Neste estudo caracterizamos a expressão de Mif nas células trofoblásticas (CT), placentárias e decidua (D) na gestação em camundongos.A imunolocalização foi realizada nos dias de gestação(dg) 7,5, 10,5, 13,5 e 17,5.Com cones ectoplacentários e placentas fetais (PL) realizou-se ensaios de Western blotting e qRT-PCR nos mesmos dg.D foram submetidas a qPCR.Aos 7,5 dg as CT gigantes e D apresentaram imunomarcação.Dos 10,5 ao 17,5 dg o Mif concentrou-se nas CTG e no espongiotrofoblasto.Na D, a imunomarcação foi menor que aos 7,5 dg.A expressão protéica na PL aumentou do 7,5 dg para o 10,5 dg (p=0,005) e para o 13,5 dg (p=0.03).A maior expressão gênica foi em 10,5 dg e diferente em 13,5 dg (p=0,048) e 17,5 dg (p=0,009).Na D, a maior expressão gênica foi em 7,5 dg e diferente dos 10,5 (0,012) e 13,5 (0,032) dg.O aumento da expressão de Mif na PL coincide com a organização em quatro camadas e com o início da circulação fetal.Esta distribuição temporal e tecido-específica sugere o MIF como modulador no início da placentação ou na sua adaptação ao ambiente uterino / The goal of this study was to characterize Mif expression by trophoblast (TC),fetal placenta (FP) and decidua (D) during mouse pregnancy.Mif was immunolocalized at TC and D on gestation days (gd) 7.5, 10.5, 13.5 and 17.5.Ectoplacental cones (EC) and FP were used for Western blotting and qPCR.D were also used for qRT-PCR.On gd 7.5,DC and TC giant (TGC) showed strong reactivity.On gds 10.517.5,were concentrated in TGC and spongiotrophoblast cells. D reactivity was weaker than 7.5 gd.Protein expression at FP increased from gd 7.5 to 10.5 (p=0.005) and to 13.5 (p=0.03).Higher mRNA expression was found on gd 10.5 and was different from gds 13.5 (p=0.048) and 17.5 (p=0.009).At D on gd 7.5 was greater than those on gds 10.5 (0,012) and 13.5 (0,032).The up-regulation of Mif coincides with the stage that placenta assumes its four-layered organization and the fetal blood circulation begins,This temporal tissue-specific distribution and expression data suggests that Mif may play a modulator role in the onset of placentation or in it adaptation to the uterine environment

O 17β-E2 estimula a expressão de receptores endometriais, ER e OXTR. A ativação de OXTR induz a ativação da cadeia de síntese de PGF2α. A hipótese do presente estudo é que as enzimas de síntese de PGF2α são reguladas pelo 17β-E2. Objetivou-se determinar os efeitos do 17β-E2 na expressão gênica e proteica, assim como na localização de proteínas envolvidas na síntese de PGF2α. Vacas Nelore multíparas, solteiras e cíclicas foram sincronizadas por aplicação de BE e inserção de dispositivo de P4. Após 8 dias realizou-se a remoção do dispositivo de P4 e aplicação de PGF2α, seguido por 4 dias de observação de estro (D0=dia do estro). Entre D14 e D27 foram realizadas avaliações diárias da área do CL (cm2), fluxo sanguíneo (%) e concentração plasmática de progesterona (P4). No D15 as vacas foram divididas em três grupos: Controle (C; não tratado;N= 10), Placebo (P; 6mL de etanol 50%; IV; N= 21) e Estradiol (E; 3mg 17β-E2; 6mL de etanol 50%; IV;N=21). Subsequente aos tratamentos, biópsias uterinas foram coletadas nos tempos 0h (C; N=10); 4h (E4h, N=11 e P4h; N=10) ou 7h (E7h, N=10 e P7h, N=11). Amostras de sangue foram obtidas nos tempos 0h a 7h, para mensuração das concentrações PGFM no D15. O grupo E apresentou acentuada diminuição da área do CL, fluxo sanguíneo e concentração de P4 (P<0,05), comparado ao grupo P. Comparado ao grupo P, as vacas do grupo E anteciparam o dia da luteólise funcional e estrutural em 2 e 3 dias, respectivamente. O grupo E apresentou maior concentração de PGFM nos tempos 4h, 6h e 7h (P<0,05), comparado ao grupo P. A quantificação dos transcritos realizada por qPCR (N=6/grupo). Na hora 4, a abundância dos genes ESR1, ESR2, PRKCα, PRKCβ, PLA2G4, AKR1B1 e AKR1C4 foi menor nas amostras E4h, enquanto OXTR foi maior nas mesmas amostras comparando-se com as amostras P4h (P<0,05). A expressão gênica de PTGS2 não diferiu entre os grupos E4h e P4h (P>0,05). Na hora 7, as amostras E7h também apresentaram menor abundância de ESR1, PRKCα, PRKCβ, AKR1B1 e AKR1C4 (P<0,05) e houve tendência para menor expressão de ESR2, comparado às amostras P7h. Contudo, não houve diferença na abundância de OXTR, PLA2G4 e PTGS2 entre as amostras E7h e P7h (P>0,05). A abundância da enzima PKCα analisada por Western Blotting (N=3/grupo) foi diminuída tanto nas amostras E4h como nas E7h, em relação às amostras P4h e P7h, respectivamente. Na avaliação por imunohistoquímica (N=5/grupo), o grupo E4h apresentou maior imunomarcação de PGR no epitélio glandular (P< 0,05) e houve tendência para maior imunomarcação de PKCϒ no epitélio luminal, comparado ao grupo P4h (P=0,08). Houve tendência para menor imunomarcação de ERα no epitélio glandular do grupo E4h comparado ao grupo E7h (P=0,1). Concluí-se que a aplicação do 17β-E2 levou a redução da maioria dos transcritos das moléculas de síntese de PGF2α, assim como da abundância de PKCα. O possível mecanismo para a estimulação de PGFM por 17β-E2 pode incluir o aumento da ativação de enzimas que participam na cascata de síntese de PGF2α. / 17β-E2 stimulates the expression of endometrial receptors, ER and OXTR. Activation of OXTR induces the activation of the synthesis of PGF2α pathway. The central hypothesis is that the enzymes involved in PGF2 synthesis are reguleted by 17β-E2. The objective of this study was to determine the effects of 17β-E2 on gene and protein expression and localization of the enzymes involved in PGF2α synthesis. Multiparous, non-lactating and cyclic Nelore cows were synchronized by BE application and P4 device insertion. After 8 days the P4 device was removed and a single dose of PGF2α applied, followed by 4 days of estrus detection (D0 = day of estrus). Daily measurements of CL area (cm2), blood flow (%), and plasma progesterone concentration (P4) were performed between D14 and D27. On D15 cows were divided into three groups: Control (C, untreated, N = 10), Placebo (P; 6mL of ethanol 50%, IV; N = 21) and Estradiol (E; 3mg 17β-E2; Ethanol 50%, IR: N = 21). After the treatments administration, uterine biopsies were collected at times 0h (C; N = 10); 4h (E4h, N = 11 and P4h, N = 10) or 7h (E7h, N = 10 and P7h, N = 11). Blood samples were obtained from time 0h to 7h for the measurement of the PGFM concentrations on D15. Group E showed a marked decrease in CL area, blood flow, and P4 concentration (P <0.05) compared to group P. Also, when compared to group P, cows from group E anticipated the day of functional and structural luteolysis in 2 and 3 days, respectively. Group E presented higher concentration of PGFM at 4h, 6h and 7h (P <0.05), compared to group P. The transcripts abundance was performed by qPCR (N = 6 / group). The transcripts abundance of ESR1, ESR2, PRKCα, PRKCβ, PLA2G4, AKR1B1, and AKR1C4 genes was lower in the E4h samples, while OXTR was higher in the same samples compared to the P4h (P <0.05) samples in the time 4h. The gene expression of PTGS2 did not differ between groups E4h and P4h (P> 0.05). At time 7h, samples E7h also had lower abundance of ESR1, PRKCα, PRKCβ, AKR1B1 and AKR1C4 (P <0.05) and there was a tendency for lower ESR2 expression, compared to samples P7h. Nevertheless, there was no difference in the abundance of OXTR, PLA2G4, and PTGS2 between samples E7h and P7h (P> 0.05). The abundance of the PKCα enzyme analyzed by Western Blotting (N = 3 / group) was decreased in both the E4h and E7h samples, relative to the samples P4h and P7h, respectively. In the evaluation by immunohistochemistry (N = 5 / group), the E4h group presented greater PGR immunostaining in the glandular epithelium (P <0.05) and there was a tendency for a greater immunostaining of PKCϒ in the luminal epithelium, compared to the P4h group (P = 0,08). There was a tendency for lower ERα immunostaining in the glandular epithelium of the E4h group compared to the E7h group (P = 0.1). It was concluded that the application of 17β-E2 led to the reduction of most of the transcripts of the PGF2α synthesis molecules, as well as the abundance of PKCα. The possible mechanism for stimulation of PGFM by 17β-E2 may include increased activation of enzymes that participate in the cascade of PGF2α synthesis.

A matriz extracelular do endométrio (ECM) é constituída por moléculas secretadas que compõem o microambiente celular e são ativadas ou suprimidas principalmente pelos hormônios esteróides ovarianos, estradiol (E2) e progesterona (P4) durante o ciclo estral. A identificação de genes envolvidos no remodelamento e receptividade pode levar à descoberta de importantes processos biológicos ligados ao sucesso gestacional. Os objetivos foram: 1. identificar a relação de diferentes tamanhos de folículos pré-ovulatórios (FPO) e corpo lúteo (CL) e de seus respectivos hormônios E2 e P4 com a expressão endometrial de genes associados com o remodelamento da ECM durante o período de pré-implantação; e 2. analisar a relação entre a expressão gênica de determinados componentes da ECM avaliada no dia 6 após inseminação artificial (IA) com sucesso gestacional. Para tal, dois experimentos foram realizados. No experimento 1, estudo 1 e estudo 2, 42 e 74 vacas Nelore (Bos indicus) adultas, respectivamente foram sincronizadas obtendo-se ao final dois grupos com distintos tamanhos FPO e CL consequentemente, distintas concentrações de E2 no proestro e P4 no diestro. Os grupos foram: Folículo Grande-CL Grande (FG-CLG; estudo 1, n=20; estudo 2, n=35) e Folículo Pequeno-CL Pequeno (FP-CLP; estudo 1, n=22; estudo 2, n=39). Amostras de tecido endometrial foram coletadas por biópsia no D0 (estro) e pós-mortem no D4 (estudo 1) e D7 (estudo 2). Concentrações de E2 e P4 foram mensuradas por radioimunoensaio (RIA) obtendo- se menores concentrações no grupo FP-CLP. No experimento 2, vacas adultas, Nelore (Bos indicus; n=33) foram sincronizadas utilizando um protocolo a base de prostaglandina F 2α (PGF2α) e observação de estro. As vacas foram inseminadas artificialmente (IA) e seis dias após, uma biópsia endometrial coletada. O diagnóstico de gestação foi realizado após 30 dias por meio de ultrasonografia (US) e então as vacas foram divididas em grupo Prenhe e Não- Prenhe (P e NP) para análise retrospectiva. Abundância de transcritos foi avaliada por sequenciamento (RNAseq) assim como qPCR em amostras de ambos experimentos. Realizaram-se também exames histológicos em amostras do D4 e D7 (estudo 2) para avaliação de colágeno total assim como espessura de fibras colágenas. Resultados determinaram uma maior abundância de transcritos relacionados ao remodelamento de MEC, em destaque TGFβ, MMPs, TIMPs e colágenos em vacas pertencentes aos grupos NP e FP-CLP. O mesmo foi observado para abundância de colágeno. No entanto, não observou-se diferença na relação entre fibras grossas e finas entre os tratamentos. Análises de correlação e regressão indicaram que folículos pré-ovulatórios de maior tamanho geram CL maiores e assim maiores concentrações de P4, a qual está negativamente associada à abundância de colágenos. Assim, de acordo com resultados aqui descritos, assume-se que a alteração da homeostase da MEC devido ao incremento na abundância de colágeno pode ser prejudicial à gestação em bovinos. / The endometrial extracellular matrix (ECM) é build up of secretory molecules that make up the cellular microenvironment and suffer activation or suppression mainly by the ovarian steroid hormones, estradiol (E2) and progesterone (P4) during the estrous cycle. The identification of genes involved in endometrial remodeling and receptivity may reveal important biological processes linked to gestational success. The objectives were: 1. identify the relationship among preovulatory follicle (POF) size and corpus luteum (CL) and its respective hormones, E2 and P4 on the endometrial expression of genes related to extracellular matrix remodeling during the pre-implantation period; and 2. analyze the relationship between endometrial ECM gene expression evaluated on day 6 post artificial insemination (AI) with pregnancy outcome. For such, two experiments were carried on. On experiment 1, study 1 and study 2, 42 and 74, respectively, adult Nelore (Bos indicus) cows were synchronized aiming to manipulate the peri-ovulatory endocrine environment, obtaining at the end of the protocol, two groups with distinct pre-ovulatory follicle (POF) and corpus luteum (CL) sizes, leading to groups with distinct E2 and P4 concentrations. The groups were: Large Follicle/CL (LF/CL; study 1, n=20, study 2, n=35) and Small Follicle/Cl (SF/CL; study 1, n=22; study 2, n=39). Endometrial samples were collected by biopsy on D0 (Estrus) and post-mortem on D4 and on study 2 post-mortem on D7. P4 and E2 concentrations were measured by RIA with a significative difference between the groups, being lower hormonal concentrations in the SF- SCL group and higher concentrations in the LF-LCL group . In experiment 2, adult Nelore (Bos indicus) cows (n=33) were synchronized using a prostaglandin 2α (PGF2α) and heat detection based protocol. The cows were AI and six days after an endometrial biopsy was collected. Pregnancy diagnosis was performed on day 30 by ultrasound (US) examination and cows were divided into pregnant and non-pregnant (P vs. NP) groups for a retrospective analysis. Histology was performed on D4 and D7 samples for total collagen abundance as well as fiber thickness. Correlation and regression analysis indicate that larger preovulatory follicles as well as higher P4 concentrations have a negative effect on collagen content. RNA- Seq analysis and confirmation by qPRC was performed on selected samples from experiment 1, study 2 and experiment 2. Comparison of mRNA levels of ECM components samples revealed higher levels of transcripts envolved in ECM remodeling, highlighting TGFβ MMPs, TIMPs and collagens in NP cows when compared with P cows as well as in the SF- SCL compared to the LF-LCL group. The same was observed for collagen abundance. However, there was no difference between thin and thick collagen fibers between treatments. Correlation and regression analysis indicate that larger POF lead to larger CL and hence, higher P4 concentrations, which has a negative effects on collagen abundance. Therefore, according to the results presented here, we can imply that an alteration in ECM homeostasis due to increased collagen abundance may be harmful to pregnancy in cows.

INTRODUÇÃO: O tratamento estrogênico isolado e contínuo por seis meses é uma opção no tratamento de sintomas climatéricos. A monitorização endometrial deve ser realizada rotineiramente; nela, a ultra-sonografia (US) e a biópsia uterina têm papel importante. A US e a Dopplervelocimetria também avaliam as mudanças circulatórias uterinas. OBJETIVO: Estudar o comportamento da vascularização uterina e do endométrio em mulheres na pós-menopausa tratadas com estrógeno contínuo por seis meses, seguido de progestógeno isolado por 14 dias, e estabelecer suas relações com a proliferação endometrial. MÉTODO: Estudo clínico, prospectivo e controlado, onde quarenta mulheres na pós-menopausa, sem contraindicações para tratamento hormonal (TH). Foram divididas em dois grupos: Estrógeno e Controle. As do Grupo Estrógeno (GE), n= 24, receberam 50 mcg de estradiol-17 beta (E2) transdérmico, duas vezes por semana, durante seis meses. As mulheres do Grupo Controle (GC), n=16, não receberam TH. Todas realizaram FSH, E2 e glicemia de jejum; US transvaginal; Dopplervelocimetria das artérias uterinas, miometriais e endometriais e biópsia aspirativa de endométrio. O GE repetiu os mesmos exames, com exceção de FSH, E2 e glicemia, no terceiro e no sexto mês de tratamento. No GC, a biópsia do endométrio foi repetida apenas no sexto mês de tratamento. As mulheres do GE utilizaram, ao fim de seis meses, 10 mg de acetato de medroxiprogesterona por dia, durante 14 dias. RESULTADOS: No GE, a resistência vascular das artérias uterinas diminuiu no terceiro e no sexto mês de tratamento. O fluxo miometrial das artérias arqueadas aumentou significantemente no sexto mês de tratamento. O aumento da espessura do endométrio ocorreu de forma significante no terceiro mês. No GE houve hiperplasia endometrial simples e sem atipias em 20,8 % das mulheres. No GE, comparando as mulheres que tiveram proliferação com aquelas que mantiveram a atrofia endometrial, observou-se que, no sexto mês de tratamento, o grupo que apresentou proliferação teve diminuição significante da resistência vascular da artéria uterina esquerda, enquanto no grupo que manteve a atrofia, a resistência vascular aumentou na artéria uterina direita. No GC não ocorreu variação da resistência vascular das artérias uterinas bilaterais; o fluxo miometrial das artérias arqueadas não se modificou e não houve proliferação endometrial. CONCLUSÃO: A terapia estrogênica isolada por seis meses diminuiu a resistência vascular das artérias uterinas bilateralmente. A proliferação endometrial precedeu o aumento de vascularização miometrial. Houve associação entre a proliferação endometrial e a diminuição da resistência vascular na artéria uterina esquerda, no final do sexto mês de tratamento estrogênico / INTRODUCTION: Isolated continuous estrogen therapy for 6 months is an option to manage climacteric symptoms. Endometrial monitoring should be performed as a routine, in which ultrasound and uterine biopsy have an important role. Ultrasound with Dopplervelocimetry also assesses uterine circulatory changes. OBJECTIVE: To study the uterine circulatory changes of women in continuous estrogen therapy for 6 months using Doppler velocimetry and to define correlations with endometrial proliferation. METHOD: Clinical prospective controlled study. Forty menopause women were studied, without contraindications to hormone therapy (HT). They were divided into 2 groups: Estrogen and Control. In the Estrogen Group (EG) n = 24, they were treated with transdermal 50mcg estradiol-17 beta (E2), changed twice a week for 6 months. Women in the Control Group (CG) n=16, were not treated with hormones. They all underwent FSH, E2, fast glucose, transvaginal ultrasound , uterine, myometrial and endometrial artery Dopplervelocimetry and aspiration biopsy of endometrium. The EG repeated the same procedures in months 3 and 6 of treatment. In CG, endometrial biopsy was repeated only in the 6th month of treatment. At the end of treatment, EG women received 10 mg of medroxyprogesterone acetate per day for 14 days. RESULTS: In EG, vascular resistance of uterine arteries reduced in the 3rd and 6th months of treatment. Myometrial flow of arcuate arteries was significantly increased in the 6th month of treatment. Increased endometrial thickness was significant in the 3rd month. In EG, the authors detected simple endometrial hyperplasia without atypias in 20.8% of the subjects. In EG, in the 6th month of treatment, upon comparing women who had proliferation and those who maintained the endometrial atrophy, we observed that the group that presented proliferation had significant reduction of vascular resistance of left uterine artery, whereas the group that maintained atrophy had increase in vascular resistance of right uterine artery. In CG, there was no vascular resistance modification, no myometrial flow diference and no endometrial proliferation. CONCLUSION: Isolated estrogen therapy for 6 months reduced vascular resistance of bilateral uterine arteries. Morphological affections to the endometrium preceded myometrial vascular abnormalities. There was association of endometrial proliferation and reduction of vascular resistance of the left uterine artery in the 6th month of estrogen treatment

Objetivo: Medir a expressão da integrina av~3 no endométrio de mulheres usuárias do DIUT200. Desenho: Estudo observacional controlado. Local realizado: Centro de saúde secundário e laboratório universitário. Pacientes: Treze mulheres sadias e férteis (controles) e treze usuárias do DIUT200 (casos). lntervençao: Biópsia endometrial realizada entre o 6°-1 0° dia pós-ovulatório do ciclo menstrual. Principal Desfecho Avaliado: A expressão da integrina av~3 através do HSCORE em amostras endometriais criopreservadas. Resultados: O HSCORE das usuárias do DIUT200 foi 0,9 ± 0,7 (média± DP), enquanto que o dos controles foi 2,13 ± 0,7 (média ± DP) (p = 0.001 Teste-t de Student). Todos os controles foram positivos para a expressão da integrina av~3. mas as usuárias do DIUT200 não apresentou positivdade para a integrina av~3 em 38,5% dos casos (p = 0,03 Teste Exato de Fisher). Conclusao: Os resultados apoiam a teoria que o DIUT200 de cobre também tem um mecanismo de ação que interfere diretamente com a receptividade uterina e a implantação. / Objective: To measure the expression of avf33 integrin in the endometrium of IUDT200 users. Design: Observational controlled study Setting: Secondary health care center and University laboratory Patients: Thirteen healthy fertile women (contrais) and thirteen IUDT200 users (cases). lntervention: Endometrial biopsy on postovulatory day 6-1 O of the menstrual cycle. Main Outcome Measure: The expression of avJ33 by HSCORE on cryopreserved endometrial sections. Results: The HSCORE for IUD users was 0.9 ± 0.7 (mean ± SEM), while for contrais was 2.13 ± 0.7 (mean ±SEM) (p < 0.001 Teste-t de Student). Ali contrais were positiva for avJ33, but women with IUD did not express avl33 integrin in 38.5% of the cases (p < 0.03 Fisher's exact test). Conclusion: These results support the theory that copper IUD T200 also has a mechanism of action that is directed at interference with uterine receptivity and implantation.

Nas éguas cíclicas, a luteólise ocorre entre os dias 14 e 16 após ovulação, pela ação da PGF2&#940 endometrial. Entretanto, durante a gestação, a luteólise deve ser bloqueada, ao mesmo passo que a ação da PGE2 deve ser estimulada. Ambos hormônios possuem baixa difusão pela membrana plasmática, sendo necessária a presença da proteína transportadora de prostaglandina (PGT) para o influxo e efluxo destes hormônios nas células. Os objetivos deste experimento foram identificar e relacionar o RNAm da PGT no endométrio de éguas cíclica e gestante aos 14 dias (experimento 1) e avaliar o perfil do RNAm para PGT no endométrio eqüino em final de diestro sob efeito de secreção embrionária (experimento 2). Para o experimento 1, um ciclo estral de 11 éguas foi acompanhado. Seis éguas não foram inseminadas e somente detectado o tempo de ovulação e cinco foram inseminadas. Biópsias endometriais foram realizadas quando detectado folículo pré-ovulatório (&#8805;35mm de diâmetro) e edema endometrial (E0; n=6), sete (E7; n=6) e quatorze (E14; n=6) dias após ovulação nas fêmeas cíclicas e aos quatorze dias de gestação (EG; n=4) nas fêmeas gestantes. No experimento 2, 5 embriões eqüinos de 13,5 dias de idade foram coletados, cultivados por 24 horas em ambiente com temperatura e CO2 controlados e o meio condicionado embrionário (MCEE) gerado foi armazenado a -80&ordm;C. Em seguida, amostras endometriais de sete éguas cíclicas aos 14 dias pós ovulação foram coletadas por biópsia uterina e cultivadas por 24 horas, em ambiente com temperatura e CO2 controlados, na presença do MCEE. O RNA total foi extraído de todas as amostras endometriais e amplificado pela reação em cadeia da polimerase em tempo real (RT-PCR), de um passo. A abundância relativa média dos transcritos foi submetida a análise de variância e as médias foram separadas pelo teste LSD (P<0,05). No experimento 1, o RNAm da PGT em tecido equino foi identificado, de maneira que as quantidades relativas deste gene foram similares entre E0, E7, E14 e EG. No experimento 2, o MCEE não modificou a quantidade de RNAm para PGT no endométrio em fase final do diestro. / In cyclic mares, luteolysis occurs between the 14th and 16th days after ovulation, due to endometrial PGF2&#940 However, in pregnant mares luteolysis must be blocked, whereas the PGE2 action must be stimulated. Both hormones have low diffusion through the plasma membrane, wherein the Prostaglandin Transporter Protein (PGT) is needed to influx and efflux of these hormones in the cells. The objectives of this experiment are to identify and to relate with the mRNA to PGT in the endometrium of cyclic and pregnant mares (experiment 1) and to evaluate the mRNA profile to PGT in equine endometrium at end of diestrous, under embryonic secretion effect (experiment 2). In the experiment 1, one estrous cycle of 11 mares (5 to 12 years old) was examined. Six mares were not inseminated and only the time of ovulation was recorded, and five mares were inseminated. Endometrial biopsies were performed when pre-ovulatory follicles (diameter &#8805; 35mm) and endometrial edema were detected (E0; n=6), seven (E7; n=6) and fourteen days (E14; n=6) after ovulation in cyclic mares, and fourteen days after ovulation in pregnant mares (EG; n=4). In the experiment 2, five embryos of 13,5 days of age were collected, cultured during 24 hours in controlled temperature and CO2 and the embrionic conditioned medium (ECM) was stored at -80&ordm;C. After that, endometrium samples of 7 mares at fourteen days after ovulation were collected by uterine biopsy and they were cultured during 24 hours, in controlled temperature and CO2, with ECM. Total RNA was extracted and submitted to amplification by one step real-time polymerase chain reaction (RT-PCR). The abundance relative average of trancripts was submitted to variance analysis and averages were separated by LSD test (P<0,05). In the experiment 1 the mRNA to equine PGT was identified so that the relative quantities of this gene were equal among E0, E7, E14 e EG. In the experiment 2, the ECM did not modify the mRNA quantity to PGT in the endometrium at end of diestrous.

Endometrial carcinoma is the most common gynaecologic cancer in the United States with about 41,200 new cases projected to occur in 2006. It often presents with abnormal uterine bleeding and spreads to the cervix in 10 to 20% of cases. Whilst early diagnosis is essential for optimal disease treatment, the best investigation for abnormal uterine bleeding remains uncertain. Although hysteroscopy has been reported to have high accuracy in predicting normal or abnormal endometrial histopathology, its accuracy varies with the underlying pathology. The highest accuracy occurs in the diagnosis of intrauterine anatomical pathology such as endometrial polyp whereas it is at its lowest in microscopic histopathology such as endometrial hyperplasia. Hysteroscopy is also potentially useful for detecting tumour spread to the uterine cervix that helps in staging and surgical planning. However, the role of hysteroscopy with guided biopsy in detecting endometrial cancer and the choice of distension medium remain to be determined. As the uterine cavity is a collapsed space, hysteroscopy requires its distension with a gaseous or liquid medium to allow complete visualization of the uterine cavity. The use of such media to rinse the uterine cavity raises the concern that when the endometrium harbours endometrial carcinoma cells, there is a potential risk of retrograde dissemination of these cells into the peritoneal cavity. The work in this thesis has addressed four major issues of diagnostic hysteroscopy in the management of patients with endometrial carcinoma. Firstly, the role of diagnostic hysteroscopy and guided biopsy is limited especially in microscopic tumours. Secondly, the role of diagnostic hysteroscopy to detect cervical invasion in preoperative staging of endometrial carcinoma is proven and the usage of normal saline is more accurate than that which uses carbon dioxide. Thirdly, hysteroscopic dissemination occurs more frequent when using normal saline as opposed to carbon dioxide as the distension medium. Lastly, complete occlusion of both fallopian tubes can effectively prevent the dissemination of endometrial carcinoma cells into the peritoneal cavity during diagnostic hysteroscopy. / Lo, Wing Kit Keith. / "May 2006." / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5873. / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (167-193). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

Wong Yuk-Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 132-152). / abstract --- p.1 / ACKNOWLEDGEMENT --- p.4 / content --- p.6 / Chapter I. --- INTRODUCTION --- p.8 / Chapter II. --- literature reviews --- p.11 / Chapter 1. --- Menstrual cycle --- p.11 / Chapter 2. --- oestrogen receptor and progesterone receptor --- p.18 / Chapter 3. --- Monoclonal antibody assays for the study of oestrogen and progesterone receptors --- p.30 / Chapter III. --- materials and methods --- p.35 / Chapter 1. --- study population --- p.35 / Chapter 2. --- sample collection and analysis JO --- p.36 / Chapter 3. --- Histological dating of endometrial biopsies --- p.37 / Chapter 4. --- Determination of oestrogen and progesterone receptors using immunocytochemical assay --- p.38 / Chapter 5. --- Determination of oestrogen and progesterone receptors using enzyme immunoassay --- p.52 / Chapter 6. --- Determination of serum oestradiol and progesterone --- p.66 / Chapter 7. --- Data handling and statistical analysis --- p.76 / Chapter IV. --- results --- p.77 / Chapter 1. --- Study population --- p.77 / Chapter 2. --- Histological dating of endometrial biopsies --- p.77 / Chapter 3. --- oestrogen and progesterone receptors in frozen section of endometrium in menstrual cycle --- p.80 / Chapter 4. --- oestrogen and progesterone receptors in paraffin section of endometrium in menstrual cycle --- p.95 / Chapter 5. --- Oestrogen and progesterone receptors in endometrium in menstrual cycle determined by enzyme immunoassay --- p.113 / Chapter 6. --- Serum oestradiol and progesterone --- p.116 / Chapter V. --- DISCUSSIONS --- p.120 / Chapter 1. --- oestrogen and progesterone receptors in frozen section of endometrium in menstrual cycle --- p.121 / Chapter 2. --- Oestrogen and progesterone receptors in paraffin section of endometrium in menstrual cycle --- p.124 / Chapter 3. --- oestrogen and progesterone receptors in endometrium in menstrual cycle determined by enzyme immunoassay --- p.127 / Chapter 4. --- Potential application of oestrogen and progesterone receptors in endometrium in menstrual cycle --- p.129 / REFERENCE --- p.132

Chan Ling Nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 143-153). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The Human Endometrium --- p.1 / Chapter 1.1.1 --- The Structure of the Endometrium --- p.1 / Chapter 1.1.2 --- Cyclic Changes in the Endometrium --- p.1 / Chapter 1.1.3 --- Physiological Roles of the Endometrium --- p.5 / Chapter 1.1.4 --- Roles of Luminal Epithelium in Implantation --- p.5 / Chapter 1.1.5 --- Exocrine Functions of the Endometrial Epithelium --- p.6 / Chapter 1.2 --- Review of Epithelial Ion Channels --- p.8 / Chapter 1.2.1 --- Epithelial Na+ Channels (ENaC) in Absorbing Epithelia --- p.9 / Chapter 1.2.2 --- Epithelial C1- Channels in Secretory Epithelia --- p.13 / Chapter 1.2.3 --- Na+ and C1- Channels in Endometrial Epithelia --- p.15 / Chapter 1.3 --- Review of the Intracellular Signal Transduction Pathways --- p.15 / Chapter 1.3.1 --- The cAMP-Mediated Signal Transduction Pathway --- p.17 / Chapter 1.3.2 --- The cAMP-Mediated Chloride Channels in Epithelial Cells --- p.17 / Chapter 1.3.3 --- Ca2+-Dependent Signal Transduction Pathway --- p.21 / Chapter 1.4 --- Physiological Roles of some Neurohormonal Agents in Uterine Functions: Selected Examples --- p.23 / Chapter 1.4.1 --- Roles of Adrenaline on the Endometrial Ion Transport --- p.23 / Chapter 1.4.2 --- Prostaglandin (PG) E2 and PGF2α --- p.24 / Chapter 1.4.3 --- Biological Effect of Extracellular Nucleotides --- p.26 / Chapter 1.5 --- Objective of this Study --- p.28 / Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Culture Media and Enzymes --- p.31 / Chapter 2.1.2 --- Drugs --- p.31 / Chapter 2.1.3 --- Chemicals --- p.32 / Chapter 2.1.4 --- Experimental Tissues and Animals --- p.32 / Chapter 2.2 --- Preparations --- p.32 / Chapter 2.2.1 --- Previous Support for Cell Growth --- p.32 / Chapter 2.2.2 --- Growth Medium --- p.33 / Chapter 2.2.3 --- Culture of Mouse Endometrial Epithelial Cells --- p.35 / Chapter 2.2.4 --- Solutions for the Short-Circuit Current Measurements --- p.36 / Chapter 2.2.5 --- Solutions for the Patch-Clamp Experiments --- p.38 / Chapter 2.2.6 --- Running Buffers for RNA and DNA Gel Electrophoresis --- p.39 / Chapter 2.2.7 --- UTP-free UDP --- p.40 / Chapter 2.2.8 --- Electrodes for the Short-Circuit Current Measurement --- p.40 / Chapter 2.3 --- Protocols --- p.41 / Chapter 2.3.1 --- Characterization of Neurohormonal Agents-induced Ion Channels --- p.41 / Chapter 2.3.2 --- Possible Interaction between CFTR and ENaC --- p.41 / Chapter 2.3.3 --- Characterization of Pyrimidinoceptors-mediated Conductances --- p.42 / Chapter 2.4 --- Methods of Measurements --- p.42 / Chapter 2.4.1 --- The Patch-Clamp Technique --- p.42 / Chapter 2.4.1.1 --- The Patch-Clamp Setup --- p.43 / Chapter 2.4.1.2 --- Shielding and Grounding --- p.45 / Chapter 2.4.1.3 --- Pipette Fabrication --- p.45 / Chapter 2.4.1.4 --- Pipette Holder and Electrodes --- p.48 / Chapter 2.4.1.5 --- Experimental Procedures --- p.49 / Chapter 2.4.1.6 --- Signal Recording and Data Acquisition --- p.54 / Chapter 2.4.1.7 --- Data Analysis --- p.54 / Chapter 2.4.2 --- The Short-Circuit Current Technique --- p.55 / Chapter 2.4.2.1 --- The Short-Circuit Current Setup --- p.56 / Chapter 2.4.2.2 --- Experimental Procedures --- p.56 / Chapter 2.4.2.3 --- Data Analysis --- p.61 / Chapter 2.4.3 --- Reverse Transciption - Polymerase Chain Reaction (RT-PCR) --- p.61 / Chapter 2.4.3.1 --- RNA Isolation --- p.61 / Chapter 2.4.3.2 --- RNA Gel Electrophoresis --- p.62 / Chapter 2.4.3.3 --- Reverse Transcription (RT) --- p.63 / Chapter 2.4.3.4 --- Polymerase Chain Reaction (PCR) --- p.64 / Chapter 2.4.3.5 --- DNA Gel Electrophoresis --- p.66 / Chapter 2.4.4 --- Statistical Analysis --- p.66 / Chapter 3 --- Results --- p.67 / Chapter 3.1 --- Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Response to Hormonal Stimuli --- p.67 / Chapter 3.2 --- Inhibition of Na+ Absorption by CFTR --- p.89 / Chapter 3.3 --- Pyrimidinoceptors-activated Ca2+-dependent C1- Conductance --- p.111 / Chapter 4 --- General Discussions --- p.132 / Appendix --- p.140 / Chapter A --- RNA Isolation --- p.140 / Chapter B --- Reverse Transcription --- p.141 / Chapter C --- Polymerase Chain Reaction --- p.142 / References --- p.143

Orientador: Rogério Dias / Coorientador: Daniel Spadoto Dias / Banca: Maria Aparecida Custódio Domingues / Banca: Waldir Pereira Modotte / Resumo: Objetivo: Avaliar a expressão imunoistoquímica de receptores de estrogênio (RE) e progesterona (RP), de proteínas relacionadas à proliferação celular (Ki-67), à neoangiogênese (endoglina - CD105), à adesão celular (claudinas 3 e 4) e proteínas da matriz extracelular (metaloproteinases 2 e 9 - MMP 2 e MMP 9) nos pólipos endometriais e no câncer de endométrio comparativamente ao endométrio normal. Tipo de Estudo: Estudo transversal comparativo com amostra de conveniência. O levantamento foi realizado através de banco de dados do Laboratório de Patologia Clínica da Faculdade de Medicina de Botucatu. Local: Hospital das Clínicas da Faculdade de Medicina de Botucatu, Universidade Estadual Paulista "Júlio de Mesquita Filho" - UNESP. Pacientes: Foram realizados estudos imunoistoquímicos de 30 amostras de pólipos endometriais sem atipias e de 30 amostras de adenocarcinoma endometrial do tipo endometrioide e confrontados com os resultados da análise de 30 amostras de endométrio normal (grupo controle). Intervenções: Dados epidemiológicos, clínicos e antropométricos foram levantados através de análise dos prontuários. Para análise dos casos de adenocarcinoma de endométrio e dos controles foi empregada a técnica de tissue microarray (TMA). Os blocos de parafina, com os cortes do maior fragmento de lesão polipoide e os blocos receptores de TMA foram utilizados para avaliação imunoistoquímica de RE, RP, CD105, Ki-67, claudinas 3 e 4, MMP-2 e MMP-9. Resultados Principais: Identificou-se diferença significativa entre os grupos na expressão de RE (P<0,001) e RP (P<0,05), do Ki-67 (P<0,001), do CD105 (P<0,001) e da claudina 3 (P<0,001). Não foram identificadas diferenças nos marcadores pesquisados entre pólipos e câncer de endométrio (P≥0,05). A expressão de MMP-2 e MMP-9 foi praticamente ausente nos três grupos. Conclusões: Nas amostras pesquisadas, não foram demonstradas diferenciações entre os... / Abstract: Objective: To evaluate the immunohistochemical expression of estrogen receptors (RE), progesterone receptors (RP), cell proliferation (Ki-67), neoangiogenesis (endoglin - CD105), cell adhesion (claudin 3 and 4) and extracellular matrix proteins (metalloproteinases 2 and 9 - MMP 2 and MMP 9) in endometrial polyps and endometrial cancer compared to normal endometrium. Design: Comparative cross-sectional study with occasional sample. The survey was conducted from the database of the Clinical Pathology Laboratory of Botucatu Medical School - Unesp. Setting: Clinics Hospital from Botucatu Medical School, São Paulo State University - UNESP. Patients: Were performed immunohistochemical studies of 30 samples of endometrial polyps without atypia and 30 samples of endometrioid adenocarcinoma of endometrium confronted with the results of 30 normal endometrial samples (control group). Interventions: Epidemiological, clinical and anthropometric data were collected through analysis of medical records. To analyze the cases of endometrial adenocarcinoma and controls we used the tissue microarray technique (TMA). Paraffin blocks, with the larger fragment of polypoid lesions and recipient blocks of TMA were used to evaluate immunohistochemical expression of ER, PR, CD105, Ki-67, claudin 3 and 4, MMP-2 and MMP-9. Main Results: We identified a significant difference between groups in ER (P <0.001) and PR expression (P<0.05), Ki-67 (P<0.001), CD105 (P<0.001) and claudin 3 (P<0.001). No difference was found between polyps and endometrial cancer (P≥0,05). The expression of MMP-2 and MMP-9 was virtually absent in all three groups. Conclusions In the studied samples according to the immunohistochemical parameters evaluated no differentiation between polyps and endometrial cancer was observed. There was no expression of MMP-2 and -9 in endometrial tissues analyzed. Further studies are necessary to better understand ... / Mestre