Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 361-374).

TITLE: IN VIVO ANTRAL FOLLICLE WALL BIOPSY: A NEW RESEARCH TECHNIQUE TO STUDY OVARIAN FUNCTION USING THE HORSE AS A MODEL Abstract Understanding hormone and growth factor receptor expression in the follicular wall associated with the intrafollicular milieu and systemic endocrine aspects in vivo is crucial for a complete discovery of mechanisms behind folliculogenesis in different species. Furthermore, the comprehension of the interrelationships existent among the preovulatory follicle (POF) and corpus luteum (CL) diameter and blood flow, and progesterone (P4) production during different seasons of the year are warranted to optimize fertility in different species. In this dissertation, four studies in mares are presented with the following general objectives: (i) develop and validate a minimally invasive in vivo technique to obtain antral follicle wall biopsy (FWB) and follicular fluid (FF) samples simultaneously and repeatedly from the same individuals, during different stages of follicle development in different seasons of the year; (ii) use the FWB technique to elucidate and compare the expression pattern of different receptors and proteins involved in folliculogenesis and ovulatory mechanisms during different seasons of the year; and (iii) use B-mode and color Doppler ultrasonography techniques to characterize POF and CL diameter and blood flow during two transitional ovulatory seasons (spring and fall), and elucidate the interrelationships among these variables with systemic P4 concentration. In the first study (Chapter 2), two experiments were conducted; in Experiment 1 (ex vivo), a proof of concept study was developed to check the feasibility of using the biopsy forceps to harvest FWB samples. Ovaries of six slaughtered mares were used, and each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from growing follicles during various developmental stages were harvested using a novel FWB transvaginal ultrasound-guided technique. In the second study (Chapter 3), antral follicle wall and FF samples were obtained from 10−, 20−, and 30−mm follicles during spring anovulatory (SAN) and spring ovulatory (SOV) seasons. Samples were collected in vivo from the same mares in both seasons, with the aims to study and compare the expression of proliferative, angiogenic, and pro-/anti-apoptotic receptors and proteins in the follicle wall among three follicle classes, and between the SAN and SOV seasons. In the third study (Chapter 4), FWB and FF samples were collected in vivo from dominant growing follicles during the SAN, SOV, summer (SU), and fall ovulatory (FOV) seasons, to study the expression patterns of different receptors and proteins involved in follicle development. In the fourth study (Chapter 5), the temporal relationships among diameter and blood flow of the POF and CL, and systemic P4 concentration during spring and fall transitional ovulatory seasons were studied. The main findings of the present studies were: (i) the histological appearance, organization, and thickness of the follicle wall layers (i.e. granulosa, theca interna, and theca externa) were not influenced by the harvesting techniques (biopsy forceps vs. scalpel blade); (ii) the overall recovery rates of FWB and FF samples were 97% and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size; (iii) follicles of the SOV season had a higher expression of proliferative, angiogenesis, and anti-apoptotic proteins compared to the SAN season; (iv) follicles of the FOV season had lower expression of proliferative, angiogenesis, and luteinizing hormone receptors; and (v) positive correlations were observed among POF and CL parameters, and P4 concentration. In conclusion, results demonstrated that the novel in vivo FWB technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function in mares. Furthermore, results presented herein showed that several proliferative, angiogenic, and pro-/anti-apoptotic proteins are differentially expressed within different layers of the antral follicle wall among different seasons of the year. Finally, results demonstrated that larger and well-vascularized POFs have the potential to produce larger CLs with greater blood flow, and subsequently greater P4 systemic levels.

H παρούσα μελέτη είχε σαν στόχο την μελέτη και τον προσδιορισμό των αιμοδυναμικών αλλαγών που συμβαίνουν στο ηπατικό παρέγχυμα με τη βοήθεια της απλής (Gray Scale Ultrasonography, GSU) και της έγχρωμης Doppler υπερηχοτομογραφίας (Color Doppler, CDU), στην διάρκεια της χρόνιας ηπατικής νόσου. Ο απώτερος σκοπός ήταν ο διαχωρισμός της χρόνιας νόσου του ήπατος από την αρχόμενη καλά αντιρροπούμενη κίρρωση σε ομάδα ασθενών με ένα καλά τεκμηριωμένο ιστολογικό profil. Για τον λόγο αυτό, έγινε προσπάθεια να απομονωθούν εκείνες οι GSU και CDU παράμετροι που με μεγάλη ακρίβεια διαχώριζαν τους ασθενείς με τη χρόνια ηπατίτιδα από αυτούς που έπασχαν από καλά αντιρροπούμενη κίρρωση αρχικού σταδίου (CIR). / To assess the value of gray scale (GS) and colour Doppler ultrasonography (CDU) in differentiating the progression of chronic viral hepatitis (CVH) and compensated liver cirrhosis (CIR).

INTRODUÇÂO: A tireoidite crônica autoimune (TCA) é a principal causa de hipotireoidismo adquirido, o qual requer tratamento contínuo com levotiroxina (LT4). Até o momento, não há terapia capaz de regenerar o tecido tireóideo lesado e melhorar sua função. Como a terapia com Laser de baixa intensidade (LILT) foi eficaz em outras doenças autoimunes, bem como na regeneração de vários tecidos, o objetivo deste estudo foi avaliar a eficácia do Laser de baixa intensidade no tratamento de pacientes com hipotireoidismo decorrente de tireoidite crônica autoimune utilizando-se os seguintes parâmetros de resposta: a) o mapeamento dúplex-Doppler colorido da tireoide; b) a função tireóidea estimada pela dose de LT4 necessária para manter as concentrações séricas de T3 total, T4 total, T4 livre e TSH normais; c) as concentrações séricas de anticorpos antiperoxidase tireóidea (TPOAb) e antitireoglobulina (TgAb). MÉTODOS: Trata-se de ensaio clínico randomizado, placebo-controlado, conduzido no Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, de março de 2006 a março de 2009, no qual foram incluídos 43 pacientes com hipotireoidismo causado por TCA. Todos eles apresentavam altas concentrações séricas de TPOAb e/ou TgAb e padrão ultrassonográfico compatível com TCA. Os pacientes foram randomizados em grupo L (submetido à LILT, n = 23) e P (submetido ao placebo, n = 20). Os limites da tireoide foram demarcados com o auxílio da ultrassonografia. Pacientes do grupo L submeteram-se à LILT (830 nm) e os do grupo P à função placebo do mesmo equipamento. Ambos os grupos foram submetidos, no total, à 10 sessões, duas vezes por semana, com a mesma técnica. Realizou-se pré e 30 dias pós-intervenção: o estudo ultrassonográfico (US) pelo modo-B, que incluiu o histograma computadorizado de escala de cinzas para estimar quantitativamente o índice de ecogenicidade; o US-Doppler colorido de amplitude atribuindo-se valores de 0 a 4 para os padrões de vascularização e o US-Doppler pulsado para estimar a velocidade de pico sistólico e o índice de resistividade das artérias tireóideas superiores e inferiores. Após o segundo US, os pacientes descontinuaram a LT4, a qual foi reintroduzida para os pacientes que apresentaram hipotireoidismo, em dose suficiente para obter normalização hormonal. Realizaram-se determinações séricas de T3 total, T4 total, T4 livre, TSH, TPOAb e TgAb pré-intervenção e no 1º, 2º, 3º, 6º e 9º meses pós-suspensão de LT4. RESULTADOS: No US modo-B pós-intervenção, verificou-se aumento estatisticamente significativo do índice de ecogenicidade no grupo L (1,24 ± 0,11) comparado ao P (0,98 ± 0,07; P < 0,001), assim como a proporção de pacientes com volume normal foi estatisticamente maior no grupo L (P = 0,005). O US-Doppler colorido de amplitude mostrou que o valor do padrão de vascularização foi estatisticamente maior no grupo P (2,3 ± 0,27) do que no L (1,87 ± 0,36; P = 0,033). Observou-se redução da dose de LT4 no grupo L (38,59 ± 20,22 g/dia) comparada à do P (106,88 ± 22,9 g/dia; p < 0,001). TPOAb foi menor no grupo L (681,91 ± 317,44 U/mL) do que no P (1176,40 ± 551,9 U/mL; p = 0,043). Não houve redução de TgAb e efeitos adversos. CONCLUSÕES: A LILT foi eficaz no tratamento da TCA, uma vez que no grupo L verificou-se: a) melhora da ecogenicidade, do volume e do padrão de vascularização da glândula tireoide no mapeamento dúplex-Doppler colorido; b) melhora da função da glândula tireoide, evidenciada pela redução da dose de LT4 necessária para tratar o hipotireoidismo c) modulação parcial da autoimunidade, demonstrada por meio da redução das concentrações séricas de TPOAb / INTRODUCTION: A chronic autoimmune thyroiditis (CAT) is the main cause of acquired hypothyroidism which requires continuous treatment with levothyroxine (LT4). So far there has been no such therapy which can make the damaged thyroid tissue regenerate, improving its function. As the low-intensity Laser therapy (LILT) was effective in other autoimmune diseases, as well as in regenerating several tissues, the objective of this study was to evaluate the efficacy of LILT in patients with hypothyroidism caused by CAT by utilizing the following response parameters: A) Color Doppler ultrasonography of thyroid; B) The thyroid function estimated by the dose of LT4 in order to keep the serum concentrations of normal T3, T4, free T4 (fT4) and TSH; C) The serum concentrations of thyroid peroxidase (TPOAb) and thyroglobulin antibodies (TgAb). METHODS: This is a placebo-controlled randomized clinical essay guided at the Institute of Radiology, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo from March 2006 to March 2009, made up of 43 patients with hypothyroidism caused by CAT. All the patients showed high serum concentrations of TPOAb and/or TgAb and ultrasound pattern compatible with CAT. The patients were randomized in L group (submitted to LILT, n = 23) and P group (submitted to placebo, n = 20). The limits of thyroid were marked off with the help of ultrasonography. The patients in L group were submitted to LILT (830 nm) and the patients in P group were submitted to the placebo function of the same equipment. Both groups were submitted a total of 10 sessions, twice a week, using the same technique. Pre- and 30 days post-intervention were applied: ultrasonographic study (US) by B-mode, which included the grey scale computerized histogram to quantitatively estimate the index of echogenicity; the amplitude color Doppler US with values given from 0 to 4 for the vascularization patterns and the pulsed Doppler US to estimate the systolic peak velocity and the index of resistivity of superior and inferior thyroid arteries. After the second US the patients discontinued the LT4, which was later re-introduced in the patients having hypothyroidism in a certain amount so as to be sufficient to obtain hormonal normalization. Serum determinations of total T3, total T4, fT4, TSH, TPOAb and TgAb pre-intervention were accomplished and also in the 1st, 2nd, 3rd, 6th and 9th month post-suspension of LT4. RESULTS: In post-intervention B-mode US a significant increase in the index of echogenicity in L group (1.24 ± 0.11) was statistically observed compared with the P group (0.98 ± 0.07; P < 0.001), as well as the proportion of patients with normal volume was shown statistically higher in L group (P = 0.005). The amplitude color Doppler US showed the standard value of vascularization was statistically greater in P group (2.3 ± 0.27) than in L group (1.87 ± 0.36; P = 0.033). Pulsed Doppler US showed an increase in the systolic peak velocity of the inferior thyroid arteries in L group (34.47 ± 4.81 cm/s) in relation to P group (26.12 ± 4.29 cm/s; P = 0.016). A reduction in the dose of LT4 in L group (38.59 ± 20.22 g/day) was observed compared with the one in P group (106.88 ± 22.9 g/day; p < 0.001). TPOAb was smaller in L-group (681.91 ± 317.44 U/mL) than in P-group (1176.40 ± 551.9 U/mL; p = 0.043). There was no reduction of TgAb and adverse effects. CONCLUSIONS: LILT was effective in the treatment of CAT, once L group showed: A) amelioration of echogenicity, of volume and of vascularization of the thyroid gland in the color Doppler ultrasonography; B) improvement of thyroid function, featured through the reduction in the necessary dose of LT4 to treat the hypothyroidism; C) partial modulation of autoimmunity demonstrated by reduction of TPOAb serum concentrations

INTRODUÇÂO: A tireoidite crônica autoimune (TCA) é a principal causa de hipotireoidismo adquirido, o qual requer tratamento contínuo com levotiroxina (LT4). Até o momento, não há terapia capaz de regenerar o tecido tireóideo lesado e melhorar sua função. Como a terapia com Laser de baixa intensidade (LILT) foi eficaz em outras doenças autoimunes, bem como na regeneração de vários tecidos, o objetivo deste estudo foi avaliar a eficácia do Laser de baixa intensidade no tratamento de pacientes com hipotireoidismo decorrente de tireoidite crônica autoimune utilizando-se os seguintes parâmetros de resposta: a) o mapeamento dúplex-Doppler colorido da tireoide; b) a função tireóidea estimada pela dose de LT4 necessária para manter as concentrações séricas de T3 total, T4 total, T4 livre e TSH normais; c) as concentrações séricas de anticorpos antiperoxidase tireóidea (TPOAb) e antitireoglobulina (TgAb). MÉTODOS: Trata-se de ensaio clínico randomizado, placebo-controlado, conduzido no Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, de março de 2006 a março de 2009, no qual foram incluídos 43 pacientes com hipotireoidismo causado por TCA. Todos eles apresentavam altas concentrações séricas de TPOAb e/ou TgAb e padrão ultrassonográfico compatível com TCA. Os pacientes foram randomizados em grupo L (submetido à LILT, n = 23) e P (submetido ao placebo, n = 20). Os limites da tireoide foram demarcados com o auxílio da ultrassonografia. Pacientes do grupo L submeteram-se à LILT (830 nm) e os do grupo P à função placebo do mesmo equipamento. Ambos os grupos foram submetidos, no total, à 10 sessões, duas vezes por semana, com a mesma técnica. Realizou-se pré e 30 dias pós-intervenção: o estudo ultrassonográfico (US) pelo modo-B, que incluiu o histograma computadorizado de escala de cinzas para estimar quantitativamente o índice de ecogenicidade; o US-Doppler colorido de amplitude atribuindo-se valores de 0 a 4 para os padrões de vascularização e o US-Doppler pulsado para estimar a velocidade de pico sistólico e o índice de resistividade das artérias tireóideas superiores e inferiores. Após o segundo US, os pacientes descontinuaram a LT4, a qual foi reintroduzida para os pacientes que apresentaram hipotireoidismo, em dose suficiente para obter normalização hormonal. Realizaram-se determinações séricas de T3 total, T4 total, T4 livre, TSH, TPOAb e TgAb pré-intervenção e no 1º, 2º, 3º, 6º e 9º meses pós-suspensão de LT4. RESULTADOS: No US modo-B pós-intervenção, verificou-se aumento estatisticamente significativo do índice de ecogenicidade no grupo L (1,24 ± 0,11) comparado ao P (0,98 ± 0,07; P < 0,001), assim como a proporção de pacientes com volume normal foi estatisticamente maior no grupo L (P = 0,005). O US-Doppler colorido de amplitude mostrou que o valor do padrão de vascularização foi estatisticamente maior no grupo P (2,3 ± 0,27) do que no L (1,87 ± 0,36; P = 0,033). Observou-se redução da dose de LT4 no grupo L (38,59 ± 20,22 g/dia) comparada à do P (106,88 ± 22,9 g/dia; p < 0,001). TPOAb foi menor no grupo L (681,91 ± 317,44 U/mL) do que no P (1176,40 ± 551,9 U/mL; p = 0,043). Não houve redução de TgAb e efeitos adversos. CONCLUSÕES: A LILT foi eficaz no tratamento da TCA, uma vez que no grupo L verificou-se: a) melhora da ecogenicidade, do volume e do padrão de vascularização da glândula tireoide no mapeamento dúplex-Doppler colorido; b) melhora da função da glândula tireoide, evidenciada pela redução da dose de LT4 necessária para tratar o hipotireoidismo c) modulação parcial da autoimunidade, demonstrada por meio da redução das concentrações séricas de TPOAb / INTRODUCTION: A chronic autoimmune thyroiditis (CAT) is the main cause of acquired hypothyroidism which requires continuous treatment with levothyroxine (LT4). So far there has been no such therapy which can make the damaged thyroid tissue regenerate, improving its function. As the low-intensity Laser therapy (LILT) was effective in other autoimmune diseases, as well as in regenerating several tissues, the objective of this study was to evaluate the efficacy of LILT in patients with hypothyroidism caused by CAT by utilizing the following response parameters: A) Color Doppler ultrasonography of thyroid; B) The thyroid function estimated by the dose of LT4 in order to keep the serum concentrations of normal T3, T4, free T4 (fT4) and TSH; C) The serum concentrations of thyroid peroxidase (TPOAb) and thyroglobulin antibodies (TgAb). METHODS: This is a placebo-controlled randomized clinical essay guided at the Institute of Radiology, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo from March 2006 to March 2009, made up of 43 patients with hypothyroidism caused by CAT. All the patients showed high serum concentrations of TPOAb and/or TgAb and ultrasound pattern compatible with CAT. The patients were randomized in L group (submitted to LILT, n = 23) and P group (submitted to placebo, n = 20). The limits of thyroid were marked off with the help of ultrasonography. The patients in L group were submitted to LILT (830 nm) and the patients in P group were submitted to the placebo function of the same equipment. Both groups were submitted a total of 10 sessions, twice a week, using the same technique. Pre- and 30 days post-intervention were applied: ultrasonographic study (US) by B-mode, which included the grey scale computerized histogram to quantitatively estimate the index of echogenicity; the amplitude color Doppler US with values given from 0 to 4 for the vascularization patterns and the pulsed Doppler US to estimate the systolic peak velocity and the index of resistivity of superior and inferior thyroid arteries. After the second US the patients discontinued the LT4, which was later re-introduced in the patients having hypothyroidism in a certain amount so as to be sufficient to obtain hormonal normalization. Serum determinations of total T3, total T4, fT4, TSH, TPOAb and TgAb pre-intervention were accomplished and also in the 1st, 2nd, 3rd, 6th and 9th month post-suspension of LT4. RESULTS: In post-intervention B-mode US a significant increase in the index of echogenicity in L group (1.24 ± 0.11) was statistically observed compared with the P group (0.98 ± 0.07; P < 0.001), as well as the proportion of patients with normal volume was shown statistically higher in L group (P = 0.005). The amplitude color Doppler US showed the standard value of vascularization was statistically greater in P group (2.3 ± 0.27) than in L group (1.87 ± 0.36; P = 0.033). Pulsed Doppler US showed an increase in the systolic peak velocity of the inferior thyroid arteries in L group (34.47 ± 4.81 cm/s) in relation to P group (26.12 ± 4.29 cm/s; P = 0.016). A reduction in the dose of LT4 in L group (38.59 ± 20.22 g/day) was observed compared with the one in P group (106.88 ± 22.9 g/day; p < 0.001). TPOAb was smaller in L-group (681.91 ± 317.44 U/mL) than in P-group (1176.40 ± 551.9 U/mL; p = 0.043). There was no reduction of TgAb and adverse effects. CONCLUSIONS: LILT was effective in the treatment of CAT, once L group showed: A) amelioration of echogenicity, of volume and of vascularization of the thyroid gland in the color Doppler ultrasonography; B) improvement of thyroid function, featured through the reduction in the necessary dose of LT4 to treat the hypothyroidism; C) partial modulation of autoimmunity demonstrated by reduction of TPOAb serum concentrations