, respectively.

La implantación embrionaria es el proceso que resulta en la unión del embrión a la pared uterina. En este estudio histoquímico, se determinó la fase inicial de implantación del embrión en ratas mediante el análisis morfológico y por la detección de proteínas totales y glicosaminoglicanos con hematoxilina-eosina, azul de toluidina a pH 4,0 (TB) y xilidina Ponceau a un pH de 2,5 (XP). En las hembras no preñadadas, las capas del útero pueden ser claramente distinguidas y mostraron la histología normal del órgano. En las hembras preñadas, se observa un incremento en el número de células y una reducción en el espacio intersticial del endometrio para cerrar los sitios de implantación. El blastocisto se implanta parcialmente en el endometrio, con presencia de masa de células internas en torno a la cavidad del blastocisto, rodeado por las células trofoblásticas. La tinción TB reveló leve basofilia metacromática, lo cual fue más evidente en el estroma endometrial alrededor del sitio de implantación. Tinción histoquímica con XP también fue más intensa próximo del estroma en el sitio de implantación. Por otro lado, la tinción histoquímica, ya sea con la tuberculosis o XP fue más leve en los lugares distantes del embrión. Este estudio demostró cambios en el estroma del endometrio en las zonas adyacentes al sitio de la implantación del embrión, con variaciones en glucosaminoglicanos y proteínas, como lo demuestra la detección de radicales aniónicos y catiónicos, respectivamente.

Immunohistochemical expression of oestrogen and progesterone receptors during experimental acute and chronic murine Schistosomiasis mansoni / Expressão imunohistoquímica de receptores para estrogênio e progesterona nas fases aguda e crônica da esquistossomose mansônica experimental em camundongos

INTRODUCTION: The responsibility of Schistosoma mansoni in female infertility is still controversial. This study was conducted to evaluate the effect of acute and chronic schistosomiasis mansoni infection on the endometrium using immunohistochemical analysis of uterine hormone receptor expression. METHODS: Twenty-four nonpregnant swiss albino mice were divided into three groups: control, noninfected; acute; and chronic Schistosoma mansoni infection. Histological sections of uterine specimens were examined by light microscope with an image analyzing system to detect structural histological, estrogen receptor (ER) and progesterone receptor (PR) expression in the endometrium. RESULTS: No secretory phase was detected in the endometrium in acute and chronic Schistosoma infection. Hormone receptor expression (ER and PR) showed statistically significant differences among the groups (p< 0.05), with significant low ER hormone expression in chronic infection, compared to control proliferative, control secretory and acute infection cases, and statistically significant high PR expression in both acute and chronic infection cases compared to the control secretory cases (p< 0.05). CONCLUSIONS: Schistosomiasis mansoni seems to have an important impact on the hormone expression of affected women. Further studies to explore the mechanism of such changes are recommended.

INTRODUÇÃO: A responsabilidade do Schistosoma mansoni em esterilidade feminina é ainda controversa. Este estudo é conduzido para avaliar o efeito da esquistossomose mansoni aguda e crônica no endométrio usando análise de imuno-histoquímíca da expressão de receptor hormonal uterina. MÉTODOS: Vinte e quatro camundongos fêmeas albinas suíças não grávidas foram divididas em 3 grupos (controle não-infectado, grupos agudos e crônicos infeccionados com Schistosoma mansoni). As seções histológicas de espécimes uterinos foram examinadas por microscópio leve com imagem, analisando sistema para detectar no endométrio expressões histológicas estruturais, receptor de estrogênio (ER) e receptor de progesterona (PR). RESULTADOS: Nenhuma fase secretora foi detectada no endométrio com infecção aguda e crônica de Schistosoma. A expressão hormonal de receptor (ER e PR) mostrou diferenças estatisticamente significantes entre grupos diferentes (p<0,05) com baixa significativa hormonal de ER com infecção crônica (comparado com controle proliferativo, controle secretório e casos agudos de infecção) e alta expressão de receptor de PR estatisticamente significativa em casos tanto agudos e crônicos de infecção como comparado com os casos de controle secretório (P <0,05). CONCLUSÕES: A esquistossomose mansoni parece ter um maior impacto em expressão hormonal das mulheres afetadas. Mais estudos para explorar o mecanismo de tais mudanças são recomendados.

Expression of cyclin D1 in endometrial hyperplasia and endometrial carcinoma.

This study investigates the role of cyclin D1 in 30 uterine surgical resection and endometrial biopsy specimens from 30 patients with simple hyperplasia (10 cases), complex hyperplasia (6 cases) and endometrial carcinoma (14 cases). Cyclin D1 immunohistochemistry was performed on 2-4 mm thick paraffin sections using labelled streptavidin biotin kit. Cyclin D1 expression was present in 2/6 (33%) cases of complex hyperplasia, 7/14 (50%) cases of endometrial carcinoma and none in simple hyperplasia. Difference in cyclin D1 immunopositivity in simple hyperplasia and endometrial carcinoma was statistically significant (p = 0.018) but the difference in cyclin D1 immunopositivity between complex hyperplasia and endometrial carcinoma was not statistically significant. Our study suggests that cyclin D1 over-expression may be an early event in endometrial carcinogensis. Since there was no difference in extent and intensity of cyclin D1 expression between complex hyperplasia and endometrial carcinoma, it appears that deregulation is maximal in complex hyperplasia.

Immunohistochemical localization of macrophage CD68+, HLA-DR+, L1+ and CD44+ subsets in uterine endometrium during different phases of menstrual cycle.

Different tissue macrophage subsets were immunohistochemically examined in normal endometrial samples collected from proliferative (n=4), peri-ovulatory (n=6) and secretory (n=8) phases of menstrual cycles in women. The different macrophage subsets, namely CD68 (pan macrophage marker), CD44 (transmembrane adhesion molecule), HLA-DR (transmembrane heterodimeric protein involved in antigen presentation) and L1 (calprotectin)-positive cells, as well as, CD45 (common leucocytic antigen)-positive cells were examined on the basis of immunohistochemical staining, and areas of immunoprecipitation were analyzed morphometrically using computer-assisted video imaging system. The stage-specific distribution of receptors for estrogen (ER) and progesterone (PR) in endometrial cells were examined and morphometrically analyzed. There was an increase in the number of CD45+ cells (P < 0.01) and CD68+ cells (P < 0.05) in secretory phase endometrium compared with proliferative and peri-ovulatory phases. There was no remarkable cycle dependent pattern in HLA-DR+ and L1+ cells. However, there was an increase in CD44 immunopositive area in peri-ovulatory (P < 0.05) and in secretory (P < 0.01) phases of endometrium compared with proliferative phase endometrium. A higher (P < 0.01) degree of immunopositivity for ER was observed during peri-ovulatory phase, and for PR, during peri-ovulatory (P < 0.05) and secretory (P < 0.01) phases compared with proliferative phase of cycle. Positive correlations between areas occupied by (i) CD68+ cells and PR (P < 0.01), (ii) HLA-DR+ and L1+ cells (P < 0.05), (iii) CD45+ and CD68+ cells (P < 0.01), (iv) CD45+ and L1+ cells (P < 0.05), and (v) PR and L1+ cells (P < 0.05) were obtained. It appears that the recruitment of different macrophage subsets in human endometrium involves a complex set of endocrine and paracrine factors.

Histological and histochemical study of endometrium in dysfunctional uterine haemorrhage.

Menstrual disorder is one of the most frequently encountered and perplexing conditions in adult women. Dysfunctional uterine haemorrhage (DUH) includes scanty, excessive or irregular bleeding, the primary cause of which is yet to become clear. A total of 100 cases of DUH were studied by histological and histochemical evaluation of endomertium with an object to find out the pathophysiology of DUH. Twenty cases of the similar age group without DUH acted as control. Irregular excessive bleeding was found to be the highest in multiparous women in their 4th decade of life. The material from endometrium was studied by haematoxylin and eosin statin, alcian blue, periodic acid Schiff's reaction and alkaline phosphatase activity. Histological examination of the study group revealed normal endometrium in 66% of cases and abnormal in 34% cases while histochemical behaviour was normal in 54% cases and abnormal in 46% cases. Hormonal imbalance is the chief factor in the pathogenesis of DUH and this abnormal hormonal status can better be revealed by a combined study of histological and histochemical evaluation of endometrium.

Effect of vaginally administered fumagillin on immunohistochemical distribution of leukemia inhibitory factor, interleukin 6, transforming growth factor beta and vascular endothelial growth factor in implantation stage endometrium of the rhesus monkey.

Intravaginal administration of an anti-angiogenic agent, fumagillin, during blastocyst implantation inhibits pregnancy establishment in a dose-related manner in the rhesus monkey. In the present study, mated female rhesus monkeys were vaginally inserted with tampons containing vehicle (group 1; n = 5) and test agent (fumagillin, 4 mg/animal; group 2; n = 6) on cycle day 20, and endometrial tissue samples were collected on cycle day 24 from all monkeys and processed for histological examination and immunohistochemical localization for LIF, IL-6, TGF-beta and VEGF. Concentrations of estradiol-17 beta, progesterone and chorionic gonadotrophin in peripheral circulation were determined. From the serum profiles of the hormones, 2 monkeys in group 1, and 1 monkey in group 2 appeared pregnant. However, endometrial morphology revealed histological evidence of pregnancy in 3 out of 6 fumagillin-treated animals. Histometric analysis of immunohistochemical staining in epithelial, stromal and vascular compartments revealed that per cent areas occupied by immunoprecipitate for the cytokines studies did not change in epithelial and stromal compartments, except that for TGF-beta which was higher (P < 0.05) in epithelial compartment in group 2. No change was observed in immunoprecipitation areas for IL-6 in epithelial, stromal and vascular compartments. On the other hand, changes (P < 0.05) for LIF, TGF-beta and VEGF were evident in the vascular compartment. It is possible that disparate responses observed in glandular, stromal and vascular compartments in implantation stage endometrium following fumagillin treatment actually caused from associated decline in progesterone concentration in peripheral circulation. It is also possible that fumagillin, an angiostatic agent, affects the synthesis and secretion of cytokines primarily in the vascular compartment of implantation stage endometrium, and thereby manifests differential responses in epithelial, stromal and vascular compartments.

Immunohistological localisation of vascular endothelial growth factor in human endometrium.

Several polypeptide growth factors regulate epithelial and stromal development in endometrium under the influence of estrogen and progesterone, and thereby regulate growth and differentiation of endometrium during menstrual cycle. However, little is known about the angiogenic growth factors that may affect endometrial vasculature throughout each menstrual cycle. Vascular endothelial growth factor (VEGF) is suggestively an important angiogenic growth factor in the female reproductive tract. The aim of the present study was to immunolocalize and assess semi-quantitatively VEGF immunostaining in cells of proliferative phase (n = 3), secretory phase (n = 6) and hyperplastic (n = 6) human endometrial samples. VEGF concentrations were significantly higher in glandular (P < 0.001) and stromal (P < 0.01) compartments of proliferative stage endometrium compared with those in secretory stage and hyperplastic endometrial samples, with no difference in the scores for glandular and stromal compartments between secretory stage and hyperplastic endometrial samples. Generally, glandular expression of VEGF was higher as compared to stromal compartment. Thus, it appears that endometrial VEGF expression and concentration are enhanced by estrogen, and may be correlated with neovascularization and increased vascular permeability during late proliferative period. Additionally, there was no enhancement in VEGF expression in hyperplastic glands, suggesting that regulation of glandular growth and that of angiogenesis in human endometrium operate through different mechanisms.

Expresion diferencial de los carbohidratos de mucina en endometrios humanos / Differential expression of mucin carbohydrates in human endometriuns

La aplicación de Lectinas para la identificación de oligosacáridos constituyentes de las Mucinas presentes en la superficie celular, es una herramienta técnica muy útil, debido a que, probablemente estas sustancias estén involucrados en procesos tales como invasión y metástasis. En este trabajo, nosotros estudiamos tejido endometrial normal com entidades benignas y malignas, para investigar la presencia de Galactosa beta 1-3 N Acetilgalactosamina (Galbeta 1-3GalNacalpha) y de Galactosa beta 1-3 N Acetilgalactosamina(Galbeta 1-3GalNa alpha y beta) empleando dos Lectinas: Agaricus bisporus (ABL) y Arachis hipogea (PNA) respectivamente. Lo controles fueron realizados com baños de galactosa para PNA y com mucina porcina de estómago de cerdo para ABL. El uso de estas dos Lectinas, permitió encontrar diferencias en los patrones de unión, ya que si bien ambas se unen a los mismos oligosacáridos, ABL realiza la unión en presencia de Acido Siálico mientras que PNA no. Se observaron significativas diferencias en los patrones de unión de ambas lectinas en tejidos, com entidades benignas, malignas y tejido normal. En este último, la marcación fue simpre continua com ambas Lectinas, mientras que fue irregular en el carcinoma.

Immunoreactivity with S100 protein as an indicator of pregnancy.

The usefulness as a pregnancy marker, of immunoreactivity with S100 protein antiserum in endometrial curettings and fallopian tubes during pregnancy was assessed. Twenty six placental tissues of various gestational age, two hydatidiform moles and four fallopian tubes removed for ectopic pregnancy were stained with S100 protein polyclonal antiserum by immunoperoxidase technique. Strong immunostaining was found in glands within the decidua and epithelial cells of fallopian tubes during early pregnancy. However, no S100 protein could be demonstrated in the endometrium in various phases, endometrial carcinomas, decidual glands beyond 12 wk of gestation and normal fallopian tubes. The results indicate a relationship between S100 protein and early pregnancy. Positive immunoreactivity with S100 protein antiserum in the decidual glands and fallopian tube epithelium may help in confirmation of doubtful cases of pregnancy.

Effect of medrogestone on endometrial histology and histochemistry in patients with luteal phase defects

Fifty infertile patients with luteal phase defects were the subject of this study. Progestrone in oil, [12.5 and 25 mg] and medrogestone, [5 and 10 mg] were administered daily, starting 3 days after the probable day of ovulation for 2 consecutive cycles. Endometrial biopsies were subjected to histlogical and histochemical examinations. Secretory endometrial patterns that were in phase were observed in all cases after progestrone therapy. Also, the same patterns were observed in 90% and 70% of case after Medrogestone in 5 mg and 10 mg dosage respectively. Undatable endometrial patterns with early glandular exhaustion and sometimes pseudodecidual reaction were observed with medrogestone therapy. These were detected in 10% and 30% of cases with the 5 mg and 10 mg dosage respectively. Also weak activity of the glandular succinic dehydrogenase was observed on histochemical examination of the undatable endometrial patterns. No Histologic or histochemical differences were noted, when both drugs produce secretory endometrium that was in-phase or 1 day out of phase of the cycle day. It is concluded that oral medrogestone is liable to produce early glandular exhaustion when given to patients with inadequte luteal phase. Such asynchronous maturation may interfer with blastocyst nidation. It can also be concluded that progestrone therapy, albeit inconvinent when administered intramuscularly, is the drug of choice in the treatment of luteal phase defects